Abstract 442: A Sphingosine Kinase-1 (SphK-1) Mutation Determines Cell Fate in Isolated Adult Mouse Cardiac Myocytes
Background: Sphingosine kinase (SphK), which has two known isoforms, is responsible for intracellular synthesis ofw sphingosine 1-phosphate (S1P), a cell survival factor. We studied adult mouse cardiac myocytes from wildtype and SphK-1 null mice.
Hypotheses: Cardiac myocytes null for the SphK-1 gene are more vulnerable to hypoxic stress. The monoganglioside GM-1, which activates Sphk via PKC-epsilon, is ineffective in SphK-1 null myocytes. Intracellular S1P generated by SphK activation requires cellular export to be cardioprotective.
Methods and Results: Wild type adult myocytes subjected to 4 hours of hypoxic stress, cell viability measured by trypan blue exclusion was significantly higher than in SphK-1 null cardiomyocytes. SphK-1 null cells also displayed more mitochondrial cytochrome C release. Hypoxia-induced cell death was substantially prevented by pretreatment with exogenous S1P in both wildtype and SphK-1 null myocytes. Hence, the absence of the Sphk-1 gene did not affect receptor coupling or downstream signal transduction. Pretreatment for 30 min with 1 μM GM-1 increased survival in wildtype cells, but not in SphK-1 null myocytes. Thus activation of SphK-1 by GM-1 leads to cell survival. In wildtype cells, enhanced survival produced by GM-1 was abrogated by pretreatment either with S1P1 antagonist VPC23019 or with pertussis toxin. As the effect of GM-1 is blocked both at the receptor and the G-protein (Gi) levels, we conclude that internally generated S1P must be exported from the cell to act in a paracrine or autocrine manner by coupling with its cognate receptor.