miR-155参与调控心房颤动Cav1.2钙通道电重构的机制研究

Atrial fibrillation (AF) is the most common sustained cardiac rhythm disorder, and is associated with pronounced morbidity, mortality, and socio-economic burden. An important acknowledgement is that AF atrial electric remodeling in a way that promotes its occurrence and maintenance. Decreased L-type Ca2+ current (ICaL) is a consistent feature of atrial remodeling and is believed to contribute importantly to the action potential duration abbreviation that shortens refractoriness and promotes AF. Evidence has proved ICaL reduction is associated with transcriptional downregulation of the Cav1.2 (ICaL α-subunit). However, the mechanisms of Cav1.2 downregulation are poorly understood. MicroRNAs (miRNAs) are ~22-nucleotide single-stranded RNAs that inhibit the expression of specific mRNA targets through Watson-Crick base pairing between the miRNA 'seed region' and sequences commonly located in the 3'-untranslated regions. Although the cellular mechanisms and gene mutations responsible for numerous cardiovascular disorders have been extensively studied, it has become apparent only recently that miRNAs have key roles in cardiovascular development and disease. In our previous study which supported by NSFC, the miRNA transcriptome was analyzed by microarray and verified by real-time reverse-transcription polymerase chain reaction with left atrial samples from nonvalvular AF patients and healthy control. MiR-155, miR-142-3p, miR-19b, miR-223, miR-146b-5p, miR-486-5p, miR-301b, miR-193b, miR-519b were found to be upregulated by >2 fold, whereas miR-193a-5p were downregulated. In particular, miR-155 level was elevated by 5.7-fold in AF patients relative to healthy control. Furthermore, computational prediction identified CACNA1C, which encode Cav1.2, as direct targets of miR-155.In the meantime, construction of reporter plasmids and reporter assays showed that miR-155 represses the Luciferase activity of the 3' untranslated regions of CACNA1C. This work reveals a potential link between the regulation of Cav1.2 and miR-155. In this application, we are going to test this hypothesis. We plan to combine in vitro culture model of human atrial cardiomyocytes and AF mice to assess (1) whether miR-155 contributes ICaL reduction and Cav1.2 downregulation in vivo cellular electrophysiological remodeling by patch clamp and single-cell RT-PCR; (2) whether forced expression of miR-155 is able to recapitulate the phenotypes of AF and Cav1.2 remodeling with a miR-155 transgene mouse model; and (3) whether downregulation of endogenous miR-155 could reduces AF vulnerability and prevent Cav1.2 remodeling with a miR-155 knock down mouse model. The accomplishment of this application will provide a new understanding of underlying mechanisms of atrial remodeling to allow for the definition of novel molecular targets in AF. And it is tempting to pave the avenue for more specific novel drug targets and potential for developing that missing pharmacopeia for AF.

电重构在心房颤动（房颤）的发生和发展过程中占有重要地位，Cav1.2钙通道是电重构的始动因素和最主要的分子基础。microRNA（miRNA）作为生命科学界研究的新焦点，在心血管系统中的关键调控作用不断得以阐明。本项目组前期通过miRNA芯片技术发现非瓣膜性房颤患者左心耳组织中miR-155的表达上调导致了预测的靶基因CACNA1C和相应Cav1.2蛋白表达下调，更重要的是体外构建报告载体实验发现miR-155可以结合到CACNA1C的3'-UTR区，提示miR-155有可能通过调控Cav1.2的表达，参与了房颤的电重构。本研究拟通过人心房肌细胞和小鼠的房颤模型，结合基因敲除和转基因小鼠，从细胞分子水平和动物实验探讨miR-155调控Cav1.2电重构的机制。本研究不仅对阐明miRNA调控电重构的机制有重要意义，而且为寻找可逆转电重构的新靶点奠定了理论基础，为房颤的上游治疗提供了新的思路。

心房颤动(房颤) 是临床上最常见的心律失常， 并且有很高的致残率和致死率，严重威胁人类的生命和健康。心房电重构促使房颤发生和延续， 改善心房电重构可以有效预防房颤。微RNA (miRNA)是新发现的基因表达调控因子，研究已表明其通过参与调控多种心脏电活动相关蛋白的表达，参与瓣膜性房颤的发生和发展，但是作为绝大多数的非瓣膜性房颤，其电重构机制和miRNA的关系的研究却未见报道。本研究目的是基于前期非瓣膜房颤心房组织芯片研究的重要发现—miR-155，阐释其通过调节靶基因CACNA1C，参与房颤Cav1.2电重构的调控机制。研究通过人胚胎干细胞衍生的心房肌细胞和小鼠的房颤模型，结合基因敲除和转基因小鼠，从细胞分子水平和动物实验探讨miR-155调控Cav1.2电重构的机制。.首先利用视黄酸信号调控人胚胎干细胞分化为经验证的人源性的心房肌细胞，转染过表达miR-155后，PCR和WB检测分别显示CACNA1C mRNA和Cav1.2表达水平显著下调，膜片钳结果同样表明L-型钙通道（ICa,L）也显著下调。其次利用过表达miR-155的转基因小鼠建立房颤模型，结果显示转基因组（TG）房颤发生率和房颤持续时间均显著高于野生鼠（WT）。获取小鼠心房肌细胞，TG组CACNA1C mRNA和Cav1.2表达水平显著抑制下调，伴随ICa,L也显著下调，使得TG组房颤易感性增高和持续时间显著延长。而在敲除miR-155的小鼠（KO）房颤模型中，与WT组相比较，房颤的发生率显著降低。而利用miR-155过表达干预的KO鼠其房颤的易感性和持续时间又显著高于WT组，说明敲除miR-155后，房颤的表型受到显著抑制。电生理进一步证实KO组较WT组心房肌细胞CACNA1C mRNA和Cav1.2表达水平显著上调，导致ICa,L也显著上调，使得KO组房颤易感性降低。而miR-155过表达干预的KO鼠组的CACNA1C mRNA和Cav1.2表达水平反而显著抑制下调，伴随ICa,L也显著下调，使得该组房颤易感性增高。.由此可见miR-155通过在转录后水平调控Cav1.2蛋白表达，进而影响了心房的电重构，最终导致了房颤的发生和发展。本研究不仅对阐明miR-155调控心肌细胞电重构的机制有重要意义，而且为寻找可逆转电重构的新靶点奠定了理论基础，为房颤的上游治疗提供了新的思路。

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