Calcineurin Pathway Is Required for Endothelin-1–Mediated Protection Against Oxidant Stress–Induced Apoptosis in Cardiac Myocytes

Abstract

Abstract-Endothelin-1 (ET-1) acts not only as a growth-promoting peptide but also as a potent survival factor against myocardial cell apoptosis. However, the signaling pathways leading to myocardial cell protection by ET-1 are poorly understood. Using a culture system of primary cardiac myocytes derived from neonatal rats, we show in the present study that ET-1 almost completely blocked the hydrogen peroxide-induced increase in the percentage of TdT-mediated dUTP-biotin nick-end labeling-positive myocytes. Apoptosis inhibition by ET-1 was confirmed by cytofluorometric analysis as well as by examination of the ladder formation, morphological features, and caspase-3 cleavage. We have found that ET-1 converts the nuclear factor of activated T lymphocytes (NFATc) in cardiac myocytes into high-mobility forms and translocates cytoplasmic NFATc to the nuclei. In addition, ET-1 stimulates the interaction between NFATc and the cardiac-restricted zinc-finger protein GATA4 in these cells. The immunosuppressants cyclosporin A and FK506, which antagonize calcineurin, negated the inhibitory effect of ET-1 on apoptosis. Calcineurin activation de novo was sufficient to inhibit hydrogen peroxide-induced apoptosis. ET-1 induced the expression of an antiapoptotic protein bcl-2 in cardiac myocytes in a cyclosporin A-dependent manner, but it did not alter the expression of bax. Cyclosporin A also attenuated the ET-1-stimulated transcription of the bcl-2 gene in these cells. These findings demonstrate that the calcineurin pathway is required for the inhibitory effect of ET-1 on oxidant stress-induced apoptosis in cardiac myocytes.

Antiapoptotic Effect of Endothelin-1 in Rat Cardiomyocytes In Vitro

“…23 In cardiac myocytes, ET-1 prevents oxidative stress-and ␤-adrenergic agonist-induced apoptosis. 24,25 In the present study, we demonstrate that ET-1 prevents apoptosis induced by serum deprivation in cultured cardiac myocytes and investigate the signaling pathways that mediate the antiapoptotic effect of ET-1.…”

Rho/ROCK Pathway Contributes to the Activation of Extracellular Signal-regulated Kinase/GATA-4 during Myocardial Cell Hypertrophy

“…The cardiac myocytes were grown on flask-style chambers with glass slides (Nalgen Nunc, Naperville, IL) and then stimulated with saline or 1.0 ϫ 10 Ϫ5 M of PE in serum-free medium for 48 h. The cells were then fixed with 3% formaldehyde in phosphate-buffered saline for 15 min at room temperature. Immunocytochemical staining for ␤-MHC was performed using the indirect immunoperoxidase or immunofluorescense method as previously described (27). As the primary antibody we used anti-␤-MHC polyclonal antibody (NovoCastra Laboratories Ltd., Newcastle, UK) at a dilution of 1:50.…”

Regulation of Cardiac Transcription Factor GATA4 by Post-Translational Modification in Cardiomyocyte Hypertrophy and Heart Failure

“…In adult cardiac myocytes, Kakita, et al reported that the calcineurin/NFAT/GATA4 pathway is required for ET-1-mediated protection against cardiac myocyte apoptosis. 19) Cardiac-specific deletion of GATA4 induces apoptosis by alteration of the gene expression implicated in cellular apoptosis after pressureoverload. 20) The overexpression of GATA4 has been shown to prevent cardiac myocyte apoptosis induced by anthracyclines Vol 57 No 6 GATA4 MODIFICATIONS IN CARDIOMYOCYTE HYPERTROPHY such as daunorubicin and doxorubicin.…”