Role of HIF-1.ALPHA./HKII signaling pathway in inhibition of hypoxia/reoxygenation-induced apoptosis in rat cardiomyocytes by hypoxic preconditioning

Abstract

Objective To investigate the role of hypoxia-inducible factor-1α/ hexokinase Ⅱ (HIF-1α/HKⅡ) signaling pathway in the inhibition of hypoxia/reoxygenation (H/R)-induced apoptosis in the rat cardiomyocytes by hypoxic preconditioning (HPC). Methods Primarily cultured cardiomyocytes obtained from the neonatal rats were seeded in culture dishes at the density of 5×105 cells/ml.The cardiomyocytes were attached to the wall for 72 h and then randomly divided into 6 groups (n=15 each) using a random number table: control group (group C); group H/R; group HPC; HIF-1α inhibitor YC-1 group (group YC-1); group HPC + YC-1; dimethyl sulfoxide (DMSO) group.The cells were exposed to D-Hank solution saturated with 95% N2 and 5% CO2 for 4 h, and then cultured in the normal culture atmosphere for 2 h. The cells in YC-1 and DMSO groups were incubated in the culture medium containing 10 μmol/l YC-1 and 0.1% DMSO (100 μl) for 24 h, respectively.HPC was induced by 3 cycles of 10 min hypoxia followed by 30 min reoxygenation, and H/R injury model was then established in group HPC.In group HPC+ YC-1, the cells were incubated for 5 min in the M199 culture medium supplemented with 10 μmol/L YC-1 and 10% fetal bovine serum, and the other treatments were similar to those previously described in group HPC.After the end of treatments, the apoptosis in cardiomyocytes was detected by TUNEL, the expression of HIF-1α, HKⅡ and cytochrome c was detected by Western blot, and the mitochondrial membrane potential was quantitatively measured using fluorescence.Apoptotic rate was calculated. Results Compared with group C, the apoptotic rate was significantly increased, the mitochondrial membrane potential was significantly decreased, and the expression of HIF-1α, HKⅡ and cytochrome c was significantly up-regulated in group H/R(P<0.05=. Compared with group H/R, the apoptotic rate was significantly decreased, and the mitochondrial membrane potential was significantly increased, and the expression of HIF-1α and HKⅡ was significantly up-regulated, and the expression of cytochrome c was significantly down-regulated in group HPC(P<0.05=. Compared with group HPC, the apoptotic rate was significantly increased, the mitochondrial membrane potential was significantly decreased, the expression of HIF-1α and HKⅡ was significantly down-regulated, and the expression of cytochrome c was significantly up-regulated in group HPC+ YC-1 (P<0.05=. Conclusion The mechanism by which HPC inhibits H/R-induced apoptosis in rat cardiomyocytes is associated with activation of HIF-1α/HKⅡ signaling pathway. Key words: Anoxias; Ischemic preconditioning; Myocardial reperfusion injury; Apoptosis; Hypoxia-inducible factor 1, alpha subunit; Hexokinase