Bez235

The H9c2 cardiomyocytes derived from rat embryonic heart were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and routinely maintained in complete Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 10% heat-inactivated foetal bovine serum (FBS; Hyclone, Logan, UT, USA) and antibiotics (100 U/ ml penicillin and 100 lg/ml streptomycin) at 37 C in a humidified incubator containing 95% air and 5% CO 2 . To establish H/R injury model, H9c2 cardiomyocytes were transferred to FBS-and glucose-free DMEM and incubated under hypoxic conditions in an incubator supplied with a gas mixture containing 1% O 2 , 94% N 2 and 5% CO 2 for 12 h at 37 C, followed by reoxygenation (95% air and 5% CO 2 ) in complete medium in a normoxic chamber for another 12 h at 37 C. H9c2 cardiomyocytes in the normal control were cultured under normoxic conditions. PK2 and BEZ235 (a dual phosphatidylinositol 3-kinase (PI3K)/the mammalian target of rapamycin (mTOR) inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulphoxide (DMSO) to a final concentration of 5 nM and 5 mM, respectively. In some experiments, H9c2 cardiomyocytes were incubated in FBS-and glucose-free DMEM supplemented with 5 nM PK2 in the presence or absence of 5 mM BEZ235 and then subjected to H/R treatment.