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

ABSTRACT: 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.

SUBMITTER: Konorev EA

PROVIDER: S-EPMC3039518 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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

Konorev Eugene A EA Vanamala Sravan S Kalyanaraman Balaraman B

Free radical biology & medicine 20080920 12

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 casp ...[more]

PMID: 18926904

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Project description:BackgroundDoxorubicin, an anthracycline chemotherapeutic known to incur heart damage, decreases heart function in up to 11% of patients. Recent investigations have implicated the Wnt signaling cascade as a key modulator of cardiac tissue repair after myocardial infarction. Wnt upregulation in murine models resulted in stimulation of angiogenesis and suppression of fibrosis after ischemic insult. However, the molecular mechanisms of Wnt in mitigating doxorubicin-induced cardiac insult require further investigation. Identifying cardioprotective mechanisms of Wnt is imperative to reducing debilitating cardiovascular adverse events in oncologic patients undergoing treatment.MethodsExposing human cardiomyocyte AC16 cells to varying concentrations of Wnt10b and DOX, we observed key metrics of cell viability. To assess the viability and apoptotic rates, we utilized MTT and TUNEL assays. We quantified cell and mitochondrial membrane stability via LDH release and JC-1 staining. To investigate how Wnt10b mitigates doxorubicin-induced apoptosis, we introduced pharmacologic inhibitors of key enzymes involved in apoptosis: FR180204 and SB203580, ERK1/2 and p38 inhibitors. Further, we quantified apoptotic executor enzymes, caspase 3/7, via immunofluorescence.ResultsAC16 cells exposed solely to doxorubicin were shrunken with distorted morphology. Cardioprotective effects of Wnt10b were demonstrated via a reduction in apoptosis, from 70.1% to 50.1%. LDH release was also reduced between doxorubicin and combination groups from 2.27-fold to 1.56-fold relative to the healthy AC16 control group. Mitochondrial membrane stability was increased from 0.67-fold in the doxorubicin group to 5.73 in co-treated groups relative to control. Apoptotic protein expression was stifled by Wnt10b, with caspase3/7 expression reduced from 2.4- to 1.3-fold, and both a 20% decrease in p38 and 40% increase in ERK1/2 activity.ConclusionOur data with the AC16 cell model demonstrates that Wnt10b provides defense mechanisms against doxorubicin-induced cardiotoxicity and apoptosis. Further, we explain a mechanism of this beneficial effect involving the mitochondria through simultaneous suppression of pro-apoptotic p38 and anti-apoptotic ERK1/2 activities.

Project description:BACKGROUND:Doxorubicin (Dox) is a chemotherapy drug with limited application due to cardiotoxicity that may progress to heart failure. This study aims to evaluate the role of cardiomyocytes derived from mouse embryonic stem cells (CM-mESCs) in the treatment of Dox-induced cardiomyopathy (DIC) in mice. METHODS:The mouse embryonic stem cell (mESC) line E14TG2A was characterized by karyotype analysis, gene expression using RT-PCR and immunofluorescence. Cells were transduced with luciferase 2 and submitted to cardiac differentiation. Total conditioned medium (TCM) from the CM-mESCs was collected for proteomic analysis. To establish DIC in CD1 mice, Dox (7.5 mg/kg) was administered once a week for 3 weeks, resulting in a cumulative Dox dose of 22.5 mg/kg. At the fourth week, a group of animals was injected intramyocardially with CM-mESCs (8 × 105 cells). Cells were tracked by a bioluminescence assay, and the body weight, echocardiogram, electrocardiogram and number of apoptotic cardiomyocytes were evaluated. RESULTS:mESCs exhibited a normal karyotype and expressed pluripotent markers. Proteomic analysis of TCM showed proteins related to the negative regulation of cell death. CM-mESCs presented ventricular action potential characteristics. Mice that received Dox developed heart failure and showed significant differences in body weight, ejection fraction (EF), end-systolic volume (ESV), stroke volume (SV), heart rate and QT and corrected QT (QTc) intervals when compared to the control group. After cell or placebo injection, the Dox + CM-mESC group showed significant increases in EF and SV when compared to the Dox + placebo group. Reduction in ESV and QT and QTc intervals in Dox + CM-mESC-treated mice was observed at 5 or 30 days after cell treatment. Cells were detected up to 11 days after injection. The Dox + CM-mESC group showed a significant reduction in the percentage of apoptotic cardiomyocytes in the hearts of mice when compared to the Dox + placebo group. CONCLUSIONS:CM-mESC transplantation improves cardiac function in mice with DIC.

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

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

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