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(Circulation Research. 2001;0:hh1201.091794.)
© 2001 American Heart Association, Inc.

Article

Calcineurin Pathway Is Required for Endothelin-1–Mediated Protection Against Oxidant Stress–Induced Apoptosis in Cardiac Myocytes

Tsuyoshi Kakita, Koji Hasegawa, Eri Iwai-Kanai, Souichi Adachi, Tatsuya Morimoto, Hiromichi Wada, Teruhisa Kawamura, Tetsuhiko Yanazume Shigetake Sasayama

From the Department of Cardiovascular Medicine (T. Kakita, K.H., E.I.-K., T.M., H.W., T. Kawamura, T.Y., S.S.), Graduate School of Medicine, Kyoto University, Kyoto, and Department of Pediatrics (S.A.), Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Correspondence to Koji Hasegawa, Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail koj{at}kuhp.kyoto-u.ac.jp

Abstract

Abstract—Endothelin-1 (ET-1) acts not only as a growth-promoting peptide but also as a potent survival factor against myocardial cell apoptosis. However, the signaling pathways leading to myocardial cell protection by ET-1 are poorly understood. Using a culture system of primary cardiac myocytes derived from neonatal rats, we show in the present study that ET-1 almost completely blocked the hydrogen peroxide–induced increase in the percentage of TdT-mediated dUTP-biotin nick-end labeling–positive myocytes. Apoptosis inhibition by ET-1 was confirmed by cytofluorometric analysis as well as by examination of the ladder formation, morphological features, and caspase-3 cleavage. We have found that ET-1 converts the nuclear factor of activated T lymphocytes (NFATc) in cardiac myocytes into high-mobility forms and translocates cytoplasmic NFATc to the nuclei. In addition, ET-1 stimulates the interaction between NFATc and the cardiac-restricted zinc-finger protein GATA4 in these cells. The immunosuppressants cyclosporin A and FK506, which antagonize calcineurin, negated the inhibitory effect of ET-1 on apoptosis. Calcineurin activation de novo was sufficient to inhibit hydrogen peroxide–induced apoptosis. ET-1 induced the expression of an antiapoptotic protein bcl-2 in cardiac myocytes in a cyclosporin A–dependent manner, but it did not alter the expression of bax. Cyclosporin A also attenuated the ET-1–stimulated transcription of the bcl-2 gene in these cells. These findings demonstrate that the calcineurin pathway is required for the inhibitory effect of ET-1 on oxidant stress–induced apoptosis in cardiac myocytes.

Key Words: apoptosis • endothelin-1 • cardiac myocyte • calcineurin

Apoptosis, or programmed cell death, is a central feature of normal tissue development in the fetus and of cell replacement in certain adult tissues (eg, the thymus). Apoptosis is most often associated with cells that are progressing through the cell cycle. However, accumulating evidence suggests that terminally differentiated adult cardiac myocytes undergo apoptosis in various animal models of heart failure. These include models of rapid ventricular pacing^{1 2} and pressure overload caused by aortic constriction³ in aged spontaneously hypertensive rats.⁴ In addition, blockage of the survival pathway by cardiac-specific disruption of gp130 results in massive myocardial cell apoptosis after pressure overload, dilatation of the heart, and heart failure.⁵ Thus, the identification of the signaling pathways that mediate cardiac myocyte cell death and survival is critical to the ultimate elucidation of the molecular basis of cardiac muscle failure.

The control of programmed cell death is dependent on a balance between inhibitors and inducers of apoptosis. A number of humoral factors activated in congestive heart failure⁶ may possibly play positive and negative roles in the regulation of myocardial cell apoptosis. Endothelin-1 (ET-1), a 21-residue peptide originally isolated from vascular endothelium,⁷ is one such factor; the levels of ET-1 in plasma and in ventricular myocardium are markedly increased in human and animal models of heart failure.^{8 9 10} ET-1 exerts diverse physiological effects, including vasoconstriction and growth promotion. ET-1 is sufficient to induce the myocardial cell hypertrophy associated with the reactivation of the fetal gene program.¹¹ It was recently reported that ET-1 is a survival factor in smooth muscle cells,¹² fibroblasts,¹³ and cardiac myocytes.¹⁴ However, the precise molecular mechanisms that mediate these survival effects of ET-1 are unclear at present.

Stimulation with ET-1 results in an increase in intracellular calcium levels.¹⁵ Calcium-activated phosphatase calcineurin is necessary for the nuclear import of the nuclear factor of activated T lymphocytes (NFAT) transcription factors, which mediate changes in gene expression in response to calcium signaling from the T-cell receptor (reviewed by Rao et al¹⁶ ). NFAT3, a member of the NFAT family, interacts with high affinity and specificity with the cardiac-restricted zinc-finger protein GATA4.¹⁷ Although calcineurin-GATA4 is involved in the transcriptional pathways that modulate cardiac hypertrophy,¹⁷ a role for this pathway in ET-1–mediated protection against myocardial cell survival is unknown. The present study was conducted to determine whether the calcineurin pathway is involved in the inhibitory effects by ET-1 on myocardial cell apoptosis induced by oxidant stress.

Materials and Methods

Detection of DNA Fragmentation
Primary ventricular cardiac myocytes were prepared from neonatal rats as previously described.^{14 18} The cells were subjected to terminal deoxynucleotidyl transfer–mediated end labeling of fragmented nuclei (TdT-mediated dUTP-biotin nick-end labeling [TUNEL] assay) and nucleosomal ladder assay as previously described.^{14 18}

Cytofluorometric Analysis
Cells were incubated for 10 minutes at 37°C in a culture medium containing 2.5 mmol/L 5,5',6,6'-tetrachloro-1,1',3,37-tetra-ethylbenzimidazolocarbocyanine iodide (JC-1) and 5 µg/mL propidium iodide (both from Molecular Probes), followed by analysis within 30 minutes of the addition of fluorochrome in a Becton Dickinson FAC-Scalibur cytofluorometer. After suitable compensation, fluorescence was recorded at different wavelengths: JC-1 at 525 nm and propidium iodide at 600 nm.

Immunoprecipitation/Western Blotting
Immunoprecipitation and Western blotting were performed as previously described.¹⁹ Aliquots of the lysates containing 100 µg were immunoprecipitated by using an anti-mouse GATA4 polyclonal antiserum (Santa Cruz Biotechnology) or normal goat IgG in low-stringency buffer for 16 hours at 4°C and incubated with protein G (Sigma) beads for 1 hour at 4°C. The precipitate was washed 4 times in the same buffer and subjected to Western blots by using a monoclonal antibody against murine NFATc (Santa Cruz Biotechnology).

As a primary antibody in Western blots for caspase-3, we used anti–caspase-3 polyclonal antibody (Santa Cruz Biotechnology). This antibody detects both the full-length and the cleaved fragment of caspase-3.

For the detection of bcl-2 and bax, we have used an anti-rat bcl-2 monoclonal antibody (Medical & Biological Laboratories) and an anti-mouse bax monoclonal antibody (Santa Cruz Biotechnology). The images were analyzed with computer-assisted densitometry (NIH Image software).

Immunocytochemistry
The cardiac myocytes were grown in a flask-style chamber with glass slides. The cells were then fixed with 3% formaldehyde in PBS for 15 minutes at room temperature. Immunocytochemical staining for NFATc was performed by using the indirect immunofluorescence method. Cells were incubated with anti-NFATc monoclonal antibody (Santa Cruz Biotechnology) at a dilution of 1:100. Signals of NFATc were detected with anti-mouse FITC-conjugated secondary antibody at a dilution of 1:500 for 45 minutes.

Plasmid Constructs
An expression vector encoding a constitutively active form of calcineurin A subunit (CaNA) and encoding a wild-type calcineurin B subunit (CaNB) were provided by Dr Stephen J. O’Keefe, Merck Research Laboratories, Rahway, NJ. The plasmid pCMVß-gal is a ß-gal expression vector and has been described prevously.¹⁹ The plasmid construct pbcl-2luc,²⁰ a firefly luciferase–reporter plasmid driven by a DNA fragment containing sequences from -1796 to -999 relative to the translation start site of the human bcl-2 gene, was kindly donated by Dr Linda M. Boxter, Stanford University School of Medicine, Stanford, Calif. pRSVCAT contains the bacterial chloramphenicol acetyl transferase (CAT) gene driven by Rous sarcoma virus (RSV) long-terminal repeat sequences.¹⁹

Transfection and Luciferase/CAT Assays
Cardiac myocytes were cotransfected with 2 µg of pbcl-2 and 0.1 µg of pRSVCAT by using Lipofectamine Plus (GIBCO BRL) and were subjected to assays for luciferase and CAT activities as described previously.¹⁹

Statistical Analysis
Data are presented as mean±SE. Statistical comparisons were performed by using unpaired two-tailed Student’s t tests or ANOVA with Scheffe’s test when appropriate, with a probability value less than 0.05 taken to indicate significance.

Results

ET-1 Inhibits H₂O₂-Induced Apoptosis in Cardiac Myocytes
To determine the effects of ET-1 on hydrogen peroxide (H₂O₂)–induced myocardial cell apoptosis, neonatal rat cardiac myocytes were treated with saline, H₂O₂ (10^-5 mol/L) alone or H₂O₂ plus ET-1 (10^-7 mol/L) in serum-free medium for 48 hours. We provide representative photographs of TUNEL staining in Figure 1A and quantitative data in Figure 1B. Under our experimental conditions, in which cells were plated at a high density, serum deprivation alone did not increase the number of TUNEL-positive cells (<10%, panel A in Figure 1A and bar 1 in Figure 1B). However, the stimulation with H₂O₂ markedly increased the number of TUNEL-positive cells (>35%, panel B in Figure 1A and bar 2 in Figure 1B). These positive cells may specifically indicate the presence of internucleosomal DNA fragmentation, because no positive cells were found when we omitted the terminal deoxytransferase treatment (panel C in Figure 1A). The cells stimulated with H₂O₂ displayed small condensed nuclei, cell shrinkage, and nuclear fragmentation, consistent with the morphological features of apoptosis. Fewer myocardial cells treated with ET-1 in addition to H₂O₂ were positive for internucleosomal cleavage by TUNEL staining (panel D in Figure 1A and bar 3 in Figure 1B) compared with the cells treated with H₂O₂ alone. Figure 1C (lane 2) shows the H₂O₂-induced typical ladder formation of fragmented internucleosomal DNA in agarose gels, a hallmark of apoptosis. As shown in lane 3 of Figure 1C, ET-1 completely inhibited the internucleosomal cleavage of genomic DNA in H₂O₂-stimulated cardiac myocytes. To confirm the inhibitory effect of ET-1 on myocardial cell apoptosis, we examined whether ET-1 will inhibit the activation of the caspase cascade by Western blot analysis of the active p17 subunit of caspase-3. As illustrated in Figure 1D, stimulation of cardiac myocytes with H₂O₂ induced an increase in p17 of caspase-3, which indicates the proteolytic activation of caspase-3. In addition to H₂O₂, ET-1 inhibited this increase. These findings suggest that ET-1 has an antiapoptotic effect on oxidant stress–induced apoptosis in cardiac myocytes.

View larger version (42K): [in this window] [in a new window]   Figure 1. ET-1 inhibits H2O2-induced apoptosis in cardiac myocytes. Neonatal rat cardiac myocytes were treated with saline as a control or with H2O2 (10-5 mol/L) in the presence or the absence of ET-1 (10-7 mol/L) for 48 hours. A, Representative photographs of TUNEL-staining. Cardiac myocytes were subjected to TUNEL staining in the absence of H2O2 or ET-1 (panel A), in the presence of H2O2 (10-5 mol/L) (panels B and C), in the presence of H2O2 plus ET-1 (10-7 mol/L) (panel D). Terminal deoxytransferase (TdT) was omitted in panel C. Arrowheads show cells with evidence of apoptosis, including chromatin condensation. B, Quantitative analysis of TUNEL staining. TUNEL-positive nuclei were counted and expressed as the percentage of total nuclei. An average of 400 to 500 nuclei were counted in random fields on each slide. The results are the mean±SE of 3 independent experiments. C, Genomic DNA fragmentation. Genomic DNA was isolated from myocytes and loaded on a 1.5% agarose gel. M indicates molecular markers. D, Caspase-3 cleavage. Lysates from cardiac myocytes were subjected to Western blotting with an antibody that recognized both the full-length and the p17 subunit of caspase-3. E, Mitochondrial transmemebrane potential. Mitochondrial transmembrane potential and cell membrane permeability were assayed by incubating these cells for 10 minutes at 37°C in a culture medium containing 2.5 mmol/L of JC-1 and 5 µg/mL propidium iodide. The x axis is JC-1 fluorescence and the y axis is propidium iodide fluorescence.

ET-1 Inhibits H₂O₂-Mediated Reduction of Mitochondrial Transmembrane Potential in Cardiac Myocytes
The reduction of mitochondrial transmembrane potential precedes DNA fragmentation in apoptosis. To further confirm the inhibitory effect of myocardial cell apoptosis by ET-1, we examined mitochondrial membrane potential and cell membrane permeability by cytofluorometric analysis at 24 hours after the stimulation with H₂O₂. Stimulation of cardiac myocytes with H₂O₂ did not alter cell membrane permeability as shown by no increase in propidium iodide binding to DNA. However, H₂O₂ stimulation increased the number of cells with low JC-1 fluorescence, indicating that H₂O₂ reduced mitochondrial transmembrane potential. It was found that 30±4.2% of the H₂O₂-treated cells and 4.6±1.3% of the saline-stimulated cells had low JC-1 fluorescence (lower left quadrant in Figure 1E). However, ET-1 reversed this effect of H₂O₂ and reduced the number of cells with low JC-1 fluorescence to 8.2±2.1%. These findings suggest that H₂O₂ stimulation specifically reduces mitochondrial membrane potential and provide further evidence for the inhibitory effect of ET-1 against H₂O₂-induced apoptosis.

ET-1 Converts NFATc Into High-Mobility Forms and Stimulates the Interaction With GATA4
To determine whether ET-1 dephosphorylates NFATc in cardiac myocytes, we performed Western blotting. Neonatal cardiac myocytes were stimulated with ET-1 or saline as a control for 3 hours, and then whole lysates derived from these cells were subjected to Western blotting with the anti-NFATc antibody. As shown in Figure 2A, the expression level of NFATc did not differ between saline- and ET-1–stimulated cardiac myocytes. However, all 3 forms of NFATc in ET-1–stimulated cardiac myocytes (lane 2) migrated faster than NFATc in saline-stimulated cells (lane 1). Because dephosphorylated NFATc migrates faster than phosphorylated NFATc, this might indicate dephosphorylation of NFATc by ET-1 stimulation. To further confirm this hypothesis, we have used the immunosuppressants cyclosporin A (Cys A), which inhibits the ability of calcineurin to activate NFAT transcription factors.^{21 22} As shown in Figure 2A, a therapeutic concentration of Cys A (4x10^-7 mol/L) reversed the effect of ET-1 on the migration of NFATc.

View larger version (51K): [in this window] [in a new window]   Figure 2. ET-1 converts NFATc into high-mobility forms and stimulates the interaction between NFATc and GATA4. A, Neonatal cardiac myocytes were cultured in serum-free medium in the presence or absence of ET-1 (10-7 mol/L) and Cys A (4x10-7 mol/L) as indicated for 3 hours. Lysates from these cells were subjected to Western blotting with an anti-NFATc antibody. B, Neonatal cardiac myocytes were stimulated with saline or 10-7 mol/L ET-1 for 3 hours. Lysates derived from these cells were immunoprecipitated with an anti-NFATc antibody (lanes 1 and 4), IgG (lanes 2 and 5), or an anti-GATA4 antibody (lanes 3 and 6). After electrophoresis and electroblotting, the membranes containing immobilized immunocomplexes were subjected to Western blotting with an anti-NFATc antibody. Lysates were subjected to Western blotting without immunoprecipitation in lanes 7 and 8.

To determine whether NFATc interacts with GATA4 in cardiac myocytes by ET-1 stimulation, we performed immunoprecipitations followed by Western blotting. Neonatal cardiac myocytes were stimulated with ET-1 or saline as a control for 3 hours, and then whole lysates derived from these cells were subjected to immunoprecipitation with an anti-NFATc antibody as a positive control (Figure 2B, lanes 1 and 4), IgG as a negative control (Figure 2B, lanes 2 and 5), or an anti-GATA4 antibody (Figure 2B, lanes 3 and 6). Western blotting with the anti-NFATc antibody showed that the anti-NFATc antibody immunoprecipitated all 3 forms of NFATc in saline- and ET-1–stimulated cardiac myocytes (lanes 1 and 4). The anti-GATA4 antibody (lane 6), but not IgG (lane 5), coprecipitated NFATc protein in lysates from cardiac myocytes stimulated with ET-1, even after extensive washing. The main form of NFATc coprecipitated was the one with the highest mobility (compare lanes 6 and 8). The anti-GATA4 antibody did not coprecipitate NFATc in lysates from saline-stimulated myocytes (lane 3). However, the expression level of GATA4 did not differ between saline- and ET-1–stimulated cells (data not shown). Thus, ET-1 stimulated the interaction between NFATc and GATA4 in cardiac myocytes.

ET-1 Translocates NFATc Into the Nucleus in a Calcineurin-Dependent Manner
To examine nuclear translocation of endogenous NFATc in response to ET-1 treatment in cardiac myocytes, we performed immunofluorescence. As shown in Figure 3A, NFATc was detected in cytoplasm of nearly all saline-stimulated cardiac myocytes. However, the stimulation of cardiac myocytes with ET-1 markedly changed this localization and caused the nuclear translocation of NFATc (Figure 3B). ET-1–mediated translocation was reversed by Cys A (Figure 3C), suggesting a requirement of calcineurin activation for this translocation.

View larger version (31K): [in this window] [in a new window]   Figure 3. ET-1 translocates NFATc into the nucleus in a calcineurin-dependent manner. A through C, Cultured cardiac myocytes were treated with ET-1 (10-7 mol/L, B), ET-1 plus Cys A (4x10-7 mol/L, C), or saline as a control (A) for 3 hours in serum-free conditions, and were then subjected to immunofluorescence with an anti-NFATc antibody. D and E, Cultured cardiac myocytes were transfected with 0.2 µg of CaNA and 0.2 µg of CaNB together in E or with 0.4 µg of pCMVß-gal alone in panel D. Forty-eight hours later, these cells were subjected to immunofluorescence with an anti-NFATc antibody.

To examine the effects of calcineurin activation de novo on cardiac myocytes, we cotransfected an expression vector encoding a constitutively active form of CaNA and CaNB. As a control, we transfected the corresponding amount of pCMVß-gal. Then, we examined the localization of endogenous NFATc in cardiac myocytes by immunofluorescence. The transfection of pCMVß-gal did not change the location of NFATc in cardiac myocytes (Figure 3D). However, cotransfecting CaNA and CaNB resulted in the nuclear translocation of endogenous NFATc in almost all cells (Figure 3E).

Cys A and FK506 Neutralize the Antiapoptotic Effect of ET-1
We examined whether the calcium-activated phosphatase calcineurin is required for the inhibition of myocardial cell apoptosis by ET-1. We exposed neonatal cardiac myocytes to H₂O₂ and ET-1 in the presence or the absence of the Cys A and FK506, which inhibit the ability of calcineurin to activate NFAT transcription factors.²³ As shown in Figure 4, ET-1 inhibits the H₂O₂-mediated increase in the number of TUNEL-positive myocytes (compare bars 1, 4, and 7). A therapeutic concentration of Cys A (4x10^-7 mol/L) or FK506 (1x10^-9 mol/L) in addition to H₂O₂ and ET-1 increased the number of TUNEL-positive myocytes (compare bars 7, 8, and 9). However, the same concentration of Cys A and FK506 did not increase the number of TUNEL-positive myocytes in saline-stimulated cardiac myocytes (compare bars 1, 2, and 3). Thus, the effect of Cys A and FK506 on ET-1 may not be explained by the cytotoxicity of these agents. To exclude the possibility that Cys A and FK506 augment proapoptotic effects of H₂O₂, we treated cardiac myocytes with Cys A and FK506 in addition to H₂O₂. However, Cys A and FK506 did not increase the number of TUNEL-positive cells in H₂O₂-stimulated states (compare bars 4, 5, and 6). These results indicate that the apoptosis-inhibitory effect of ET-1 is Cys A– and FK506-sensitive and therefore involves calcineurin activation.

View larger version (64K): [in this window] [in a new window]   Figure 4. Cys A and FK506 neutralize ET-1–mediated inhibition of apoptosis. Cultured cardiac myocytes were treated for 48 hours in serum-free medium in the presence or absence of H2O2 (10-5 mol/L), ET-1 (10-7 mol/L), Cys A (4x10-7 mol/L), and FK506 (1x10-9 mol/L). TUNEL-positive nuclei were counted and expressed as the percentage of total nuclei. An average of 400 to 500 nuclei were counted in random fields on each slide. The results are the mean±SE of 3 independent experiments.

To examine the effects of calcineurin/NFATc activation de novo on myocardial cell apoptosis, we cotransfected an expression vector encoding a constitutively active form of CaNA and CaNB. As a control, we transfected the corresponding amount of pCMVß-gal. Transfection of CaNA and CaNB but not that of pCMVß-gal results in the nuclear translocation of NFATc in cardiac myocytes as described earlier (Figures 3D and 3E). As shown in Figure 5, the treatment of pCMVß-gal–transfected cardiac myocytes with H₂O₂ markedly increased the number of TUNEL-positive nuclei. In contrast, H₂O₂ did not increase the TUNEL-positivity in cells cotransfected with CaNA and CaNB. These findings demonstrate that the activation of calcineurin/NFATc is sufficient to inhibit H₂O₂-induced myocardial cell apoptosis.

View larger version (43K): [in this window] [in a new window]   Figure 5. Calcineurin activation de novo inhibits H2O2-induced apoptosis. Cultured cardiac myocytes were transfected with 0.2 µg of CaNA and 0.2 µg of CaNB together (bars 3 and 4) or with 0.4 µg of pCMVß-gal alone (bars 1 and 2). These cells were treated for 48 hours in a serum-free medium in the presence or absence of H2O2 (10-5 mol/L) as indicated and were then subjected to TUNEL staining. The results are the mean±SE of 3 independent experiments.

Cys A Inhibits ET-1–Induced Transcription of bcl-2 in Cardiac Myocytes
Bcl-2 is a proto-oncogene–encoded protein that prevents apoptosis induced by various stimuli.²⁴ To determine whether stimulation of cardiac myocytes with ET-1 induces the expression of bcl-2, and if so, whether a calcineurin pathway is involved in this process, neonatal cardiac myocytes were treated with ET-1 in the presence or absence of Cys A (4x10^-7 mol/L). Forty-eight hours later, lysates from these cells were subjected to Western blotting with anti–bcl-2 antibody. As shown in Figures 6A and 6B, ET-1 stimulation induced the expression of bcl-2 protein in cardiac myocytes (lane 2) by 3.6±0.3-fold compared with saline simulation (lane 1). Blockage of calcineurin activation by Cys A inhibited the induction of expression of bcl-2 by ET-1 in these cells (lane 3). However, neither ET-1 nor Cys A altered the expression of the proapoptotic molecule bax. These results indicate that calcineurin activation is required for the induction of the expression of an antiapoptotic molecule, bcl-2, by ET-1. To determine whether ET-1 stimulates the transcriptional activity of the upstream regulatory sequences of the bcl-2 gene, we evaluated the expression of a luciferase-reporter gene driven by DNA sequences from -1796 to -999 relative to the translation start site of the human bcl-2 gene in saline- and ET-1–stimulated cardiac myocytes. These sequences contain the major transcriptional promoter P1.²⁰ Neonatal cardiac myocytes were transfected with pbcl-2luc and a small quantity of pRSVCAT as an internal control to normalize for transfection efficiency and were then stimulated with saline or ET-1 in the presence or the absence of Cys A (4x10^-7 mol/L). Forty-eight hours later, luciferase and CAT activities were assessed in lysates from these cells. As shown in Figure 7, ET-1 increased the relative luciferase activity of pbcl-2luc by 3.7-fold. Cys A inhibited the ET-1–stimulated increase in the bcl-2 promoter activity by 34% (P<0.01), suggesting that calcineurin activation is, in part, involved in ET-1–responsive bcl-2 transcription.

View larger version (19K): [in this window] [in a new window]   Figure 6. Cys A inhibits ET-1–induced expression of bcl-2. Cardiac myocytes were cultured in serum-free medium in the presence or absence of ET-1 (10-7 mol/L) and Cys A (4x10-7 mol/L) as indicated for 48 hours. Cell lysates were subjected to Western blotting with an anti–bcl-2 antibody, anti-bax antibody, or anti–ß-actin antibody.

View larger version (47K): [in this window] [in a new window]   Figure 7. Cys A inhibits ET-1–mediated activation of the bcl-2 promoter. Two micrograms of pbcl-2 and 0.1 µg of pRSVCAT were cotransfected into primary cultures of neonatal rat cardiac myocytes, which were subsequently stimulated with saline or 10-7 mol/L ET-1 in the presence or absence of Cys A (4x10-7 mol/L) as indicated for 48 hours. The relative luciferase activities (luc/CAT) of pbcl-2 in the saline-stimulated state was set at 1.0 in each experiment. Values are mean±SE of 2 independent experiments, each carried out in duplicate.

Discussion

Accumulating evidence suggests that myocyte apoptosis occurs in failing hearts.^{1 2 3 4} Because adult cardiac muscle cells are terminally differentiated and have lost their proliferative capacity, the maintenance of cardiac muscle cell survival is critical for the maintenance of systolic cardiac function. Recently, it has been shown that ET-1 is not only a growth-promoting peptide, but it is also a potent protective factor against apoptosis in various cell types, including cardiac myocytes.^{12 13 14} However, the precise signaling pathways leading to antiapoptotic effects of ET-1 are poorly understood. The present study has demonstrated that the calcineurin pathway is required for the inhibitory effect of ET-1 on oxidant stress–induced apoptosis in cardiac myocytes. These findings provide further insights into the role of calcineurin in heart failure in vivo.

Using primary neonatal rat cardiac myocytes, we showed that ET-1 blocked an H₂O₂-induced increase in the TUNEL-positive cells. We cannot rule out the possibility that TUNEL-positive cells contain a subset of false-positive cells. However, inhibition of apoptosis in rat cardiac myocytes was demonstrated by another 4 lines of evidence: (1) the inhibition of nucleosomal ladder formation detected by agarose gel electrophoresis, (2) the decrease in the number of cells showing nuclear condensation, a morphological feature of apoptosis, (3) the inhibition of cleavage of caspase-3, and (4) the increase of mitochondrial transmembrane potential shown by cytofluorometric analysis. These findings are consistent with the idea that ET-1 is a potent protective factor against myocardial cell apoptosis.

The levels of ET-1 in plasma and in ventricular myocardium markedly increase in close association with systolic dysfunction.^{8 9 10} Blockade of the myocardial ET-1 pathway by ET-1 receptor antagonists has been shown to improve cardiac function and survival in experimental animal models of heart failure.^{8 9 10} These data indicate that activation of myocardial ET-1 pathway worsens cardiac function in the development of heart failure. Our data that ET-1 protects cardiac myocytes from apoptosis are paradoxical to data of these previous studies, assuming that myocardial cell apoptosis is the only factor that determines cardiac function in the development of heart failure. However, an apoptosis-independent model of heart failure has been reported,²⁵ and we speculate that ET-1 could worsen cardiac function through a mechanism other than apoptosis. For example, ET-1 receptors bind with Gi as well as Gq and decrease cAMP levels in cardiac myocytes.²⁶ Thus, an increase of myocardial ET-1 may impair cardiac pump function by decreasing intracellular cAMP levels in the development of heart failure. Although the precise mechanisms by which ET-1 impairs systolic function should be further investigated, endogenous ET-1 may provide self-protection against myocardial cell apoptosis as well as function as a harmful factor under the conditions of heart failure.

ET-1 signaling is coupled with an increase in intracellular calcium levels.¹⁵ Increased calcium activates calcineurin, a ubiquitously expressed serine/threonine phosphatase, by binding the 19-kDa regulatory subunit of calcineurin.²⁷ The activated form of calcineurin dephosphorylates NFAT transcription factors, which enables them to translocate to the nucleus.^{16 17} NFAT transcription factors interact with high affinity and specificity with the cardiac-restricted zinc finger protein GATA4.¹⁷ Cys A and FK506 bind the immunophilin cyclophilin and FK506-binding protein (FKBP12), respectively, forming complexes that bind the calcineurin catalytic subunit and inhibit the ability of calcineurin to activate NFAT transcription factors.²³ The present study demonstrated that ET-1 translocated NFATc into the nucleus and stimulated the interaction between NFATc and GATA4. These findings clearly indicate that ET-1 activates calcineurin. Cys A and FK506 antagonized the protective effects of ET-1. However, the same concentration of Cys A and FK506 alone increased the number of apoptotic cells in neither saline-stimulated cells nor H₂O₂-stimulated cells. This indicates that these agents might block the downstream signaling pathway by which ET-1 prevents apoptosis. These results demonstrate that calcineurin activation requires the antiapoptotic effect of ET-1. Several possibilities should be taken into account when the data of this study are applied to the in vivo setting in the adult. First, because myocardial development is not complete at birth, differences may exist between neonatal and adult cardiac myocytes. Second, the biological properties of disassociated myocytes in culture and myocytes in the organized heart in vivo may differ. However, a recent study showed that transgenic mice overexpressing an activated form of calcineurin exhibited less myocardial cell apoptosis after myocardial ischemia than wild-type mice.²⁸ Thus, the inhibition of cardiomyocyte apoptosis through calcineurin-dependent signaling pathways is not confined to in vitro assays in neonatal cells but may also occur in adult cardiac myocytes in vivo.

Recently, it has been reported that Cys A and FK506 significantly inhibit myocardial cell apoptosis induced by the ß-adrenergic agonist isoproterenol.²⁹ Calcineurin has also been implicated in proapoptotic signaling in thymocytes through mechanisms that are thought to involve the activation of the nur77 gene³⁰ ; thus, the calcineurin pathway is involved in the induction of apoptosis in some situations. One possible explanation of the opposing effects of calcineurin on apoptosis is the crosstalk with other signaling pathways. The activation of calcineurin by isoproterenol stimulation results in the dephosphorylation of Bad, which may be involved in the induction of apoptosis.²⁹ In contrast, because ET-1 is functionally related to the activation of Akt/Bad, calcineurin activation by ET-1 does not result in dephosphorylation of Bad. Despite the opposing effects of calcineurin activation on apoptosis, our findings demonstrate that calcineurin activation de novo inhibits H₂O₂-induced myocardial cell apoptosis, in agreement with a previous report.²⁵ However, precise mechanisms by which calcineurin could have diametrically opposing effects in different contexts should be further investigated.

The bcl-2 gene product is a 25-kDa membrane protein that functions to prevent apoptosis by various stimuli.²⁴ Prevention of apoptosis by increased bcl-2 expression has also been shown in adult cardiac myocytes.²¹ The present study demonstrated that stimulation of cardiac myocytes with ET-1 increased the expression of bcl-2 protein. ET-1 signaling is functionally linked to phospholipase C to induce phosphoinositide breakdown.²² It is also becoming clear that an ET-1 pathway cross-talks with Ras and MAP kinase cascades.³¹ Because ET-1 affects multiple signaling pathways, the upregulated bcl-2 expression may not be the only mechanism for the antiapoptotic effects of ET-1. However, the potent ability of bcl-2 to block apoptosis in many cell types suggests that this upregulation is, at least in part, involved in the protective effects of ET-1.

Calcineurin-GATA4 is involved in the transcriptional pathways that modulate cardiac hypertrophy.¹⁷ Calcineurin inhibition by Cys A is sufficient to block cardiac hypertrophy in a transgenic animal model of hypertrophic cardiomyopathy.³² Although calcineurin seems to be involved in the development of compensating hypertrophy evoked by pressure overload, Cys A instead worsens systolic function.²⁸ Whether the deterioration of systolic function caused by Cys A is attributable to an increase of myocardial cell apoptosis should be further investigated. Further elucidation of signaling pathways leading to myocardial cell hypertrophy and survival by calcineurin activation will be useful for understanding the role of this pathway in the development of heart failure in vivo.

Acknowledgments

This work was supported in part by grants to K.H. from the Ministry of Education, Science and Culture of Japan. We thank N. Sowa for his excellent technical assistance.

Footnotes

Original received November 13, 2000; resubmission received March 7, 2001; revised resubmission received April 20, 2001; accepted April 20, 2001.

References

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