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目的:通过建立拟交感性心房颤动(房颤)模型,探讨心肌缝隙连接蛋白(Cx)43和Cx40表达水平的变化。方法:选择杂种犬15只,随机分为3组,即:对照组(N组)、快速心房起搏组(RAP组)和快速心房起搏+异丙肾上腺素(ISO)灌流组(RAP+ISO组),每组5只,处死后取出心脏,建立langendorff心脏离体灌流模型。3组分别检测心房有效不应期(AERP)及房颤诱发率,免疫组化检测神经生长因子、酪氨酸羟化酶在细胞内的表达及分布,蛋白免疫印记法检测Cx43和Cx40总蛋白水平,透射电镜检测线粒体形态,荧光比色法检测线粒体内活性氧簇(ROS)生成量。结果:与N组AERP[(166±5.1)ms]比较,RAP组AERP[(160±3.2)ms]无明显改变,无法诱发出房颤,RAP+ISO组AERP[(148±3.7)ms]明显缩短(P<0.05),并可成功诱发房颤。与N组比较,RAP组线粒体轻度肿胀,基质基本完整,RAP+ISO组线粒体明显肿胀,部分基质透明。RAP组和RAP+ISO组Cx43和Cx40总蛋白含量均低于N组(P<0.05),且RAP+ISO组蛋白含量低于RAP组(P<0.05)。RAP+ISO组神经生长因子、酪氨酸羟化酶分布和含量以及线粒体内ROS生成量显著高于RAP组和N组(P<0.05),而RAP组则高于N组(P<0.05),差异均有统计学意义。结论:交感性房颤的发生与Cx43和Cx40蛋白含量的变化有关,交感神经可能通过氧化应激下调Cx43和Cx40的蛋白含量来介导房颤的发生。
Objective: To investigate the changes of the expression of Cx43 and Cx40 in myocardium by establishing a model of sympathetic atrial fibrillation (atrial fibrillation). Methods: Fifteen mixed dogs were randomly divided into three groups: control group (N group), rapid atrial pacing group (RAP group) and rapid atrial pacing + isoproterenol group (RAP + ISO group), 5 in each group. After sacrifice, the heart was removed and the langendorff cardiac perfusion model was established. The AERP and the induced rate of atrial fibrillation were detected in the three groups. The expression and distribution of NGF and tyrosine hydroxylase in the cells were detected by immunohistochemistry. The total Cx43 and Cx40 protein The morphology of mitochondria was detected by transmission electron microscopy and the formation of reactive oxygen species (ROS) in mitochondria was detected by fluorescence colorimetry. Results: AERP [(160 ± 3.2) ms] in RAP group showed no significant change compared with AERP in group N [(166 ± 5.1) ms] Significantly shortened (P <0.05), and successfully induced atrial fibrillation. Compared with the N group, the mitochondria of RAP group swelled slightly and the matrix was basically intact. The mitochondria of RAP + ISO group were obviously swollen and some of the matrix was transparent. The total protein contents of Cx43 and Cx40 in RAP group and RAP + ISO group were lower than those in N group (P <0.05), and RAP + ISO group protein content was lower than that in RAP group (P <0.05). The distribution and content of NGF, tyrosine hydroxylase and ROS in mitochondria in RAP + ISO group were significantly higher than those in RAP group and N group (P <0.05), but higher in RAP group than those in N group (P <0.05) , The differences were statistically significant. CONCLUSIONS: The occurrence of sympathetic atrial fibrillation is related to the changes of Cx43 and Cx40 protein contents. Sympathetic nerves may mediate the occurrence of atrial fibrillation by down-regulating the protein levels of Cx43 and Cx40 in oxidative stress.