The G12/13family of G protein regulates the low molecular weight G proteins Ras homolog gene member A (RhoA) through direct stimulation of guanine exchange factors [2125]. of PKD, and this was verified in studies using isolated hearts coming from S1P3knockout (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 S1P3KO mouse heart. CYM-51736, an S1P3-specific agonist, also decreased infarct size after ischemia/reperfusion to a degree just like that achieved by S1P. The finding that S1P3receptor- and G13-mediated RhoA activation is SIS3 responsible for protection against ischemia/reperfusion suggests that selective concentrating on of S1P3receptors could offer therapeutic benefits in ischemic heart disease. Keywords: sphingosine-1-phosphate (S1P), G protein-coupled receptor (GPCR), SIS3 Ras homolog gene family member A (RhoA), protein kinase D (PKD), phospholipase C (PLC), ischemia/reperfusion (I/R), cardioprotection == Graphical abstract == == 1 . Introduction == Restoration of blood flow after an ischemic episode (e. g. myocardial infarct) is necessary to prevent catastrophic heart failure but reperfusion can by itself increase cardiomyocyte death, a process referred to as reperfusion injury [1]. Previous studies have demostrated that the circulating bioactive lysophospholipid sphingosine-1-phosphate (S1P) is endogenously released in response to cardiac damage [2, 3] and that S1P helps to guard the center from the oxidative damage that leads to reperfusion injury [47]. S1P is a large affinity ligand for five G protein-coupled receptor (GPCR) subtypes denoted S1P15[8, 9]. The S1P13receptor subtypes are indicated in cardiomyocytes. S1P1is the predominant subtype expressed in the heart as well as in cardiomyocytes and it specifically couples to Gi[10, 11]. Coupling to Gileads to inhibition of cyclic AMP formation and accounts for the ability of S1P to decrease cardiac contractility [9, 10, 12, 13]. The S1P2and S1P3receptors can couple to Gisignaling in the center [10] but these subtypes are, in addition , capable to couple to Gq, G12, and G13[9, 16, 15]. Activation of Gqstimulates phospholipase C-beta (PLC) and has been exhibited to play a significant role in the development of cardiac hypertrophy [1620]. Whilst S1P2and S1P3have been shown to couple to Gqin other systems [9, 14], it has not been determined whether stimulation of these receptors in cardiomyocytes activates Gqand PLC to elicit signals that lead to cardiomyocyte hypertrophy. The G12/13family of SIS3 G proteins regulates the low molecular weight G protein Ras homolog gene member A (RhoA) through direct stimulation SIS3 of guanine exchange factors [2125]. Recently we demonstrated that cardiac expression of RhoA protects the SIS3 heart against oxidative stress and ischemia/reperfusion (I/R) injury whereas gene deletion of RhoA decreases tolerance to ischemic damage [26]. We also showed that S1P could confer cardioprotection through RhoA and its downstream effectors [6]. In this study, we examined whether S1P regulates PLC activation and hypertrophy through S1P2or S1P3receptors and/or whether these receptor subtypes regulate activation of RhoA and in turn S1P-mediated cardioprotection. The data presented here demonstrate that S1P primarily signals through coupling to G13and activation of RhoA, that this pathway does not strongly activate PLC or contribute Rabbit Polyclonal to Cofilin to development of cardiac hypertrophy, and that it is the S1P3receptor that regulates RhoA activation to mediate cardioprotection. == 2 . Materials and Methods == == 2 . 1 . Animals == All animal procedures were performed in accordance with NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the University of California San Diego. Generation of global homozygous C57BL/6 S1P2KO and S1P3KO mice has been previously described [27]. All experiments were performed on age-matched male WT and KO littermates. == 2 . 2 . NRVM cell culture and reagents == Neonatal rat ventricular myocytes (NRVMs) were isolated from.
