in Skeletal Muscle and Heart

Monday, February 13, 2017 subdomain. While the known Ca handling proteins were among the most abundant proteins, most of the highly abundant and highly enriched cardiac SR proteins were proteins that have received little research attention. In summary, this SERCA-based enrichment and sorting methodology may represent a complete, semi-quantitative functional analysis of the cardiac proteome, providing a useful tool for studying the intracellular structure and function of ventricular muscle tissue. 792-Plat Cellular, Biochemical and Molecular Changes in Muscles from Patients with X-Linked Myotubular Myopathy due to MTM1 Mutations Susan Treves1,2, Christoph Bachmann1, Heinz Jungbluth3, Francesco Muntoni4, Adnan Y. Manzur4, Francesco Zorzato2. 1 Departments of Research and Anaesthesia, Kantonsspital Basel, Basel, Switzerland, 2Life Sciences, University of Ferrara, Ferrara, Italy, 3 Departments of Pediatric Neurology, Evelina Children’s Hospital, St Thomas’ Hospital, London, United Kingdom, 4Dubowitz Neuromuscular Centre, Institute of Child Health, London, United Kingdom. Centronuclear myopathies are early-onset muscle diseases caused by mutations in several genes including MTM1, DNM2, BIN1, RYR1 and TTN. The most severe and often fatal X-linked form or myotubular myopathy (XLMTM) is caused by mutations in the gene encoding the ubiquitous lipid phosphatase myotubularin 1, an enzyme specifically dephosphorylating phosphatidylinositol-3-phosphate and phosphatidylinositol- 3,5-bisphosphate. Because XLMTM patients have a predominantly muscle-specific phenotype a number of pathogenic mechanisms have been proposed, including a direct effect of the accumulated lipid on the skeletal muscle calcium channel ryanodine receptor 1, a negative effect on the structure of intracellular organelles and defective autophagy. Animal models knocked out for MTM1 show severe reduction of ryanodine receptor 1 mediated calcium release but, since knocking out genes in animal models does not necessarily replicate the human phenotype, we considered it important to study directly the effect of MTM1 mutations on patient muscle cells. The results of the present study show that at the level of myotubes MTM1 mutations do not dramatically affect excitation contraction coupling and calcium homeostasis though they do affect myotube size and nuclear content. On the other hand, mature muscles such as those obtained from patient muscle biopsies exhibit a significant decrease in expression of the ryanodine receptor 1, a decrease in muscle -specific microRNAs and a considerable up-regulation of histone deacetylase-4. We hypothesize that the latter events consequent to the primary genetic mutation, are the cause of the severe decrease in muscle strength that characterizes these patients. 793-Plat Assembly of Calcium Entry Units Improves Muscle Resistance to Fatigue Antonio Michelucci, Laura Pietrangelo, Claudia Pecorai, Simona Boncompagni, Feliciano Protasi. University G. d’Annunzio of Chieti, Chieti, Italy. We discovered that treadmill exercise in-vivo promotes formation in skeletal fibers of new junctions between sarcoplasmic reticulum (SR) and transversetubules (TTs) containing STIM1 and Orai1, the two main players in storeoperated Ca2þ entry (SOCE). We proposed that these new SR-TT junctions may function as Calcium Entry Units (CEUs) during repetitive muscle activity. Interestingly, in mice lacking Calsequestrin-1 (CASQ1-null) CEUs are constitutively present. In extensor digitorum longus (EDL) of mice, we quantified using electron-microscopy the number of CEUs/100mm2: 2.050.3 9.950.7; and 39.652.1, respectively in control (ctr)WT, exercised (ex)WT (subjected to 1 h of running at increasing speed: from 5 m/min to 25 m/min), and CASQ1-null mice. Higher number of CEUs/area in exWT and CASQ1-null mice correlates with higher expression levels of STIM1 and Orai1 detected by western-blot. We then used a repetitive stimulation protocol (30 x 1s-60Hz pulses every 5 seconds) to compare fatigue resistance in EDL muscles in presence or absence of extracellular Ca2þ, or in presence of SOCE inhibitors (BTP-2, 2-APB and SKF96365). Results of these experiments indicated: a) in 2.5 mM Ca2þ external solution, EDL muscles from exWT and CASQ1-null mice exhibited an increased capability to maintain contractile force compared to ctrWT mice (residual force after 15 tetani: 42.953.7%, 69.353.1% and 128.855.4% respectively for ctrWT, exWT, and CASQ1-null EDL muscles); b) in Ca2þ-free external solution, muscles from exWT and CASQ1-null mice showed a greater decay of contractile force than ctrWT (residual force after 15 tetani: 30.452.4%, 34.753.3% and 63.552.2% respectively for ctrWT, exWT, and CASQ1null EDL muscles); c) virtually identical results were obtained in presence of SOCE inhibitors. These data suggest that CEUs provide a preferential pathway for Ca2þ entry during repetitive muscle activity, likely important to limit muscle fatigue.

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794-Plat RyR2 Tetramer Distributions in Ventricular Myocytes from Phosphomutant Mice Parisa Asghari1, David R.L. Scriven1, Yanting Zhao2, Roberto Ramos Mondragon2, Hector Valdivia2, Xander Wehrens3, Edwin D.W. Moore1. 1 Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada, 2University of Michigan, Ann Arbor, MI, USA, 3 Baylor College of Medicine, Houston, TX, USA. Malfunction of the type 2 Ryanodine Receptor (RyR2) has been implicated in heart failure while mutations have been shown to lead to stress-induced cardiac arrhythmias and sudden cardiac death. It is therefore important to understand how the receptors function and how they are regulated. Recently we discovered that RyR2 in ventricular myocytes are mobile and move relative to each other in response to a cocktail of phosphorylation kinases and phosphatase inhibitors. Using correlation microscopy we recorded, in the same cells, Ca2þ sparks (line scan confocal imaging) and receptor positions (dual-tilt electron tomography) and discovered a correlation between the receptors’ open probability (Po) and their distribution: side-by-side tetramers had the lowest open probability (Po) which increased as the tetramers moved farther apart. To determine whether phosphorylation of RyR2 was required for these responses we have acquired transgenic mice expressing serine to alanine mutations of S2030, S2808, and S2814. Hearts are either fixed and examined with dual-tilt electron tomography or exposed to 300 nM isoproterenol for 2 minutes prior to fixation and examination. We will be reporting the results of these experiments. 795-Plat Measuring Electrical Conductivity of the Cardiac T-Tubular System Marina Scardigli1, Claudia Crocini1, Cecilia Ferrantini2, Tecla Gabbrielli1, Ludovico Silvestri1, Raffaele Coppini3, Chiara Tesi2, Elisabetta Cerbai3, Corrado Poggesi2, Francesco Pavone1, Leonardo Sacconi1. 1 European Laboratory for Non-linear Spectroscopy, University of Florence, Sesto Fiorentino (FI), Italy, 2Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy, 3Department ‘‘NeuroFarBa’’, University of Florence, Florence, Italy. Cardiomyocytes are characterized by a complex network of membrane invagination (the transverse axial tubular system, TATS) that propagates the action potential to the cell core guarantying a synchronous and uniform cell contraction. Pathological settings are commonly associated with structural remodelling of TATS that may cause failures of action potential propagation at single T-tubular level. Here, we investigate whether structural alterations occurring in TATS are linked to overall changes in its electrical conductivity which in turn hinder the regular propagation of action potentials across the network. Exploiting the formal analogy between diffusion and electrical conductivity we linked the conductivity with the diffusion properties of TATS. Fluorescence recovery after photo-bleaching (FRAP) microscopy is used to probe the diffusion properties of the tubular system in isolated cardiomyocytes: the fluorescent dextran inside TATS lumen is photo-bleached and the diffusion of unbleached dextran from extracellular space to TATS is monitored. We designed a mathematical model that correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the dextran. Then, taken advantage of the analogy between diffusion and conductivity, the apparent diffusion is used to assess TATS conductivity. This value is used to evaluate the efficiency of the passive spread of voltage changes along TATS. To probe how TATS conductivity is influenced by geometrical features, we used a model of acutely detubulated cells. We found that the overall reduction of TATS halves tubular conductivity. Then we tested our method in a pathological setting characterized by structural alteration, i.e. the heart failure rat model. We found that the tubular conductivity is not changed as compared to control. Actually, this result should not be surprising since heart failure is characterized by compound alterations that potentially compensate each other in their individual effect on global conductivity. 796-Plat DHBP Reversibly Inhibits Calcium Release from Sarcoplasmic Reticulum (SR) in Skeletal Muscle and Heart Yuriana Aguilar-Sanchez1, Marino DiFranco2, Yuanzhao L. Darcy3, Marbella Quin˜onez2, Ariel L. Escobar1, Julio A. Copello3. 1 School of Engineering, University of California Merced, Merced, CA, USA, 2 Department of Physiology, David Geffen School of Medicine - University of California Los Angeles, Los Angeles, CA, USA, 3Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL, USA. We characterized the effects of 1,10 -diheptyl-4,40 -bipyridinium dibromide (DHBP), a putative inhibitor of ryanodine receptors (RyRs), on Ca2þ signaling in striated muscle cells and on single RyR channels. DHBP (10-50 mM) significantly inhibited RyR-mediated Ca2þ release from sarcoplasmic reticulum (SR)

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in intact single fibers enzymatically isolated from murine flexor digitorum brevis (FDB) muscles. DHBP (10-50 mM) also inhibited SR Ca2þ release in ventricular epicardium of intact hearts. DHBP effects peaked after a few minutes of superfusion and dissipated within minutes upon washout. Unexpectedly, DHBP was a weak inhibitor of RyR-mediated Ca2þ release from skeletal SR microsomes. DHBP was also less effective on heart and skeletal muscle RyR’s reconstituted into planar bilayers, where the drug did not affect open probability and required high doses (~1 mM) to reduce open channel conductance by 50% (which suggest flicker block of the open channel). Our preliminary results suggest that DHBP is an attractive water-soluble, cell-permeable and reversible experimental probe to understand the role of RyR’s in cellular processes. The drug action seems relatively specific (DHBP was also without effects on SERCA-mediated SR Ca2þ loading or ATPase activity). Also, in skeletal muscle DHBP may be an alternative to inhibitors of myosin (BTS, blebbistatin) to arrest muscle contraction. Further research is still required to fully understand DHBP mechanism of action. The larger cellular effects of DHBP (an organic divalent cation) versus isolated RyRs may suggest drug accumulation in the cytosol at very high levels. However, DHBP synergized the effects of imperatoxin and peptide blockers on RyRs reconstituted into bilayers. These results may indicate that DHBP inhibits RyR’s in cells by acting on ancillary proteins, which may dissociate from the channels during the process of cell subfractionation and/or isolation of SR microsomes. 797-Plat Optical Stimulation of iPS Cardiomyocytes allows Brand New Insights into Contractility and Electropyhsiology Conjunctions Sonja Stoelzle-Feix1,2, Matthias Beckler1,2, Patrick Mumm1,2, Ulrich Thomas1,2, Leo Doerr1,2, Elena Dragicevic1,2, Krisztina Juhasz1,2, Corina T. Bot3, Michael George1,2, Andrea Br€uggemann1,2, Niels Fertig1,2, Jean-Francois Rolland2,4, R. Rizzetto2,4, L. Redaelli2,4, Philipp Sasse2,5. 1 Nanion Technologies Gmbh, Munich, Germany, 2OPTEL Consortium, funded by EuroTransBio Initiative, Germany, 3Nanion Technologies Inc, Livingston, NJ, USA, 4Axxam S.p.A., Milan, Italy, 5Life and Brain Center, Inst. f€ ur Physiologie I, Univ. Bonn, Bonn, Germany. Optical in-vitro platforms will be of particular relevance in the early stages drug discovery processes. We show recordings on impedance and extracellular field potential (EFP)-based devices with induced pluripotent stem cell (iPSC)derived cardiomyocytes as well as automated patch clamp data. Optogenetic stimulation and the recording of electrophysiological and contractile parameters of ChR2 (channelrhodopsin 2) transfected iPS Cor.4U cardiomyocytes were performed in a new assay approach, which allows a parallel investigation of impedance and EFP signals. This allowed a mechanistic understanding of cardiomyocyte cell physiology, which has been investigated over a physiological frequency range (60-180 ppm). Frequency dependent effects on cell physiology with reference compounds such as Ranolazine and Mexiletine will be presented. Furthermore, automated patch clamp investigations in the voltage-and currentclamp mode on blue-light activated ChR2 (channelrhodopsin 2) transfected cells will be presented and discussed in association with impedance/EFP results. 798-Plat Human Mesenchymal Stem Cell Paracrine Signaling Counteracts Heterocellular Coupling Effects on Cardiac Contractility and Arrhythomgenicity Joshua Mayourian1, Timothy J. Cashman1, Bryce V. Johnson2, David M. Sachs1, Deepak A. Kaji3, Eric A. Sobie3, Kevin D. Costa1. 1 Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 2Department of Medicine, University of Washington Seattle, Seattle, WA, USA, 3Icahn School of Medicine at Mount Sinai, New York, NY, USA. Myocardial delivery of human mesenchymal stem cells (hMSCs) is an emerging therapy for treating the failing heart. Demonstrated benefits include reduced fibrosis and enhanced contractile function, with predominant mechanisms thought to involve paracrine signaling (PS) and heterocellular coupling (HC) between hMSCs and host myocardium. In this study, we utilized mathematical modeling and three-dimensional human engineered cardiac tissues (hECTs) to test the hypothesis that hMSC-mediated PS enhances cardiac contractility and minimizes arrhythmogenicity, counterbalancing the unfavorable effects of direct HC. Based on published studies, our previous hMSC-cardiomyocyte HC model was modified to incorporate hMSC PS effects on single-cell cardiomyocyte ion channel activity and tissue-level fibrosis. Incorporating an established excitation-contraction model, our simulations of PS-only and combined HCþPS effects of hMSCs on human cardiomyocytes replicated our measurements of contractile function of hECTs under matched experimental hMSCmediated treatments. For example, model simulations and hECTs both demonstrated that the hMSC-mediated effects were most beneficial under PS-

only conditions, where developed force significantly increased by 3.5-fold compared to non-hMSC-supplemented controls during physiologic 1-Hz pacing. Similarly, maximum rates of contraction and relaxation were enhanced by PS-only conditions, and diminished by HC. Counteracting PS and HC effects of hMSCs were also revealed in a vulnerable window (VW) analysis of tissuelevel arrhythmogenicity in simulated cardiac tissue with moderate (21%) and high (40%) diffuse fibrosis; hMSC HCþPS conditions had variable effects on VW dependent on the percent of hMSCs delivered, while PS-only conditions consistently decreased the VW, thus minimizing arrhythmogenicity. Together, these findings support our hypothesis, and suggest identifying key hMSC paracrine signaling factors as an alternative hMSC-based cardiac therapy.

Platform: Voltage-gated K Channels and Mechanisms of Voltage Sensing and Gating II 799-Plat Non-Canonical Interactions between Voltage Sensors and Pore Domain in Shaker KD-Channel Joa˜o Carvalho-de-Souza, Francisco Bezanilla. Biochem and Mol Biology, The University of Chicago, Chicago, IL, USA. W434F mutation in the pore domain (PD) of Shaker Kþ-channels yields nonconductive channels, useful for gating currents studies. Comparisons of Q-Vs recorded with W434F with those recorded with absence of Kþ show virtually indistinguishable data. When a mutation in the S3-S4 linker, L361R, is introduced, the Q-V recorded with W434F, and the curve of Kþ conductance activation by voltage (G-V) are both strongly shifted to more negative voltages, and they unexpectedly cross each other. This effect is typical when the channel has more than one open state, which does not seem to be the case according to our single channels recordings. As expected, Q-V curve recorded in the conductive Shaker containing L361R mutation does not cross its G-V. This unprecedentedly showed that W434F-containing PD can potentially affect mutant VSDs although it is virtually silent for WT VSDs. To investigate this noncanonical coupling between VSD and PD, other than via S4-S5 linker, we produced dimers of Shaker that would show a mutant VSD (L361R) CLOSE, or FAR from a mutant PD (W434F). According to the Kv1.2 crystal structure, VSDs are near the PD from the neighbor subunit and we consider that would happen similarly in dimers of shaker. VSD mutations shift the slowinactivation curves to more negative voltages, with the effect being more intense in CLOSE compared to FAR channels. Furthermore, current peaks progressively increase at þ60 mV after 100-ms pre-pulses from 180 to 100 mV differently on CLOSE and FAR channels, indicating the VSD mutation interfere with the PD inactivation according to its relative position. Our data show that VSD and PD are in close communication beyond what is predicted by the S3-S4 connection, especially when mutations are present in both domains of the protein. Support: NIH-GM030376. 800-Plat Voltage Sensing in Hyperpolarization Activated Cyclic Nucleotide Gated (HCN) Channels Karen M. Callahan, Nazzareno D’Avanzo. Pharmacology et Physiologie, Universite´ de Montre´al, Montre´al, QC, Canada. The voltage-sensing domains (VSDs) of HCN channels have topologies similar to other voltage-dependent ion channels, including a series of positively charged residues in their S4 helix that are predicted to move with the same directionality as in other channels. However, HCN channels activate very slowly at hyperpolarized potentials. Intriguingly, despite identical S4 and S4S5 linkers between mammalian isoforms, HCN1-4 activate with different voltage-dependencies and gating kinetics. Here we begin to examine the molecular details of VSD movement in HCN channels through molecular dynamics simulations. Although the gating charge of sea urchin HCN (spHCN) channels has been estimated to be very small compared to that of Kv channels, our data indicates this is obtainable by a similar displacement of the S4 helix. We also compare gating charge estimates of human HCN isoforms, which have been experimentally unattainable to date. Lastly, we examine key interactions between isoforms that may underlie differences in the voltage-gating and kinetics. 801-Plat The Tarantula Toxin Guangxitoxin-1E Traps KD Channel Voltage Sensor in a Fully Resting Conformation Drew C. Tilley, Kenneth S. Eum, Jon T. Sack. Physiology and Membrane Biology, University of California Davis, Davis, CA, USA. Allosteric ligands modulate protein activity by altering the energy landscape of conformational change in ligand-protein complexes. Here, we investigate how