Deletion of the Sphingosine Kinase-1 gene influences cell fate during hypoxia and glucose deprivation in adult mouse cardiomyocytes

Objectives: Activation of sphingosine kinase (SphK), which has two known isoforms, is responsible for the synthesis of sphingosine 1-phosphate (S1P), a cell survival factor. We tested the following hypotheses: 1] cardiac myocytes null for the SphK1 gene are more vulnerable to the stress of hypoxia+glucose deprivation; 2] the monoganglioside GM-1, which activates SphK via protein kinase C epsilon, is ineffective in SphK1-null myocytes; 3] SIP generated by SphK activation requires cellular export to be cardioprotective. Methods: We cultured adult mouse cardiac myocytes from wildtype and SphK1-null mice (deletion of exons 3-6) and measured cell viability by trypan blue exclusion. Results: In wildtype adult mouse cardiomyocytes subjected to 4 h of hypoxic stress + glucose deprivation, cell viability was significantly higher than in SphK1-null cardiomyocytes. SphK1-null cells also displayed more mitochondrial cytochrome C release. Cell death induced by hypoxia+ glucose deprivation was substantially prevented by pretreatment with exogenous S1P in both wildtype and SphK1-null myocytes, but S 1 P was effective at a lower concentration in wildtype cells. Hence, the absence of the Sphk1 gene did not affect receptor coupling or downstream signal transduction. Pretreatment for 1 h with 1 mu M of the monoganglioside GM-1 increased survival in wildtype cells, but not in SphK1-null myocytes. Thus, activation of SphK1 by GM-1 leads to cell survival. In wildtype cells, enhanced survival produced by GM-1 was abrogated by pretreatment either with 300 nM of the S1P(1) receptor-selective antagonist VPC23019 or with 100 ng/ml of pertussis toxin for 16 It before exposure to hypoxia + glucose deprivation. Conclusion: As the effect of GM-1 is blocked both at the receptor and the G-protein (Gi) levels, we conclude that S I P generated by GM-1 treatment must be exported from the cell and acts in a paracrine or autocrine manner to couple with its cognate receptor. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.