Hypoxic preconditioning of cardiomyocytes and cardioprotection: phophorylation of HIF-1alpha induced by p42/p44 mitogen-activated protein kinases is involved.

ABSTRACT

OBJECTIVE: To elucidate the molecular mechanisms involved in hypoxic preconditioning (HPC) of neonatal rat cardiomyocytes against hypoxia/reoxygenation (H/R) injury. METHODS: Cardiomyocytes from neonatal Sprague-Dawley rats were randomly distributed into the following experimental groups (1) HPC group 20 min of hypoxia was performed to induce hypoxic preconditioning. Twenty four hours after HPC, cardiomyocytes were exposed to lethal hypoxia for 3 h followed by 3 h normoxia (reoxygenation). (2) Hypoxia/reoxygenation (H/R) group cardiomyocytes were directly subjected to hypoxia (3 h) followed by reoxygenation (3 h). (3) PD98059+HPC (PD+HPC) group cardiomyocytes were preincubated with PD98059 (a selective MEK-1/2 inhibitor, 50 mumol/l) 10 min prior to HPC. (4) BDM+HPC group cardiomyocytes were pretreated with an activator of protein phosphatase 2,3-butanedione monoxide (BDM, 20 mmol/l) 10 min prior to HPC. (5) Control group cardiomyocytes were incubated in cell incubator for 30 h. Viability of cardiomyocytes was assessed by MTT assay. Lactate dehydrogenase (LDH) activity in medium was determined using a LDH assay kit. Activity of p42/44 mitogen-activated protein kinases (p42/44 MAPKs) was detected using Western blotting method. SDS-PAGE mobility shift experiments were performed to determine phosphorylation of Hypoxia-inducible factor-1alpha (HIF-1alpha). RESULTS: HPC promoted survival and membrane integrity of cardiomyocytes subjected to subsequent sustained H/R. The protective effects of HPC were completely abolished either by PD98059 [a selective inhibitor of MEK-1/2 (upstream activators of p42/44 MAPKs)], or by BDM (an activator of protein phosphatase). Western blot analysis showed activated p42/44 MAPKs in whole cell extracts from hypoxic preconditioned cardiomyocytes. SDS-PAGE mobility shift experiments showed increased phophorylation level of HIF-1alpha in HPC group, and the phosphorylation can be blocked by PD98059 or BDM. CONCLUSIONS: HPC protects neonatal cardiomyocytes against H/R injury by promoting cardiomyocyte survival and membrane integrity. The protective mechanism might be attributed to upregulation of HIF-1alpha phosphorylation which may be induced by P42/44 MAPKs.