Abstracts from the 34th Annual Meeting, European

Journal of Molecular and Cellular Cardiology 109 (2017) 1–62

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Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc

Abstract publication

Abstracts from the 34th Annual Meeting, European Section of the International Society for Heart Research, July 24–37, 2017, Hamburg, Germany 1. TRIM24 promotes while TRIM32 inhibits cardiomyocyte hypertrophy via regulation of dysbindin protein levels

TRIM24/32. The present study also implies that TRIM32 is a key regulator of apoptosis in cardiomyocytes via simultaneous activation of p53 and Caspase-3/-7, and inhibition of XIAP. 2.

A. Borlepawar1,2, A.Y. Rangrez1,2, A. Bernt1,2, L. Christen1,2, S. Sossalla1,2, D. Frank1,2, N. Frey1,2

MicroRNA hsa-miR-665 prevents cardiomyocyte hypertrophy and preserves normal cardiac function after pressure overload

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Department of Internal Medicine III (Cardiology, Angiology, Intensive Care), University Medical Center Kiel, Kiel, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Lübeck, Germany Background: We have previously shown that Dysbindin is a potent inducer of cardiomyocyte hypertrophy via activation of Rho-dependent SRF signaling. Methods and results: We now performed a yeast-two hybrid screen using Dysbindin as bait against a cardiac cDNA library to identify the cardiac Dysbindin interactome. Amongst several putative binding proteins, we identified Tripartite motif-containing protein 24 (TRIM24) and confirmed this interaction by co-immunoprecipitation and co-immunostaining. Like other TRIM family proteins, TRIM32, which targets Dysbindin for degradation in skeletal muscle, degraded Dysbindin in cardiomyocytes as well. Surprisingly however, TRIM24 did not promote Dysbindin decay but rather protected Dysbindin against degradation by TRIM32. Correspondingly, TRIM32 attenuated the activation of SRF-signaling and hypertrophy due to Dysbindin, whereas TRIM24 promoted these effects in cardiomyocytes. Moreover, TRIM32 dramatically reduced cell viability and accelerated apoptosis via activation of p53, Caspase-3 and -7 signaling and inhibition of XIAP (X-linked inhibitor of apoptosis). Conclusion: We here provide a novel mechanism of post-translational regulation of Dysbindin in cardiomyocytes via its interaction partners

http://dx.doi.org/10.1016/j.yjmcc.2017.06.002 0022-2828/© 2017 Published by Elsevier Ltd.

L. Braga1, M. Dal Ferro1,2, M. Mano1, A. Eulalio1, S. Moimas1, L. Zentilin1, S. Zacchigna1 and M. Giacca1 1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy 2 Department of Cardiology, Azienda Sanitaria Universitaria Integrata Trieste, Trieste, Italy

Background: The adult heart is capable of remodelling in response to different pathological stimuli; in most cases, a phase of compensated hypertrophy evolves into frank dysfunction and heart failure. Methods and results: To identify microRNAs able to prevent cardiac hypertrophy and preserve cardiac function, we performed a highcontent microscopy, high-throughput functional screening for human miRNAs able to reduce neonatal cardiomyocyte (CM) cell size using a whole-genome miRNA library. The most effective anti-hypertrophic miRNAs was hsa-miR-665. In a model of transverse abdominal aortic constriction (TAC) in 8 weeks old CD1 mice (n = 14 per group), AAV9-mediated delivery of miR-665 showed remarkable capacity to protect against pathological cardiac hypertrophy and preserve function over time. This effect was observed when the vectors were delivered either before (LVEF at 60 day after TAC: 51.3% ± 5.8 in treated vs 34.82% ±0.77 in controls; P b 0.005) or after hypertrophy onset (LVEF at 60 days after TAC: 57.5% ±5.60 in treated vs 28.4% ±15 in controls; P b 0.001). Global mRNAs changes in hearts treated with miR-665

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were evaluated by mRNA deep sequencing. All the 90 genes that were found to be expressed ≤2 fold over control were individually downregulated by specific siRNAs and tested for being direct miR-665 targets. This approach identified three sarcomeric proteins as direct mediators of miR-665 activity, namely Enah, Fhl1 and Xirp2, which are known to be involved in sarcomeric I-band mechanotransduction and myofibrillar remodelling. Conclusion: miR-665 represents an important tool to decipher the molecular mechanisms of hypertrophy and offer a potential lead for the development of new biotherapeutics. 3. Expression levels of zinc transporters in human failing heart A. Durak1, Y. Olgar1, E. Tuncay1, C.V. Bitirim1, E. Ozcinar2, M.B. Inan2, K.C. Akcali1, A.R. Akar2, B. Turan1 1

Department of Biophysics, Ankara University School of Medicine, Ankara, Turkey 2 Department of Cardiovascular Surgery, Heart Center, Ankara University School of Medicine, Turkey Background: Heart failure (HF) is an irreversible process while numerous signaling mechanisms are involved during its development. Defective cardiac activity is well-characterized in HF. However, association between impaired Zn2+-homeostasis and cardiac dysfunction still remains obscure. Zn2 +-homeostasis is regulated through Zn2 +transporters. Therefore, evaluation of subcellular distributions of Zn2+-transporters may provide a better understanding of their contributions to cytosolic free Zn2+ ([Zn2+]i) in cardiomyocytes. Methods and results: We tested above hypothesis in human HF and first monitored markedly increased [Zn2 +]i in failing ventricularcardiomyocytes induced with doxorubicin. Additionally, we observed decreased expression of Zn2+-transporter ZIP8 and increased expression levels of ZIP14 and ZnT8 in both modelled-cardiomyocytes and left ventricle from human HF. We also, for the first time, showed subcellular localizations of these transporters in mammalian cardiomyocytes. Confocal examinations with calculated-Pearson's coefficients revealed that ZIP8, ZIP14 and ZnT8 were localized to both sarcolemma and S(E)R in cardiomyocytes. We also measured markedly high expressions of ER-stress markers GRP78, Gadd153 and calnexin in both HF samples and directly cytosolic Zn2+-increased cardiomyocytes. Furthermore, we detected markedly increased PKCα phosphorylation in heart preparations, which functions in regulating cardiac contractility and HF.

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Department of Cardiology, Angiology, and Pneumology, University Hospital Heidelberg, Innere Medizin III, Heidelberg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner Site Heidelberg/Mannheim, Heidelberg, Germany 3 SDSU Heart Institute and Department of Biology, San Diego State University, San Diego, CA 92182, USA Background: Sarco/endoplasmic reticulum (SR/ER) integrity is crucial for cardiac function. Cardiac diseases induce ER protein misfolding and activate ER stress. ATF6, an ER-transmembrane protein, is a sensor of ER protein misfolding. Upon ER stress, ATF6 becomes a transcription factor that induces adaptive proteins that restore ER protein folding capacity. We previously showed that several ATF6-inducible gene products protect from progression towards heart failure during pathological remodeling. However, the molecular mechanisms of this protection are unknown.

Results: In neonatal rat ventricular myocytes, ATF6 knockdown decreased cell size and phenylephrine-induced growth. Increased protein synthesis during growth led to the increased expression of ATF6-induced chaperones in adult rat cardiac myocytes. To examine the role of ATF6 in cardiac growth, in vivo, ATF6 knockout (KO) and wild type (WT) mice were treated with isoproterenol in osmotic minipumps to induce a hypertrophic response to β-adrenergic stimulation. Minipumps with saline in WT and ATF6 KO mice served as controls. Compared to WT, hearts from isoproterenol-treated ATF6 KO mice exhibited reduced cardiac hypertrophy, as assessed by heart weightto-body weight ratios and myocyte cross-sectional area. Echocardiography revealed significantly decreased fractional shortening in hearts from isoproterenol-treated ATF6 KO, compared to WT mice, suggesting that ATF6 contributes to the cardiac growth seen in isoproterenolinduced cardiac hypertrophy. RNA-seq and molecular signaling analyses revealed increased expression of pathology-associated genes in the absence of ventricular hypertrophy in the hearts of ATF6 KO mice treated with isoproterenol. Conclusion: ATF6 is required for balancing protein quantity and quality during cardiac growth. 5. Re-expression of foetal transcription factor POU4F2/Brn3b in the stressed heart – Beneficial or detrimental to cardiac function? L. Maskell, D. Stuckey, V. Budhram-Mahadeo UCL Institute of Child Health, London, UK

The stress response transcription factor ATF6 is necessary for compensatory cardiac hypertrophy

Background: Cardiovascular disease contributes to a high mortality rate in the UK. A key feature of this is cardiac hypertrophy, whereby terminally differentiated cardiomyocytes increase in size in response to stress. This can be beneficial or detrimental to cardiac function depending on the type of stress. For example, exercise induces a physiological hypertrophic response which is reversible, enhancing cardiac function. Pathological stimuli such as hypertension induce cardiomyocyte cell death, which reduces function and leads to heart failure. The fate of cardiomyocytes during these processes is highly dependent on changes in gene expression. Hence, factors that control such changes will be important for facilitating the hypertrophic response. The molecular mechanisms however are not fully understood. The transcription factor (TF) POU4F2/Brn3b is expressed in cardiomyocytes and regulates several target genes involved in cardiac development. It is highly expressed in the foetal heart and reduced in adult cardiomyocytes but is re-expressed in response to injury.

C. Hofmann1,2,3, E.A. Blackwood3, T. Jakobi1,2, C. Dieterich1,2, M. Völkers1,2, C.C. Glembotski3, S. Doroudgar1,2,3

Methods and results: In this study, male Brn3b-KO mutant mice and wild type (WT) controls were used to assess the role of this TF in

Conclusions: Our present data, regarding association between Zn2+transporters, [Zn2 +]i, PKCα activation and ER-stress, suggest that these Zn2 +-transporters together with PKCα activation may play an important role in HF via induction of ER-stress, providing a novel therapeutic approach in the treatment of human HF. Overall, our present data provide an important insight to the HF in humans, in part, due to the importance of intracellular free Zn2+ and Zn2+-transporters. Acknowledgments: This study was supported through TUBITAK for grant SBAG-113S466 and COST Action TD1304. 4.


hypertrophic responses induced by Angiotensin II infusion (pathological) or exercise (physiological), by analysing for changes in cardiac morphology, function and genetic changes. Functional parameters such as cardiac output, fractional shortening, ventricular mass, etc. were measured using echocardiography and histological changes were assessed (e.g. AFOG and Masson's Trichrome staining) at baseline and 4 weeks post exercise/Angiotensin II treatment. Both pathological (Angiotensin II) and physiological stress (exercise) caused reduced cardiac function in male Brn3b-KO mice.

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delay disease progression, in part by upregulation of RGS2. Thus, CRTC1 represents a novel player of cardiac hypertrophy. 7. The Zn2+ transporters in hypertrophied rat heart Y. Olgar1, A. Durak1, E. Tuncay1, S. Ozdemir2, B. Turan1 1

Conclusion: These data suggest complex roles for Brn3b in the hypertrophic response/stressed heart.

Department of Biophysics, Ankara University School of Medicine, Ankara, Turkey 2 Department of Biophysics, Akdeniz University, Faculty of Medicine, Antalya, Turkey

6. Crtc1-deficient mice show cardiac hypertrophy and decreased cardiac function K. Morhenn1,2, T. Quentin1, S. Schröder1, A. Pahl1, M. Steinmetz3,4, B. Geertz5, T. Eschenhagen2,5, M. Prondzynski2,5, S. Schlossarek2,5, L. Carrier2,5, W.-H. Zimmermann3,6, S. Lutz3,6, E. Oetjen1,2,7

Background: It is well accepted that numerous proteins, involved in cellular signaling pathways and playing a pivotal role in controlling cardiac contractility, are common targets of cytosolic free Zn2+ in mammalian cells. Studies indicate that regulation of cellular Zn2+ and mediation of Zn2 +-transporters in this regulation can provide a novel means against cardiac dysfunction under any pathological condition. We, herein, aimed to demonstrate possible contribution of Zn2+-transporters to hypertrophied cardiomyopathy via a remodelling of Zn2+ homeostasis.

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Department of Clinical Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner Site Hamburg/Kiel/Lübeck, Germany 3 DZHK (German Centre for Cardiovascular Research), partner Site Göttingen, Germany 4 Department of Pediatric Cardiology and Intensive Medicine, University Medical Center Göttingen, Göttingen, Germany 5 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Hamburg, Germany 6 Department of Pharmacology, University Medical Center Göttingen, Göttingen, Germany 7 Institute of Pharmacy, University of Hamburg, Hamburg, Germany Aim: We studied the effect of the transcriptional coactivator CRTC1 on maladaptive cardiac hypertrophy in human and murine hearts. Results: CRTC1 was enhanced in human and murine heart tissue under conditions of acquired and inherited maladaptive hypertrophy. In cardiomyocytes, treatment with isoprenaline, but not with endothelin or angiotensin II, dephosphorylated, thus activated CRTC1. Activation was prevented by propranolol. Crtc1-deficient (KO) mice showed a higher ratio of heart weight to tibia length and increased cardiomyocyte size than their wild-type littermates (WT). The ejection fraction and fractional shortening were reduced in KO mice while the left ventricular volume was increased. KO hearts were not fibrotic with unchanged mRNA level of Col1a1, Col3a1, and the pro-fibrotic Ctgf while mRNA expression of the metalloproteinase inhibitor Timp4 was decreased. By reducing Gαq-protein-induced signals in the heart, the Regulator of G-Protein Signaling 2 (RGS2) decreases hypertrophy. In KO mice Rgs2 mRNA and protein levels were reduced. In a luciferase reporter gene assay overexpression of CRTC1 stimulated Rgs2 promoter transcriptional activity, which was prevented by mutation of the CRTC1-DNAinteraction site. In addition, chromatin immunoprecipitation showed the recruitment of endogenous CRTC1 to the Rgs2 promoter in cardiac tissue. In neonatal mouse cardiomyocytes, this recruitment was increased by isoprenaline. The phosphorylation of the Gαq-proteindownstream kinase ERK1/2 was enhanced in KO adult mouse ventricular myocytes. Conclusion: Our data suggest that increased CRTC1 protein content in maladaptive cardiac hypertrophy is a compensatory mechanism to

Methods and results: We examined the expression levels of these transporters in heart samples from chronic hypertrophy induced with transverse aortic constriction (TAC) in rats. Hypertrophy development was confirmed by increased (around 30%) heart weight and heart weight to tibia length in TAC group. The expression level of ZIP7, ZIP14 and ZnT8 were increased significantly while the ZIP8 and ZnT7 level was decreased in TAC group rat heart and further confirmed by expression levels of these transporters in doxorubicin-induced modelled cardiomyocytes. We also monitored markedly increased cytosolic free Zn2+ level in failing modelled ventricular-cardiomyocytes. Biochemical analysis exhibited marked apoptotic status with increased ratio of Bax to Bcl-2, increased ERK phosphorylation and a decreased ratio of phosphorylated Akt to total Akt in failing heart tissue and failing-modelled cardiomyocytes. Conclusion: We therefore conclude that beyond Ca2+-dishandling in this heart model, Zn2 +-homeostasis is affected significantly being associated with the protein expression levels of ZIP-ZnT transporters, which may underlie the observed decreased contractile activity in hypertrophic cardiomyopathy. Acknowledgments: This study was supported by grant of TUBITAK SBAG-113S466 and COST Action TD1304. 8. Controlling the maturation of human engineered heart tissues with alterations in afterload M. Rodriguez1,2, T. Werner1,2, M. Hirt1,2, T. Eschenhagen1,2 1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany

Background: The engineered heart tissue (EHT) system enables three-dimensional growth of geometrically well-defined cardiac tissues and is compatible with biochemical and electrical stimulation, making it an ideal platform for the maturation of cardiac tissues. However, this system currently lacks a means of altering tissue load, which plays a critical role in cardiac growth. Here, EHTs were grown on silicone

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posts of different stiffnesses to assess the effect of afterload on tissue maturation. Methods: EHTs made from neonatal rat cardiomyocytes were grown on colored posts of four different stiffnesses (white = 0.60, yellow = 0.80, green = 1.24, and blue = 2.11 mN/mm) for 35 days. Over this period, the tissues were assessed for differences in contractile, structural, and calcium handling properties. Results: At the height of their growth, tissues on blue posts produced 3× the force than those on white or yellow posts, and those on green posts produced 2 × higher forces. However, tissues on blue posts exhibited significantly lower tissue shortening, contractile velocity, and relaxation velocity and significant increases in the percentage of ANP positive area. No significant differences in sarcomere length, cardiomyocyte cross-sectional area, cardiomyocyte number, or calcium response were found between any of the tissues. Though, qualitatively, cellular distribution appeared more even in green and blue tissues. Conclusions: In EHTs, increased afterload results in elevated contractile forces. However, tissues with the highest degree of afterload exhibited some signs of pathological growth, suggesting that intermediate values in afterload are most beneficial for cardiac maturation. 9. Structural and molecular changes associated with experimental hypertension-induced compensatory left ventricular hypertrophy G. Tanko, M. Dumitrescu, A. Constantin, M. Nemecz, E. Dragan, D. Popov, A. Georgescu, M. Simionescu Institute of Cellular Biology and Pathology “Nicolae Simionescu”, Bucharest, Romania Background: Hypertension-associated left ventricular hypertrophy evolves initially as an adaptive response meant to minimize ventricular wall stress. The mechanisms involved in cardiac function maintenance in compensatory hypertrophy are still uncertain. Purpose: to uncover the fine processes by which the heart adjusts the hypertension-induced stress. Methods: Male golden Syrian hamsters were given NG-nitro-L-arginine methyl ester (L-NAME) for 16 weeks. Structural features of the left ventricles were examined by electron microscopy. In left ventricular homogenates, proteins expression was assessed by Western blotting, the activity of MMP-2 and -9 was evidenced by zymography, and protein carbonyl content was determined spectrophotometrically. Results: L-NAME-treated hamsters became hypertensive and developed left ventricular hypertrophy without fibrosis or impaired contractility. As compared to aged-matched controls, left ventricles in hypertensive hamsters presented: (i) accumulation of lipid droplets within cardiomyocytes; (ii) relocation of gap junctions to lateral membranes, or close to mitochondria; (iii) increased expression of CD36, PKC and MMP-2, activation of PI3K/AKT pathway and unaltered expression of Cx43 and N-cadherin; (iv) higher carbonyl content, suggestive of an enhanced reactive oxygen species (ROS) formation, and (v) enhanced MMP-2 activity. Conclusion: These changes denoting the adaptations of the hypertrophied left ventricles in HT hamsters could explain the ability of the heart to maintain its contractile function. Taken together, these findings provide support for designing future approaches meant to improve the performance of the hypertensive heart.

Acknowledgements: This work was supported by the Romanian Academy and from the Romanian National Authority for Scientific Research, CNCS-UEFISCDI, through grants no. PN-II-RU-TE-2014-40525 and PN-II-PT-PCCA-2013-4-2154. 10. Activating the PGC-1α -HO-1 axis is required for attenuation of cardiomyopathy in hypertensive diabetic mice M. Waldman1,2, K. Cohen1,2, V. Nudelman1, D. Gorfil1, M. LaniadoSchwartzman3, R. Kornwoski1, N.G. Abraham3, D. Aravot1, A. Shainberg4, M. Arad2, E. Hochhauser1 1

Felsenstein Research Center, Rabin Medical Center, Sackler School of Medicine, Tel Aviv University, Heart Research, Petach Tiqwa, Israel 2 Leviev Heart Center, Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 3 New York Medical College, Department of Pharmacology, Valhalla, USA 4 The Mina and Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat Gan, Israel Background: Diabetes mellitus leads to cardiomyopathy, aggravated in the presence of hypertension. PGC-1α is a transcriptional coactivator that modulates mitochondrial biogenesis and oxidative metabolism. Caloric restriction (CR) activates SIRT1, PGC-1α and HO-1 signaling pathway. We aimed to assess the effect of CR on the development of cardiomyopathy in diabetic mice as related to activation of SIRT1, PGC-1α and HO-1 signaling. Methods: Obese diabetic db/db mice, were treated with, angiotensin II (AT) for 4 weeks to induce cardiomyopathy. Mice were concomitantly treated with CR (65% of calories) or fed ad libitum. Results: Diabetic mice treated with AT developed left ventricular hypertrophy, increased oxidative stress (MDA) and inflammation that were associated with reduction in SIRT1, HO-1 and PGC-1α levels. CR reduced MDA levels (p = 0.02) and prevented the development of cardiomyopathy. CR concomitantly normalized the reduction in SIRT activity, PGC-1α and HO-1 levels (p b 0.04). Importantly, HO-1 inhibition with SnMP in the calorically restricted mice abolished the protective effect of CR. Cultured cardiomyocytes exposed to high glucose levels exhibited increased ROS production (p b 0.05). Treatment with HO-1 inhibitor (SnMP) promoted ROS production (p b 0.04) and lead to a marked reduction in SIRT1 and PGC-1α (p b 0.03). To the contrary, induction of HO-1 with CoPP prevented the glucose-induced elevation in ROS and augmented these proteins. Conclusion: CR attenuated the development of cardiomyopathy in diabetic mice through SIRT1, HO-1 and PGC-1α.The results of this study reveal a link between SIRT1, PGC-1α and HO-1 signalling in diabetic cardiomyopathy. Targeting of PGC-1α and HO-1 may facilitate the development of therapies for cardiomyopathy in diabetes. 11. Protein expression, contractile behavior and the effect of left ventricular assist devices in pediatric cardiomyopathy I.A.E. Bollen1, M. van der Meulen2, K. de Goede1, D. Kuster1, M. Dalinghaus2, J. van der Velden1 1 Department of Physiology, Institute for Cardiovascular Research, VU University medical center, Amsterdam, The Netherlands 2 Department of Pediatric Cardiology, Erasmus Medical Center, Rotterdam, The Netherlands


Background: Typically inherited cardiomyopathies manifest themselves in adulthood, however a subset of patients experience severe heart failure at childhood (pediatric cardiomyopathy, PCM). These patients are often difficult to treat since treatments are extrapolated from adult treatment schemes as little is known about pathogenesis in PCM. The aim of this study was to assess protein expression, modification and contractile behavior in PCM patients. In addition, we defined the effect a left ventricular assist device (LVAD) on cellular changes and cardiac function. Methods: We included 11 patients between 3 and 16 years old. Most patients suffered from dilated cardiomyopathy (N = 10) and one from restrictive cardiomyopathy. Samples were taken from the left ventricle (LV) prior to implantation of a LVAD or during heart transplantation. Control samples were LV tissue from non-failing donors. Protein expression and modification was assessed by gel electrophoresis and contractile performance was measured in membrane-permeabilized cardiomyocytes.

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were disappeared after washout or nitric oxide synthase (NOS) inhibition with L-NAME application. In equal concentration of another P2Y12 inhibitor ticagrelor, fibrillation was not detected despite its negative inotropic effect on ventricular myocytes. Furthermore, the reduced contractility of ventricular myocytes in response to ticagrelor application didn't reverse with perfusion of L-NAME. Conclusion: In conclusion, findings of this study suggest that commonly used antithrombotic agent prasugrel may cause depolarization in membrane potential of ventricular myocytes and induces fibrillation via presumably NO mediated pathway. 13. The role of heterochromatin protein 1 binding partner 3, HP1BP3, in regulating systolic and diastolic function in the murine heart failure model C.X. Chan1,2, Y. Li 3, D.A.T. Luu3, K.Z. See2, J. Jiang3, R.S.Y. Foo1,2,3

Results: Maximal force was lower (p b 0.01) in PCM (23.7 ± 1.2 kN/m2, N = 8 patients, n = 43 cardiomyocytes) compared to controls (30.6 ± 2.2 kN/m2, N = 9, n = 31), a feature not observed in adult DCM. Myofilament calcium-sensitivity was higher (pb0.001) in PCM (N = 8, n = 26) compared to controls (N = 3, n = 8). Hypophosphorylation of cardiac troponin I was observed. Increased calcium-sensitivity could be normalized in PCM to controls with protein kinase A treatment which is in line with adult DCM. A large variation in titin isoform composition was observed in PCM (N = 11) and length-dependent activation was only impaired in patients with increased compliant titin. Conclusion: LVAD restored SERCA expression but did not reverse myofilament properties. 12. Prasugrel affects membrane potential and contractile functions of rat ventricular myocytes M.C. Celen1, M. Kücük2, B.E. Yamasan3, S. Kucukseymen4, S. Ozdemir3 1

NE University Meram Medical Faculty Department of Biophysics, Konya, Turkey 2 Akdeniz University Faculty of Medicine Department of Cardiology, Antalya, Turkey 3 Akdeniz University Faculty of Medicine Department of Biophysics, Antalya, Turkey 4 Training and Research Hospital Department of Cardiology, Antalya, Turkey Background: Prasugrel is an important and widely used P2Y12 inhibitor for antiplatelet therapy, nevertheless knowledge is very limited about how it is affecting the contractility and membrane potential of ventricular myocytes. This study examined the acute effect of prasugrel on contractile function and membrane potential of isolated ventricular myocytes. Methods and results: Freshly isolated ventricular myocytes from 3-month-old Wistar rat hearts were used for this study. During field stimulation at 0.5 Hz frequency, alteration in sarcomere length of ventricular myocytes was detected and analyzed. Action potential (AP) recordings were acquired with current clamp mode of patchclamp amplifier at 1 Hz. Although perfusion of myocytes with 1 μM of prasugrel didn't change AP duration and fractional shortening of ventricular myocytes, there was remarkable depolarization in resting membrane potential of myocytes which was followed by apparent fibrillation. When concentration of prasugrel was increased to 10 μM, repeated fibrillations were observed (in all tested cells n = 8) which

1 NUS Graduate School for Integrative Sciences and Engineering, NUS, Singapore 2 Genome Institute of Singapore, Singapore 3 Cardiovascular Research Institute, NUS, Singapore

Background: Heterochromatin protein 1 binding partner 3 (HP1BP3) has recently been shown to influence development and cellular proliferation in mice and cancer cells. However, this epigenetic protein is unexplored in the cardiovascular setting, and identifying it in our lab's single cell transcriptomics of healthy and diseased cardiomyocytes (CMs), led to the hypothesis if it could have a role in the progression of heart failure. Methods and results: Though the up-regulation of Hp1bp3 transcripts is only observed in a subset of diseased CMs, an in vitro knockdown performed in isolated murine CMs revealed a global down-regulation of genes, with apoptosis and cell-cycle pathways being affected. These were validated via RT-qPCR, and an apoptosis assay coupled with activated caspase3 staining. In vivo Hp1bp3 knockdown performed in mice followed by the transverse aortic constriction (TAC) procedure showed maintenance of heart function via ejection fraction (compared to non-knockdown controls). Significantly, ventricular septal walls were thickened and ventricular chamber diameters were less dilated. Classical hypertrophy and heart failure marker genes like Nppa, Nppb and Myh6/7 ratios remained high and were not significantly different between knockdowns and controls. When the diastolic function was assessed, several parameters like the E/A and E′/A′ ratios, and myocardial performance index, suggest altered diastolic function in the knockdown mice prior to TAC. Conclusion: Taken together, our data suggest that Hp1bp3 has a crucial role in regulating the systolic, and possibly diastolic, function in mice challenged with pressure overload. More work is currently being done to validate the observations and elucidate the underlying mechanism. 14. Impact of cGMP-PKG pathway modulation on titin phosphorylation and titin-based myocardial passive stiffness M. Herwig1, S. Hoelper2, M. Krueger2,3, D. Koesling4, M. Kuhn5, W.A. Linke1, N. Hamdani1 1 Department of Cardiovascular Physiology, Ruhr University Bochum, Bochum, Germany

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2 Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany 3 Institute for Genetics and Cologne Excellence Cluster, University of Cologne, Germany 4 Institute for Pharmacology and Toxicology, Ruhr University Bochum, Germany 5 Institute for Physiology, University of Würzburg, Germany

Question: The crucial contribution of the giant myofilament protein titin to diastolic and cardiomyocyte stiffness (Fpassive) is studied to elucidate which elements of the nitric oxide-cGMP-PKG signaling network are critical for titin phosphorylation and stiffness in vivo. Methods and results: We utilized genetic knockout (KO) mouse models (cardiomyocyte-specific deletion of the guanylyl cyclase (GC)-A receptor, cGMP-dependent PKG (cGKI), and global deletion of soluble GC). Using immunoblotting we found in all models reduced all-titin phosphorylation and reduced PKG-dependent phospho-site compared to WT. Unexpectedly, quantitative mass spectrometry analysis revealed that most class 1 titin phosphosites within the molecular spring segment, including the Ig-domain regions, were hyperphosphorylated. Only a few sites showed a phosphorylation deficit or remaining unchanged. Particularly in the cGKI model many class 1 phospho-sites were hyperphosphorylated compared to WT hearts, indicative of the presence of compensatory processes following loss of PKG. Indeed, this was associated with upregulation of CaMKII and MAPK and a clear rise in Fpassive in KO vs. WT cardiomyocytes, which was corrected after PKG treatment. In addition, we explored the effect of oxidative stress on cGMPdependent PKG signalling in myocardial biopsies of HF with preserved ejection fraction (HFpEF) patients and the regulatory action of PKG on CaMKII and MAPK activity. In both HFpEF biopsies and cGKI KO mice oxidative stress correlates significantly with increased CaMKII activity and reduced PKG activity. Conclusions: Our novel findings suggested that cGMP-PKG/oxidative stress/CaMKII/MAPK signaling pathway plays an important role in regulation of cardiomyocyte and diastolic stiffness (a new aspect in HFpEF). 15. Elastic titin properties and protein quality control in aging hearts S. Sahin1, I. Efimov2, T. Rassaf3, M. Krüger1, S. Kötter1 1

Cardiovascular Physiology, Heinrich-Heine-University, Düsseldorf, Germany Department of Biomedical Engineering, George Washington University, Washington, USA 3 Clinic for Cardiology, University Hospital Essen, Essen, Germany 2

Background: Cardiac aging is accompanied with morphological and structural changes resulting in left ventricular hypertrophy and fibrosis. As another consequence of aging the activity of protein-quality-control (PQC) systems decreases in the heart, leading to accumulation and aggregation of defective or misfolded proteins. Here we studied whether aging affects elastic titin properties and cardiac titin turnover by intracellular degradation systems. Methods and results: Titin isoform composition, elastic I-band-domain phosphorylation, titin-based passive cardiomyocyte stiffness, titin and general protein ubiquitination were analyzed in left ventricular tissues from healthy human donor patients in the age of 15–75 years and in young and aged mice (6 and 20 months). Titin isoform composition was not significantly altered during aging of human heart and mouse heart tissue. PKA-dependent phosphorylation of the N2-Bus (S4010)

was significantly reduced and PKG-dependent phosphorylation of N2-Bus (S4099) was significantly increased in human donor and old mouse hearts. Phosphorylation of S11878 in the PEVK segment was not altered in human or mouse hearts. Phosphorylation at S12022 was significantly reduced in human donor hearts and unaltered in mouse hearts. The observed changes in N2-Bus phosphorylation have the opposite effect on cardiomyocyte tension and therefore did not significantly alter titin-based passive stiffness in young and old human and mice tissue. Proteasomal subunits were downregulated in human donor hearts while autophagic flux was diminished in aged mouse hearts. Conclusions: Altered elastic titin properties in cardiomyopathy patients are mainly based on the pathology and not significantly influenced by aging. Protein degradation systems are differentially downregulated in aged human and mice hearts. 16. Diabetes affects adaptive titin modification in response to acute myocardial ischemia/reperfusion M. Kazmierowska1, S. Kötter1, D. Semmler2, J.P. Schmitt2, M. Krüger1 1

Department of Cardiovascular Physiology, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany 2 Department of Pharmacology and Clinical Pharmacology, Medical Faculty, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany Background: Acute myocardial infarction is a leading cause of mortality worldwide, particularly in patients with diabetes mellitus. Our group recently reported that in the acute phase after myocardial ischemia/ reperfusion (I/R) posttranslational modification of the sarcomeric protein titin increases cardiac myocyte stiffness in the viable nonischemic myocardium and plays a significant role in the maintenance of ventricular stability. Here, we hypothesized that due to preexisting diabetes-induced alterations of titin properties diabetic leptin receptor deficient mice (Leprdb) lack this important mechanism for rapid cardiac adaptation after I/R. Material and results: I/R was induced in heterozygous controls (Leprdb/+) and Leprdb mice by 60 min reversible ligature of the left anterior descending artery. Hearts were excised and cardiac tissue was collected 24 h after reperfusion. Western Blot analysis was performed using phosphospecific antibodies. In non-diabetic Leprdb/+ mice I/R significantly increased PKCα activity and phosphorylation of the residues S12022 and S11878 in the elastic titin PEVK region. These changes were similar to those observed in diabetic Leprdb mice without I/R. In line with our hypothesis I/R did not further increase PKCα activity in the diabetic animals. In diabetic Leprdb mice, but not in non-diabetic controls I/R caused a significant activation of ERK1/2 and phosphorylation of residue S4010 in the titin N2-B region. Conclusions: In summary cardiac titin of diabetic Leprdb mice was differentially modified after myocardial I/R compared to non-diabetic controls. We speculate that these changes alter the adaptational response of the sarcomeres to myocardial injury and may contribute to the worsened outcome of type 2 diabetes patients after I/R. 17. The role of cardiac telethonin phosphorylation in vivo H. Lewis, S. Eminaga, S.L. Puhl, M. Gautel, M. Avkiran King's College London, London, UK


Background: Telethonin (T-cap) is a muscle-specific sarcomeric protein which is constitutively bis-phosphorylated at C-terminal residues S157 and S161 in rat and mouse cardiomyocytes. Moreover, mutations in telethonin have been associated with human cardiomyopathies and further data suggests that telethonin expression is altered in response to pathological remodelling in vivo. Thus, in order to determine the functional significance of telethonin phosphorylation in vivo, we have generated a novel knock-in (KI) mouse model expressing non-phosphorylatable telethonin (S157/161A).

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(MV DecT) and diastolic force from intact cardiomyocytes (r2 = 0.72, p = 0.006) was identified, establishing a link between whole heart and single cell diastolic function. Conclusion: Evaluation of intact cardiomyocyte force development, using glass fiber attachment provides a measure of cardiomyocyte stiffness which can be related to in vivo cardiac function. Specifically, in diabetes, cardiac diastolic dysfunction is associated with increased cardiomyocyte mechanical stiffness. 19.

Results: At baseline, TCAPS157/161A KI mice do not reveal any variances in cardiac structure or function, when compared to wild-type littermate controls. Additionally, the mutated telethonin protein maintains co-localisation at the sarcomeric Z-disc with titin. However, a gene dosage-dependent decrease in telethonin protein expression was revealed in mice carrying the S157/161A KI alleles, implying that telethonin phosphorylation influences protein stability. When challenged with sustained β-adrenergic stimulation, via a two week isoprenaline infusion, TCAPS157/161A KI mice develop mild systolic dysfunction, accompanied by increased expression of fibrotic genes. Therefore, telethonin phosphorylation at Ser157 and Ser161 may be necessary to preserve cardiac function during sustained β-adrenergic stimulation. Furthermore, immunoblot analysis revealed an increase in telethonin protein expression in wild-type mice only, following isoprenaline stimulation. Conclusion: These data indicate that the stability, and therefore expression, of telethonin is strongly influenced by its phosphorylation state and further work will aim to reveal the mechanisms underlying this regulation. 18. Linking diastolic dysfunction in rat diabetic hearts with mechanical stiffness in derived intact cardiomyocytes

Investigating the interactome of cardiac myosin-binding protein C during enhanced afterload treatment of rat engineered heart tissue by BioID and mass spectrometry S. Schulz1,2,3, T. Werner1,2, I. Wittig3,4, T. Eschenhagen1,2, M.N. Hirt1,2, F. Cuello1,2 1

Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck 3 Department of Functional Proteomics, Goethe University Frankfurt, Frankfurt, Germany 4 DZHK (German Centre for Cardiovascular Research), partner site RheinMain, Germany Background: Cardiac myosin-binding protein C (cMyBP-C) is a multimodular myosin-associated protein of the immunoglobulin superfamily. It is essential for maintaining the structure of normal thick filaments and to ensure unperturbed contractile function. Its physiological importance is emphasised by the fact that mutations that lead to expression of truncated proteins that do not integrate into the myofilament lattice are causative in patients with autosomaldominant familial hypertrophic cardiomyopathy.

A.J. Raaijmakers, J.V. Janssens, J.R. Bell, C.L. Curl, L.M.D. Delbridge Department of Physiology, University of Melbourne, Vic, Australia Background: Diastolic heart failure is characterized by ventricular stiffness and inadequate filling of the ventricles. In addition to collagen deposition, evidence suggests that cardiomyocyte specific intrinsic pathologies may also underlie ventricular stiffness, but the mechanisms are not well understood. The aim of this study was to evaluate intact cellular stiffness properties in a diabetic model of diastolic dysfunction and also to relate these findings directly to the in vivo functional properties of the origin hearts. Methods: Echocardiography was performed in 15-week male Sprague Dawley rats treated or not for 8 weeks with Streptozotocin (STZ; 55 mg/kg) treatment (GE Vivid 9). Isolated cardiomyocytes were prepared by collagenase dissociation. Glass fibers were attached (MyoTak) at the cell longitudinal surface, and paced cardiomyocytes (1 Hz, 2.0 mM Ca2 +, 37 °C) were serially stretched (0–11.2%, piezomotor). Sarcomere length/shortening, force development and intracellular Ca2+ transients (Fura-2AM, 5 μM) were simultaneously measured (Myostretcher, IonOptix). Results: Hearts from diabetic animals displayed diastolic dysfunction (E/E′ 14.8 ± 0.9 vs 10.4 ± 0.6; STZ vs Control; p b 0.05). When cardiomyocytes were uniformly stretched, measured force was significantly higher in myocytes from the STZ hearts compared to Control (6.88 ± 0.3 vs 5.55 ± 0.4 μN; N = 4&5, n = 4&8, p b 0.05). A positive correlation between diastolic function measured in intact hearts

Hypothesis: cMyBP-C is considered as a signalling hub, with its physiological function regulated by protein-protein interactions and posttranslational modifications. We aimed to investigate diseasespecific changes in the cMyBP-C interactome during cardiac hypertrophy development. Methods and results: Engineered heart tissues (EHTs) from rat cardiac cells were transduced with an adeno-associated virus encoding for mouse cMyBP-C N-terminally fused to a E. coli biotin ligase. EHTs were left either non-transduced or transduced with the AAV6-BioID-cMyBP-C at an MOI of 500. After 14 days, EHTs received 50 μmol/L biotin into the culture medium. AAV-transduced EHTs were allocated into two groups: a control group and a group subjected to an enhanced afterload protocol (AE) [1]. After three days, EHTs were harvested and analysed by western immunoblotting and immunocytochemistry. Importantly, biotinylated proteins from all three treatment groups were enriched by streptavidine-pulldown and biotinylated proteins representing potential novel cMyBP-C interaction partners analysed by mass spectrometry. This identified 56 proteins present exclusively in the AE group, as novel putative interaction partners of cMyBP-C during hypertrophy development. Interestingly, the serine/threonine kinase mammalian target of rapamycin (mTOR) and the Ca2 + dependent protease calpain were amongst them. Conclusion: BioID was established as a versatile tool to identify dynamic alterations in cardiac myocytes during development of cardiac hypertrophy.

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Reference [1] Hirt MN, Sörensen NA, Bartholdt LM, Boeddinghaus J, Schaaf S, Eder A, Vollert I, Stöhr A, Schulze T, Witten A, Stoll M, Hansen A, Eschenhagen T (2012) Basic Res Cardiol 6:307. 20. Studying cell membrane nanomechanics in normal and failing hearts – New approach P. Swiatlowska, J. Sanchez-Alonso, P. Wright, P. Novak, J. Gorelik Imperial College London, National Heart and Lung Institute, London, UK Background: Myocytes sense different mechanical signals from the surrounding environment, and any abnormal stimulation leads to adaptive changes that may be detrimental to their function. Previously we observed structural membrane disorganization in cells from rat heart failure model, which could represent a potential substrate for changes in the mechanosensation of these cells. Membrane compliance alterations were also found in failing cells; rat and human cardiomyocytes exhibited a higher elasticity as compared to healthy cells. These changes are likely to be connected to the mitochondrial derangement and microtubule disorganization leading to alteration in calcium propagation that could contribute to arrhythmogenesis. Methods: We modified the high resolution Scanning Ion Conductance Microscopy (SICM) to examine membrane elasticity. When a live cell topography image is generated, constant, positive pressure (15 kPa) is applied to check membrane compliance. The topography image can be superimposed to the elasticity map (expressed as elastic modulus, EM) to show domains of variable mechanical properties with nanoscale resolution. Results: Our results confirm that mechanical properties of adult cardiac cells are not uniform. Z-groove areas are softer than the areas in between, crests (EM = 0.7 kPA; EM = 1.5 kPa, respectively). Disrupting actin filaments and/or microtubular network decreases cell elasticity, whereas, removal of caveolae increases it. In failing rat cardiac cells (after myocardial infarction) surface elasticity is increased (EM = 8 kPa). Interestingly, cell elasticity is relatively low in infarcted cardiomyocytes from mechanically unloaded rat hearts, reaching values similar to control cells. This correlated with higher expression of more compliant titin isoform (N2BA) in these cells. Obtained data show that cell elasticity might have implication in cardiac disease. In order to shed more light on this phenomenon studies are necessary to find potential intracellular mechanosensors. 21. Comparison of the effects of a truncating and a missense MYBPC3 mutation on contractile parameters of engineered heart tissue P.J.M. Wijnker1,2,3, F.W. Friedrich1,2, A. Dutsch1,2, S. Reischmann1,2, A. Eder1,2, I. Vollert1,2, G. Mearini1,2, T. Eschenhagen1,2, J. van der Velden3, L. Carrier1,2 1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands

Background: Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease. The most frequently mutated HCM gene is MYBPC3, encoding cardiac myosin-binding protein-C (cMyBP-C). We compared the pathomechanisms of a truncating mutation (c.2373_2374insG) and a missense mutation (c.1591GN C) in MYBPC3 in engineered heart tissue (EHT). Methods and results: EHTs were generated from Mybpc3-targeted knock-out (KO) and wild-type (WT) mouse cardiac cells. MYBPC3 WT and mutants were expressed in KO EHTs via adeno-associated virus. KO-EHTs displayed higher maximal force and sensitivity to external [Ca2+] than WT-EHTs. Expression of WT-Mybpc3 at MOI-100 resulted in ~73% cMyBP-C level but did not prevent the KO phenotype, whereas MOI-300 resulted in ≥95% cMyBP-C level and prevented the KO phenotype. Expression of the truncating or missense mutation (MOI-300) or their combination with WT (MOI-150 each), mimicking the homozygous or heterozygous disease state, respectively, failed to restore force to WT level. Immunofluorescence analysis revealed correct incorporation of WT and missense, but not of truncated cMyBP-C in the sarcomere. Conclusion: This study provides evidence in KO EHTs that i) haploin sufficiency affects EHT contractile function if WT cMyBP-C protein levels are ≤ 73%, ii) missense or truncating mutations do not fully restore the disease phenotype and have different pathogenic mechanisms, e.g. sarcomere poisoning for the missense mutation, iii) the direct impact of (newly identified) MYBPC3 gene variants can be evaluated. 22. Aged mice with endothelial deletion of muscarinic M3 receptors exhibit normal body weight and preserved cardiac function despite impaired ACh-induced dilation A. Eder1,2, B. Geertz1,2, J. Uebeler1,2, J. Speiser2,3, K. Schmidt2,3, C. de Wit2,3, T. Eschenhagen1,2 1

Institut für Experimentelle Pharmakologie und Toxikologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Germany 3 Institut für Physiologie, Universität zu Lübeck, Lübeck, Germany Background: Muscarinic receptors (M1-M5) are fundamental receptors for the neurotransmitter acetylcholine (ACh). Moreover, ACh is the prototypical agonist stimulating vascular endothelial NO release, but blood vessels lack parasympathetic innervation, raising the question as to the physiologic role of endothelial muscarinic receptors in vivo. Global M3−/− mice exhibit blunted ACh-induced vasorelaxation in vitro, hypophagy and low body weight, but no apparent cardiac phenotype or altered blood pressure. Methods: To investigate the role of endothelial M3 receptors, we deleted M3 specifically in endothelial cells using the Cre-Lox system driven by the endothelial promoter TIE2 (EC-M3−/−). Mice carrying only the floxed M3 gene served as controls. We studied arteriolar dilation in 12-week-old mice in vivo. Aged mice (74–78 weeks) were studied to evaluate long-term effects on heart and body weight (HW, BW) and left-ventricular function (echocardiography). Results: ACh-induced vasodilation in cremaster arterioles was significantly reduced in EC-M3−/− (24.2 ± 3.9%, n = 6 vs. 78.9 ± 3.3%, n = 5) with preserved NO-induced dilation. Aged mice did not differ in BW (~30 g). Likewise, HW/BW ratio and parameters of cardiac function were not different between genotypes (Control (n = 6) vs. EC-M3−/− (n = 10): HW/BW 5.2 ± 0.4 vs. 4.7 ± 0.1 mg/g; ejection


fraction 31.0 ± 4.2 vs. 31.5 ± 2.8%; fractional shortening 14.7 ± 2.3 vs. 15.0 ± 1.5%). Conclusion: The endothelial M3 receptor is crucial for AChinduced dilation. However, this defect did not lead to a cardiac phenotype at advanced age arguing against a major role of parasympathetic innervation in blood vessels. The growth deficit in M3−/− mice is most likely related to the function of the M3 receptor in pancreatic β-cells. 23. Impaired Ca2 + handling precedes overt heart failure in right ventricular hypertrophy A.S. Power, A.J. Hickey, D.J. Crossman, D.S. Loiselle, M.-L. Ward Department of Physiology, University of Auckland, New Zealand Background: Pulmonary hypertension progresses to right heart failure, with evidence of altered cardiomyocyte Ca2+ handling and impaired energetics underlying the contractile dysfunction. However, the early stage of the disease is poorly studied. Aims: To examine Ca2+ handling, t-tubule organization, and energetic sufficiency in a rat model of right ventricular hypertrophy (RVH), prior to the onset of heart failure. Methods: Pulmonary hypertension and RVH were induced in rats (I.P. 60 mg mL− 1 monocrotaline) and controls were given an equivalent volume of saline. [Ca2 +]i (fura-2/AM) and stress were measured in isolated right ventricular trabeculae. Two energydemanding scenarios were investigated: increased stimulation frequency, and β-adrenergic stimulation. Trabeculae were subsequently permeabilised and mitochondrial energy sufficiency investigated. Finally, trabeculae were fixed and processed for confocal imaging and analysis (TTpower) of the t-tubular system regularity.

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3 Excellence Cluster Cardio-Pulmonary System, Universities of Giessen and Marburg Lung Centre Member of the German Lung Canter Justus-LiebigUniversity Giessen, Giessen, Germany 4 Faculty of Medicine, Department of Medicine, Imperial College London, London, SW7 2AZ, UK

Background: Pulmonary hypertension (PH) resulting from chronic vasoconstriction contributes to pulmonary arterial remodelling, leading to right ventricle (RV) failure and increased mortality. PKGI expression is upregulated during hypoxia, while PKG knock-out mice develop spontaneous PH. PKGIα is susceptible to oxidation, forming a disulfide homodimer associated with kinase targeting that mediates vasodilation. In this study, we investigated the redox state of pulmonary PKGIα during chronic hypoxia and its role in the pathogenesis of hypoxic PH. Results: Chronic hypoxia caused lung PKGIα oxidation in mice, as determined at 3 or 28 days compared to basal normoxia. The H2O2producing enzymes NOX4 and SOD3 were increased in the lungs at the 28-day time-point, thus potentially contributing to PKGIα oxidation. Cystathionine gamma-lyase (CSE) which produces the vasorelaxant H2S, was also upregulated at both time-points of hypoxia. Increased lung PKGIα oxidation, NOX4 and CSE protein expression were observed in human PAH patients. Redox-dead Cys42Ser PKGIα knockin (KI), mice which are resistant to PKGIα oxidation, had increased pulmonary vascular resistance (PVR), myosin light chain phosphorylation, potentiated RV hypertrophy and higher RV pressure, as well as excessive muscularisation of small pulmonary vessels and enhanced endothelial-to-mesenchymal cells transition at the 28-day time-point, compared to WT littermates. Chronic pharmacological CSE inhibition with propargylglycine potentiated RV pressure and hypertrophy, and attenuated lung PKGIα oxidation after 14 days of hypoxia. Conclusion: Our findings suggest that chronic PKGIα oxidation serves an important novel intrinsic adaptive mechanism that offsets increased PVR in PH, thus reducing RV after-load and so limiting progression to RV failure. 25.

Results: Ca2 +-transients were 26% smaller in RVH trabeculae, with a 45% slower time-to-peak, but no difference in the decay of the transients. RVH had a negative force-frequency response in comparison to the controls. Beta-adrenergic stimulation altered the time course, but not the amplitude, of Ca2+-transients, and increased spontaneous Ca2 + release. No difference was found in maximum Ca2 +-activated stress in trabeculae reliant on mitochondrial ATP production and supply. T-tubular regularity (TTpower) was decreased from 0.11 ± 0.02 a.u. in control trabeculae to 0.07 ± 0.01 a.u. (p = 0.02) in RVH trabeculae. Conclusions: We conclude that impaired Ca2+ handling is present in RVH before mitochondrial energetic deficits occur. This is explained, in part, by t-tubular remodelling which impairs excitation-contraction coupling. 24. PKGIα oxidation during chronic hypoxia provides an adaptive vasodilatory mechanism that limits pulmonary hypertension O. Rudyk1, S. Krasemann2, K. Hartmann2, O. Prysyazhna1, M. Zhang1, A. Weiss3, L. Zhao4, R. Schermuly3, P. Eaton1 1 BHF Centre of Research Excellence, King's College London, London, SE1 7EH, UK 2 Core Facility for Mouse Pathology, University Medical Centre HamburgEppendorf, Hamburg 20246, Germany

Autoimmune disease mouse model exhibits pulmonary arterial hypertension K. Sugimoto, Y. Takeishi Department of Cardiovascular Medicine, Fukushima Medical University, Fukushima, Japan Background: Pulmonary arterial hypertension is often associated with connective tissue disease. Although there are some animal models of pulmonary hypertension, an autoimmune disease-based model has not yet been reported. MRL/lpr mice, which have hypergammaglobulinemia, produce various autoimmune antibodies, and develop vasculitis and nephritis spontaneously. However, little is known about pulmonary circulation in these mice. In the present study, we examined the pulmonary arterial pressure in MRL/lpr mice. Methods and results: We used female MRL/lpr mice aged between 12 and 14 weeks. Fluorescent immunostaining showed that there was no deposition of immunoglobulin or C3 in the lung tissue of the MRL/lpr mice. Right ventricular systolic pressure, and the ratio of right ventricular weight to left ventricular weight in the MRL/lpr mice were significantly higher than those in wild type mice with same background (C57BL/6). The medial smooth muscle area and the proportion of muscularized vessels in the lung tissue of the MRL/lpr mice were larger than those of the C57BL/6 mice. Western blot analysis demonstrated markedly elevated levels of endothelin-1 and survivin as well as

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decreased endothelial nitric oxide synthase phosphorylation in the lung tissue of the MRL/lpr mice. Conclusion: We showed that MRL/lpr mice were complicated with pulmonary hypertension. MRL/lpr mice appeared to be a useful model for studying the mechanism of pulmonary hypertension associated with connective tissue diseases. 26. Fibrosis biomarkers in peripartum cardiomyopathy F. Azibani1, W. Dowling1, J. Baard1, O. Briton1, T. Adam1, S. Kraus2, J. Anthony3, K. Sliwa1 1 Hatter Institute for Cardiovascular Research in Africa, Department of Medicine, Groote Schuur Hospital, University of Cape Town, Cape Town, South Africa 2 Department of Medicine, Groote Schuur Hospital, University of Cape Town, Cape Town, South Africa 3 Division of Obstetrics & Gynaecology, Groote Schuur Hospital, University of Cape Town, Cape Town, South Africa

Background and objective: Peripartum cardiomyopathy (PPCM) is a new onset of heart failure in late pregnancy and up to the first five months postpartum. Fibrosis biomarkers such as the amino-terminal peptide of type III procollagen PIIINP, Galectin-3 and soluble ST2 (sST2), together with microRNA-21 were described as prognostic biomarkers in heart failure. We aimed to determine the expression levels of these factors and whether they are associated with poor outcome in PPCM. Methods and results: We performed a subgroup biomarker study on 40 consecutive patients with PPCM and 10 age-matched healthy subjects. Circulating biomarkers were measured at baseline. Echocardiograms were performed at baseline and six-months postpartum. Poor outcome in PPCM patients was defined by NYHA ≥ 3 or death at 6 months. At baseline, PPCM patients had significantly higher NT-proBNP, PIIINP, sST2, Galectin-3 and microRNA-21 levels than healthy controls (p ≤ 0.01). Six-months postpartum, three patients did not improve their cardiac function (EF, 26.7 ± 7.4%) and eight died. Baseline NT-proBNP (3973.1 ± 955 vs. 1835.2 ± 268pmol/l, p = 0.02), sST2 (67.01 ± 14.2 vs. 28.5 ± 4.3 ng/ml, p = 0.011) and Galectin-3 (15.37 ± 2.8 vs. 8.55 ± 0.66ng/ml, p = 0.01) levels were higher in patients with poor outcome compared to patients that improved their cardiac function (EF, 45.7 ± 11.3%). PIIINP and microRNA-21 levels were not different between recovered and poor outcome patients. Conclusion: For the first time we demonstrated that PIIINP, sST2, Galectin-3 and microRNA-21 are upregulated in PPCM. Galectin-3 and sST2 may be clinically useful biomarkers that identify a subset of PPCM patients at highest risk of myocardial dysfunction due to fibrosis. These findings should be confirmed in a larger cohort. 27. Adeno-associated virus-mediated reexpression of myopalladin mutants in myopalladin knockout mice results in dilated cardiomyopathy, confirming myopalladin as a cardiomyopathy-associated gene M.C. Filomena1,2, D.L. Yamamoto3, P. Carullo2,4, N. Piroddi5, C. Tesi5, B. Scellini5, R. Crispino6, J. Zhang7, R. Knöll8,9, R. Polishchuk6, C. Poggesi5, V. Nigro6,10, M. Giacca11,12, J. Chen7, M.-L. Bang2, 4 1 Department of Experimental Medicine, “La Sapienza”, University of Rome, Rome, Italy

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Humanitas Research Hospital, Rozzano, Milan, Italy Institute of Biomedical Technologies (ITB) and 4Institute of Genetic and Biomedical Research (IRGB), UOS Milan, National Research Council, Milan, Italy 5 Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy 6 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy 7 Department of Medicine, University of California San Diego, La Jolla, CA, USA 8 Department of Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden 9 AstraZeneca Research and Development Mölndal, Sweden 10 Department of General Pathology, Second University of Naples, Naples, Italy 11 University of Trieste 12 ICGEB, Trieste, Italy 3

Background: Myopalladin (MYPN) is a 145 kDa striated musclespecific sarcomeric protein belonging to a family of actin-associated immunoglobulin-containing proteins in the Z-line. MYPN gene mutations are causative for dilated (DCM), hypertrophic, and restrictive cardiomyopathy. However, the role of MYPN in the heart remains elusive. Thus, we generated MYPN knockout (MKO) mice, which have a normal life span but are smaller compared to WT controls. Results: Cardiac analyses of MKO mice showed the development of slowly progressive cardiac dilation and decreased fractional shortening starting from 4 months of age, associated with reduced sarcomere contractility (Ionoptix analysis). Furthermore, mechanical measurements on ventricular myofibrils revealed decreased tension generation and increased resting tension in MKO mice compared to WT controls. Following transaortic constriction, MKO mice quickly developed severe cardiac dilation and reduced cardiac function associated with altered AKT and calcium signaling. To study the pathways leading from MYPN gene mutations to DCM, we injected neonatal MKO mice with adenoassociated virus 9 (AAV9) expressing WT or mutant MYPN to obtain reexpression of MYPN at physiological levels in the heart. While transduction of MKO mice with AAV9-MYPN WT rescued the MKO phenotype, transduction with AAV9 expressing MYPN DCM-associated mutants resulted in DCM of various severity, in all cases more severe than observed in MKO mice, consistent with a pathological role of the mutations. Several of the mutants were associated with cardiac fibrosis and RNA-Seq analyses showed significant molecular changes. Conclusion: Our studies demonstrate that the studied MYPN gene mutations are causative for DCM and have dominant gain-of-function effects. 28. Alterations in cardiac cytoarchitecture due to cellular stress in dilated cardiomyopathy M. Pluess1,2, O. Koldyka1, T. Iskratsch1, P. Bennett1, J. Dwyer1, P. Pandey1, C. dos Remedios3, E. Ehler1,2 1 Randall Division of Cell and Molecular Biophysics, King's College London, London, UK 2 Cardiovascular Division, King's College London, London, UK 3 Bosch Institute, University of Sydney, Sydney, Australia

Background: Heart tissue from patients with dilated cardiomyopathy (DCM) can look histologically relatively normal, lacking the obvious areas of myocyte and myofibril disarray that characterise hypertrophic cardiomyopathy. However, there is often pronounced fibrosis, which results in a stiffer mechanical environment for the cardiomyocytes.


Results: We have over the years characterised alterations at the cellular level in DCM and find dramatic changes in intercalated disc composition, the specialised type of cell-cell contact in heart. This leads to an increased concentration of actin anchoring and polymerising proteins and also affects the ultrastructure of the intercalated disc. These changes are correlated with a redistribution of PKCalpha to the intercalated disc, where it is held in a complex together with CARP1. Removal of CARP1 prevents continuous activation of PKCalpha and the characteristic alterations at the intercalated disc fail to develop in a mouse model for DCM, the MLP knockout mouse. The second area of the cell, where we find changes in organisation is the nuclei. A subset of the nuclei of cardiomyocytes in DCM patient samples displays an increased size and a changed composition of their nuclear lamina. This includes disruptions and can also be detected at the ultrastructural level as increased convolution of the nuclear membrane and the appearance of membrane stacks. There is also a change in phosphorylation of nuclear lamina components. Conclusions: We propose that the altered mechanical environment caused by the increased amount of extracellular matrix deposition is fed back to the interior of the cell and eventually leads to changes in the nucleoskeleton, which may affect gene transcription. 29. Arrhythmias and electrophysiological abnormalities in Mybpc3-targeted mouse models of hypertrophic cardiomyopathy F. Flenner1,2, F.W. Friedrich1,2, C. Jungen2,3, H. Kniep3, M. Kohlhaas4, N. Küpker1, K. Gurr1, K.-D. Soehren1, T. Eschenhagen1,2, C. Maack4, C. Meyer2,3, T. Christ1,2, L. Carrier1,2 1

Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Cardiology-Electrophysiology, cardiac Neuro- and Electrophysiology Research Group (cNEP), University Heart Center Hamburg, Hamburg, Germany 4 Working group for Cardiovascular Physiology and Bioenergetics, Saarland University Faculty of Medicine, Homburg, Germany Background: Hypertrophic cardiomyopathy (HCM) often causes sudden cardiac death (SCD) due to ventricular fibrillation. The underlying causes for the fatal arrhythmias are not well understood and pharmacological HCM treatment strategies do not significantly lower SCD risk. Here, we show that cardiomyocytes, ventricular muscle strips and hearts of mice genetically modified in Mybpc3, one of the major HCM-disease genes, have increased arrhythmia susceptibility and are therefore a helpful tool to understand arrhythmia mechanisms and test therapy options in HCM. Methods and results: Arrhythmia susceptibility was examined by β-adrenergic stimulation, electrical stimulation protocols and pharmacological interventions in isolated ventricular cardiomyocytes, ventricular muscle strips and Langendorff-perfused hearts in Mybpc3targeted mouse models. Mybpc3-targeted cells, tissues and hearts had a higher arrhythmia inducibility than wild-type (WT) controls. Pharmacological intervention recapitulating phenotypic HCM aspects, namely elevation of myofilament Ca2+ sensitivity and action potential (AP) prolongation, increased arrhythmia incidence in WT cells and tissue. Molecular analysis underlined abnormalities in pathways involved in hypertrophy and electrophysiology in Mybpc3-targeted mouse models.

tissue and whole heart level. Until now, several possible causes for arrhythmia development such as fibrosis, myocardial disarray, prolonged APs, abnormal ion channel mRNA levels, increased Ca2 + myofilament sensitivity and intracellular oxidative stress have been discovered. These potential causes may add up and increase the arrhythmia risk. Further experiments will be done to dissect which findings show primary and which show secondary effects to decipher mechanisms of the observed arrhythmias and discover new treatment strategies. 30. Insights into disease mechanisms of cardiomyopathy from the Csrp3 C58G mouse model M. Ehsan1, M. Kelly1, C. Hooper1, J. Beglov1, B. Davies2, K. Fleischanderl3, E. Ehler3, E. Makeyev3, C. Redwood1, H. Watkins1, K. Gehmlich1 1 Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK 2 Transgenic Core, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK 3 King's College London, London, UK

Background: Missense mutations in CSRP3, the gene coding for muscle LIM protein (MLP), are known to cause hypertrophic cardiomyopathy (HCM). We hypothesise that mutations in proteins involved in biomechanical stress signalling such as MLP can cause perturbations in hypertrophic signalling cascades in the heart, resulting in cardiomyopathy. Methods: A knock-in mouse model was generated by homologous recombination, introducing the C58G mutation into the mouse MLP protein. Heterozygous and homozygous knock-in mice were generated and their cardiac function assessed in vivo by echocardiography and invasive haemodynamic measurements. Tissues harvested from these animals were analysed for molecular markers of hypertrophy and heart failure. Results: Heterozygous MLP C58G knock-in mice display no overt phenotype and show a similar hypertrophic response to trans-aortic constriction when compared to wildtype littermates. Homozygous MLP C58G knock-in mice have a phenotype resembling dilated cardiomyopathy, with compensatory activation of hypertrophy. At the molecular level, markers of heart failure and hypertrophic signalling are induced in these mice. Total MLP protein is significantly reduced (approximately 20% of wild type levels) and the protein destabilisation is caused by proteasomal activity. Further in vitro experiments suggest that protein destabilisation is a common feature of HCM-causing MLP mutations. Conclusion: The mouse model recapitulates aspects of human hypertrophic cardiomyopathy caused by MLP missense mutations (such as protein destabilisation) and is a valuable tool to study molecular mechanisms of aberrant biomechanical stress signalling resulting in cardiomyopathy. 31. Functional impact of a novel α-Actinin 2 mutation associated with hypertrophic cardiomyopathy in human iPSC-derived cardiomyocytes M. Prondzynski1,2,3, M. Lemoine1,2, E. Krämer1,2, S. Laufer4, J. Münch2,5, C. Redwood6, T. Christ1,2, M. Patten2,5, A. Hansen1,2, T. Eschenhagen1,2, G. Mearini1,2, L. Carrier1,2 1

Conclusions and Outlook: We demonstrate that Mybpc3-targeted mouse models display a higher arrhythmia inducibility on single cell,

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Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

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2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Fraunhofer IME Screening-Port, Hamburg, Germany 4 Department of Experimental Pharmacology and Toxicology, Stem Cell facility, University Medical Center Hamburg-Eppendorf, Hamburg, German 5 Department of General and Interventional Cardiology, University Heart Center Hamburg, Hamburg, Germany 6 University of Oxford, Oxford, UK

Background and objective: Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease caused by mutations in genes encoding for sarcomeric proteins, such as α-Actinin 2. This protein plays a major role as an anchor and cross linker of actin filaments in the sarcomere. Goal of this study was to elucidate the functional impact of α-Actinin 2 in HCM using induced-pluripotent stem cells (iPSCs) from a HCM patient and a healthy donor. Methods and results: DNA sequencing with a cardiac 19-gene panel of the HCM patient revealed a novel heterozygous missense mutation in the ACTN2 gene, encoding α-Actinin 2. Human skin fibroblasts were reprogrammed into iPSCs and further differentiated into cardiomyocytes (CMs). 2D-cultured CMs were measured by the Opera® High Content Screening System and cell areas were 4.9-fold higher in HCM than in control cell line. As a 3D model, engineered heart tissues (EHTs) were generated and paced at 1 Hz in 1.8 mM Tyrode solution at 37 °C. HCMEHTs showed no significant difference in force, but a significantly prolonged relaxation time, when compared to Control-EHTs. Action potential duration (APD) measurements using EHTs and sharp electrodes resulted in a significantly prolonged APD for the HCM-EHTs. To validate these findings, isogenic control cell lines using CRISPR/Cas9 were generated. Those cell lines are currently under investigation. Conclusion: This work provides evidence for successful HCM disease modeling using human iPSC-CMs in 2D and 3D models. Further experiments are needed to evaluate the underlying disease mechanism of this novel α-Actinin 2 mutation in HCM. 32. Lusitropic and anti-hypertrophic effects of the sphingosine-1-phosphate receptor modulator, FTY720, in hypertrophic cardiomyopathy D.M. Ryba1, C.M. Warren1, C.N. Karam1, R.T. Davis 3rd1, S.A.K. Chowdhury1, M.G. Alvarez1, D.F. Wieczorek2, R.J. Solaro1, B.M. Wolska1,3 1

Department of Physiology and Biophysics and the Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA 2 Department of Molecular Genetics, Biochemistry, & Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA 3 Department of Medicine, Section of Cardiology, University of Illinois at Chicago, Chicago, IL, USA Background: Hypertrophic cardiomyopathy (HCM) patients present with progressively worsening hypertrophy, diastolic dysfunction and fibrosis. FTY720 is an immunomodulating drug, used in the treatment of multiple sclerosis, which we have previously identified to have anti-fibrotic and anti-hypertrophic effects in a model of pressureoverload-induced hypertrophy. We sought to understand whether modulation of the sphingosine-1-phosphate receptor by FTY720 would be of therapeutic benefit in established HCM. Methods: We treated an HCM mouse model linked to a mutation in tropomyosin (Tm-E180G) and NTG littermates with FTY720 or

vehicle for six weeks. We assessed cardiac function and structure using echocardiography and pressure-volume (PV) relations and the myofilament-Ca-response using detergent-extracted fibers. We also investigated if signaling pathways were altered. Results: We found that FTY720-treated Tm-E180G mice had a significant reduction in left atrial size, E/A and E/Em ratios compared to significantly increased baseline measurements assessed by echocardiography. PV relations revealed significant improvements in EDPVR, PRSW, and Tau. Skinned fiber bundles revealed a significant decrease in myofilament-Ca-responsiveness in FTY720-treated Tm-E180G mice. We attributed these functional improvements to a down-regulation of S-glutathionylation of cMyBP-C in FTY720-treated Tm-E180G mice. The increases in oxidative modification in vehicle-treated Tm-E180G mice were due to an up-regulation of NOX2. We attributed the and fibrosis and hypertrophy to increased periostin and TGF-beta expression, and GATA4 phosphorylation, respectively. These maladaptive changes were reversed by FTY720 treatment. Conclusions: FTY720 improves diastolic function and morphology by reversing oxidative modification of myofilament proteins via downregulation of NOX2 and maladaptive remodeling signals. 33. Effect of mutant protein dose and mutation location on disease severity in hypertrophic cardiomyopathy M. Schuldt1, D.W.D. Kuster1, J.R. Pinto2, J. van der Velden1 1 Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, the Netherlands 2 Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA

Background: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease with a prevalence of 1:200. It is caused by N 1400 mutations in contractile genes and characterized by increased wall thickness, diastolic dysfunction, fibrosis and an increased risk of sudden cardiac death. However, variability in disease progression between patients is huge, and we are not yet able to predict who will become symptomatic and when. We hypothesize that the dose of mutant protein and the mutation location are main determinants of disease burden in HCM. Methods: To assess the effect of mutation location, we used the HCM causing Troponin T mutants I79N and R94C. The effect of dose was tested by performing troponin exchange in detergent-permeabilized human cardiomyocytes whereby we can control the amount of mutant troponin. We tested the ability of the mutants to successfully incorporate into the sarcomere, measured force development, calcium sensitivity and length-dependent activation to determine the direct influence on cardiomyocyte function at different dosages. Results: Preliminary results have shown that the mutants differ in their ability to incorporate into the sarcomere. By using 1.5 mg/ml troponin complex with the TnT-R94C mutation we replaced around 40% endogenous troponin, whereas we could replace 70% with TnT-I79N. Compared to controls (EC50: 1.78 ± 0.18 μM) both mutants showed an increased calcium sensitivity of force development, with TnT-R94C (1.17± 0.13 μM) showing an even higher increase than I79N (1.53 ± 0.13 μM), even with the lower degree of incorporation. This effect was already present at a very low TnT-R94C dosage of 18%. Conclusion: The mutation location determines the degree of cardiomyocyte dysfunction which can already be present at a very low mutant protein dosage.


34. PhysioZoo: Open source software for heart rate variability analysis of mammal electrophysiological data J. Behar, A. Rosenberg, A. Alexandrovich, Y. Elul, O. Shemlas, Y. Yaniv Biomedical Engineering Faculty, Technion-IIT, Haifa, Israel Background: The normal variation of the time between heartbeats is commonly referred as heart rate variability (HRV). Loss of complexity in HRV has been documented in several cardiovascular diseases and has been correlated with an increase in morbidity and mortality. However, there is a limited understanding of the mechanisms which control HRV and how these patterns could be predictive of disease related functional or structural complications. Animal experiments at various scales, from a single cell to the whole body, are a key tool in investigating this question. However, to date, there is no centralized public database for researchers to access, and the availability of tools to quantify HRV of different mammal ECG are sparse. Results: We created a public standard animal database containing a series of recordings for a variety of wild type healthy mammals (mice, rabbits, dogs and humans) with reference annotations. We provide a new open source software and associated algorithms for beat detection, correction and HRV analysis. The algorithms are tailored to the mammalian biology, which can significantly differ from human biology. Conclusion: The PhysioZoo project provides a new standard platform and reference databases for heart rate variability analysis in mammals. 35. Phenotype differences of valve interstitial cells in healthy and calcified aortic valves M. Bogdanova1,2, A. Rutkovskiy3, K. Žihlavníková Enayati1, A. Malashicheva2, A. Zabirnyk1, K.O. Stensløkken1, A. Fiane,4,5, J. Vaage3,5 1 Division of Physiology, Institute of Basic Medical Sciences, University of Oslo, Norway 2 Almazov Federal Heart Centre, Saint Petersburg, Russian Federation 3 Department of Emergency and Critical Care, Oslo University Hospital, Oslo, Norway 4 Department of Cardiothoracic Surgery, Oslo University Hospital, Oslo, Norway 5 Institute of Clinical Medicine, University of Oslo, Oslo, Norway

Background: Aortic valve calcification is caused by differentiation of valve interstitial cells (VICs) into osteoblasts and myofibroblasts. We hypothesize that VICs from calcified aortic valves have changed their phenotype into a pro-calcification state. Methods: VICs were isolated from human aortic valves with or without calcification harvested during surgery. VICs were cultured in osteogenic medium for 21 days to induce osteoblast differentiation and calcification. Calcification was quantified using Alizarin Red staining. To differentiate VICs into myofibroblasts we stimulated them with TGFβ1. The expression of myofibroblastic marker alpha smooth muscle actin (aSMA) was evaluated by immunostaining and rqPCR. Cells were also cultured in collagen gel for seven days with TGFβ1. Gel contraction is a measurement of the presence of myofibroblasts. Results: VICs from calcified valves, but not VICs from healthy valves, accumulate calcium deposits during culture for 21 days even without osteogenic medium. After exposure to osteogenic medium cells from

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both types of donors accumulated calcium, but significantly more with VICs from calcified valves (P b 0.0001). Upon stimulation with TGFβ1 VICs from both calcified and normal valves differentiate into myofibroblasts, but expression of aSMA was highest in VICs from the healthy ones (P b 0.0001). VICs from healthy valves also had the strongest gel contraction. Conclusions: VICs from healthy and calcified aortic valves have significant differences in phenotype. The VICs from calcified valves have a stronger ability to differentiate into osteoblasts and myofibroblasts, they have a more pro-calcification state. By elucidating the cellular and molecular mechanisms of calcification, new therapeutic options may be developed. 36. Modification of SERCA pump sub-cellular localization in cardiac Purkinje cells can be a possible origin for the triggers of ventricular tachycardias associated with myocardial ischemia Y. Guo1,2,3, S. Chaigne1,2,3,4, S. Charron1,2,3,, G. Bru-Mercier1,2,3, O. Bernus1,2,3, M. Hocini1,2,3,4, M. Haissaguerre1,2,3,4, C. Cros1,2,3, F. Brette1,2,3, B. Stuyvers1,2,3,5 1

University of Bordeaux, Bordeaux, France INSERM, France 3 IHU-LIRYC, Electrophysiology and Heart Modeling Institute, Pessac, France 4 University Hospital of Bordeaux, Bordeaux, France 5 Faculty of Medicine, Memorial University, St. John's, NL, Canada 2

Background: Increased incidence of electrogenic Ca2+ waves in cardiac Purkinje cells (Pcells) account for a cellular trigger of tachyarrhythmias after myocardial infarction (MI). A change in gating properties of SR-Ca2+ release channels (CRC) and SR-Ca2+ overload are 2 potential explanations for this increase. The current study intended to clarify whether MI is accompanied by a change in Ca2 + uptake function of P-cells by addressing the expression of principal proteins involved in intracellular Ca2+ signalling. Methods: P-cells were dispersed enzymatically from sheep, and human Purkinje fibers. The fibers were dissected from normal hearts and from hearts with MI. MI was induced by coronary occlusion under fluoroscopic imaging in 6 adult sheep. Specific antibodies were used to map the SR Ca2 + release channels, RyR2 and IP3R and SERCA2 and gene expression was measured by qPCR. Results: No change was observed in CRC expression in Pcells of MI hearts. However a marked SERCAs redistribution to the cell periphery was detected in our MI animal model and in human heart with large MI. Compared to the centre, the cell periphery showed a 4.5fold increase of SERCA protein expression while SERCA2 transcript level was increased by 2.3 times after MI. Conclusion: Our results are consistent with previous functional measures of Ca2+ cycling in other models of MI and, therefore, support the idea that a dramatic increase of SERCA density at the cell periphery is a direct cause of pro-arrhythmic spontaneous Ca2+ release in Pcells of human heart after MI. 37. Essential but partially redundant roles for POU4F1/Brn-3a and POU4F2/Brn-3b transcription factors in the developing heart L. Maskell, K. Qamar, A. Babakr, T.A. Hawkins, R.J. Heads, V.S. Budhram-Mahadeo

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MMBU, Institute of Cardiovascular Sciences, University College London, London, UK Background: Congenital heart defects contribute to embryonic or neonatal lethality but due to the complexity of cardiac development the molecular changes associated with such defects are not fully understood. Brn-3a/POU4F1 and Brn-3b/POU4F2 transcription factors (TFs) are expressed in cardiomyocyte and may control cell fate under different conditions by eliciting similar effects on some target genes e.g. Hsp27 but have antagonistic effects on cell survival. Thus, Brn-3a blocks p53 mediated apoptosis but enhances cell differentiation whereas Brn-3b promotes growth but when coexpressed with p53 can lead to cooperative transactivation of pro-apoptotic genes e.g. Bax, thereby increasing cell death. Both TFs were detected in the developing heart but with previously unknown functions.

Discussion: Based on the beneficial observations through our experimental arrangement, the new molecule synthetized in our University is a powerful vasodilator with no significant toxic effects. It may contribute to new therapeutic approaches against ischemic heart diseases and possibly related syndromes, but the molecular mechanisms needs to be clarified. Acknowledgements: NTP-NFTÖ-16, OTKA-PD-111794, TÁMOP 4.2.4. A/2-11-1-2012-0001 and GINOP-2.3.2-15-2016-00043. 39. Increased cytosolic free Zn2+ alters action potential parameters via activation of KATP-channels in rat ventricular cardiomyocytes S. Degirmenci, Y. Olgar, E. Tuncay, B. Turan

Results: Brn-3a and Brn-3b can compensate for each other in the developing heart because double knock-out (KO) (Brn-3a−/−:Brn-3b−/−) mutant mice are embryonically lethal by embryonic day (e)9.5 whereas single knock-out (KO) mutants survive throughout gestation. Furthermore, reducing both Brn-3a and Brn-3b in zebrafish (ZF) using morpholino oligonucleotides (MO) caused significant cardiac defects e.g. failure to loop and valve defects in double morphants compared with single or control morphants. However, elevated Brn-3b in Brn-3a KO hearts increased target genes such as cyclin D1 and caused hyperplastic growth in valve/septum during mid-gestation but at later stages, Brn-3b cooperates with p53 to increase cardiomyocyte apoptosis, resulting in ventricular wall/septal thinning and non-compaction that may cause death of Brn-3a KO mutants soon after birth. Conclusion: Our results suggest essential but partially redundant roles for Brn-3a/POU4F1 and Brn-3b/POU4F2 transcription factors (TFs) in the developing heart. 38. Vasoactive effects of a new nitric oxide donating acetylsalicylic derivative A. Czompa, I. Bak, K. Szoke, I. Lekli, A. Borbas, P. Herczegh, A. Tosaki Department of Pharmacology, Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary

Department of Biophysics, Ankara University School of Medicine, Ankara, Turkey Background: Recent studies showed that the [Zn2+]i is increased mostly under pathophysiological conditions. In this study, we aimed to investigate the effects of the increased [Zn2+]i on action potential parameters, together with voltage-gated K+-channel currents (IK) and ATPdependent K+-channel currents (IKATP), in cardiomyocytes from rats and H9c2 cell lines. Methods and results: Action potentials were recorded on currentclamp mode and total K+-currents were recorded on voltage-clamp mode of the patch-clamp technique by using whole-cell configuration. The [Zn2 +]i increase is achieved by acute application of ZnPT or ZnCl2 in the pipette solution during recordings. The acute ZnPT application induced significant prolongation in the APD parameters. Furthermore, the acute ZnPT application (10 nM–1 μM) as well as the ZnCl2 (10–30 nM) markedly inhibited the voltage-gated K+-channel currents and increased IKATP in both the freshly isolated rat cardiomyocytes and the H9c2 cells. The histological investigation of either ZnPT or ZnCl2 treated cardiomyocytes by using transmission electron microscopy showed marked increases in mitochondrial-matrix/cristae-area and matrix volume together with increased numbers of lysosomes. Moreover, we measured marked depolarization in mitochondrial membrane potential during ZnPT minute-application by using confocal microscopy.

Aims: According to the latest WHO mortality rates ischemic heart conditions are the main cause of death among non-communicable diseases. In the following investigation we have aimed to test the properties of a nitric oxide (NO) donating acetylsalicylic acid (ASA) derivative, which is stable, water soluble compound newly synthetized at the University of Debrecen, Hungary. Based on our hypothesis this molecule could combine the positive effects of the anticoagulant ASA and the vasodilator effect of NO, therefore, it could be a more powerful medication against ischemic heart diseases.

Conclusion: Our data exert that increased level of free Zn2 + causes markedly prolonged action potential duration via activated IKATP and reduced IK as well as detrimental effect on mitochondrial structure and function in cardiomyocytes. In this regard, one can suggest that these observed events may underlie the depressed mechanical activity of the heart under increased cytosolic free Zn2+ level in cardiomyocytes.

Materials and methods: In the first part of our experiments we were tested the possible cytotoxic effects of our ASA derivative in vitro in 10−4–10−7M concentrations. MTT cell viability assay and hemolysis tests were performed on H9c2 cells and rat erythrocytes, respectively. Thereafter, we were measured the vasoactive effects on female Sprague Dawley rats ex vivo according to the Langendorff method in the presence or absence of the NO donating molecule.

40.

Results: Our new NO donating ASA derivative is not affecting negatively to the cell viability in the tested concentrations. Furthermore, we have noticed a dose-dependent, out washable vasodilation on coronaries.

Acknowledgments: This study was supported by grant of TUBITAK SBAG-113S466 and COST Action TD1304.

Acute effects of thymoquinone on electrophysiological parameters of rat ventricular myocytes T. Mercan, B.E. Yamasan, O. Erkan, S. Özdemir Akdeniz University Faculty of Medicine Department of Biophysics, Antalya, Turkey Background: Thymoquinone is one of the components of Nigella sativa. It has been used for many years to treat various diseases


such as asthma, hypertension and diabetes. Studies show that thymoquinone has protective effects against cardiac injuries and cardiotoxic impacts of chemicals. In this study, we tested whether thymoquinone modulates action potential (AP) and ionic currents of ventricular myocytes. Methods and results: Ventricular myocytes were isolated from male rats. Then, AP and relevant ionic currents were recorded during whole-cell configuration of patch-clamp. Thymoquinone had dose dependent effect on AP peak and duration (APD). Application of thymoquinone decreased (48.32 ± 1.51 mV vs. 42.33 ± 1.96 mV n = 14 for 10−6 M and 48.01 ± 1.98 mV vs. 39.47 ± 2.77 mV, n = 12 for 10− 5 M) peak value of AP significantly. Repolarization times to 75% and 90% of AP was also prolonged with thymoquinone application (for 10−5 M APD75: 15.63 ± 2.36 vs. 21.97 ± 2.41 p b 0.001 and APD90: 29.68 ± 2.41 vs. 36.32 ± 2.77 p b 0.001, n = 12). When transient outward potassium (Ito) and L-type Ca2 + currents (ICaL) were assessed, dose-dependent reduction was measured in these currents with thymoquinone application. During perfusion with 10-5 M it decreased both Ito (18.35 ± 3.33 vs. 17.19 ± 2.72 p b 0.001 n = 12) and ICaL (-12.07 ± 1.32 vs. -10.47 ± 1.18 p b 0.001 n = 12) significantly. Conclusion: We have shown that thymoquinone modulates action potential characteristics and ionic currents of cardiomyocytes. Apparently, the reduction in K+ currents predominates Ca2 + currents which result in longer AP duration. However, Na+ currents may be responsible for the reduced AP peak. Further studies are needed to figure out this point and potential mechanisms of these changes. 41. Prominent differences in cardiac performance and myocardial properties between right ventricular and left ventricular-based pacing modes in rats W. Mulla1, S. Etzion2, S. Elyagon1, Y. Konstantino3, N. Liel-Cohen3, Y. Etzion1,2 1

Cardiac Arrhythmia Research Laboratory, Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel 2 Regenerative Medicine & Stem Cell Research Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel 3 Cardiology Department, Soroka University Medical Center, Beer-Sheva, Israel Background: The biological effects of cardiac resynchronization therapy (CRT) are still elusive and require insights from animal models. Recently, we introduced a device for long-term double-site epicardial pacing in rats, and found marked dyssynchrony during RV pacing compared with both, LV and Biventricular (BIV) pacing modes. Here we characterized this model by hemodynamic recordings and analysis of myocardial properties following sustained tachypacing. Objective: To elucidate the effects of RV vs. LV-based pacing modes in rats.

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p b 0.001). In contrast, LV and BIV pacing modes markedly enhanced cardiac performance compared with RV pacing (n = 8, p b 0.001). Sustained RV tachypacing (n = 6), but not BIV tachypacing (n = 6), increased the QT interval by 6.63 ± 3.1 ms relative to baseline and dispersed refractoriness between the right and left pacing sites (10.0 ± 3.8 ms, p b 0.05). In addition, increased expression of Osteopontin and activation of CAMKII and JNK signaling (Figure) were found in the early-activated septal tissue of RV-paced group compared with the BIV-paced group. Conclusion: Our rat model is an attractive new tool to study the biological consequences of dyssynchrony and CRT. The new biochemical findings can shade light on fundamental effects of the different pacing modes on the tissue level. 42. Cerebral amyloid β (42) deposits and microvascular pathology in ageing baboons M.N. Ndung'u, W. Härtig, F. Wegner, J.M. Mwenda, R.W. Low, R.O. Akinyemi, R.N. Kalaria Institute of Primate Research, National Museums of Kenya, Nairobi, Kenya Background: Previous studies have extensively reported the deposition of amyloid β (Aβ) peptide with carboxyl- and amino-terminal heterogeneity in cortical and cerebrovascular deposits in Alzheimer's disease (AD) and in non-human primates except baboons. Methods: We examined the immunocytochemical distribution of Aβ peptides and Aβ oligomers in brain tissue from three subspecies of 18- to 28-year-old baboons (Papio) and in other monkeys including the squirrel (Saimiri sciureus) and rhesus (Macaca mulatta) for comparison. Results: A general preponderance of Aβ(42) in parenchymal deposits and many vascular deposits in all cortical lobes was evident in the baboons. Aβ oligomeric immunoreactivity was also apparent like to amyloid plaques. We found that the amino acid sequence of the Aβ domain of the baboon amyloid precursor protein is similar to that of man. In contrast to Aβ, immunoreactivity to hyperphosphorylated tau protein was largely intracellular and rare in these baboons. Brain tissues from squirrel and rhesus monkeys examined in parallel exhibited mostly vascular and parenchymal deposits containing Aβ(42) peptides. Our results were comparable to AD, but showed that even in younger monkeys exhibiting few deposits, Aβ(42) was evident in both parenchymal deposits and cerebral amyloid angiopathy. Perivascular amyloid deposits were frequent and often accompanied by microvascular abnormalities in form of collapsed degenerated capillaries. Conclusions: Similar to other primates above and below in the phylogenetic order, our observations and evaluation of the literature implicate pathogenicity of Aβ(42) peptide associated with microvascular degeneration in baboons. We suggest baboons are useful animals to investigate the dynamics of AD-related pathology. 43.

Methods: Electrodes were implanted either on the RA and RV or on the RV and LV. Following recovery, rats were either used for admittancebased pressure-volume loop measurements or exposed to sustained RV vs. BIV tachypacing for 3 days.

Langendorff-free method for isolation of mouse cardiomyocytes – Characterisation and comparison with standard Langendorff cardiomyocyte isolation method

Results: Sequential RA-RV pacing compromised systolic parameters including dP/dt max and stroke work, compared with RA pacing (n = 8,

D. Pavlovic1, M.A. Ackers-Johnson2,3, P.Y. Li2,3, S.-M. O'Brien1, A.P. Holmes1, C. Hepburn1, L. Fabritz1, P. Kirchhof1, R.S. Foo2,3

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1

Institute of Cardiovascular Sciences, University of Birmingham, UK Cardiovascular Research Institute, Centre for Translational Medicine MD6, National University Health System, Singapore, Singapore 3 Genome Institute of Singapore, Singapore 2

Background and aims: Recently we developed a novel Langendorff-free method for cardiomyocyte isolation whereby coronary perfusion and enzymatic cell dissociation is achieved through intraventricular injection. Here, we compare calcium handling, contractile and signaling characteristic of cardiomyocytes isolated by intraventricular injection and standard Langendorff methods. We also investigate whether intraventricular injection technique can be used for isolation of (i) ventricular cardiomyocytes from infarcted hearts, where a large and thin scar may rupture the myocardium and (ii) atrial cells from healthy hearts.

Methods and results: Fluorescence recovery after photo-bleaching (FRAP) microscopy is used to probe the diffusion properties of T-tubules in isolated rat cardiomyocytes: a fluorescent dextran inside tubular lumen is photo-bleached and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along T-tubules. We applied this method to detect alterations of T-tubule conductivity in a pathological setting, characterized by compromised T-tubule integrity. We found that in heart failure T-tubule conductivity is significantly reduced compared to healthy cardiac cells.

Methods: Cardiomyocytes were isolated by intraventricular injection or standard Langendorff method, and calcium transients and contractility were measured by photometry/contractility Ionoptix system and protein expression/phosphorylation by western blotting. Myocardial infarction (MI) was induced by left coronary artery ligation.

Conclusion: A reduction in conductivity can impair the propagation of action potential across the network and may explain the presence of conduction defects found at single tubular level.

Results: Ventricular cardiomyocytes isolated via the injection method displayed comparable responses to field-stimulation (1–3 Hz), on diastolic calcium, calcium transient morphology and sarcomere length, to those isolated by standard Langendorff (n ≥ 8 cells from n ≥ 3 hearts). No difference in the stress-response was detected between the two methods, as assesed by PKC-α activation and ERK1/2 phosphorylation in quiescent cardiomyocytes (n ≥ 5 hearts). Myocytes were successfully isolated from MI hearts via the intraventricular injection method, with expected decrease in rod-shaped cells, compared to shams (sham = 77.4 ± 4.1%; MI = 64.2 ± 6.4%; n = 4). Preliminary experiments demonstrate that reducing the collagenase perfusion time allowed isolation of atrial cells.

Impairment of ventricular repolarization and induction of arrhythmias by mechanical unloading in a rodent model of heterotopic heart transplantation

Conclusions: We show that ventricular cardiomyocytes isolated by the intraventricular injection method retain key contractile and signaling characteristics and demonstrate the utility of the method for isolation of cells from infarcted hearts. 44. Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy M. Scardigli1,2, C. Crocini1,2, C. Ferrantini3, T. Gabbrielli1, L. Silvestri1,2, R. Coppini4, C. Tesi3, E. A. Rog-Zielinska5, P. Kohl5, E. Cerbai4, C. Poggesi3, F.S. Pavone1,2,6, L. Sacconi1,2 1

European Laboratory for Non-Linear Spectroscopy (LENS), Florence, Italy National Institute of Optics (INO), National Research Council (CNR), Florence, Italy 3 Division of Physiology, Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy 4 Division of Pharmacology, Department “NeuroFarBa”, University of Florence, Florence, Italy 5 Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen, Faculty of Medicine, University of Freiburg, Freiburg, Germany 6 Department of Physics and Astronomy, University of Florence, Sesto Fiorentino, Italy

45.

A. Schwoerer1,2, M. Jelinek1,2, M. Paske1,2, D. Biermann3, H. Reichenspurner2,3, H. Ehmke1 1 Department of Cellular and Integrative Physiology, University Medical Center Hamburg Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany

Background: Support of failing hearts with ventricular assist devices (VADs) is associated with a high incidence of newly occurring ventricular arrhythmias. While underlying mechanisms are unresolved, data obtained in these patients and in astronauts suggest that a reduction in cardiac workload itself impairs ventricular repolarization. To test this hypothesis, nonfailing rat hearts were unloaded using the model of heterotopic cardiac transplantation (hHTX) and ventricular electrophysiology was monitored in vivo by telemetric ECG recording. Methods: hHTX was performed in syngeneic male Lewis rats (n = 16, 269 ± 16 g). ECG was recorded either of the orthotopic hearts (n = 8) or of the unloaded hearts (n = 8) for 56 days and was analysed using D.S.I. Ponemah. Statistical analysis was conducted using Graphpad Prism, data are given as mean ± SEM.

2

Background: Although myocardial conduction of excitation has been widely described, the electrical properties of T-tubules remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of T-tubules.

Results: Unloading induced a pronounced reduction in LV weight illustrating the reduced cardiac workload (control: 748 ± 24 mg, n = 8; unloaded: 345 ± 33 mg, n = 8; P b 0.0001). Despite comparable heart rates, repolarization was severely impaired in the unloaded hearts. The mean QT interval was ~65% longer in the unloaded hearts (control: 55 ± 1 ms, n = 8; unloaded: 91 ± 4 ms, n = 8; P b 0.0001). The incidence of ventricular arrhythmias was significantly higher in the unloaded than in the control hearts (control: 2 ± 2 events/56 days, n = 8; unloaded: 225 ± 112 events/56 days, n = 8; P b 0.01). Conclusions: This study demonstrates that a reduction of LV workload in the absence of underlying heart disease impairs repolarization and induces ventricular arrhythmias, most likely via an ion channel remodeling. This implicates that a reduction in LV workload could indeed underlie ventricular arrhythmias during space travel and in patients with VAD support.


46. Aortic valve calcification: the role of interaction between valve endothelial and interstitial cells A. Zabirnyk1, M. Bogdanova1,2, A. Rutkovskiy3, K. Žihlavníková Enayati1, A. Malashicheva2, A. Kostareva2, A. Fiane4,5, KO. Stensløkken1, J. Vaage3,5 1

Division of Physiology, Institute of Basic Medical Sciences, University of Oslo, Norway 2 Almazov Federal Heart Centre, Saint Petersburg, Russian Federation, Russia 3 Department of Emergency and Critical care, Oslo University Hospital, Oslo, Norway 4 Department of Cardiothoracic Surgery, Oslo University Hospital, Oslo, Norway 5 Institute of Clinical Medicine, University of Oslo, Oslo, Norway Background: Aortic valve calcification is caused by differentiation of valve interstitial cells (VIC) into osteoblasts and myofibroblasts. Endothelial cells produce an abundance of bioactive substances. We hypothesize that the valve endothelial cells (VEC) may influence the phenotype and the calcification process of the VIC. Methods: VIC and VEC were isolated from human aortic valves with or without calcification harvested during surgery. VEC were seeded over a 3D culture of VIC embedded in collagen gel and cultured for 21 days in osteogenic differentiation medium. All four possible combinations of VIC and VEC from calcified and noncalcified valves were investigated. The expression of osteogenic markers BMP2, BCAT and RUNX2 was evaluated by RQ-PCR. To achieve myofibroblast differentiation co-cultures were stimulated by TGFβ1. Their ability to contract the gel size was used as a quantitative measurement of myofibroblast differentiation. Results: The expression of mRNA of BMP2, BCAT and RUNX2 was most prominent in co-cultures of VEC from calcified valves together with VIC from non-calcified valves compared to all other groups. Compared to co-culture of VIC and VEC from calcified valves, BMP2 increased 5.6 fold (p = 0.02), BCAT increased 4.8 fold (p = 0.03), and RUNX2 increased 3.9 fold (p = 0.02). Gel co-cultures of the same cell combination also contracted more strongly compared to all other groups (1.8 fold, p b 0.0001) suggesting stronger myofibroblast differentiation. Conclusion: VEC are able to influence osteogenic and myofibroblast differentiation of VIC. This suggests that cross-talk between VEC and VIC may have a regulatory function in the development of valve calcification. 47. Inhalation therapy delivers nanoparticle-based therapeutic peptides to the diseased heart M. Miragoli, P. Ceriotti, M. Iafisco, M. Vacchiano, N. Salvarani, A. Alogna, P. Carullo, G.B. Ramirez-Rodríguez, T. Patrício, S. Pinelli, R. Alinovi, M. Erreni, S. Rossi, G. Condorelli, H. Post, A. Tampieri, D. Catalucci Institute of Genetics and Biomedical Research, Milan Unit, National Research Council, Milan, Italy Background: Peptides are recognized for being highly selective and efficacious for the treatment of cardiovascular as well as other diseases but their administration via a non-invasive procedure is currently not possible, thus representing scientific and technological challenges.

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Methods and results: Here we demonstrate that inhalation of small sized (b 50 nm) biocompatible and biodegradable calcium phosphate nanoparticles (CaPs) allows for rapid translocation of CaPs from the pulmonary tree to the blood stream and thus to the myocardium where their cargo is quickly released. In particular, treatment of a rodent model of diabetic cardiomyopathy through inhalation of CaPs loaded with a therapeutic mimetic peptide that we previously demonstrated to improve myocardial contraction resulted in restoration of cardiac function. When translated to large animals, tangible evidence that inhalation of a peptide-loaded CaP-formulation is a safe and valid method of cardio-targeted administration was provided. Conclusion: Altogether, these results demonstrate that inhalation of biocompatible tailored peptide nanocarriers represents a pioneering approach for the pharmacological treatment of heart failure. 48. CRISPR/Cas9-derived isogenic hiPSC controls reveal a mutation-specific contractile and arrhythmic phenotype of hiPSC-cardiomyocytes from PLN-R14del carriers A.E. Knaust1,2, M. Prondzynski1, B. Klampe1, U. Saleem1, A.T.L. Zech1, F. Flenner1, I. Mannhardt1, S. Laufer2,3, A. Shibamiya2,3, I. Braren2,3, I. van Rijsingen4, Y. Pinto4, S. Harding5, T. Eschenhagen1,2, A. Hansen1,2 1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Hamburg Centre for Experimental Therapy Research (HEXT), Stem Cell Core Unit and Vector Facility, University Medical Centre HamburgEppendorf, Hamburg, Germany 4 Department of Cardiology, Heart Failure Research Centre, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands 5 Faculty of Medicine, National Heart & Lung Institute, Imperial College, London, UK

Introduction: Phospholamban (PLN) regulates cardiomyocyte calcium homeostasis, and PLN mutations are a frequent cause of dilated cardiomyopathy (DCM) in The Netherlands. We aimed to establish a human in vitro model for the PLN-R14del mutation with disease-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCM) and CRISPR/Cas9-generated isogenic controls. Force-generating, three-dimensional engineered heart tissues (EHT) were used to define disease-specific contractility pattern. Methods: hiPSCs were generated from skin fibroblasts of a mother and daughter with a heterozygous PLN-R14del-mutation. Gene-corrected isogenic hiPSC-controls were generated by homology-directed repair using both DNA- and ribonucleoprotein-based CRISPR approaches. CM were differentiated with an embryoid body, growth factor-based three-stage protocol, dissociated and used for the generation of EHTs in a 24-well format. Contractile parameters were determined by automated video-optical recording. Results: Sequencing of the CRISPR-line revealed seamless genecorrection and lack of off-target effects (top 10). The intervention did not affect CM differentiation efficiency. Electrically paced PLN-R14del (n = 27) compared to isogenic control EHTs (n = 9) developed significantly lower peak force (0.08 ± 0.03 mN vs. 0.21 ± 0.03 mN) with shorter contraction time (105 ± 10 ms vs. 127 ± 3 ms), but similar relaxation time (124 ± 17 ms vs. 131±3 ms). PLN-R14del-EHTs were less sensitive to extracellular calcium concentrations (EC50 0.71 mM, n = 27, vs. 0.32 mM, n = 9) and showed a time- and concentration-dependent

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increase in arrhythmias with increasing extracellular calcium concentration (1.0, 1.8, 3.0 mM). Conclusion: EHTs from PLN-R14del-mutation carriers showed a robust contractile and arrhythmic phenotype which was absent in isogenic, CRISPR-corrected controls. Further investigations will focus on molecular mechanisms of PLN-R14del. 49. Comparison between structure and function of rare coding variants identified in coronary artery disease: insights into BAY41-2272 binding mode S. Tennstedt1,2,3, J. Wobst4, Z. Aherrahrou1,2,3, H. Schunkert4,5, J. Erdmann1,2,3 1

Institute for Cardiogenetics, University of Lübeck, Lübeck, Germany DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck/, Germany 3 University Heart Center Lübeck, Lübeck, Germany 4 Deutsches Herzzentrum München, Technische Universität München, Munich, Germany 5 DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Germany

3

Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China 4 Ankara University Biotechnology Institute, Ankara, Turkey 5 Ankara University Stem Cell Institute, Ankara, Turkey 6 Department of Cardiovascular Surgery, Ankara University School of Medicine, Turkey Background: Atrial fibrillation (AF), the most common persistent clinical tachyarrhythmia, is associated with transcriptional changes leading to cardiomyocyte dysfunction and AF susceptibility and progression. Recent research showed class I and class IIa histone deacetylases (HDACs) to regulate pathological and fetal gene expression, which causes cardiac contractile dysfunction and hypertrophy. Whether class I and class IIa HDACs are involved in AF progression is unknown. Therefore, we investigated their role in tachypacing-induced contractile dysfunction and pathological fetal gene expression in experimental model systems for AF and in clinical AF.

2

Background: We showed previously that genetic alterations in the gene GUCY1A3, which encodes the α1 subunit of soluble guanylyl cyclase (sGC), are strongly associated with coronary artery disease (CAD) (Erdmann et al. Nature (2013)). Moreover, we found a relationship between coding variants of GUCY1A3 and reduced cGMP levels (Wobst et al. Basic Res Cardiol. (2016)). In vitro studies show that reduced cGMP levels triggered by genetic alterations in GUCY1A3, can be rescued by the sGC stimulator BAY 41-2272 (Wobst et al. Basic Res Cardiol. (2016)). Aim: To identify structural effects of rare coding variants on BAY 412272-sGC complex by in silico analysis and to get insight in the binding mode of BAY 41-2272, which is not fully understood yet. Method: We calculated the 3D protein structure by homology modeling. To investigate the effect on protein-ligand complex we performed docking analysis and molecular dynamic simulations. Results: In silico analysis showed that each mutation causes local structural changes that compromise the catalysis of cGMP. A comparison of our latest in vitro study (Wobst et al. Basic Res Cardiol. (2016)) with the results of the docking analysis and molecular dynamic simulations offer a structure activity relationship that gives insights in the binding mode of BAY41-2272. Treating patients harbouring genetic alterations in GUCY1A3 with sGC stimulators such as BAY 41-2272 might be a novel approach to reducing the risk of cardiovascular diseases. Stimulators of sGC are emerging therapeutic agents for cardiovascular diseases, but in-depth studies are lacking (Wobst et al. Basic Res Cardiol. (2016).

Methods and results: Tachypacing (TP, 5Hz) of HL-1 cardiomyocytes resulted in a significant reduction in Ca2+ transient (CaT) amplitude. In control normal-paced (1 Hz) cardiomyocytes, overexpression of class I HDACs, HDAC1 or HDAC3, caused a significant loss of CaT. TP aggravated the CaT loss in HDAC3, but not in HDAC1 overexpressing cardiomyocytes. Knockdown of HDAC3 but not HDAC1, significantly protected against TP-induced CaT loss. Consistently, HDAC3 specific inhibitor RGFP966 also protected against TP-induced CaT loss. Overexpression of class IIa HDACs, HDAC5 or HDAC7, protected against TP-induced CaT loss, while HDAC4 or HDAC9 did not. Notably, the protective effect of HDAC5 and HDAC7 was abolished in cardiomyocytes overexpressing a dominant negative HDAC5 or HDAC7 mutant, bearing a mutation in the binding domain for myosin enhancer factor 2 (MEF2). Furthermore, TP induced the phosphorylation of HDAC5, promoted its translocation from the nucleus to cytoplasm, and consequently increased MEF2-related fetal gene expression (β-MHC, BNP). The PKCα inhibitor Go6983, the HDAC5-MEF2 stabilizer MC1568, and the HDAC5 phosphorylation dominant mutant (S498A) inhibited HDAC5 nuclear export and thus protected against TP-induced CaT loss. In line with these experimental findings, patients with AF showed a significant increase in HDAC3, phosphorylated HDAC5 and fetal gene expression. Conclusion: Blocking HDAC5 nuclear exportation by reducing its phosphorylation or inhibiting HDAC3 protects against tachypacing-induced CaT reduction in HL-1 cardiomyocytes. As phosphorylated HDAC5 and HDAC3 are both increased in AF patients, HDAC3 and HDAC5 are interesting therapeutic targets in clinical AF. 51. Generalized compound heterozygosity analysis highlights coronary artery disease associated genetic loci in genetic and exome data M.L. Munoz Venegas1,2,3, M. Munz1,2,3, D. Gola2,4, L. Zeng5,6, T. Kessler5,7, I.R. König2,4, H. Schunkert5,6, J. Erdmann1,2,3

50. 1

Diverse role of class I and class IIa HDACs in atrial fibrillation D. Zhang1,2, X. Hu1, F. Hoogstra-Berends2, Q. Zhuang3, M.A. Esteban3, G.C. Deni4,5, S. Durd4,5,6, R. Akar5, B.J.J.M. Brundel1 1 Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands 2 Department of Clinical Pharmacy and Pharmacology, University Medical Centre Groningen, University of Groningen, The Netherlands

Institute for Cardiogenetics, University of Lübeck, Lübeck Germany DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Germany 3 University Heart Center Lübeck, Lübeck, Germany 4 Institute of Medical Biometry and Statistic, University of Lübeck, Lübeck, Germany 5 Deutsches Herzzentrum München, Technical University of Munich, München, Germany 6 DZHK (German Center for Cardiovascular Research), partner site München Heart Alliance, Germany 2


Background: The active search for genetic factors contributing to coronary artery disease (CAD) has seen major progress from (hypothesis-free) GWAS of common SNPs. However for CAD, a complex disease, GWAS's only explain a small portion of phenotypic variance. A new proposed approach is to look at the generalized form of compound double heterozygosity (GCDH) to detect the genetic associations caused by compound heterozygosity (CH). Thereby aid in explaining some of the ‘missing heritability’ and possible underlying (patho-) mechanisms. Our study aimed to use the GCDH to discover genetic loci associated with CH of CAD in genetic and exome data of European ancestry. Methods: The CollapsABEL software tool was used to conduct the GWA scan and the GCDH test on the German Myocardial Infarction Family Study V (GerMIFS V) whereby all cases had myocardial infarction (Ncases = 2459; Ncontrols = 1611); and the German CAD North and South Studies (Ncase| Ncontrol for North: 4464 |2886 and South: 5255 | 2921) which were our genetic and exome discovery samples, respectively. Results: In our genetic discovery sample thus far, we found loci on chromosome 1, 6, 9 and 12 to be significantly associated with CH in heart related phenotypes. Interestingly some SNPs were found to be significant using the “traditional” single-SNP based method (≤5 × 10−8) while other SNPs were only found by using GCDH method (≤ 1.25 × 10−10). We are currently undertaking ‘post-GCDH’ analysis to find the functional meaning of our results, in addition to follow-up with replication cohorts. Conclusions: Based on this preliminary study, GCDH was able to reveal loci that was associated with CH which could have otherwise been ‘missed’ using single-SNP based method.

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1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany

Background: Engineered heart tissues (EHTs) are promising to study/ treat cardiac pathologies associated with loss of cardiac function/tissue. Despite major advances, studies of EHT-maturated cardiomyocytes (CMs) as single cells are still limited by the difficulty to isolate living myocytes with intact elongated shape and intracellular structures. Here we describe a new papain-based protocol to isolate living elongated CMs from EHTs. Methods and results: 3D strip-format fibrin-based EHTs were generated from freshly isolated neonatal rat heart cells or human induced pluripotent stem cell-derived CMs and kept in culture for N2 weeks to allow CMs maturation as assessed by maximal EHT force developed. Mature EHTs were dissociated into single myocytes with Earle's balanced salt solution, 10 U/mL papain, 1 mM EDTA, 5.5 mM L-cystein, 10 μM Y-27632, and 30 μM N-benzyl-p-toluene-sulphonamide. Enzymatic action was stopped by a 10% foetal calf serum balanced salt solution. Cells viability and shape were evaluated directly after dissociation and compared with results obtained with the standard collagenase II-based protocol. Cells were then either fixed for intracellular structure integrity evaluation via immunofluorescence, or loaded with the intracellular fluorescent Ca2+ probe Fura-2-AM for Ca2+ transients recording. Papain protocol gave higher yields than collagenase protocol for living and elongated CMs, which exhibited Ca2+ transients under electrical pacing and preserved sarcomeres organisation.

Integrative analysis of psoriasis and cardiovascular disease unfolds pathway convergence

Conclusion: We report a new protocol based on papain to isolate living rod shape cardiomyocytes with preserved intracellular structure. This protocol, faster than collagenase-based protocols, will increase the possibilities to study cardiomyocytes maturated within EHTs at the single cell level.

S. Sharma1, M. Akhter1, S. Ali2

54.

1 Department of Pharmaceutical Chemistry, School of Pharmacy and Bioinformatics Facility, Jamia Hamdard University, New Delhi, India 2 Department of Biochemistry, Faculty of Science, Jamia Hamdard University, New Delhi, India

The role of monoamine oxidases in differentiation and maturation of iPSC-derived cardiomyocytes

52.

Background: In the present study, we employed system biology approaches to reveal the most significant events and key molecular modulators of cardiovascular disease and induced psoriasis disease. Methods and results: Literature mining was used to identify the proteins involved in cardiovascular disease and psoriasis. A proteinprotein interaction (PPI) network was prepared using literature mining seed nodes.Topological analysis of network and network nodes were carried out using cytoscape and annotation, visualization and integration of database respectively, in order to identify most developed pathways and processes involved in inter-linking them. Conclusion: Our study presents composite picture of main pathways linking cardiovascular disease induced psoriasis disease and possible targets. 53. Papain to isolate living cardiomyocytes with preserved shape and intracellular structure from engineered heart tissues P. Bobin1,2, T. Christ1,2, A. Hansen1,2, T. Eschenhagen1,2

M. Di Sante1, S. Deshwal2, S. Antonucci2, C. Troiano2, E. Greotti1,2, D. Pendin1,2, F. Di Lisa1,2, N. Kaludercic1 1

CNR Neuroscience Institute, Padova, Italy Department of Biomedical Sciences, University of Padova, Padova, Italy

2

Background: Monoamine oxidases (MAOs) are mitochondrial flavoenzymes that exist in two isoforms, MAO-A and -B, and are responsible for neurotransmitter and biogenic amines catabolism. In pathological conditions MAO activity contributes to cardiac damage, mainly due to excessive formation of H2O2 and aldehydes. Yet, the role and contribution of MAOs during early development and cardiomyogenesis has not been investigated and remains elusive. Therefore, we established a human model based on cardiomyocytes derived from human induced pluripotent stem cells (iPSCs), in which MAO expression was genetically manipulated. Methods and results: During cardiomyocyte differentiation, mRNA and protein levels of both isoforms increased progressively. MAO-A appeared to be the most abundant isoform, suggesting that the presence of this protein is essential in the early stages. In order to assess whether MAOs might affect cardiomyocyte differentiation and function, iPSCs were treated with siRNA against either MAO-A or MAO-B. Genetic inhibition of either MAO isoform expression resulted in lower frequency

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and amplitude of calcium transients in iPSC-derived cardiomyocytes. Moreover, only cells treated with siRNA against MAO-A displayed dyssynchronous beating and loss of the regular striated staining pattern of α-actinin. Conclusion: Taken together, these results highlight the physiological role of MAOs in human cardiomyogenesis. Further studies are necessary to clarify whether derangements in calcium homeostasis, rhythm and ultrastructure resulting from MAO-A ablation in iPSC-derived cardiomyocytes are due to the accumulation of MAO substrates or whether products of MAO activity, i.e. H2O2 and aldehydes, may play a role in the signalling cascade involved in cardiomyogenesis. 55. Properties of the sodium-calcium exchanger and the Na+/K+-ATPase in human induced pluripotent stem cell-derived cardiomyocytes A. Horváth1,2,3, K. Gurr1, D. Ismaili1, I. Mannhardt1,2, B. Ulmer1,2, A. Hansen1,2, A. Varró3, T. Eschenhagen1,2, T. Christ1,2 1

Department of Experimental Pharmacology and Toxicology, University Medical Center HamburgEppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Szeged, Szeged, Hungary Background: Suitability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac repair and for cardiovascular drug testing critically depends on whether hiPSC-CM share properties of adult human cardiomyocytes (CM). The sodium-calcium exchanger (NCX) is a major regulator in the Ca2+-homeostasis. Na+/K+-ATPase removes Na+ from the cytoplasm, and can affect NCX-function on a beat-to-beat basis. We investigated properties of NCX and Na+/K+-ATPase currents in hiPSC vs. adult human left ventricular (LV) and right atrial (RA) CM. Methods: HiPSC-CM were dissociated from monolayer (ML) cultures and from engineered heart tissues (EHT). Adult CM were obtained from human samples obtained during open-heart surgery. Currents were recorded, by the whole-cell patch-clamp technique at 37 °C and were defined as Ni2+- and ouabain-sensitive. Results: In forward mode, Ni2+-sensitive NCX-current densities were larger in CM from EHT than in ML, RA and LV (1.22 ± 0.2 pA/pF n = 29 ML vs. 2.44 ± 0.3 pA/pF n = 28 EHT vs. 1.1 ± 0.23 pA/pF n = 18 RA vs 1.22 ± 0.3 n = 15 LV at –80 mV p b 0.05), also in reverse mode (0.75 ± 0.1 pA/pF n = 29 ML vs. 1.44 ± 0.1 pA/pF n = 28 EHT vs. 0.37 ± 0.1 pA/pF n = 18 RA vs 0.73 ± 0.1 pA/pF n = 15 LV at 20 mV p b 0.05). Na+/K+-ATPase in ML and EHT was similar to RA and LV (64.2 ± 21.4 pA n = 18 ML vs. 101.9 ± 16.4 pA n = 28 EHT vs. 106.6 ± 15.4 pA n = 5 RA vs 149.8 ± 34.1 pA n = 4 LV at –30 mV). Conclusion: NCX and Na+/K+-ATPase were present in ML and EHT and they were similar to RA and LV, however NCX was larger in EHT. To decide whether Na+/K+-ATPase influences NCX activity, further studies are needed. 56. HiPSC-cardiomyocytes are an oversensitive model for IKr block compared to human ventricular tissue M.D. Lemoine1,2, T. Krause1,2, F. Flenner1,2, M. Prondzynski1,2, I. Mannhardt1,2, M. Schulze1,2, E. Girdauskas3, S. Willems2,4, A. Hansen1,2, T. Eschenhagen1,2, T. Christ1,2

1

Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany 4 Department of Cardiology and Electrophysiology, University Heart Center Hamburg, Hamburg, Germany Objectives: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising test bed for electrophysiological drug effects. Since animal models show a lesser dependence of repolarization on IKr, the repolarization reserve in hiPSC-CM might be an important model for safety pharmacology. Methods: We measured action potentials (AP) with sharp microelectrode in four control cell-lines of hiPSC-CMs cultured in engineered heart tissue (EHT) compared to human left ventricular tissue (LV). Results: AP shape of hiPSC-CMs in EHT resembled LV with similar resting membrane potential, upstroke velocity and AP amplitude, whereas AP duration was slightly smaller and spontaneous depolarization occurred only in hiPSC-CMs. Spontaneous depolarization could be diminished by the funny channel inhibitor ivabradine in hiPSC-CMs. IK1 block by Barium evoked a similarly small AP prolongation in hiPSC-CMs and LV. IKr block by E-4031 revealed similar IC50 values for hiPSC-CMs (44 nM) and LV (52 nM), but hiPSCCMs showed a larger maximum AP prolongation and stronger reverse use-dependence than in LV. APs in hiPSC-CMs frequently showed EADs upon full IKr block (10/21), whereas it does not for LV (0/12). Conclusion: Spontaneous activity in hiPSC-CMs is mainly driven by the funny current. Similar to LV, the contribution of IK1 to final repolarization is small in hiPSC-CMs. However, hiPSC-CMs showed an exaggerated AP prolongation and EAD development upon IKr block, indicating hiPSC-CM as a high/oversensitive test system for QT-prolongation and arrhythmic triggers in hERG-assays. 57. What a difference a cell makes – Comparison of 7 healthy control hiPSC-CM cell lines for drug screening I. Mannhardt1,2, U. Saleem1,2, D. Mosqueira3, M.D. Lemoine1,2,4, M. Prondzynski1,2, C. Denning3, A. Hansen1,2, T. Eschenhagen1,2 1

Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Stem Cell Biology, Centre for Biomolecular Sciences, University of Nottingham, UK 4 Department of Cardiology-Electrophysiology, University Heart Center Hamburg, Hamburg, Germany Background: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are commercially available, and cardiac differentiation became routine in many laboratories interested in drug screening and disease modeling. The question arises to which extent the cardiac phenotype of healthy control hiPSC-CM differs and what the consequences are. Here we investigated 7 different healthy control hiPSC-CM cell lines and analyzed their function and suitability for drug screening in the engineered heart tissue (EHT) format.


Methods: 5 commercial hiPSC-CM cell lines, 1 hiPSC-CM cell line from Hamburg and 1 hiPSC-CM cell line from Nottingham were casted in fibrin-based EHT. Spontaneously and electrically stimulated EHT contractions were analyzed with a video-optical setup and 7 indicator compounds (BayK-8644, nifedipine, EMD-57033, isoprenaline, digoxin, thapsigargin, ryanodine) were investigated in cumulative concentrationresponse curves. Results: Baseline contractility varied between the different control cell lines with relaxation time spanning from 149 to 445 ms. Nevertheless, canonical drug responses were observed in EHTs of all cell lines. Only in 5/49 concentration-response curves, did the drugs fail to provoke the expected effect in one or the other cell line: No lusitropic effect was observed after cumulative beta-adrenergic stimulation in 4/7 cell lines, no force increase by calcium sensitizer in 1/7 cell lines. Conclusion: Our results support the request for isogenic controls in disease modeling as baseline phenotypes of healthy control cell lines differ considerably. For drug screening though, baseline variability appears less relevant. 58. Genetically encoded calcium indicators for drug screening in human engineered heart tissues U. Saleem1,2, I. Mannhardt1,2, I. Braren2,3, T. Eschenhagen1,2, A. Hansen1,2 1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Centre, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Hamburg Centre for Experimental Therapy Research (HEXT), Vector Facility, Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Centre, University Medical Centre HamburgEppendorf, Hamburg, Germany

Background: Investigating excitation contraction coupling in human iPS-derived cardiomyocytes (hiPSC-CM) can give important insight into the mechanisms of action of drugs. Our study aimed at establishing an in-vitro system for measurement of contraction force and calcium transient (CaT) in human engineered heart tissues (EHT) using genetically encoded calcium indicators (GECI). Methods: Three-dimensional, force-generating fibrin-based EHTs were prepared from hiPSC-CM and transduced with GECI (GCaMP5G or GCaMP6f). We measured contraction force (video-optical recording) and CaT (fluorescent light intensity) sequentially (10 sec delay) and analysed 6 compounds: EMD-57033 (myofilament Ca2 + sensitizer), Bay K-8644, nifedipine (L-type calcium channel agonist/antagonist), ryanodine (ryanodine receptor antagonist), beta-adrenergic agonist (isoprenaline) and SR Ca2+-ATPase inhibitor (thapsigargin). Results: GCaMP6f showed faster kinetics than GCaMP5G, better reflecting physiological CaT kinetics. EMD-57033 (10 μM) produced an increase in force (+ 170%) but no change of CaT. Bay K-8644 (300 nM) increased amplitude of force (+ 55%), relaxation time (+ 64%) and Calcium decay time (+ 55%). Nifedipine (100 nM) decreased force (- 79%), contraction time (-30%), CaT (- 76%) and calcium rise time (-26%). Ryanodine (10 μM) decreased force (-37%) and CaT (- 49%). Isoprenaline (10 nM) increased force (+ 32%), decreased relaxation time (- 17%), increased CaT (+13%) and decreased calcium decay time (- 21%). Thapsigargin decreased force (- 28%) and CaT (-33%). Force/calcium loops revealed compound-specific changes illustrating the mechanism of action.

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Conclusion: The use of GCaMP6f in EHTs provides an efficient approach to optically analyse contractile parameters and calcium handling in stably beating engineered human heart muscles. 59. CRISPR-Cas knock-out of nexilin in hiPSC and cardiomyocyte differentiation B. Schmidt1,2, A. Trillhaase1,2, S. Stölting1,2, M. Trilck3, T. Webb4, J. Erdmann1,2, Z. Aherrahrou1,2 1

Institute for Cardiogenetics, University of Lübeck, Lübeck, Germany DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Lübeck, Germany 3 Institute of Neurogenetics, University of Lübeck, Lübeck, Germany 4 Department of Cardiovascular Sciences, University of Leicester, UK 2

Background: The protein Nexilin was identified to be a structural protein in the Z-disk in the sarcomere of cardiomyocytes. It is known to play an important role in sarcomere integrity as it was shown that loss of Nexilin leads to blurry Z-disks in zebrafish. Mutations in Nexilin were found to lead to dilated cardiomyopathy (DCM) in humans, zebrafish and mice. The underlying pathomechanisms are still unknown. The main aim of this study is to generate hiPSCs lacking Nexilin and functionally characterize derived cardiomyocytes to get a better understanding of the molecular mechanisms leading to DCM in humans. Methods and results: In a first step of the project we aimed to establish the protocol of differentiation and characterize cardiomyocytes derived from human iPSCs. Therefore, modulation of canonical Wnt signaling is performed, leading to efficient differentiation of hiPSC to cardiomyocytes. Characterization of cells was performed before differentiation at RNA level using the markers Oct4, Klf4 and Nanog to show pluripotency of hiPSCs. After differentiation, characterization was performed using heart specific expression markers such as MYL2, Troponin T, Desmin, GATA4 and HCN4 at RNA level. Outlook: The next step was the generation of Nexilin knockout (Nexn-KO) iPSCs using the CRISPR/Cas system. The resulted cell lines will then be differentiated into cardiomyocytes and the same aforementioned markers will be used for detailed molecular characterization of Nexn-KO iPSC-derived cardiomyocytes and compared to controls. Additionally, phenotypic characterization, sarcomere integrity and expression levels of cardiac transcription factors such as Smad proteins and Tbx5 will be analysed to understand the function of Nexilin better. 60. Generation of multicoloured engineered heart tissue containing human iPSC-derived cardiomyocytes, endothelial cells, smooth muscle cells and fibroblasts T. Werner1,2, M.N. Hirt1,2, J. Stenzig1,2, K. Riecken3, B. Fehse3, T. Eschenhagen1,2 1 Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Research Department of Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

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Background: Engineered heart tissue (EHT) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used for drug screening and disease modelling. The addition of defined populations of non-myocytes may improve EHT function and cardiomyocyte maturity. The aim of the current study was to generate hEHTs comprising the main four cardiac cell types marked with different colours to investigate cellular composition dynamics and tissue organization with the goal to establish a tissue model close to the native human heart.

Acknowledgments: OTKA PD 111794, ‘National Excellence Program’: TÁMOP-4.2.4.A/2-11/1-2012-0001, GINOP-2.3.2-15-2016-00043. 62. Oxidation of Atg3 and Atg7 during aging inhibits autophagy K. Frudd, J. Burgoyne King's College London

Methods and results: HiPSCs were transduced with lentiviral vectors encoding four different fluorescent proteins. Following the common mesodermal induction, blue hiPSCs were differentiated to cardiomyocytes via inhibition of wnt-signaling, while green mesodermal progenitors were treated with VEGF to generate endothelial cells (ECs). For the differentiation of orange smooth muscle cells (SMCs) and red fibroblasts, wnt-signaling was further activated, followed by treatment with either a combination of TGFβ and bFGF or bFGF only. All four were differentiated successfully and functionally characterized using cell type-specific properties such as the ability for contraction, tube formation or collagen production. Fibrin-based multicellular hEHTs were generated from 50% ECs, 30% CMs, 10% SMCs and 10% fibroblasts. The stable transduction of hiPSCs allowed for the analysis of cell organization in living tissue via confocal microscopy. Flow cytometry revealed that especially endothelial cells have low survival rates in 3-dimensional cardiac tissue culture. Conclusion: Colour-coded hiPSC are feasible for the generation of multicellular hEHTs and live-analysis of the fate of different hiPSC-derived cell types. Future experiments will systematically investigate the impact of different cell composition on contractile function, cell-cell interactions and tissue organization.

Background: Autophagy provides essential bioenergetic intermediates during hypoxia and nutrient deprivation, as well as degrading defective macromolecules and organelles. As this process is essential in maintaining cellular homeostasis its dysfunction is causatively linked to prevalent diseases including those of the cardiovascular system. Aim, Methods and results: Our aim was to assess the impact of oxidative stress, associated with cardiovascular disease, on the induction of autophagy, by monitoring LC3 lipidation in cultured cells exposed to hydrogen peroxide (H2O2). This led to the novel observation that LC3 lipidation in human embryonic kidney and primary rat aortic smooth muscle cells was inhibited by H2O2 during amino acid deprivation. As LC3 lipidation is essential for autophagosome maturation, loss of this process by H2O2 prevents induction of autophagy. The two conjugase enzymes, Atg3 and Atg7, essential for LC3 lipidation have catalytic thiols that we hypothesised were sensitive to inhibitory oxidation, a process that would prevent autophagosome maturation. The sensitivity of Atg3 and Atg7 to oxidative inhibition was observed in in vitro lipidation assays by loss of LC3 lipidation when enzymes were exposed to oxidised glutathione, and detection of an intracellular catalytic thiol bound disulphide heterodimer.

61. Autophagy and ventricular fibrillation in isolated rat hearts A. Czegledi, A. Gyongyosi, R. Zilinyi, A. Tosaki, I. Lekli Department of Pharmacology, Faculty of Pharmacy, University of Debrecen, Hungary Background: Pathomechanisms of many diseases are promoted by disruption of cellular homeostasis through the accumulation of toxic detritus produced by altered cellular metabolisms. The autophagy degrades toxic aggregates, enabling cells to maintain structural, biochemical and functional integrity. The objective of the study was to determine whether electrically-induced ventricular fibrillation (VF) can elicit autophagic processes in the absence of ischemia in the myocardium.

Conclusion: Consistent with impaired autophagy and increased oxidative stress associated with aging, we observed increased formation of an inhibitory disulphide between the catalytic thiols of Atg3 and Atg7 in aorta of aged, starved mice. This loss in autophagy through Atg3 and Atg7 oxidation during aging may predispose individuals to increased risk of cardiovascular disease, therefore providing a potential novel target for therapy. 63. Myocardial signaling alterations induced by chronic systemic hypoxia and hyperoxia A. Gyongyosi1, L. Terraneo2, I. Lekli1, P. Bianciardi2, A. Tosaki1, M. Samaja2 1

Methods and results: Isolated working rat hearts were aerobically perfused and VF was induced by pacing 20 Hz for 1, 3, 10 min, followed by 120 min of VF-free aerobe perfusion. Since there were no significant changes in coronary flow rates, the changes in autophagic markers could not be attributed to a pacing-induced ischemic event. The deterioration of the cardiac function (heart rate, aortic flow, coronary flow, cardiac output, stroke volume) was registered with the increase of the duration of the VF. Increased expression of autophagic proteins including, Atg5-12, p62 and P-mTOR/mTOR ratio were detected in hearts subjected to VF. However, a decline of the autophagic protein LC3BII/ LC3I ratio was observed in hearts. Parallel to the VF-induced autophagic processes, an increased expression of apoptotic signaling was detected indicating by the level of cleaved caspase-3. Conclusion: The results suggest that during a prolonged duration of VF the autophagic process is unable to control the stress and may induce apoptotic activity.

Department of Pharmacology, Faculty of Pharmacy, University of Debrecen, Hungary 2 Department of Health Science, University of Milan, Italy Background: The redox imbalance and the consequent oxidative stress areimplicated in many pathological conditions, including cardiovascular diseases. Moreover, it has been associated with autophagy and apoptosis processes. The aim of the present study was to understand the heart tissue responses to prolonged hypoxia or hyperoxia, and whether such situations might lead to the activation of survival mechanisms or to trigger cell death. Methods and results: Seven-week old Foxn1 mice were exposed to hypoxia (10% O2), normoxia (21% O2) or hyperoxia (30% O2) for 28 days. Myocardial alterations in redox balance, housekeeping protein levels, autophagic and apoptosis process regulation were studied. The level of hypoxia inducible factor (HIF-1α) was increased by hypoxia while (HIF-2α) was not affected. The altered O2 fractions in breathed


air inversely elevated the housekeeping protein levels, the oxidative stress and autophagy process. Surprisingly, our results revealed alterations in the level of housekeeping proteins. The expressions of α-tubulin, actin and GAPDH were increased in the hypoxic while were decreased in the hyperoxic group. D-ROMs test demonstrated increased oxidative stress in hypoxic group compared with hyperoxic. Chronic hypoxia activated autophagy: we observed elevated levels of Beclin-1 and LC3B-II in hypoxia but p62 was constant. Nevertheless, we measured significantly enhanced apoptosis positive cells by the Tunel assay and higher Bax/Bcl2 ratio in hyperoxia compared with hypoxia. Conclusion: These finding suggest that autophagy is induced in hearts under hypoxia, which may serve as a protective mechanism in response to enhanced oxidative stress. Furthermore, on one hand prolonged hypoxia-induced autophagy leads to reduced heart apoptosis, on the other hand low autophagic level in hyperoxia could not fend off the excessive apoptosis. Acknowledgments: Campus Mundi programme (Tempus Public Foundation, Hungary). 64. Possible connection between heme-oxygenase-1/CO system and autophagy in cardiomyocytes A. Gyongyosi, K. Szoke, A. Czompa, I. Bak, A. Tosaki, I. Lekli Department of Pharmacology, Faculty of Pharmacy, University of Debrecen, Hungary Background: Several groups have demonstrated that separately the increased activity of heme-oxygenase-1/CO system and autophagy could protect the myocardium against ischemic events. However, it is still not clear whether a connection between HO-1/CO system and autophagy processes exists in the myocardium. In the current study we aimed to examine the possible connection between the two systems. Material and methods: H9c2 cardiomyoblast cells were treated with different dose of hemin or cobalt-protoporphyrin IX (CoPPIX) or vehicle (20 mM NaOH solution) for 24-h. After the treatment of the cells cytotoxicity was measured by MTT assay. Furthermore, staining was carried out to determine the alterations in cell size. To study the autophagic process CytoID staining was carried out and cells were studied by fluorescence microscope. Moreover, Western blot analysis was performed to analyse the level of HO-1, certain autophagy related proteins and apoptosis markers. Results: We have detected a slight decrement in cell viability in the hemin and CoPPIX treated groups, respectively, which may indicate a toxic effect of high concentration of heme-oxygenase-1 inducers. The cell size did not alter. As it was expected a robust induction of HO-1 were detected with both of inducer. An enhanced number of autophagosome were detected by CytoID staining, and elevated level of LC3B-II was found in the highest hemin and CoPPIX treated groups. Conclusion: Taken together, our results show that, there is a connection between HO-1/CO system and autophagy process, but further experiments needs to be carried out to precisely understand the nature of the connection. Acknowledgments: OTKA PD 111794, ‘National Excellence Program’: TÁMOP-4.2.4.A/2-11/1-2012-0001, NTP-NFTÖ-16, GINOP-2.3.2-152016-00043.

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65. Screening modulators of the ubiquitin-proteasome system and the autophagy-lysosomal pathway - Towards novel targets to treat proteotoxicity S.R Singh1,2,3, W.C Bacon1, G. Davis1, J. Gulick1, H. Osinska1, P. McLendon1, S. Schlossarek2,3, J. Robbins1, L. Carrier2,3 1

Division of Molecular Cardiovascular Biology, the Heart Institute, Cincinnati Children's Hospital, Cincinnati, Ohio, USA 2 Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3 DZHK (German Centre of Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany Background: The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Whereas initially considered as two independent pathways, there is emerging evidence that both work in concert. With many diseases such as neurodegenerative disorders, cancer and cardiac diseases being defective in one of the two or even both systems, there is a great interest in finding targets that modulate these systems. Objective: To identify novel modulators of the UPS and ALP in a lentiviral shRNA library, unbiased, total genome high-throughput screen (HTS). Methods and results: We generated a stable HEK293 cell line expressing a UPS reporter (ubiquitinG76V-mCherry) and an ALP reporter (GFP-LC3) under the control of two different promoters. This cell line was screened with a genome-wide shRNA library for genes that increase ubiquitinG76V-mCherry intensity (ubiquitinated substrates) and GFP-LC3 puncta (autophagosomes). Our screen identified 310 hits of which 12 were previously associated with the UPS and 9 with the ALP. Twenty-four of the hits were previously found to be downregulated in Alzheimer's disease and 5 to be associated with heart disease. We successfully tested some of the most efficient candidates on their ability to modulate protein aggregation in a cardiomyocyte protein aggregation model. Conclusion: We identified several novel modulators of UPS and ALP that could be evaluated as targets in various pathologies involving protein aggregation and/or deficient protein degradation systems. 66. Possible connection between the anti-aging klotho protein and autophagy K. Szoke, A. Czompa, A. Gyongyosi, I. Lekli, A. Tosaki Department of Pharmacology, Faculty of Pharmacy, University of Debrecen, Hungary Background: According to the WHO, the average age of the population is increasing. Age is considered as main risk factor for the common diseases including cardiovascular disease and cancer in the developed countries. Several studies show that autophagy plays an important role in aging. Numerous studies revealed that autophagy decreases with aging. In this study we have investigated the connection between autophagic process and the level of klotho protein. Material and methods: To induce the klotho protein aged mice (2 years old) were treated with intraperitoneal injection of rapamycin

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(1 mg/bwkg) weekly for 12 weeks. We have monitored the body weight during the treatment. Thereafter the organs were isolated. The expression levels of anti-aging klotho protein and autophagic, apoptopic proteins such as LC3B-II and p62 were evaluated by Western blot. Furthermore, we have also visualized the immunohistochemical localization of klotho protein in the different organs. Results: We have noticed decreased lifespan and body weight at the control group compared to mice exposed to rapamycin, where the extended lifespan suggests that klotho functions as an anti-aging protein. We have investigated that the expression level of the klotho increased in most of the organs, especially in the kidney. Furthermore, a decreased p62 protein level was found in the treated group, which is involved in the regulation of apoptosis and autophagy.

inhibition of ER stress and autophagy in tachypaced Drosophila confirm their role in derailing cardiomyocyte function. In vivo treatment with Na-PBA protected atrial-tachypaced dog cardiomyocytes from electrical remodeling (action potential duration shortening, L-type Ca 2 +-current reduction), cellular Ca 2 +-handling/contractile dysfunction, ER stress and autophagy, and attenuated AF progression. Finally, atrial tissue from persistent AF patients reveals activation of autophagy and induction of ER stress, which correlates with markers of cardiomyocyte damage. Conclusions: These results identify ER stress-associated autophagy as an important pathway in AF progression and demonstrate the potential therapeutic action of the ER-stress inhibitor 4PBA. 68. Withdrawn

Conclusion: Taken together our results suggest that in case of rapamycin induced klotho protein autophagy is increasing, which may help to reduce damaged protein organelles. Acknowledgements: OTKA-PD-111794, TÁMOP 4.2.4 A/2-11-1-20120001, GINOP-2.3.2-15-2016-00043. 67. Endoplasmic reticulum stress is associated with autophagy and cardiomyocyte remodeling in experimental and human atrial fibrillation M. Wiersma1,2,⁎, R.A.M. Meijering2,⁎, X.Y. Qi3, D. Zhang1,2, T. Liu4, F. Hoogstra-Berends2, O.C.M. Sibon5, R.H. Henning2, S. Nattel3, B.J.J.M. Brundel1,2 1

Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands 2 Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands 3 Department of Medicine, Montreal Heart Institute and Université de Montréal, the Department of Pharmacology and Therapeutics, McGill University, Montreal, Québec, Canada and Institute of Pharmacology, West German Heart and Vascular Center, Faculty of Medicine, University Duisburg-Essen, Essen, Germany 4 Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, China 5 Department of Cell Biology, University Medical Center Groningen, University of Groningen, The Netherlands *contributed equally Background: Derailment of proteostasis, the homeostasis of production, function and breakdown of proteins, contributes importantly to the self-perpetuating nature of atrial fibrillation (AF), the most common heart rhythm disorder in man. Autophagy plays a key role in proteostasis by degrading aberrant proteins and organelles. Since the role of autophagy in AF is unknown, we investigated its role in experimental and clinical AF. Methods and results: Tachypacing of HL-1 atrial cardiomyocytes causes a gradual and significant activation of autophagy, as evidenced by enhanced LC3B-II expression, increased autophagic flux, enhanced autophagosome formation and degradation of p62, resulting in reduction of Ca 2 +-amplitude. Autophagy is activated downstream of endoplasmic reticulum (ER) stress: blocking ER stress by the chemical chaperone 4-phenyl butyrate (4PBA), overexpression of the ER chaperone-protein HSPA5 or overexpression of a phosphorylation-blocked mutant of eIF2α, an ER-regulated autophagy inducer, prevent autophagy activation and Ca2 +-transient loss in tachypaced HL-1 cardiomyocytes. Moreover, pharmacological

69. Evaluation of the autophagy-lysosomal pathway in cardiac tissue and hiPSC-derived cardiomyocytes from patients with inherited cardiomyopathies A.T.L. Zech1,2, S.R. Singh1,2, E. Krämer1,2, C. Redwood3, J. van der Velden4,5, T. Eschenhagen1,2, S. Schlossarek1,2, L. Carrier1,2 1 Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom 4 Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, the Netherlands 5 ICIN-Netherlands Heart Institute, Utrecht, the Netherlands Background: One of the major proteolytic systems within cells is the autophagy-lysosomal pathway (ALP) that keeps cellular homeostasis by engulfing damaged proteins and organelles in a double-membraned vesicle that matures into an autophagosome and later eventually fuses with a lysosome for degradation of its content. If this system is defective, it can cause rare diseases (e.g. Danon disease). Here, we evaluate putative ALP alterations in human hypertrophic and dilated cardiomyopathies (HCM, DCM). Methods: Protein levels of major ALP players were determined in septal myectomies from HCM individuals carrying MYBPC3 mutations, in explanted hearts of DCM patients, and in healthy donor hearts. The autophagic flux was evaluated in human inducedpluripotent stem cell (hiPSC)-derived cardiomyocytes from a healthy donor. Results: LC3 (microtubule-associated protein light chain 3)-II levels were higher in HCM and DCM than non-failing individuals, whereas phosphorylated S6 ribosomal protein levels were markedly lower only in HCM than in non-failing samples. Contrary, protein levels of lysosome-associated membrane protein 2 (LAMP-2) and p62 were markedly higher only in DCM. The autophagic flux was evaluated in hiPSC-derived cardiomyocytes. Treatment with autophagy modulators induced a marked accumulation of LC3-II protein levels. Similar results were obtained in hiPSC-derived cardiomyocytes from a HCM and a DCM patient. Preliminary data suggests a blunted autophagic flux in HCM and DCM hiPSC-derived cardiomyocytes. Conclusion: These data suggest a lysosomal dysfunction in human HCM and DCM characterized either by a loss of lysosomes or its accumulation. Experiments in hiPSC-derived cardiomyocytes indicate that it is feasible to evaluate the autophagic flux.


70. Effects of DNA methylation inhibition in pathological cardiac hypertrophy in vitro and in vivo A. Löser1,2, J. Stenzig1,2, Y. Schneeberger1,2,3, M.N. Hirt1,2, A. Hansen1,2, R. S.-Y. Foo4, T. Eschenhagen1,2 1

Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Cardiovascular Surgery, University Heart Center, Hamburg, Germany 4 Genome Institute of Singapore, Singapore BAckground: DNA methylation has gained recent attention as a mechanism of gene transcription regulation and possible target for heart failure therapy. Here we studied DNA methylation and the effect of DNA methyltransferase (DNMT) inhibition in two models of cardiac hypertrophy, rat engineered heart tissue (EHT) subjected to afterload enhancement (AE) and rats subjected to transverse aortic constriction (TAC). Methods: To induce experimental hypertrophy in vitro, EHTs cultured between flexible hollow silicone mountings were subjected to an acute increase in resistance by insertion of metal braces into the mounting posts (AE). Cardiac hypertrophy in adolescent rats was provoked by TAC surgery with partial clip ligation of the thoracic artery. DNA methylation was analyzed by microarray, pull-down assay, bisulfite treatment and subsequent high resolution melting (EHT) or by reduced representation bisulfite sequencing (TAC). Results: DNA methylation data from both models suggested the existence of a generic methylation signature of pathological hypertrophy. Several genes associated with contractile function were differentially methylated both in vivo and in vitro. Pharmacological DNMT inhibition (RG108) reduced promoter hypermethylation and partially restored the hypertrophy-associated impairment of contractile function. The effect on methylation was more obvious in purified cardiomyocytes, emphasizing the importance of cell type-specific analysis. Conclusion: Our results suggest that dynamic DNA methylation plays a role in pathological cardiac remodeling and may be a target for future therapy. 71. High-content screening identifies microRNAs modulating cardiac fibrosis M.G. Bexiga, R. Videira, M. Mano Functional Genomics and RNA-based Therapeutics laboratory, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal Background: Cardiac fibrosis is a maladaptive remodelling process of the myocardium, which results from an over-activation of cardiac fibroblasts leading to excessive extracellular matrix deposition and fibrotic scar formation. Upon injury, activated fibroblasts differentiate into myofibroblasts, cells that play a role in collagen turnover and that are able to contract, thereby aiding in the maintenance of heart wall integrity. However, prolonged persistence of cardiac myofibroblasts and excessive deposition of extracellular matrix proteins leads to stiffening of the heart wall. In this context, the

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identification of the cellular factors that modulate cardiac fibrosis and a better understanding of the underlying molecular mechanisms is of the utmost importance to reveal novel opportunities for therapeutic intervention. Recent studies have shown that microRNAs control several features of cardiovascular diseases, including cardiac fibrosis. Methods and results: To systematically identify microRNAs that positively or negatively modulate human primary cardiac fibroblast proliferation and myofibroblast differentiation, we performed a highcontent microscopy functional screening using a library of microRNA mimics corresponding to all the annotated microRNAs (2,588 mature microRNAs). Using this approach, we identified 145 microRNAs that increase and 45 microRNAs that decrease myofibroblast differentiation (α-SMA + fibroblasts) by more than 4-fold relative to control. To pinpoint the most relevant microRNAs, we performed a secondary screening focused on the analysis of the impact of the identified microRNAs on extracellular matrix deposition. Conclusion: Finally, through the combination of comparative transcriptomic analysis and bioinformatics, we are identifying the key targets of the most interesting microRNAs, in order to infer their mechanisms the action. 72. Revealing transcriptomic changes in response to chronic angiotensin II infusion in atria versus ventricles of the mouse by nanostring analysis S. Schulz1,2, O. Prysyazhna3, P. Eaton2, F. Cuello1,2 1

Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf; Cardiovascular Research Center, Hamburg, Germany 2 DZHK partner site Hamburg/Lübeck/Kiel, Hamburg, Germany 3 King's College London, Cardiovascular Division, The British Heart Foundation Centre of Excellence, The Rayne Institute, St Thomas' Hospital, London, SE1 7EH, UK Background: Angiotensin II (AngII) is the most potent endogenous vasoconstrictor. Chronically high levels of AngII are contributing to hypertension, which is an important risk factor for heart failure development in patients. We aimed to investigate the transcriptomic changes induced by chronic AngII infusion in atria and ventricles to gain a better understanding of the cardiac remodelling processes induced by chronic blood pressure elevation. Methods and results: C57BL/6 mice (30g) were infused with AngII 1.1 mg/kg in saline (n = 6) or saline only (n = 6) by Alzet osmotic minipumps for three days. Mice were sacrificed and the atria and ventricles prepared and snap frozen. RNA preparation was performed using the PureLink RNA Mini Kit from Invitrogen and the RNA quality investigated by Bioanalyzer (RIN N 8). RNA samples were pooled (n = 6) from each intervention to finally obtain 4 treatment groups: atria saline, ventricle saline, atria AngII, ventricle AngII. Transcriptomic analysis was performed using a PanCancer Pathways Mouse Panel on a Nanostring nCounter Sprint. The panel allows simultaneous analysis of 751 genes important for cell division and growth. Of the 751 analyzed genes, 96 genes were upregulated (2-fold) in the atrium group after AngII and 27 downregulated (2-fold). In the ventricle group AngII induced upregulation of 57 genes and downregulation of 8 genes compared to the respective saline group. Conclusion: AngII exposure induces distinct changes in gene transcription in atria versus ventricles. Amongst the genes changed in

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transcription were those involved in extracellular matrix formation (tenascin), fibrosis (collagen) development and metalloproteinases (MMP9). 73. The m6A mRNA-methylome in healthy and diseased myocardium V. Serbent1, E. Riechert1, X. Li1, C. Dieterich2, H. Katus1, M. Völkers1 1 Innere Med. III, Kardiologie, Angiologie und Pneumologie, Universitätsklinikum Heidelberg, Heidelberg, Germany 2 Klaus-Tschira-Institute für Computational Cardiology, Universitätsklinikum Heidelberg, Heidelberg, Germany

Background: Conceptually similar to modifications of DNA, mRNAs undergo chemical modifications affecting RNA activity, localization, and stability. Methylation of the adenosine to N6-Methyladenosine (m6A) is crucial for several aspects of biology. Still, the role of mRNA modifications in cardiac diseases has never been studied. Hence the goal of this study was to define the m6A-RNA-methylome from healthy and diseased myocardium in murine and human samples. Additionally, the effect of manipulation of overall m6A abundance by knocking down or overexpressing the m6A “writer” Mettl3 in cardiomyocytes on growth and survival in vitro and on cardiac function in vivo was studied. Results: Overall m6A-RNA quantification as well as m6A and RNA sequencing were performed on total RNA from human DCM, ICM and healthy heart tissue. Hundreds differentially methylated transcripts were identified. Transcripts encoding for genes involved in heart development and cardiac muscle function are differentially methylated in human DCM samples. Moreover, an increase of m6A levels by overexpressing METTL3 reduced cell growth, whereas a decrease by Mettl3 knockdown led to an increased cell growth in vitro. In vivo cardiomyocyte specific overexpression of METTL3 resulted in a reduced heart growth but also to reduced cardiac function after TAC surgery. The loss of m6A after TAC surgery led to higher translation efficiency of those transcripts in mouse myocardium suggesting m6A's main effects by affecting translation of the carrying mRNAs. Conclusion: Thus, mRNA modifications might represent an additional way of controlling gene expression in the myocardium, which has not been studied before. 74. Sex differences in oestrogen-dependent macrophage activation in cardiac chronic inflammation M.L. Barcena de Arellano1,2, M. Niehues1,2, A. Beling2,3, V. Regitz-Zagrosek1,2 1 Institute of Gender in der Medicine, Center of Cardiovascular Research, Charité – Universitätsmedizin Berlin, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Berlin, Berlin, Germany 3 Institute for Biochemistry, Charité Universitätsmedizin Berlin, Berlin, Germany

Background: Sex hormones act on the immune system. Most autoimmune diseases are more prevalent in women than in men, which has been attributed to the immune stimulatory effects of oestrogen (E2). IFN-γ-positive-CD4 cells and activated T-cells were decreased in the oestrus phase, while the amount of regulatory T-cells was increased.

mIRNA are known to positively/negatively inflammatory processes in a sex-dependent manner. Hypothesis: We propose sex differences in the macrophagedifferentiation/activation in a chronic myocarditis and that the differently macrophage activation is regulated be sex-differences in the expression of miRNA in the heart of myocarditis animals. Methods: Male/female ABY/SnJ-mice were infected with COXV3. We analyzed the miRNA expression in female/male macrophages. Male/ female macrophage cell line and male/female bone marrow macrophages were differentiated into pro-inflammatory-M1 or into antiinflammatory-M2 macrophages. M1/M2-macrophages were treated with E2/E2 + ICI-182,780. Results: We found sex differences in the expression of let7a-d, miR27b and miR199a and a reduction in the expression of 199a in male mice. ERK activation was only reduced in males. IKB-α was decreased in both sexes. NFκB and c-fos expression were increased in both sexes. TNF-α, IL-1β, MCP-1 were upregulated in male and downregulated female mice. Male M1-macrophages showed a reduction of miR199a after E2 treatment. E2 modulated differently the expression of MCP-1 in female and male pro-inflammatory macrophages. Conclusion: Downregulation of miR199a in male, but not female mice with chronic myocarditis leads to c-fos regulation/upregulation of its downstream targets. E2 in decreases the expression of miR199a males and modulates MCP-1/IL-1β, potentially promoting modulation of the immune response in the heart during inflammation. 75. Shaping of cardiomyocyte metabolism by myofibroblasts: a new paradigm for the derangement of energy metabolism in heart failure? E. Lazzarini1, C. Ruggeri1, P. Altieri1, S. Garibaldi1, C. Brunelli1,2, S. Ravera3, P. Ameri1,2 1 Laboratory of Cardiovascular Biology, Department of Internal Medicine, University of Genova, Genova, Italy 2 Cardiovascular Disease Unit, IRCCS AOU San Martino – IST, Genova, Italy 3 Laboratory of Biochemistry, Department of Pharmacy, University of Genova, Genova, Italy

Background: Cardiac fibroblasts (Fib) activated to myofibroblasts (myoFib) have been shown to regulate aspects of cardiomyocyte biology in a paracrine way. Here, we hypothesized that myoFib-secreted factors might affect energy metabolism of cardiomyocytes. Methods and results: Primary mouse neonatal cardiomyocytes incubated with the conditioned medium (CM) of transforming growth factor-beta (TGFβ)-primed myoFib, but not quiescent Fib, exhibited decreased oxygen consumption and ATP synthesis in response to exogenous pyruvate/malate or succinate and a drop in the intracellular ATP/AMP ratio, despite heightened glycolysis with lactate production. These alterations were not observed when cardiomyocytes were treated with TGFβ alone. Furthermore, mitochondrial membrane potential and apoptosis markers were unaffected by the myoFib-CM, excluding structural damage. Since depressed mitochondrial oxidative metabolism and enhanced glycolysis are features of heart failure (HF), we asked whether factors chiefly involved in HF pathogenesis might activate Fib to modulate cardiomyocyte metabolism. We thus focused on doxorubicin, which has been demonstrated to cause HF in experimental models and patients. Fib exposed to sub-apoptotic concentrations of the drug


displayed increased α-smooth muscle actin expression and collagen synthesis. These results were confirmed with primary human Fib isolated from atrial appendages collected during cardiac surgery. Similar to the CM of TGFβ-treated myoFib, the CM of doxorubicinchallenged Fib inhibited mitochondrial oxygen consumption and ATP synthesis and stimulated the activity of glycolytic enzymes and lactate dehydrogenase in cardiomyocytes. Conclusions: Cardiomyocyte metabolism is reorganized by the myoFib secretome towards reduced mitochondrial oxidation and augmented glycolysis with lactate release. Further studies are needed to understand the role of myoFib-initiated metabolic shift in HF. 76. Novel software package for high-throughput processing and analysis of cardiac optical mapping data – Development of a conduction velocity analysis module C. O'Shea1,2,3, S. Wells2, A.P. Holmes2, T.Y. Yu2, J. Correia4, P. Kirchhof2, L. Fabritz2, K. Rajpoot3, D. Pavlovic2

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77. Mitochondrial N-formyl peptides in sterile inflammation after acute myocardial infarction M.-K. Torp1,3, Trine Ranheim2,3,4, Torun Flatebø1, Christina Heiestad1, Arne Yndestad2,3,4, and Kåre-Olav Stensløkken1,3 1

Division of Physiology, Institute of Basic Medical Sciences, University of Oslo, Norway 2 Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Norway 3 Center for Heart Failure Research, University of Oslo, Norway 4 KG Jebsen Center for Inflammation Research, University of Oslo, Norway Background: Acute myocardial infarction results in necrosis and initiates a local sterile inflammation activated through DamageAssociated Molecular Patterns (DAMPs). Mitochondrial proteins are highly immunogenic due to their bacterial origin. Moreover, the cardiomyocyte volume consists of ~30% mitochondria. We aim to investigate if mitochondrial N-formyl peptides influence healthy cardiomyocytes in co-culture with tissue resident macrophages.

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EPSRC Centre for Doctoral Training in Physical Sciences for Health, School of Chemistry, University of Birmingham, Birmingham, UK 2 Institute of Cardiovascular sciences, University of Birmingham, Birmingham, UK 3 School of Computer Science, University of Birmingham, Birmingham, UK 4 Cairn Research, Graveney Road, Faversham, UK Background: Optical mapping is a powerful research tool allowing unique visualisation of action potential conduction speed across cardiac tissue. However, activation and conduction analysis is computationally intensive, camera specific and not standardised post-acquisition. We have developed and tested a novel method where tissue activation is visualised as an ‘activation curve’. This new method is packaged into a standalone module for quantifying conduction velocity, along with established single- and multivector methods. Methods and results: We first validated single- and multi-vector analysis algorithms by measurement of conduction velocity in computationally simulated data (single-vector: 30 cm/s = 30.43 cm/s; 50 cm/s = 50.12 cm/s; 70 cm/s = 78.54 cm/s and multi-vector: 30 cm/s = 29.67 ± 1.66 cm/s; 50 cm/s = 50.47 ± 2.02 cm/s; 70 cm/s = 71.51 ± 5.74 cm/s;). To generate “activation curve”, whole tissue activation is plotted as a function of time and 50% activation point defined as activation constant. Activation constant decreased at faster speeds (30 cm/s = 4.40 ms; 50 cm/s = 2.70 ms; 70 cm/s = 1.91 ms;). To validate software package and novel “activation constant” method with experimental data, MF1 mouse atria (n = 8) were loaded with di-4-ANEPPS and paced for 10 minutes under normoxia (95%O2/5%CO2) and hypoxia (95%N2/5%CO2). As expected, in normoxia, increase in pacing frequency from 3 Hz to 10 Hz caused conduction velocity slowing from 54.85 ± 4.45 cm/s to 48.71 ± 4.75 cm/s (multi-vector method, P b 0.05). Activation constant increased from 1.95 ± 0.53ms to 2.37 ± 0.81 ms (P b 0.05). Hypoxia decreased velocity to 39.33 ± 4.14 cm/s (3 Hz, multi-vector method, P b 0.0001) and increased activation constant to 3.26 ± 1.06 ms (P b 0.01). Conclusions: We report development of new, standalone software for measurement of conduction velocity across the myocardium, compatible with different camera parameters and animal species. Software accurately measures conduction velocity through multiple customisable quantification methods, including a novel activation curve method.

Methods: Cardiomyocytes and bone marrow-derived macrophages were isolated from adult C57BL/6N male mice, co-cultured in a Corning Transwell system, and treated with N-formyl peptide receptor (FPR) agonists. Cell death was investigated with HighThroughput microscopy. Expression of the FPRs was investigated using absolute quantitative qPCR, western blotting, and immunohistochemistry. Tissue resident macrophages were identified in mouse heart sections by immunohistochemistry. Release of cytokines was measured by ELISA. Results: Absolute quantification of FPR genes revealed high expression of FPR2 in macrophages and low expression in cardiomyocytes. However, the receptors were expressed in PBS perfused mouse hearts possibly indicating presence of tissue resident macrophages. Macrophages were confirmed present in the heart by immunohistochemistry with Mac-2 antibody. No significant changes in viability in cardiomyocyte alone or cardiomyocytes in co-culture were observed when treated with the FPR2 agonist, fMMYALF. Moreover, IL-6 release was significantly increased with 10 μM fMMYALF in both cardiomyocytes and in the co-culture, but Mip-2 release remained unaffected. Conclusion: Mitochondrial N-formyl peptides appear to have limited roles in cardiomyocytes after myocardial infarction with the interaction of tissue resident macrophages. 78. Cardiac repair with human engineered heart tissue from induced pluripotent stem cells K. Breckwoldt1,2, F. Weinberger1,2, S. Pecha2,3, A. Kelly4, B. Geertz1,2, J. Starbatty1,2, G. Smith4, A. Hansen1,2, T. Eschenhagen1,2 1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Cardiovascular Surgery, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 4 Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, Scotland

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Background: In this study we transplanted human Engineered heart tissues (hEHTs) from human induced pluripotent stem cell (hiPSC)derived cardiomyocytes and hiPSC-endothelial cells on cryo-injured guinea-pig hearts and investigated whether hEHTs support left ventricular function. Methods and results: Left ventricular myocardial cryo-injury was induced in adult guinea pigs (n = 72) and resulted in large transmural scars (22 ± 8% of the LV wall, n = 52). 7 days after injury hEHTs (n = 35), cell-free constructs (n = 29) or constructs with ECs only (hEET, n = 8) were implanted. Echocardiographic evaluation 7 days after injury showed a 35% decline in fractional area change (FAC). 28 days after transplantation, left ventricular function remained essentially unchanged in animals that had received cellfree constructs or hEETs, whereas hEHT transplantation improved FAC by 31%. Evidence of electrical coupling was successfully demonstrated in a subset of hEHT hearts. Within the remote myocardium, hEHT-transplanted hearts demonstrated shorter AP duration and faster transmural conduction velocity compared to control hearts, compatible with a reversal of the heart failure phenotype in the infarcted hearts. ECG recordings performed throughout the study showed no pro- or anti-arrhythmic effect of hEHT transplantation. Histological and immunohistochemical analyses 28 days after transplantation demonstrated compact human muscle islands, positive for human Ku80, within the scar only in the hEHT group, amounting to 11.9 ± 23.1% of the scar area (n = 21). The new myocardium was infiltrated by vessels containing erythrocytes, demonstrating connection to the guinea pig vasculature. Conclusion: This study demonstrated for the first time that hEHTs are able to electrically couple to host myocardium and improve left-ventricular and electrophysiological function after transplantation. 79. Generating a single cell gene expression atlas of the healthy and diseased heart M.M. Gladka1, B. Molenaar1, H. de Ruiter1, D. Versteeg1,2, S. van der Elst1, G. Lacraz1, A. van Oudenaarden1, E. van Rooij1,2 1

Hubrecht Institute, KNAW, 3584CT Utrecht, The Netherlands Department of Cardiology, University Medical Centre, 3584CX Utrecht, The Netherlands 2

Rationale: Genome-wide transcriptome analysis of healthy and diseased tissues has greatly advanced our understanding of the regulatory networks driving remodeling responses in the heart. However, so far these approaches have been hampered by the use of whole tissue samples and cell populations. Recent developments in RNA amplification strategies provide the opportunity to use small amounts of input RNA for genome-wide sequencing. Single-cell transcriptome analysis of the adult heart would allow for identification of cell type composition and transcriptome heterogeneity across cells of the same type. This unbiased view of cellular gene expression changes will greatly help to improve our understanding of the relevant molecular mechanisms that underlie cardiac remodeling. Objective: To perform transcriptome analysis at a single cell level of both the healthy and injured heart to identify changes in cellular gene expression and composition in response to ischemic damage. Methods and results: To obtain high quality RNA from digested cardiac tissue we first optimized our method for enzymatic digestion and subsequent RNA isolation. Next we adjusted our gating

strategy based on several scatter properties to allow us to sort living cells from both healthy and injured hearts into 384-well plates. Cell lysis followed by robotics liquid handling for barcoding was used to perform automated single-cell-sequencing using the CELseq2 protocol. This powerful methodology provides us with gene expression data for all cardiac cell types and allows us to study inter- and intracellular changes induced by cardiac injury. Conclusion: Here we present a reliable method for obtaining a single cell gene expression signature in individual cells from both the healthy and diseased heart. We anticipate that this new approach will be useful for developing a deeper understanding of cardiac biology and the pathophysiology of heart disease. 80. The gender divide: H-Y-mismatched embryonic stem cell transplantation causes rejection of nucleus matched cells in mice S.T. Kueppers1,2, X. Hu1,2,3, T. Deuse1,2,3,5, M. Alawi4, D. Wang1,2,3, H. Reichenspurner2,5, S. Schrepfer1,2,3,5 1

Transplant and Stem Cell Immunobiology (TSI) Laboratory, University Heart Center Hamburg, University of Hamburg, Cardiovascular Research, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Transplant and Stem Cell Immunobiology (TSI) Laboratory, Department of Surgery, University of California, San Francisco, CA, USA 4 Center for Bioinformatics, University of Hamburg, Hamburg, Germany 5 Department of Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany Background: Due to their pluripotency, embryonic stem cells (ESCs) are promising candidates for cell-based regenerative therapies for heart failure. Rejection of ESC-based grafts due to major histocompatibility complex mismatch is a known limitation of these approaches. However, less is known about the relevance of the H-Y antigens, a class of minor histocompatibility antigens encoded by the Y chromosome. Therefore, we sought to investigate the antigenicity of H-Y in a mouse model. Methods: Male ESCs (BALB/c) were transplanted into syngeneic male or female recipients. Immune activation was measured using unidirectional ELISPOT and mixed lymphocyte assays, and acquired tolerance was analyzed using Medawar assays. Results: Our results show an increased immune activation in the HY mismatched although syngeneic model. ELISPOT assays showed a significantly stronger response in females than males (p b 0.0001). T cells seem necessary for this H-Y-dependent immune response since female recipients lacking T cells (BALB/c-nu) generated a significantly lower ELISPOT frequency than female wildtypes (p b 0.0001). Female recipients showed no teratoma formation, whereas teratoma formation occurred in all male, female BALB/c-nu, and immunodeficient SCID beige recipients. In the Medawar experiments, we could induce tolerance against H-Y in female recipients by exposing them to male mESCs during their neonatal period. Conclusion: Our results indicate that H-Y-apssociated antigens by themselves are strong enough to cause rejection of MHCidentical ESC grafts. Due to relevant implications for both experimental as well as clinical applications, the immunobiology of the Y-chromosome has to be considered in the development of ESC-based tissue and organ regenerative strategies and therapies.


81. The brain natriuretic peptide improves cardiac function after myocardial infarction through vascular regeneration N. Li, C. Bielmann, S. Rignault-Clerc, N. Rosenblatt-Velin Département Cœur-Vaisseaux, Laboratoire Physiopathologie Clinique, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland Background: Brain natriuretic peptide (BNP) treatment was shown to protect the adult heart after myocardial infarction (MI). However, the mechanisms were not yet determined. Adult endothelial cells are able to express BNP receptors and BNP was shown to increase angiogenesis in mouse ischemic hindlimb. Thus the aim of this study was to determine the role of BNP on vascular regeneration after MI. Methods: MI was induced in C57BL/6 mice by ligation of the left anterior descending coronary artery. BNP was injected directly into the myocardium after the surgery, then intraperitoneally every 2 days up to 14 days. Mice were sacrificed 10 days or 4 weeks after surgery. The infarction zone (ZI) was separated from the rest of the heart (remote zone, RZ).

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more significant decrease in CV of healthy hearts when positioned in the more conductive compared to less conductive orientation (More conductive: 84.0 ± 4.3%; Less conductive; 103.1 ± 6.25% of baseline; p b 0.05; n = 7), due to the conductive anisotropy of the patch, although no differences were seen between the two orientations when applied to infarcted hearts (More conductive: 96.6 ± 9.6%; Less conductive; 106.5 ± 14.5% of baseline; n = 5). Conclusions: Micropatterning offers a unique way to modulate the mechanical anisotropy of the patch so that it better matches that of the heart. However, this results in less conductive material and a longer conduction path which could explain the smaller effects seen on cardiac conduction. Future work will focus on identifying the mechanism by which conductive patches exert their effect on the heart and determining the optimal mechanical and conductive properties. References 1 Mawad D, et al., Sci Adv. 2(11), 2016. 2 Kapnisi M, et al., (In preparation). 83. Inhibition of miR-21 improves outcome after acute myocardial infarction

Results: Interestingly, 10 days after MI induction, mRNA levels coding for eNOS (+63%, p = 0.04), Ve-Cad (+42%, p = 0.01), CD34 (+70%, p = 0.03), FIk-1 (+109%, p = 0.03) were increased in BNP-treated ZI when compared to infarcted untreated hearts. This led to an increase of the number of CD31+ cells in both ZI (+ 69%, p b 0.001) and RZ (+ 73%, p b 0.001) 4 weeks after BNP treatment. Furthermore, the level of mRNA coding for Wt1 (epicardial progenitor marker) was reduced (- 75%, p = 0.03) in ZI, while the number of Wt1+ cells was increased in RZ after BNP injections.

D. Ramanujam1,4, R. Hinkel2,3,4, V. Kazcmarek3, A. Howe2,3, K. Klett2,3, S. Engelhardt1,4, C. Kupatt2,4

Conclusion: Our results suggest that the beneficial effect of BNP after MI could be due in part to enhanced angiogenesis. Whether BNP modulates the migration and differentiation of cardiac vascular and/or epicardialderived progenitors is under investigation.

Rationale: Ischemic heart disease and heart failure are characterized by an adverse remodeling of the heart, featuring cardiomyocyte hypertrophy, increased fibrosis and capillary rarefication. For heart failure miRNAs have been identified as disease-relevant and dysregulated. Therefore, we aimed to investigate the therapeutic potential of a cardiac treatment of LNA-21.

82. Characterising auxetic micropatterning of a conductive cardiac patch C. Mansfield1, M. Kapnisi2, D. Mawad3, I. Bardi1, F. Perbellini1, C.M. Terracciano1, M.M. Stevens2, S.E.Harding1 1

National Heart and Lung Institute, Imperial College London, London, UK Department of Materials, Imperial College London, London, UK 3 University of New South Wales, Sydney, Australia 2

Background: We have developed a polyaniline-based conductive patch with enhanced electronic biostability. We have previously shown that this patch is able to modulate myocardial conduction1. Methods and results: Epicardial voltage mapping of Langendorffperfused rat hearts showed that the patch causes a significant reduction in conduction velocity (CV) in healthy hearts (72.4 ± 5.6% of baseline; p b 0.02, n = 5) and a significant increase in CV in infarcted hearts (129.5 ± 7.9% of baseline; p b 0.05, n = 5) with similar trends seen when the patch was applied to viable myocardial slices. We have applied auxetic micropatterning to modulate the mechanical and conductive properties of the patch2. By altering the size of the auxetic micropattern we aim to mimic the mechanical and conductive anisotropy of the native myocardium. The micropatterned patch showed a

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Institut für Pharmakologie und Toxikologie, TU München Medizinische Klinik und Poliklinik, Klinikum Rechts der Isar der TU München 3 Institute for Cardiovascular Prevention, LMU Munich 4 DZHK (German Center for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany 2

Methods: Pigs (n = 4/group) underwent 60 min LAD occlusion at day 0. Locked nucleic acid-modified anti-sense miR-21 (LNA-21) was regionally applied at days 5 and 14 post MI. Left ventricular end-diastolic pressure (LVEDP) and ejection fraction (EF) were obtained at days 0, 28 and 56. Moreover, subendocardial segment shortening (SES) was conducted at day 56. Tissue samples were harvested and sirius red oil staining was performed for quantitative analysis of fibrosis. Results: Regional application of LNA-21 was capable of reducing miR-21 expression in the heart. Global myocardial function significantly improved after miR-21 inhibition: EF at day 33 was enhanced after LNA-21 treatment (EF: 22 ± 1% control vs. 33 ± 2 LNA-21). Moreover, the expected increase of LVEDP was reduced after miR-21 inhibition (LVEDP: d33 19 ± 1mmHg control vs. d33 14 ± 1mmHg LNA-21). In addition, regional myocardial function in ischemic region was enhanced after LNA-21 treatment (SES 28 ± 2% control vs. 52 ± 7% LNA-21). In addition, LNA-21 reduced myocardial fibrosis and heart weight to body weight ratio, both signs for the development of heart failure after acute myocardial infarction. Conclusion: Regional LNA-21 delivery reduces miR-21 levels, postischemic loss of function and fibrosis. miR-21 inhibition might be a novel therapeutic tool to preserve cardiac function and inhibit fibrosis/ hypertrophy after myocardial ischemia.

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84. Targeting the miR-106b ~ 25 cluster as a potential regenerative therapeutic approach for myocardial injury E. Dirkx1, A. Raso1, S. Olieslagers1, H. El Azzouzi1, L. Braga2, L. Zentilin2, S. Zacchigna2, P. da Costa Martins1, M. Giacca2, L.J. de Windt1 1

Department of Cardiology, CARIM School for Cardiovascular Diseases, Maastricht University, Maastricht, The Netherlands 2 Department of Molecular Medicine, International Center for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy Background: Myocardial infarction (MI) causes cell death and triggering cardiac hypertrophic remodeling of the remaining cells, resulting in heart failure. A therapeutic approach aiming at induction of cardiomyocyte proliferation while inhibiting the hypertrophic response would improve cardiac function and prevent MI-induced heart failure. By bio-informatics analysis, we identified the miR-106b ~ 25 cluster to potentially target both a network of negative cell cycle regulators, as well as series of pro-hypertrophic factors, which indicates this microRNA cluster to possess a yet unexplored role in recovering the heart muscle after myocardial injury. Hypothesis: Overexpression of the miR-106b ~ 25 cluster boosts cardiomyocyte proliferation, inhibits hypertrophy and regenerates the myocardial damage after MI. Methods and results: Knock-out of miR-106b ~ 25 leads to spontaneous hypertrophy by derepressing the expression of prohypertrophic transcription factors such as Hand2 and Mef2d, while overexpression of miR-106b ~ miR-25 using adenoassociated virus (AAV9) in neonatal mice induces cardiomyocyte proliferation, by derepressing cell cycle inhibitors Cdk1a, Cdk1c and E2F5, which was associated with an increased incorporation of EdU, combined with increased levels of the proliferative markers Aurora-B and phosho-histone 3. Furthermore, in adult mice, which received a myocardial infarction, echocardiographic measurements demonstrate improvement of cardiac function upon AAV9miR106b ~ 25 treatment versus AAV9-MCS (empty vector), which is associated with a reduced infarction size and a clear decrease in fibrotic damage. Conclusion: Our data demonstrate that AAV9-miR106b ~ 25 treatment induces cardiac muscle regeneration after MI, by boosting cardiomyocyte proliferation and inhibiting hypertrophic remodeling. These findings might give rise to a new gene therapeutic approach aiming at mending the cardiac muscle after myocardial injury. 85. Biological pacing of neonatal rat engineered heart tissue by human iPSC-derived cardiomyocyte organoids M.L. Schulze1,2, M. Lemoine1,2, B. Ulmer1,2, A. Fischer1,2, J. Uebeler1,2, T. Schulze1,2, A. Hansen1,2, T. Eschenhagen1,2 1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany

Background: Biological pacemakers may represent a lifelong alternative to electronic pacemakers by functioning without battery and by their principal ability to adapt to the developing heart during adolescence and to physical exercise.

Methods and results: For creating a biological pacemaker three critical components of pacemaker function are needed: (i) a trigger, (ii) a substrate to which the signal of the trigger is conveyed and (iii) connectivity between trigger and substrate for proper signal transmission. We used the highly arrhythmic rat 3D engineered heart tissue (EHT) model as a substrate to study the potential of spontaneously and regularly beating (~ 1 Hz) human iPSC-derived organoids (~ 80% cardiomyocytes) to take over pacemaker function. Incorporation of one human organoid per rat EHT during casting was sufficient to revert the usual burst beating (mean 4.5 Hz in the burst, 20–30 s pause between bursts) into a slow (~ 1 Hz) and regular beating over the entire culture time of the rat EHTs. Connectivity between the human organoid and rat tissue was substantiated by calcium transient measurements, immunohistochemistry and action potential measurements. Conclusion: Taken together, human iPSC-cardiomyocyte organoids comply with the prerequisites for biological pacemakers in a model of 3D engineered rat heart tissue. 86. Chronic treatment with quercetin prevents negative consequences of doxorubicin treatment in the heart tissue and improves postischemic recovery of heart function in doxorubicin-treated rats M. Bartekova1,3, I. Dovinova2, N. Tribulova1, L. Okruhlicova1, J. Radosinska1,3, K. Ferenczyova1, M. Fogarassyova1, M. Barancik1 1

Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovak Republic 2 Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic 3 Institute of Physiology, Faculty of Medicine, Comenius University in Bratislava, Slovak Republic Background: The aim of the present study was to explore effects of prolonged administration of plant flavonoid quercetin on changes induced by repeated application of doxorubicin. Methods and results: We focused on the ischemic tolerance of hearts, changes of ultrastructure, MMP activities, apoptosis induction and redox status in rat hearts, as well as on determining possible mechanisms underlying effects of quercetin. Rats were treated with doxorubicin in cumulative dose of 15 mg/kg i.p. divided into 7 doses applied in 3 weeks. Quercetin was applied for 6 weeks (20 mg/kg/day), starting at the same day as the doxorubicin administration and continued for further 3 weeks after the completion of the doxorubicin treatment. Our results showed that quercetin-treated rat hearts were more resistant to ischemia/reperfusion injury in both control and doxorubicintreated rats. Effects of quercetin on modulation of ischemic tolerance were linked to Akt kinase activation followed by GSK-3β downregulation and elevation of β-catenin. Moreover, we showed that quercetin reversed effect of doxorubicin on heart ultrastructure, tissue MMP-2 activity, changes in connexin-43, changes in SOD activity and apoptosis induction in the heart tissue. Conclusion: Our results demonstrate that prolonged treatment with quercetin prevented negative chronic effects of doxorubicin in the heart tissue, and positively influenced myocardial responses to acute ischemic stress. These findings bring new insights into mechanisms of quercetin action in the hearts exposed to chronic effects of doxorubicin. Acknowledgments: This study was supported by VEGA SR 2/0108/15; 2/0061/16; and APVV-0348-12.


87. Acute but not repeated inotropic activation by levosimendan prevents doxorubicin-induced cardiomyopathy: implication of the critical role of inotropic action P. Efentakis1, E.K. Iliodromitis2, A. Varela3, D. Benaki3, E. Chavdoula3, F. Sigala4, N. Kostomitsopoulos3, A. Papapetropoulos1, A. Tasouli6, D. Farmakis2, E. Mikros3, A. Klinakis3, T. Suter7, D. Cokkinos3, I. Andreadou1 1 National and Kapodistrian University of Athens, Faculty of Pharmacy, Athens, Greece 2 National and Kapodistrian University of Athens Medical School, Second University Cardiology Department, Athens, Greece 3 Biomedical Research Foundation, Academy of Athens, Athens, Greece 4 National and Kapodistrian University of Athens Medical School, First Department of Surgery, Athens, Greece 5 Onassis Cardiac Surgery Center, Athens, Greece 6 Bern University Hospital, Department of Cardiology, Bern, Switzerland

Aim: We investigated the role of levosimendan (LEVO) on acute and chronic doxorobucin (DXR)-induced cardiomyopathy, the underlying signaling mechanisms and its effects on an in vivo breast cancer model. Methods: For the acute DXR-induced cardiotoxicity, rats were randomized into 4 groups [Control, DXR (20 mg/kg), DXR+LEVOA and DXR+ LEVOB]. LEVO was administered at 12 mg/kg and 24 mg/kg for seven consecutive days. For the chronic experiments the rats underwent the following interventions for 14 consecutive days: Control, DXR (18 mg/kg, 6 equal doses), DXR + LEVO (LEVO 24 mg/kg, 4 equal doses), DXR + LEVO (ac) (LEVO 24 mg/kg acutely on the first day). Cardiac function was assessed at baseline, at the 7th and 14th days. Female mice injected with MMTV-Neu cells were randomized as Control, DXR (18 mg/kg, 6 equal doses), DXR + LEVO (ac) (LEVO 24 mg/kg acutely on the first day) and LEVO(ac). DXR was given for 14 additional days in order to assess the effect of LEVO on survival. Echocardiography was performed at the 14th and the 28th days.

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diseases. Unfortunately, the use of anthracyclines is limited due to their cumulative dose-dependent cardiotoxicity (CTX). Doxorubicin (Doxo) is one of the standard anti-tumoral therapies among anthracyclines. Chronic administration of Doxo induces CTX due to its inability to discriminate tumor from normal cardiac cells. The molecular mechanisms involved in Doxo-mediated CTX are emerging and include the induction of ROS-triggered cell death. Thus, anti-oxidant and prosurvival agents seem to be attractive candidates as cardioprotective molecules for future cancer patients. Among pro-survival factors the hepatocyte growth factor (HGF) is recognized to exert cardioprotective function. In our work we have investigated the cardioprotective role of HGF/Met tyrosine kinase receptor signaling against Doxo-mediated CTX in vivo. Methods and results: Analysis of cardiac function by magnetic resonance imaging (MRI) show that activation of Met receptor through an agonist antibody (MetamAb) leads to protection of cardiac damage in animal models of Doxo cardiomiopathy. In particular, the presence of MetamAb improves the heart systolic activity through a thickening of contractile fibers. Mechanistically, Met activation counteracts Doxoinduced cell death through its anti-oxidant function, as we have demonstrated by in vitro and in vivo analysis on cardiac models. Conclusion: All together, these results suggest that MetamAb can be used as novel therapeutic strategy for cardioprotection. For this reason, we are engineering our antibody to develop a therapeutic molecule for patients that will create cardio-safer anti-cancer therapies. 89. Characterization of in vitro pharmacological properties of dexrazoxane analogues for the protection from the anthracycline cardiotoxicity A. Jirkovska1, G. Karabanovich1, E Jirkovsky2, J Bures1, J. Roh1, P. Kovarikova1, T. Simunek1, M. Sterba2 1

Results: High dose LEVO abrogated morphological deficits induced by acute DXR, regulated redox signaling, prevented apoptosis and restored metabolic dysregulation. Acute LEVO, on the contrary to repeated one, prevented chronic DXR-induced cardiomyopathy, via restoration of cardiac function and myocardial morphology mainly through restoration of Ca2+ homeostasis. LEVO abrogated reduced cardiac contractility in the breast cancer model and increased their survival. Conclusions: Acute LEVO prevents long term Ca2+ dysregulation and contractility deficits. Its dose and the frequency of administration are pivotal for the cardioprotection, rendering LEVO appropriate for clinical use in DXR-induced cardiomyopathy. 88. Activation of Met tyrosine kinase receptor to protect the heart against anthracycline cardiotoxicity S. Gallo1,2, G. Ferrauto3, E. Di Gregorio3, M. Spilinga2, A. Bonzano1, P.M. Comoglio1, T. Crepaldi1,2 1

Candiolo Cancer Institute, Candiolo (TO), Italy 2 Department of Oncology, University of Turin, Turin, Italy 3 Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Background: Anthracyclines are highly effective and widely used cytotoxic agents with applications in the treatment of different cancer

Faculty of Pharmacy, Charles University in Prague, Prague, Czech Republic Faculty of Medicine in Hradec Kralove, Charles University I Prague, Prague, Czech Republic 2

Background: Dexrazoxane is the only drug approved so far as a protective against chronic anthracycline cardiotoxicity in clinics. In this study, we synthesized dexrazoxane analogs with a modification on the connective chain of the bis-dioxopiperazine structure to characterize their pharmacological properties and help to elucidate the structure-function relationship of bis-dioxopiperazines in cardioprotection. Methods: We tested the analogs (in comparison with dexrazoxane) for their ability to protect the isolated neonatal cardiomyocytes from the toxicity induced by daunorubicin (1.2 μM), to inhibit the catalytic activity of topoisomerase II alpha and beta and to modify their content in cardiomyocytes and cancer cells, to inhibit the proliferation of human promyelocytic leukemia cell line (HL-60) and impact the growth inhibition caused by daunorubicin. Moreover, the in vitro pharmacokinetics has been studied using HPLC-MS analysis. Results and conclusion: We found that, compared to dexrazoxane, (4,4′-(1-oxoethane-1,2-diyl)bis(piperazine-2,6-dione)) inhibited the topoisomerase II activity similarly, but lacked the antiproliferative and cardioprotective effect and weren't able to deplete topoisomerase II beta from cardiomyocytes. An in vitro pharmacokinetic study revealed that JR-311 has comparably shorter half-life than DEX, with virtually no detectable parent structure after 3 h, and thus the faster decay of JR-311 to the structure could be attributed to the loss of activity,

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implying the parent structure importance for the cardioprotection. The analogue 4,4′-((2R,3S)-butane-2,3-diyl)bis(piperazine-2,6-dione) (ICRF-193), which was studied previously for the antiproliferative activity, had stronger inhibition activity on the topoisomerase II and has also stronger cardioprotective activity than dexrazoxane. Acknowledgement: This study was supported by GACR 13-15008S and UNCE 204019/304019/2012.

oxidants and other therapies have generally failed to attenuate the development of this condition, leading to calls for more innovative adjuvant therapies. We identified the ubiquitin proteasome system (UPS), as a potentially ground-breaking therapeutic avenue since it regulates proteins which control mitochondrial morphology, apoptosis and physiological anti-oxidants. Investigating the role deubiquitinating enzymes (DUBs), which reverse or modify ubiquitination status of proteins play in this context, could prove the breakthrough needed in this scientific quest against cardiotoxicity.

90. Interaction with topoisomerase II-beta, instead of metal chelation, seems to be responsible for effective cardioprotection against chronic anthracycline cardiotoxicity E. Jirkovsky1, P. Brazdova1, Z. Pokorna1, J. Roh2, G. Karabanovich2, A. Jirkovska2, P. Kovarikova2, K. Vavrova2, T. Simunek2, M. Sterba1 1

Faculty of Medicine, Charles University, Hradec Kralove, Czech Republic Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic

2

Background: To date, only dexrazoxane (DEX) has been unequivocally proven in clinical practice as a protective measure against anthracycline (ANT) cardiotoxicity. DEX has been understood as a prodrug of metal-chelating active metabolite ADR-925, but newer data implicate interaction possibility with topoisomerase II-beta (TOP2b). This study compares the ability of DEX, GK-627 (dimethylated DEX analogue) and ADR-925 (the chelating metabolite) to interact with TOP2b in the hearts in vivo and its connection to cardioprotective efficacy against chronic ANT cardiotoxicity. Methods: ANT cardiotoxicity was induced in rabbits with daunorubicin (DAU, 3 mg/kg, once weekly for 10 weeks). DEX, ADR-925 or GK-627 (60 mg/kg) were administered before each DAU dose. Cardioprotective efficacy was determined by LV performance, cTnT plasma levels, histopathological analysis, and molecular hallmarks of heart failure (e.g. expression of ANP in the LV). In other experiments, plasma/ myocardial concentrations of studied drugs were determined by HPLC-MS, and Top2b in the LV myocardium by WB. Results: In sharp contrast to DEX, both ADR-925 and GK-627 showed no protection of rabbits against DAU-induced mortality, myocardial degeneration, LV dysfunction (N 40% dP/dtmax decrease) and congestive heart failure. The lack of cardioprotective of both GK-627 and ADR-925 was linked to inability of these compounds to deplete TOP2b in the LV, which contrasted to DEX, although the exposures of the heart to the drugs were similar. Conclusions: Our results show that interaction of DEX with TOP2b, but not chelation by its metabolite, seem crucial for effective cardioprotection against ANT cardiotoxicity. Acknowledgments: This study was supported by GACR-13-15008S, UNCE-204019/304019/2012.

Methods: Chronic Doxorubicin (DOX) cardiotoxicity was induced in H9C2 cardiomyoblasts and male Sprague-Dawley rats, samples harvested and protein expression determined through Western Blots. DUB down-regulation was achieved through SiRNA knockdowns and confirmed with western blots. Cell and mitochondrial health was evaluated by determining mitochondrial morphology, a toxicity assay, an ATP assay, and protein determination. Results and discussion: DOX increased expression of USP9x (103.7 ± 4,7%, p b 0.01), a regulator of MCL1, whose pro-apoptotic short form was also up-regulated at the expense of its anti-apoptotic form. DOX also reduced USP30 expression (27.54 ± 5.9%, p b 0.001); a regulator of mitofusin proteins also influenced by DOX treatment. DOX influenced anti-oxidant expression regulated by USP36. Downregulating specific DUBs was able to improve mitochondrial morphology, ATP production, and reduce mitochondrial toxicity in the presence of DOX. Showing mechanisms through which DOX exerts its effects, and that down-regulating these DUBs in this context was beneficial. Conclusion: De-ubiquitinating enzymes show promise as a potential therapy for DOX-induced cardiotoxicity. 92. The cardioprotective effect of metformin on doxorubicin-induced cardiotoxicity R. Zilinyi, I. Lekli, A. Czompa, A. Czegledi, A. Tosaki Department of Pharmacology, Faculty of Pharmacy, University of Debrecen, Hungary Background: Doxorubicin (DOX) is an effective chemotherapeutic agent, but its cardiotoxicity has been an important clinical limitation. However, molecular mechanisms underlying DOX cardiotoxicity are still being uncovered, but known to involve, mitochondrial dysfunction, oxidative stress and apoptosis.Recently, a number of studies have investigated the role of autophagyon DOX-induced cardiotoxicity but to date it is not clear how DOX alters that process and its consequence on cardiomyocytes. The aim of our study was to investigate the possible protective role of metformin (MET) and its effect on autophagy in a model of DOX-induced cardiotoxicity.

Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South Africa

Methods: Sprague-Dawley rats were divided into four group. The DOX group rats received doxorubicin (3 mg/kg every second day) intraperiotoneally. The MET group rats received 250 mg/kg/day metformin for two weeks. The DOX + MET group rats received both at the same dose. Control group received vehicle. After the last dose of doxorubicin isolated working hearts were prepared and heart function parameters were evaluated. Serum level of LDH, CK-MB enzymes, Troponin-T, and cardiac MDA were measured. Heart tissue samples were histopathologically examined. Western blot analysis was conducted for autophagy-associated proteins.

Background: Cardiotoxicity, a complication induced by anthracycline administration, has puzzled scientists for decades. The use of anti-

Results: Our results revealed that metformin produced cardioprotection manifested by significant decrease in serum Troponin-T and cardiac

91. Deubiquitination: does the answer to cardiotoxicity lie within the ubiquitin proteasome pathway? T.R. Ogundipe, B.J.N. Sishi


MDA level, and remarkable improvement in the heart function and in the histopathological features. Furthermore, we have found that metformin induced autophagy and increased the expression of AMPK which may help cardiomiocytes to survive during doxorubicin treatment. Conclusion: These results may suggest using metformin would be preferable drug for patients receiving DOX in their chemotherapy. Acknowledgments: OTKA PD 111794, GINOP-2.3.2-15-2016-00043. 93. Establishment of an optogenetic human engineered heart tissue platform M. Lemme1,2, M. Lemoine1,2, B. Ulmer1,2, T. Christ1,2, T. Eschenhagen1,2 1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany

Background: Engineered heart tissue (EHT) could be used to study effects of persistent tachyarrhythmias. However, long term electrical pacing is complicated by substantial electrolysis, leading to toxicity. Expression of channelrhodopsin-2 (ChR2), a light-activated nonselective cation channel allows optical pacing. Here we have investigated whether this technique could be applied in EHT from hiPSC-CM. Methods: EF1a-hChR2 (H134R) lentivirus was added to hiPSC-CM cultured in 2D monolayers and during casting of EHT. Ionic currents were recorded by the whole-cell patch clamp technique at 37 °C. We developed an optogenetic platform that can be integrated into the experimental setup for video-optical recording to measure EHT contractility. In addition, APs were measured with sharp microelectrodes at 36 °C during optical pacing. Results: Transduced hiPSC-CM showed uniform expression of ChR2 in the membrane. Short blue light pulses (10 ms, 8 mW/mm 2 ) could evoke inward currents in the range of 1 nA. Even EHT transduction was effective. On average 30% of cardiomyocytes were positive as assessed by flow cytometry. Light pulses (rheobase of 0.6 mW/mm2, chronaxie 12 ms) could pace EHT up to 3.5 Hz. As expected contraction dynamics speeded up at higher pacing rate. Optical pacing decreased upstroke velocity (165 ± 3.41 vs 90 ± 1.17 v/s, p b 0.0001) and increased APD90 (208 ± 1.4 vs 237 ± 3.4 ms, p b 0.0001). The depolarization induced by ChR2 could slow down the spontaneous AP firing. Conclusions: ChR2 transduction allows pace EHT. Lower upstroke velocity and feasibility of long-term optical pacing need to be investigated. Optogenetics might be a promising tool to study mechanisms of tachymyopathy in EHT. 94. Sorting out Ca2 + regulation by calmodulin dependent kinase II (CaMKII) and calmodulin C. N. Johnson1, N. Gomez-Hurtado1, D.O. Kryshtal1, R. Pattanayek2, J. Haynes2, S.M. Damo2, and B.C. Knollmann1 1

Vanderbilt University Medical Center, Nashville TN, USA Fisk Univeristy, Nashville TN, USA

2

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Background: Calmodulin dependent protein kinase (CaMKII) is responsible for regulating numerous protein processes within a cardiomyocyte. Heart disease is often associated with increased kinase activity and phosphorylation. Despite much investigation, the atomic underpinnings and functional outcome(s) of many CaMKII regulatory processes are controversial and poorly understood. Method and results: Here we present preliminary data demonstrating that the widely utilized CaMKII inhibitor KN93 and its control KN92 both interact with the Ca2 + sensing protein Calmodulin, while the Autocamtide Inhibitor Peptide (AIP) does not. Importantly these KN93 and KN92 interactions alter the regulatory function of the CaM-RYR2 interaction as well as other CaM-regulatory processes. Conclusion: Our findings provide an explanation for conflicting reports on CaMKII regulation and allow for appropriate considerations in future investigations that will enhance understanding of Ca2 + signaling in excitable cells such as cardiomyocytes. 95. Critical role of Phosphodiesterase 2A in congenital heart defects M.R. Assenza1, F. Barrios1, F. Barbagallo2, M. Cornacchione3, A.M. Isidori2, M. Pellegrini4, F. Naro1 1

Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Sapienza University of Rome, Rome, Italy 2 Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy 3 SDN Istitute of Diagnostic and Nuclear Research, Naples, Italy 4 Institute of Cell Biology and Neurobiology, CNR, Monterotondo, Rome, Italy Background: cAMP and cGMP levels are regulated by cyclic nucleotide degrading phosphodiesterases (PDEs). Among the different PDEs, Pde2A is unique because it responds to elevated cGMP levels by increasing its cAMP hydrolytic activity. Pde2A is essential for mouse development and Pde2A null mice die at E17.5 (1). Pde2A −/− embryos display nuchal edema which is frequently associated with congenital heart defects (CHD) in other mouse models (2). Methods and results: To investigate if Pde2A absence is associated with heart defects, histological observations of hearts isolated from Pde2A-/- E14.5 embryos were performed. Pde2A-/- hearts displayed interventricular septum and myocardial wall defects, hypertrabeculation and atrial trabeculae loss. Increased apoptosis and decreased cell proliferation was detected in specific areas of Pde2A-/- hearts. Altered expression pattern of critical genes involved in heart development (T-box genes) and cAMP-dependent transcriptional repressor (Icer) activation in Pde2A-/- hearts was observed. To test if T-box gene expression was directly regulated by cAMP increase, fetal cardiomyocytes were treated with a PDE2A inhibitor (EHNA) and the β-adrenergic agonist Isoproterenol. Transcripts down-regulation was observed by qPCR in treated cardiomyocytes. Furthermore, Pde2A-/- cardiac phenotypes were recapitulated in WT mice in which PDE2 was pharmacological inhibited. Conclusion: Taken together these data suggest that PDE2A activity is critical for heart development by modulating T-box gene expression and cannot be compensated by other PDEs. To our knowledge, this is the first time that the genetic deletion of a phosphodiesterase is associated with the establishment of CHD.

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96. Beta-blockade restores myocardial function in type 2 diabetic rats via CaMKII-independent mechanisms C.T. Bussey, R.S. Wallace, R.R. Lamberts, J.R. Erickson Department of Physiology, HeartOtago, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand Background: Type 2 diabetes, and associated heart disease, continue to climb to epidemic proportions; while beta-blockers remain best treatment practice despite reduced effectiveness in diabetes. The second generation beta-blocker metoprolol is commonly prescribed, despite indications that the third generation carvedilol may have added benefits such as blunting CaMKII hyperactivity. We hypothesized that betablockade improves myocardial function in type 2 diabetes, and that carvedilol is more effective than metoprolol due to inhibition of CaMKII hyperactivity. Methods: Male Zucker Diabetic Fatty (DM) rats and their nondiabetic (ND) littermates were randomly assigned to metoprolol (100 mg/kg/day), carvedilol (10 mg/kg/day) or control groups at 16 weeks of age. Drugs were administered via food for four weeks, before myocardial function was assessed in isolated right ventricular trabeculae. Results: Myocardial force development was reduced in diabetes (ND 18.2 ± 2.6 vs. DM 10.6 ± 1.9 mN/mm2, p b 0.05). Beta-blockade in diabetic rats restored myocardial function to control levels (DM metoprolol 20.3 ± 7.2 and DM carvedilol 15.8 ± 4.7 mN/mm2, NS vs. ND). However, beta-blockade in non-diabetic rats impaired myocardial function to a similar degree as diabetes (ND metoprolol 8.0 ± 2.1 and ND carvedilol 8.0 ± 1.3 mN/mm2, p b 0.05 vs. ND). Metoprolol and carvedilol were similarly effective, and total and phosphorylated CaMKII protein levels were not different between groups, inconsistent with carvedilol better inhibiting CaMKII. Conclusion: Type 2 diabetes impairs myocardial function, an effect reversed by chronic beta-blockade. However, beta-blockade in the absence of sympathetic over-activation may be detrimental, promoting caution in administering these widely prescribed drugs.

Methods: miRs were blindly transfected in adult rat cardiomyocytes using Lipofectamine 3000. Percentage shortening was measured using an Ionoptix system. Calcium transients were obtained using Fluo-4-AM and sarcoplasmic reticulum (SR) calcium content measured with caffeine micro-application. N numbers displayed as n/N (n/N = cells/rats). Results: Increased miR-16/-26a significantly reduced baseline contractility in cardiomyocytes (miR-16 = 3.52 ± 0.34% versus control = 4.91 ± 0.46%; n/N = 30/6; p b 0.05 and miR-26a = 2.77 ± 0.21% versus control = 4.30 ± 0.43%; n/N = 50/10; p b 0.01). The effect was seen in apical but not basal cardiomyocytes. miR-16/-26a did not alter β2AR-response. Inhibiting Gi with pertussis toxin (PTX) prevented the decrease in contraction with either miR. Calcium transient amplitude was decreased with miR-16/-26a, along with a decrease in SR calcium content. Conclusion: Increased miR-16/-26a reduced contractility of apical cardiomyocytes through a shared Gi-dependent mechanism. Downstream pathways may include decreased calcium transient amplitude and SR calcium content. This suggests miR-16/-26a may be mechanistically involved in TTS, but further work is needed to investigate specific signalling pathways. 98. Exposure of adult rat ventricular myocytes to nitroxyl induces activation of cAMP-dependent protein kinase through interdisulfide formation and substrate targeting S. Diering1,2, M. Goetz1,2, S. Schobesberger2,3, K. Stathopoulou1,2, A. Piasecki1,2, V.O. Nikolaev2,3, P. Eaton4, F. Cuello1,2 1

Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 4 King's College London, Cardiovascular Division, The British Heart Foundation Centre of Excellence, The Rayne Institute, St Thomas' Hospital, London, UK

97. Takotsubo syndrome associated miR-16 and miR-26a reduce contractility of apical cardiomyocytes in vitro by an inhibitory G-protein dependent mechanism L. Couch1, R. Clayton1, A.A. Derda2, L. Wienecke1,2, T. Thum2, C. Terracciano1, S.E. Harding1 1

National Heart and Lung Institute, Imperial College London, London, UK Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School, Hannover, Germany

Background: Heart failure (HF) is associated with a reduction in contractile force and on cardiac myocyte level with a desensitization of the β-adrenoceptor signaling cascade. Pharmacological nitroxyl (HNO) donors have recently emerged as a promising addition to the treatment regime for HF patients, with clinical trials ongoing. Amongst the beneficial actions is their positive lusitropic and inotropic effect, which is maintained also under HF conditions. The underlying molecular mechanisms remain incompletely understood.

2

Background: Takotsubo syndrome (TTS) is a severe but reversible acute heart failure affecting predominantly post-menopausal women. Extreme adrenaline levels arising with stress cause ventricular apical akinesis with basal function preserved. This occurs due to β2AR-Gi coupling, which is dysregulated in TTS. A microRNA (miR) profile of increased miR-16 and miR-26a has been identified that distinguishes TTS from acute coronary syndrome. Our preliminary data favour miR 16 and 26a increases as preceding TTS, rather than being triggered by adrenaline release. We hypothesise that miR-16 and miR-26a functionally interact to predispose to, or exacerbate, the cardiodepression seen in TTS.

Methods and results: FRET measurements were performed in adult rat ventricular myocytes (ARVMs) infected with an adenovirus encoding the FRET biosensor AKAR3 at MOI100 for 48 h. Exposure to the HNO donor 1-nitrosocyclohexyl acetate (NCA) induced β-adrenoceptor-independent enhancement of the FRET signal (39% of the maximal response, n = 27), suggesting activation of cAMP-dependent protein kinase (PKA). Western immunoblot analysis under non-reducing conditions revealed NCA-mediated interprotein disulfide formation of the regulatory subunit RIα of PKA, which has been previously shown to activate the kinase (1). Fractionation of ARVMs after NCA treatment into cytosolic, membrane and myofilament fraction showed translocation and accumulation of the regulatory and catalytic subunit of PKA in the myofilament containing fraction. This was paralleled by increased


phosphorylation of the myofilament protein cardiac myosin-binding protein C. Conclusion: In ARVMs, exposure to NCA activates PKA by inducing (A) interprotein disulfide formation in RIα and (B) translocation of the kinase to its substrates. This observation could partially explain the positive inotropic and lusitropic actions of HNO donors.

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in three distinct subcellular microdomains formed around Ca2+ handling proteins, such as L-type-calcium-channels (LTCCs), sarco/endoplasmic reticulum Ca2+-ATPase(SERCA) and ryanodine receptors(RyRs), to understand their impact on heart function and disease.

Diastolic [Ca2+]i is controlled by systolic [Ca2+]i in rat ventricular myocytes

Methods: Transgenic mice expressing three Förster resonance energy transfer(FRET) based cAMP biosensors targeted to caveolin-rich plasma membrane, SERCA and RyR microdomains, were crossed with PDE4B and PDE4D knockout mice. Using FRET-imaging in ventricular cardiomyocytes isolated from wildtype and knockout mice, direct analysis of the specific effect of both PDE subfamilies could be performed by measuring the kinetics of local cAMP degradation.

K. Kistamas1, R. Sankaranarayanan2, D.J. Greensmith3, L.A. Venetucci1, D.A. Eisner1 1 Unit of Cardiac Physiology, Division of Cardiovascular Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK 2 Department of Cardiology, Aintree University Hospital NHS Trust, Liverpool, UK 3 School of Environment and Life Sciences, Biomedical Research Centre, University of Salford, Salford, UK

Results: Our results indicate that the cAMP kinetics around the LTCC microdomain is critically regulated by PDE4B and PDE4D. We found PDE4B to be involved in regulating the cAMP signaling in the RyR microdomain. PDE4D deletion also revealed the critical role of this subfamily for the control of cAMP dynamics in the SERCA. Basal levels of cAMP were elevated when PDE4B was absent from any of the PDE4Bregulated microdomains, whereas no such alterations were detected for PDE4D knockout cells.

Background: For the heart to function as a pump, [Ca2+]i must increase during systole and must be low enough in diastole so that the ventricle is relaxed and can refill with blood; as impairment of relaxation may lead to diastolic heart failure. The mechanisms responsible for control of diastolic Ca remain poorly understood.

Conclusions: All three microdomains are differentially regulated by PDEs. Even within one organelle, we could show the existence of at least two distinct cAMP microdomains, i.e. around RyR and SERCA which are preferentially controlled by PDE4B and PDE4D, respectively. We aim to systematically analyze biochemical composition of the three microdomains, their distinct roles in cardiac function and disease as well as ways of their pharmacological modulation.

99.

Methods: Cardiac myocytes isolated from rat hearts were loaded with Fluo-3-AM. Ca transients and membrane currents were recorded with the perforated patch clamp technique. Results: Increasing stimulation frequency increased diastolic [Ca2+]i. When sarcoplasmic reticulum function was impaired (either by making the RyR leaky with caffeine or ryanodine or alternatively by decreasing SERCA activity with thapsigargin) this increase of diastolic [Ca2+]i was further enhanced. This effect was accompanied by a decrease of the amplitude of the systolic Ca transient. Importantly, time-averaged [Ca2+]i was unaffected. Time-averaged [Ca2+]i was increased by β-adrenergic stimulation (isoprenaline) and increased in a saturating manner with increased stimulation frequency; average [Ca2+]i was a linear function of Ca entry per unit time. Conclusion: These results suggest that diastolic [Ca2+]i is controlled by the balance between Ca entry and Ca efflux during systole. Any manipulations that decrease the amplitude of the Ca transient will increase the diastolic [Ca2 +]i. Our results identify a novel mechanism whereby changes of the amplitude of the systolic Ca transient control diastolic [Ca2+]i. 100. Impact of phosphodiesterases 4B and 4D on cAMP signal compartmentation around calcium handling proteins A.E. Kraft1,2, M. Conti3, V.O. Nikolaev1,2 1 Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Center for Reproductive Sciences, UCSF School of Medicine, San Francisco, CA, USA

Aim: The aim of this project was to systematically investigate specific roles of PDE4B and PDE4D in regulating cAMP dynamics

101. Patient-specific induced pluripotent stem cell-derived cardiomyocytes to model, screen drugs and decipher molecular mechanisms of CPVT1 syndrome Y. Sleiman1, M.M. Refaat2, V. Scheuermann1, M. Scheinman3, A. Lacampagne1, A.C. Meli1 1 PhyMedExp, Inserm U1046, CNRS UMR9214, University of Montpellier, Montpellier, France 2 Cardiology Division, Cardiac Electrophysiology Section, American University of Beirut Medical Center, Beirut, Lebanon 3 University of California, San Francisco Medical Center, San Francisco, CA, USA

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a highly lethal inherited arrhythmogenic disorder predominantly caused by mutations in the cardiac ryanodine receptor gene (RYR2). Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. We have identified a 25 years-old athletic woman with exercise-induced syncope associated with ventricular tachycardia (VT), harboring the novel single mutation RyR2-D3638A. The ECG revealed no improvement on the β-blocker metoprolol (METO) while flecainide treatment did. We aimed at deriving functional cardiomyocytes (CMs) from the proband via hiPSCs and investigating the response to standard therapy administrated to CPVT patients as well as to new Rycal compounds. Methods and results: When focusing on the intracellular calcium (Ca2+) handling in CPVT hiPSC-CMs, we observed no preventing effect of METO on the aberrant Ca2+ transients under isoproterenol (ISO), a β-adrenergic receptor agonist, which was consistent with the clinical data. However, both S107 and flecainide applications preceding ISO were able to suppress abnormalities of the Ca2 + transients in CPVT hiPSC-CMs with reduced RyR2 Ca2+ leak and increased Ca2 + release

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velocity and amplitude. Co-immunoprecipitation of the RyR2 macromolecular complex showed that CPVT hiPSC-CMs exhibited higher basal RyR2 PKA phosphorylation at Ser2809 and less PP2A bound to RyR2 when compared to healthy control (HC) hiPSC-CMs. Unlike METO, S107 treatment was able to prevent the depletion of calstabin2, a stabilizing RyR2 partner under stress conditions which is likely associated with its stabilizing effect on the Ca2+ transients. Conclusion: This work provides new evidence of CPVT modeling, drug screening and molecular mechanism deciphering, using patientspecific hiPSC-CMs. 102. Regulation of cardiac pacemaker activity by phosphodiesterases D. Mika, A.M. Gomez, G. Vandecasteele, R. Fischmeister INSERM UMR-S 1180, Univ. Paris-Sud, Université Paris-Saclay, ChâtenayMalabry, France Background: Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP, leading to a positive chronotropic effect. Among the cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for cardiac PDE4s: pde4a, pde4b and pde4d. Their respective contribution to the regulation of pacemaker activity remains ill-defined. Methods: The total enzymatic PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including PDE4 inhibitor Ro-20-1724. The in vitro pacemaker activity was assessed by measuring the spontaneous Ca2+-transients in Fluo4-loaded-SAN intact tissue. Images were obtained using confocal microscopy. Results: Ro-20-1724 increased beating rate of intact SAN and increased PKA-phosphorylation levels of key ECC actors. PDE4 enzymatic activity accounts for 60% of the total cAMP-PDE activity in SAN. The 3 isoforms PDE4A, 4B and 4D are expressed in mouse SAN. In PDE4D-, but not in PDE4B-deficient mice, Ca2+-homeostasis was altered in control conditions and after βAR-stimulation. Indeed, ablation of PDE4D induced decreased beating rate and increased Ca2+-spark frequency in control and βAR-stimulated conditions. Conclusion: Our results reveal that PDE4 controls pacemaker function in mice and that PDE4D ablation strongly perturbs normal SAN activity. 103. Dysfunction of the mitochondrial Optic Atrophy-1 protein (Opa1) alters the neurogenic control of heart function V. Prando1,2, N. Pianca1,2, A. Di Bona2,3, M. Franzoso1,2, A. Incensi4, L. Scorrano2,5, V. Donadio4, V. Carelli4, M. Mongillo1,2,6, T. Zaglia1,2,3 1

Department of Biomedical Science, University of Padova, Padova, Italy 2 Venetian Institute of Molecular Medicine (VIMM), Padova, Italy 3 Department of Cardiac, Thoracic and Vascular Sciences, University of Padova, Padova, Italy 4 IRCCS Institute of Neurological Sciences, Bologna, Italy

5

Department of Biology, University of Padova, Padova, Italy CNR Institute of Neuroscience, Padova, Italy

6

Purpose: Cardiac sympathetic neurons (cSNs) are the main extrinsic regulators of heart function in physiologic and stress conditions. Neuronal viability and activity depend on the correct mitochondrial function. Opa1 controls mitochondrial fusion, ATP production and apoptosis and its haploinsufficiency causes Autosomal Dominant Optic Atrophy (ADOA), a neurodegenerative disease characterized by retinal ganglion cell death, leading to visual loss already in childhood. ADOA patients also display peripheral neuropathy and cardiac arrhythmias but the mechanism linking Opa1 dysfunction and heart abnormalities has not been investigated. Our purpose is to establish the role of Opa1 in the neurogenic regulation of heart function. Methods: We generated a mouse model haploinsufficient for Opa1 selectively in cSNs (TOHcre-Opa1+/-). We performed telemetrybased ECG recording and pharmacological assays in vivo, confocal immunofluorescence and morphometric analysis to characterize sympathetic innervation in mouse hearts and skin biopsies from ADOA patients. Results: Opa1 haploinsufficiency leads to a decrease in cSN density, which starts in the adulthood and worsens during ageing accompanied to alterations in cSN distribution patterning and morphology. Cardiac dysinnervation in TOHcre-Opa1+/- mice results in a significant decrease in heart rate variability, reduced exercise tolerance and increased propensity to arrhythmias, all events exacerbating during ageing. Consistently, we detected decreased SN density in skin biopsies from ADOA patients, which progresses during ageing. Conclusion: Opa1 haploinsufficiency leads to precocious cSN degeneration and dysfunctional extrinsic control of the cardiac rhythm. These data support a key role of Opa1 in peripheral sympathetic neurons. 104. β3 adrenergic signaling alters compartmentation and NO/cGMP/ PDE2 signaling in heart failure S. Schobesberger1,2, P. Wright3, C. Poulet3, C. Mansfield3, A. Friebe4, V.O. Nikolaev1,2, J. Gorelik3 1

Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Germany 2 DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Medicine, Imperial College London, Centre for Translational and Experimental Medicine, Hammersmith Hospital, London, UK 4 University Würzburg, Institute of Physiology, Würsburg, Germany Background: Cardiomyocyte β3-adrenoceptors (β3-AR) which signal via 3′,5′-cyclic guanosine monophosphate (cGMP) could prevent pathological hypertrophy and cellular remodelling in heart failure, where their levels appear to be unaffected. It is however unclear if subcellular compartmentation of the β3-AR/cGMP signal and its regulation by phosphodiesterases (PDEs) also remain unaffected. Aim: To investigate β3-AR/cGMP signaling in health and heart failure and to study β3-AR microdomain regulation by PDEs as well as receptor interaction partners. Methods and results: Adult rat ventricular cardiomyocytes were transduced to express the cGMP biosensor red cGES-DE5 for Förster


Resonance Energy Transfer (FRET) measurements using Isoprenaline and selective β-AR or PDE inhibitors. This revealed β3-AR/cGMP signals which occurred via NO/soluble guanylyl cyclase interaction. The β3-AR/cGMP signal was mainly regulated via PDEs 2 and 5. By combining FRET with agonist delivery via a scanning ion conductance pipette it was revealed that β3-AR/cGMP signalling moves out of the Ttubules of healthy cardiomyocytes to unstructured membrane areas of cardiomyocytes in heart failure. This signal relocation was reproduced in control cells by disrupting the scaffolding domain of Caveolin 3 (C3SD) in caveolae via the TAT-C3SD peptide. Also in heart failure, a reduction in β3-AR/cGMP levels with simultaneously increased PDE2 activity and a decreased sGC/Caveolin 3 association was discovered. Conclusion: In health β3-AR/cGMP signaling is localized in T-tubules and β3-AR/cGMP levels are regulated via PDE 2 and 5. Heart failure shifts this β3-AR/cGMP to non-tubular membrane domains. Caveolae disruption generates a similar shift of β3-AR/cGMP signals. This shift combined with altered PDE2 activity disrupts the potentially protective β3-AR/cGMP signaling. 105. Role of phosphodiesterase 1 in controlling intracellular cAMP and cGMP concentrations in rat aortic smooth muscle cells L. Zhang, G. Vandecasteele, R. Fischmeister, V. Leblais UMR-S 1180, Inserm, Univ. Paris-Sud, Université Paris-Saclay, ChâtenayMalabry, France Background: The phosphodiesterase 1 (PDE1) family has been demonstrated to play important roles in cardiovascular system. Here, we evaluated a new selective PDE1 inhibitor, PF-04471141 (PF1, 1 μM), on intracellular cAMP and cGMP concentrations ([cAMP]i, [cGMP]i) in cultured rat aortic smooth muscle cells (RASMCs). Methods: cAMP- and cGMP-PDE activities were measured by a radioenzymatic assay in the protein extracts, in the absence or presence of Ca2+ + calmodulin (Ca2 + − CaM) as PDE1 activator. [cAMP]i and [cGMP]i were recorded by fluorescence resonance energy transfer (FRET) imaging technique in single living cells. Results: In RASMCs, PF1 significantly decreased the basal cGMP-PDE activity and the [Ca2+-CaM]-stimulated cAMP- and cGMP-PDE activities. PF1 had no effect on the FRET cAMP response induced by isoproterenol (Iso, 100 nM), but partially blocked the inhibitory effect of Angiotensin II (AngII, 400 nM; used to increase [Ca2+]i) on this Iso response. PF1 altered the FRET cGMP responses to a NO donor (DEA/NO) by delaying its recovery phase and to the C-type natriuretic peptide (CNP) by increasing its amplitude, but had no effect on the A-type natriuretic peptide response. AngII transiently decreased and delayed the cGMP response to 10 μM DEA/NO, but did not alter the dynamics of ANP- and CNP-induced cGMP responses. Conclusions: These results suggest that in RASMCs, PDE1 controls cAMP pools produced by β-adrenergic stimulation only under high [Ca2+]i conditions. By contrast, under basal conditions, PDE1 controls cGMP pools generated by the soluble guanylate cyclase (sGC) and the particulate GC-B, but not the particulate GC-A.

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Faculty Research Centre – Applied Biological and Exercise Science (FRC-ABES), Coventry University, Coventry, UK Background: Long acting muscarinic-receptor antagonists (LAMA) relieve bronchoconstriction in chronic obstructive pulmonary disease (COPD), however clinical studies have correlated LAMAs, particularly Tiotropium bromide, with increased risk of stroke and cardiovascularrelated mortality. Calcium is tightly regulated and drives cardiac contractility; however, pathological increases trigger cell death. Ca2+/calmodulin-dependent kinase II (CaMKII) is involved in calcium regulation and sensing oxidative stress, which is activated in conditions such as heart failure and arrhythmia. Methods: Isolated perfused hearts from male Sprague-Dawley rats were subjected to stabilisation (20 min), followed by perfusion (155 min) ± Tiotropium (10 nM–0.1 nM) or Tiotropium (1 nM) ± the CaMKII inhibitor, KN-93 (400 nM). Hearts were then stained using triphenyl-tetrazolium chloride (TTC) for infarct/risk ratio (%), or snapfrozen for Western blotting to determine p-CaMKII (Thr286) expression. Data was analysed using one-way ANOVA and LSD, presented as mean ± SEM. Results: Tiotropium significantly increased infarct/risk ratio compared with controls at all concentrations. Administration of KN-93 showed no significance with respect to controls (9.5 ± 0.9% vs. 10.3 ± 1.9%), however co-administration with Tiotropium attenuated infarct development (12.8 ± 2.8% vs. 18.7 ± 1.8%, p b 0.02, n = 3–5). Western blotting showed significantly increased expression of p-CaMKII following Tiotropium (1 nM) administration compared to controls (86.5 ± 8.43% vs. 43.73 ± 5.71%, p b 0.0001, n = 3), coadministration of KN-93 attenuated this (59.58 ± 8.94% vs 86.46 ± 8.43%, p b 0.01). Conclusion: This is the first pre-clinical study to suggest that Tiotropium increases infarct/risk ratio in an isolated perfused heart via CaMKII upregulation; this may account for the adverse cardiac sideeffects presented clinically. 107. Oxidant activation of PKG controls vascular tone by regulating Ca2+ spark frequency V. Csató, S. Zamiah, M. Ahmed, H. Bennett, A. Greenstein University of Manchester, Division of Cardiovascular Sciences Background: We have recently shown that oxidant activated Protein Kinase G (PKG) contributes to small artery contractility by regulation of the Ca2+ spark/Large conductance Ca2+-activated K+ (BK) channel axis. To explore characteristics of this signalling pathway in more detail, a transgenic mouse model was used, in which PKG is resistant to oxidant induced dimerization. Methods: Vascular function was examined in vitro with pressure myography. Ca2+ sparks and caffeine-induced Ca2+ transients (indicative of sarcoplasmic reticulum (SR) Ca2+ load) were imaged with highspeed spinning-disc confocal microscopy. Western blot protocols examined phospholamban and ryanodine phosphorylation.

106. 2+

Role of Ca /calmodulin kinase II in tiotropium bromide induced cardiotoxicity S. Cassambai, S. Dean, K.L. Harvey, C.J. Mee, A. Hussain

Results: In wild type (WT) arteries, Ca2+ spark frequency increased with intraluminal pressure but reached a ‘Ca2 + spark frequencyceiling’ at 80 mm Hg. There was no effect of pressure on the Ca2 + spark frequency in oxidant-resistant (OR) arteries. Similarly, exogenous H2O2 increased Ca2 + spark frequency in WT arteries at low

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pressures but did not have this effect at higher intraluminal pressures. Conversely, caffeine-induced Ca2+ transients showed very little relationship with intraluminal pressure, although at higher pressures SR Ca2 + content was reduced in WT compared with OR arteries. Both exogenous H2O2 and the BK agonist NS11021 vasodilated pressureconstricted mesenteric arteries equivalently between WT and OR arteries. H2O2 caused phosphorylation of the ryanodine receptor but not phospholamban. Conclusion: Oxidant activated PKG appears to target the ryanodine receptor to increase Ca2 + spark frequency and maintain BK function during pressure induced constriction. However, once activated, further increases pressure or exogenous oxidants cannot additionally increase the activity of this vasodilatory pathway, despite only low-level activity of the BK channel.

by an increase of cGMP, suggesting that intradisulfide formation mimics cGMP binding. Consistent with our previous observations, HNO induced concentration-dependent vasorelaxation in vitro in isolated mesenteric arteries and in vivo as shown in cremaster arterioles by intravital microscopy. Vasorelaxation in vitro and in vivo was correlated with enhanced intradisulfide formation in PKGIα. Conclusion: HNO induces an intradisulfide in the high affinity cGMPbinding site of PKGIα, leading to the same effect as cGMP binding, namely kinase activation followed by vasorelaxation. 109. P90 ribosomal S6 kinase as a nodal point regulating the crosstalk between α1- and β1-adrenoceptor signalling pathways

108. Nitroxyl mediates vasodilation by inducing an activating intradisulfide bond in the high affinity cGMP-binding site of PKGIα M. Goetz1,2, S. Donzelli1,2, K. Schmidt2,3, M. Wolters4, K. Stathopoulou1,2, O. Prysyazhna5, J. Scotcher5, C. Dees6, H. Subramanian2,7, E. Butt6, A. Kamynina5, S.B. King8, V.O. Nikolaev2,7, C. de Witt2,3, L.I. Leichert9, R. Feil4, P. Eaton5, F. Cuello1,2 1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University, Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany 4 King´s College London, Cardiovascular Division, British Heart Foundation Centre of Excellence, the Rayne Institute, St Thomas´ Hospital, London, UK 6 Institute of Experimental Biomedicine II, University Medical Center Würzburg, Würzburg, Germany 7 Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 8 Department of Chemistry, Wake Forest University, Winston-Salem, NC, USA 9 Institute of Biochemistry and Pathobiochemistry - Microbial Biochemistry, Ruhr University Bochum, Bochum, Germany 10 Department of Physiology, University of Lübeck, Lübeck, Germany

Introduction: Nitroxyl (HNO), the reduced sibling of nitric oxide, has emerged as a novel therapeutic strategy to treat patients suffering from acute decompensated heart failure with clinical trials ongoing. cGMP-dependent protein kinase I-alpha (PKGIα) as a key regulator of smooth muscle tone and cardiac hypertrophy is emerging as a cellular target of HNO. Our aim is to understand the underlying mechanisms of observed therapeutic effects. Methods and results: Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors induced disulfides within PKGIα: an interdisulfide between Cys42 of two adjacent monomers, as well as a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity in response to HNO was monitored in cells separately transfected with WT or mutant PKGIα. Whilst WT kinase activity was enhanced, this effect was significantly attenuated in inter- or intradisulfide-deficient PKGIα. To determine, whether HNO-induced intradisulfide formation modulates cGMP binding, a FRET-based biosensor comprising the cGMP-binding sites of PKGIα was employed. Real-time imaging revealed that HNO induced FRET changes similar to those elicited

K. Stathopoulou1,2, S. Schobesberger2,3, H. Sotoud1,2, A. Piasecki1,2, T. Christ1,2, K.-D. Söhren1,2, P.-P. David4, V.O. Nikolaev2,3, M. Avkiran5, F. Cuello1,2 1

Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 4 Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Germany 5 Cardiovascular Division, King's College London British Heart Foundation Centre of Research Excellence, The Rayne Institute, St Thomas' Hospital, London, UK Background: Crosstalk between signalling pathways participates in the regulation of cardiac myocyte function. During the development of heart failure (HF), desensitization of the β1-adrenoceptor/cAMPdependent protein kinase (PKA) signalling pathway is paralleled by enhanced or unchanged activation of the α1-adrenoceptor signalling pathway. Stimulation of α1-adrenoceptors leads to activation of the MAPK signalling cascade including p90 ribosomal S6 kinase isoforms (RSKs). RSK activity has been shown to increase during the development of HF. Aim: To investigate a potential crosstalk between α1- and β1adrenoceptor signalling pathways. Methods and results: Exposure of adult rat ventricular myocytes to the neurohumoral agonist noradrenaline (NA) induced phosphorylation of PKA substrates involved in excitation-contraction coupling such as cardiac myosin-binding protein C (cMyBP-C) at Ser282, troponin I (TnI) at Ser22/23 and phospholamban (PLN) at Ser16. Interestingly, pharmacological inhibition of RSK using BI-D1870 (1 μmol/L) prior to neurohumoral activation of PKA significantly enhanced PLN Ser16 phosphorylation without affecting the phosphorylation status of cMyBP-C and cTnI. This suggests the involvement of RSK activity in the regulation of compartmentalized PKA activity. This was confirmed in isolated cardiac myocytes from Rsk2-targeted knockout (KO) mice, excluding a potential off-target effect of BI-D1870. FRETexperiments performed in isolated cardiac myocytes from Rsk2 KO mice transgenically expressing a FRET-biosensor localized in vicinity to PLN, showed enhanced cAMP production compared to wildtype cardiac myocytes (60.2% vs 36.4% of maximum response by IBMX/ forskolin). Concentration-response experiments to NA in rat ventricular strips displayed increased force generation (EC50: 1.19 μmol/L), which was significantly enhanced (EC50: 0.88 μmol/L) after exposure to BI-D1870.


Conclusion: Pharmacological or genetic ablation of RSK improved force production significantly by increasing cellular cAMP concentrations, leading to activation of PKA and phosphorylation of PLN at Ser16. 110. Kinomic array analysis to identify druggable kinases limiting atrial fibrillation remodeling M. Wiersma1,⁎, R.A.M. Meijering2,⁎, D. Zhang1, F. Hoogstra-Berends2, J. Scholma3, S. Diks4, X.Y. Qi5, S. Nattel5, R.H. Henning2, B.J.J.M. Brundel1 1 Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands 2 Department of Clinical Pharmacy and Pharmacology, University Institute for Drug Exploration (GUIDE), University of Groningen, University Medical Center Groningen, The Netherlands 3 Department of Developmental BioEngineering, University of Twente, Enschede, The Netherlands 4 Department of Pediatric Oncology, Beatrix Children's hospital, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 5 Department of Medicine, Montreal Heart Institute and Université de Montréal, and the Department of Pharmacology and Therapeutics, McGill University, Montreal, Québec, Canada and the Institute of Pharmacology, West German Heart and Vascular Center, Faculty of Medicine, University Duisburg-Essen, Essen, Germany *Contributed equally

Background: Dysregulation of protein kinase-mediated signaling is an early event in many diseases, including the most common clinical cardiac arrhythmia, atrial fibrillation (AF). Kinomic profiling represents a promising technique to identify candidate kinases. Here we employ kinomic profiling to identify kinases involved in AF remodeling using atrial tissue from a canine AF-model (atrial tachypacing). To detect potentially druggable kinases, AF and control dog data were contrasted with results from tachypaced dogs protected from atrial remodeling by treatment with geranylgeranylacetone (GGA). Methods and results: Three groups of dogs were included: nonpaced controls (C), atrial tachypaced (TP) dogs, and TP/GGAtreated dogs. Left atrial tissue was used for kinase array (containing 1024 kinase pseudo-substrates) analysis. TP changed activity of 50 kinases, which for 40 (involved in differentiation and proliferation, contraction, metabolism, immunity, development, cell cycle (CDK4) and survival (Akt)) was prevented by GGA. Inhibitors of Akt (MK2206) and CDK4 (PD0332991) and overexpression of a dominant negative phosphorylation mutant of CDK4 protected against tachypacing-induced contractile dysfunction in HL-1 cardiomyocytes. Conclusion: Kinomic array analysis offers a versatile tool to identify kinases involved in atrial remodeling due to tachypacing, which include Akt and CDK4. Pharmacological inhibition of Akt and CDK4 protects against tachypacing-induced contractile dysfunction, pointing to the value of kinomic assays in identifying novel therapeutic possibilities to treat clinical AF. 111. Greater vulnerability to atrial arrhythmias is linked with augmented pericardial adipose deposition and estrogen synthesis capacity G. Bernasochi1, W.C. Boon2, S. Pepe3, S. Harrap1, L. Delbridge1, J. Bell1

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1

School of Biomedical Sciences, University of Melbourne, Australia The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Australia 3 Murdoch Children's Research Institute, Royal Children's Hospital, Department of Paediatrics, University of Melbourne and Department of Surgery, Monash University, Australia 2

Background: A link exists between atrial fibrillation (AF) risk and the extent of pericardial adipose accumulation. The underlying mechanisms are poorly understood. Regional adipose depots highly express aromatase and may be an important source of local estrogen. Pericardial adipose as a local cardiac estrogen source has not been investigated. Clinically, estrogen-only hormone supplementation has been linked with increased AF risk. The extent to which both endogenous (locally synthesized) and exogenous estrogens contribute to AF substrate is unknown. The aim of this study was to determine the relationship between pericardial adipose accumulation, local estrogen synthesis capability and atrial arrhythmia vulnerability. Methods and results: Pericardial adipose expression was determined by Western immunoblot. Atrial arrhythmias were assessed in agedcontrol (60 weeks) & high-fat diet (HFD) fed mice, using bipolar electrograms recorded from isolated hearts subjected to programmed pulse stimulation. Aromatase was detected in human atrial appendage and epicardial adipose, and aromatase expression was substantially increased in the myocardium and pericardial adipose of aged rodents (N30-fold vs young control). Analysis of control and HFD fed animals generated a positive correlation between the total aromatase content of pericardial adipose and the incidence (r = 0.74, p b 0.05) and duration (r = 0.61, p b 0.05) of triggered atrial arrhythmias. Perfusion of hearts with 10nM estradiol also significantly increased the incidence/ duration of triggered arrhythmias (p b 0.05). Conclusion: This study provides the first evidence linking atrial arrhythmia with augmented local estrogen biosynthetic capacity in pericardial adipose. These findings provide new leads for the targeting of anti-arrhythmic therapies. 112. Atrial arrhythmia susceptibility in a plakoglobin-deficient murine model with endurance exercise C.Y. Hepburn1,⁎, F. Syeda1,⁎, T. Yu1, A. Holmes1, T. Wright1, S.N. Kabir1, S. Wells1, E. Vloumidi1, C. Apicella1, L. Fortmueller2, A. Isaacs4, M. Stoll3,4, D. Pavlovic1, P. Kirchhof1, 5,6, L. Fabritz1,2,5 1

University of Birmingham, Institute of Cardiovascular Sciences, Birmingham, UK 2 Cardiovascular Medicine, Div. Rhythm. Univ. Hospital Muenster, Germany 3 Human Genetics, Department of Genetic Epidemiology, Germany 4 CARIM, University of Maastricht, The Netherlands 5 University Hospitals Birmingham Foundation NHS Trust, UK 6 Sandwell and West Birmingham Hospitals NHS Service Trust, UK ⁎contributed equally Background: Endurance training increases atrial fibrillation (AF) risk and provokes ventricular arrhythmias in patients with desmosomal dysfunction. We studied the effect of desmosomal dysfunction on atrial arrhythmia susceptibility in trained and sedentary mice with reduced plakoglobin expression (Plako+/-). Methods: Wildtype (WT) and Plako+/− young adult littermates underwent 8 weeks of endurance swim training or sedentary lifestyle. The effects of training and Plako+/− on left atrial (LA) function were assessed using echocardiography, monophasic action potentials

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(MAP), transmembrane action potentials (TAPs), optical mapping, calcium transients, histology, and RNA sequencing. Data are expressed as mean ± SEM. Results: Training increased atrial arrhythmias in Plako+/− hearts only (Plako+/−-sedentary 1/9 hearts; Plako+/−-trained 9/17; WTsedentary 3/11; WT-trained 1/11). Training increased LA size in both genotypes (WT-sedentary 3.1 ± 0.1 mm2; WT-trained 3.8 ± 0.2 mm2; Plako+/−-sedentary 3.2 ± 0.1 mm2; Plako+/−-trained 4.1 ± 0.2 mm2, p b 0.05; n = 21 per group sedentary vs. training). Training shortened APD and ERP in both genotypes (APD; WT-sedentary 22.6 ± 0.7 ms; WT-trained 20.3 ± 0.7 ms, p b 0.05; Plako+/−-sedentary 23 ± 0.6 ms; Plako+/−-trained 21.3 ± 0.9 ms, p b 0.05; ERP; WT-sedentary 41 ± 5 ms; WT-trained 28 ± 2 ms; Plako+/−-sedentary 31 ± 3 ms; Plako+/−-trained 28 ± 2 ms, p b 0.05; n = 9–17 per group sedentary vs. trained). Calcium relaxation times were prolonged in trained Plako+/− LA (WT-trained 24.5 ± 0.9 ms; Plako+/−-trained 30.5 ± 1.6 ms, p b 0.05; n = 5–10). RNA sequencing confirmed decrease in plakoglobin (JUP). Fibrosis-related genes and CAMKII were up-regulated between in Plako+/− LA samples compared with WT, after training. Conclusion: Plakoglobin deficiency predisposes to atrial arrhythmias post-training. Plako+/− is associated with altered expression of genes involved in calcium homeostasis and pro-fibrotic signaling in response to training, and subsequent changes in atrial function. 113. Left atrial sodium channel inhibition by propafenone and dronedarone is enhanced at more positive resting membrane potentials: relevance to personalised therapy for patients with reduced atrial PITX2 mRNA A.P. Holmes1, C. Apicella1, S.N. Kabir1 J.F. Martin2, S.-M. O'Brien1, D. Pavlovic1, L. Fabritz1,3,5, P. Kirchhof1,3,4 1

Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, UK 2 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA 3 Department of Cardiology, UHB NHS Trust, Birmingham, UK 4 Division of Rhythmology, University Hospital, Muenster, Germany 5 SWBH NHS Trust, Birmingham, UK Background: PITX2 is a left atrial (LA) transcription factor that regulates the resting membrane potential (RMP). We have previously defined a cohort of atrial fibrillation (AF) patients with reduced PITX2 mRNA expression. Reduced PITX2 levels and the associated depolarised RMP could help to select effective antiarrhythmic drugs in patients with AF (personalised therapy). We have already identified that flecainide is more effective at more positive RMPs and terminates atrial arrhythmia in Pitx2c deficient hearts. Methods: The inhibitory effect of propafenone (300 nM) and dronedarone (5 μM), on whole cell sodium current was examined at different, defined RMPs (- 100 to - 70 mV) in WT and Pitx2c+/− murine LA cardiomyocytes. Results: Sodium current inhibition by propafenone was enhanced at more positive RMPs in WT (e.g. - 100 mV: 35 ± 3%, - 80 mV: 52 ± 3%, P b 0.05, n = 12) and Pitx2c+/− (e.g. - 100 mV: 38 ± 2%, - 80 mV: 55 ± 3%, P b 0.05, n = 21) LA cardiomyocytes. Dronedarone was also more effective at more positive RMPs in WT (e.g. - 100 mV: 21 ± 2%, - 80 mV: 54 ± 4%, P b 0.05, n = 19) and Pitx2c+/− (e.g. - 100 mV: 26 ± 4%, - 80 mV: 65 ± 8%, P b 0.05, n = 4). Drug

sensitivity to RMP was greater for dronedarone than propafenone (WT: 1.0 ± 0.08%block/mV propafenone, 1.8 ± 0.1%block/mV dronedarone, P b 0.05; Pitx2c+/− 0.88 ± 0.06%block/mV propafenone 2.0 ± 0.1%block/mV dronedarone, P b 0.05). RMP sensitivity for propafenone was similar to flecainide. Pitx2c+/− and Pitx2 null+/− have depolarised RMP. Conclusion: Propafenone and dronedarone inhibit sodium currents more effectively at more positive RMPs. Dronedarone is more sensitive to RMP than propafenone and flecainide. Since Pitx2 deficiency causes RMP depolarisation, these agents may provide effective AF treatment in patients with reduced atrial PITX2 mRNA. 114. Heat shock protein inducer accelerates recovery from contractile dysfunction in experimental atrial fibrillation X. Hu, D.M.S. van Marion, M. Wiersma, D. Zhang, B.J.J.M. Brundel Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands Background: Atrial fibrillation (AF) is the most common tachyarrhythmia associated with electrical, structural remodeling and contractile dysfunction, rooted in an impaired protein quality control system (PQC). PQC regulates the correct folding, trafficking and degradation of proteins in cells. Heat shock proteins (HSPs) play a critical role in facilitating the PQC. Our previous studies showed that the HSP inducers GGA and its derivatives prevent contractile dysfunction in tachypaced atrial cardiomyocyte and Drosophila model for AF. Here we tested whether HSP inducers also restore contractile dysfunction in tachypaced cardiomyocytes and explored the underlying mechanisms. Methods: HL-1 atrial cardiomyocytes were tachypaced (TP) for 10 h at 5 Hz, to induce calcium transient (CaT) loss, followed by a posttreatment with HSP inducer Nyk41(10 μM) for 8 h and 24 h. Contractile function was indicated by CaT amplitudes. To study contractile and structural protein expression levels, Western blot analysis was utilized. Results: Cardiomyocytes that were post-treated with Nyk41 significantly recovered from TP induced CaT loss compared to the nontreated cardiomyocytes. Furthermore, mRNA and protein expression levels of HSP25 were significantly induced during 8 h posttreatment. In addition, the expression level of the microtubule proteins α-tubulin and acetylated α-tubulin, contractile proteins troponin I (TnI) and troponin T (TnT) were significantly reduced after TP and recovered by Nyk41 posttreatment. Conclusions: Our results imply that the HSP inducer Nky41 improves recovery from AF-induced contractile dysfunction and structural remodeling in cardiomyocytes. Nyk41 increases HSP25 expression and normalizes contractile and microtubule protein expression after TP. Future experiments are needed to elucidate the molecular mechanisms underlying recovery. 115. Retinoic acid-treated hiPSC-CM: a suitable model for human atrium? M. Lemme1,2, B. Ulmer1,2, M. Garcia1,2, A. T. L. Zech1,2, T. Christ1,2, T. Eschenhagen1,2 1

Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Hamburg, Germany

Journal of Molecular and Cellular Cardiology 109 (2017) 1–62 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany

Background: Current pharmacological therapies of atrial fibrillation (AF) are limited by low efficacy and disastrous side effects. Human atrial tissue is an invaluable model to develop new therapeutic options. However, access is limited. Cardiomyocytes (CM) generated from human induced pluripotent stem cells (hiPSC) could fill the gap. However, they are typically a heterogeneous mix of ventricular-, atrial-, and nodal-like cells. Alltrans retinoic acid (RA) favours atrial CMs differentiation from human embryonic stem cells (hESC-CM). Here we investigated whether the same holds true for hiPSC-CM. Methods: HiPSC-CMs were treated with RA after mesodermal induction. Atrial specific genes and proteins expression was studied. Moreover, voltage and contractility measurements were obtained from RA treated and untreated hiPSC-CM on multiparametric CellOptiq platform. Results: RA (0.1 and 1 μM) increased the mRNA expression of myosin light chain 2A and the expression of atrial-specific ion channel genes KCNA5 and KCNJ3. Contractility analysis demonstrated that RA significantly decreases the duration of contraction from 380.6 ± 15.3 ms to 242 ± 7.5 ms. Finally, RA treated cells showed a significant shorter AP and higher repolarization fraction than untreated cells (206 ± 24.6 ms vs. 126.3 ± 10.6 ms p b 0.05, n = 140 and 0.28 ± 0.02 vs. 0.15 ± 0.018, p b 0.05, n = 140). Conclusions: The increase of atrial specific genes together with smaller contraction time and higher repolarization fraction might suggest a shift towards atrial-like phenotype. More data are needed before RA-treated hiPSC-CM can be considered as a suitable model to study human atrial physiology and pharmacology. 116. Heart failure increases the susceptibility to atrial cellular alternans: a role for t-tubule loss? G.W.P. Madders, D.C. Hutchings, L. Woods, A.W. Trafford, D.A. Eisner, K.M. Dibb University of Manchester, Manchester, UK Background: Atrial fibrillation (AF) increases stroke risk five-fold and is prevalent in heart failure (HF)1–3. Alternans, a beat-to-beat oscillation in the shape of the action potential and/or Ca transient, has been implicated in AF initiation1; however, the aetiology of alternans is not fully understood. Evidence suggests changes in intracellular Ca cycling play an important role in cellular alternans. The Ca handling machinery is concentrated around a network of membrane invaginations or transverse (t)-tubules. T-tubules are lost in AF and HF, thus t-tubule loss may predispose to cellular alternans. The aim of this work was to determine if alternans could be induced more easily in HF and if this was due to t-tubule loss. Methods and results: Left atrial cardiac myocytes were isolated from control and HF sheep. The loss of t-tubules we have observed in HF5 was mimicked in control cells by formamide detubulination. Myocytes loaded with Fluo-5F were paced between 1 and 3 Hz under perforated patch current clamp control to induce alternans. The lowest frequency at which alternans was detected was deemed the threshold. The threshold for calcium alternans was decreased in HF vs. control (1.25 ± 0.18 vs. 2.40 ± 0.16 Hz, p b 0.05) while in detubulated cells threshold decreased it did not reach significance 1.75 ± 0.34 vs. 2.40 ± 0.16 Hz).

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Conclusion: Our data suggests alternans occurs more readily in atrial cells from animals at the point of HF potentially providing a mechanism for the increased prevalence of AF in HF. Further work is required to establish the role for atrial t-tubule loss in reducing the alternans threshold in HF. References 1. Narayan, S.M., et al., Repolarization alternans reveals vulnerability to human atrial fibrillation. Circulation, 2011. 123(25): p. 2922-30. 2. Wang, T.J., et al., Temporal relations of atrial fibrillation and congestive heart failure and their joint influence on mortality – The Framingham Heart Study. Circulation, 2003. 107(23): p. 2920-2925. 3. Choi, H.W., J.A. Navia, and G.S. Kassab, Stroke Propensity Is Increased under Atrial Fibrillation Hemodynamics: A Simulation Study. Plos One, 2013. 8(9). 4. Wolf, P.A., et al., Epidemiologic assessment of chronic atrial fibrillation and risk of stroke: the Framingham study. Neurology, 1978. 28(10): p. 973-7. 5. Dibb, K.M. et al. Characterization of an extensive transverse tubular network in sheep atrial myovytes and its depletion in heart failure. Circulation. Heart Failure, 2009. 2(5): p 482-9. 117. Biophysical properties of cardiac voltage-gated sodium channels: differences between mouse atrial and ventricular cardiomyocytes S.-M. O'Brien, A.P. Holmes, G. Parnell, C. Apicella, N.S. Kabir, L. Fabritz, D. Pavlovic, P. Kirchhof Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, UK Background: Cardiac voltage-gated sodium current (INa) is a target of several anti-arrhythmic drugs for treatment of atrial fibrillation. As current drugs do not specifically target atrial INa, there is increasing interest in development of atrial-specific ion channel blockers. Differences in the INa biophysical properties in atria and ventricles could help to identify targets for atrial-selective antiarrhythmic drugs for treatment of atrial fibrillation. Aim: We compared biophysical properties of INa between mouse left atrial and ventricular cardiomyocytes. Methods: Na+ channel currents were measured using whole-cell voltage clamp in left atrial and ventricular cardiomyocytes. Expression of Nav1.5 proteins and Navβ-subunits was measured by Western blotting in left and right atrial and left ventricular tissue of wild-type 129/sv mice (15–20 weeks). Protein levels were normalised against calnexin. Results: Peak INA was increased in atrial compared to ventricular cardiomycoytes (LA = -29 ± 1.766 pA/pF; n = 15/4 cells/mice; LV = - 19.83 ± 4.186 pA/pF; n = 5/2 cells/mice; *p b 0.05) and V50 for INa inactivation was significantly more negative in atrial cardiomyocytes (LA = - 92.4 ± 1.877 mV; n = 16/4 cells/mice; LV = - 81.77 ± 2.413 mV; n = 5/2 cells/mice; **p b 0.01). Nav1.5 expression was not different, however, expression of β2 and β4 subunits was lower in atrial tissue (LA = 0.257 ± 0.0286; RA = 0.411 ± 0.0453; LV = 1.00 ± 0.0975; *p b 0.05; n = 4) and (LA = 0.142 ± 0.0493; RA = 0.186 ± 0.0535; LV = 1.00 ± 0.165; ****p b 0.0001; n = 7) respectively. Conclusion: Mouse left atrial cardiomyocytes display increased INA compared to left ventricular cardiomyocytes. Alterations in biophysical properties of INA in atrial myocytes may be attributable to reduced expression of the Nav1.5 β2 and β4 subunits. The interactions between

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Nav1.5 and its β subunits may provide a novel target for antiarrhythmic drug therapy. 118. Postnatal development of atrial t-tubules and excitation contraction coupling C.E.R. Smith, D.A. Eisner, A.W. Trafford, K.M. Dibb University of Manchester, Manchester, UK Background: T-tubules ensure a synchronous rise in systolic calcium to generate contraction. In the ventricles of small mammals t-tubules are absent at birth and develop postnatally resulting in changes in calcium induced calcium release (CICR). At present nothing is known about the postnatal development of t-tubules and calcium handling in the atria. Methods and results: 1 week, 1 month, 3 months and adult sheep were euthanased and cells isolated from the left atrial appendage. T-tubule density assessed using di-4-ANEPPS and confocal microscopy showed that t-tubule density increased during development up until 3 months of age where density was greater than adult (p b 0.01) despite adult cells being wider and having greater need for t-tubules (p b 0.001). Expression of t-tubule regulatory proteins amphiphysin-II and junctophilin-2 was increased in 1 week and 1 month animals in line with t-tubule formation (p b 0.001). Despite fewer t-tubules, L-type current density was greater at 1 week and 1 month vs 3 months and adult (p b 0.01). SR content was also increased in early postnatal life (p b 0.05) but there was no difference in calcium transient amplitude between time points. Conclusions: T-tubules are present at birth in the sheep atria and develop until ~ 3 months of age where there is no further ttubule development despite cells continuing to increase in size. Expression of t-tubule regulatory proteins correlates with ttubule development with levels declining as development slows. The lack of change in transient amplitude despite increased Ltype current and SR content indicates reduced CICR efficiency in early life with maturation coinciding with complete t-tubule development. 119. A method sustaining the bioelectric, biophysical, and bioenergetic function of cultured atrial cells N. Kirschner Peretz1, S. Segal1, L. Arbel-Ganon1, R. Ben Jehuda2,3, Y. Shemer2,3, B. Eisen2,3, M. Davoodi1, O. Binah2,3, Y. Yaniv1 1

Biomedical Engineering Faculty, Technion-IIT, Haifa, Israel Department of Physiology, Biophysics and Systems Biology, Technion-IIT, Haifa, Israel 3 The Rappaport Institute, The Ruth and Bruce Rappaport Faculty of Medicine, Technion-IIT, Haifa, Israel 2

Introduction: Cultured atrial cells lose their ability to be externally paced at physiological rates and to maintain their shape.

Methods and results: Rabbit atrial cells were maintained in culture for 24 h in a medium enriched with a myofilament contraction inhibitor, BDM. The morphology and volume of the cells, including their ability to contract in response to 1–3 Hz electrical pacing, was maintained at the same level as fresh cells. Importantly, the cells could be successfully infected with a GFP adenovirus. Removing BDM from the culture medium led to changes in cell volume and in its ability to be externally paced. In the cells that were cultured with BDM, action potentials, Ca2+ transients, and local Ca2 + spark parameters were similar in the cultured and in fresh cells. Finally, these cultured cells' flavoprotein autofluorescence was maintained at a constant level in response to electrical pacing, a response similar to that of fresh cells. Computational model predicts small changes in ionic currents of fresh and cultured cells. Conclusions: Eliminating contraction during the culture period preserves the bioelectric, biophysical and bioenergetic properties of rabbit atrial myocytes. This method therefore has the potential to further improve our understanding of energetic and biochemical regulation in the atria. 120. DNA damage-induced PARP1 activation confers cardiomyocyte dysfunction through NAD+ depletion in atrial fibrillation D. Zhang1,2, X. Hu1, M. Wiersma1, D.M.S. van Marion1, M. Tolouee2, F. Hoogstra-Berends2, E.A.H Lanters3, N.M.S. de Groot3, R.H. Henning2, B.J.J.M. Brundel1 1 Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands 2 Department of Clinical Pharmacy and Pharmacology, University Medical Centre Groningen, University of Groningen, The Netherlands 3 Department of Cardiology, Erasmus Medical Center, Rotterdam, The Netherlands

Background: Atrial fibrillation (AF) is the most common clinical tachyarrhythmia with a strong tendency to progress from a self-terminating paroxysmal condition into persistent AF. AF progression is driven by derailment of protein homeostasis of cardiomyocytes, which causes contractile dysfunction of the atria. Methods and results: Here, we report that tachypacing-induced functional loss of HL-1 cardiomyocytes is precipitated by excessive poly(ADP)-ribose polymerase 1 (PARP1) activation caused by oxidative stress initiated DNA damage. PARP1 mediated synthesis of ADP ribose chains in turn depletes nicotinamide adenine dinucleotide (NAD+), induces oxidative stress and DNA damage, and contributes to contractile dysfunction. Accordingly, replenishment of NAD+ or depletion of PARP1 precludes tachypacing-induced functional loss. Moreover, inhibition of PARP1 protects against tachypacing-induced NAD+ depletion, oxidative stress, DNA damage and contractile dysfunction in HL-1 cardiomyocytes and Drosophila. Consistent with these findings, cardiomyocytes of patients with persistent AF show significant DNA damage, which correlates with PARP1 activity. Conclusion: The findings uncover a hitherto unrecognized mechanism by which tachypacing impairs cardiomyocyte function and implicates PARP1 as a therapeutic target to preserve cardiomyocyte function in clinical AF. 121.

Objective: We aim to develop a culture method that sustains the shape of atrial cells along with their biophysical and bioenergetic properties in response to physiological pacing. We hypothesize that adding 2,3Butanedione 2-monoxime (BDM), which inhibits contraction during the culture period, will preserve these biophysical and bioenergetic properties.

The control of transverse tubules by Amphiphysin II (Bin 1) J. Caldwell, R. Taylor, D. Eisner, K. Dibb, A. Trafford University of Manchester. Manchester, UK


Background: Transverse (T)-tubules are vital for maintaining normal cardiac contractility through the regulation of excitation coupling. In heart failure t-tubule loss is associated with dysynchronous calcium release resulting in impaired contractility. Thus, determining the mechanisms that control t-tubule formation may provide a treatment of cardiac disease. Evidence suggests that Amphiphysin II (AmpII) controls cardiac t-tubule formation and may play a role in calcium regulation. This study therefore aims to determine if AmpII is sufficient to drive t-tubule formation in the heart. Methods and results: Vectors encoding isoforms 5, 8 and 9 of the AmpII gene (Bin1) with an mKate2 fluorescent protein tag were transiently expressed in neonate rat ventricular myocytes. A vector containing only the mKate2 fluorescent tag was used as control. After 48 h, overexpression of Bin1 was confirmed at the mRNA and protein level. Of cells successfully transfected with Bin1, 95% had developed tubule structures. Tubules were absent in cells expressing the fluorescent tag control. When loaded with a calcium indicator and field stimulated, myocytes transfected with Bin1 isoforms 5, 8 and 9 had larger systolic calcium transient amplitudes than untransfected myocytes. Furthermore, expression of Bin1 isoforms 5 and 9 led to faster rise and decay of the systolic calcium transient. Conclusion: These data suggest that Bin1 plays a vital role in tubule formation in cardiac myocytes and specific Bin1 isoforms may enhance calcium kinetics. Given the importance of t-tubules to normal excitation contraction coupling and their perturbation in heart failure we therefore suggest that Bin1 might be a novel therapeutic target. 122. The impact of t-tubule disruption on excitation-contraction coupling and calcium handling in rat ventricular trabeculae A. Power, S. Kaur, C. Dyer and M.-L. Ward Department of Physiology, Faculty of Medical & Health Sciences, University of Auckland, Auckland, New Zealand Background: Reduced contractility is a hallmark of failing hearts, often linked to depressed Ca2 + transients, since Ca2 + is the key activator of contraction. Impaired excitation-contraction coupling has been observed in conjunction with structural alterations to the ttubular system. However, many sub-cellular changes occur in heart failure, making it difficult to determine the contribution of disrupted t-tubules. Our aim was to quantify the impact of detubulation on Ca 2 + handling and contraction in otherwise healthy ventricular tissue. Methods: Measurement of [Ca2+]i (fura-2) and stress were made in RV trabeculae from rat hearts before and after detubulation following formamide treatment (1.5 M, 5 min). The effect of detubulation on two important regulatory mechanisms was also investigated: the response to stretch, and to β-adrenergic stimulation (0.1 μM isoproterenol). Results: De-tubulation of trabeculae slowed the timecourse of the Ca2+ transients and twitch force: time-to-peak, maximum rate-of-rise, and relaxation were prolonged in both. The slow force response to stretch was decreased following detubulation, and the response to β-adrenergic activation blunted. Conclusions: This study enabled us to examine the effect of detubulation on Ca2+ transients and twitch force in tissue from non-failing hearts in which expression of the different Ca2+ handling proteins was not altered by any disease process. Our results demonstrate the importance of an intact t-tubular system on the kinetics of excitation-contraction coupling,

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as well as on mechanisms that regulate contractility in vivo: the response to stretch and β-adrenergic stimulation. 123. Stabilization of Na+-K+-ATPase with an antibody against its DR region protects heart against oxidative injury F. Hua, Z. Wu, J.-S. Bian Department of Pharmacology, SOM, National University of Singapore, Singapore Background: The activity and expression of Na +/K+-ATPase (NKA) are down-regulated in the failing hearts. An antibody against the DRregion of NKA (DR-Ab) stimulated its activity. The present study was designed to investigate the beneficial effects of this antibody against cardiac injury and the underlying mechanisms. Results: We found in the present study that DR-Ab improved viability of AC16 human cardiomyocytes and primary cultured rat cardiomyocytes under oxidative challenge. It, too, reduced the infarct size in isolated rat hearts subjected to ischemia-reperfusion injury and alleviated cardiac hypertrophy in isoproterenol treated rats. Corresponding to the observation of the reduced NKA in the hypertrophy rat hearts, NKA abundance on plasma membrane was lowered during oxidative stress. Suppressed activity of protein phosphatase 2A (PP2A) was responsible for the loss of membrane NKA due to the increased phosphorylation of key serine residues that triggered endocytosis. Incubation with DR-Ab restored PP2A activity and stabilized NKA expression on the plasma membrane through enhanced interaction between PP2A and NKA. Inhibitors of PP2A abolished the protective effect of DR-Ab against oxidative stress. Conclusion: In summary, our data indicate that loss of membrane NKA may contribute to cardiac pathologies in heart failure. DR-Ab, by stabilizing membrane NKA, protects cardiomyocytes against oxidative injury and improves cardiac function in failing hearts, suggesting a novel approach to heart failure therapy. 124. Visualization of cardiomyocyte cGMP dynamics in hypoxia/reoxygenation N.I. Bork1,2, V.O. Nikolaev1,2 1

Institute of Experimental Cardiovascular Research, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany Background: Cyclic guanosine 3′5′-monophosphate (cGMP) is an important second messenger in multiple physiological processes including the cardiovascular system. In the heart, hypoxia activates maladaptive signaling cascades and can finally lead to heart failure. However, cellular responses to hypoxia/reoxygenation (H/R) are incompletely understood and little is known about cGMP dynamics in the context of H/R. Aim of this project was to investigate the effects of H/R on cGMP dynamics in mammalian cardiomyocytes. Methods: We used transgenic mice with cardiomyocyte-specific expression of the cytosolic Förster resonance energy transfer (FRET)based cGMP sensor red cGES-DE5 to study cGMP dynamics in single adult cardiomyocytes exposed to H/R. Additionally, we

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performed FRET measurements in whole-hearts using a Langendorff system.

on the cell crest induce much stronger nuclear cAMP signals than the receptors located in the T-tubules.

Results: Basal cGMP levels were increased in single adult cardiomyocytes exposed to H/R. This increase was generated during hypoxia and was maintained during reoxygenation. PDE3 protein level and activity were significantly downregulated after H/R, however RNA expression level was not significantly changed. At the same time, protein levels of the soluble guanylyl cyclase β-subunit were downregulated during H/R. In whole-heart measurements, cGMP levels increase during anoxia and decrease during reoxygenation. This indicates that there is also an influence of other cell-types on cGMP dynamics or on cGMP cell-cell transfer.

Conclusion: in the present study, we have uncovered a mechanism regulating cAMP signaling at the T-tubules in healthy and diseased cardiomyocytes. Our findings also provide the evidence for two distinct populations of β1AR, one is located on the crest and signals stronger to the nucleus and the second one located in the T-tubule and produce local signal.

Conclusion: In conclusion, we found an increase of intracellular cGMP levels in hypoxia. The development of FRET-based cGMP measurements in single cardiomyocytes and whole-hearts enables us to dissect the molecular mechanism of cGMP signaling regulation during ischemic injury. 125. Two different microdomains of β1-adrenoreceptor signalling revealed by scaning ion conductance microscopy/fluorescence resonance energy transfer A. Froese1,2, N.O. Nikolaev1,2 1

Institute of Experimental Cardiovascular Research, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany Background: 3′,5′-cyclic adenosine monophosphate (cAMP) is a intracellular second messenger critically involved in the regulation of heart function. Signalling by cAMP in cardiomyocytes is organized in discrete functionally relevant microdomains formed for example around calcium-handling proteins. Previously, we have shown that in adult mouse cardiomyocytes, the β1-adrenergic receptor (β1AR) is localized across the whole cell membrane, including transverse (T)-tubules and the crest regions. However, it is unknown whether these two β1AR pools, one is located in T-tubules and another in the crest, contribute differentially to regulation of the cardiac contraction and gene expression. Methods and results: To measured microdomain-specific receptor/ cAMP signals in cardiomyocytes, we used a combination of scanning ion conductance microscopy (SICM) with transgenically expressed targeted Förster resonance energy transfer (FRET)-based cAMP biosensors. This technique allows measurements of local cAMP responses in different microdomains (such as subsarcolemmal space, cytosol and nucleus) after specific and precise local stimulation of βARs on T-tubule and crest regions of healthy and diseased cardiomyocytes. Following visualization of the cell membrane topography by SICM, simultaneous local stimulation of a various membrane structures by scanning nanopipette and FRET-based real-time cAMP recordings in microdomains were performed. Using a plasma membrane targeted cAMP biosensor in this approach we has uncovered mechanisms regulating local cAMP signaling in healthy myocytes as well as in compensated cardiac hypertrophy induced by transverse aortic constriction (TAC). These data show that β1AR stimulation in the T-tubules generated smaller subsarcolemmal cAMP signals compared to crest stimulation. cAMP in the proximity of T-tubules of healthy cardiomyocytes is mainly compartmentalized by PDE3, while after TAC, local PDE3 regulation is partially lost and PDE4 becomes the major cAMP-hydrolyzing phosphodiesterase. Next, using nucleus targeted cAMP biosensor we could demonstrate that β1ARs located

126. A cardiac Wnt-nuclear complex promotes epigenetic changes controlling tissue remodeling in the adult heart L.M. Iyer1, S. Nagarajan2, 3, M. Woelfer1, S. Khadjeh4, E. Schoger1, S.T. Pang1, V. Kari2, M.-P. Zafeiriou1, J. Herting4, T. Fischer4, K. Toischer4, G. Hasenfuss4, C. Noack1, S.A. Johnsen2, L.C. Zelarayán1 1

Institute of Pharmacology and Toxicology, University Medical Center, Göttingen, Germany 2 Department of Visceral and Pediatric Surgery, University Medical Center, Göttingen, Germany 3 Cancer Research UK (CRUK-CI), Cambridge, UK 4 Department of Cardiology and Pneumology, University Medical Center, Göttingen, Germany Background: Activation of the developmental Wnt pathway has been reported during maladaptive cardiac remodeling. Methods and results: We show that β-catenin and transcription factor 7-like 2 (TCF7L2), the main nuclear components of the Wnttranscriptional cascade, and dependent-transcription are increased upon pathological remodeling in both murine and human hearts. To understand the consequences of this increased Wnt activity, we mimicked this activation in an in vivo mouse model, in which β-catenin is stabilized in adult cardiomyocytes (CM), leading to increased ventricular TCF7L2 expression. These mice displayed cardiac hypertrophy and impaired cardiac function. They showed a reactivation of Wntdependent developmental programs; including activation of the Wnt non-canonical pathway, increased cardiomyocyte cell cycling with poly-nucleation and cytoskeletal disorganization, underscoring a central role in adult tissue remodeling. By integrating transcriptomic and genome-wide analyses (ChIP-seq) of the endogenous ventricular TCF7L2, we show that upon aberrant Wnt activation, TCF7L2 induces context and Wnt-specific gene regulation in pathological remodeling. Interestingly, β-catenin stabilized ventricles showed increased active enhancer mark H3K27ac and TCF7L2 recruitment to novel diseaseassociated enhancers. In a similar mouse model with β-catenin loss of function, upregulation of these identified disease-associated genes was abrogated upon induced hypertrophy, confirming their association to cardiac Wnt signaling. Importantly, using integrative motif analyses and experimental evidences, our data uncovered a role for GATA4 as a cardiogenic modulator of TCF7L2/β-catenin complex and established a paradigm for cell-specific effects of Wnt signaling. Conclusion: Altogether, our studies unraveled the nuclear Wnt-TCF7L2associated chromatin landscape and its role in heart failure progression. 127. Identification of necroptosis in failing hearts following myocardial infarction M. Lichý1, A. Szobi1, J. Hrdlička2, M. Kocmalová3, K. Kopásková1, T. Rajtík1, S. Fraňová3, J. Neckář2, F. Kolář2, A. Adameová1

Journal of Molecular and Cellular Cardiology 109 (2017) 1–62 1 Faculty of Pharmacy, Commenius University in Bratislava, Bratislava, Slovak Republic 2 Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic 3 The Jessenius Faculty of Medicine in Martin, Commenius University in Bratislava, Bratislava, Slovak Republic

Background: As pathomechanisms of heart failure (HF) due to myocardial infarction (MI) are still incompletely understood and necroptosis, a cell death type sharing features of necrosis and apoptosis, has been detected in ischemic/reperfused hearts, the aim was to study the subcellular expression of main necroptotic proteins (RIP1, RIP3, MLKL) in failing hearts and thereby propose signaling pathway leading into cell death and heart dysfunction under such conditions. Methods: MI was induced by ligation of the left anterior descending artery which resulted in ischemic cardiomyopathy in rats. After 42 days, left ventricles of the heart were subjected to subcellular fractionation and protein expression was identified by immunoblotting analysis. Echocardiography and collagen content were used to determine alterations in heart function and remodeling. To get a complex view on the proposed mechanisms of necroptosis in HF, inflammatory response (cytokines) and oxidative stress (MDA) were assessed.

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Methods and results: Using cardiomyocytes and adult inducible SIRT1 knockout mice, we demonstrate that SIRT1 inhibition or deficiency increases ER stress-induced cardiac injury, whereas activation of SIRT1 by the sirtuin-1-activating compound STAC-3 is protective. Analysis of the expression of markers of the three main branches of the unfolded protein response (i.e. PERK/eIF2α, ATF6 and IRE1) showed that SIRT1 protects cardiomyocytes from ER stress-induced apoptosis by attenuating PERK/eIF2α pathway activation. We also present evidence that SIRT1 physically interacts with and deacetylates eIF2α. Mass spectrometry analysis identified lysines K141 and K143 as the acetylation sites on eIF2α targeted by SIRT1. Furthermore, mutation of K143 to arginine to mimic eIF2α deacetylation confers protection against ER stressinduced apoptosis. Conclusion: Collectively, our findings indicate that eIF2α deacetylation on lysine K143 by SIRT1 is a novel regulatory mechanism for protecting cardiac cells from ER stress and suggest that activation of SIRT1 has potential as a therapeutic approach to protect the heart against ER stress-induced injury. 129. Mitochondrial function in heart failure: A role for nitric oxide signaling E. Radcliffe, J. Sun, A. Aponte, D. Eisner, E. Murphy, A. Trafford

Results: In post-ischemic failing hearts with serious remodeling as assessed by increased LVDs, LVDd, and decrease in FS along with higher collagen content and oxidative stress, the expression of total MLKL and RIP3 was significantly increased. Importantly, the content of membrane MLKL was found to be increased in HF. Likewise, its upstream activator and necroptosis marker, pSer229–RIP3, was higher in cytosolic and membrane fractions of the diseased hearts. Conclusions: Identification of increased levels and subcellular relocalization of key necroptosis proteins in post-ischemic failing hearts indicates that cardiomyocyte necroptosis might, at least in part, underlie cardiac dysfunction and remodeling in this pathology. Acknowledgments: This study was supported by APVV15-607, VEGA 1/0271/16. 128. SIRT1 protects the heart from endoplasmic reticulum stress-induced apoptosis through eIF2α deacetylation J. Pires Da Silva1, A. Prola1, A. Guilbert1, L. Lecru3, J. Piquereau1, M. Ribeiro1, P. Mateo1, M. Gressette1, C. Gallerne3, H. François3, D.A. Sinclair4, P. Eid3, R. Ventura-Clapier1, A. Garnier1, C. Lemaire2 1

UMR-S 1180, INSERM, Univ Paris-Sud, Université Paris-Saclay, ChâtenayMalabry, France 2 Université Versailles St-Quentin, Inserm UMR-S 1180, Univ Paris-Sud, Université Paris Saclay, 92296 Chatenay-Malabry, France 3 UMR-S 1014, INSERM, Univ Paris-Sud, Université Paris-Saclay, Hôpital Paul Brousse, Villejuif, France 4 Glenn Labs for the Biological Mechanisms of Aging, Department of Genetics, Harvard Medical School, Boston, MA, USA Background: Over the past decade, endoplasmic reticulum (ER) stress has emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases including heart failure. Cardiac therapy based on ER stress modulation is viewed as a promising avenue towards effective therapies for the diseased heart. Here, we tested whether sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, participates in modulating ER stress response in the heart.

University of Manchester, Manchester, UK Background: Heart failure, in roughly 50% of patients, is associated with reduced cardiac contractility. The electron transport chain (ETC) is the principle source of ATP within cardiac muscle. Consequently, the regulation of the ETC in heart failure represents a potential rate limiting step to contraction in the disease state. This study will therefore investigate mitochondrial respiratory function in heart failure and the potential regulatory role of nitric oxide signalling using an ovine tachypaced model of heart failure. Results: Cardiac contractility, measured as m-mode fractional shortening was reduced from 0.47 ± 0.01 pre-pacing to 0.20 ± 0.03 postpacing (p b 0.01, n = 5). Changes in S-nitrosylation (S-NO) were assessed using S-NO resin assisted capture and mass spectrometry. The number of S-NO proteins increased from 232 ± 18 in control to 314 ± 28.3 in heart failure (p = 0.02, n = 6). When normalised for changes in expression, 79% of proteins also showed an increase in S-NO abundance in heart failure. Among these, several ETC proteins displayed increased S-NO in the disease state (eg. NDUFS1 ↑2.9 fold, SDHA ↑1.2 fold, UQCRH ↑1.1 fold, n = 6) in the absence of corresponding expression changes (eg. NDUFS1 ↓ 0.2 fold, SDHA ↓ 0.07 fold, UQCRH ↓ 0.2 fold, n = 5). In vitro the nitric oxide donor S-nitrosoglutathione reduces oxidative phosphorylation by approximately 25% (0.2 mM, p b 0.01, n = 8). Conclusion: Together this data indicates an upregulation of nitric oxide signalling in heart failure and that this may have a deleterious functional consequence on respiration. This work therefore provides a novel insight into how nitric oxide signalling and mitochondrial dysfunction may contribute to the deterioration of contractility in heart failure. 130. Direct visualization of cGMP dynamics in plasma membrane microdomain of adult cardiomyocytes H. Subramanian1,2,⁎, A. Froese1,2,3,⁎, H. Schmidt4, J. Gorelik5, V.O. Nikolaev1,2

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1 Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Clinic of Cardiology and Pulmonology, University Medical Center Göttingen, Göttingen, Germany 4 Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany 5 Myocardial Function, National Heart and Lung Institute, Imperial College London, ICTEM, Hammersmith Hospital, Du Cane Road, London, UK *contributed equally

Purpose: cGMP play several roles in cardiomyocytes by acting in distinct subcellular microdomains. Previously, we measured cGMP in the cytosolic compartment of adult myocytes using highly sensitive Förster resonance energy transfer (FRET) sensor (red cGES-DE5). Our aim is to identify the regulation of cGMP in plasma membrane (PM) microdomain. Methods: PM-targeted sensor (pmDE5) was developed by fusing the red cGES-DE5 with 10-aminoacid palmitoylation and myristoylation motif. cGMP was measured in freshly isolated cardiomyocytes from transgenic mice expressing pmDE5 specifically in myocardium. Results: Transgenic mice showed normal cardiac morphology and unaltered heart function, and the localization of the sensor in the PM was verified by confocal imaging. Atrial Natriuretic Peptide (ANP) and C-type Natriuretic Peptide (CNP) activating Guanylate cyclases GC-A and GC-B, respectively increased cGMP in PM. While ANP induced a reversible cGMP increase, CNP-induced cGMP was consistent. Interestingly in ANP pre-stimulated cells, PDE2 inhibitor (BAY 60-7550) showed strong cGMP increase indicating tight PDE2 regulation in GC-A associated microdomain, while in CNP-prestimulated cells PDE3 inhibitor (Cilostamide) significantly increased cGMP. Using Scanning Ion Conductance Microscopy in myocytes expressing red cGES-DE5, we identified ANP stimulates cGMP response at T-tubules, but not in crest. However, CNP induced cGMP response in both T-tubules and crest. These data indicate that GC-A is exclusively localized in T-tubules, while GC-B is expressed in both T-tubules and crest.

Hypothesis: Heterotopic heart transplantation (hHTX) in transgenic mice would be an adequate model of unloading. Since the procedure is technically highly demanding with low error tolerance it is hardly used. We present our experience establishing the abdominal hHTX in mice and trouble shootings. Methods: The transplantation-technique is based on internationally accepted hHTX models in rodents, leading to complete mechanical unloading. Initial technical training was performed using video-education, rat hHTX that is well-established in our institution, and training in institutes that successfully established the method in mice. The transplantation was initially performed in dead mice (one-mouse-training-transplantation). Reaching technical confidence, transplantation of a donor heart from one mouse to the abdomen of another (two-mouse-transplantation) was performed. Results: 20 one-mouse-training-operations were performed. The first successful transplantation with a well-beating donor-heart and recipient-survival N 24 h was achieved after 20 two-mousetransplantations. 90%-success rate was reached after 60 twomouse-transplantations. Operation time decreased in correlation with the increasing number of procedures from 200 min to 45 min after 60 operations. Cold/warm ischemia time improved from 45/100 to 10/20 min. Key factors for success and trouble shootings were identified. Conclusion: Establishing abdominal hHTX in mice is laborious, timeconsuming and costly, but can be improved by sharing expertise. A structured education-programme and avoiding presented pitfalls reduces expenses and animals. 132. The Insulin-like growth factor binding protein 5 (IGFBP5) - a potential developmental gene is regulated upon heart failure M. Wölfer1, S. Khadjeh2, M. Tiburcy1, C. Noack1, L.M. Iyer1, G. Hasenfuß2, W.-H. Zimmermann1, K. Toischer2, L.C. Zelarayán1 1

Conclusion: Our data strongly suggest that GC-A and GC-B are distinct microdomains in adult myocytes due to their differential localization and compartmentation by different PDEs. 131. Establishing heterotopic heart transplantation in a mouse model to study mechanical unloading - learning curve and troubleshooting S. Westhofen1, A.P. Schwoerer2,3, M. Jelinek2,3, D. Biermann1, H. Reichenspurner1,3, H. Ehmke2,3 1

Department of Cardiovascular Surgery, University Heart Center, Hamburg, Germany 2 Department of Cellular and Integrative Physiology, University Medical Center Hamburg Eppendorf, Hamburg, Germany 3 DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany Background: Unloading of failing hearts by left ventricular assist devices can lead to negative alterations in cardiomyocyte physiology associated with increased mortality and morbidity. Observations in patients and translational animal models indicate that reduction in workload itself induces impaired calcium-handling and cellular electrophysiology with unclear pathophysiological relevance.

Department of Pharmacology and Toxicology, University Medical CenterGeorg-August-University-Göttingen, Göttingen, Germany 2 Department of Cardiology and Pneumology, University Medical CenterGeorg-August-University-Göttingen, Göttingen, Germany Background: Transition from compensatory hypertrophy to heart failure is accompanied by a re-activation of embryonic gene expression. We identified the Insulin-like growth factor binding protein 5 (IGFBP5) to be re-expressed during this transition. IGFBP5 is expressed in the pre-cardiac region in mouse embryos and its upregulation impairs cardiac progenitor differentiation. The role of this factor in cardiac remodeling is still unknown. Methods and results: Analysis of Igfbp5 in murine cardiac tissue at different developmental stages showed a decreasing mRNA expression with progressing heart maturation, becoming very low in the adult heart. Expression analysis of Igfbp5 in different cell populations of the adult heart showed very low Igfbp5 levels in cardiomyocytes in contrast to high levels in undifferentiated Sca-1 positive cells, indicating a role of IGFBP5 in cardiac progenitor homeostasis. A conserved reactivation of expression was found in the failing heart in mouse (TAC) and in human (aortic stenosis), whereas IGFBP5 targeting microRNAs were found regulated antagonistically in patients with aortic stenosis. Concerning the responsive cell type, cardiomyocytes and fibroblasts both showed increased mRNA levels of IGFBP5 in vivo and in vitro upon stress, whereas the protein levels were unchanged. This might


be explained with increased serum levels of IGFBP5 found in mice with cardiac dysfunction, indicating an autocrine or paracrine function of IGFBP5. Conclusion: We hypothesize that IGFBP5 may participate in the developmental gene program, becoming reactivated in the diseased heart, where it might regulate cardiomyocyte-fibroblast crosstalk at the transition to heart failure. The functional role and regulation of IGFBP5 is under investigation. 133. Alteration in the composition and function of high-density lipoproteins in hypertensive patients with heart failure N. Woudberg1, R. James2, O. Billyrose3, D. Ojji3, M. Frias2, S. Lecour1 1 Hatter Institute for Cardiovascular Research in Africa, Department of Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa 2 Department of Internal Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland 3 University of Abuja Teaching Hospital, Abuja, Nigeria

Background: Hypertension related complications account for 9.4 million worldwide deaths and is a major contributor to cardiovascular disease (CVD), including heart failure. The pathophysiology of the disease involves endothelial dysfunction, an effect that may be enhanced with high-density lipoprotein (HDL) dysfunction. We aim to compare HDL composition and function in healthy and hypertensive patients with/without heart failure. Methods: Nigerian patients (n = 90) were divided into healthy controls, hypertensive patients and hypertensive patients with heart failure (HHF) (inclusion criteria measured by echocardiography and blood pressure). HDL function was assessed by measuring paraoxonase activity, HDL anti-inflammatory function and HDLmediated activation of endothelial nitric oxide synthase (eNOS). ApoA1 and ApoM in HDL were measured using Western blot techniques whilst sphingosine-1-phosphate (S1P) was assessed using mass spectroscopy. HDL subclass distribution was measured using Lipoprint® system.

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1

Laboratory of Cardiovascular Biology, Department of Internal Medicine, University of Genova, Genova, Italy 2 Laboratory of Nephrology, Department of Internal Medicine, University of Genova, Genova, Italy 3 Laboratory of Biochemistry, Department of Pharmacy, University of Genova, Genova, Italy 4 Cardiovascular Disease Unit, IRCCS AOU San Martino – IST, Genova, Italy Background: Indoxyl sulphate (IS) accumulates early in chronic kidney disease (CKD) and has been implicated in CKD-related cardiomyopathy, in which myocardial fibrosis is being recognized as important as cardiomyocyte hypertrophy. Thus, we investigated the effects of IS, at a concentration found in moderate CKD, on cardiac fibroblasts (Fib). Methods and results: Treatment of mouse neonatal Fib with 50 μM IS triggered mild oxidative stress, enhanced immunopositivity for αSMA, and stimulated type 1a collagen synthesis. Moreover, IS increased mRNA and protein levels of monocyte chemoattractant protein-1, tumor necrosis factor-α and myostatin, the latter being an emerging mediator of IS action. The genes encoding angiotensinogen, angiotensin (Ang)-converting enzyme, and Ang type 1 receptor (AT1R) were also upregulated by IS, as well as the one for neprilysin, an endopeptidase that cleaves AngII and – mosty – natriuretic peptides. Compared with control cells, mouse neonatal cardiomyocytes incubated with the conditioned medium of IS-primed Fib exhibited higher levels of β-myosin heavy chain and atrial natriuretic peptide, and heightened glycolysis and lactate dehydrogenase activity with lactate production. Part of IS effects were counteracted by the AT1R antagonist, valsartan. At the time of this abstract, we are setting an animal model of IS exposure: preliminary data suggests that greater Fib activation and cardiomyocyte hypertrophy occur in the hearts of adult C57BL/6 mice given IS in drinking water (1 mg/mL) for 7 weeks than water alone. Conclusion: IS activates Fib to a secretory pro-fibrotic, pro-inflammatory and pro-hypertrophic phenotype, including a cell-autonomous Ang/ neprilysin system that may further promote myocardial fibrosis and hypertrophy. 135. Effects of LDL and oxidized LDL on toll-like receptor 4 expression and activity in the hearts of rats with myocardial infarction

Results: ApoM and S1P content in HDL were lower in HFF patients compared to controls (ApoM: 3.6 ± 0.5 AU vs 4.8 ± 0.4 AU, p b 0.05; S1P: 178 ± 13.8 pmol/mg vs 209 ± 9 pmol/mg, p b 0.05). ApoA1 was unchanged. HDL subclass distribution shifted in HFF patients from large (51 ± 3 vs 62 ± 3%, p b 0.005) to small HDL (7.6 ± 1 vs 2.7 ± 1%, p b 0.005) compared to controls. Paraoxonase activity and antiinflammatory function did not differ between groups. In contrast to HDL from control patients, HDL from all hypertensive patients failed to activate eNOS.

N. Maleki-Dizaji, A. Khorrami, M. Ziaee, H. Soraya, S. Andalib, A. Garjani

Conclusion: HDL composition, subclass and function are modified in patients with hypertension and heart failure. Our data suggest that these changes in HDL may contribute to the pathophysiology of heart failure associated with hypertension.

Methods and results: Male Wistar rats were allocated in to 3 groups and received standard, cholesterol-rich, and oxidized cholesterolrich diets. Fourteen weeks after MI induction, rats were received ISO (100 mg/kg) for 2 consecutive days subcutaneously. Serum levels of oxidized-LDL remarkably were high in the group fed with oxidizedcholesterol (P b 0.001). High serum level of oxidized-LDL caused a notable fibrotic damage to the heart tissues even in the absence of MI. The mean infarct size in the heart of rats with iso-induced MI which were fed with standard diet was 32.1 ± 2.9%, while feeding with diets containing cholesterol and specially oxidized-cholesterol increased the size significantly up to 40.33 ± 2.3% (P b 0.05) and 52.16 ± 3.8% (P b 0.01), respectively. Serum and tissue levels of TNFα and IL6 were significantly higher in the oxidized-LDL group (P b 0.001). Oxidized

134. Indoxyl sulphate activates cardiac fibroblasts with enhanced collagen synthesis, upregulated angiotensin-neprilysin, and paracrine induction of cardiomyocyte hypertrophy C. Barisione1, E. Lazzarini1, P. Fabbi1, S. Garibaldi1, C. Ruggeri1, D. Verzola2, S. Ravera3, C. Brunelli1,4, G. Ghigliotti1,4, P. Ameri1,4

Department of Pharmacology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran Aim: In the present study, we examined the effects of high-cholesterol and high-oxidized cholesterol diets on myocardial injuries, TLR4 expression, and cytokines production in the hearts with myocardial infarction.

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cholesterol considerably increased TLR4 mRNA level in the rats myocardium (3.35±0.22 vs 4.47 ± 0.34; P b 0.05). Dietary oxidized cholesterol significantly (P b 0.01) increased MYD88, TLR adaptor protein, content in the same way that increased TLR4 expression.

137.

Conclusion: Cholesterol and much more than that oxidized cholesterol potentiated TLR4 activity and TNFα and IL6 production in the hearts of rats with myocardial infarction.

S. Zaouidi, F. Cusin, S. Clerc-Rignault, C. Bielmann, N. Rosenblatt-Velin

136. Modulatory role of serum bilirubin in training induced-bradycardia I. Musa1, M. Abdul-Aziz Mabrouk2 1

Department of Human Physiology, Kogi state University, Anyigba, Nigeria Department of Human Physiology, Bayero University, Kano, Nigeria

Characterisation of endogenous cardiac inflammatory cells displaying a cardiogenic potential

Département Cœur-Vaisseaux, Laboratoire de Physiopathologie Clinique, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland Background: During the culture of non-myocyte cells (NMCs) isolated from neonatal or adult mouse hearts, round weakly adherent cells growing on the fibroblast and endothelial cell layer appeared. The aim of this study was to identify the nature and function of these weakly adherent cardiac cells (WACs).

2

Background: The mechanisms responsible for exercise-induced reductions in baseline heart rate (HR), known as training bradycardia, remain controversial. Decades ago, Studies had shown that, a low level of serum bilirubin was associated with high risk for cardiovascular disease in human by mitigating inflammation processes. However, one potential modifiable behavior to increase bilirubin levels is physical activity. The purpose of this study was to examine the association between physical activity and serum bilirubin levels and its potential modulatory role in training induced-bradycardia. Methods: A total number of 40 males (20 Trained and 20 Untrained) between 18 and 25 years of age were recruited. The 20 apparently healthy untrained males were taken as control and 20 trained males comprise the study group. Anthropometric parameters, serum total bilirubin and leukocyte counts were measured in fasting state at rest, from all the participants. The VO2max was estimated from a standard regression equation. Statistical analysis was done on SPSS/win.20.0 using Student's unpaired t-test. Linear regression analysis was also used for prediction of any association. The level of significance was set at p b 0.05. Results: There was no significant difference (p N 0.05) in age between the untrained and the trained group. Our result showed that VO2max exhibited significant differences between the trained and untrained. Mean heart rate, and leukocyte counts were significantly (p b 0.05) lower in trained than those of untrained group. In contrast, the mean total bilirubin was significantly (p b 0.05) higher in trained when compared with untrained group. The associations of Heart rate, with leucocytes and total bilirubin, for both groups was determined by univariate regression analysis. The VO2max, correlated positively with serum total bilirubin (p b 0.0001) in the trained group (R2 = 0.088, + 0.297). Heart rate, correlated positively with serum total bilirubin (R2 = 0.003, + 0.052, p b 0.0001), and with serum leukocyte counts (R2 = 0.005, +0.074, p N 0.05), in the untrained group. In the trained group, Heart rate, correlated negatively with serum total bilirubin (R2 = 0.013, − 0.115, p b 0.0001) and with leukocyte counts (R2 = 0.000489, −0.022, p b 0.05), respectively. In addition, Leukocyte counts correlated negatively with serum total bilirubin (p b 0.0001), in the untrained group (R2 = 0.162, − 0.403) and correlated positively in the trained group (R2 = 0.032, +0.178; p b 0.0001). Conclusions: These data suggest that, training bradycardia could be influence from increase serum bilirubin in the trained. In addition, we showed that potential modulating effect of the bilirubin might be on inflammation process because we observed lower leukocyte counts in the trained compared to the untrained. This study is novel, as, to our knowledge, no studies to date have examined this association.

Methods and results: We first characterized WACs by flow cytometry analysis. These cells were CD45+ F4/80+ CD11b+ CD206+ CD64+ cells, demonstrating their appartenance to the M2 macrophage cell subset. This was confirmed by qPCR analysis. In vivo, in neonatal or adult unmanipulated hearts, cells with the same phenotype represent about 5 to 6% of the total non-myocyte cells. We then tested the function of these cells in vitro. When cultured alone in adequate medium during 3 weeks, WACs were able to express mRNAs coding for cardiomyocyte specific markers: Nkx2.5 (×4), Troponin I (×19), beta (× 4) and alpha (× 9) Myosin Heavy Chain when compared to the WACs after isolation. mRNAs coding for Sca-1 (× 5.5) and c-kit (× 3.5) were also increased. WAC's supernatant inhibited neonatal NMC proliferation (−25%, p b 0.001) and stimulated the proliferation of the neonatal cardiomyocytes (+ 13%, p = 0.06) after 7 days of culture. Conclusions: We identified in neonatal and adult non-myocyte cell cultures, macrophages belonging to the M2 subset able to modulate the proliferation of neonatal cardiomyocytes and non-myocyte cells. Furthermore, these cells are able to express some cardiogenic markers. Their role in vivo in physiological as well as in pathophysiological conditions must be investigated. 138. Withdrawn 139. Developmental origin of cardiac macrophages in steady state and under inflammatory conditions T. Weinberger, R. Thaler, V. Schneider, O. Hoti, A. Titova, S. Massberg, C. Schulz Medizinische Klinik und Poliklinik I, Ludwig-Maximilians-Universität, Munich, Germany DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany Background: Macrophages are the most prominent immune cells in the myocardial tissue in steady state and play a critical role under pathological conditions. Recently, it has become clear that macrophages have a dual developmental origin from embryonic and bone marrow (BM) hematopoiesis. However, the quantitative contribution of either source to the cardiac macrophage pool has remained unclear. We therefore revisited the development of cardiac macrophages. Methods and results: In this study we used multiple models of lineage tracing in mice during steady state and following exposure to angiotensin (AT)-II induced inflammation. We found that at steady state a


considerable amount (approximately 70%) of cardiac macrophages developed independently of definitive hematopoiesis from embryonic precursors. After AT-II infusion, a transient increase in the number of BM-derived macrophages was observed in the heart, which declined over time and could not be detected after 30 days. Further, the developmental origin of cardiac macrophages after AT-II infusion was related to the extent of local inflammation. We found the most profound influx of BM-derived macrophages in areas of myocardial and perivascular fibrosis. Conclusion: Under steady conditions the pool of cardiac tissue-resident macrophages is mainly composed of embryonic macrophages. In response to AT-II inflammation BM-derived macrophages entered the myocardium, however the influx was transient and limited to tissues with fibrotic changes. Our findings are of potential importance for understanding the cardiac immune response and for therapeutic targeting of macrophages in inflammatory conditions. 140. Beneficial and detrimental consequences of a primary increase in mitochondrial ROS formation on mitochondrial function, Ca2+ homeostasis and cardiomyocyte viability S. Antonucci1, M. Di Sante1,2, S. Deshwal1, R.C. Hartley3, T. Krieg4, M.P. Murphy5, F. Di Lisa1,2 1

Department of Biomedical Sciences, University of Padova, Padova, Italy Neuroscience Institute, National Research Council of Italy (CNR), Padova, Italy 3 Centre for the Chemical Research of Ageing, WestCHEM School of Chemistry, University of Glasgow, Glasgow, UK 4 Department of Medicine, University of Cambridge, Cambridge, UK 5 Mitochondrial Biology Unit, MRC, University of Cambridge, Cambridge, UK 2

Background: Since changes in intracellular [Ca2+] and reactive oxygen species are generally intertwined, it is difficult to establish causal relationships. Here, we aimed at elucidating the consequences of a primary increase in mitochondrial ROS formation induced by a paraquat analogue targeted to mitochondria (MitoPQ). This compound, that elicits mitochondrial ROS formation by means of redox cycling, was investigated in neonatal ventricular cardiac myocytes (NRVMs). Results: (i) High doses (≥500 nM) induce an increase in ROS levels (p b 0.001) and a decrease of mitochondrial membrane potential (mmp) (p b 0.001) followed by cell death (p b 0.001); (ii) Moderate doses (between 50 nM and 100 nM) induce an increase in H2O2 levels (p b 0.01), still resulting in a decrease of mmp (p b 0.01). Both frequency and amplitude of Ca2 + transients are decreased and cells become unexcitable after caffeine addition. Notably, cell viability is not affected; (iii) Low doses (10 nM) induce a mild accumulation of H2 O2 (p b 0.01) without affecting mmp. Ca2+ amplitude is increased (p b 0.01) while frequency is not modified. Importantly, not only cell viability is not affected, but also 2 h pretreatment with this low dose decreases the susceptibility to anoxia/ reoxygenation injury (p b 0.001). Conclusions: Cardioprotection, derangements in Ca2+ homeostasis and loss of cell viability are caused by low, moderate and high doses of MitoPQ, respectively. These findings demonstrate that mitochondrial ROS exert both beneficial and detrimental effects that have been previously suggested only by indirect evidence.

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141. The role of demethylase FTO and adipokines in the heart: effect of chronic hypoxia D. Benak1, D. Kasparova1, M. Cyprova1, P. Telensky2, Z. Bendova2, J. Neckar1, F. Kolar1, M. Hlavackova1,2 1

Institute od Physiology, Czech Academy of Sciences, Prague, Czech Republic Department of Physiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic 2

Bacground: Adaptation to hypoxia renders the heart more tolerant to ischemia/reperfusion injury. This adaptation is enabled by physiological changes on the cellular level. One of these changes is a shift in the energy metabolism. This process can be regulated by fat mass and obesity associated (FTO) protein, demethylase epigenetically regulating cell protein synthesis. Heart metabolism can be also modulated by adipokines leptin and adiponectin. This project aimed to study the role of FTO and adipokines in the chronically hypoxic heart. Methods and results: Adult male Sprague-Dawley rats were adapted to continuous normobaric hypoxia (CNH; 12% O2; 3 weeks). The FTO protein level was measured in the left (LV) and right (RV) ventricles of both hypoxic and normoxic animals. Under the normoxic conditions, the level of FTO in the RV was by 50% higher than in the LV. CNH led to a significant increase of FTO protein level in the heart by 21% in the LV and by 27% in the RV. We observed lower levels of adiponectin (45% decrease) in the plasma of hypoxic rats and slightly increasing trend of leptin levels in the LV (25% increase), but these changes were not statistically significant. Conclusion: We showed that CNH increases level of FTO in the heart, which may potentially participate in the ischemia-resistant phenotype. 142. Development of matrix metalloproteinase-2 inhibitors against acute myocardial infarction P. Bencsik1,2, K. Kupai3, A. Görbe1,2, Z.V. Varga4, J. Pálóczi4, I. Hajdú5, S. Cseh5, G. Dormán5, P. Ferdinandy2,6 1 Cardiovascular Research Group, Department of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary 2 Pharmahungary Group; Szeged, Hungary 3 Department of Physiology, Faculty of Science and Informatics, University of Szeged, Hungary 4 Laboratory of Cardiovascular Physiology and Tissue Injury, National Institutes of Health/NIAAA, Bethesda, MD, USA 5 Targetex Inc, Dunakeszi, Hungary 6 Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Budapest, Hungary Background: Acute activation of matrix metalloprotease-2 (MMP-2) during acute myocardial infarction (AMI) has been demonstrated to contribute to myocardial dysfunction. Inhibiting MMP-2 is therefore a potent tool to treat AMI patients. Despite promising preclinical data, adverse events derived from non-selective MMP inhibition have rendered clinical trials unsuccessful. Therefore, the aim of this study was to identify and test novel selective MMP-2 inhibitors against AMI. Methods: Hydroxamic acid compounds, as first generation of MMP inhibitors, were taken as basic structures, however, during in silico analyses, we found that imidazole- and thiazole-carboxylic acid

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compounds exerts higher MMP-2 selectivity. After in silico screening, MMP inhibitor compounds were synthetized and subjected to a biological screening cascade. IC50 values for MMP-2 were determined by gelatin zymography. Then, stimulated ischemia/reperfusion (I/R) injury on neonatal cardiomyocytes was performed to assess the cardioprotective effects of the most potent compounds. Finally, the compound with the highest cytoprotective effect during cell culture experiments, MMPI-1154, was tested in an isolated rat heart model of AMI. Results: After in silico screening, the synthesis of 30 compounds was available, from that 12 compounds showed a significant MMP-2 inhibition under 100 μM concentration. Eight from the 12 compounds exhibited 100% inhibition to MMP-2 as examined by gelatin zymography. Six compounds increased cell viability significantly in response to simulated I/R injury. In isolated hearts subjected to global I/R injury, MMPI1154 significantly reduced infarct size at 1 μM as compared to the vehicle-treated hearts. Conclusion: MMPI-1154 can be a promising lead candidate for further steps of drug development since it exhibited high selectivity for MMP-2, and significant cardioprotection in cell culture as well as in an isolated heart model of AMI. 143. A1 adenosine receptor activation can protect the myocardium from ischaemia reperfusion injury throughout post reperfusion J. Bhandal, D. Renshaw, J. Buckley, H. Maddock, A. Hussain Coventry University, Coventry, UK Background: A 1 adenosine receptors (A 1 AR), have previously been shown to limit injurious infarct development. The purpose of this current study was to determine if delayed activation of A1ARs from 0, 15 or 30 min post-reperfusion can still limit infarct development.

144. Runx1 deficiency in cardiomyocytes preserves cardiac contractile function post-MI A. Bradley, C. Nixon, A. Riddell, C.S. McCarroll, S.M. McKenzie, K. Blyth, S.A. Nicklin, E. Cameron, C.M. Loughrey Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, UK Background: Myocardial infarction (MI) is a leading cause of heart failure and death worldwide. Adverse cardiac remodelling underlies the progression from MI to heart failure and can manifest clinically as reduced contractility. Novel therapeutic targets that can preserve contractile function post-MI are therefore required urgently. Runx1 is a transcription factor minimally expressed in adult cardiomyocytes but is re-activated at 3 weeks post-MI in humans and animals. The functional role and expression of Runx1 at earlier time-points (b14 days) remains unknown. Methods and results: Here we show the importance of Runx1 at earlier time point's post-MI using a tamoxifen-inducible cardiomyocyte-specific Runx1 deficient mouse (Runx1Δ/Δ). A novel in situ hybridisation technique (RNAscope®) using probes for cardiomyocyte-specific nuclei (peri-centriolar material-1) together with Runx1 demonstrated detectable Runx1 expression in 43% and 59% of cardiomyocytes in the border zone post-MI at 1 and 14 days respectively (cf. 10% in sham). Furthermore, Runx1 was detectable in 59% and 47% of cardiomyocytes in the infarct region post-MI at 1 and 14 days respectively (cf. 10% in sham). These data supported separate RT-qPCR experiments demonstrating an increased Runx1 expression level at 1 and 14 days post-MI in isolated cardiomyocytes. Whilst LV fractional shortening as measured by echocardiography in control (Runx1fl/fl and Runx1wt/wt) mice decreased at 1 and 7 day post-MI, Runx1Δ/Δ mice demonstrated a remarkable preservation of LV contractile function ([35% vs. 45%; 1 day] [30% vs. 44%; 7 days];P b 0.05).

Methods: Male Sprague-Dawley rats (300 ± 50 g) were mounted on the Langendorff system. A1AR agonist, 2-chloro-N-cyclopentyl-2′methyladenosine (2′-MeCCPA) (0.1 nM–1 μM) was administered at the onset of reperfusion as well as 15 and 30 min post-reperfusion (10 nM) in the presence and absence of the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 200 nM) as well as in the presence and absence of Wortmannin (100 nM).

Conclusion: Collectively, these results demonstrate the therapeutic potential of Runx1 as a novel translatable target to limit the progression of adverse cardiac remodelling post-MI. 145.

Results: Administration of 2'-MeCCPA (1 nM–1 μM) at reperfusion significantly reduced infarct size to risk ratio. Furthermore, delaying the activation of A1AR to 15 or 30 min post-reperfusion significantly reduced infarct size compared to IR control. The co-administration of DPCPX (200 nM) with 2′-MeCCPA (10 nM) at the onset of reperfusion significantly attenuated the 2′-MeCCPA mediated infarct. The concomitant administration of 2′-MeCCPA with DPCPX at either 15 or 30 min post-reperfusion also abrogated 2′-MeCCPA induced cardioprotection. Co-administration of Wortmannin (100 nM) with 2′-MeCCPA (10 nM) at the onset of reperfusion also significantly reduced the 2′-MeCCPA mediated infarct size as well as the co-administration at 15 or 30 min post-reperfusion reversed the cardioprotection.

G. Brenner, A. Makkos, A. Görbe, Z. Giricz, P. Ferdinandy

Conclusion: This is the first study to display how 2′-MeCCPA, a highly selective A1 AR agonist, when administered at reperfusion, 15 or 30 min post-reperfusion can limit the infarct size development and how the PI3K cell signaling pathway is also associated with cardioprotection.

Rofecoxib is cardioprotective and does not show hidden cardiotoxic properties

Cardiometabolic Research Group, Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary Background: Unexpected ischemia-related cardiac adverse effects are the leading causes of discontinuation of clinical trials and withdrawal of drugs from the market. Drugs may exert unwanted effects by interfering with ischemic tolerance and inhibiting endogenous cardioprotective pathways which led to novel concept of “hidden cardiotoxicity”. Hidden cardiotoxicity cannot be revealed by currently available toxicity screening methods. Moreover, the mechanism of hidden cardiotoxocity is not known. Therefore, we aimed to explore the effect of drugs withdrawn from the market due to unexpected myocardial ischemic events on ischemia-reperfusion injury and cardioprotection by ischemic preconditioning. In the present study rofecoxib, the use of which was associated with increased risk of myocardial infarction was investigated in a rat model of myocardial infarction and ischemic preconditioning.


Methods: Male Wistar rats were treated with rofecoxib (5.12 mg/kg/day) and vehicle (control group) for 28 days. Both groups of animals were subjected to 30 min LAD occlusion followed by 120 min reperfusion or ischemic preconditioning elicited by 3 cycles of 5-min LAD occlusion and 5-min reperfusion before the 30 min LAD occlusion/reperfusion. After the 120 min reperfusion, area at risk and infarct size were measured by Evans blue and triphenyltetrazolium chloride staining. Results: Chronic rofecoxib treatment reduced infarct size per se (rofecoxib + Isch: 23.29 ± 3% vs. control: 42.97 ± 4.4%) and did not affect the cardioprotective effect of ischemic preconditioning (rofecoxib + IPC 13.59 ± 1.3% vs. control + IPC: 19.20 ± 5.2%). Conclusion: This is the first demonstration that rofecoxib exert direct cardioprotective effect. Moreover, it does not show hidden cardiotoxic properties. 146. Regulation of cardiomyocyte DNA damage and apoptosis by the type 2A protein phosphatase regulatory protein alpha4 J. Cowan1, M.R. Longman1, P. Eaton2, A.K. Snabaitis1

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1

Department of Pharmacology, Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary 2 Department of Bioanalytical Chemistry, Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary Background: Nowadays there is a growing interest in the components of plants as potential pharmaceutical raw materials. Among these beta-carotene is one of most intensively studied compound. Numerous clinical studies have examined the cardiovascular effects of it, but the results are quite contradictory. Beta-carotene could function as an antioxidant, however, many evidence show that it can act as pro-oxidant under increased oxidative circumstances. In the present study we investigated the cardiovascular effects of long-term, low and high dose beta-carotene treatment in hearts isolated from Zucker obese rats. Methods: The animals were treated with a daily dose of 30 and 150 mg/kg beta-carotene for 4 weeks. Before, at the 2nd and 4th week of treatment oral glucose tolerance tests were carried out. After the treatment period, hearts were isolated, mounted on a "working-heart" apparatus and subjected to 30 min of global ischemia and 120 min of reperfusion. During the measurements heart function was registered. Finally infarcted area and HO-1 enzyme levels of the hearts were determined.

1

Kingston University, London, UK King's College London, UK

2

Background: Cardiomyocyte cell death associated with heart disease is thought to involve apoptosis and it is therefore important to understand which mechanisms drive apoptosis in the heart. In this study, we demonstrate that the non-catalytic type 2A protein phosphatase regulatory protein alpha4 behaves as a novel anti-apoptotic protein in cardiomyocytes. Methods and results: Short interfering RNA (siRNA) molecules specific to rat alpha4 mRNA were used to knockdown alpha4 protein (alpha4 KD) in cultured H9c2 cardiomyocytes. Western analysis revealed that expression of the anti-apoptotic B-cell lymphoma 2 (BCL-2) protein was reduced by 54.8 ± 3.6% (p b 0.05) following alpha4 KD, whereas expression of the pro-apoptotic binding partner BCL-2 associated X (BAX) protein was increased by 54.5 ± 2.2%. Hence, the apoptotic index BAX:BCL-2 ratio significantly (p b 0.05) peaked (2.7-fold) after 3 days of alpha4 KD, when compared to control (CTR) cardiomyocytes. Expression of full length BH3-interacting domain (BID) protein was reduced by 58.7 ± 11.0% (p b 0.05) following alpha4 KD when compared to CTR. Alpha4 KD reduced expression of full length procaspase-3 by 58.8 ± 19.6% (p b 0.05) and the caspase-3 substrate poly ADP ribose polymerase-1 by 80.3 ± 16.6% (p b 0.05) compared to CTR. Sensitivity to doxorubicin-induced DNA damage (H2A.X Ser139 phosphorylation) and cytotoxicity (MTT assay) was enhanced (p b 0.05) by alpha4 KD (235.4% and 45.0%, respectively) compared to CTR. Furthermore, cardiomyocyte apoptosis and cell death were confirmed by positive TUNEL staining and light microscopy after 8 days of alpha4 KD, respectively. Conclusion: This study illustrates the novel significance of the type 2A protein phosphatase regulatory protein alpha4 as an anti-apoptotic protein in cardiomyocytes. 147. Cardiovascular effects of low- and high-dose beta-carotene treatment in Zucker obese rats E. Csepanyi1,2, A. Czompa1, P. Szabados-Furjesi1,2, I. Lekli 1, A. Tosaki1, I. Bak1,2

Results: The glucose tolerance tests showed dose-independent reduction in blood glucose level. Furthermore, low dose treatment resulted in a significant increase in postischemic cardiac function, which was followed by decreased infarcted area. Moreover, high-dose betacarotene treatment singnificantly increased the level of HO-1 in hearts subjected to ischemia/reperfusion. Interestingly, the observed cardioprotective effects are disappeared in case of high dose treatment, in spite of increased HO-1 level. Conclusion: The observed controversial effects would be the result of formation of harmful oxidative products of beta-carotene during reperfusion. Acknowledgements: ‘National Excellence Program’: TÁMOP-4.2.4.A/211/1-2012-0001, GINOP-2.3.2-15-2016-00043. 148. MAO-dependent ER-mitochondria dysfunction and mast cell degranulation lead to adverse cardiac remodeling in diabetes S. Deshwal1, M. Forkink2, C.-H. Hu2, G. Buonincontri2, S. Antonucci1, M. Di Sante3, M.P. Murphy4, N. Paolocci5,6, D. Mochly-Rosen7, T. Krieg2, N. Kaludercic3, F. Di Lisa1,3 1

Department of Biomedical Sciences, University of Padova, Padova, Italy Department of Medicine, University of Cambridge, Cambridge, UK 3 Neuroscience Institute, National Research Council of Italy (CNR), Padova, Italy 4 Mitochondrial Biology Unit, MRC, Cambridge, UK 5 Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD, USA 6 Department of Experimental Medicine, University of Perugia, Perugia, Italy 7 Department of Chemical and Systems Biology, Stanford University, School of Medicine, Stanford, CA, USA 2

Background: Cardiovascular complications are the major cause of mortality among diabetic patients. Reactive oxygen species (ROS), inflammation, mitochondrial dysfunction and endoplasmic reticulum (ER) stress contribute to the structural and functional alterations in the diabetic heart. Although numerous reports hint towards a cross-talk

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occurring between these factors, the precise interplay remains unknown. Here we investigated whether monoamine oxidases (MAO), mitochondrial flavoenzymes capable of generating H2O2, might represent the missing link.

Conclusion: The results of this pilot study suggest that moderate and chronic consumption of reduced-alcohol red wine may confer cardioprotective benefit in PAH. 150.

Methods and results: Using both genetic and pharmacological approaches we show that exposure of primary cardiomyocytes to high glucose and pro-inflammatory stimuli leads to MAO-dependent increase in ROS formation (≥1.5-fold vs control, p ≤ 0.05). This is accompanied by reduced mitochondrial membrane potential and perturbed ER homeostasis, the latter evidenced by the upregulation of ER stress markers ATF4, GRP78 and GADD34 (≥ 2-fold vs control, p ≤ 0.05). Remarkably, MAO inhibition prevented mitochondrial dysfunction and ER stress, highlighting the novel role of these flavoenzymes in the cross-talk between mitochondria and ER in diabetes. In vivo, administration of the MAO inhibitor pargyline to streptozotocin (STZ)-induced type 1 diabetic mice abolished oxidative stress (4-hydroxynonenal), reduced the upregulation of ER stress markers ATF4 and GADD34, and prevented LV diastolic dysfunction (2-fold decrease in diastolic stiffness vs STZ mice, p ≤ 0.05). Furthermore, we report that MAO inhibition reduced cardiac mast cell degranulation in STZ-mice, likely accounting for the decreased fibrosis observed in STZ-mice following MAO inhibition. Conclusion: Taken together, these results highlight the critical role of MAO in diabetic cardiomyopathy and provide novel insights into the mechanisms underlying MAO-induced changes in the diabetic heart. 149. Beneficial effect of reduced-alcohol red wine in a rat model of pulmonary arterial hypertension: A pilot study P. Diaba-Nuhoho1, M. Cour2, N. Hadebe2, S. Lecour2, D. Marais1, D. Blackhurst1 1

Division of Chemical Pathology, Department of Pathology, University of Cape Town Health Science Faculty, Cape Town, South Africa 2 Hatter Institute & LOPI, Cardioprotection Group, University of Cape Town Health Science Faculty, Cape Town, South Africa Background: Pulmonary arterial hypertension (PAH), is a severe disorder that causes right ventricular (RV) dysfunction and death. Pathophysiological mechanisms remain unclear but oxidative stress may contribute. Antioxidants in red wine may thus be cardioprotective. The aim of this study was to test if reduced-alcohol (5.5%) red wine (RARW) consumption limits monocrotaline (MCT)-induced PAH in rats. Methods: Rats were grouped as follows: (1) Control; (2) MCT-group (80 mg/Kg, s.c. at day 0) and (3) MCT-Treated with RARW (diluted 1 in 7 in drinking water, for 5 days before MCT and for 28 days after MCT). Echocardiography measured pulmonary artery acceleration time (PAAT) and RV thickness. Oxidative stress was evaluated by measuring conjugated dienes (CD). Results: Baseline echocardiography was similar amongst all groups. RV hypertrophy was observed after 28 days in the MCT- group compared to the control group (1.23 ± 0.06 mm and 0.52 ± 0.04 mm; p b 0.001). PAAT in the MCT- group was reduced compared to the control group (14.33 ± 0.88 ms and 26.00 ± 1.16 ms; p b 0.001). Chronic treatment with RARW in PAH animals improved hypertrophy and PAAT (0.81 ± 0.07 mm and 24.50 ± 1.50 ms, respectively; p b 0.001 versus MCT-induced PAH). CD increased in MCT- animals compared to controls (659 ± 27 nmol/L and 486 ± 46 nmol/L; p = 0.02). Chronic treatment with RARW reduced CD: 444 ± 40 nmol/L; p = 0.01 versus MCT-group).

Improved post-ischemic function by necrostatin-1S in ex-vivo perfused mice hearts exposed to ischemia-reperfusion C. Mathisen Heiestad1, M.-K. Torp1,2, Y. Li1, K.O. Stensløkken1,2 1

Division of Physiology, Institute of Basic Medical Sciences, University of Oslo. Oslo, Norway 2 Center for Heart Failure Research, University of Oslo, Oslo, Norway Background: During myocardial infarction (MI), insufficient blood supply to the tissue results in necrotic cell death. Release of cytotoxic content from necrotic cells causes a local sterile inflammation and cell death may spread through the remaining myocardium. It is now clear that necrotic cell death can be driven by defined molecular pathways and different types of regulated necrosis are identified. Our aim was to study the molecular mechanisms of regulated necrosis in cardiac cells, with the main goal of finding an approach to reduce necrotic cell death after MI and thereby limit the following sterile inflammation. We investigated the potentially protective effect of necrostatins in the setting of myocardial ischemia-reperfusion. Methods: C57BL6 male mice hearts were isolated, retrogradely perfused, and subjected to global ischemia followed by 60 min of reperfusion in a Langendorff-perfusion system. Hearts were exposed to Necrostatin-1 and Necrostatin-1S. After reperfusion infarct size and post-ischemic function was analyzed. In addition, we investigated the effect of necrostatins in primary adult cardiomyocytes and fibroblasts isolated from C57BL6 male mice. Results: We found no differences in infarct size between the groups, but we showed that Necrostatin-1S improved post-ischemic cardiac function, supported by reduced p38-MAPK phosphorylation. We also found elevated levels of Bcl-2-associated-X- protein, which can be an indication of increased apoptosis, in hearts subjected to necrostatins. Conclusion: The improved function despite similar infarct size may indicate a change in the type of cell death in cardiac cells after myocardial infarction. 151. LNA-132 application is cardioprotective in a model of percutaneous transverse aortic constriction (pTAC) R. Hinkel1,2,4, K. Klett1,2,4, S. Straub1, A. Howe1,2, S. Baktai3, R. Kozlik-Feldmann5, F. Freudenthal6, T. Thum3, C. Kupatt1,2,4 1 Medizinische Klinik und Poliklinik, Klinikum Rechts der Isar der TU München, Munich, Germany 2 Institute for Cardiovascular Prevention, LMU, Munich, Germany 3 IFB, Molekulare und Translationale Therapiestrategie, Medizinische Hochschule Hannover, Hannover, Germany 4 DZHK (German Center for Cardiovascular Research), partner site Munich Heart Alliance, Munich Germany 5 University Medical Center Hamburg-Eppendorf, Hamburg, Germany 6 PFM, La Paz, Bolivia

Background: Hypertrophy, fibrosis, capillary rarefaction and reduced myocardial function are hallmarks of pressure-induced heart failure. Considering the lack of specific therapies, the need for a pre-clinical large animal model and novel therapies is evident. Therefore, we


established a model hypertrophy in pigs and evaluated the relevance of miR-132 in cardiac hypertrophy. Methods: Pigs (n = 5/group) underwent percutaneous transverse aortic constriction (pTAC, 60% area stenosis) on day 0. LNA-132 or LNA-scr were applied regionally to the heart (day 0 and 28). LVEDP and EF were obtained at days 0, 28 and 56. Moreover, regional myocardial function (SES) was amalysed at day 56. Tissue samples were harvested for hypertrophy and fibrosis. Results: pTAC induced an increase of aortic pressure (85 ± 3 mm Hg before, 98 ± 2 mm Hg after stent implantation), which further increased over 56 days (133 ± 5 mm Hg). pTAC induced an impaired global myocardial function (LVEDP 8 ± 1% d0 vs. 15 ± 1, d56). In addition, regional myocardial function was impaired in the pressureoverload group. Inhibition of miR-132, a miR up-regulated in hypertrophy, via LNA-132 significantly improved global myocardial function (LVEDP 10 ± 1 mm Hg, d56) as well as regional myocardial function (SES at 150 bpm: 23 ± 2%). Hypertrophy was observed in pTAC, whereas LNA 132 treatment reduced this effect (4.1 ± 0.1 g/kg pTAC vs. 3.4 ± 0.2 g/kg in pTAC + LNA-132). Fibrosis was increased in pTAC (32 ± 1% vs. control 21 ± 1%) and reduced in pTAC + LNA 132 (23 ± 1%). Conclusion: We successfully established a model of percutaneous transverse aortic constriction (pTAC) leading to pressure-induced heart failure. LNA-132 was capable of significantly improving myocardial function and reducing cardiac hypertrophy. 152. Effect of continuous normobaric hypoxia and exercise training on acute myocardial ischemic tolerance and post-infarction heart failure J. Hrdlicka, J. Neckar, P. Alanova, M. Hlavackova, F. Papousek, J. Vasinova, F. Kolar Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic Background: Adaptation to continuous normobaric hypoxia (CNH) or exercise training (ET) protect the heart against acute ischemia/reperfusion (I/R) injury but it is unknown whether these cardioprotective interventions are additive. Their potential therapeutic effects are also unclear. Methods and results: Our aims were to determine effects of CNH (12% O2), ET (treadmill; 60 min/day, 5 days/week) and CNH combined with ET on acute I/R injury and to find out whether ET or CNH can attenuate the progression of post-infarction heart failure (HF). Animals (2-month-old male Wistar rats) assigned to acute I/R study were subjected to 20-min coronary occlusion and 3-h reperfusion after 3-week exposure to CNH or 2 weeks of ET (30 m/min) in either CNH or room air (normoxia). Rats assigned to HF study were subjected to CNH or ET either before 60-min coronary occlusion or during 3-week period starting 7 days after occlusion (ET 15 m/min). CNH, ET and ET in hypoxia decreased acute infarct size to 36.7 ± 3.3%, 44.3 ± 2.7% and 37.4 ± 3.7% of are at risk vs. 54.1 ± 4.0% in sedentary normoxic controls. Chronic infarction resulted in 38% left ventricle (LV) scar circumference and progressive HF development. CNH before occlusion reduced acute mortality (to 26% vs. 55% in normoxic group) and post-infarction exposure to CNH attenuated LV dilatation; ET had no effect. Conclusion: Our data suggest that cardioprotective effects of CNH and ET in acute I/R are not additive. Adaptation to CNH improves acute I/R survival, and therapeutic exposure to CNH can attenuate the progression of unfavorable changes in LV geometry in post-infarction HF.

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153. The effect of hydrogen sulfide (H2S) on reperfusion-induced ventricular arrhythmias Q.G. Karwi1,2, M. Whiteman3, M. Wood4, G. Baxter1 1 School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK 2 College of Medicine, University of Diyala, Diyala, Iraq 3 University of Exeter, Medical School, Exeter, UK 4 School of Biosciences, University of Exeter, Exeter, UK

Background: We have demonstrated that pharmacological intervention with either slow-generating1 or mitochondrial-targeted2 H2S donors at reperfusion limited myocardial infarction following ischaemia/ reperfusion injury in vivo. This study aimed to characterise the potential anti-arrhythmic activities of different H2S donors against reperfusioninduced arrhythmias in a rat infarct model. We have conducted a comprehensive analysis of electrocardiograms traces during the early phase of reperfusion following H2S postconditioning. Methods: The left coronary artery was occluded for 30 min to induce ischaemia followed by 2 h of reperfusion in thiobutabarbital-anesthetized Sprague-Dawley rats. Slow-generating (GYY4137) and mitochondrial targeted (AP39 and AP123) H2S donors were employed along with their controls, namely decomposed-GYY4137, ADT-OH, AP39's H2Sreleasing moiety, HTB, AP123's H2S-releasing moiety and AP219, mitochondrial-targeting scaffold of AP39 and AP123. Pharmacological interventions were administrated 10 min prior to reperfusion. Ischaemic preconditioning (IPC, 2 × 3 min) was employed as a positive control. Ventricular premature beats (VPBs), ventricular tachycardia (VT), sustained VT(N30 s) and ventricular fibrillation(VF) during the first 10 min of reperfusion were detected using Lead-II electrocardiograms. Results: AP39 significantly limited reperfusion-induced VPBs by 56% compared to the vehicle group (p b 0.01) while GYY4137 and AP123 reduced VPBs by 41% and 31%, respectively (not significant). AP39 also attenuated the incidence of reperfusion-induced sustained VT and VF by 69% and 67%, respectively (p b 0.05). Conclusion: We here reported for the first time the potential antiarrhythmic effect of selective mitochondrial delivery of H2S by AP39 on reperfusion-induced ventricular arrhythmia in vivo. Characterising the mechanism of AP39's anti-arrhythmic effect is the subject of ongoing investigation. References 1. Pharmacol Res 2016;111:442–451. 2. Br J Pharmacol 2017;174:287–301. 154. Pre- and post-conditioning with hydrogen sulfide in vivo: a systematic review and meta-analysis Q.G. Karwi, J.S. Bice, G.F. Baxter School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK College of Medicine, University of Diyala, Diyala, Iraq Background: Enhanced level of hydrogen sulfide (H2S) before ischaemia or at reperfusion is known to exert a conditioning-like myocardial infarct limitation in vivo. Here, we conducted a comprehensive systematic review followed by meta-analysis of preclinical

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studies investigating pharmacological pre- and post-conditioning with H2S enhancers to critically assess the infarct-limiting effect of exogenous H2S.

response to such humoral signal transfer cardiomyocyte ROS formation was reduced. 156.

Methods: The study was conducted in accordance with Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guideline. Medline, Embase and Web of Science databases were searched for in vivo reports which characterised infarct limitation by pharmacological intervention with H2S enhancer(s). Returned reports (6031) were screened at the title and abstract levels for eligibility assessment. Eligible reports (135) were critically appraised at the full-text level, using a critical appraisal tool, after which thirty six reports were included in the final analysis. Data were pooled in a random-effects meta-analysis with the weighted mean difference in the infarct size between H2S-treated group and the control group as the main outcome. Results: Preconditioning (pre-ischaemic treatment) with H2S limited myocardial infarction by 21.6% (95% Cl 26.4–16.9, n = 217) compared to untreated controls (n = 128, p b 0.001,). The overall reduction in infarct size exerted by H2S-induced postconditioning (pre-reperfusion treatment) was 22.8% (95%Cl 25.1–20.5, n = 382) when compared with the control animals (n = 188, p b 0.001). A high degree of heterogeneity was observed in both categories (90% and 58% for preconditioning and postconditioning groups, respectively). Conclusion: This study confirms that cardioprotection against acute myocardial infarction is observed when enhancing the bioavailability of H2S bioavailability either before ischaemia onset or at reperfusion. 155. Plasma dialysate from pigs with remote ischemic perconditioning preserves the viability and reduces ROS formation of isolated adult ventricular mouse cardiomyocytes following hypoxia/reoxygenation P. Kleinbongard, L. Stoian, N. Gedik, H.R. Lieder, A. Skyschally, G. Heusch Institut für Pathophysiologie, Westdeutsches Herz- und Gefäßzentrum, Universitätsklinikum Essen, Essen, Germany Background: Brief episodes of ischemia/reperfusion (I/R) in a tissue remote from the heart reduce infarct size after sustained myocardial I/R. The signal transfer from the periphery to the heart and its signal transduction within the myocardium remain largely enigmatic. In isolated cardiomyocytes, we here identify the humoral transfer of cardioprotection by using plasma dialysate from pigs with remote ischemic perconditioning or placebo (RPER or PLA).

Cardioprotection from hypoxia/reoxygenation injury in engineered human myocardium E. Levent1,2, M. Tiburcy1,2, L. Rodriguez-Borlado3, I. Palacious Lopez3, C. Noack1,2, K. Sharkova1, L.-C. Zelarayan1,2, D. Katschinski2,4, W.-H. Zimmermann1,2 1 Institute of Pharmacology and Toxicology, University Medical Center, Georg-August University Göttingen, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Göttingen, Göttingen, Germany 3 Coretherapix S.L., Madrid, Spain 4 Institute of Cardiovascular Physiology, University Medical Center, GeorgAugust University Göttingen, Göttingen, Germany

Background: Cardioprotection from myocardial reperfusion injury after sustained ischemia remains to be an unmet clinical need in cardiology. We hypothesized that engineered human myocardium (EHM) may serve as a pre-clinical test-bed to investigate cardioprotective strategies in a human model of hypoxia/reoxygenation (H/R) injury. Methods and results: The effect of hypoxia was first tested in 2D monolayer human embryonic stem cell (hESC)-derived cardiomyocytes (CM). Exposure to 1% O2 led to the stabilization of hypoxia-inducible factor-1 (HIF)α followed by a metabolic switch to glycolysis. In addition, a hypoxia reporter hESC-line genetically modified for the expression of a fusion protein composed of oxygen dependent degradation domain of HIF-1α and firefly luciferase enabled us monitor the degree of hypoxia sensing in intact cells. Next, we investigated the effect of hypoxia in EHM generated from hESC-derived cardiomyocytes and human dermal fibroblasts in a collagen hydrogel. Human EHMs subjected to hypoxia shifted their energy metabolism to glycolysis with an increase in lactate production. Furthermore, not hypoxia alone, but reoxygenation after 72 h hypoxia resulted in a significant loss of force of contraction in EHM. We then tested medium conditioned by human cardiac progenitors or human dermal fibroblasts for their potential paracrine cardioprotective activity. Interestingly, fibroblast-derived conditioned medium better protected cardiomyocytes and maintained heart muscle functionality after H/R injury. Conclusion: EHM was established as a model system for the evaluation of cardioprotective strategies in a human model of H/R injury. Fibroblasts secreted beneficial factors to protect from H/R injury in EHM.

Methods: Pigs were subjected to RPER or PLA (n = 6) during occlusion of the left anterior descending coronary artery and subsequent reperfusion. Arterial blood was taken at 10 min reperfusion. Cardiomyocytes were isolated from adult mouse hearts (n = 6) and incubated with saline buffer and dialysate of pig plasma (dRPER and dPLA, 1:10; b12–14 kDa). After exposure to hypoxia/reoxygenation (H/R) or to normoxic time-control (TC), cardiomyocyte viability and extracellular ROS was quantified.

157.

Results: dRPER preserved cardiomyocyte viability following H/R, whereas dPLA has no effect. In TC experiments, there was no difference between dPLA and dRPER. Increased extracellular ROS concentration during H/R was attenuated by dRPER, but not by dPLA.

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Mitohormesis: cardioprotection by the superoxide generator MitoParaquat in a mouse model of acute myocardial infarction J. Mulvey1, A. Hall2, A. Logan2, S. Arndt2, S. Caldwell3, R.C. Hartley3, M. Murphy2, T. Krieg1 1

Conclusion: Cardioprotection induced by RPER can be transferred with plasma dialysate b12–14 kDa to isolated cardiomyocytes and in

Department of Medicine, University of Cambridge, Cambridge, UK MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK 3 WestCHEM School of Chemistry, University of Glasgow, Glasgow, UK Background: Acute myocardial ischaemia reperfusion injury (IRI) occurs when the blood supply to the heart is disrupted and then restored, leading to a burst of reactive oxygen species (ROS) production from the mitochondria and thus tissue damage. It is increasingly being


understood that ROS contribute not only to pathology but also to normal physiology and some studies have even suggested that the addition of exogenous ROS may limit infarct size. Such a biphasic response, characterized by detrimental effects at high doses and beneficial effects at low doses, has been termed mitohormesis. MitoParaquat is a mitochondria-targeted compound that redox cycles at complex I to produce superoxide, closely mimicking the endogenous production of ROS. Aim and methods: Here it is used to investigate the role of ROS in acute myocardial IRI. Further, we use the db/db mouse model of type 2 diabetes mellitus (T2DM) as a clinically relevant comorbidity to examine how this dose response relationship may be modulated. Results and conclusion: MitoParaquat produced ROS in a dose dependent manner and this was found to be protective against acute myocardial IRI in vivo. The dose response curve was found to be biphasic, with infarct size not significantly different from control at low doses, cardioprotection observed in an intermediate range and high doses found to be lethal. This dose-response curve was found to be shifted in the db/db model of T2DM, with a differing dose required to confer cardioprotection. 158. The feasibility and superiority of high frame rate strain imaging compared to ejection fraction in a rat model of ischamia-reperfusion myocardial infarction using cardiac MRI S. Narodden1, N. Baxan2, S.E. Harding3 1 National Heart and Lung Institute, Imperial Centre of Translational Medicine, Imperial College London, Hammersmith Campus, London, UK 2 Biomedical Imaging Centre, Department of Medicine, Imperial Centre of Translational Medicine, Imperial College London, Hammersmith Campus, London, UK 3 National Heart and Lung Institute, Imperial Centre of Translational Medicine, Imperial College London, Hammersmith Campus, London, UK

Background: In the fields of cardiac regeneration and cardioprotection, robust quantification of the cardiac function is paramount for the assessment of the effects of experimental interventions on the heart. We hypothesized the feasibility of performing cardiac MRI strain imaging and assessed its superiority to ejection fraction (EF) in I/R MI rats. Methods: Lewis rats (normal versus 24–48-hour post I/R MI, each n = 6) were imaged using cine FLASH and high-frame rate IntraGate cine (Bruker BioSpec 9.4T MRI, Ettlingen) conforming to AHA's planes recommendations. MI was confirmed using multi-slice IR LGE. Segment (Medviso AB, Lund) and 2D CPA MR (Tomtec) were used respectively for volumes measurements and strain analysis. Results: Normal rat versus MI rats: EF (mean 64.0%, SD 2.3% versus mean 59.5%, SD 5.1%), p = 0.089; endo peak GLS (global longitudinal strain) (mean -21.9.0%, SD 1.1% vs. - 14.1%, SD 5.5%), p = 0.017; endo peak GCS (global circumferential strain) (mean -34.0%, SD 1.4% versus mean -26.1%, SD 6.4%), p = 0.028; myo peak GCS (mean - 23.5%, SD 1.7% vs - 17.0%, SD 5.4%), p = 0.032; GRS (global radial strain) (mean 45.8%, SD 5.3% versus mean 33.7%, SD 9.8%), p = 0.029. Conclusion: Strain imaging indices show statistically significant changes after MI where EF does not. A small change in EF was observed despite large infarct sizes. Increase in heart rate and alterations of remote regional strain and tissue velocities (data not shown) are suggested to be compensatory mechanisms which preserve EF.

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159. Investigation of the potential cardioprotective roles of a mitochondria-targeted persulfidation agent, mitosulf, in ischemia reperfusion injury T. Nishimura1,2,3, K.-L. Shih1, I. Ivanov1, O. Sauchanka1, S. Arndt2, M. Forkink1, A. Norman4, R.C. Hartley4, M.P. Murphy2, T. Krieg1 1

Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK 2 Medical Research Council Mitochondrial Biology Unit, Cambridge, UK 3 CNS DDU Innovative Biology Laboratories, Takeda Pharmaceutical Company Limited, Japan 4 Centre for the Chemical Research of Ageing, WestCHEM School of Chemistry, University of Glasgow, Glasgow, UK Background: It has been reported that hydrogen sulfide has protective effects against myocardial ischemia reperfusion injury (IRI). One of the major functions of hydrogen sulfide is considered to be protein thiol modification, known as persulfidation. However, the underlying IRI-protective effects of persulfidation and the target thiols remain unclear. It is also known that excessive generation of reactive oxygen species (ROS) during IRI contributes to tissue damage at reperfusion and is caused at least in part by acute reactivation of mitochondrial complex I. This reactivation has been shown to lead to excessive ROS production by enabling reverse electron transport (RET) driven by the rapid oxidation of succinate accumulated during ischemia. Indeed, the inhibition of succinate accumulation or complex I reactivation by thiol modification of the ND3 subunit has been shown to be cardioprotective. Therefore, we hypothesized that the protein persulfidation and its inhibitory effect on mitochondrial complex I contribute to the cardioprotection by hydrogen sulfide. Methods and results: In order to selectively target mitochondrial thiol persulfidation and unravel its protective effects on IRI, we developed a novel mitochondria-targeted persulfidation agent MitoSulf. This compound was successfully uptaken into mitochondria and reacted with mitochondrial glutathione. In addition, MitoSulf inhibited complex I-derived respiration and RET-induced ROS production in isolated mitochondria. Finally, protective effects on cell death after simulated IRI of rat cardiomyoblasts and on infarct size in an in vivo model of myocardial IRI were observed. Conclusion: These data suggest that persulfidation of mitochondrial protein such as the ND3 subunit is cardioprotective and, hence, could be a promising drug target. 160. Withdrawn 161. Insulin supplementation attenuates cancer-induced cardiac atrophy and lowers tumor volume S. Pietzsch1, B. Stapel1, J.T. Thackeray2, A. Haghikia1, M. Ricke-Hoch1, J. Heineke1, G. Scharf1, S. Erschow1, F.M. Bengel2, D. Hilfiker-Kleiner1 1 Department of Cardiology and Angiology, Hannover Medical School, Hannover, Germany 2 Department of Nuclear Medicine, Hannover Medical School, Hannover, Germany

Background: Advanced cancer induces fundamental changes in metabolism, promotes cardiac atrophy and heart failure.

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Results: We discovered systemic insulin deficiency in cachectic tumor patients. Similarly, mice with advanced B16F10-melanoma or C26colon cancer display decreased systemic insulin associated with marked cardiac atrophy, metabolic impairment and function. B16F10 and C26 tumors decrease systemic insulin via high glucose consumption, lowering pancreatic insulin production and producing insulin-degrading enzyme. As tumor cells consume glucose in an insulin-independent manner they shift glucose away from cardiomyocytes. Since cardiomyocytes in both tumor models remained insulin responsive, low-dose insulin supplementation by subcutaneous implantation of insulin-releasing pellets improved cardiac glucose-uptake, atrophy and function with no adverse side effects. In addition, by redirecting glucose to the heart and also other organs the systemic insulin treatment lowered glucose usage by the tumor and thereby decreased tumor growth and volume. Insulin corrected the cancer-induced reduction in cardiac Akt activation and the subsequent over-activation of the proteasome and autophagy. Conclusion: Thus, cancer-induced systemic insulin depletion contributes to cardiac wasting and failure and may promote tumor growth. Low-dose insulin supplementation attenuates these processes and may be supportive in cardio-oncologic treatment concepts. 162.

Conclusion: TBX5 enhances the transcription of cardioprotective genes like Fhl2, Fgf16 and Gpr22. Upon cardiac stress TBX5KO mice show exacerbated cardiac remodelling and reduced function. Since TBX5 is reduced in DCM and ICM patients, the re-expression of TBX5 is an attractive therapeutic target not only for patients with congenital heart disease but also for patients upon cardiac remodelling. 163. Stimulation of endogenous H2S synthesis inhibits oxidative and nitrosative stress, restores cNOS activity and endothelium-dependent vasorelaxation in old rats. V. Sagach, L. Mys, N. Strutynska Bogomolez Institute of Physiology, Kiev, Ukraine Aim: In view of the fact that aging increases the risk of cardiovascular diseases, the purpose of the work was to determine the effect of administration of pyridoxal-5-phosphate (P-5-P), hydrogen sulfide (H2S)synthesizing enzyme cofactor, on H2S biosynthesis, cNOS activity, NO synthesis in the heart tissues of old (22 months old) and adult (6 months old) animals, as well as on the endothelium-dependent vascular relaxation.

TBX5 protects the heart upon cardiac remodelling F. Rathjens1,2, A. Renger1,2, L.M. Iyer1,2, C. Noack1,2, F. Syeda3, M. Dewenter4, A. El-Armouche5, L. Fabritz3,6, W.-H. Zimmermann1,2, L. Zelarayán1,2, M.P. Zafeiriou1,2 1 Institut für Pharmakologie und Toxikologie, Universitätsmedizin Göttingen, Göttingen, Germany 2 DZHK (German Centre for Cardiovascular Research), Partner site Göttingen, Göttingen, Germany 3 Institute of Biomedical Research, Institute of Cardiovascular Sciences, Birmingham, UK 4 Department of Molecular Cardiology and Epigenetics, University of Heidelberg, Heidelberg, Germany 5 Institut für Pharmakologie und Toxikologie, Medizinische Fakultät Carl Gustav Carus, Dresden, Germany 6 College of Medical and Dental Sciences, Institute of Cardiovascular Sciences, Birmingham, UK

Methods and results: Administration (per os) of P-5-P (0.7 mg/kg) for 14 days resulted in restitution of reduced by half H2S content in the heart tissues of old rats to the level of adult animals (control). It promoted the decrease in the rate of superoxide (•O− 2 ) generation 3.1-fold, usually increased in old rats. The activity of cNOS, reduced in old rats twice, increased 3.4-fold after the activation of H2S synthesis. At that, P-5-P decreased iNOS activity twice, which was increased in old rats 8-fold. Endothelium-dependent relaxation of the blood vessels was reduced in old rats from 67 ± 4.8% (control) to 17 ± 2.3%, was restored up to 51 ± 2.8% following P-5-P administration. Conclusion: Thus, P-5-P-induced restoration of H2S synthesis in old animals leads to the increase in cNOS activity, NO synthesis, and improved endothelium-dependent relaxation of vascular smooth muscles. 164. Myocardial necroptosis is limited by classic ischemic preconditioning

Background: Transcription factor T-box 5 (TBX5) is essential for cardiac development. Patients with a non-functional TBX5 allele present cardiac malformations and conduction disorders (Holt-Oram-Syndrome). Preliminary data: We found reduced TBX5 expression in human dilated and ischemic cardiomyopathy samples, suggesting TBX5 loss may contribute to cardiac deterioration upon remodelling. Results: By transcriptomic and epigenetic analysis of a cardiomyocytespecific inducible TBX5 Knock-Out (KO), we identified 97 TBX5-bound genes downregulated in KO ventricles. Among others, gene clusters related to heart homeostasis and cardioprotection were overrepresented. Validation of three cardioprotective transcripts, showed a reduction of Fhl2 (3-fold, p b 0.05), Gpr22 (5-fold, pb 0.05) and Fgf16 (4-fold, p b 0.05, all n N 5). The loss of these factors is detrimental only upon cardiac remodelling since unstressed KO hearts have normal cardiac function. Similarly, we found that TBX5 loss does not affect cardiac function. To assess whether TBX5 KO mice are more sensitive to cardiac stress, they were subjected to stimulation with Angiotensin-II (AngII, 1,44 mg/kg/day for 14 days). Indeed the KO mice show reduced ejection fraction (21%, p b 0.01, n N 6) and increased heart-to-body-weight (27%, p b 0.01, n N 6) upon AngII treatment compared to CT. KO mice also presented increased levels of fibrosis (2.5-fold, p b 0.01, n N 11).

A. Szobi1, V. Farkašová-Ledvényová2, M. Muráriková2, S. Čarnická2, M. Molitorisová1, T. Ravingerová2, A. Adameová1 1

Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovak Republic

2

Background: Necroptosis, a form of regulated necrosis dependent on RIP1, RIP3 and MLKL, is extensively involved in various pathologies, including myocardial damage due to ischemia/reperfusion (I/R). While molecular mechanisms of necroptosis execution are being elucidated, little is known about its means of modulation. Classic ischemic preconditioning (IPC) is a well-known cardioprotective phenomenon known to limit necrosis after I/R. Therefore, we studied whether necroptosis can be inhibited by IPC to identify potential pathways capable of ameliorating cell loss. Methods: Langendorff-perfused rat hearts were subjected to I/R and the effects of IPC, necroptosis inhibitor Nec-1s or their combination were investigated. To monitor necroptosis activation, whole left ventricular tissue homogenates and density centrifugation fractionated membrane, nuclear and cytosolic fractions were analyzed by Western blotting for the


expression of necroptotic proteins. Additionally, infarct size was determined by TTC staining and postreperfusion heart function was evaluated. Results: Total content of all investigated necroptotic proteins was increased by I/R while Nec-1s reduced pSer229-RIP3 and MLKL levels. Contrarily, IPC alone nor in combination with Nec-1s had no effect on any of them. Subcellular fractionation revealed that IPC, Nec-1s and their combination all suppressed MLKL membrane translocation and oligomerization suggesting shared mechanism of action. In accordance, infarct size and postreperfusion mechanical function were improved to the same degree by all approaches showing no additive protective effects. Conclusion: This work demonstrates myocardial necroptosis is limited by IPC at the level of MLKL activation. Furthermore, necroptosis abrogation is likely to be an important component of IPC-induced cardioprotection. Acknowledgments: This study was supported by APVV-15-607, VEGA1/0271/16.

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Conclusions: Exercise training as well as ACE-I treatment improved ACE/ACE2 balance in mice at the end stage of heart failure and their influence was potentiated when they were combined. Additionally, ACE-I treatment improved inotropic reserve. 166. Oral geranylgeranylacetone treatment induces heat shock protein expression in human left and right atria D.M.S. van Marion1, L. Baks-te Bulte1, X. Hu1, F. Hoogstra-Berends2, T. Kukuchini3, L. Bockeria3, E.H. Lanters4, N.M.S. de Groot4, B.J.J.M. Brundel1 1 Deparment of Physiology, Vrije Universiteit MC, Amsterdam, The Netherlands 2 Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, Groningen, The Netherlands 3 Department of Cardiology, Bakoulev Scientific Center for Cardiovascular Surgery, Moscow, Russia 4 Department of Cardiology, Erasmus MC, Rotterdam, The Netherlands

165. Effects of spontaneous physical activity when applied alone or when combined with ACE inhibitor on cardiac function and angiotensin profile in mouse model of heart failure U. Tyrankiewicz1,2, M. Olkowicz3,4, M. Jablonska2, T. Skórka2, A. Osiak2, K. Jasinski2, A. Bar1,2, M. Wojewoda1, R.T. Smolenski3, J. Zoladz5, S. Chlopicki1,6 1

Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow, Poland Department of Magnetic Resonance Imaging, Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland 3 Medical University of Gdansk, Department of Biochemistry, Gdansk, Poland 4 Department of Biotechnology, Poznan University of Life Sciences, Poznan, Poland 5 The Department of Muscle Physiology, Faculty of Rehabilitation, Krakow, Poland 6 Jagiellonian University Medical College, Department of Experimental Pharmacology, Krakow, Poland 2

Background: The aim of this work was to analyze the effects of spontaneous physical activity combined or not with ACE-I treatment on cardiac function in murine model of dilated cardiomyopathy (Tgαq*44 mice). Methods: Tgαq*44 mice at the advanced stage of heart failure were running spontaneously in spinning wheel or were treated with ACE-I (perindopril, 10 mg/kg) or both. After two months cardiac performance was assessed by in vivo MRI and blood samples were taken for angiotensin profile characterization. Following parameters at rest and under dobutamine stimulation (from one midventricular LV slice) were assessed: ejection fraction, end systolic/diastolic volume, stroke volume, left atrial dimensions and heart rate. Results: Time spending in spinning wheel as well as distance that mice run not differ between groups. After two months of therapy basal cardiac performance was not different between groups, however inotropic reserve was preserved only in mice treated with ACE-I. All treated mice expect the controls presented increased concentration of plasma alamandine, Ang-(1–7), Ang-(1–9) and Ang-(1–10), and decreased concentration of Ang II and Ang III with the most powerful effect in group with combined therapy. Mice treated ACE-I and combined therapy had additionally increased levels of Ang-(1-12), Ang-(1–10), Ang-(1–7), Ang-(1–9), and (together with exercised group) decreased concentration of Ang-II, Ang III and Ang A in aorta.

Background: Heat shock proteins (HSPs) are important chaperones in the maintenance of a healthy protein quality control in the cell. Impairment of HSPs is related to ageing-related diseases such as Parkinson's, Huntington's, and Alzheimer's disease, as well as cardiac diseases, including atrial fibrillation (AF) and myocardial infarction. While shown to be protective in experimental models for AF, so far it is unknown whether the well-known HSP-inducing compound geranylgeranylacetone (GGA) can induce HSP expression in human heart tissue. Methods: HSP27, HSP70, HSP60, GRP78, HSF1 and phosphorylated HSF1 levels were measured by Western blot analysis in right and left atrial appendages (RAAs and LAAs, respectively) collected from patients undergoing coronary artery bypass grafting (CABG) surgery that were treated with placebo (n = 13) or GGA 400 mg/day (n = 13) 3 days before surgery at the Bakoulev Center in Moscow, Russia. Results: GGA significantly induced HSP27 (42% ± 18%, p b 0.01) and HSP70 (81% ± 40%, p b 0,05) expression levels in LAA and RAA compared to the placebo group. GGA did not induce HSF1, phosphorylated HSF1, HSP60 and GRP78, which is not HSF1 regulated. Interestingly, 3 patients from the placebo treated group developed post-surgery AF, whereas none of the GGA treated patients. Conclusions: Three days of GGA treatment induces HSP27 and HSP70 expression levels in RAA and LAA of patients undergoing CABG surgery. This finding indicates that GGA can be used to test its effect against development of post-surgery AF. Furthermore, future studies with GGA may elucidate protective effects against other cardiac diseases including AF. 167. Heat shock proteins 27 and 70 levels are reduced in atrial tissue and induced in serum from patients with in atrial fibrillation D.M.S. van Marion1, L. Baks-te Bulte1, Eva L. H. Lanters2, Natasja M. S. de Groot2, Bianca J. J. M. Brundel1 1

Department of Physiology, Vrije Universiteit MC, Amsterdam Department of Cardiology, Erasmus MC, Rotterdam

2

Background: There is strong evidence that derailment of proteostasis underlies progression of atrial fibrillation (AF), resulting in structural remodelling, impaired electrical coupling and contractile dysfunction. Previously, we showed that exhaustion of the most important chaperones in the maintenance of proteostasis, the heat shock proteins

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(HSPs), contribute to the remodeling processes. So far it is unclear whether HSP levels in blood and atrial tissue correlate and can predict the stage of AF. Methods: 100 patients (N18 y) scheduled for elective cardiothoracic surgery for structural heart disease without (n = 47) or with a history of paroxysmal (n = 14), persistent (n = 23) or long-standing persistent (n = 16) AF were included (EMC, Rotterdam, NL). Before surgery blood samples were taken and during surgery, tissue samples of the right atrial appendage were obtained from the patients for HSP27 and HSP70 measurements, by ELISAs and Western blot analyses. Results: Compared to tissue HSP27 levels in sinus rhythm patients, tissue HSP27 are significantly lower in patients with longstanding persistent AF (p = 0.020). For HSP70 this was not significant (p = 0.45). Tissue HSP27 and HSP70 levels significantly correlate (p = 3.5 × 10−9). Serum HSP27 positively correlates with the stage of AF (p = 0.023). Serum and tissue HSP27 (AF patients) and HSP70 (all patients) levels inversely correlate (p = 0.003 and p = 0.034, resp.). Conclusions: Tissue HSP27 is lower in longstanding persistent AF. Serum and tissue HSP27 and HSP70 levels inversely correlate, indicating that HSPs might be released from tissue into the blood. Serum HSP27 levels predict the stage of AF and may serve as a potential biomarker for AF progression. 168. Rofecoxib is cardioprotective and does not show hidden cardiotoxic properties G. Brenner, A. Makkos, A. Görbe, Z. Giricz, P. Ferdinandy Cardiometabolic Research Group, Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary Background: Unexpected ischemia-related cardiac adverse effects are the leading causes of discontinuation of clinical trials and withdrawal of drugs from the market. Drugs may exert unwanted effects by interfering with ischemic tolerance and inhibiting endogenous cardioprotective pathways which led to novel concept of “hidden cardiotoxicity”. Hidden cardiotoxicity cannot be revealed by currently available toxicity screening methods. Moreover, the mechanism of hidden cardiotoxocity is not known. Therefore, we aimed to explore the effect of drugs withdrawn from the market due to unexpected myocardial ischemic events on ischemia-reperfusion injury and cardioprotection by ischemic preconditioning. In the present study rofecoxib, the use of which was associated with increased risk of myocardial infarction was investigated in a rat model of myocardial infarction and ischemic preconditioning.

169. Distinct cardiac metabolic shifts in two mouse models of diseased heart A.L. Gundler1,2, K. Morhenn1,2, E. Oetjen1,2,3 1 Department of Clinical Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Institute of Pharmacy, University of Hamburg, Hamburg, Germany

Background: Cardiac hypertrophy is associated with changes in cardiac metabolism. The cAMP regulated transcriptional coactivators (CRTC) are known to regulate metabolism. In the present study, the contribution of CRTC1 to cardiac metabolism was investigated in Crtc1deficient (KO) mice and in Mybpc3-targeted knock-in (Mut) mice. Methods and results: Mice globally deficient in CRTC1 develop cardiac hypertrophy and dysfunction. Mybpc3-targeted knock-in mice are a well-accepted model for HCM, overexpressing CRTC1. mRNA content, analyzed by RT-qPCR, of PGC-1α, a transcriptional coactivator contributing to the expression of enzymes involved in β-oxidation, was reduced by 57.6 ± 5.7% (n = 4) in Mut mice but remained unchanged in KO mice compared to WT mice. The insulin-dependent glucose transporter GLUT4 mRNA level was reduced by 46 ± 3.5% (n = 4–6) in Mut mice, and did not differ in KO mice. The mRNA level of the γ2-subunit of the energy sensing enzyme AMPK was unchanged in Mut mice, but increased by 32.1 ± 7.1% (n = 5) in KO mice compared to WT mice. No increased phosphorylation of the AMPK target Acetyl-CoA-Carboxylase was observed by immunoblot analysis. The mitochondrial transcription factor A mRNA level was reduced by 26.3 ± 7.2% (n = 4–9) in Mut mice and in contrast increased by 29.2 ± 6.8% in KO mice compared to WT mice. Conclusion: Our data show distinct metabolic patterns in the hearts of Mybpc3-targeted knock-in mice and in Crtc1-deficient mice, hinting at an involvement of CRTC1 in the regulation of cardiac metabolism. Acknowledgments: We thank Jean René Cardinaux (Lausanne) for the kind gift of the KO mice and Lucie Carrier (Hamburg) for the kind gift of the Mut mice. 170. Components of height and blood pressure among Ellisras rural children: Ellisras Longitudinal Study N. Ramoshaba1, K. Monyeki1, L. Hay2

Methods: Male Wistar rats were treated with rofecoxib (5.12 mg/kg/day) and vehicle (control group) for 28 days. Both groups of animals were subjected to 30 min LAD occlusion followed by 120 min reperfusion or ischemic preconditioning elicited by 3 cycles of 5-min LAD occlusion and 5-min reperfusion before the 30 min LAD occlusion/reperfusion. After the 120 min reperfusion, area at risk and infarct size were measured by Evans blue and triphenyltetrazolium chloride staining. Results: Chronic rofecoxib treatment reduced infarct size per se (rofecoxib + Isch: 23.29 ± 3% vs. control: 42.97 ± 4.4%) and did not affect the cardioprotective effect of ischemic preconditioning (rofecoxib + IPC 13.59 ± 1.3% vs. control + IPC: 19.20 ± 5.2%). Conclusion: This is the first demonstration that rofecoxib exert direct cardioprotective effect. Moreover, it does not show hidden cardiotoxic properties.

1

University of Limpopo, Johannesburg, South Africa Sefako Makgatho Health Sciences, Johannesburg, South Africa

2

Background: There is no study done to investigate the relationship between the components of height and blood pressure (BP) in rural South African children to date. Therefore, the aim of the study was to investigate the relationship between height, sitting height (SH), leg length (LL) and SH to height ratio (SH/H) with BP in Ellisras rural children. Methods: All children underwent anthropometric and BP measurements using standard procedure. Linear regression was used to assess the relationship between height, SH, LL, SH/H and BP. Results: The regression showed a positive significance (P b 0.001 and 0.05) association between systolic BP (SBP) with height, SH (β ranged


from 0.127 to 0.134 and 95% CI ranged from 0.082 0.172 to 0.210 0.415). Diastolic BP (DBP) also showed a positive significant (P b 0.001 and 0.05) association with Height and SH (β ranged from 0.080 to 0.088 and 95% CI 0.042 0.118 to 0.086 0.259). After having been adjusted for age, gender, body mass index and waist circumference, DBP showed a positive significant (P b 0.05) association with height. Conclusion: There was a positive significant association between DBP and SBP together with the components of height amongst Ellisras rural children. We recommend that further studies be conducted on the relationship between components of height and CVD risk factors overtime in rural South African children. 171. Class effects of SGLT2 inhibitors (SGLT2i) in the heart: SGLT2i inhibit NHE, reduce [Na+]c and activate vasorelaxation in isolated mouse cardiomyocytes/hearts L. Uthman1, A. Baartscheer2, B. Bleijlevens3, C.A. Schumacher2, J.W.T. Fiolet2, A. Koeman1, M.W. Hollmann1, N.C. Weber1, R. Coronel2, C.J. Zuurbier1 1 Department of Anesthesiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 2 Clinical and Experimental Cardiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 3 Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Background: The SGLT2i empagliflozin (EMPA) reduced cardiovascular diseases in T2D patients, yet the cardioprotective mechanism of this kidney-targeted diabetes drug remains unknown. We recently reported that EMPA lowers cytosolic sodium ([Na+]c) and calcium, while increasing mitochondrial calcium, through inhibition of the myocardial sodium/hydrogen exchanger (NHE). Cardiac NHE has been shown to be upregulated in, and contribute to, heart failure and diabetes. We therefore investigated 1) how SGLT2i can interact with NHE, 2) whether two other SGLT2i inhibitors (dapagliflozin (DAPA) and canagliflozin (CANA)) block cardiac NHE and reduce [Na+]c as well, and 2) how SGLT2i affect mechanical, vascular and energetic parameters of mouse hearts. Methods and results: Molecular docking simulations on a homology model of the NHE-1 protein structure predicted that all three compounds bind with relatively high binding affinity to the extracellular Na+-binding site of NHE-1. Subsequently, NHE activity was indirectly measured from recovery of intracellular pH after loading of NH4 +pulse in isolated mice cardiomyocytes. All three SGLT2i inhibited NHE compared to vehicle (7.09 ± 0.04), with EMPA showing the strongest inhibition of NHE (1 μM EMPA 6.69 ± 0.03, p b 0.001; 1 μM DAPA 6.79 ± 0.03, p b 0.001; 3 μM CANA; 6.87 ± 0.06, p = 0.015). Moreover, measurements of [Na+]c with SBF-1 fluorescent probe showed that all three SGLT2i significantly lowered [Na+]c (EMPA: 10.0 ± 0.2, DAPA: 10.7 ± 0.2 and CANA: 11.0 ± 0.3 vs. vehicle: 12.7 ± 0.3 mM). Finally, Langendorff-perfused mouse hearts, perfused with 7 mM glucose, 1 mM lactate, 0.1 mM pyruvate, 0.5 mM glutamine and 50 mU/L insulin and 0.1% albumin, were subjected to either vehicle, 1 μM EMPA, 1 μM DAPA or 3 μM CANA for 30 minutes. EMPA and CANA activated vasorelaxation, determined by a 21% and 19% reduction in perfusion pressure respectively (p b 0.05), whereas no effects on cardiac mechanical performance or cardiac energetic status (PCr/ATP) were observed. Conclusions: Here, we show that SGLT2i exhibit a class effect by blocking NHE and reducing [Na+]c. Additionally, CANA and EMPA induce vasorelaxation of coronary circulation of the intact heart. These

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direct cardiac effects may contribute to reduced cardiovascular death and hospitalization for heart failure by SGLT2i. 172. Exercise intervention alters HDL subclass distribution and function in obese women N.J. Woudberg1, A.E. Mendham2, J.H. Goedecke2,3, S. Lecour1 1 Hatter Institute for Cardiovascular Research in Africa and South African Medical Research Council Inter-University Cape Heart Group, Department of Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa 2 Division of Exercise Science and Sports Medicine, Department of Human Biology, University of Cape Town, Cape Town, South Africa 3 Non-Communicable Disease Research Unit, South African Medical Research Council

Background: Obesity is a major risk factor for cardiovascular disease and alters HDL function and subclass distribution. Exercise benefits obese patients by reducing cardiovascular risk. However, the effect of exercise on HDL function and subclass is unknown. We therefore aimed to investigate how an exercise intervention may improve HDL function and alter HDL subclass in obese women. Methods: Thirty-two obese black South African women were recruited and randomly assigned to exercise (40–60 min of aerobic and resistance exercise 4 times/week, n = 20) or control (no exercise, n = 12) conditions for 12-weeks. HDL functionality was assessed by measuring reverse cholesterol efflux capacity, anti-inflammatory function, paraoxonase (PON) activity and platelet activating factor acetylhydrolase (PAF-AH) activity. PON-1 and PAF-AH expression were determined in serum using Western blotting. Levels of large, intermediate and small HDL subclasses were measured using Lipoprint®. Results: Independent of effects on body composition, exercise lowered PON activity (-0.08 ± 0.02 vs +0.01 ± 0.02 U/L, p b 0.05 for interaction) and PAF-AH serum expression compared to the control (-0.27±0.08 vs 0.13 ± 0.10 Arbitrary Units (AU), p b 0.05 for interaction). Exercise decreased the distribution of small HDL subclasses compared to the control (- 2.0 ± 0.7% vs + 1.7 ± 0.9%, p b 0.05 for interaction). Exercise did not alter HDL reverse cholesterol efflux capacity and antiinflammatory function. Conclusions: A 12-week aerobic intervention was associated with improvements in HDL functionality and decreases in small HDL subclasses. The beneficial effect of exercise on HDL function and composition is a possible mechanism by which exercise reduces cardiovascular risk in obese women. 173. Defective dicarbonyl detoxification in the senescent human myocardium underlies ryanodine receptor glycation and mitochondrial dysfunction D. Bou-Teen, M. Minguet, E. Miro-Casas, J. Castellano, D. Garcia-Dorado, M. Ruiz-Meana Vall d´Hebron University Hospital and Research Institute, Barcelona, Spain Background: Advanced age is a major determinant of myocardial infarct size; it increases mitochondrial damage that underlies higher vulnerability to cell death, but the mechanisms remain controversial and have not been tested in humans. Because advanced glycation

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end-products (AGEs) accumulate throughout life, we investigated whether intracellular glycation occurs in human aged myocardium and its potential impact on mitochondria. Methods and results: Human myocardium was obtained from right atrial appendages of surgical patients. AGEs content was increased in the myocardium of patients ≥ 75 years, whereas glyoxalase activityinvolved in detoxification of dicarbonyl intermediates- and its metabolite D-lactate were reduced. Myocardial aerobic capacity (mitochondrial O2 consumption/g) was impaired in the elderly due to reduced number of viable mitochondria and increased content of mitochondrial calcium. Aging mice (N 20 months) recapitulate glyoxalase deficit, myocardial AGE accumulation and mitochondrial calcium overload. Because mitochondrial calcium mainly depends on SR calcium release, we investigated the impact of AGEs on RyR. Simultaneous immunolabeling disclosed a higher colocalization between RyR and AGEs in cardiomyocytes from aging mice that was associated with increased RyR-dependent calcium leak. Long-term exposure to glyoxalase inhibitor in cultured H9c2 myotubes mimicked age-dependent mitochondrial damage, and was associated with reduced cell survival. Conclusions: Senescent human and murine myocardium develops glyoxalase deficit and mitochondrial calcium overload. Glycative damage of RyR, one of the targets of AGEs, and increased RyR-dependent calcium leak, expose mitochondria to high calcium environment and results in excessive calcium accumulation and damage in the mitochondria of aged hearts. 174. MicroRNA-1 targets MCU translation and modulates its mitochondrial content in physiologic and pathologic cardiac hypertrophy T. Zaglia1,2,3,⁎, A. Campo2,3,⁎, P. Ceriotti4,⁎, G. Borile2,3,⁎,, A. Armani3, P. Carullo4, R. Coppini5, V. Vida6, T.O. Stølen7, W. Ulrik7, J. Olivotto5, G. Stellin6, G. Faggian8, D. De Stefani2, R. Rizzuto2, M. Sandri2,3,9, F. Di Lisa2, T. Pozzan2,3,9, D. Catalucci4, M. Mongillo2,3,9 1 Department of Cardiac, Thoracic and Vascular Sciences, University of Padua Medical School, Padova, Italy 2 Department of Biomedical Sciences, University of Padua, Padova, Italy 3 Venetian Institute of Molecular Medicine, Padova, Italy 4 Humanitas Clinical and Research Center, Rozzano Milan, and National Research Council, Institute of Genetic and Biomedical Research - UOS Milan, Rozzano (Milan), Italy 5 Division of Pharmacology, Department "NeuroFarBa", University of Florence, Florence, Italy 6 K.G. Jebsen Center of Exercise in Medicine at Department of Circulation and Medical Imaging, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway 7 University Hospital of Verona, Division of Cardiac Surgery, Verona, Italy 8 CNR Institute of Neuroscience, Padova Section, Padova, Italy *contributed equally

Background: The myocardium adapts heart contractility and cell size during acute or chronic stresses causing increased workload and energy request. Mitochondria are cardiomyocyte powerhouse and Ca2 +, by entering into the mitochondria via the uniporter complex (MCUC) is a primary regulator of ATP production. MCU is one of the MCUC poreforming subunits, and its deletion impairs exercise capacity and βadrenergic heart responses. At present, the regulation of MCU is poorly understood. MyomiRs are involved in the structural and functional heart remodeling. We thus sought to determine whether myomiRs, may impact on mitochondrial function by modulating the expression of MCUC components.

Methods and results: In silico analysis identified miR-1 as specific interactor with the MCU-3′-UTR. These data were confirmed using a MCU-3′-UTR-Luc construct expressed in HEK293T cells and MiR-1 overexpression in HL-1 cardiomyocytes reduced MCU content with no effects on its transcription. Consistently, the maximal mitochondrial Ca 2 + uptake was reduced in miR-1 overexpressing cardiomyocytes. Given that chages in miR-1 levels occur during cardiac hypertrophy, we investigated the miR-1/MCU axis using biochemical and molecular analysis of murine and human samples from physiologic and pathologic hypertrophic hearts. The MCU density increased in exercise- and pressure-overload hypertrophy, mirroring the decline in miR-1. Conclusions: These data indicate that the miR-1/MCU axis shapes the molecular MCUC architecture, during adaptation to increased cardiac workload, with a mechanism conserved from rodents to humans. Interestingly, beta-blockers prevented the decrease in miR-1 expression, and the increase in MCU protein content in pressure-overloaded hearts, suggesting that β-AR signaling may be an upstream regulator of the mir-1/MCU axis. 175. Alternative NAD+ biosynthesis pathways activated by energetic stress in cardiac cells R. Deloux1,2, C. Tannous1,2, N. Diguet2, Z. Li2, M. Mericskay1 1 Université Paris-Saclay, Université Paris-Sud, INSERM UMR-S 1180 Signalisation et Physiopathologie Cardiovasculaire, Châtenay-Malabry, France 2 Sorbonne Universités, UPMC Université Paris 6, CNRS INSERM UMR-S 8256 Adaptation biologique et Vieillissement, Paris, France

Background: NAD+ is a key coenzyme in energy metabolism and a signaling substrate used by the sirtuins deacetylases and poly(ADPribose) polymerases, regulating energy metabolism and oxidative stress response. Myocardial NAD+ levels are reduced in various mouse models of heart failure in association with the repression of the NAMPT enzyme involved in NAD+ synthesis. This perturbation could affect bioenergetics systems and importantly also NAD+ signaling. Purpose: We aim to understand the metabolism of two major vitamins B3 precursors of NAD+ in cardiac cells to restore NAD+ levels: nicotinamide, the substrate of NAMPT, and nicotinamide riboside (NR), the substrate of NMRK kinases. Methods: We treated neonatal (NRC) and adult rat cardiomyocytes (ARC) with FK866, a specific inhibitor of NAMPT and evaluated the impact on NAD+ level and gene expression. We evaluated the ability of NR to sustain NAD+ synthesis when NAMPT is repressed and its impact on NRC metabolism using the Seahorse cell analyser. Results: We show in NRC and ARC that i/FK866 treatment depletes NAD + to low levels in cultured cardiomyocytes and triggers a robust induction of the Nmrk2 kinase; ii/NR supplementation increases NAD + level at baseline and is able to maintain the NAD+ in presence of FK866. We show in NRC that, i/Nmrk2 gene is induced by energy stress through the AMP-activated kinase, ii/NR supplementation increases oxygen consuption linked to ATP production. Conclusion: NR supplementation represents an efficient alternative to NAM Vitamin B3 to sustain NAD+ levels in cardiac cells when NAMPT enzyme is repressed.


176.

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1

IHU-LIRYC, Université de Bordeaux_INSERM_U1045, Pessac, France Human Sciences, Université de Bordeaux, Pessac, France 3 Cardiothoracique, CHU-Bordeaux, Pessac, France 2

Early metabolic and mitochondrial remodeling in a pig model of atrial fibrillation P. Pasdois1,⁎, H. Nasrallah3,⁎, M. Kuiper3, B. Beauvoit4, J. Luiken3, V. Loyer1, P. Dos Santos1,2, U. Lendeckel5, L. Schild6, U. Schotten3, P. Jaïs1,2, S. Verheule3 1

IHU-LIRYC, Université de Bordeaux_INSERM_U1045, Pessac, France 2 Cardiothoracique, CHU-Bordeaux, Pessac, France 3 Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, the Netherlands 4 INRA, UMR 1332 Biologie du Fruit et Pathologie, France Université de Bordeaux, France 5 Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald, Germany 6 Department of Pathological Biochemistry, Institute of Clinical Chemistry and Pathological Biochemistry, Otto-von-Guericke-University, Magdeburg, Germany ⁎Contributed equally Background: The high activation rates during atrial fibrillation (AF) increase atrial oxygen demand. We have shown that acute AF causes supply/demand ischemia. Objective: Characterize early AF-induced remodeling of atrial mitochondrial function after one week of induction. Methods: AF was mimicked in Landrace pigs by one week of rapid atrial pacing (RAP). Subsequently, pigs were anesthetized and the chest was opened to freeze and collect fresh tissue to perform structural, biochemical and functional studies. Results: After one week of RAP, the atrial energy status was altered as evidenced by a decrease in phosphocreatine to inorganic phosphate ratio when compared to control. This was associated with a reduction in mitochondrial size, a decrease of the total cardiolipin (CL) content, and a trend towards an increased oxidized CL to total CL ratio. Mitochondrial hydrogen peroxide production was decreased secondary to a twofold increase in its total peroxidase activity. RAP decreased mitochondrial basal oxygen consumption in presence of fatty acid, while their ADPstimulated respiration was increased in presence of glutamate + malate (GM). During fatty acid oxidation, RAP induced 1) depolarization of mitochondrial membrane potential under phosphorylating conditions and 2) increase the sensitivity of the mitochondrial permeability transition pore to calcium in presence of cyclosporin A. Moreover, RAP mitochondria showed an increase in their rate of calcium uptake in presence of GM. RAP atria showed glucose6-phosphate accumulation and increased activity of bound mitochondrial hexokinase. Conclusion: One week of AF causes early remodeling of atrial mitochondrial function and structure. Although mitochondrial function is largely preserved, the energy production capacity is insufficient to meet demand, possibly contributing to structural remodeling and AF progression. 177. Evidence for a contribution of mitochondrial permeability transition pore opening to ventricular arrhythmia maintenance P. Pasdois1, R. Walton1, L. Arsac2, V. Deschodt-Arsac2, O. Bernus1, P. Dos Santos1,3

Background: Diastolic calcium “leak” from the sarcoplasmic reticulum (SR) caused by RyR2 dysfunction can promote delayed after depolarization and arrhythmia. Objective: To test whether the SR diastolic calcium “leak” can influence ventricular arrhythmia susceptibility and/or maintenance by a mechanism involving the mitochondrial permeability transition pore (mPTP). Methods: Long or short burst pacing sequences were used to respectively induce sustained or non-sustained ventricular arrhythmia in the perfused rat heart. Surface electrocardiogram (ECG), left ventricular pressure, oxygen consumption, mitochondrial membrane potential and intracellular calcium were monitored. Surface ECG signals were used to assess arrhythmia duration and complexity. MPTP activity was assessed by the calcein entrapment method. Hearts were perfused with tacrolimus (FK506) alone, or in combination with cyclosporin A (CsA) to promote diastolic SR calcium “leak” or inhibit mPTP, respectively. Results: FK506 perfusion promoted a CsA-sensitive mitochondrial depolarization. In presence of FK506 mPTP was activated following a short burst-pacing sequence. Susceptibility to arrhythmia was increased following mPTP activation. Long burst-pacing sequence in the presence of FK506 induced sustained (N300 s) ventricular arrhythmias, while co-perfusion with CsA lead to spontaneous defibrillation in less than 9 s. This defibrillating effect of CsA was associated to a decrease in mitochondrial membrane potential depolarization and a decrease in intracellular calcium overload induced by FK506. Importantly, the FK506induced increase in susceptibility to arrhythmia and maintenance were counteracted by inhibition of the ATP-dependent potassium channel (IKATP). Conclusions: SR calcium leak associated to an external stress leads to mPTP activation. Our data suggest that the consequent energetic depression opens IKATP thus favoring ventricular arrhythmia maintenance. 178. Mitochondrial energy transfer in left ventricular hypertrophy A.S. Power, T. Pham, D.S. Loiselle, D.J. Crossman, M.-L. Ward, A.J. Hickey School of Biological Sciences and the Department of Physiology, University of Auckland, Auckland, New Zealand Background: Systemic hypertension initially promotes compensatory cardiac hypertrophy, yet it progresses to heart failure, and energetic deficits appear to be central to this failure. However, the transfer of energy between the mitochondria and the myofibrils is not often considered as part of the energetic homeostasis. Aim: To measure mitochondrial ATP production and ADP transfer in hypertrophic hearts from old spontaneously hypertensive rats (SHRs) in comparison to normotensive Wistar controls. Methods: Hearts were excised from 20-month-old SHR and Wistar rats and left ventricle (LV) tissue dissected. Mitochondrial function was measured by high-resolution respirometry in LV homogenates while following ATP or reactive oxygen species (ROS) production fluorometrically. Transfer of ADP between myofibrils and mitochondria was measured in situ in permeabilised fibres. Distances between the myofibrils and mitochondria were then determined by confocal

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imaging of fixed LV tissue labelled with phalloidin (myofibrils) and Tom20 antibody (mitochondria). Results: SHR hearts showed a 35% depression of mitochondrial function in comparison to controls, yet produced at least double the amount of ROS. Impaired transfer of ADP in permeabilised fibres suppressed respiration and elevated ROS production more in the SHR fibres relative to controls. Confocal imaging identified a 34% increase in the mean distance from the centres of myofibrils to the nearest mitochondrion in the SHR hearts. Conclusions: We propose that impaired connectivity between mitochondria and myofibrils may contribute to elevated ROS production in SHR hearts. This could be the result of sub-cellular structural changes that occur with hypertrophy in this model of hypertension.

mitochondria and S(E)R and contribute to cellular Zn2 + muffling, at most, between these two subcellular organelles in mammalian heart under pathological conditions will provide new insight for prevention/ therapy of mammalian failing heart. Acknowledgments: This study was supported through TUBITAK for grant SBAG-113S466 and COST Action TD1304. 180. 3D culture drives maturation of energy metabolism in hiPSC-derived cardiomyocytes mimicking developmental hypertrophy B.M. Ulmer1,2, A. Stoehr3, I. Mannhardt1,2, S. Patel4, M. Gucek4, M. Schulze1,2, E. Murphy3, T. Eschenhagen1,2, A. Hansen1,2

179. Mitochondrial localization and function of Zn2+-transporters ZIP7 and ZnT7 in mammalian heart E. Tuncay1, V.C. Bitirim1, Y. Olgar, A. Durak1, G.A. Rutter2, B. Turan1 1 Department of Biophysics, Ankara University School of Medicine, Ankara, Turkey 2 Section of Cell Biology and Functional Genomics, Division of Diabetes Endocrinology and Metabolism, Department of Medicine, Imperial College London, London, UK

Background: Excess free Zn2+ can be detrimental to cells, particularly for cardiomyocytes, and cytosolic Zn2 + has an important role in excitation-contraction coupling in cardiomyocytes by shaping Ca2 + dynamics. Changes in cellular free Zn2+ concentration, including those in the sarco(endo)plasmic reticulum, S(E)R and mitochondrail matrix are primarily coordinated by Zn2 +-transporters, whose identity and role in the heart is not well established. Since we previously have shown that these two transporters are responsible for Zn2+ transport from S(E)R into cytosol and visa-versa in cardiomyocytes, and that any change in their activity could play an important role in the development of cardiac dysfunction, herein, we first monitored the subcellular mitochondrial-localization of ZIP7 and ZnT7 in rat cardiomyocytes and in isolated mitochondrial-preparations. Methods and results: Under pathological conditions such as hyperglycemia or heart failure, the protein expression level of ZIP7 measured in isolated mitochondria is decreased while ZnT7 is increased. Additionally, the mitochondrial free Zn2+ level is increased significantly under pathological conditions although its level is low comparison to those of cytosolic or S(E)R levels under physiological condition. Electron and confocal microscopy data also have shown that increased intracellular free Zn2 + could induce depolarization in mitochondrial membrane potential, increases in mitochondrial matrix/cristae area and matrix volume in cardiomyocytes. Conclusions: Taken together previous and present data, one can suggest that the Zn2 +-transporters ZIP7 and ZnT7 localize to both

1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Systems Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA 4 Proteomics Core Facility, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

Background: Energy metabolism is a key aspect of cardiomyocyte biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising tool for biomedical application but they are immature and have not undergone changes related to postnatal developmental hypertrophy. Cultivation of hiPSC-CM in 3D engineered heart tissue (EHT) format leads to morphological maturation. The focus of this study was to determine the metabolic state of hiPSC-CM in the EHT format. Methods and results: HiPSC-CM in the 3D format exhibited higher mitochondrial number (transition electron miscroscopy), mitochondrial mass (Mitotracker®), mitochondrial DNA (MT-ND1, MT-ND2), and abundance of proteins involved in mitochondrial oxidative metabolism (label-free proteome) than cells cultured for the same time and under identical medium conditions in 2D. HiPSC-CM used glucose, lactate and fatty acids as energy substrates irrespective of culture format. However, hiPSC-CM cultured in EHT format performed more oxidation of glucose, lactate and fatty acid and less anaerobic glycolysis than hiPSC-CM in 2D. Ingenuity pathway analysis and quantitative assessment of gene expression showed upregulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a master regulator of postnatal and physiological mitochondrial biogenesis and metabolism. Conclusion: HiPSC-CM in the EHT format replicate metabolic aspects of developmental hypertrophy.