Cardiac preconditioning with sphingosine-1-phosphate requires activation of signal transducer and activator of transcription-3

Aims: Sphingosine-1-phosphate (S1P) is a cardioprotective agent. Signal transducer and activator of transcription 3 (STAT-3) is a key mediator of many cardioprotective agents. We aimed to explore whether STAT-3 is a key mediator in S1P-induced preconditioning. Methods: Langendorff-perfused hearts from Wistar rats and wild-type or cardiomyocyte-specific STAT-3 knockout mice were pre-treated with S1P (10 nmol/l), with or without the STAT-3 pathway inhibitor AG490, before an ischaemia-reperfusion insult. Triphenyltetrazolium chloride and Evans blue staining were used for the determination of infarct size. Western blot analysis was carried out on the S1P pre-treated hearts for detection of cytosolic, nuclear and mitochondrial phosphorylated and total STAT-3 proteins. Results: Pre-treatment with S1P decreased the infarct size in isolated rat (5 +/- 3% vs control 26 +/- 8%, p < 0.01) and wild- type mouse hearts (13 +/- 1% vs control 33 +/- 3%, p < 0.05). This protective effect was abolished in the rat hearts pre-treated with AG490 (30 +/- 10%, p = ns vs control) and in the hearts from STAT-3 knockout mice (35 +/- 4% vs control 30 +/- 3%, p = ns). Levels of phosphorylated STAT-3 were significantly increased in both the nuclear (p < 0.05 vs control) and mitochondrial (p < 0.05 vs control) fractions in the S1P pre-treated hearts, but remained unchanged in the cytosolic fraction (p = ns vs control). Conclusion: These novel results demonstrate that pharmacological preconditioning with S1P in the isolated heart is mediated by activation of mitochondrial and nuclear STAT-3, therefore suggesting that S1P may be a novel therapeutic target to modulate mitochondrial and nuclear function in cardiovascular disease in order to protect the heart against ischaemia-reperfusion.