Changes And Effects Of Autophagy And Mitochondrial Cx43 In Cardiomyocytes During Hypoxia/reoxygenation Injury

Objective:To investigate the effects of hypoxia/reoxygenation on autophagy and mitochondrial Cx43 in cardiomyocytes,and to explore the relationship between autophagy and mitochondrial Cx43 in cardiomyocytes during hypoxia/reoxygenation injury.Methods:Primary rat cardiomyocytes and H9c2 cardiomyocytes were cultured separately,and H9c2 cardiomyocytes were selected for subsequent experiments ultimately according to the culture results.H9c2 cardiomyocytes were cultured with the serum-free and low-glucose DMEM medium and placed in an anoxic(5%C02,2%02)incubator for 12h of hypoxia.After the end of this period,the culture medium was replaced with high-glucose DMEM supplemented with serum containing,and cells were transferred to a normoxic incubator for 12h of reoxygenation.In order to establish the hypoxia/reoxygenation model of cardiomyocytes,the morphology of the cells was observed with an inverted microscope,the cell viability was assessed by CCK-8,and the LDH activity in the culture medium was measured by a lactate dehydrogenase(LDH)kit.Cardiomyocytes were randomly divided into five groups:control group,H/R group,geldanamycin group(GA group),3-methyladenine group(3-MA group)and geldanamycin+3-methyladenine group(GA+3-MA group).GA treatment was performed on cardiomyocytes for 30 min(1 ?mol/L)prior to hypoxia-reoxygenation.3-MA treatment was performed by adding 3-MA(5 mmol/L)into the medium while hypoxia-reoxygenation.Cell viability was assessed by CCK-8.Mitochondrial membrane potential(MMP)was messured by JC-1.Protein levels of Beclin-1,LC3B,mitochondrial Cx43 and mitochondrial phosphorylation Cx43(p-Cx43)were determined by Western blot,and autophagosomes were observed by electron microscopy.Results:1.Compared with the control group,morphologically,the dead cells increased and the cell density decreased in H/R group.In addition,cell viability of H/R group decreased,while the concentration of LDH in the culture medium increased(P<0.01).Hypoxia/reoxygenation(H/R)model of H9c2 cardiomyocytes was established.2.Cardiomyocyte injury:Compared with the control group,MMP and cell viability decreased in H/R group(P<0.05);Compared with H/R group,MMP and cell viability decreased in GA group,3-MA group and GA+3-MA group(P<0.05);MMP and cell viability in GA+3-MA group are both lower than that in GA group and 3-MA group respectively(P<0.05).3.Autophagy activity assay:Compared with the control group,the autophagosomes significantly increased and levels of Beclin-1 and ratio of LC3B?/LC3BI also increased in 1H/R group(P<0.05);Compared with H/R group,the autophagosomes and levels of Beclin-1 and ratio of LC3B?/LC3BI all decreased in GA group,3-MA group and GA+3-MA group(P<0.05);levels of Beclin-1 and ratio of LC3B?/LC3BI in GA+3-MA group are both lower than that in GA group and 3-MA group respectively(P<0.05).4.Protein level of mitochondrial Cx43:Compared with the control group,content of mitochondrial Cx43 and p-Cx43 decreased in H/R group(P<0.05);Compared with H/R group,content of mitochondrial Cx43 and p-Cx43 decreased in GA group and GA+3-MA group,but only the content of mitochondrial p-Cx43 decreased in 3-MA group(P<0.05);content of mitochondrial p-Cx43 in GA+3-MA group are both lower than that in GA group and 3-MA group respectively(P<0.05).Conclusion:Up-regulation of autophagy,decrease in the content of mitochondrial Cx43 and increase in the dephosphorylation of mitochondrial Cx43 in the cardiomyocytes were induced by hypoxia/reoxygenation.A significant decrease and dephosphorylation of mitochondrial Cx43 maybe an important mechanism of ischemia/reperfusion injury.Autophagy is involved in the maintenance of mitochondrial Cx43 phosphorylation,and mitochondrial Cx43,especially p-Cx43,is also involved in the activation of autophagy.Autophagy and mitochondrial Cx43 both contribute to the cardioprotection after hypoxia/reoxygenation.Targetiong upregulation of autophagy and mitochondrial Cx43 and improve the phosphorylation of mitochondrial Cx43 maybe an important treatment of myocardial ischemia/reperfusion injury.