Cocaine Induces Apoptosis in Fetal Myocardial Cells through a Mitochondria-Dependent Pathway1

Abstract

In the present study, we examined the direct cytotoxic effects of cocaine on fetal cardiac myocytes. Cocaine treatment of cultured fetal rat (21 days) myocardial cells (FRMCs) induced a time- and concentration-dependent increase in apoptotic cells in FRMCs. Cocaine induced surface exposure of phosphatidylserine in FRMCs at 12-h treatment and increased apoptotic cells up to 96 h. Corresponding DNA fragmentation induced by cocaine in these cells was demonstrated in situ by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay and by electrophoresis of labeled DNA fragments, showing the characteristic apoptotic ladders. The pD₂ and maximum increase of cocaine-induced apoptosis in FRMCs were 4.3 and 3.2-fold, respectively. Both caspase-9 and caspase-3 inhibitors (Z-LEHD-FMK and Ac-DEVD-CHO, respectively) blocked cocaine-induced apoptosis. In addition, cyclosporin A inhibited cocaine-induced apoptosis in a concentration-dependent manner with an IC₅₀ value of 0.1 μM. The maximum of 86% inhibition was obtained with 3 μM cyclosporin A. Cocaine induced the release of cytochromec from the mitochondria and increased its levels in the cytosol by 3.1-fold. In accordance, the level of cytochromec in the mitochondria fraction decreased by ∼60%. Cocaine-induced translocation of cytochrome c was inhibited by cyclosporin A. The results indicate that cocaine has a direct cytotoxic effect on fetal cardiomyocytes by inducing apoptosis in the cells. Furthermore, the release of cytochrome cfrom the mitochondria and its subsequent activation of caspase-9 and caspase-3 play a key role in cocaine-induced apoptosis.