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RhoA Regulates PINK1 Accumulation and Mitophagy through PKD Activation

RhoA is a small GTPase that regulates myriad cellular events, including adhesion, migration, invasion, cell cycle progression, gene expression, and cellular protection. Our previous research demonstrated that expression of RhoA in the heart protects cardiomyoctes from ischemia/reperfusion injury. RhoA is activated by various GPCRs, including those for sphingosine‐1‐phosphate (S1P); activation of RhoA contributes to S1P‐mediated cardioprotection. We recently determined that RhoA activation in the heart increases mitochondrial autophagy (mitophagy), a process that eliminates compromised mitochondria and thereby diminishes induction of mitochondrial death pathways. The involvement of RhoA induced mitophagy in the cardioprotective response to RhoA has not been examined. The accumulation of PTEN‐induced putative kinase 1 (PINK1) at damaged mitochondria plays an important role in induction of mitophagy. Increasing RhoA expression through adenovirus in neonatal rat ventricular myocytes (NRVMs), or in the mouse heart by AAV9 RhoA injection, leads to increased PINK1 expression at mitochondria. Importantly, this also increases Parkin recruitment to mitochondria, ubiquitination of mitochondrial proteins, and induction of autophagosomes containing mitochondria. Interestingly, there is no concomitant mitochondrial membrane depolarization associated with these responses. qRT‐PCR analysis also showed no difference in PINK1 mRNA levels in control and RhoA‐expressing cells. Degradation of exogenously expressed PINK1 protein in the presence of cycloheximide was, however, greatly inhibited by RhoA expression. ROCK is one of the most established RhoA downstream effectors, but inhibition of ROCK did not affect PINK1 accumulation induced by RhoA. We previously showed that RhoA activates PKD to confer cardioprotection. Studies using siRNA‐mediated knockdown of PKD or pharmacological inhibition showed that this largely attenuated RhoA mediated PINK1 stabilization. These results suggest that RhoA increases PINK1 levels at mitochondria by preventing its degradation through PKD and in a mitochondrial membrane depolarization‐independent fashion. We plan to further investigate the functional significance of RhoA‐dependent PINK1 induction and mitophagy in cardioprotection against sustained ischemia in vivo (e.g. myocardial infarction).