β-Adrenergic Pathway Induces Apoptosis through Calcineurin Activation in Cardiac Myocytes

Shuichi Saito,Yukio Hiroi,Yunzeng Zou,Ryuichi Aikawa,Haruhiro Toko,Futoshi Shibasaki,Yoshio Yazaki,Ryozo Nagai,Issei Komuro

doi:10.1074/jbc.m002844200

Abstract

Apoptosis of cardiac myocytes is one of the causes of heart failure. Here we examine the mechanism by which the activation of beta-adrenergic receptor induces cardiomyocyte apoptosis. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling and DNA ladder analyses revealed that isoproterenol (Iso) induced the apoptosis of cardiac myocytes of neonatal rats through an increase in intracellular Ca(2+) levels. The Iso-induced cardiomyocyte apoptosis was strongly inhibited by the L-type Ca(2+) channel antagonist nifedipine and by the calcineurin inhibitors cyclosporin A and FK506. Iso reduced the phosphorylation levels of the proapoptotic Bcl-2 family protein Bad and induced cytochrome c release from mitochondria to the cytosol through calcineurin activation. Infusion of Iso increased calcineurin activity by approximately 3-fold in the hearts of wild-type mice but not in the hearts of transgenic mice that overexpress dominant negative mutants of calcineurin. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling analysis revealed that infusion of Iso induced apoptosis of cardiac myocytes and that the number of apoptotic cardiomyocytes was significantly less in the hearts of the transgenic mice compared with the wild-type mice. These results suggest that calcineurin plays a critical role in Iso-induced apoptosis of cardiac myocytes, possibly through dephosphorylating Bad.

Highlights

Heart failure is the final clinical manifestation of a variety of human heart diseases, including idiopathic dilated cardiomyopathy, hypertrophic cardiomyopathy, and coronary artery disease
When cardiomyocytes were treated with the selective Ltype Ca2ϩ channel antagonist nifedipine (1 ␮M) for 1 h, Isoinduced cardiomyocyte apoptosis was significantly suppressed (Fig. 2A), suggesting that the Ca2ϩ influx through L-type Ca2ϩ channels plays a critical role in Iso-induced cardiomyocyte apoptosis
These results suggest that the Ca2ϩ-calcineurin pathway plays a critical role in ␤-adrenergic receptor-induced apoptosis in cardiac myocytes

Summary

EXPERIMENTAL PROCEDURES

Materials—Iso and nifedipine were obtained from Sigma, ionomycin was from Calbiochem, and cyclosporin A was from Wako Chemical Industries, Ltd. (Osaka, Japan). Anti-Bcl-2 and anti-Bcl-xL monoclonal antibodies were from Transduction Laboratories (Lexington, KY), and anti cytochrome c polyclonal antibody was from Santa Cruz Biotechnology, Inc. Cell Culture—Primary cultures of cardiac myocytes were prepared from the ventricles of 1-day-old Wistar rats as described previously [16]. Immunocytochemical study revealed that more than 90% of cells were cardiac myocytes. Terminal Deoxynucleotide Transferase-mediated dUTP Nick End Labeling (TUNEL)—Cardiomyocytes plated on a cover glass were fixed with 4% paraformaldehyde solution for 30 min at room temperature. After a rinse with phosphate-buffered saline, the samples were first incubated with phalloidin-rhodamine for 1 h and with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and fluorescein isothiocyanate-dUTP. The sections were incubated with proteinase K (20 ␮g/ml), washed in phosphate-buffered saline, and incubated with terminal deoxynucleotidyl transferase for 90 min and fluorescein isothiocyanate-dUTP for 30 min at 37 °C using an apoptosis detection kit

The abbreviations used are

RESULTS

A Role of Calcineurin in Cardiomyocyte Apoptosis

DISCUSSION