This Article Abstract Full Text (PDF) All Versions of this Article: 41/5/1156    most recent 01.HYP.0000064342.30653.24v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ogata, Y. Articles by Kobayashi, E. Search for Related Content PubMed PubMed Citation Articles by Ogata, Y. Articles by Kobayashi, E. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH Related Collections Apoptosis

(Hypertension. 2003;41:1156.)
© 2003 American Heart Association, Inc.

Scientific Contributions

Antiapoptotic Effect of Endothelin-1 in Rat Cardiomyocytes In Vitro

Yukiyo Ogata; Masafumi Takahashi; Shuichi Ueno; Koichi Takeuchi; Takashi Okada; Hiroyuki Mano; Shigeo Ookawara; Keiya Ozawa; Bradford C. Berk; Uichi Ikeda; Kazuyuki Shimada; Eiji Kobayashi

From the Division of Cardiovascular Medicine (Y.O., M.T., S.U., U.I., K.S.), Department of Medicine; the Divisions of Organ Replacement Research (M.T., E.K.), Functional Genomes (S.U., H.M.), and Genetic Therapeutics (T.O., K.O.), Center for Molecular Medicine; and the Department of Anatomy (K.T., S.O.), Jichi Medical School, Tochigi, Japan, and the Center for Cardiovascular Research (B.C.B.), University of Rochester, Rochester, NY.

Correspondence to Masafumi Takahashi, MD, PhD, Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, Minamikawachi-machi, Tochigi 329-0498, Japan. E-mail masafumi{at}jichi.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Apoptosis of cardiac myocytes is thought to be a feature of many pathological disorders, including congestive heart failure (CHF) and ischemic heart disease (IHD). Because recent investigations indicate that endothelin-1 (ET-1) plays an important role in CHF and IHD, we investigated the effect of ET-1 on cardiomyocyte apoptosis. The presence of apoptosis in rat cardiomyocytes (H9c2 and neonatal) was evaluated by morphological criteria, electrophoresis of DNA fragments, 4',6'-diamidine-2'-phenylindole staining, and TUNEL analysis. ET-1, but not angiotensin II, prevented apoptosis induced by serum deprivation via ET_A receptors in a dose-dependent manner (1 to 100 nmol/L). ET-1 also prevented cytochrome c release from mitochondria to the cytosol. The use of specific pharmacological inhibitors demonstrated that the antiapoptotic effect of ET-1 was mediated through a tyrosine kinase pathway (genistein and AG490) but not through protein kinase C (PKC; calphostin C), mitogen-activated protein kinases (PD98059 and SB203580), or PKA (KT5270) pathways. Adenovirus-mediated gene transfer of kinase-inactive (KI) c-Src reversed the antiapoptotic effect of ET-1. We further investigated whether Bcl-x_L, an antiapoptotic molecule, would be upregulated by using a luciferase-based reporter system. ET-1 upregulated Bcl-x_L, and this upregulation was inhibited by genistein or AG490 but not by calphostin C. The experiments with KI mutants for various tyrosine kinases revealed that c-Src and Pyk2 (but not JAK1, Jak2, Syk, and Tec) are involved in ET-1–induced upregulation of Bcl-x_L expression. These findings suggest that ET-1 prevents apoptosis in cardiac myocytes through the ET_A receptor and the subsequent c-Src/Bcl-x_L–dependent pathway.

Key Words: signal transduction • kinase • endothelin • apoptosis • myocardium

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Cardiac myocyte cell death by apoptosis accompanies several heart diseases.^1,2 It has been demonstrated in the myocardium from failing human hearts,³ in patients with dilated cardiomyopathy and arrhythmogenic right ventricular dysplasia,^4,5 and in association with myocardial infarction.⁶ Apoptosis causes loss of contractile cells, compensatory hypertrophy of myocardial cells, and reparative fibrosis.⁷ Because a reduction of contractile material is a prominent feature in heart failure, modification of apoptosis in the myocardium might provide a new therapeutic target for cardiovascular diseases.

A number of stimuli induce a hypertrophic response in cardiac myocytes, including -adrenergic agents, heparin-binding epidermal growth factor–like growth factor, insulin-like growth factor-1, leukemia inhibitory factor, neuregulin, cardiotropin-1, angiotensin II (AII), and interleukin-1ß.^8–12 Several of these factors have also been shown to be proapoptotic, whereas others have an antiapoptotic role in cardiac myocytes.^13,14

Endothelin-1 (ET-1), a family of 21–amino acid peptides, is 1 of the most potent hypertrophic stimuli for cardiac myocytes.^15,16 Furthermore, a number of clinical and experimental investigations have demonstrated that ET-1 might play an important role in the pathophysiology of cardiovascular diseases, including congestive heart failure (CHF) and ischemic heart disease.¹⁷ The plasma and myocardial tissue levels of ET-1 increase in patients with CHF.^18,19 In patients with acute myocardial infarction, plasma ET-1 levels are elevated^20,21 and are correlated with 1-year mortality.²¹ We therefore hypothesized that ET-1 regulates apoptosis in the myocardium.

The effect of ET-1 on apoptosis is controversial. ET-1 has been reported to be an antiapoptotic factor in endothelial cells.²² On the other hand, there are studies of smooth muscle cells in which ET-1 causes apoptosis.²³ In cardiac myocytes, ET-1 prevents oxidative stress– and ß-adrenergic agonist–induced apoptosis.^24,25 In the present study, we demonstrate that ET-1 prevents apoptosis induced by serum deprivation in cultured cardiac myocytes and investigate the signaling pathways that mediate the antiapoptotic effect of ET-1.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture and Materials
The embryonic rat heart–derived myogenic cell line H9c2 was obtained from American Type Culture Collection (Rockville, Md). Rat neonatal cardiomyocytes were prepared from ventricles of 1-day-old Sprague-Dawley rats as described previously.²⁶ The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin solution. The investigation was performed in accordance with the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act, 1986 (Her Majesty’s Stationary Office, London, UK).

The cDNA for a kinase domain–deleted Tec was subcloned into an expression vector, pSRa.²⁷ The kinase-deleted forms of porcine Syk (amino acids 1 to 504), human c-Src (amino acids 1 to 253), and mouse Jak1 (amino acids 1 to 879) were amplified by the polymerase chain reaction and inserted individually into the same vector. The expression plasmid for the kinase-deleted Jak2 was constructed as described previously.²⁷ Expression plasmids for a kinase-inactive (KI) form of Pyk2 were described previously.²⁸ Adenovirus containing either the ß-galactosidase cDNA (Ad.LacZ) or a cDNA encoding chicken KI–c-Src was prepared, amplified, and purified as described previously.²⁹ Human ET-1 was purchased from the Peptide Institute Inc. Antibodies against Bcl-x_L, Bcl-2, c-Src (clone GD11, ED10), and an activated form of c-Src ([pY418] phosphospecific antibody) were purchased from Santa Cruz Inc, Upstate Biotechnology Inc, and Biosource International, respectively. Antibodies against phosphosignal transducer and activator of transcription 3 (Stat3 [Tyr705]) and Stat3 were purchased from Cell Signaling Technology Inc. BQ123, BQ788, genistein, AG490, calphostin C, KT5270, PD98059, SB203580, and PP2 were purchased from Calbiochem. The remaining reagents including AII were obtained from Sigma unless otherwise indicated.

DNA Laddering
To evaluate DNA fragmentation, cellular fragmented DNA was extracted by the Triton X-100 lysis method, which efficiently eliminates intact chromatin. Floating and/or adherent cells were collected, and DNA fragments were extracted, fractionated by 1.8% agarose gel electrophoresis, and stained with ethidium bromide.³⁰

DAPI Staining
Cells were fixed in 3% paraformaldehyde in phosphate-buffered saline for 20 minutes and stained with a solution of 4',6-diamidino-2-phenylindole (DAPI; 10 mmol/L Tris-HCl, pH 7.4, 10 mmol/L EDTA, 100 mmol/L NaCl, 500 ng/mL DAPI) for 10 minutes at room temperature. The apoptotic cells were evaluated under a fluorescent microscope.³¹

TUNEL Analysis
Cells were fixed and then labeled using terminal deoxyribonucleotidyl transferase according to the manufacturer’s instructions (in situ apoptosis detection kit, Wako).

Detection of Cytochrome C Release
Cell lysates were prepared for the detection of cytochrome c in cytosolic and mitochondrial fractions, and detection of cytochrome c release was performed by Western blot analysis with an anti–cytochrome c antibody according to the manufacturer’s instructions (cytochrome c releasing apoptosis assay kit, Biovision).

Western Blot Analysis
Expression levels of Bcl-x_L, Bcl-2, c-Src, the activated form of c-Src, Stat3, and the phosphorylated form of Stat3 were analyzed by Western blot analysis. In brief, cells were lysed in a modified radioimmunoprecipitation assay buffer (10 mmol/L HEPES, pH 7.4, 5 mmol/L EDTA, 50 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 50 mmol/L NaCl, 100 µmol/L Na₃VO₄, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, and fresh 0.5 mmol/L PMSF and 10 µg/mL leupeptin). Cell lysates were prepared by scraping, sonication, and centrifugation for 20 minutes at 14 000 rpm in a microfuge at 4°C. Cell lysates were subjected to 5% to 20% SDS-polyacrylamide gradient gel electrophoresis. The separated proteins were electrophoretically transferred onto nitrocellulose membranes, and the resultant blots were incubated with the first antibody for 2 hours, followed by incubation for 1 to 2 hours with the secondary antibody (horseradish peroxidase conjugated). Immunoreactive bands were visualized by chemiluminescence (ECL, Amersham Pharmacia Biotech UK Ltd).

Transfection and Luciferase Assay
Transfections were performed with the Tfx-50 lipofectin reagent (Promega). In brief, cells were plated at 10⁵ cells/well in DMEM supplemented with 10% fetal calf serum (FCS) in 6-well plates and allowed to attach overnight. Transfections were performed 1 day after seeding by using a combination of 1.5 µg of expression plasmids, 1.5 µg Bcl-x_L-luc, 0.3 µg pRL-TK (Promega), and 9.9 µL Tfx-50. Cells were deprived of serum for 16 hours and then treated with 100 nmol/L ET-1 for 5 hours. Cell lysates were prepared, and the activity of Photinus pyralis luciferase was measured with the dual-luciferase reporter assay system (Promega) and normalized by the activity of Runilla reniformis luciferase.

Statistical Analysis
Data are expressed as the mean±SD. For comparisons between multiple groups, we determined the significance of differences between group means by ANOVA with the least significant difference for multiple comparisons. P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of ET-1 on Serum Deprivation– Induced Apoptosis
We first examined the effects of ET-1 on apoptosis in cultured cardiomyocytes. Electrophoresis of DNA fragments showed that 100 nmol/L ET-1 prevented formation of the characteristic apoptosis ladder induced by serum deprivation in both H9c2 cells and rat neonatal cardiomyocytes (Figure 1A). In contrast, AII, which is a vasoconstrictive peptide similar to ET-1, had no effect on apoptosis in H9c2 cells, although these cells express the angiotensin II type 1 (AT₁) receptor.³² The antiapoptotic effect of ET-1 was dose dependent over the range used (1 to 100 nmol/L; Figure 1B). We confirmed that ET-1 prevented serum deprivation–induced apoptosis by TUNEL analysis and DNA-binding dye (DAPI) staining (Figures 1C through 1E). Serum deprivation reduced cell viability, as measured by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (data not shown). Furthermore, ET-1 prevented serum deprivation–induced cytochrome c release from mitochondria to the cytosol (Figure 2).

View larger version (46K): [in this window] [in a new window]   Figure 1. Effect of ET-1 on serum (FCS) deprivation-induced apoptosis. A, H9c2 or rat neonatal cardiomyocytes (CM) were deprived of serum for 24 hours in the presence or absence of 100 nmol/L ET-1 or 100 nmol/L AII. B, H9c2 cells were deprived of serum for 24 hours in the presence or absence of indicated concentrations of ET-1. Genomic DNA was extracted by the Triton X-100 lysis method. DNA (10 µg/lane) was loaded onto 1.8% agarose gel containing ethidium bromide. After electrophoresis, DNA bands were visualized under ultraviolet light. M indicates DNA size markers. Results are representative of 3 independent experiments. C to E, H9c2 cells were deprived of serum for 24 hours in the presence or absence of 100 nmol/L ET-1. TUNEL analysis (C) and DAPI staining (D) were performed as described in Methods. Results are representative of 3 independent experiments. E, Bar graphs show mean±SD of 1200 to 1600 cells of 3 independent experiments (DAPI staining). *P<0.01 vs control.

View larger version (26K): [in this window] [in a new window]   Figure 2. Prevention of cytochrome c release by ET-1. H9c2 cells were deprived of serum for 24 hours in the presence or absence of 100 nmol/L ET-1 and prepared for detection of cytochrome c in mitochondrial (Mito.fr.) and cytosolic (Cyto.fr.) fractions, as described in Methods. Cytochrome c release was measured by Western blotting (A and B). Relative expression of cytochrome c was quantified by densitometry (n=3). Bar graphs show mean±SD of 3 independent experiments. *P<0.01 and **P<0.05.

The effects of ET-1 are initiated by their binding to G protein–coupled heptahelical receptors, ET_A and ET_B, expressed in a wide variety of tissues and cells.³³ To identify which ET receptor (ET_A or ET_B) is responsible for the antiapoptotic effect of ET-1 in H9c2 cells, we used selective ET_A and ET_B receptor antagonists. The antiapoptotic effect of ET-1 was almost completely inhibited by treatment with the selective ET_A receptor antagonist BQ123, but not by the ET_B receptor antagonist BQ788 (Figures 3A and 3B). These observations indicate that ET-1 participates in the survival of cardiac myocytes by preventing apoptosis by way of the ET_A receptor in myocytes.

View larger version (27K): [in this window] [in a new window]   Figure 3. Antiapoptotic effect of ET-1 is mediated via ETAreceptor. H9c2 cells were deprived of serum (FCS) for 24 hours with 100 nmol/L ET-1 in the presence or absence of 100 nmol/L BQ123, BQ788, or BQ123 plus BQ788. DNA fragmentation (A) and DAPI staining (B) were performed as described in Methods. Genomic DNA was extracted from cells as described in the legend to Figure 1. M indicates DNA size markers. Bar graphs show mean±SD of 1200 to 1600 cells of 3 independent experiments (DAPI staining). *P<0.01 vs control.

Effects of Various Signaling Inhibitors on ET-1–Prevented Apoptosis
Because the intracellular protein kinases mediate the prevention of apoptosis in cardiac myocytes,^1,2,34 we next examined whether protein kinases were involved in the antiapoptotic effect of ET-1 in H9c2 cells by using tyrosine kinase inhibitors (genistein and AG490), protein kinase C inhibitors (calphostin C), mitogen-activated protein (MAP) kinase inhibitors (PD98059 for extracellular-regulated kinase and SB20380 for p38-MAP kinase), and a cAMP-dependent kinase inhibitor (KT5270). Genistein and AG490 inhibited the effect of ET-1, whereas calphostin C, PD98059, SB20380, and KT5270 failed to show any effect (Figures 4A and 4B). In addition, treatment with the phosphatidyl inositol-3 kinase inhibitor wortmannin showed no effect on ET-1–prevented apoptosis (data not shown). These results suggest that ET-1 prevents serum deprivation–induced apoptosis in cardiac myocytes through a tyrosine kinase–dependent mechanism.

View larger version (30K): [in this window] [in a new window]   Figure 4. Effects of various inhibitors on ET1-prevented apoptosis. After H9c2 cells were pretreated with 0.1% dimethyl sulfoxide (cont), 10 µmol/L genistein (Geni), 100 µmol/L AG490 (AG), 1 µmol/L calphostin C (CalC), 1 µmol/L KT 5720 (KT), 10 µmol/L PD98059 (PD), or 10 µmol/L SB203580 (SB) for 1 hour, cells were deprived of serum (FCS) for 24 hours in the presence of ET-1. DNA fragmentation (A) and DAPI staining (B) were performed as described in Methods. Genomic DNA was extracted from cells as described in the legend to Figure 1. M indicates DNA size markers. Bar graphs show mean±SD of 1200 to 1600 cells of 3 independent experiments (DAPI staining). *P<0.01 vs control.

c-Src Involved in the Antiapoptotic Effect of ET-1
Recent evidence suggests that the c-Src family of protein tyrosine kinases is involved in apoptotic cell death in certain types of cells.^35–37 We therefore hypothesized that c-Src might participate in the antiapoptotic effect of ET-1. To determine whether c-Src activity was regulated by ET-1, cells were stimulated by 100 nmol/L ET-1 for varying amounts of time, and c-Src activity was analyzed by Western blotting by using an antibody that selectively recognizes the activated form of c-Src. The activity of c-Src clearly increased by 4.5-fold within 30 seconds in response to ET-1 stimulation and then declined (Figure 5, top). We confirmed that there were no significant changes in c-Src protein levels of the same amount of cell lysates (Figure 5, bottom).

View larger version (28K): [in this window] [in a new window]   Figure 5. ET-1 stimulates c-Src activation. After H9c2 cells were deprived of serum in the presence or absence of 100 nmol/L ET-1 for the indicated periods, cell lysates were prepared. Anti-phosphospecific–c-Src (top, active c-Src) and c-Src antibody (bottom, c-Src) were used to quantify these proteins by Western blotting of whole lysates that were obtained in the same experiments. Relative c-Src kinase activity was quantified by densitometry. Results are representative of 2 independent experiments.

To further investigate the role of c-Src in the antiapoptotic effect of ET-1, adenoviruses were used to overexpress either ß-galactosidase (Ad.LacZ) or a KI–c-Src. Transfection of H9c2 cells with a KI–c-Src, but not with Ad.LacZ, increased c-Src in a concentration-dependent manner (Figure 6A). Expression of KI–c-Src significantly inhibited the antiapoptotic effect of ET-1, whereas expression of ß-galactosidase with Ad.LacZ had no effect (Figures 6B and 6C). These results indicate that c-Src is required for the antiapoptotic effect of ET-1.

View larger version (27K): [in this window] [in a new window]   Figure 6. Overexpression of Ad.KI–c-Src inhibits antiapoptotic effect of ET-1. A, H9c2 cells were infected with either Ad.LacZ (10 and 100 m.o.i.) or Ad.KIc-Src (1, 10 and 100 m.o.i.) for 1 hour at 37°C and then incubated with DMEM supplemented with 10% FCS for 48 hours. Cell lysates were prepared and analyzed by Western blotting with anti–c-Src antibody. B, Cells were infected with 100 m.o.i. of either Ad.LacZ or Ad.KI–c-Src for 1 hour at 37°C, incubated with DMEM supplemented with 10% FCS for 48 hours, and then deprived of serum (FCS) in the presence of 100 nmol/L ET-1 for 24 hours. Fragmented DNA was extracted from cells as described in the legend to Figure 1. M indicates DNA size markers. Results are representative of 3 independent experiments. C, Bar graphs show mean±SD of 1200 to 1600 cells of 3 independent experiments (DAPI staining). *P<0.01 vs control.

Effects of KI Mutants for Various Tyrosine Kinases on Bcl-x_LExpression
Because c-Src has been shown to negatively regulate apoptosis via Bcl-x_L, an antiapoptotic molecule, in several cell types,^35–37 we investigated whether Bcl-x_L was involved by using a luciferase-based reporter system. Western blot analysis showed that ET-1 stimulated Bcl-x_L expression (Figures 7A and 7B). ET-1 upregulated Bcl-x_L gene expression, which was inhibited by treatment with genistein or AG490, but not by calphostin C (Figure 7C). The experiments with KI mutants for various tyrosine kinases showed that KI–c-Src completely inhibited ET-1–induced Bcl-x_L gene expression (Figure 7D). In addition, KI-Pyk2 partially inhibited its expression, whereas KI-JAK1, KI-Jak2, KI-Syk, or KI-Tec showed no effect. These findings suggest that c-Src and Pyk2 are involved in Bcl-x_L expression induced by ET-1.

View larger version (13K): [in this window] [in a new window]   Figure 7. Effects of KI mutants for various tyrosine kinases on Bcl-xL expression. A, H9c2 cells were deprived of serum for 24 hours in the presence or absence of ET-1. Cell lysates were prepared, and expression of Bcl-xL and Bcl-2 was analyzed by Western blotting, as described in Methods. B, Relative Bcl-xL expression was quantified by densitometry. Results are representative of 2 independent experiments. C, After pBcl-xL/luc (1.5 µg) and pRL-TK (0.3 µg) were introduced into cells, they were deprived of serum for 16 hours and pretreated with 0.1% dimethyl sulfoxide (cont), 10 µmol/L genistein (Geni), 100 µmol/L AG490 (AG), or 1 µmol/L calphostin C (CalC) for 1 hour; then cells were treated with 100 nmol/L ET-1 for 5 hours. Cell extracts were subjected to luciferase assay as described in Methods. The activity of Photinus pyralis luciferase was normalized by activity of Runilla reniformis luciferase. Bar graphs show mean±SD (n=4). *P<0.01 vs control. D, After pBcl-xL/luc (1.5 µg) and pRL-TK (0.3 µg) were introduced into cells together with 1.5 µg each of blank vector (Vector), expression plasmid for control vector (vector), or the KI form of each protein tyrosine kinases (Src, Jak1, Jak2, Tec, Pyk2, Syk), cells were deprived of serum (FCS) for 16 hours, and then treated with 100 nmol/L ET-1 for 5 hours. Cell extracts were analyzed as described above. Bar graphs show mean±SD (n=6). *P<0.01 vs control.

ET-1 Stimulates STAT3 Phosphorylation
Because it has been reported that STAT3 regulates Bcl-x_L expression in cardiac myocytes,³⁸ finally we examined whether ET-1 stimulates STAT3 phosphorylation. ET-1 clearly stimulated STAT3 phosphorylation in a time-dependent manner, and this STAT3 phosphorylation was inhibited by treatment with a specific c-Src inhibitor, PP2 (Figure 8).

View larger version (57K): [in this window] [in a new window]   Figure 8. ET-1 stimulates STAT3 phosphorylation. A, After H9c2 cells were deprived of serum (FCS) in the presence or absence of 100 nmol/L ET-1 for indicated periods, cell lysates were prepared. B, After cells were pretreated with 10 µmol/L PP2 for 1 hour, they were stimulated with ET-1, and then cell lysates were prepared. Anti-phospho-STAT3 (pSTAT3) and STAT3 antibody (STAT3) were used to quantify these proteins by Western blotting. Results are representative of 2 independent experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major findings of this study are that ET-1 prevents apoptosis induced by serum deprivation in a dose-dependent manner via an ET_A receptor in H9c2 cardiomyocytes and that the antiapoptotic effect of ET-1 is mediated through a c-Src/Bcl-x_L pathway. Evidence for this proposal includes the following: (1) ET-1, but not AII, prevented mitochondrial cytochrome c release and apoptosis induced by serum deprivation in a dose-dependent manner, and this antiapoptotic effect was inhibited by an ET_A receptor antagonist (BQ123) but not by an ET_B receptor antagonist (BQ788); (2) the inhibitory effects of ET-1 on apoptosis were inhibited by tyrosine kinase inhibitors and adenovirus-mediated overexpression of KI–c-Src; (3) ET-1 stimulated c-Src activation; and (4) ET-1 upregulated an antiapoptotic molecule, Bcl-x_L, and this upregulation was inhibited by tyrosine kinase inhibitors or cotransfection with KI–c-Src.

Recent evidence suggests that apoptosis of cardiac myocytes is a feature in cardiovascular diseases, including CHF and myocardial infarction.^1,2,39 The levels of plasma and myocardial ET-1 increase in patients with CHF and myocardial infarction,^18–21 suggesting the critical role of ET-1 in these cardiovascular disease states. Therefore, we investigated whether ET-1 affects myocardial apoptosis in this study. We showed here that ET-1 prevents serum deprivation–induced mitochondrial cytochrome c release and apoptosis, suggesting that the antiapoptotic effect of ET-1 is mediated through a mitochondrial apoptotic pathway. We further demonstrated that ET-1 prevents apoptosis in a dose-dependent manner via the ET_A receptor. The effects of ET were mediated through 2 distinct receptor subtypes of G protein–coupled receptors, termed ET_A and ET_B, expressed in a wide variety of cells and tissues.^40,41 In myocardium, ET_A receptors are mainly expressed, and small amounts of ET_B receptors are expressed.³³ Consistent with the expression levels in the myocardium, ET_A receptors act as a major pathway for several effects of ET-1, such as myocardial contraction and hypertrophy.¹⁷ Similar to these effects of ET-1, our findings indicate that the antiapoptotic effect of ET-1 in cardiac myocytes is also mediated via the ET_A receptor.

A number of proapoptotic and antiapoptotic signaling pathways in cardiac myocytes have been demonstrated.^1,2 To investigate the molecular mechanisms of the antiapoptotic effect of ET-1 in cardiac myocytes, we used an adenovirus-based vector system that allows for highly efficient DNA transfection in many cell types. The efficiency of expression examined with Ad.LacZ in H9c2 cells infected by adenovirus was found to be almost 100% (data not shown). Because tyrosine kinase inhibitors, such as genistein and AG490, reversed the antiapoptotic effect of ET-1, we examined whether protein tyrosine kinase c-Src is involved in the antiapoptotic effect of ET-1 by using the KI–c-Src–expressing adenovirus. We demonstrated that overexpression of KI–c-Src reversed the antiapoptotic effect of ET-1, suggesting that c-Src plays a critical role in the ET-1–mediated antiapoptotic pathway in cardiac myocytes.

Bcl-x_L plays a critical role in the antiapoptotic signaling pathway in a variety of cells, including cardiac myocytes.⁴² In addition, recent investigations have suggested that c-Src regulates Bcl-x_L in several cell types.^35–37 Therefore, we next focused on Bcl-x_L expression in the antiapoptotic pathway by ET-1. We demonstrated that ET-1 upregulated Bcl-x_L expression, and this upregulation was completely inhibited by both KI–c-Src and tyrosine kinase inhibitors, which inhibited the antiapoptotic effect of ET-1. These findings suggest that c-Src is an upstream molecule for Bcl-x_L expression in cardiac myocytes. c-Src itself has also induced the activation of several signaling molecules, including MAP kinase and STAT3,⁴³ and both can stimulate Bcl-x_L expression.^36,38 Regarding this, Araki et al²⁵ recently reported that ET-1 prevents apoptosis induced by ß-adrenergic agonists, and this effect is inhibited by treatment with the MAP kinase inhibitor PD98059. This difference might be due to apoptosis-inducing stimuli, because the ß-adrenergic agonist itself stimulates MAP kinase activity in cardiac myocytes.⁴⁴ Downstream from c-Src, STAT3 is another molecule that might stimulate Bcl-x_L expression. Karni et al³⁷ reported that c-Src positively regulates Bcl-x_L expression via STAT3 activation. In addition, Negoro et al⁴⁵ recently reported that pretreatment with AG490 significantly inhibited STAT3 phosphorylation and increased apoptosis in rat hearts after infarction. In our study, AG490 was found to inhibit the antiapoptotic effect of ET-1. In addition, ET-1 stimulated STAT3 phosphorylation through a c-Src–dependent mechanism. Thus, STAT3 is a possible molecule that participates in a c-Src/Bcl-x_L pathway. Another signaling molecule responsible for ET-1–prevented apoptosis is Pyk2, because KI-Pyk2 significantly inhibited ET-1–induced Bcl-x_L expression in part. At present, however, the role of Pyk2 in cardiomyocyte apoptosis is unknown. Further investigation is required to understand the precise mechanisms of the antiapoptotic signaling pathway by ET-1 in the myocardium.

In summary, we demonstrated a novel signaling pathway for the antiapoptotic effect of ET-1 in cardiac myocytes. ET-1 prevents serum deprivation–induced apoptosis in cardiac myocytes via the ET_A receptor. c-Src is activated by ET-1, upregulates Bcl-x_L expression, and shows an antiapoptotic effect in cardiac myocytes. Collectively, these findings indicate a potentially important role for the c-Src/Bcl-x_L pathway in the antiapoptotic effect of ET-1. Because the loss of contractile cardiac myocytes due to apoptosis results in a further decrease of cardiac function, identification of the signaling pathway that mediates survival and/or apoptosis in cardiac myocytes is important. Thus, our data provide new insight into the molecular basis and therapeutic target for several cardiovascular diseases, including CHF and ischemic heart disease.

	Acknowledgments

 
This study was supported by grants from the Ministry of Education, Science, Sports and Culture and the Kanae Foundation for Life and Socio-Medical Science. We thank Toshiko Kambe, Yuki Onuma, and Megumi Hata for their expert technical assistance.

Received July 10, 2002; first decision September 23, 2002; accepted February 18, 2003.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Haunstetter A, Izumo S. Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ Res. 1998; 82: 1111–1129.[Free Full Text]

2. MacLellan WR, Schneider MD. Death by design: programmed cell death in cardiovascular biology and disease. Circ Res. 1997; 81: 137–144.[Abstract/Free Full Text]

3. Narula J, Haider N, Virmani R, DiSalvo TG, Kolodgie FD, Hajjar RJ, Schmidt U, Semigran MJ, Dec GW, Khaw BA. Apoptosis in myocytes in end-stage heart failure. N Engl J Med. 1996; 335: 1182–1189.[Abstract/Free Full Text]

4. Kanoh M, Takemura G, Misao J, Hayakawa Y, Aoyama T, Nishigaki K, Noda T, Fujiwara T, Fukuda K, Minatoguchi S, Fujiwara H. Significance of myocytes with positive DNA in situ nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy: not apoptosis but DNA repair. Circulation. 1999; 99: 2757–2764.[Abstract/Free Full Text]

5. Mallat Z, Tedgui A, Fontaliran F, Frank R, Durigon M, Fontaine G. Evidence of apoptosis in arrhythmogenic right ventricular dysplasia. N Engl J Med. 1996; 335: 1190–1196.[Abstract/Free Full Text]

6. Saraste A, Pulkki K, Kallajoki M, Henriksen K, Parvinen M, Voipio-Pulkki LM. Apoptosis in human acute myocardial infarction. Circulation. 1997; 95: 320–323.[Abstract/Free Full Text]

7. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev. 1999; 79: 215–262.[Abstract/Free Full Text]

8. Buerke M, Murohara T, Skurk C, Nuss C, Tomaselli K, Lefer AM. Cardioprotective effect of insulin-like growth factor I in myocardial ischemia followed by reperfusion. Proc Natl Acad Sci U S A. 1995; 92: 8031–8035.[Abstract/Free Full Text]

9. Fujio Y, Kunisada K, Hirota H, Yamauchi-Takihara K, Kishimoto T. Signals through gp130 upregulate bcl-x gene expression via STAT1-binding cis-element in cardiac myocytes. J Clin Invest. 1997; 99: 2898–2905.[Medline] [Order article via Infotrieve]

10. Zhao YY, Sawyer DR, Baliga RR, Opel DJ, Han X, Marchionni MA, Kelly RA. Neuregulins promote survival and growth of cardiac myocytes: persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes. J Biol Chem. 1998; 273: 10261–10269.[Abstract/Free Full Text]

11. Sheng Z, Knowlton K, Chen J, Hoshijima M, Brown JH, Chien KR. Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen-activated protein kinase-dependent pathway: divergence from downstream CT-1 signals for myocardial cell hypertrophy. J Biol Chem. 1997; 272: 5783–5791.[Abstract/Free Full Text]

12. Rokosh DG, Stewart AF, Chang KC, Bailey BA, Karliner JS, Camacho SA, Long CS, Simpson PC. 1-Adrenergic receptor subtype mRNAs are differentially regulated by 1-adrenergic and other hypertrophic stimuli in cardiac myocytes in culture and in vivo: repression of 1B and 1D but induction of 1C. J Biol Chem. 1996; 271: 5839–5843.[Abstract/Free Full Text]

13. Kajstura J, Cigola E, Malhotra A, Li P, Cheng W, Meggs LG, Anversa P. Angiotensin II induces apoptosis of adult ventricular myocytes in vitro. J Mol Cell Cardiol. 1997; 29: 859–870.[CrossRef][Medline] [Order article via Infotrieve]

14. Singh K, Balligand JL, Fischer TA, Smith TW, Kelly RA. Regulation of cytokine-inducible nitric oxide synthase in cardiac myocytes and microvascular endothelial cells: role of extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) and STAT1. J Biol Chem. 1996; 271: 1111–1117.[Abstract/Free Full Text]

15. Shubeita HE, McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Brown JH, Chien KR. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes: a paracrine mechanism for myocardial cell hypertrophy. J Biol Chem. 1990; 265: 20555–20562.[Abstract/Free Full Text]

16. Bogoyevitch MA, Glennon PE, Andersson MB, Clerk A, Lazou A, Marshall CJ, Parker PJ, Sugden PH. Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes: the potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy. J Biol Chem. 1994; 269: 1110–1119.[Abstract/Free Full Text]

17. Spieker LE, Noll G, Ruschitzka FT, Luscher TF. Endothelin receptor antagonists in congestive heart failure: a new therapeutic principle for the future? J Am Coll Cardiol. 2001; 37: 1493–1505.[Abstract/Free Full Text]

18. McMurray JJ, Ray SG, Abdullah I, Dargie HJ, Morton JJ. Plasma endothelin in chronic heart failure. Circulation. 1992; 85: 1374–1379.[Abstract/Free Full Text]

19. Wei CM, Lerman A, Rodeheffer RJ, McGregor CG, Brandt RR, Wright S, Heublein DM, Kao PC, Edwards WD, Burnett JC Jr. Endothelin in human congestive heart failure. Circulation. 1994; 89: 1580–1586.[Abstract/Free Full Text]

20. Miyauchi T, Yanagisawa M, Tomizawa T, Sugishita Y, Suzuki N, Fujino M, Ajisaka R, Goto K, Masaki T. Increased plasma concentrations of endothelin-1 and big endothelin-1 in acute myocardial infarction. Lancet. 1989; 2: 53–54.[Medline] [Order article via Infotrieve]

21. Omland T, Lie RT, Aakvaag A, Aarsland T, Dickstein K. Plasma endothelin determination as a prognostic indicator of 1-year mortality after acute myocardial infarction. Circulation. 1994; 89: 1573–1579.[Abstract/Free Full Text]

22. Shichiri M, Kato H, Marumo F, Hirata Y. Endothelin-1 as an autocrine/paracrine apoptosis survival factor for endothelial cells. Hypertension. 1997; 30: 1198–1203.[Abstract/Free Full Text]

23. Cattaruzza M, Dimigen C, Ehrenreich H, Hecker M. Stretch-induced endothelin B receptor-mediated apoptosis in vascular smooth muscle cells. FASEB J. 2000; 14: 991–998.[Abstract/Free Full Text]

24. Kakita T, Hasegawa K, Iwai-Kanai E, Adachi S, Morimoto T, Wada H, Kawamura T, Yanazume T, Sasayama S. Calcineurin pathway is required for endothelin-1–mediated protection against oxidant stress–induced apoptosis in cardiac myocytes. Circ Res. 2001; 88: 1239–1246.[Abstract/Free Full Text]

25. Araki M, Hasegawa K, Iwai-Kanai E, Fujita M, Sawamura T, Kakita T, Wada H, Morimoto T, Sasayama S. Endothelin-1 as a protective factor against ß-adrenergic agonist- induced apoptosis in cardiac myocytes. J Am Coll Cardiol. 2000; 36: 1411–1418.[Abstract/Free Full Text]

26. Kamitani T, Ikeda U, Muto S, Kawakami K, Nagano K, Tsuruya Y, Oguchi A, Yamamoto K, Hara Y, Kojima T, Medford RM, Shimada K. Regulation of Na,K-ATPase gene expression by thyroid hormone in rat cardiocytes. Circ Res. 1992; 71: 1457–1464.[Abstract/Free Full Text]

27. Yamashita Y, Watanabe S, Miyazato A, Ohya K, Ikeda U, Shimada K, Komatsu N, Hatake K, Miura Y, Ozawa K, Mano H. Tec and Jak2 kinases cooperate to mediate cytokine-driven activation of c-fos transcription. Blood. 1998; 91: 1496–1507.[Abstract/Free Full Text]

28. Takaoka A, Tanaka N, Mitani Y, Miyazaki T, Fujii H, Sato M, Kovarik P, Decker T, Schlessinger J, Taniguchi T. Protein tyrosine kinase Pyk2 mediates the Jak-dependent activation of MAPK and Stat1 in IFN-, but not IFN-, signaling. EMBO J. 1999; 18: 2480–2488.[CrossRef][Medline] [Order article via Infotrieve]

29. Okuda M, Takahashi M, Suero J, Murry CE, Traub O, Kawakatsu H, Berk BC. Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation. J Biol Chem. 1999; 274: 26803–26809.[Abstract/Free Full Text]

30. Takahashi M, Okazaki H, Ogata Y, Takeuchi K, Ikeda U, Shimada K. Lysophosphatidylcholine induces apoptosis in human endothelial cells through a p38-mitogen-activated protein kinase-dependent mechanism. Atherosclerosis. 2002; 161: 387–394.[CrossRef][Medline] [Order article via Infotrieve]

31. Takahashi M, Ogata Y, Okazaki H, Takeuchi K, Kobayashi E, Ikeda U, Shimada K. Fluvastatin enhances apoptosis in cytokine-stimulated vascular smooth muscle cells. J Cardiovasc Pharmacol. 2002; 39: 310–317.[CrossRef][Medline] [Order article via Infotrieve]

32. Hatch GM, Lee D, Man RY, Kroeger EA, Choy PC. On the mechanism of the losartan-mediated inhibition of phosphatidylcholine biosynthesis in H9c2 cells. Biochim Biophys Acta. 1997; 1347: 183–190.[Medline] [Order article via Infotrieve]

33. Russell FD, Molenaar P. The human heart endothelin system: ET-1 synthesis, storage, release and effect. Trends Pharmacol Sci. 2000; 21: 353–359.[CrossRef][Medline] [Order article via Infotrieve]

34. Haunstetter A, Izumo S. Future perspectives and potential implications of cardiac myocyte apoptosis. Cardiovasc Res. 2000; 45: 795–801.[Abstract/Free Full Text]

35. Rosen K, Coll ML, Li A, Filmus J. Transforming growth factor- prevents detachment-induced inhibition of c-Src kinase activity, Bcl-XL down-regulation, and apoptosis of intestinal epithelial cells. J Biol Chem. 2001; 276: 37273–37279.[Abstract/Free Full Text]

36. Jost M, Huggett TM, Kari C, Boise LH, Rodeck U. Epidermal growth factor receptor-dependent control of keratinocyte survival and Bcl-xL expression through a MEK-dependent pathway. J Biol Chem. 2001; 276: 6320–6326.[Abstract/Free Full Text]

37. Karni R, Jove R, Levitzki A. Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors. Oncogene. 1999; 18: 4654–4662.[CrossRef][Medline] [Order article via Infotrieve]

38. Yamauchi-Takihara K, Kishimoto T. A novel role for STAT3 in cardiac remodeling. Trends Cardiovasc Med. 2000; 10: 298–303.[CrossRef][Medline] [Order article via Infotrieve]

39. Kang PM, Izumo S. Apoptosis and heart failure: a critical review of the literature. Circ Res. 2000; 86: 1107–113.[Free Full Text]

40. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature. 1990; 348: 730–732.[CrossRef][Medline] [Order article via Infotrieve]

41. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, Masaki T. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 1990; 348: 732–735.[CrossRef][Medline] [Order article via Infotrieve]

42. Nakamura T, Mizuno S, Matsumoto K, Sawa Y, Matsuda H. Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF. J Clin Invest. 2000; 106: 1511–1519.[Medline] [Order article via Infotrieve]

43. Abram CL, Courtneidge SA. Src family tyrosine kinases and growth factor signaling. Exp Cell Res. 2000; 254: 1–13.[CrossRef][Medline] [Order article via Infotrieve]

44. Singh K, Communal C, Sawyer DB, Colucci WS. Adrenergic regulation of myocardial apoptosis. Cardiovasc Res. 2000; 45: 713–719.[Abstract/Free Full Text]

45. Negoro S, Kunisada K, Tone E, Funamoto M, Oh H, Kishimoto T, Yamauchi-Takihara K. Activation of JAK/STAT pathway transduces cytoprotective signal in rat acute myocardial infarction. Cardiovasc Res. 2000; 47: 797–805.[Abstract/Free Full Text]

This article has been cited by other articles:

				D. K. Singla, R. D. Singla, and D. E. McDonald Factors released from embryonic stem cells inhibit apoptosis in H9c2 cells through PI3K/Akt but not ERK pathway Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H907 - H913. [Abstract] [Full Text] [PDF]

				M. Kang, K. Y. Chung, and J. W. Walker G-Protein Coupled Receptor Signaling in Myocardium: Not for the Faint of Heart Physiology, June 1, 2007; 22(3): 174 - 184. [Abstract] [Full Text] [PDF]

				R. McWhinnie, D. V. Pechkovsky, D. Zhou, D. Lane, A. J. Halayko, D. A. Knight, and T. R. Bai Endothelin-1 induces hypertrophy and inhibits apoptosis in human airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L278 - L286. [Abstract] [Full Text] [PDF]

				M.-N. Raymond, C. Bole-Feysot, Y. Banno, Z. Tanfin, and P. Robin Endothelin-1 Inhibits Apoptosis through a Sphingosine Kinase 1-Dependent Mechanism in Uterine Leiomyoma ELT3 Cells Endocrinology, December 1, 2006; 147(12): 5873 - 5882. [Abstract] [Full Text] [PDF]

				J. Sun, G. Yan, A. Ren, B. You, and J. K. Liao FHL2/SLIM3 Decreases Cardiomyocyte Survival by Inhibitory Interaction With Sphingosine Kinase-1 Circ. Res., September 1, 2006; 99(5): 468 - 476. [Abstract] [Full Text] [PDF]

				X.-S. Zhao, W. Pan, R. Bekeredjian, and R. V. Shohet Endogenous Endothelin-1 Is Required for Cardiomyocyte Survival In Vivo Circulation, August 22, 2006; 114(8): 830 - 837. [Abstract] [Full Text] [PDF]

				N. Shimojo, S. Jesmin, S. Zaedi, M. Soma, S. Maeda, I. Yamaguchi, K. Goto, and T. Miyauchi Changes in important apoptosis-related molecules in the endothelin-1-induced hypertrophied cardiomyocytes: effect of the pretreatment with eicosapentaenoic Acid. Experimental Biology and Medicine, June 1, 2006; 231(6): 932 - 936. [Abstract] [Full Text] [PDF]

				M. B. Marrero, A. K. Banes-Berceli, D. M. Stern, and D. C. Eaton Role of the JAK/STAT signaling pathway in diabetic nephropathy Am J Physiol Renal Physiol, April 1, 2006; 290(4): F762 - F768. [Abstract] [Full Text] [PDF]

				M. Masaki, M. Izumi, Y. Oshima, Y. Nakaoka, T. Kuroda, R. Kimura, S. Sugiyama, K. Terai, M. Kitakaze, K. Yamauchi-Takihara, et al. Smad1 Protects Cardiomyocytes From Ischemia-Reperfusion Injury Circulation, May 31, 2005; 111(21): 2752 - 2759. [Abstract] [Full Text] [PDF]

				T. M. Casey, P. G. Arthur, and M. A. Bogoyevitch Proteomic Analysis Reveals Different Protein Changes during Endothelin-1- or Leukemic Inhibitory Factor-induced Hypertrophy of Cardiomyocytes in Vitro Mol. Cell. Proteomics, May 1, 2005; 4(5): 651 - 661. [Abstract] [Full Text] [PDF]

				J. Melendez, C. Turner, H. Avraham, S. F. Steinberg, E. Schaefer, and M. A. Sussman Cardiomyocyte Apoptosis Triggered by RAFTK/pyk2 via Src Kinase Is Antagonized by Paxillin J. Biol. Chem., December 17, 2004; 279(51): 53516 - 53523. [Abstract] [Full Text] [PDF]

				T. Tokudome, T. Horio, M. Fukunaga, H. Okumura, J. Hino, K. Mori, F. Yoshihara, S.-I. Suga, Y. Kawano, M. Kohno, et al. Ventricular Nonmyocytes Inhibit Doxorubicin-Induced Myocyte Apoptosis: Involvement of Endogenous Endothelin-1 as a Paracrine Factor Endocrinology, May 1, 2004; 145(5): 2458 - 2466. [Abstract] [Full Text] [PDF]

				M. P. Aoki, N. L. Guinazu, A. V. Pellegrini, T. Gotoh, D. T. Masih, and S. Gea Cruzipain, a major Trypanosoma cruzi antigen, promotes arginase-2 expression and survival of neonatal mouse cardiomyocytes Am J Physiol Cell Physiol, February 1, 2004; 286(2): C206 - C212. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) All Versions of this Article: 41/5/1156    most recent 01.HYP.0000064342.30653.24v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ogata, Y. Articles by Kobayashi, E. Search for Related Content PubMed PubMed Citation Articles by Ogata, Y. Articles by Kobayashi, E. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH Related Collections Apoptosis