交感神经介导心房颤动与氧化应激下胞膜和线粒体Cx43的相关机制

Sympathetic nervous system (SNS) is an important physiological regulation system for human. Inasmuch as simple increase stimulation in SNS would not culminate in atrial fibrillation (AF) in majority of people, sympathetic-mediated AF should be associated with some kind of structural disorders. Our previous studies have shown that gap junction (GJ) and connexin (Cx) could trigger AF; besides, many researches have proved that oxidative stress imbalance also plays an important role in mechanism of AF. In our recent studies, there is a close relationship among the cell membrane, mitochondrial connexin 43 (Cx43) and oxidative stress imbalance. Thus, SNS might have effects in triggering and maintaining AF through affecting oxidative stress resulting in changes between cell membrane and mitochondrial Cx43 and remodeling of GJ. In this study, Cx43mRNA are made silent at the transcriptional level using RNA interference (RNAi) then build Cx43 low expression of canine isolated myocytes, Langendorff and in vivo models; changing sympathetic tone by sympathetic ganglion or neurotransmitter interventions; and intervention with specific membrane transduction blocker and ROS scavenger. The distribution, function and expression level of Cx43; and the condition of oxidative stress and AF induction are detected in cellular and tissue levels separately. Exploring the relationship between sympathetic-mediated AF and change in the membrane and mitochondrial Cx43 due to oxidative stress could deepen the understanding of significant mechanisms of triggering and maintenance of sympathetic-mediated AF.

交感神经是重要的生理调节体系，对于大多数正常人，其单纯的张力增高不会导致房颤。故交感神经介导的房颤中可能存在相应的结构病变。我们既往研究证实缝隙连接（GJ）和连接蛋白（Cx）促进房颤发生；此外，许多研究显示氧化应激也可影响房颤；最近，我们的研究进一步发现细胞膜和线粒体连接蛋白43（Cx43）与氧化应激关系密切。因此，假设交感神经通过影响氧化应激下胞膜和线粒体Cx43之间的变化及GJ的重构而介导房颤的发生和维持。本课题采用RNA干扰技术在转录水平将Cx43mRNA沉默，构建Cx43低表达犬离体心肌细胞、离体心脏灌流及活体模型；予神经节或递质干预改变交感张力；并采用特异性膜转导蛋白阻滞剂和ROS清除剂干预。在细胞、组织水平，检测Cx43的表达、分布和功能，氧化应激状态及房颤诱发情况；探讨交感神经介导房颤与氧化应激下胞膜和线粒体Cx43的相关性，这对深化认识交感性房颤的发生与维持机制有重要意义。

缝隙连接蛋白介导心肌细胞之间的电偶联。其中，缝隙连接蛋白43（connexin 43，CX43）在心肌中表达最丰富。心房颤动是临床上最常见的心律失常。自主神经张力的改变是房颤发生和维持的重要因素。研究表明交感神经与房颤的发生密切相关，但其内在机制目前尚不明确。氧化应激和缝隙连接蛋白43的变化及交感神经张力增高均有密切关系。假设交感神经介导心房颤动与氧化应激下胞膜和线粒体Cx43变化密切相关。. 首先，提取犬心房肌原代细胞，采用RNA干扰技术将Cx43mRNA在转录水平沉默，并在细胞水平构建了交感性房颤模型。观察Cx43低表达在异丙肾上腺素灌流及快速电场起搏心房肌细胞模型中的影响。结果显示，异丙肾上腺素及电场高频刺激可抑制Cx43的表达， Cx43低表达可导致细胞间缝隙连接通道信号传导功能障碍，可能对交感性房颤的发生和维持起到重要作用。. 接着，我们在在体水平采用神经节干预或递质干预，结合快速心房起搏方法构建交感性房颤犬模型，观察交感神经对心房有效不应期、房颤诱发率的影响；比较神经节干预与递质干预在房颤犬模型建立中的差异。结果提示：交感神经活性与心房电重构有一定联系，单纯刺激交感神经不能改变房颤的诱发频率，但交感神经递质可使房颤更易于诱发。. 接着，我们用药物干预交感性房颤犬的细胞膜和线粒体Cx43介导的氧化应激反应。发现膜转导蛋白阻滞剂可以下调磷酸化Cx43的表达量，增加房颤易感性，当ROS清除剂与膜转导蛋白阻滞剂共同使用时，可减轻Cx43的下降程度，同时降低房颤的易感性。提示Cx43在胞膜和线粒体膜的转移可起到拮抗细胞电重构的改变，ROS可能通过降低磷酸化Cx43表达诱导房颤的发生。. 最后，我们成功构建了Cx43低表达的犬心脏模型，并利用langendorff建立交感性房颤模型。于Cx43正常表达组及低表达组均分别予以快速心房起搏及异丙肾上腺素灌流+快速心房起搏刺激。结果发现，单纯心房起搏无法诱发房颤，而Cx43低表达时，则使房颤易于诱发。交感兴奋状态加剧了房颤的易感性，且Cx43显著下降，提示了交感神经可能通过下调Cx43介导房颤的发生。