The lysophospholipids sphingosine-1-phosphate and lysophosphatidic acid enhance survival during hypoxia in neonatal rat cardiac myocytes

The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) stimulate cellular proliferation and affect numerous cellular functions by signaling through G protein-coupled endothelial differentiation gene-encoded (Edg) receptors. S1P and LPA also act as survival factors in many cell types, but have not previously been studied in cardiac myocytes. We incubated neonatal rat cardiac myocytes either in room air/1% CO2 (normoxia) or in an atmosphere of 99% N2/1%CO2 (hypoxia) at 37 degrees C for 18-20 h in the absence of glucose. Cell viability was measured using a calcein ester green fluorescence assay. Under normoxic conditions 88.7+/-1.0% of the cells were viable after 18-20 h. Severe hypoxia reduced viability to 61.3+/-4.3% (n=6, P<0.05). In myocytes preincubated with either 10 microM S1P or 1 microM LPA for 2 h, the effects of severe hypoxia on cell viability were prevented resulting in survival equivalent to normoxia. Neither the protein kinase C inhibitor chelethyrine (1 microM) nor the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoic acid, (5-HD, 100 microM) had any effect on myocyte survival during severe hypoxia, but both agents completely abolished the ability of S1P to rescue cardiac myocytes from hypoxic cell death. We also tested the effects of dimethylsphingosine (DMS), which inhibits sphingosine kinase synthesis of S1P. Incubation of neonatal rat cardiac myocytes with 10 microM DMS for 2 h in the presence of serum resulted in 25-30% cell death during 18-20 h of normoxia. DMS-induced cell death was prevented by concurrent preincubation with either S1P or GM-1, a ganglioside that activates sphingosine kinase to increase intracellular levels of S1P. We conclude that both S1P and LPA are cardioprotective for hypoxic neonatal rat ventricular myocytes. S1P acts through cellular membrane receptors by signaling mechanisms involving protein kinase C and mitochondrial K(ATP) channels. Both endogenous and exogenously applied S1P are effective in preventing cell death induced by inhibition of sphingosine kinase.