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

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.