Csx/Nkx2-5 is required for homeostasis and survival of cardiac myocytes in the adult heart

Csx/Nkx2-5, which is essential for cardiac development of the embryo, is abundantly expressed in the adult heart. We here examined the role of Csx/Nkx2-5 in the adult heart using two kinds of transgenic mice. Transgenic mice that overexpress a dominant negative mutant of Csx/Nkx2-5 (DN-TG mice) showed degeneration of cardiac myocytes and impairment of cardiac function. Doxorubicin induced more marked cardiac dysfunction in DN-TG mice and less in transgenic mice that overexpress wild type Csx/Nkx2-5 (WT-TG mice) compared with non-transgenic mice. Doxorubicin induced cardiomyocyte apoptosis, and the number of apoptotic cardiomyocytes was high in the order of DN-TG mice, non-transgenic mice, and WT-TG mice. Overexpression of the dominant negative mutant of Csx/Nkx2-5 induced apoptosis in cultured cardiomyocytes, while expression of wild type Csx/Nkx2-5 protected cardiomyocytes from doxorubicin-induced apoptotic death. These results suggest that Csx/Nkx2-5 plays a critical role in maintaining highly differentiated cardiac phenotype and in protecting the heart from stresses including doxorubicin.     

Overexpression of Nd1, a novel Kelch family protein, in the heart of transgenic mice protects against doxorubicin-induced cardiomyopathy

Doxorubicin is one of the most effective drugs available for cancer chemotherapy. However, the clinical use of doxorubicin has been greatly limited because of severe side effects on cardiomyocytes. Since Nd1-L, a novel actin-binding protein, is expressed most abundantly in the heart of adult mice, we examined a role of Nd1-L in doxorubicin-induced cardiomyopathy. When doxorubicin (5 mg/kg × 4 times) was injected into adult mice at a 3-day-interval, approximately 50% of injected mice died within 4 weeks of the first injection. Nd1-L mRNA expression in the heart decreased within 3 weeks after the first injection and many cardiomyocytes of injected mice died by apoptosis. Overexpression of Nd1-L in the heart of transgenic mice protected the cardiomyocytes from apoptosis and improved survival rate after doxorubicin injection. Furthermore, activation of Erk1/2 was observed in cultured cells overexpressing Nd1-L. Thus, Nd1-L plays a critical role in protecting the heart from doxorubicin-induced cardiomyopathy.     

NADPH oxidases participate to doxorubicin-induced cardiac myocyte apoptosis

Cumulative doses of doxorubicin, a potent anticancer drug, lead to serious myocardial dysfunction. Numerous mechanisms including apoptosis have been proposed to account for its cardiotoxicity. Cardiac apoptosis induced by doxorubicin has been related to excessive reactive oxygen species production by the mitochondrial NADH dehydrogenase. Here, we explored whether doxorubicin treatment activates other superoxide anion generating systems such as the NADPH oxidases, membrane-embedded flavin-containing enzymes, and whether the subsequent oxidative stress contributes to apoptosis. We showed that doxorubicin treatment of rat cardiomyoblasts H9c2 triggers increases in caspase-3 like activity and hypoploid cells, both common features of apoptosis. Doxorubicin exposure also leads to a rapid superoxide production through NADPH oxidase activation. Inhibition of these enzymes using diphenyliodonium and apocynin reduces doxorubicin-induced reactive oxygen species production, caspase-3 like activity and sub-G1 cell population. In conclusion, NADPH oxidases participate to doxorubicin-induced cardiac apoptosis.     

Melatonin as an effective protector against doxorubicin-induced cardiotoxicity

The present study was designed to explore the protective effects of melatonin and its analogs, 6-hydroxymelatonin and 8-methoxy-2-propionamidotetralin, on the survival of doxorubicin-treated mice and on doxorubicin-induced cardiac dysfunction, ultrastructural alterations, and apoptosis in mouse hearts. Whereas 60% of the mice treated with doxorubicin (25 mg/kg ip) died in 5 days, almost all the doxorubicin-treated mice survived when melatonin or 6-hydroxymelatonin (10 mg/l) was administered in their drinking water. Perfusion of mouse hearts with 5 microM doxorubicin for 60 min led to a 50% suppression of heart rate x left ventricular developed pressure and a 50% reduction of coronary flow. Exposure of hearts to 1 microM melatonin or 6-hydroxymelatonin reversed doxorubicin-induced cardiac dysfunction. 8-Methoxy-2-propionamidotetralin had no protective effects on animal survival and on in vitro cardiac function. Infusion of melatonin or 6-hydroxymelatonin (2.5 microg/h) significantly attenuated doxorubicin-induced cardiac dysfunction, ultrastructural alterations, and apoptosis in mouse hearts. Neither melatonin nor 6-hydroxymelatonin compromised the antitumor activity of doxorubicin in cultured PC-3 cells. These results suggest that melatonin protect against doxorubicin-induced cardiotoxicity without interfering with its antitumor effect.     

Bicarbonate exacerbates oxidative injury induced by antitumor antibiotic doxorubicin in cardiomyocytes

Doxorubicin, a broad-spectrum antitumor antibiotic, causes dose-dependent cardiomyopathy and heart failure. Although the exact molecular mechanisms of cardiotoxicity are not well established, oxidative mechanisms involving doxorubicin-induced superoxide anion production have been proposed. In this study, we show that bicarbonate, a physiologically relevant tissue component, greatly amplified doxorubicin-induced cardiomyocyte injury. Bicarbonate also enhanced inactivation of aconitase, a crucial tricarboxylic acid cycle enzyme, in cardiomyocytes exposed to doxorubicin. The cell-permeable superoxide dismutase mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin, reversed doxorubicin-induced cardiomyocyte injury. Bicarbonate enhanced the inactivation of purified mitochondrial aconitase in the xanthine/xanthine oxidase system, generating superoxide. The results suggest that bicarbonate amplifies the prooxidant effect of superoxide. Bicarbonate also caused an increased loading of cardiomyocytes with doxorubicin. We conclude that the bicarbonate-mediated increase in doxorubicin toxicity is due to increased intracellular loading of doxorubicin in cardiomyocytes and subsequent exacerbation of superoxide-mediated cardiomyocyte injury.     

LncRNA FOXC2-AS1 protects cardiomyocytes from doxorubicin-induced cardiotoxicity through activation of WNT1-inducible signaling pathway protein-1

Doxorubicin (Dox) is an anthracycline antibiotic that has been used to treat different cancers. Dox-induced cardiotoxicity is common in clinical practice, while its mechanism is unknown. It has been proved that lncRNA FOXC2-AS1 may promote doxorubicin resistance and WNT1-inducible signaling pathway protein-1 (WISP1) blocks doxorubicin-induced cardiomyocyte death. Our study aimed to investigate the involvement of lncRNA FOXC2-AS1 and WISP1 in doxorubicin-induced cardiotoxicity and to explore their interactions. In our study we observed that FOXC2-AS1 and WISP1 mRNA were downregulated in heart tissues of mice with Dox-induced cardiotoxicity. FOXC2-AS1 and WISP1 mRNA expression were positively correlated in mice with Dox-induced cardiotoxicity but not in healthy mice. Overexpression of FOXC2-AS1 promoted to viability of mice cardiomyocytes under Dox treatment and also increased the expression level of WISP1. In contrast, WISP1 overexpression showed no significant effect on FOXC2-AS1. We therefore conclude that lncRNA FOXC2-AS1 may upregulate WISP1 to protect cardiomyocytes from doxorubicin-induced cardiotoxicity.     

Involvement of Yes‐associated protein 1 (YAP1) in doxorubicin‐induced cytotoxicity in H9c2 cardiac cells

Cardiac cell death is one of the major events implicated in doxorubicin‐induced cardiotoxicity, which leads to heart failure. We recently reported that Yes‐associated protein 1 (YAP1) regulates cell survival and apoptosis. However, it is unclear whether YAP1 regulates doxorubicin‐induced cell death in cardiomyocytes. We investigated whether YAP1 is involved in doxorubicin‐induced cell death using H9c2 cardiac cells and mouse heart. In an in vivo study, YAP1 protein expression was significantly decreased in hearts of doxorubicin‐treated mice with increased caspase‐3 activation. Doxorubicin also caused cell death by increasing caspase‐3 activation in H9c2 cells. Doxorubicin reduced YAP1 protein expression and messenger RNA expression accompanied by increased phosphorylation of YAP1 at Ser127. Doxorubicin further increased cell death with increased caspase‐3/7 activation in the absence of YAP1 when compared with doxorubicin or siYAP1 treatment alone. Overexpression of constitutively active YAP1 (YAP1–5SA) using an adenovirus gene transfer technique significantly reversed doxorubicin‐induced cell death by decreasing caspase‐3/7 activation in H9c2 cells. Akt, a potential prosurvival factor, decreased in doxorubicin‐ and YAP1 short interfering RNA (siRNA)‐treated cells. Doxorubicin further significantly decreased Akt protein expression when YAP1 was silenced. Overexpression of YAP1 canceled decreased Akt protein expression induced by doxorubicin treatment in H9c2 cells. In conclusion, these results suggest that doxorubicin‐induced cardiac cell death is mediated in part by down‐regulation of YAP1 and YAP1‐targeted gene, Akt. Modulating YAP1 and its related Hippo pathway on local cardiomyocytes may be a promising therapeutic approach for doxorubicin‐induced cardiotoxicity.     

Differences in doxorubicin-induced apoptotic signaling in adult and immature cardiomyocytes

A proposed mechanism for the cardiotoxicity of doxorubicin (DOX) involves apoptosis in cardiomyocytes. In the study described here, we investigated the molecular basis for the differences in DOX-induced toxicity in adult rat cardiomyocytes (ARCM), neonatal rat cardiomyocytes (NRCM), and rat embryonic H9c2 cardiomyoblasts. Activation of caspase-9 and -3 was considerably lower in DOX-treated ARCM as compared with NRCM and H9c2 cardiomyoblasts. Addition of cytochrome c caused the activation of caspase-9 and -3 in permeabilized NRCM and H9c2 cardiomyoblasts but not in permeabilized ARCM. Expression of proapoptotic proteins, apoptotic protease activating factor-1 (Apaf1), and procaspase-9 was significantly lower, and abundance of antiapoptotic X-linked inhibitor of apoptosis protein (XIAP) was higher in ARCM, as compared with immature cardiac cells. Despite the abundance of XIAP in ARCM, its role in the inhibition of apoptosome function was dismissed, as second mitochondria-derived activator of caspases (Smac)-N7 peptide, had no effect on caspase activation in response to cytochrome c in these cells. Adenoviral expression of Apaf1 exacerbated the activation of caspase-9 and -3 in DOX-treated NRCM, but did not increase their activities in DOX-treated ARCM. This finding points to a major difference in the apoptotic signaling between immature and adult cardiomyocytes. The mitochondrial apoptotic pathway is limited in ARCM treated with DOX.     

Suppression by metallothionein of doxorubicin-induced cardiomyocyte apoptosis through inhibition of p38 mitogen-activated protein kinases

Cardiomyopathy induced by doxorubicin (DOX) has long been a major impediment of clinical applications of this effective anticancer agent. Previous studies have shown that cardiac-specific metallothionein (MT)-overexpressing transgenic mice are highly resistant to DOX-induced cardiotoxicity. To investigate cellular and molecular mechanisms by which MT participates in this cytoprotection, transgenic mice containing high levels of cardiac MT and non-transgenic controls were treated intraperitoneally with DOX at a single dose of 15 mg/kg and sacrificed on the 4th day after treatment. Myocardial apoptosis was detected by a terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay and confirmed by electron microscopy of immunogold staining of apoptotic nuclei. Dual staining of cardiac alpha-sarcomeric actin using an immunohistochemical method further identified apoptotic myocytes. Apoptosis was significantly inhibited in the transgenic myocardium. The anti-apoptotic effect of MT was further revealed in primary cultures of neonatal mouse cardiomyocytes. Furthermore, DOX activated p38 mitogen-activated protein kinase (MAPK), which was critically involved in the apoptotic process, as demonstrated by inhibition of DOX-induced apoptosis by a p38-specific inhibitor, SB203580. Both DOX-induced p38 MAPK activation and apoptosis were dramatically inhibited in the transgenic cardiomyocytes. The results thus demonstrate that DOX induces apoptosis in cardiomyocytes both in vivo and in vitro and MT suppresses this effect through at least in part inhibition of p38 MAPK activation.     

Differences in doxorubicin-induced apoptotic signaling in adult and immature cardiomyocytes

A proposed mechanism for the cardiotoxicity of doxorubicin (DOX) involves apoptosis in cardiomyocytes. In the study described here, we investigated the molecular basis for the differences in DOX-induced toxicity in adult rat cardiomyocytes (ARCM), neonatal rat cardiomyocytes (NRCM), and rat embryonic H9c2 cardiomyoblasts. Activation of caspase-9 and -3 was considerably lower in DOX-treated ARCM as compared with NRCM and H9c2 cardiomyoblasts. Addition of cytochrome c caused the activation of caspase-9 and -3 in permeabilized NRCM and H9c2 cardiomyoblasts but not in permeabilized ARCM. Expression of proapoptotic proteins, apoptotic protease activating factor-1 (Apaf1), and procaspase-9 was significantly lower, and abundance of antiapoptotic X-linked inhibitor of apoptosis protein (XIAP) was higher in ARCM, as compared with immature cardiac cells. Despite the abundance of XIAP in ARCM, its role in the inhibition of apoptosome function was dismissed, as second mitochondria-derived activator of caspases (Smac)-N7 peptide, had no effect on caspase activation in response to cytochrome c in these cells. Adenoviral expression of Apaf1 exacerbated the activation of caspase-9 and -3 in DOX-treated NRCM, but did not increase their activities in DOX-treated ARCM. This finding points to a major difference in the apoptotic signaling between immature and adult cardiomyocytes. The mitochondrial apoptotic pathway is limited in ARCM treated with DOX.     

ERKs/p53 signal transduction pathway is involved in doxorubicin-induced apoptosis in H9c2 cells and cardiomyocytes

The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine whether extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, annexin V-fluorescein isothiocyanate, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at 6 h, and these changes are associated decreased expression of Bcl-2, increases in Bax and p53 upregulated modulator of apoptosis-alpha expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at Ser15 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U-0126 and p53 inhibitor pifithrin (PFT)-alpha, which abrogated the changes in Bcl-2 family proteins and cell death produced by doxorubicin. U-0126 blocked the phosphorylation and nuclear translocation of both ERK1/2 and p53, whereas PFT-alpha blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2-p53, PARP, and caspase-3 in neonatal rat cultured cardiomyocytes. Thus we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity.     

Metformin protects against doxorubicin-induced cardiotoxicity: involvement of the adiponectin cardiac system

Doxorubicin has cardiotoxic effects that limit its clinical benefit in cancer patients. Metformin exerts cardioprotective actions via AMP-activated protein kinase (AMPK) and increases the expression of adiponectin and its receptors (adipoR1 and adipoR2) in skeletal muscle and adipose tissue, but its effect on cardiac tissue is still unknown. This work aimed to study whether metformin exerts any protective action against the cardiotoxicity of doxorubicin and whether the cardiac system of adiponectin is involved in any such action. The addition of doxorubicin (5μM) to adult mouse cardiomyocytes (HL-1 cell line) induced apoptosis, which was characterized by a loss of cell viability, activation of caspases, and fragmentation of the genetic material. Doxorubicin treatment also caused a decrease in the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase. Pretreatment with metformin (4mM, 24h) provided protection against doxorubicin-induced damage. This pretreatment significantly increased cell viability, attenuated the activation of caspases and the fragmentation of genetic material, and restored the antioxidant activity. In addition, metformin up-regulated the expression of adiponectin and its receptors, adipoR1 and adipoR2, in cardiomyocytes. In contrast, silencing either adipoR1 or adipoR2 with siRNA inhibited the AMPK activation and the protective effects of metformin. Taken together, these results demonstrate that metformin protects cardiomyocytes from doxorubicin-induced damage and that the cardiac adiponectin system plays an important role in this protective action.     

Doxorubicin Impairs the Insulin-Like Growth Factor-1 System and Causes Insulin-Like Growth Factor-1 Resistance in Cardiomyocytes

BackgroundInsulin-like growth factor-1 (IGF-1) promotes the survival of cardiomyocytes by activating type 1 IGF receptor (IGF-1R). Within the myocardium, IGF-1 action is modulated by IGF binding protein-3 (IGFBP-3), which sequesters IGF-1 away from IGF-1R. Since cardiomyocyte apoptosis is implicated in anthracycline cardiotoxicity, we investigated the effects of the anthracycline, doxorubicin, on the IGF-1 system in H9c2 cardiomyocytes.ConclusionsDoxorubicin down-regulates IGF-1R and up-regulates IGFBP-3 via p53 and oxidative stress in H9c2 cells. This leads to resistance to IGF-1 that may contribute to doxorubicin-initiated apoptosis. Further studies are needed to confirm these findings in human cardiomyocytes and explore the possibility of manipulating the IGF-1 axis to protect against anthracycline cardiotoxicity.     

Doxorubicin requires the sequential activation of caspase-2, protein kinase Cdelta, and c-Jun NH2-terminal kinase to induce apoptosis

Here, we identified caspase-2, protein kinase C (PKC)delta, and c-Jun NH2-terminal kinase (JNK) as key components of the doxorubicin-induced apoptotic cascade. Using cells stably transfected with an antisense construct for caspase-2 (AS2) as well as a chemical caspase-2 inhibitor, we demonstrate that caspase-2 is required in doxorubicin-induced apoptosis. We also identified PKCdelta as a novel caspase-2 substrate. PKCdelta was cleaved/activated in a caspase-2-dependent manner after doxorubicin treatment both in cells and in vitro. PKCdelta is furthermore required for efficient doxorubicin-induced apoptosis because its chemical inhibition as well as adenoviral expression of a kinase dead (KD) mutant of PKCdelta severely attenuated doxorubicin-induced apoptosis. Furthermore, PKCdelta and JNK inhibition show that PKCdelta lies upstream of JNK in doxorubicin-induced death. Jnk-deficient mouse embryo fibroblasts (MEFs) were highly resistant to doxorubicin compared with wild type (WT), as were WT Jurkat cells treated with SP600125, further supporting the importance of JNK in doxorubicin-induced apoptosis. Chemical inhibitors for PKCdelta and JNK do not synergize and do not function in doxorubicin-treated AS2 cells. Caspase-2, PKCdelta, and JNK were furthermore implicated in doxorubicin-induced apoptosis of primary acute lymphoblastic leukemia blasts. The data thus support a sequential model involving caspase-2, PKCdelta, and JNK signaling in response to doxorubicin, leading to the activation of Bak and execution of apoptosis.     

Cardiomyocyte specific ablation of p53 is not sufficient to block doxorubicin induced cardiac fibrosis and associated cytoskeletal changes

Doxorubicin (Dox) is an anthracycline used to effectively treat several forms of cancer. Unfortunately, the use of Dox is limited due to its association with cardiovascular complications which are manifested as acute and chronic cardiotoxicity. The pathophysiological mechanism of Dox induced cardiotoxicity appears to involve increased expression of the tumor suppressor protein p53 in cardiomyocytes, followed by cellular apoptosis. It is not known whether downregulation of p53 expression in cardiomyocytes would result in decreased rates of myocardial fibrosis which occurs in response to cardiomyocyte loss. Further, it is not known whether Dox can induce perivascular necrosis and associated fibrosis in the heart. In this study we measured the effects of acute Dox treatment on myocardial and perivascular apoptosis and fibrosis in a conditional knockout (CKO) mouse model system which harbours inactive p53 alleles specifically in cardiomyocytes. CKO mice treated with a single dose of Dox (20 mg/kg), did not display lower levels of myocardial apoptosis or reactive oxygen and nitrogen species (ROS/RNS) compared to control mice with intact p53 alleles. Interestingly, CKO mice also displayed higher levels of interstitial and perivascular fibrosis compared to controls 3 or 7 days after Dox treatment. Additionally, the decrease in levels of the microtubule protein α-tubulin, which occurs in response to Dox treatment, was not prevented in CKO mice. Overall, these results indicate that selective loss of p53 in cardiomyocytes is not sufficient to prevent Dox induced myocardial ROS/RNS generation, apoptosis, interstitial fibrosis and perivascular fibrosis. Further, these results support a role for p53 independent apoptotic pathways leading to Dox induced myocardial damage and highlight the importance of vascular lesions in Dox induced cardiotoxicity.     

Involvement of ferritin heavy chain in the preventive effect of metformin against doxorubicin-induced cardiotoxicity

Doxorubicin is a wide-spectrum chemotherapeutic agent, although a cumulative dose may cause cardiac damage and lead to heart failure. Doxorubicin cardiotoxicity is dependent on the intracellular iron pool and manifests itself by increasing oxidative stress. Our group has recently shown the ability of metformin, an oral antidiabetic with cardiovascular benefits, to protect cardiomyocytes from doxorubicin-induced damage. This work aimed to study whether metformin is able to modulate the expression of ferritin, the major intracellular iron storage protein, in cardiomyocytes and whether it is involved in their protection. The addition of metformin to adult mouse cardiomyocytes (HL-1 cell line) induced both gene and protein expression of the ferritin heavy chain (FHC) in a time-dependent manner. The silencing of FHC expression with siRNAs inhibited the ability of metformin to protect cardiomyocytes from doxorubicin-induced damage, in terms of the percentage of cell viability, the levels of reactive oxygen species, and the activity of antioxidant enzymes (catalase, glutathione peroxidase, and superoxide dismutase). In addition, metformin induced the activation of NF-κB in HL-1 cells, whereas preincubation with SN50, an inhibitor of NF-κB, blocked the upregulation of the FHC and the protective effect mediated by metformin. Taken together, these results provide new knowledge on the protective actions of metformin against doxorubicin-induced cardiotoxicity by identifying FHC and NF-κB as the major mediators of this beneficial effect.     

Inhibition of hypoxia/reoxygenation-induced apoptosis in metallothionein-overexpressing cardiomyocytes

To study possible mechanisms for metallothionein (MT) inhibition of ischemia-reperfusion-induced myocardial injury, cardiomyocytes isolated from MT-overexpressing transgenic neonatal mouse hearts and nontransgenic controls were subjected to 4 h of hypoxia (5% CO2-95% N2, glucose-free modified Tyrode's solution) followed by 1 h of reoxygenation in MEM + 20% fetal bovine serum (FBS) (5% CO2-95% air), and cytochrome c-mediated caspase-3 activation apoptotic pathway was determined. Hypoxia/reoxygenation-induced apoptosis was significantly suppressed in MT-overexpressing cardiomyocytes, as measured by both terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling and annexin V-FITC binding. In association with apoptosis, mitochondrial cytochrome c release, as determined by Western blot, was observed to occur in nontransgenic cardiomyocytes. Correspondingly, caspase-3 was activated as determined by laser confocal microscopic examination with the use of FITC-conjugated antibody against active caspase-3 and by enzymatic assay. The activation of this apoptotic pathway was significantly inhibited in MT-overexpressing cells, as evidenced by both suppression of cytochrome c release and inhibition of caspase-3 activation. The results demonstrate that MT suppresses hypoxia/reoxygenation-induced cardiomyocyte apoptosis through, at least in part, inhibition of cytochrome c-mediated caspase-3 activation.