Protective RhoA Signaling Regulates Drp1 and Mitochondrial Fission in Cardiomyocytes

Abstract

Cardiac ischemia/reperfusion injury occurs due to oxidative stress resulting from loss of blood flow and its subsequent restoration. Oxidative stress leads to dysfunction and death of cardiomyocytes, increasing risk of progression to heart failure. We have shown previously that signaling through activation of the small G‐protein RhoA protects against ischemia/reperfusion injury by blocking pro‐apoptotic mitochondrial signals in cardiomyocytes. Alterations in mitochondrial dynamics, in particular mitochondrial fission, have been suggested to play a role in cardioprotection. We tested the hypothesis that activated RhoA signaling also regulates mitochondrial fission in cardiomyocytes. The protein Drp1 translocates from the cytosol to the mitochondria to promote fission. Our studies show that adenoviral expression of active RhoA in cardiomyocytes stimulates phosphorylation of Drp1 at serine‐616, a phosphorylation residue associated with increased translocation of Drp1 to the mitochondria. Accordingly, active RhoA also increases Drp1 protein levels in mitochondrial fractions. Both responses are blocked by inhibition of ROCK, a kinase downstream of active RhoA. Endogenous RhoA activation by the GPCR agonist sphingosine‐1‐phosphate (S1P), a lysophospholipid signaling ligand associated with cardioprotection, also increases Drp1 phosphorylation and mitochondrial Drp1 through the S1P3 receptor subtype. Consistent with the role of mitochondrial Drp1 in fission, expression of active RhoA in cardiomyocytes promotes accumulation of smaller mitochondria through ROCK and Drp1, as assessed by fluorescence confocal microscopy. A Drp1 S616A mutant is not phosphorylated and, when expressed in cardiomyocytes, did not increase in mitochondrial fractions in response to active RhoA. In addition, expression of mutant Drp1 inhibits mitochondrial fission in response to active RhoA. Oxidative stress, induced by hydrogen peroxide, leads to cardiomyocyte cell death which is attenuated by activated RhoA. This protective effect of RhoA activation is blocked by siRNA knockdown of Drp1. Taken together, our findings demonstrate that RhoA activation can regulate Drp1 to induce mitochondrial fission, and that this response is involved in cardioprotection due to RhoA. We propose regulation of Drp1 by RhoA activation as a novel mechanism to provide protection in response to acute cardiomyocyte injury.Support or Funding InformationThis research was funded by NIH grants T32HL007444 (CSB) and R37HL028143 (JHB).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.