Background: The major amount of energy generated by mitochondria enables cardiomyocytes to maintain excitability and contractility. Therefore, perturbations of mitochondrial functions have a fundamental impact on cardiomyocyte survival and performance, and are essential characteristics of ischemia/reperfusion injury and heart failure. Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator, which has important functions in cardiac and vascular homeostasis that are exerted by its five G protein-coupled receptors: it is a major player in the pathogenesis of I/R injury and heart failure, impacts cardiac contractility and contributes to ischemic preconditioning. However, S1P has also intracellular functions as a second messenger that are, however, still poorly understood. The impact of S1P on mitochondrial function in cardiomyocytes has not been addressed yet.

Hypothesis: S1P levels of mitochondria are essential for efficient mitochondrial respiration and ATP production in cardiomyocytes.

Methods: Mitochondria were isolated from mouse hearts by an established density centrifugation protocol resulting in highly viable preparations. Mitochondrial membrane potential and respiration were measured by an OROBOROS O2K-Oxygraph. S1P content of native and S1P-treated mitochondria was measured by LC-MS/MS. In vivo increases of S1P were achieved by feeding C57BL/6J mice a diet containing 4′-deoxypyridoxine (DOP; 3 mg/KG body weight), an inhibitor of the S1P lyase as the sole enzyme responsible for S1P degradation. In addition, cardiac mitochondria of sphingosine kinase 1 (Sphk1) and 2 (Sphk2) KO mice (the kinases that generate S1P from its precursor sphingosine) were studied.

Results: S1P loading of cardiomyocyte mitochondria increased their S1P concentration 10-fold without any signs of structural damage as measured by cytochrome c release. S1P lyase inhibition in vivo for 63 days also increased the S1P concentration in cardiomyocyte mitochondria by 5-fold. We observed that S1P “loading” both in vitro and in vivo led to an accelerated depolarization of mitochondria in the presence of the inhibitor of the ATP synthase inhibitor oligomycin. On the contrary, mitochondria of Sphk2 KO mice had 5-fold reduced S1P and exhibited a prolonged depolarization (hyperpolarization). Oxygen consumption in Sphk2 KO mitochondria were decreased compared. Currently, we are investigating which mitochondrial respiratory complex are involved in these effects and whether their pharmacological targeting may be beneficial in mitochondrial dysfunction during cardiovascular diseases.

Conclusion: S1P concentration in cardiac mitochondria affects membrane potential and oxygen consumption. Pharmacological intervention by modulating mitochondrial S1P and mito-targeting S1P analogues, some of which have entered the clinics for multiple sclerosis may constitute a novel approach to prevent mitochondrial dysfunction in cardiac disease.