Hypoxic preconditioning protects cardiomyocytes against hypoxia/reoxygenation-induced cell apoptosis via sphingosine kinase 2 and FAK/AKT pathway

Previous studies have demonstrated that hypoxic preconditioning (HPC) alleviates hypoxia/reoxygenation (H/R) injury. However, the impact and mechanism involved were not fully understood. This study aimed to evaluate the effect of HPC on H/R injury in cardiomyocytes and investigate the molecular mechanisms involved. In our study, primary neonatal rat cardiomyocytes were isolated and characterized by immunofluorescence staining. We established H/R models in vitro to mimic ischemia/reperfusion (I/R) injury in vivo. Primary cardiomyocytes were exposed to HPC and then subjected to H/R. SphK2 expression was determined by quantitative Real-Time PCR and Western blotting. Cell Apoptosis was measured by Hoechst staining. H9c2 cells were transfected with SphK2 siRNA or pcDNA3.1-SphK2 plasmid. The transfection efficiency was evaluated 48h post-transfection. After H/R, cell Apoptosis rate was determined by Annexin V-FITC/PI and Caspase-3/-9 activity was measured. The activation of FAK/Akt pathway was evaluated by Western blotting. Our results showed that HPC significantly increased SphK2 expression in primary cardiomyocytes under normal or H/R condition and protected against H/R-induced cell Apoptosis, whereas SphK2 Inhibitor K145 abolished the cardioprotective effect of HPC. HPC markedly reduced the cell Apoptosis rate of H9c2, decreased the activities of Caspase-3 and -9 and increased p-FAK and p-AKT levels, which were reversed by SphK2 knockdown. Additionally, SphK2 overexpression exerted a similar effect with HPC on cell Apoptosis and FAK/Akt. Inhibition of H9c2 cell Apoptosis induced by HPC and SphK2 overexpression was abolished by PI3K/Akt Inhibitor LY294002. These results indicate that HPC may protect cardiomyocytes against H/R injury via SphK2 and the downstream FAK/Akt signaling pathway. Our findings provided important evidences for the protective role of HPC in ameliorating myocardial H/R injury.