Ca2+激活Cl-通道TMEM16A促进心梗后纤维化的机理研究

Healing of myocardial infarction is accompanied by a process of fibrosis, leading to impairment of cardiac function and eventually heart failure. Differentiation of cardiac fibroblasts into myofibroblasts with increased production of extracellular matrix (transdifferentiation) is central in cardiac fibrosis. TGF-β/Smad pathway is known to play a key role in the transdifferentiation but how the signal transduction is precisely regulated remains to be fully defined. This study for the first time aims to explore the role of chloride (Cl-) channel in the pathogenesis of cardiac fibrosis post-myocardial infarction. The preliminary results revealed that Ca2+-activated Cl- channel TMEM16A was upregulated in expression and function in post-myocardial-infarction fibrosis, knockdown of TMEM16A decreased TGF-β-induced increase in smad phosphorylation and collagen expression, and, importantly, TMEM16A physically interacted with TGF-β receptor I (TGFβRI) in transdifferentiated cardiac fibroblasts. We thus put forth the hypothesis: deceased Cl- concentration surrounding TGFβRI, due to TMEM16A/ TGFβRI interaction and increased TMEM16A-mediated Cl- efflux, enhances smad phosphorylation, aggravating cardiac fibroblast transdifferentiation and resulting in fibrosis. This study will conduct cell culture and animal experiments employing TMEM16A-targeting small interfering RNA and fibroblast-specific TMEM16A knockout mice, in order to validate the significance of TMEM16A in fibrosis post-myocardial infarction and dissect the precise mechanisms. This study will evaluate if TMEM16A is a novel pharmacologic target and provide novel insights into the pathogenesis of myocardial infarction-induced fibrosis.

心梗愈合伴随纤维化（心梗后纤维化）损害心功能至心衰。心脏成纤维细胞分化为肌成纤维细胞（转分化）后增加基质是纤维化发生关键。TGFβ/Smad通路在转分化中至关重要，但调节机制有待阐明。本研究以Cl-通道在转分化中的作用为新切入点，首次发现①Ca2+激活Cl-通道TMEM16A在心梗后纤维化时表达和功能增强；②沉默TMEM16A降低TGFβ诱导的Smad2/3磷酸化及胶原表达；③心梗后纤维化时TMEM16A与I型TGFβ受体结合。申请人因此假设：转分化时TMEM16A与TGFβ受体结合，因其表达升高而增强Cl-外流进而降低TGFβ受体局部Cl-浓度，从而增强Smad2/3磷酸化促发心梗后纤维化。本研究拟采用心脏成纤维细胞特异TMEM16A敲除小鼠及基因沉默进行动物及细胞实验，探讨TMEM16A在心梗后纤维化中的作用及机理。本研究从TMEM16A功能异常这一新角度为防治心梗后纤维化提供新思路。

心梗后纤维化损害心功能至心衰。TGFβ/Smad通路在纤维化中至关重要，但调节机制有待阐明。本研究拟探讨Ca2+激活Cl-通道TMEM16A在心梗后纤维化中的作用。小鼠心梗模型及TGF-β干预心脏成纤维细胞时， TMEM16A通道表达显著增加；同时smad2/3的磷酸化及collagen表达升高；提示TMEM16A可能参与心梗后纤维化的过程；siRNA敲减TMEM16A表达后，smad2/3的磷酸化及collagen表达水平与假手术组没有统计学差异，进一步说明TMEM16A参与心梗后纤维化过程；TGF-β干预时TMEM16A与I型TGF-β受体结合增加，提示两者相互作用可能参与心梗后纤维化。遗憾的是由于初次使用转基因技术（Transgenic）制备的FSP1-Cre小鼠，未了解转基因小鼠随着繁殖代数增加，可能出现不可逆地Cre逐渐丢失。导致心脏成纤维细胞TMEM16A特异性敲除小鼠（KO小鼠）的实验未能按计划展开。申请人前期工作主要围绕TMEM16A通道和TRPM7通道展开，在KO小鼠的实验搁浅后，申请人展开了如下两项工作：1）检测一种新型多肽P2是否具有抗心梗后纤维化作用。前期实验证明多肽P2对肝、肺、肾等多器官纤维化具有保护作用，我们证明多肽P2对心梗后纤维化具有保护作用。2）检测TRPM7通道在血管衰老中的作用，发现：1）RT-qPCR和Western blot检测证实TRPM7通道表达升高；2）IL-1β、IL-6、TNF-α等因子表达显著升高；3）NF-κB信号通路被激活；4）Pyk2磷酸化水平升高。进一步shRNA敲低TRPM7通道表达或者BAPTA鳌和Ca2+后，NF-κB信号通路的激活和Pyk2磷酸化水平被抑制。说明TRPM7-Pyk2-NF-κB参与了血管衰老过程中IL-1β、IL-6、TNF-α等因子的释放。

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