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(Circulation. 1999;99:2934-2941.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Signaling Pathways in Reactive Oxygen Species–Induced Cardiomyocyte Apoptosis

Rüdiger von Harsdorf, MD; Pei-Feng Li, PhD; Rainer Dietz, MD

From the Department of Cardiology, Franz Volhard Clinic, Humboldt-University (R.v.H., R.D.), and the Max-Delbrück-Center for Molecular Medicine (P.-F.L.), Berlin, Germany.

Correspondence to Rüdiger von Harsdorf, MD, Franz-Volhard-Klinik, Medizinische Fakultät der Charité, Humboldt-Universität, Wiltbergstr. 50, 13125 Berlin, FRG. E-mail rharsdo{at}mdc-berlin.de

	Abstract

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Background—The importance of free radical homeostasis and apoptosis in normal and diseased hearts and their interrelationships are poorly defined. We tested whether reactive oxygen species can trigger apoptosis in cardiomyocytes, and we explored the underlying pathways.

Methods and Results—A cell culture model of isolated cardiac cells and different reactive oxygen species (ROS)–generating systems were used. Apoptosis became evident when cardiomyocytes were exposed to either H₂O₂ or superoxide anion (O₂^-). Both H₂O₂- and O₂^--induced apoptosis of cardiomyocytes were associated with an increase in p53 protein content, whereas protein levels of Bax and Bcl-2 were unaltered. H₂O₂, but not O₂^-, induced an increase in the protein content of Bad. Furthermore, H₂O₂ elicited translocation of Bax and Bad from cytosol to mitochondria, where these factors formed heterodimers with Bcl-2, which was followed by the release of cytochrome c, activation of CPP32, and cleavage of poly(ADP-ribose) polymerase. Interestingly, this pathway was not activated by O₂^-. Instead, O₂^- used Mch2 to promote the apoptotic pathway, as revealed by the activation of Mch2 and the cleavage of its substrate, lamin A.

Conclusions—Taken together, these results indicate that ROS may play an important pathophysiological role in cardiac diseases characterized by apoptotic cell death and suggest that different ROS-induced activations of the apoptotic cell death program in cardiomyocytes involve distinct signaling pathways.

Key Words: myocytes • apoptosis • reactive oxygen species • cytochrome c

	Introduction

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An increasing body of evidence suggests that apoptosis plays an important role in cardiac development and diseases. This is based on the observation that in animals and humans, the ontogenesis of the normal heart is characterized by the appearance of apoptosis, which peaks in the perinatal period¹ and in specific regions of the heart.² Furthermore, myocardial infarction is associated with apoptosis in rats³ and humans.⁴ Additionally, apoptosis is evident in the hearts of patients suffering from certain conductance disturbances² ; it is also evident in various human cardiomyopathies, including arrhythmogenic right ventricular dysplasia⁵ and dilated cardiomyopathy.⁶ These studies providing proof of apoptotic cell death in the heart are also important because they show us that the capacity for apoptotic cell death exists in both mitotic cells and in postmitotic cells. However, little information exists regarding the identification of stimuli responsible for the induction of apoptosis in the heart.

Interestingly, apoptosis occurs during reperfusion after ischemia in several organs, including the heart.⁷ However, it is not known how reperfusion triggers apoptosis. Experimental studies using isolated organ preparations or in vivo animal models have demonstrated the generation of reactive oxygen species (ROS) during ischemia and reperfusion.⁸ Also, several clinical procedures are frequently associated with ischemia and reperfusion injury on one side and production and release of ROS on the other, including clinical bypass surgery,⁹ thrombosis,¹⁰ and coronary balloon angioplasty.¹¹ That represents a ROS threat, even for the ontogenesis of the normal heart, was demonstrated recently by the induction of cardiomyopathies and early lethality in knockout mice lacking the manganese superoxide dismutase (SOD), which acts as an intracellular ROS-scavenger.¹² However, in all these cases, it remains unclear how ROS induce the pathological phenotype.

We used a cell culture model of isolated cardiac cells and different ROS-generating systems to determine whether ROS are able to induce apoptosis in cardiomyocytes, to evaluate the expression patterns of apoptosis-related genes, and to explore the apoptotic pathways in cardiomyocytes exposed to ROS.

	Methods

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Cell Cultures
Monolayer cultures of neonatal rat cardiac cells were prepared by modifying the method of Simpson et al.¹³ Briefly, hearts from 1-day-old Wistar rats were dissected, minced, and placed in PBS. The tissue was trypsinized at 37°C in a HEPES-buffered saline solution ([in mmol/L] HEPES-NaOH 20 [pH 7.6], NaCl 130, KCl 3, NaH₂PO₄ 1, and glucose 4 and 0.15% trypsin). After centrifugation, cells were resuspended in Dulbecco's modified Eagle medium/F-12 (Gibco) containing 5% heat-inactivated horse serum, 100 µmol/L ascorbate, 1 µg/mL insulin, 1 µg/mL transferrin, 10 ng/mL selenium, 100 U/mL penicillin, and 100 µg/mL streptomycin. The dissociated cells were preplated at 37°C for 1 hour. The cells were then diluted to 1x10⁶ cells/mL, plated in 94-mm cultured dishes (for MTT assay, in 96-well plates) and, before use, were cultured for 62 to 72 hours in a medium containing 0.1 mmol/L bromodeoxyuridine to prevent proliferation of nonmyocytes. More than 95% of cells were cardiomyocytes, as detected by immunostaining with the -sarcomeric actin antibody.

Exposure of Cells to ROS-Generating Systems
Cultured cells were washed twice with Hanks's balanced salt solution (HBSS) at 37°C. Washed cells were incubated at 37°C for 1 hour in HBSS containing the indicated concentration of xanthine oxidase plus xanthine (XO/X) or H₂O₂ plus ferrous sulfate, as described elsewhere.¹⁴ When superoxide dismutase (SOD), catalase (CAT), or 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) were used (all from Sigma), they were added simultaneously with XO/X. To demonstrate the specificity of the effect of SOD and CAT, exposures with heat-inactivated SOD or CAT (100°C for 1 hour before addition) were also performed; all resulted in the complete abrogation of the effect of these enzymes on cell viability or protein expression in the presence of XO/X. Furthermore, incubation of cells with SOD or CAT in the absence of XO/X had no significant effect on cell viability, as detected by MTT and trypan blue exclusion, or protein expression, as detected by immunoblotting (data not shown). Z-VAD-fmk (Calbiochem) was used 2 hours before and immediately after treatment.

Cell Viability Assay, Cell-Death Detection ELISA, and In Situ Nick-End Labeling
These procedures were performed as described elsewhere.¹⁴ >

Immunoblot Analysis
Cells were lysed for 1 hour at 4°C in a lysis buffer ([in mmol/L] Tris 20 [pH 7.5], EDTA 2, EGTA 3, DTT 2, sucrose 250, and PMSF 0.1; 1% Triton X-100; and 10 µg/mL each of leupeptin, aprotinin, and pepstatin A). Samples containing 50 µg of protein were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. Equal-protein loading was controlled by Ponceau red staining of membranes. Blots were probed using primary antibodies. These included polyclonal Bcl-2 antibody, polyclonal Bax antibody, cytochrome c monoclonal antibody (all from Pharmingen), p53 monoclonal antibody (Calbiochem), poly(ADP-ribose) polymerase (PARP) monoclonal antibody (Clontech), Bad monoclonal antibody (Transduction Laboratories), CPP32 polyclonal antibody, Mch2 polyclonal antibody, lamin A polyclonal antibody (all from Santa Cruz), and cytochrome oxidase (subunit II) monoclonal antibody (Molecular Probes). Blots were then probed by horseradish peroxidase–conjugated goat anti-rabbit IgG, rabbit anti-mouse IgG, or donkey anti-goat IgG (all from Amersham). Antigen-antibody complexes were visualized by enhanced chemiluminescence.

Preparation of Subcellular Fractions
Cells were washed twice with PBS, and the pellet was suspended in 0.5 mL of buffer ([in mmol/L] HEPES 20 [pH 7.5], KCl 10, MgCl₂ 1.5, EGTA 1, EDTA 1, DTT 1, and PMSF 0.1, and 10 µg/mL each of leupeptin, aprotinin, and pepstatin A) containing 250 mmol/L sucrose. The cells were homogenized by 5 strokes in a Dounce homogenizer. Subcellular fractions were prepared as described elsewhere.¹⁵ In brief, the homogenates were centrifuged twice at 750g for 5 minutes at 4°C to collect nuclei and unbroken cells. The supernatants were centrifuged at 10 000g for 15 minutes at 4°C to collect the heavy-membrane pellet (HM). The resulting supernatants were centrifuged at 100 000g for 1 hour at 4°C to yield light-membrane pellets (LM). The final supernatants were called cytosolic fractions. The samples were kept at -80°C. To verify the distribution of mitochondria and lysosomes in the subcellular fractions, we determined the activities of monoamine oxidase, a marker enzyme of mitochondria, and of acid phosphatase, a lysosomal marker enzyme. Monoamine oxidase activity was assayed using a method described previously.¹⁶ The assay was performed at room temperature in a mixture containing 50 mmol/L Tris-HCl (pH 7.4), 0.22 mmol/L kynuramine, and 80 µmol/L MgCl₂. The reaction was stopped by adding 0.5 mol/L NaOH and 10% ZnSO₄. The reaction product was determined by measuring the absorbance at 330 nm, and 4-hydroxyquinoline was used as the standard. The activity of acid phosphatase was detected as described elsewhere.¹⁷ The assay was started by incubating the sample with 100 mmol/L acetic acid–sodium acetate buffer (pH 4.5), which contained 4.5 mmol/L p-nitrophenyl phosphate and 0.05% Triton X-100. The assay was conducted at room temperature, and the reaction was stopped with 2 mol/L NaOH. The absorbance was measured at 405 nm. Monoamine oxidase activity represented 92.5±2.2% (n=3) of total enzyme activity in HM and 6.3±3.8% (n=3) in LM, whereas acid phosphatase activity was 24.7±8.5% (n=3) of total enzyme activity assessed in HM and 72.2±8.4% (n=3) in LM. These data indicate that the majority of mitochondria were in HM.

Immunoprecipitation
HM or LM was resuspended in a buffer (10 mmol/L HEPES [pH 7.4]; 38 mmol/L NaCl; 0.1 mmol/L PMSF; and 10 µg/mL each of leupeptin, aprotinin, and pepstatin A); they were then homogenized in a Dounce homogenizer. To perform immunoprecipitations, the cytosol, HM, or LM lysates were precleared with 10% (vol/vol) protein A–agarose for 1 hour on a rocking platform. Specific antibodies were added and rocked for 1 hour. Immunoprecipitates were captured with 10% (vol/vol) protein A–agarose for another hour. The agarose beads were spun down and washed 3 times with NET buffer. The antigens were released and denatured by adding SDS sample buffer. Immunoblot analysis was performed as described above.

Statistical Analysis
All results are expressed as mean±SEM of at least 3 independent experiments unless stated otherwise. Paired data were evaluated by Student's t test. A 1-way ANOVA was used for multiple comparisons. A value of P<0.05 was considered significant.

	Results

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ROS-Induced Death in Cardiac Cells
Exposure of isolated cardiac cells to H₂O₂ led to a dose-dependent decrease in cell viability, as assessed by MTT assay (Figure 1A). XO plus 0.1 mmol/L xanthine (XO/X) was used to generate superoxide anion (O₂^-), and cell viability was assessed by trypan blue exclusion because XO/X itself may interfere with the MTT reaction. Again, a dose-dependent decrease in cell viability could be observed (Figure 1B). However, because XO/X yields both O₂^- and H₂O₂, the ROS scavengers SOD, Tiron, and CAT were used to determine the contribution of each of these ROS in the XO/X-induced death of cardiac cells (Figure 1C). In the presence of SOD, the effect of XO/X on cell viability was partially reversed, indicating that the generation of O₂^- contributed to XO/X-induced cell death; this finding was corroborated by replacing SOD with tiron, a cell membrane–permeable scavenger of O₂^-. The presence of CAT had a similar effect on cell viability as SOD. The combination of CAT with SOD or with Tiron resulted in the complete prevention of XO/X-induced cell death. Taken together, these data suggest that H₂O₂ or O₂^- or both are able to induce death in cardiac cells.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of H2O2 or XO/X on viability of cardiac cells. A, Cell viability was determined by MTT assay after cells were treated for 1 hour with indicated doses of H2O2 plus 0.1 mmol/L FeSO4 and then further cultured in ROS-free medium for another 19 hours. Each bar represents mean±SEM of 3 separate experiments (n=16 wells in each individual experiment). *P<0.05, **P<0.01 compared with unstimulated control cells. B, Cell viability was determined by trypan blue exclusion after cells were treated for 1 hour with indicated doses of XO plus 0.1 mmol/L xanthine and then further cultured in ROS-free medium for another 7 hours. *P<0.05, **P<0.01 compared with control. C, Cells were exposed to 0.04 U/mL XO plus 0.1 mmol/L xanthine in presence of 1000 U/mL SOD, 500 U/mL CAT, or 1 mmol/L tiron. Each bar represents mean±SEM of 4 separate experiments in duplicate. *P<0.05, **P<0.02 compared with XO/X.

ROS-Induced Apoptosis in Cardiomyocytes
We hypothesized that exposure to ROS might lead to cell death through the induction of apoptosis in cardiac cells. As determined by cell-death detection ELISA, which specifically detects histone-associated DNA fragments within the cytoplasmic fraction of stimulated cells, there was a dose-dependent increase of oligonucleosomes in the cytoplasmic fraction after H₂O₂ treatment (Figure 2A). Similar results were obtained in cells exposed to XO/X (Figure 2B). Administration of SOD, CAT, or Tiron attenuated the effect of XO/X, whereas the presence of SOD or Tiron in conjunction with CAT almost completely abrogated the effect of XO/X (Figure 2C).

View larger version (23K): [in this window] [in a new window]   Figure 2. Detection of ROS-induced apoptosis in cardiac cells. DNA fragmentation was determined by cell-death ELISA, and cells were treated as described in Figure 1 with indicated doses of (A) H2O2, (B) XO/X, or (C) 0.04 U/mL xanthine oxidase plus 0.1 mmol/L xanthine in presence of SOD (1000 U/mL), Tiron (1 mmol/L), and/or CAT (500 U/mL). The histone-associated DNA fragments are presented as optical density at 405 nm. The results are expressed as mean±SEM of 3 independent determinations in triplicate. *P<0.05 compared with control. D, TUNEL and immunofluorescence with anti--sarcomeric antibody of control and stimulated cardiac cells. Cultured cells were treated with 0.1 mmol/L H2O2 plus 0.1 mmol/L FeSO4, as described in Figure 1. Arrows indicate TUNEL-positive cardiomyocytes.

To determine whether apoptosis was occurring in cardiomyocytes compared with cardiac nonmyocytes (which are always present in cultures of neonatal rat cardiomyocytes), we used in situ nick-end labeling together with immunofluorescence (Figure 2D). The results show that in contrast to the control, apoptotic cardiomyocytes could be detected, simultaneously stained with -sarcomeric actin and terminal deoxynucleotidyl transferase–mediated dUTP biotin nick-end labeling (TUNEL), when cultures were treated with 0.1 mmol/L H₂O₂. Similar results were obtained using 0.04 U/mL xanthine oxidase in the presence of 0.1 mmol/L xanthine plus CAT (500 U/mL). Taken together, these results suggest that H₂O₂, or O₂^-, or both are able to induce apoptosis in cardiomyocytes.

Effect of ROS on the Expression of Apoptosis-Related Factors
The induction of apoptosis is associated with the expression and/or activation of specific proteins, resulting in the execution of the apoptotic program within the affected cells. In general, a plethora of different signaling pathways could be involved in apoptosis, depending on the stimulus and/or type of cells affected. To specify the signaling pathway in ROS-induced apoptosis in cardiomyocytes, we first determined the protein levels of well-known apoptosis-related factors such as Bcl-2, Bax, Bad, and p53 (Figure 3). Cardiomyocytes were treated with H₂O₂ or XO/X plus CAT to yield O₂^-. Both H₂O₂ and O₂^- induced the expression of p53, which was apparent by 1 to 2 hours after treatment. However, H₂O₂, but not O₂^-, was able to induce the expression of Bad. Surprisingly, neither H₂O₂, nor O₂^- led to detectable changes in protein levels of Bcl-2 or Bax throughout the investigated time interval of 8 hours (data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 3. Western blot analysis of p53 and Bad expression in cardiomyocytes exposed to 0.1 mmol/L H2O2 plus 0.1 mmol/L FeSO4 or 0.04 U/mL XO plus 0.1 mmol/L xanthine in presence of 500 U/mL CAT. Cells were treated for 1 hour and further cultured in normal culture medium until indicated time. Representative blots of at least 3 independent experiments are shown.

Differential Effect of H₂O₂ and O₂^- on Cytochrome c Release and Caspase Activation
Because increasing evidence recently indicated that mitochondrial cytochrome c release and subsequent CPP32 activation played an important role in the execution of apoptosis in a number of different cell types, we next determined the distribution of cytochrome c in cardiomyocytes stimulated with either H₂O₂ or O₂^-. At 1 to 2 hours after exposure to H₂O₂, cytochrome c appeared in the cytosol of cultured cardiomyocytes (Figure 4A). To exclude the possibility of a significant contamination of our cytosolic fraction with mitochondria, we reprobed our blots with an antibody directed against mitochondrial cytochrome oxidase (Figure 4A). Cytochrome oxidase was nearly undetectable throughout the investigated time interval. Release of cytochrome c may lead to the activation of CPP32, which can be detected by its cleavage from the inactive pro-CPP32 to the active 17-kDa product, as we observed 3 hours after exposure to H₂O₂ (Figure 4A). A 85-kDa fragment of PARP, also indicating CPP32 activation, became visible 3 hours after H₂O₂ treatment (Figure 4A). These data indicate that mitochondrial release of cytochrome c, with subsequent activation of caspase CPP32, is involved in H₂O₂-induced cardiomyocyte apoptosis.

View larger version (28K): [in this window] [in a new window]   Figure 4. Involvement of cytochrome c release and CPP32 activation in ROS-induced apoptosis. Levels of cytochrome c (cyt c) and cytochrome oxidase (cox) in cytosolic fractions, CPP32 activation, and PARP cleavage (as detected by immunoblot analysis) are depicted. Cardiomyocyte cultures were exposed to (A) 0.1 mmol/L H2O2 plus 0.1 mmol/L FeSO4 or to (B) 0.04 U/mL XO plus 0.1 mmol/L xanthine in presence of 500 U/mL CAT. Cells were treated for 1 hour and further cultured in normal culture medium until indicated time. Representative blots of at least 3 independent experiments are shown.

Intriguingly, O₂^--induced cardiomyocyte apoptosis was not accompanied by the release of cytochrome c, activation of CPP32, or cleavage of PARP (Figure 4B). However, Z-VAD-fmk, a pan-caspase inhibitor, could significantly inhibit O₂^--induced histone-associated DNA fragmentation (Figure 5A). This indicates that O₂^- uses caspase pathways other than CPP32 to induce apoptosis in cardiomyocytes. Using anti-Mch2 antibody revealed that Mch2 was activated, as indicated by the formation of p20 (the active form of Mch2) 2 hours after the stimulation of cardiomyocytes with XO/X plus CAT (Figure 5B). At the same time point, lamin A, which is a substrate of Mch2,^{18 19} was cleaved into a 46-kDa fragment in cardiomyocytes treated with XO/X plus CAT (Figure 5C). Z-VAD-fmk could inhibit Mch2 processing, thereby preventing the cleavage of lamin A. In addition, treatment with tiron prevented Mch2 activation and lamin A cleavage. Thus, it seems that O₂^- uses Mch2 to promote the apoptotic pathway involving the cleavage of lamin A.

View larger version (22K): [in this window] [in a new window]   Figure 5. Immunoblot detection of O2--induced Mch2 activation in cardiomyocytes. Cells were exposed to 0.04 U XO/mL plus 0.1 mmol/L xanthine in presence of 500 U/mL, as described in Figure 1. A, DNA fragmentation was assessed by cell-death ELISA. Z-VAD-fmk was added at indicated doses 2 hours before and immediately after treatment. *P<0.05 compared with XO/X+CAT alone. B, Immunoblot analysis of Mch2 and its active form p20 and (C) of lamin A and its cleavage product p46; Tiron was at 1 mmol/L and Z-VAD-fmk was at 50 µmol/L. Representative blots of at least 3 independent experiments are shown.

H₂O₂ Induces Translocation of Bax and Bad from Cytosol to Mitochondria and Their Interaction With Bcl-2
To identify the factors responsible for controlling cytochrome c release in cardiomyocytes, we prepared subcellular fractions before and after treatment with H₂O₂; these fractions included cytosol, mitochondria-enriched HM, or LM. Bad, Bax, and Bcl-2, all of which are involved in the regulation of cytochrome c release from mitochondria in apoptosis, were immunoprecipitated by their antibodies and then subjected to SDS-PAGE for immunoblot detection. Before H₂O₂ treatment, Bad and Bax were found predominantly in the cytosolic fractions, with only faint signals in mitochondria-enriched HM, and they were undetectable in LM. At 1 hour after H₂O₂ treatment, both Bad and Bax translocated from the cytosol to HM but not to LM. Before and after H₂O₂ treatment, Bcl-2 could be observed only in HM but not in the cytosolic or LM fractions (Figure 6A). In contrast to H₂O₂, O₂^- did not change the subcellular localization patterns of Bad, Bax, or Bcl-2 (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 6. Subcellular distribution of Bad, Bax, and Bcl-2 and interaction of Bad or Bax with Bcl-2 in cardiomyocytes that were treated for 1 hour with 0.1 mmol/L H2O2 plus 0.1 mmol/L FeSO4 and further cultured in normal culture medium until indicated time. A, After preparations of cytosolic fractions, HM and LM, Bad, Bax, and Bcl-2 were immunoprecipitated by their specific antibodies and then subjected to SDS-PAGE for immunoblot detection. B, Immunoprecipitations of Bad or Bax were carried out in heavy membrane fractions, and immunoprecipitated samples were analyzed by immunoblot with anti-Bcl-2 antibody. Representative blots of at least 3 independent experiments are shown.

To determine whether the translocation of Bad and Bax to the mitochondria involves the interaction of these 2 factors with Bcl-2 (a factor that inhibits apoptosis by preventing cytochrome c release^{15 20} ), immunoprecipitates of Bad or Bax were blotted against an anti-Bcl-2 antibody. Both Bad and Bax appeared in Bcl-2 complexes in cardiomyocytes 1 hour after stimulation with H₂O₂ (Figure 6B). As expected, no dimerization could be observed in cardiomyocytes stimulated by O₂^- (data not shown).

These data indicate that cytochrome c release in cardiomyocytes exposed to H₂O₂ is paralleled by the translocation of Bad and Bax from the cytosol to the mitochondria, in which they form heterodimers with the antiapoptotic Bcl-2, suggesting a functional role of these 2 factors in apoptosis-related cytochrome c release. The lack of their translocation to the mitochondria in O₂^--induced apoptosis may explain why we could not observe cytochrome c release in O₂^--stimulated cardiomyocytes.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Growing evidence shows that ROS play an important role in the pathogenesis of a variety of cardiac diseases. However, little information exists about the mechanisms by which ROS elicit their effect on the structure and function of the heart. The present work reveals that ROS induce apoptosis in cardiomyocytes and that different ROS may use distinct apoptotic pathways.

Several lines of evidence indicate a tight interrelationship between p53 and oxidative stress. Conformation and DNA binding activity of p53 are modulated by intracellular redox potential.²¹ Furthermore, a potent transactivator of p53 is Ref-1, a redox/repair protein.²² Additionally, overexpression of p53 leads to the transactivation of gene-encoding proteins that are able to respond to oxidative stress.²³ Our results indicate that, despite the fact that H₂O₂ and O₂^- induce very distinct apoptotic pathways in cardiomyocytes, both H₂O₂ and O₂^- led to the immediate expression of p53. p53 may directly induce apoptosis by the activation of the Bax gene, which contains p53-binding sites and encodes an apoptosis-inducing factor.²⁴ Strikingly, in both instances, p53 upregulation was not followed by the induction of Bax expression. Two possible explanations exist for this observation. First, although its expression is increased, p53 may not be functionally active in our model. This possibility is supported by a study that demonstrated p53-independent activation of p21^WAF1/CIP1 by oxidative stress.²⁵ Second, p53 may induce apoptosis independent of the transactivation of Bax, a possibility that has been observed in other cell-culture systems.²⁶ Bcl-2, a well-known antiapoptotic factor, inhibited apoptosis in rat cardiomyocytes overexpressing p53.²⁷ Neither H₂O₂ nor O₂^- induced significant changes in Bcl-2 expression. To determine the functional activity of the Bcl-2 family members, we investigated the release of cytochrome c, which is tightly regulated by the equilibrium between antiapoptotic Bcl-2 and proapoptotic Bad and Bax.^{15 20} Cytochrome c participates in the execution of apoptosis.^{28 29} Our data show that cytochrome c is released in H₂O₂-induced cardiomyocyte apoptosis, implying a predominant activity of proapoptotic members of the Bcl-2 family. Therefore, we detected the subcellular distribution of Bax and Bad, which we found migrated from the cytosol to mitochondria. This led us to speculate that they may form heterodimers with Bcl-2 to counteract its antiapoptotic function. This assumption was confirmed by the appearance of Bad and Bax in Bcl-2 complexes in cardiomyocytes exposed to H₂O₂. Thus, the redistribution of Bad and Bax and their interactions with Bcl-2 may play an important role in regulating cytochrome c release, which sub-sequently leads to the activation of CPP32 and cleavage of PARP. This indicates the existence of cytochrome c–related apoptotic pathways in cardiomyocytes. However, as observed in the present study, O₂^--induced apoptosis was not accompanied by cytochrome c release, suggesting that there also are other cytochrome c–independent pathways of apoptosis in cardiomyocytes. O₂^- uses a pathway that includes Mch2 activation. The precise substrate specificity of different caspase family members is not yet clear. Mch2 is the only known laminase.^{18 19} It also can cleave PARP in vitro, but such an activity occurs infrequently.¹⁸ This may explain why the activation of Mch2 is not accompanied by PARP cleavage in our present study.

Apoptosis occurs in the heart during processes associated with the production and release of ROS, including ischemia and reperfusion.⁷ However, until now, only a few factors could be identified that triggered apoptosis in the heart during these and other processes. Our data provide the first link between pathophysiological events resulting in the production of ROS on one side, and the induction of apoptosis in cardiomyocytes on the other.

ROS elicit an array of damages to the cell, including membrane lipid peroxidation, cross-linking and degradation of proteins, and the nicking of DNA, resulting in the impairment of cellular integrity and function. Thus, one might speculate that the induction of apoptosis in cardiomyocytes exposed to ROS represents an evolutionarily-conserved protective mechanism of disposing of those cells that do not conform and function properly and, thus, might put cardiac function and integrity at risk.

In summary, our study identified ROS as potential inducers of cardiomyocyte apoptosis. H₂O₂ and O₂^- trigger distinct apoptotic signaling pathways in cardiomyocytes, including the release of cytochrome c and the activation of CPP32 by H₂O₂ and Mch2 activation by O₂^-. Future studies are needed to determine whether the cytochrome c/CPP32 signaling pathway is triggered by other apoptotic stimuli in the heart and whether the specific inhibition of this pathway prevents cardiomyocyte apoptosis in vivo.

Received October 16, 1998; revision received February 23, 1999; accepted March 16, 1999.

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