拟交感效应对快速起搏离体心脏心房缝隙连接蛋白43重构的影响

目的:通过建立离体拟交感心房颤动模型,探讨缝隙连接蛋白43(Cx43)水平的变化。方法:15只杂种犬随机分为3组(每组5只):对照组(control组)、快速心房起搏组(RAP组)和异丙肾上腺素灌流+快速心房起搏组(ISO+RAP组)。经胸骨正中切开术,快速取出心脏,建立心脏Langendorff离体灌流模型。各组分别检测心房有效不应期(AERP)及房颤诱发率,免疫组化检测酪氨酸羟化酶(TH)在细胞内的表达,Western blot检测Cx43和磷酸化Cx43的蛋白含量,免疫荧光检测Cx43在心肌组织的变化,TUNEL法检测心肌细胞凋亡,荧光比色法检测线粒体活性氧簇(ROS)的生成量。结果:与control组比较,RAP组的AERP无明显变化,且仅出现电紊乱,ISO+RAP组的AERP明显缩短(P0.05),并可成功诱发房颤。与control组比较,RAP组和ISO+RAP组的TH表达量、细胞凋亡指数和线粒体ROS生成量均逐渐增多(P0.05),Cx43蛋白表达量和磷酸化水平逐渐减少(P0.05)。免疫荧光显示,与control组相比,RAP组的Cx43荧光强度降低,且呈明显侧链化,ISO+RAP组则呈点状散在分布。结论:交感神经可能通过氧化应激效应,引起Cx43的重构与下调,从而介导心房颤动的发生。

Effects of sympathomimetic agent on remodeling of connexin 43 in atrium of isolated heart with rapid atrial pacing

AIM: To investigate the changes of connexin 43 (Cx43) via establishing a model of sympathomimetic atrial fibrillation (AF).METHODS: The mongrels (n=15) were randomly divided into control group, rapid atrial pacing (RAP) group and isoprenaline (ISO) perfusion+RAP group (ISO+RAP group). All mongrels' hearts were taken out rapidly by median sternotomy to establish the cardiac model with Langendorff perfusion in vitro. The atrial effective refractory period (AERP) and AF inducability were tested. The expression and distribution of tyrosine hydroxylase (TH) were analyzed by immunohistochemistry. Total protein level of Cx43 and phosphorylation of Cx43 were determined by Western blot. The distribution of Cx43 were also observed by immunofluorescence staining. The cell apoptosis was analyzed by TUNEL staining. The generation of reactive oxygen species (ROS) in the mitochondria was measured by fluorescence spectrophotometry.RESULTS: No significant change of AERP was found between control group and RAP group, while that in ISO+RAP group was significantly decreased (P<0.05) and induced AF. Compared with control group, the expression of TH, apoptotic index and the generation of ROS increased gradually (P<0.05), while the content of Cx43 decreased gradually both in the total protein and the phosphorylation levels in RAP group and ISO+RAP group (P<0.05). The fluorescence intensity of Cx43 was also attenuated and Cx43 were lateralized apparently in RAP group, while Cx43 were characterized as punctate distribution in ISO+RAP group.CONCLUSION: Sympathetic nerves may activate autophagosome at intercalated discs and trigger cell apoptosis, resulting in remodeling and downregulation of Cx43 via oxidative stress, thus having effects on mediating and maintaining AF.