Selective coupling of the S1P 3 receptor subtype to S1P-mediated RhoA activation and cardioprotection

Abstract

Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P1, S1P2, and S1P3 receptor subtypes to affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to Gq. We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα13 but not Gα12, Gαq, or Gαi. Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P3 in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P3 knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P3 KO mouse heart. CYM-51736, an S1P3-specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P3 receptor- and Gα13-mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P3 receptors could provide therapeutic benefits in ischemic heart disease.

RhoA regulates Drp1 mediated mitochondrial fission through ROCK to protect cardiomyocytes

“…1D). We previously demonstrated that the S1PR 3 is the S1P receptor subtype that selectively couples to RhoA activation in cardiomyocytes [10], and show here that the S1PR 3 specific agonist CYM-51736 also increases phosphorylation of Drp1 at serine-616 (Fig. 1E), a response abolished by functional inhibition of RhoA signaling with C3.…”

“…Our findings with expression of active RhoA are recapitulated by GPCR stimulation with S1P or an S1PR 3 agonist, both of which we have shown to lead to activation of RhoA [9, 10]. Both agonists increase phosphorylated Drp1 in a RhoA/ROCK dependent manner.…”

“…This contrasts with a study using podocytes subjected to hyperglycemia where ROCK was implicated in phosphorylation of Drp1 at serine-637 [37]. PKD, another downstream target of RhoA explored in our previous work [3, 9, 10], has also been suggested to phosphorylate Drp1 at serine-637 in cardiomyocytes [38]. However, serine-637 phosphorylation of Drp1 was not affected by activation of RhoA nor was the RhoA-induced increase in phosphorylation of Drp1 at serine-616 blocked by a PKD inhibitor (data not shown).…”

“…Notably, studies showing responses to agonists for these other GPCRs in primary cardiomyocytes have examined changes in Drp1 phosphorylation, localization, or function after 6–48 h of stimulation, when additional signaling cascades or changes in gene expression could be activated [35, 40]. In contrast, S1PR 3 couples through a different family of heterotrimeric G-proteins, Gα 12/13 , to activate RhoA signaling in both cardiomyocytes and other cardiac cells [10, 41], and the finding that S1P can stimulate RhoA-mediated Drp1 phosphorylation and translocation within 15 min of treatment provides important new insights into the role of acute activation of a GPCR signaling pathway in regulation of Drp1 in cardiomyocytes.…”

“…RhoA regulates downstream phosphorylation cascades involved in protection from ischemic or oxidative stress through kinases such as Rho-associated Protein Kinase (ROCK), Protein Kinase B (Akt) and Protein Kinase D (PKD) [2, 3, 6]. Sphingosine-1-phosphate (S1P), a ligand released in response to injury, also elicits cardioprotection [7, 8] which we have shown to be associated with activation of RhoA [9, 10]. …”