Hsp10 and Hsp60 modulate Bcl-2 family and mitochondria apoptosis signaling induced by doxorubicin in cardiac muscle cells

doi:10.1016/S0022-2828(03)00229-3

Journal of Molecular and Cellular Cardiology

Volume 35, Issue 9, September 2003, Pages 1135-1143

https://doi.org/10.1016/S0022-2828(03)00229-3 Get rights and content

Abstract

The development of doxorubicin cardiomyopathy involves apoptosis of cardiac muscle cells. This study was carried out to define the roles of two heat-shock proteins, Hsp10 and Hsp60, on doxorubicin-induced apoptosis in primary cardiomyocytes. Doxorubicin induces apoptosis of cardiomyocytes by activating mitochondria apoptosis signaling. Transducing cardiomyocytes with Hsp10 or Hsp60 with adenoviral vector suppressed the occurrence of apoptosis in the doxorubicin-treated cardiomyocytes. Overexpression of Hsp10 and Hsp60 increased the abundance of the anti-apoptotic Bcl-xl and Bcl-2, and reduced the protein content of the pro-apoptotic Bax. Hsp60 overexpression also significantly reduced doxorubicin induction of Bad, whereas overexpression of Hsp10 did not alter the expression of Bad in the doxorubicin-treated cells. Overexpression of Hsp10 and Hsp60, respectively, stabilized mitochondrial cross-membrane potential, inhibited Caspase 3, and suppressed PARP. These findings indicate that overexpression of Hsp10 and Hsp60 differentially modulated Bcl-2 family and in turn attenuate doxorubicin-induced cardiac muscle death. The effects of Hsp10 and Hsp60 on Bcl-2 family could not be explained by the abundance of Bcl-2 family mRNA levels. Hsp60 interacted with Bcl-xl and Bax in the cardiomyocytes in vivo. The effect of Hsp10 and Hsp60 on the abundance of Bcl-xl could not be blocked by cycloheximide. Moreover, Hsp10 and Hsp60 inhibited ubiquitination of Bcl-xl. These findings suggest that Hsp10 and Hsp60 modulated post-translational modification of Bcl-xl. Antisense Hsp60 reduced the abundance of endogenous Hsp60 in cardiomyocytes and amplified the cytotoxicity of doxorubicin. These data provide a novel link between Hsp10/Hsp60 and cardiac protection in doxorubicin cardiomyopathy.

Introduction

Recent studies have suggested an anti-apoptosis role of two heat-shock proteins, Hsp10 and Hsp60, in various types of cells. These two proteins can be induced when cells are under stress [1], [2]. Hsp60 expression is induced in the myocardium that underwent atrial fibrillation, and in the brain during cerebral ischemia [2], [3]. Hsp60 and Hsp10 may form mitochondrial-chaperoning complexes and are believed to play a role in maintaining normal mitochondrial function [4]. Overexpression of Hsp10 and Hsp60 inhibits myocardial apoptosis in response to ischemic injury [5]. A recent study showed that reducing Hsp60 expression with antisense oligonucleotides is associated with an increase in Bax and a reduction in Bcl-2, and induces apoptosis of cardiomyocytes, thus raising the possibility that Hsp60 may regulate apoptosis through modulation of Bcl-2 family [6].

Doxorubicin is a commonly used chemotherapy agent that also causes heart failure [7]. We and other laboratories have shown that doxorubicin induces cardiomyocytes apoptosis and causes cardiomyopathy in experimental animals [8], [9], [10]. Since myocardial apoptosis contributes to the development of myocardial dysfunction in heart failure, inhibition of doxorubicin-induced cardiomyocytes apoptosis may provide new opportunities for the prevention and treatment of doxorubicin-induced heart failure. It is not yet known whether Hsp60 and Hsp10 can modulate doxorubicin-induced cardiomyocytes apoptosis. The aims of this study were to investigate whether overexpression of Hsp10 and Hsp60 can modulate the abundance of Bcl-2 protein family and suppress activation of mitochondria-mediated apoptosis signaling in the cardiac muscle cells treated with doxorubicin. The results show that transduction of Hsp10 and Hsp60 independently modulated the abundance of Bcl-2 protein family and suppressed induction of apoptosis signaling in the cardiac muscle cells. These experiments also provide evidence that Hsp10 and Hsp60 may modulate the abundance of Bcl-2 protein family through post-translational modification. These data revealed novel anti-apoptosis effects of Hsp10 and Hsp60 in an experimental model of doxorubicin cardiomyopathy, and provide new insight into the biological role of Hsp10 and Hsp60 in cardiac muscle cells.

Section snippets

Materials

Mouse anti-Hsp60 monoclonal antibody and rabbit anti-Cpn10 (Hsp10) peptide polyclonal antibody were purchased from StressGen Biotechnologies Crop. (Victoria, BC Canada). Monoclonal Bcl-2, polyclonal Bad, monoclonal Bcl-xl, monoclonal Bax, monoclonal ubiquitin, and polyclonal β-actin antibodies and anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) were from Santa Cruz Biolabs (Santa Cruz, CA). Cleaved Caspase-3 (Asp175) and cleaved PARP (D214) antibodies were from

Overexpression of Hsp10 and Hsp60 independently suppressed apoptosis of cardiomyocytes induced by doxorubicin

Our laboratory has shown that doxorubicin induces apoptosis of cardiomyocytes [8]. To determine whether Hsp10 and Hsp60 can suppress apoptosis in cardiomyocytes, Hsp10 and Hsp60 were overexpressed in neonatal cardiomyocytes via adenoviral vectors. Hsp10 and Hsp60 were, respectively, overexpressed in the cardiomyocytes transduced with the adenovirus carrying Hsp10 or Hsp60, whereas the expression of Hsp10 and Hsp60 was not altered in the cells transduced with the control virus (Fig. 1). To

Discussion

This study defined the effects of Hsp10 and Hsp60 on critical steps of apoptosis signaling triggered by doxorubicin in cardiomyocytes. There are growing interests in the role of HSP in cardiac muscle biology [1]. HSPs were originally discovered as a set of proteins induced by temperature increase, but recent studies have identified an array of biological actions for HSPs, including protein folding/unfolding, protein degradation, anti-oxidative stress, and anti-apoptosis. Despite growing

Acknowledgements

This work is supported by grants from NHLBI and NIDDK (to P.H.W.).

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Original ArticleHsp10 and Hsp60 modulate Bcl-2 family and mitochondria apoptosis signaling induced by doxorubicin in cardiac muscle cells

Abstract

Introduction

Section snippets

Materials

Overexpression of Hsp10 and Hsp60 independently suppressed apoptosis of cardiomyocytes induced by doxorubicin

Discussion

Acknowledgements

Brain Res Mol Brain Res

Semin Oncol

J Biol Chem

Method Enzymol

Exp Cell Res

J Biol Chem

J Am Coll Cardiol

Heat shock proteins and cardiac protection

Cardiovasc Res

The expression of heat shock protein 60 in myocardium of patients with chronic atrial fibrillation

Basic Res Cardiol

Original Article
Hsp10 and Hsp60 modulate Bcl-2 family and mitochondria apoptosis signaling induced by doxorubicin in cardiac muscle cells