Abstract P222: Signaling Sphingolipids in Regulation of Cardiomyocyte Ischemia/Reperfusion-Induced Injury
Introduction: While therapeutic hypothermia (TH) remains a highly promising treatment for improving cardiac function and survival after cardiac arrest or acute myocardial infarction, its mechanisms of action remain unknown. We used two different cardiomyocyte models of I/R injury and TH protection to study changes in the signaling sphingolipids sphingosine-1-phosphate (S1P) and ceramides, part of a counteracting signaling network linked to cell survival (S1P) or death (ceramides).
Methods: Cardiomyocytes were isolated from 1–2-day old C57Bl6/J mice and were exposed to 90 min simulated ischemia (20% CO2, 2-deoxy-D-glucose 20 mM, K+ 8 mM, pH 6.8, without glucose) and 3 h reperfusion. For TH, cells were cooled from 37°C to 32°C within the last 20 min ischemia, with cooling extended for 1 hr after reperfusion. Embryonic chick cardiomyocytes were exposed to a similar protocol with 60 min ischemia. Cell viability was evaluated by propidium iodide (PI) uptake. S1P, ceramides and their stable isotope-labeled analogs were quantified by LC/MS/MS.
Results: Cardioprotective TH in murine cardiomyocytes was associated with an increased S1P levels seen at 2 hr reperfusion, suggesting that TH may work in part via enhanced sphingosine kinase 1 (SphK1) activity. To mimic this TH protection, overexpression of SphK1 was tested. SphK1 overexpression significantly increased cellular levels of S1P and its endogenous analog dihydro-S1P (DHS1P), downregulated ceramide levels, and decreased cell death at 3 hr reperfusion from 48.2±10.1% to 20.3±7.9%, n=3, p<0.05, a level of cardioprotection similar to that seen with TH. In parallel studies, targeted inhibition of ceramide synthase (CerS) with fumonisin B1 significantly decreased ceramide levels, upregulated levels of both S1P and DHS1P, and significantly decreased chick cardiomyocyte death in response to I/R (from 48.5±3,8% to 32.6±2.4%, n=3, p<0.01). Stable isotope pulse-labeling experiments revealed that fumonisin B1 blocks ceramide de novo biosynthesis and re-routs de novo-synthesized sphingoid bases towards DHS1P.
Conclusion: Overall, our data suggest that S1P and its endogenous analog DHS1P may play important roles in affecting cardiac reperfusion injury and may be involved in TH cardioprotection.
This research has received full or partial funding support from the American Heart Association, National Center.