Host: The Japanese Pharmacological Society, The Japanese Society of Clinical Pharmacology
Name : WCP2018 (18th World Congress of Basic and Clinical Pharmacology)
Location : Kyoto
Date : July 01, 2018 - July 06, 2018
RhoA is a small G-protein and intracellular signal transducer that modulates a range of cellular processes including cell survival. RhoA serves as a proximal downstream effector of numerous GPCRs and is also responsive to oxidative stress. With stress, mitochondria become damaged, and induce cell death, thus their elimination by mitochondria-selective autophagy (mitophagy) is important in preventing cellular damage and this process is also modulated by mitochondrial dynamics, specifically mitochondrial fusion/fission. We hypothesize that RhoA signaling preserves mitochondrial integrity to provide protection. RhoA expression in neonatal rat ventricular myocytes (NRVMs) lead to increased PINK1 expression at mitochondria. This also increased Parkin recruitment to mitochondria, ubiquitination of mitochondrial proteins and induction of autophagosomes containing mitochondria. Interestingly, there was no concomitant mitochondrial membrane depolarization associated with these responses and qRT-PCR analysis 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. Studies using siRNA-mediated knockdown of PKD or pharmacological inhibition demonstrated that PKD inhibition largely attenuated RhoA-mediated PINK1 protein stabilization. In addition, analysis using fluorescent microscope showed that mitochondria are smaller in cells expressing RhoA and this was quantitatively confirmed by further analysis using electron microscopy. Drp1 phosphorylation at S616 and its mitochondrial distribution were increased by RhoA. We observed that Y-27632, a ROCK inhibitor, as well as expression of dominant-negative ROCK blocks RhoA-induced phosphorylation of Drp1 at S616 and translocation of Drp1 to mitochondria in NRVMs. Importantly, RhoA activity is increased in the heart subjected to LAD ligation. To determine whether increased activation of RhoA confers cardioprotection against MI, constitutively active RhoA was expressed in the adult mouse heart using AAV9 driven by the cardiac-specific MLC2v promoter. RhoA expression significantly decreased the infarct size in the heart subjected to LAD ligation for 2 hrs. We are currently investigating whether RhoA activation regulates mitochondrial quality control in the heart in vivo. These results suggest that RhoA limits cardiac damage induced by ischemia through the coordinated preservation of mitochondrial integrity.
