Hypoxic preconditioning attenuates hypoxia/reoxygenation injury of cardiomyocytes in SD rats via activating AMPK / 解放军医学杂志

ABSTRACT

[Abstract] Objective To investigate the protective effects of hypoxic preconditioning (HPC) on cardiomyocytes after hypoxia/reoxygenation (H/R) by activating AMP-activated protein kinase (AMPK). Methods The primary cardiomyocytes were prepared from neonatal SD rats. The model of H/R of cardiomyocytes was established and randomly divided into control group, H/R group, HPC group, H/R+A-769662 (AMPK agonist) group and HPC+compound C (AMPK inhibitor) group. The survival rate of cardiomyocytes was detected by chromatometry with Cell Counting Kit-8 (CCK-8); the reactive oxygen species (ROS) and ATP contents in the cells were detected respectively by dihydroethidium (DHE) dyeing and ATP Detection Assay Kit measured by fluorescence microplate reader; the phosphorylation level of AMPK and activation level of caspase-3 were detected by Western blotting; the concentration of free Ca2+ in cardiomyocytes was detected by Fluo-3AM dyeing using laser scanning confocal microscopy; the apoptotic rate of cardiomyocytes was detected by TUNEL staining. Results CCK-8 assay showed that compared with H/R group, the survival rate of cardiomyocytes in HPC group and H/R+A-769662 group increased obviously (P0.05); compared with H/ R group, the AMPK phosphorylation levels elevated significantly in HPC group and H/R+A-769662 group respectively (P<0.05, P<0.01). Meanwhile, the activation levels of caspase-3 in H/R group and HPC+compound C group were increased obviously compared with that in control group (P<0.01), but in HPC group and H/R+A-769662 group were significantly lower than that in H/R group (P<0.01, P<0.05). The results of Fluo-3AM staining showed that compared with control group, the concentrations of intracellular free Ca2+ increased obviously in H/R group and HPC+compound C group (P<0.01); the concentrations of free Ca2+ in cardiomyocytes of HPC group and H/R+A-769662 group were less than that in H/R group (P<0.01), while the concentrations of free Ca2+ in HPC+compound C group much more increased than in HPC group (P<0.01). The results of TUNEL staining showed that compared with H/R group, the apoptotic rate of cardiomyocytes decreased obviously in HPC group and H/R+A-769662 group (P<0.01, P<0.05); while compared with HPC group, the apoptotic rate of cardiomyocytes increased significantly in HPC+compound C group (P<0.05). Conclusions H/R may lead to the increase of ROS production, the decrease of ATP contents, and increase the levels of free Ca2+ in cardiomyocytes, and thus activate caspase-3, lead to apoptosis eventually. While HPC may reduce the production of ROS by activating AMPK to ensure the energy supply of cardiomyocytes after H/R treatment, prevent apoptosis of cardiomyocytes, and then reduce the H/R injury to cardiomyocytes.