Activated RhoA Induces Cardiomyocyte Apoptosis via a Mitochondrial Death Pathway

Abstract

The small G-protein RhoA is a transducer of signals from both GPCRs and the extracellular matrix, influencing cell morphology as well as gene expression. A role for RhoA has been firmly established in cultured cardiomyocyte hypertrophy, however the possibility that RhoA can influence myocyte survival or apoptosis has not been examined. Previous work from our lab employing cardiac specific RhoA transgenic mice demonstrated that RhoA overexpression leads to premature death, apparently secondary to the development of heart failure. We have now determined that adenoviral overexpression of activated RhoA in neonatal rat cardiomyocytes induces early changes indicative of hypertrophy which transitions to apoptosis, with the accompanying hallmarks of caspase activation and nucleosomal DNA fragmentation. To delineate the mechanism of RhoA-mediated apoptosis we tested the effects of the Rho kinase inhibitors Y-27632 and HA-1077. Treatment with Y-27632 or HA-1077 prevented nucleosome fragmentation and caspase-3 cleavage. Activated RhoA increases caspase-9 cleavage but not that of caspase-8, and a caspase-9 selective inhibitor prevented DNA fragmentation whereas inhibition of caspase-8 did not, consistent with involvement of a mitochondrial death pathway. Further, RhoA induces a 3-4 fold upregulation in protein levels of the proapoptotic Bcl family protein Bax, which is sensitive to Rho kinase inhibition. Bax involvement in this apoptotic effect was tested by treatment with a Bax inhibitory peptide, which markedly attenuated RhoA-induced DNA fragmentation and caspase-9 and -3 activation. The mechanism of Bax upregulation by RhoA is currently under investigation.