Calcineurin pathway is required for endothelin-1-mediated protection against oxidant stress-induced apoptosis in cardiac myocytes

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-I 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-I 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-I 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-I on apoptosis, Calcineurin activation de novo was sufficient to inhibit hydrogen peroxide-induced apoptosis. ET-I 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 bar. 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.