miR-34a参与调控Gremlin-2在增龄性心房重构中的机制研究

Atrial fibrillation (AF) is the most common sustained cardiac rhythm disorder, and is associated with pronounced morbidity, mortality, and socio-economic burden. The prevalence of AF increases with age. As the population ages, the burden of AF increases. AF is a final common endpoint of atrial remodeling caused by ageing and itself causes important remodeling that contributes to the progressive nature of the arrhythmia. There are two principal pathophysiological mechanisms contributing to atrial remodeling: electrical remodeling and structural remodeling, each of which can result from ageing- related cardiac conditions. Gremlin 2 (GREM2), which is regulated by the ageing, is an inhibitor in the TGF beta signaling pathway, and it is a crucial regulator of the cardiac rhythm gene network that acts upstream of Sarcolipin (SLN) and transforming growth factor β1(TGFB1). Meanwhile, SLN is a specifically expressed in the atria, and may play an important role in modulating intracellular Ca2+ homeostasis in the atria; and TGFB1 is an essential mediator of atrial fibrosis in AF. .MicroRNAs (miRNAs), which are ~22-nucleotide single-stranded RNAs that inhibit the expression of specific mRNA targets, have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. MiRNAs are deregulated during aging and some miRNAs have been implicated in age-associated decline of organ functions. Although inhibition of miR-34a may protect against the decline in cardiac function with aging, it is less clear whether this may be beneficial in models of ageing-related atrial remodeling in AF. In our previous study, we identified miR-34a was upregulated, whereas its target gene GREM2 was downregulated during ageing in the atrial samples of patients with AF. The validated results revealed that after transfection of miR-34a into NIH/3T3 cells, GREM2 and SLN expression were markedly reduced and TGFB1 was increased. Moreover, the luciferase results confirmed that GREM2 was a target of miR-34a. This work reveals miR-34a probably acts as an intracellular mediator via GREM2/SLN/TGFB1 in the ageing-related atrial remodeling of AF. .In this application, we are going to test this hypothesis. We plan to combine in vitro cellular model of human atrial myocytes differentiated from human embryonic stem cells and animal model to assess (1) whether inducing expression of miR-34a and inhibition of its target GREM2 results in intracellular Ca2+ overloaded and TGFB1 upregulated in vitro cellular model by patch clamp and laser scanning confocal microscopy; (2) whether forced expression of miR-34a is able to recapitulate the phenotypes of ageing-related atrial remodeling and AF with a miR-34a transgene mouse model; and (3) whether downregulation of endogenous miR-34a could reduce AF vulnerability and prevent ageing-related atrial remodeling with a miR-34a knock down mouse model. .The accomplishment of this application will provide a new understanding of a key mechanism that regulates atrial remodeling during ageing in AF to allow for the definition of novel molecular targets in ageing-related AF. And it is tempting to pave the avenue for more specific novel drug targets and potential for developing that missing pharmacopeia for AF.

增龄性心房重构在心房颤动（房颤）的发生和发展过程中占有重要地位。microRNA（miRNA）已经成为生命科学界研究的新焦点，其中miR-34a被证实是心脏增龄性改变的重要调控因子。本项目组前期发现房颤患者伴随增龄出现miR-34a的表达上调和预测的靶基因GREM2表达下调；体外构建报告载体等实验证实miR-34a可以结合到GREM2的3'-UTR区，而且过表达miR-34a使GREM2表达降低，导致下游SLN和TGFB1蛋白表达异常，发生电重构和组织重构，提示miR-34a有可能通过调控GREM2参与心房重构。本研究拟利用人胚胎干细胞分化的心房肌细胞，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-34a调控增龄性心房重构的分子机制。本研究不仅对阐明miRNA调控增龄性心房重构的机制有重要意义，而且为寻找可逆转心房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。

增龄性心房重构在心房颤动（房颤）的发生和发展过程中占有重要地位。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心房重构相关蛋白的表达，参与房颤的发生和发展，但是作为增龄性房颤，其机制和miRNA的关系却未见报道。而miR-34a被证实是心脏增龄性改变的重要调控因子。本研究目的是基于前期发现房颤患者伴随增龄出现miR-34a过表达，进而使其靶基因GREM2表达降低，导致下游SLN和TGFB1蛋白表达异常，发生电重构和组织重构，提示miR-34a有可能通过调控GREM2参与心房重构。研究通过人胚胎干细胞分化的心房肌细胞，结合基因敲除和转基因小鼠，从细胞分子水平和动物实验阐释miR-34a调控增龄性心房重构的分子机制。首先通过通过miRNA干扰技术，利用人胚胎干细胞分化的心房肌细胞，转染过表达miR-34a，膜片钳技术证实上调miR-34a引起心房肌细胞钙电流降低，缩短APD，产生电重构。其次，通过小鼠模型，证实18周龄与年轻组小鼠（6月龄）相比较心脏表现出增龄性心肌重构：年轻组小鼠心功能明显优于老年组；房颤诱发率年轻组显著低于老年组；心肌纤维化和胶原蛋白容积指数在老年组更为严重 ；电生理发现老年组心肌细胞APD时程显著缩短；老年组心肌细胞ICa,L，Ito， IKur电流密度显著降低；PCR结果证实心肌重构相关基因的表达在两组之间也存在显著性差异。而miR-34a基因敲除老龄小鼠模型中，GREM2，SLN和CACNA1C在KO鼠显著增高，而TGFB1和COL1 A显著降低，蛋白水平的变化与mRNA水平一致。同时发现KO组和KO+过表达NC组纤维化程度最轻，房颤诱发率也是最低。最后采用miR-34a过表达的转基因小鼠模型，证实内源性上调miR-34a，通过调控 GREM2所导致的心房重构对年轻小鼠的影响。即：GREM2，SLN和CACNA1C在TG鼠和TG鼠/A-34aNC组显著降低，而TGFB1和COL1 A显著增高；AF诱发率在TG组也显著增加。由此可见miR-34a增龄性表达增高后通过转录后水平调控GREM2蛋白表达，产生钙通道重构和组织纤维化，影响了心房的重构，最终导致了增龄性房颤的发生和发展。本研究不仅对阐明miRNA调控增龄性心房重构的机制有重要意义，而且为寻找可逆转心房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。