Abstract 19576: Identification of a Novel Signaling Pathway for S1P Mediated Cardioprotection

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Background — Recently, we demonstrated that activation of the small GTPase RhoA in cardiomyocytes induces striking protection against ischemia/reperfusion (I/R) injury. The GPCR agonist, sphingosine 1-phosphate (S1P), well known for its profound protective effects on I/R injury, activates RhoA. Here we asked whether S1P protects cardiomyocytes through RhoA signaling and further characterized the mechanism and downstream targets responsible. Methods and Results — We determined that stimulation of the isolated perfused mouse heart with S1P activates RhoA and protein kinase D (PKD). Phospholipase Cε (PLCε) is a newly identified RhoA target. Genetic deletion of PLCε fully abolishes PKD activation by S1P and blocks S1P mediated protection from I/R injury. Cardioprotection by S1P is also abolished in PKD1 -/- (KO) mice. We used the neonatal rat ventricular myocytes (NRVMs) model to confirm these findings and further elucidate molecular mechanisms. S1P elicits robust activation of PKD in NRVMs through effects on RhoA (blocked with C3) and PLCε (blocked with siRNA). PKD is required for S1P mediated protection against H 2 O 2 induced cell death implicating PKD as a pro-survival target of S1P/RhoA/PLCε signaling. We further identified the PKD substrate Slingshot 1L (SSH1L) as an S1P target that is regulated through PKD mediated phosphorylation. Importantly, regulation of SSH1L by S1P reduces translocation of Cofilin2, an SSH1L substrate, to the mitochondrion in response to H 2 O 2 . In addition, we demonstrate that Cofilin2 co-IPs with the pro-death protein Bax and that mitochondrial translocation of Bax is inhibited through this pathway by S1P. Finally, we demonstrate that S1P significantly reduces translocation of Cofilin2 and Bax to mitochondria, in parallel with its ability to protect the isolated heart against I/R injury. Conclusion — These studies reveal previously unknown mechanisms through which GPCR agonists that activate RhoA induces cardioprotection. In addition they define multiple new molecular players (PLCε, PKD, SSH1L and Cofilin2) that converge at the level of the mitochondrion to protect cardiomyocytes from oxidative stress.