缺氧-复氧对H9c2心肌细胞自噬和线粒体缝隙连接蛋白43的影响及其相互作用

Changes and interaction of autophagy and mitochondrial connexin 43 in H9c2 cardiomyocytes after hypoxia-reoxygenation

目的探究心肌细胞系H9c2在缺氧-复氧(H/R)后自噬与线粒体缝隙连接蛋白43(Cx43)的变化,以及两者间的相互关系。方法通过缺氧处理12 h、复氧处理12 h建立H9c2细胞H/R模型。将H9c2细胞随机分为5组:对照组、H/R组、格尔德霉素(GA)组、3-甲基腺嘌呤(3-MA)组、GA+3-MA组。应用线粒体膜电位检测试剂盒检测各组线粒体膜电位,细胞计数试剂盒(CCK-8法)检测各组细胞活力,采用Western印迹法检测各组Beclin-1、LC3B、线粒体Cx43和磷酸化Cx43(p-Cx43)的蛋白质水平;应用电子显微镜观察各组自噬体情况。结果 H/R组的细胞活力显著低于对照组(P<0.01),乳酸脱氢酶(LDH)水平显著高于对照组(P<0.01),H-R模型建立成功。H/R组的线粒体膜电位、细胞活力均显著低于对照组(P值均<0.05);GA组、3-MA组和GA+3-MA组的线粒体膜电位、细胞活力为均显著低于H/R组(P值均<0.05);GA+3-MA组线粒体膜电位、细胞活力均分别显著低于GA组和3-MA组(P值均<0.05)。H/R组自噬相关蛋白质Beclin-1水平和LC3BⅡ/LC3BⅠ分别为0.89±0.05和4.37±0.50,均显著高于对照组的0.26±0.03和0.77±0.09(P值均<0.05);与H/R组相比,GA组、3-MA组和GA+3-MA组的Beclin-1水平(0.75±0.06、0.47±0.03、0.31±0.02)、LC3BⅡ/LC3BⅠ(3.29±0.18、1.37±0.09、0.85±0.14)均显著降低(P值均<0.05);GA+3-MA组的Beclin-1水平、LC3BⅡ/LC3BⅠ又显著低于GA组和3-MA组(P值均<0.05)。H/R组线粒体Cx43和p-Cx43蛋白质水平分别为0.75±0.03和0.41±0.03,均显著低于对照组的0.86±0.02和0.58±0.01(P值均<0.05);GA组、3-MA组和GA+3-MA组的线粒体p-Cx43蛋白质水平分别为0.27±0.06、0.33±0.05、0.18±0.04,均显著低于H/R组(P值均<0.05);GA组和GA+3-MA组的线粒体Cx43蛋白质水平(0.62±0.09、0.59±0.07)均显著低于H/R组(P值均<0.05);GA+3-MA组的线粒体p-Cx43蛋白质水平显著低于GA组和3-MA组(P值均<0.05)。结论 H/R引起的线粒体Cx43含量下降和脱磷酸化增加可能是缺血-再灌注损伤发生的重要机制。自噬参与线粒体Cx43磷酸化状态的维持,线粒体Cx43特别是p-Cx43也参与了自噬的激活过程,对H/R心肌起保护作用。

Objective To investigate the changes and interaction of autophagy and mitochondrial connexin 43(Cx43)in H9c2 cardiomyocytes after hypoxia/reoxygenation(H/R).Methods H/R model of H9c2 cardiomyocytes was established by hypoxia treatment for 12 h and reoxygenation for 12 h.H9c2 cardiomyocytes were randomly divided into five groups:control group,H/R group,geldanamycin(GA)group,3-methyladenine(3-MA)group,and GA+3-MA group.Mitochondrial membrane potential(MMP)was measured by JC-1 test.Cell viability was detected by cell counting kit-8(CCK-8).Protein levels of Beclin-1,LC3 B,mitochondrial Cx43 and mitochondrial phosphory-lation of Cx43(p-Cx43)were determined by Western blot.Autophagosomes were observed by electron microscope.Results Cell viability of H/R group was significantly lower than that in control group(P<0.01),while the level of lactate dehydrogenase(LDH)of H/R group was significantly higher than that in control group(P<0.01),indicating H/R model of H9c2 cardiomyocytes was successfully established.The proportion of cells with decreased MMP and cell viability in H/R group were both significantly lower than those in control group(all P<0.05).The proportion of cells with decreased MMP and cell viability in GA group,3-MA group and GA+3-MA group were all significantly lower than those in H/R group(all P<0.05).The proportion of cells with decreased MMP and cell viability in GA+3-MA group were both lower than those in GA group and 3-MA group(all P<0.05).Beclin-1 level and LC3 BⅡ/LC3 BⅠin H/R group were 0.89±0.05 and 4.37±0.50,respectively,which were both significantly higher than those in control group(0.26±0.03 and 0.77±0.09,both P<0.05).Beclin-1(0.75±0.06,0.47±0.03,0.31±0.02)and LC3 BⅡ/LC3 BⅠ(3.29±0.18,1.37±0.09,0.85±0.14)in GA group,3-MA group and GA+3-MA group were all significantly lower than those in H/R group(all P<0.05).Moreover,Beclin-1 and LC3 BⅡ/LC3 BⅠin GA+3-MA group were lower than those in GA group and 3-MA group(all P<0.05).Contents of mitochondrial Cx43 and p-Cx43 in H/R group were 0.75±0.03 and 0.41±0.03,respectively,which were significantly lower than those in control group(0.86±0.02 and 0.58±0.01,both P<0.05).Content of mitochondrial p-Cx43 was 0.27±0.06 in GA group,0.33±0.05 in 3-MA group and 0.18±0.04 in GA+3-MA group,which were lower than that in H/R group(all P<0.05).Content of mitochondrial Cx43 was 0.62±0.09 in GA group and 0.59±0.07 in GA+3-MA group,which were lower than that in H/R group(both P<0.05).Content of mitochondrial p-Cx43 in GA+3-MA group was lower than that in GA group and 3-MA group(both P<0.05).Conclusion A significant decrease and dephosphorylation of mitochondrial Cx43 may be important mechanisms of ischemia/reperfusion injury induced by hypoxia/reoxygenation in H9c2 cardiomyocytes.Autophagy is involved in the maintenance of mitochondrial Cx43 phosphorylation.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.