title: Hypoxic preconditioning protects cardiomyocytes against hypoxia/reoxygenation-induced cell apoptosis via sphingosine kinase 2 and FAK/AKT pathway.
abstract: 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.
authors.affiliation: Department of Emergency Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, People's Republic of China;; Department of Emergency Medicine, The Fourth Affiliated Hospital of China Medical University, Shenyang 110032, People's Republic of China.
authors.forename: Ruxin
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Emergency Medicine, The Fourth Affiliated Hospital of China Medical University, Shenyang 110032, People's Republic of China.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Emergency Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, People's Republic of China;
authors.forename: Li
authors.initials: L
authors.lastname: Yuan
authors.affiliation: Department of Emergency Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, People's Republic of China;. Electronic address: minzhao0216@163.com.
authors.forename: Min
authors.initials: M
authors.lastname: Zhao
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D000860:Hypoxia; D019194:Ischemic Preconditioning; D009025:Morpholines / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D051058:Oncogene Protein v-akt / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism; D015398:Signal Transduction; D000099157:Sphingosine Kinase
chemical_list: D053148:Caspase 3; D002867:Chromones; D051417:Focal Adhesion Kinase 1; D009025:Morpholines; D051058:Oncogene Protein v-akt; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D000099157:Sphingosine Kinase; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C496052:Ptk2 protein, rat; C505410:Casp3 protein, rat
keywords: Apoptosis; FAK/AKT signaling pathway; Hypoxia/reoxygenation injury; Hypoxic preconditioning; Sphingosine kinase 2