Activation of Notch signaling in cardiomyocytes during ...

Scandinavian Cardiovascular Journal, 2010; 00: 1–9

ORIGINAL ARTICLE

Activation of Notch signaling in cardiomyocytes during post-infarction remodeling

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ERIK ØIE1,2,3,5, WIGGO J. SANDBERG2,6, MOHAMMAD SHAKIL AHMED3,5, ARNE YNDESTAD2,5, OLE DIDRIK LÆRUM7,8, HÅVARD ATTRAMADAL3,5, PÅL AUKRUST2,4 & HANS GEIR EIKEN6,8 1Department

of Cardiology, Oslo University Hospital, Rikshospitalet, N-0027 Oslo, Norway, 2Research Institute for Internal Medicine, Oslo University Hospital, Rikshospitalet, N-0027 Oslo, Norway, 3Institute for Surgical Research, Oslo University Hospital, Rikshospitalet, N-0027 Oslo, Norway, 4Section of Clinical Immunology and Infectious Diseases, Oslo University Hospital, Rikshospitalet, N-0027 Oslo, Norway, 5Center for Heart Failure Research, University of Oslo, N-0027 Oslo, Norway, 6Center for Medical Genetics and Molecular Medicine and Department of Clinical Medicine, Haukeland University Hospital, N-5021 Bergen, Norway 7Department of Pathology, Haukeland University Hospital, N-5021 Bergen, Norway and 8Section of Pathology, the Gade Institute, University of Bergen, N-5021 Bergen, Norway

Abstract Objective. Notch signaling is crucial for cell-to-cell interaction during cardiovascular development and may influence differentiation, proliferation, and apoptotic events. We investigated whether Notch signaling is activated during myocardial remodeling in heart failure (HF). Design. Myocardial gene expression and localization of Notch receptors (Notch1-4) and ligands (Jagged1-2, and Delta-like (Dll)-1 and 4) were investigated in rats with HF after induction of myocardial infarction and in humans with HF. Results. All Notch receptors and ligands investigated and Notch intracellular domain (NICD) were present in rat and human myocardial tissue and in cardiomyocytes with differences in their relative expression levels and altered expression levels in failing vs. non-failing myocardium. In isolated rat cardiomyocytes, Notch3 and Notch4 appeared to be the major Notch receptors, and Notch3 and Notch4 mRNA levels and NICD-3 and -4 in cardiomyocytes were upregulated in chronic HF (p 0.05), indicating increased Notch3 and Notch4 signaling. Conclusion. The Notch signaling system is present in the cardiomyocytes and activated in HF, indicating a role of Notch signaling during myocardial remodeling in HF. Key words: Cardiomyocytes, heart failure, immunohistochemistry, myocardial infarction, myocardial remodeling, Notch

Cardiac remodeling is an essential process during development of heart failure (HF) (1,2). A variety of humoral factors including vasoactive peptides, growth factors, and inflammatory mediators that act in an autocrine, paracrine, or endocrine manner have been demonstrated important in these processes (3–7). However, a more direct communication between neighboring myocardial cells, both of the same and of different cell types, involving cell-to-cell contact is plausible and may be of importance for myocardial remodeling. The Notch signaling system, consisting of four Notch homologs, Notch1-Notch4, along with at least 4 ligands [Jagged1, Jagged2, Delta-like-1 (Dll1) and

Dll4] has recently been recognized as crucial for cellto-cell interaction during development of organisms ranging from worms to man (8) and has been reported to be involved in cell fate specification, embryonic patterning, and cellular proliferation and differentiation (9,10). The transmembrane ligands activate Notch receptors on neighboring cell. Upon activation, the Notch receptor undergoes a complex cleavage resulting in the liberation and nuclear translocation of its carboxyl terminal domain denominated Notch intracellular domain (NICD) (8). Nuclear NICD promotes formation of a transcriptional activator complex that induces the transcription of Notch target genes (e.g., members of the Hes and Hey gene families).

Correspondence: Erik Øie, Research Institute for Internal Medicine, Oslo University Hospital, Rikshospitalet, N-0027 Oslo, Norway. Tel: 47 23070000. E-mail: [email protected] (Received 2 May 2010 ; accepted 21 July 2010 ) ISSN 1401-7431 print/ISSN 1651-2006 online © 2010 Informa Healthcare DOI: 10.3109/14017431.2010.511256


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Interestingly, Notch signaling appears to be crucial for embryonic development of the cardiovascular system, including cardiomyocyte differentiation, boundary formation in the atrioventricular canal, valve development, outflow tract remodeling, and ventricular trabeculation (11–13). The embryonic processes involved in development of the cardiovascular system have several similarities with the myocardial remodeling process during HF development. Recently reported data indicate a protective role for signaling through one of the Notch receptors, Notch1, in cardiac hypertrophy and failure (14,15). The aim of the present study was to investigate 1) whether genes encoding the different Notch receptors and ligands are expressed in adult rat and human myocardial tissue with special focus on the cardiomyocytes, 2) whether myocardial expression of Notch signaling components is altered in ischemic and nonischemic myocardium during development of experimental HF and in failing human myocardium, and 3) the localization of the different Notch signaling components in the myocardial tissue.

Material and methods Rat model of experimental HF Male Wistar rats (∼300 g) were subjected to left coronary artery ligation or sham operation during isoflurane anesthesia (1% isoflurane in a mixture of one-third O2 and two-thirds N2O) as previously described (16). Surviving rats were euthanized in the acute and subacute phase after MI (2 days and 1 week after MI, respectively) and in the more chronic phase of HF (4 and 8 weeks after MI). Sham-operated rats euthanized at the same time points served as controls. In rats with MI, only those with left ventricular end-diastolic pressure (LVEDP) 10 mmHg and transmural infarction of the left ventricular (LV) free wall comprising 40% of LV circumference were included in the HF groups (2 days after surgery: sham, n 4; HF, n 5; 1 week after surgery: sham, n 16; HF, n 17; 4–8 weeks after surgery: sham, n 16, HF; n 20). The “Principle of laboratory animal care” (NIH publication No. 86-23, revised 1985) was followed as well as national laws regarding animal care. Hemodynamic measurements In the rats, arterial blood pressure, LVEDP, and LV systolic pressure (LVSP) were measured under isoflurane anesthesia using a 2F micromanometertipped catheter (model SPR-407, Millar Instruments, TX, USA).

Tissue sampling in rats The hearts were sectioned separating the LV (LV free wall and interventricular septum) from the rest of the heart. The LV of HF rats was further divided into infarcted and non-infarcted areas. Except in rats euthanized two days after MI, cardiomyocytes were isolated from non-cardiomyocytes as described below. A 2 mm transverse section through the middle portion of the LV from one sham rat and two HF rats euthanized in the subacute phase and in the chronic phase after MI/ sham operation were fixed in Bouin’s solution [saturated aqueous picric acid (1.2% w/v) and glacial acetic acid in formaldehyde (40% w/v)] for immunohistochemical analysis.

Myocardial cell isolation Cardiomyocytes were isolated from the LV of a group of rats euthanized in the subacute phase (sham, n 11, HF; n 10) and in the chronic phase (sham, n 11, HF; n 13) after MI or sham operation by retrogradely perfusion ex vivo with collagenase (Collagenase type II, Worthington Biochem. Corp., Lakewood, NJ, USA). Non-cardiomyocytes and cardiomyocytes were then separated by differential centrifugation (17). Immunocytochemical analysis using monoclonal anti-rabbit sarcomeric actin antibody (alpha-Sr-1, 1:30, Dako, Glostrup, Denmark) demonstrated that 95% of the cells in the cardiomyocyte fraction were sarcomeric actin-positive cardiomyocytes. Human myocardial tissue samples Tissue aliquots from human failing myocardium were removed from hearts immediately after explantation from patients with end-stage HF (New York Heart Association class III or IV; LV ejection fraction 34%; 1/3 non-ischemic HF, 2/3 ischemic HF) undergoing cardiac transplantation (n 16). Control (nonfailing) human LV tissue was obtained from sex- and age-matched subjects whose hearts were rejected as cardiac donors for surgical reasons (n 3). Due to shortage of sufficient numbers of non-failing hearts rejected as donors, additional control tissue was obtained from autopsy of subjects without a history or pathological signs of heart disease (n 5). The mRNA levels of the different Notch components in these hearts were within the same range as in the tissue from hearts rejected as cardiac donors for surgical reasons, supporting the use of such tissue as control. The study was approved by the local ethical committee and the investigation complies with the Declaration of Helsinki.

Notch signaling in post-infarction remodeling


Real-time quantitative reverse-transcription polymerase chain reaction Relative quantification of different mRNAs was performed by real-time quantitative reverse-transcription polymerase chain reaction (PCR) using ABI Prism 7700 Sequence Detector (Applied Biosystems). 18S RNA (TaqMan rRNA control reagents, Applied Biosystems) was used as an internal standard for normalization of target mRNA in rat cardiomyocytes and in myocardial tissue from humans. A geometric mean (18) of 18S RNA and mRNA encoding P0 and GAPDH was used for normalization of mRNA levels in myocardial tissue from rats. The data acquired were analyzed with the Sequence Detector software (version 1.6.3, Applied Biosystems). Primer sequences can be provided on request. Western blot analysis Western blotting was performed with equal amounts of protein separated from each sample by SDSPAGE (12%) before being transferred to PVDF membranes. Filters were incubated with cleaved Notch (NICD)-1 antibody (Cell Signaling, Beverly, MA, USA). Proteins were detected by enhanced chemiluminescence with horseradish peroxidaseconjugated anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA). Immunohistochemistry Heart sections of rat and human myocardial tissue were prepared as previously described (16) and immunostained with purified polyclonal anti-Notch1, anti-Notch2, anti-Notch3, anti-Notch4 (all Santa Cruz Biotechnology, Santa Cruz, CA, USA), antiJagged1 (R&D Systems, Minneapolis, MN, USA), anti-jagged2 (R&D Systems), anti-Dll1 (Santa Cruz Biotechnology), anti-Dll4 (Santa Cruz Biotechnology), and anti-cleaved Notch1 (Cell Signaling) IgG. The immunoreactivities were amplified by the avidin-biotin-peroxidase system (Vectastain Elite kit, Vector Laboratories). Diaminobenzidine was used as the chromogen in a commercial metal enhanced system (Pierce Chemical Co., Rockford, IL, USA). The sections were counterstained with hematoxylin. Omission of the primary antibody served as a negative control. Statistical analysis All the data are presented as mean SEM. Statistical analysis was assessed by the Mann-Whitney nonparametric test. Value of p 0.05 was considered to be statistically significant.

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Results Myocardial mRNA expression of Notch signaling component in the acute, subacute, and chronic phase in post-MI HF in rats As shown in Figure 1, mRNAs encoding all Notch receptors (Notch1-4) and the Notch ligands (Jagged1, Jagged2, Dll1, and Dll4) were detected in rat myocardium. Analysis of mRNA expression levels of the different Notch receptors and ligands as well as the Notch target gene Hes1 in ischemic and nonischemic regions of the LV in the acute, subacute, and more chronic phase in post-MI HF revealed different patterns. First, Notch2, Notch3, Notch4, Jagged1, and Hes1 mRNA levels were substantially higher in the ischemic region of HF rats than in non-ischemic myocardium and in sham-operated rats. Second, while Notch2, Jagged 1, and Hes1 mRNA levels increased throughout the study period in the ischemic area, Notch3 and Notch4 mRNA expression was more transient with maximal expression seven and two days after MI, respectively. Third, while Notch1 and Dll4 mRNA levels were not altered compared to sham neither in the ischemic nor in the non-ischemic regions two and seven days after MI, mRNA levels of these components were increased in the non-ischemic regions 28 days post-MI in HF. Finally, in contrast to up-regulation of several components of the Notch system during post-MI HF, decreased mRNA levels of Dll1 and Jagged2 were found in the non-ischemic (Dll1) and ischemic (Jagged2) LV throughout the study period. Expression of Notch receptors and ligands in failing human myocardium As shown in Figure 2, mRNA expression of all Notch receptors and ligands investigated was also detected in human myocardium. Moreover, consistent to mRNA expression in the non-infarcted myocardium in the chronic phase of HF in rats, LV of patients with chronic HF showed increased mRNA levels of Notch2 and Dll4 as compared to non-failing LV. In addition, Notch1 mRNA levels were decreased in LV of patients with severe chronic HF compared to nonfailing control myocardium. Localization of Notch receptors and ligands and NICD in rat and human myocardial tissue Immunohistochemical analysis demonstrated fairly strong immunoreactivities against Notch1, Notch3, Notch4, Jagged2, Dll1, and Dll4 in cardiomyocytes in rat (Figure 3A) and human (data not shown) myocardium. However, only very weak expression of

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Figure 1. Left ventricular mRNA levels of different Notch receptor subtypes, Notch ligands, and the Notch target gene Hes1 of sham-operated rats (white bars, n 4 at all time points) and post-MI rats in the acute (2 days), subacute (1 week), and more chronic phase (4 weeks) of HF after MI in non-ischemic (hatched bars) and ischemic (black bars) myocardium (n 5 at all time points). The mRNA levels were analyzed by real-time quantitative PCR. Data are presented as ratios of the mRNA levels of the different components relative to the geometric mean (GM) of 18S rRNA, GAPDH mRNA, and P0 mRNA expression, mean SEM. ∗p 0.05 vs. sham-operated group.

Notch signaling in post-infarction remodeling Notch2, Jagged1, and NICD were found (data not shown). Expression of Notch receptors, Notch ligands, and NICD in rat cardiomyocytes Based on gene expression, Notch3 and Notch4 appear to be the major Notch receptors in cardio-

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myocytes (Figure 3B). Notch1, Notch2, and Jagged1 mRNA were increased 1.7, 4.1, and 3.7-fold, respectively, in cardiomyocytes from HF rats in the subacute phase after MI compared to sham-operated rats (Figure 4A), whereas both Notch3 and Notch4 mRNA as well as the levels of Jagged 1 and Dll4 mRNA were increased 1.5–1.6-fold in the cardiomyocytes isolated from HF rats in the chronic phase

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Figure 2. mRNA levels of different Notch receptor subtypes and Notch ligands in LV from patients with chronic HF (n 16) and controls (n 8, Ctr). The mRNA levels were analyzed by real-time quantitative PCR. Data are presented as ratios of the mRNA levels of the different components relative to the levels of 18S rRNA, mean SEM. ∗p 0.05 vs. controls.

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Figure 3. A. Representative photomicrographs of myocardial tissue sections from a rat with HF in the subacute phase after MI (7 days), demonstrating immunostaining of Notch1, Notch3, Notch4, Jagged2, Dll1, and Dll4. Scale bar, 100 μm. B. Relative expression of mRNAs encoding Notch receptor subtypes and Notch ligands in isolated cardiomyocytes from rats without HF. The mRNA levels were determined by real-time quantitative PCR. The mRNA levels are presented as ratios relative to the levels of 18S rRNA, mean SEM.

after MI (Figure 5A). To examine whether altered gene regulation was translated into similar alteration in Notch signaling, protein levels of the different NICD isoforms in the cardiomyocytes were investigated. The antibody used detects cytosolic domain of Notch1 only when cleaved between Gly 1743 and Val 1744, i.e. NICD. Due to sequence homology, the NICD antibody also recognizes NICD2, -3, and -4, and due to differences in molecular weight, the different NICD isoforms are easily distinguished on SDS-PAGE. In the subacute phase of post-MI HF, the protein expression of NICD1 and NICD4 in cardiomyocytes was very weak and was not differentially expressed between the groups, whereas NICD2 was not detectable by Western blotting (data not shown). NICD3, however, was readily detectable and 3.8fold higher levels were found in cardiomyocytes of HF rats compared to sham-operated rats (Figure 4B). In the chronic phase of post-MI HF, NICD1, NICD3, and NICD4 levels in cardiomyocytes were

increased by 3.1-, 5.7-, and 2.7-fold, respectively (Figure 5B). Discussion The present study is to our knowledge the first to demonstrate the expression of all four Notch receptors and the four definite Notch ligands in both myocardial tissue and in cardiomyocytes and also the first study to demonstrate components of the Notch signaling system in adult human failing myocardium. The present study also demonstrates differential expression of Notch receptors and ligands in failing vs. non-failing myocardium. In particular, increased levels of NICD isoforms in cardiomyocytes of failing myocardium during experimental HF indicate increased Notch signaling. Gude et al. recently reported nuclear accumulation of Notch1 in surviving cardiomyocytes restricted to the border zone of the infarct region 4 days after

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Figure 4. A. mRNA levels of different Notch receptor subtypes, Notch ligands, and the Notch target gene Hes1 in cardiomyocytes isolated from LV in the subacute phase (7 days) in post-MI HF rats (non-ischemic tissue; n 10, black bars) or sham-operated rats (n 11, white bars). The mRNA levels were analyzed by real-time quantitative PCR. Data are presented as ratios of the mRNA levels of the different components relative to the levels of 18S rRNA, mean SEM. B. Levels of Notch intracellular domain (NICD)-3 analyzed by Western blot analysis and histograms of the densitometric analysis, mean SEM. ∗p 0.05 vs. shamoperated group.

coronary occlusion, and that signaling through Notch1 limits the extent of the hypertrophic response (15). In addition, Croquelois and colleagues reported that myocardial Notch1 and Jagged1 mRNA levels were increased in a transgenic mice model of cardiac hypertrophy and failure (14). We investigated myocardial expression profiles of all Notch receptors and four Notch ligands in ischemic and non-ischemic tissue from the acute to the more chronic phase of post-MI HF. Different expression profiles were found giving further indications of diverse roles of the Notch components during myocardial remodeling. Three of the Notch receptors (Notch2, Notch3, and Notch4) but only one of the Notch ligands (Jagged1) as well as the Notch target gene Hes1 displayed increased expression whereas Jagged2 showed decreased expression in the infarcted region compared to sham myocardium. Since Notch signaling may influence cell differentiation and proliferation as well as apoptotic events, we speculate that these Notch receptors and ligands are important for tissue healing and neoangiogenesis in the granulation tissue after MI. In addition, the observed increase in mRNA

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Figure 5. A. mRNA levels of different Notch receptor subtypes, Notch ligands, and the Notch target gene Hes1 in cardiomyocytes isolated from LV in the chronic phase (56 days) in post-MI HF rats (non-ischemic tissue; n 13, black bars) or sham-operated rats (n 11, white bars). The mRNA levels were analyzed by real-time quantitative PCR. Data are presented as ratios of the mRNA levels of the different components relative to the levels of 18S rRNA, mean SEM. B. Levels of Notch intracellular domain (NICD)-1, NICD3, and NICD4 analyzed by Western blot analysis and histograms of the densitometric analysis, mean SEM. ∗p 0.05 vs. sham-operated group.

levels of Notch1, Notch2, and Dll4 and decrease in Dll1 mRNA levels in non-ischemic myocardium compared to sham, may indicate a role for these Notch signaling components during myocardial remodeling of non-ischemic tissue. To study the potential relevance of our findings in experimental HF in post-MI rats to clinical HF, we examined the expression of Notch signaling components in human myocardial tissue. All Notch receptors and ligands were detected in human myocardium, indicating Notch signaling also in human hearts. Consistent with failing rat myocardial tissue, mRNA levels of Notch2 and Dll4 were increased in myocardial tissue from patients with HF compared to control tissue. However, in contrast to the findings in experimental HF as reported by others (14,15), Notch1 mRNA levels were decreased in myocardial


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tissue from patients with HF. The difference in the regulation of Notch1 may reflect that the human failing myocardium was obtained from explanted hearts with end-stage HF with presumably less ongoing remodeling compared to the myocardial tissue in experimental HF models. The immunohistochemical analysis demonstrated fairly strong myocardial expression of Notch1, Notch3, Notch4, Jagged2, Dll1, and Dll4 but only very weak expression of Notch2 and Jagged1. Next, we examined whether mRNA expression of the Notch receptors and ligands could be detected in isolated adult rat cardiomyocytes. We found gene expression of all Notch receptors and ligands in cardiomyocytes. This observation indicates Notch-mediated cell-to-cell communication between adjacent cardiomyocytes. Moreover, altered levels of mRNA encoding Notch receptors and ligands in cardiomyocytes during development of HF suggest Notch-mediated cell-to-cell communication between cardiomyocytes during the remodeling process. To obtain stronger indications of Notch signaling in cardiomyocytes, we investigated the expression of NICD isoforms, the released and intracellular translocated forms of the Notch receptor after binding of a ligand. We found expression of NICD in cardiomyocytes by immunohistochemical analysis (although very weak) and by Western blotting, demonstrating Notch signaling in these cells. Our data demonstrate that Notch3 and Notch4 are the two most highly expressed Notch receptor mRNAs in cardiomyocytes, suggesting their significance in cardiomyocytes. Although there was only a trend toward increased Notch3 mRNA levels in cardiomyocytes one week after MI, a substantial increase of NICD3 levels was found. The marked increase of Jagged1 in subacute post-MI HF (7 days) may also contribute to increased levels of NICD3 through binding to Notch3. The increase of Notch3 and Notch4 mRNA levels accompanied by an increase in NICD3 and NICD4 protein levels in cardiomyocytes in the more chronic phase of HF after MI, further support the importance of Notch3 and Notch4 in cardiomyocytes during post-MI myocardial remodeling. In conclusion, our data demonstrate the presence of Notch receptors and ligands and NICD in postnatal myocardium and in isolated cardiomyocytes, indicating Notch signaling. Moreover, Notch signaling components showed altered expression in ischemic versus non-ischemic myocardial tissue as well as in myocardial tissue and cardiomyocytes from failing compared to non-failing hearts. Our data may suggest distinct roles of the different myocardial Notch receptors and ligands in HF and should stimulate further examination of the functional role of myocardial Notch signaling during development of HF and

myocardial remodeling with focus on mechanisms causing activation of Notch and the structural and functional consequences of altered Notch signaling.

Acknowledgements We thank Benedikte Rosenlund, MSc, and Birthe Mikkelsen, MSc, for excellent technical assistance. This study was supported by Joh H. Andresen’s medical fund, University of Oslo, Helse Sør, and Oslo University Hospital, Rikshospitalet. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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17. De Young MB, Giannattasio B, Scarpa A. Isolation of calcium-tolerant atrial and ventricular myocytes from adult rat heart. Methods Enzymol. 1989;173:662–76. 18. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034.