Abstract 313: Sphingosine-1-Phosphate and RhoA Signaling Regulate Dynamin-Related Protein 1 and Cardiac Mitochondrial Fission

Abstract

In cardiomyocytes, signaling through RhoA is protective against ischemia/reperfusion injury. Cardioprotective receptor agonists such as sphingosine-1-phosphate (S1P) provide protection through RhoA activation. Mitochondrial fission has been suggested to play a role in cardioprotection, allowing for selective degradation of smaller damaged mitochondria. Our previous studies showed that RhoA signaling can protect cardiomyocytes against ischemia/reperfusion injury by blocking mitochondrial death pathways under oxidative stress, however it is not known if RhoA activation also regulates mitochondrial quality control. We tested the possibility that activated RhoA regulates cardiac mitochondrial fission. Adenoviral expression of constitutively active RhoA in cardiomyocytes caused an increase in small, fragmented mitochondria observed by both fluorescent confocal microscopy and electron microscopy. This mitochondrial fission phenotype was attenuated by inhibition of the downstream RhoA target Rho-associated Protein Kinase (ROCK), or by siRNA knockdown of the primary fission protein Dynamin-related Protein 1 (Drp1). Activated Drp1 causes mitochondrial fission when it translocates from the cytosol to the mitochondria, a process regulated by Drp1 phosphorylation. We determined that expression of active RhoA in cardiomyocytes stimulated phosphorylation of Drp1 at serine-616, and increased mitochondrial levels of Drp1. Both phosphorylation and mitochondrial translocation of Drp1 by active RhoA could be blocked by ROCK inhibition. Endogenous RhoA activation by S1P also increased Drp1 phosphorylation and mitochondrial translocation in a RhoA-dependent and ROCK-dependent mechanism. In conclusion, RhoA activation in cardiomyocytes can increase Drp1 phosphorylation and Drp1 mitochondrial localization, and induce mitochondrial fission. We propose that mitochondrial fission, elicited in response to increased availability of S1P and activation of RhoA in response to ischemia/reperfusion injury, represents a previously unknown pathway for cardioprotection.