LRRC8A介导AT1R-NOX-ROS信号通路刺激心肌肥大的机制

CCardiac hypertrophy is an important pathological change in the progression of cardiovascular disease. It is the main cause of heart failure and even sudden death. However, there is no effective treatment. So it is of great significance to explore a new mechanism of development of myocardial hypertrophy so that to find new targets for intervention. LRRC8A is a core component that belongs to volume-regulating anion channels (VRAC) and is the major regulator of cell volume changes. It is unknown whether the LRRC8A protein involves in the enlargement of the volume which is characterized in the process of cardiac hypertrophy. If it does, what is the mechanism? Our former studies showed that blocking of VRAC with drugs and silencing the gene of lrrc8a all could effectively inhibit ROS production and myocardial hypertrophy induced by angiotensin II. Angiotensin II receptors AT1R and NOX oxidase have been identified as key molecules in the process of cardiac hypertrophy and they located in the caveolae with LRRC8A. It is known that AT1R can bind to potassium channel proteins and NOX bind to LRRC8A. Therefore, our study intends to investigate the effect of LRRC8A on cardiac hypertrophy caused by angiotensin II and elucidate the molecular mechanism of LRRC8A-mediated AT1R-NOX-ROS signaling pathway in hypertrophy. To provide a new theoretical basis and an intervention target for the prevention and treatment of cardiac hypertrophy.

心肌肥大是心血管疾病进展过程中的重要病理改变，是导致心力衰竭甚至猝死的主要原因，目前尚无特效治疗手段。探索心肌肥大发生的新机制，寻找干预的新靶点具有重要意义。LRRC8A是组成体积调节性阴离子通道（VRAC）的核心结构蛋白，是细胞体积变化的主要调节者。那么，它是否参与了以体积增大为病理特征的心肌肥大过程？机制如何？我们前期实验发现，应用药物阻断VRAC及沉默lrrc8a可有效抑制AngⅡ诱导的心肌细胞ROS生成与肥大。特别是文献支持LRRC8A与调节心肌肥大的关键分子AT1R、NOX均定位于膜窖，且有AT1R与钾通道、NOX1与LRRC8A结合的证据。据此，本研究拟在AngⅡ诱导心肌肥大过程中，通过干预LRRC8A探讨其对细胞肥大的影响，并阐明LRRC8A介导AT1R-NOX-ROS信号通路的分子机制，为防治心肌肥大提供新的理论依据与干预靶点。

摘要：心肌肥大是心血管疾病进展过程中的重要病理改变，是导致心力衰竭甚至猝死的主要原因，目前尚无特效治疗手段。探索心肌肥大发生的新机制，寻找干预的新靶点具有重要意义。LRRC8A是组成体积调节性阴离子通道（VRAC）的核心结构蛋白，是细胞体积变化的主要调节者。本研究首次证实了LRRC8A在心肌肥大中的作用，并阐述了相关调控机制。本研究通过AngII刺激在体动物心肌组织和离体原代心肌细胞，成功构建了心肌组织（细胞）肥大模型，并检测AngII对心肌LRRC8A表达的影响；通过应用RNAi技术敲低心肌LRRC8A基因后，研究LRRC8A在AngII诱导心肌肥大中的作用及相关机制，主要研究结果如下：①AngII诱导心肌肥大的过程中伴有LRRC8A表达的增强，沉默LRRC8A可以抑制AngII诱导的心肌肥大，证明LRRC8A参与了AngII诱导的心肌肥大过程；② AngII可以激活心肌细胞VRAC电流，敲低LRRC8A、ROS清除剂、NADPH氧化酶抑制剂均可阻断或抑制VRAC电流。提示VRAC与NADPH氧化酶之间存在着功能上的相互调控；③沉默LRRC8A通过抑制NADPH氧化酶胞膜亚基Nox2、Nox4、p22phox的表达及胞质亚基p47phox和p67phox向胞膜转位，从而抑制AngII诱导的NADPH氧化酶活性增加及ROS的生成，提示LRRC8A可能通过NADPH氧化酶-ROS通路参与了心肌肥大的调控；④免疫荧光、免疫共沉淀、和过表达LRRC8A Δ91/+35实验首次证实LRRC8A通过LRRD与Nox2、Nox4、p22phox亚基直接结合，此为VRAC与NADPH氧化酶相互调控的结构基础。结论：VRAC的核心蛋白LRRC8A通过LRRD与NADPH氧化酶直接结合，调控NADPH氧化酶活性及ROS的产生，从而参与AngII诱导的心肌肥大。

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