ClC-3氯通道蛋白在肾上腺素能受体介导心肌肥厚中的功能研究

Adrenergic receptor stimulation is closely related to clinical myocardial hypertrophy, but the exact mechanism is unclear. ClC-3 has been proposed as a candidate for the volume-activated chloride channel and is involved in cell volume regulation. Our exploratory study found that adrenaline reduced ClC-3 expression in the hypertrophied cardiomyocytes, suggesting that ClC-3 is likely involved in the adrenergic receptor-mediated cardiac hypertrophy. Firstly, in this project, we will analyse the expression differences of ClC-3 between hypertrophic myocardium and normal myocardium in clinical pathology specimens and mouse models induced by adrenergic receptor stimulation, and with the observation of the effects of ClC-3 expression up-regulated in the primary hypertrophic myocardial cells on cell volume, so that we could determine the important role of ClC-3 involved in adrenoceptor stimulation-induced cardiac hypertrophy. Then, in the heart-specific ClC-3 knockout mice and primary cardiac myocyte, we’ll explore the mechanism of ClC-3 down-regulation induced by adrenergic receptor activator through G protein/AC/cAMP/PKA signaling pathway, combined with the study for ClC-3 down-regulation resulted in volume-activated chloride channel deficiency and cell volume regulation disorders, clarify the underlying molecular and cellular mechanism for ClC-3 involved in adrenoceptor stimulation-induced cardiac hypertrophy. The implementation of the project is expected to provide a new theoretical basis for the study of the mechanism of adrenoceptor stimulation-induced cardiac hypertrophy, will also extend the research of the ClC-3 chloride channel protein multifunction.

肾上腺素能受体激动与心肌肥厚密切相关，但机制未明确。ClC-3是容积激活性氯通道的构成分子，参与细胞容积调节。我们探索性研究发现肾上腺素下调心肌细胞ClC-3的表达，提示ClC-3很可能参与肾上腺素能受体介导的心肌肥厚。本项目拟深入研究：首先，分析临床病理标本和鼠肥厚心肌与正常心肌ClC-3表达差异，并观察恢复肥厚心肌细胞ClC-3表达对细胞体积的影响，确定ClC-3在肾上腺素能受体激动诱导心肌肥厚中的重要作用；然后，利用培养的心肌细胞和心脏ClC-3基因特异性敲除的小鼠，探讨肾上腺素能受体激动下调ClC-3表达及其下调导致容积激活性氯通道缺失和细胞容积失调的机理，明确肾上腺素能受体激动→G蛋白/AC/cAMP/PKA信号通路活化→ClC-3表达↓→容积激活性氯电流↓→细胞容积失调→心肌肥厚的分子和细胞机制。项目的实施有望为心肌肥厚的防治提供新的理论依据，也将ClC-3的功能研究拓展视野。

本项目研究发现ClC-3氯通道蛋白的表达量与几例临床心肌肥厚标本具有一定的负相关性；在肾上腺素能受体激动诱导的肥厚H9c2细胞、原代心肌细胞及心肌组织中发现ClC-3的表达量明显下降，而其联合拮抗剂在改善心肌肥厚的同时能明显抑制ClC-3表达量的下调；发现ClC-3上调的转基因小鼠和AAV介导的ClC-3转染小鼠给予肾上腺素能受体激动剂后小鼠的心肌肥厚情况明显得到抑制，而ClC-3表达下调能明显诱导心肌细胞肥大的发生，进一步的机制研究发现其可能与AC-cAMP-PKA通路的激活、容积激活性Cl电流的改变有关。本项目为心肌肥厚的防治提供新的视角，同时也开辟了ClC-3多功能研究的新领域，为以后专利的申请及其他推广应用奠定了前期基础。目前课题组已经发表相关研究论文3篇，其中一篇名为《ClC-3 chloride channel is involved in isoprenaline-induced cardiac hypertrophy》的论文已经被GENE杂志（SCI收录）于2018年2月发表。此外，在该项目的支持下，1名项目主要成员晋升为副教授，2名研究生顺利毕业，目前在读研究生3名，而且还指导3名本科生获得“2018年度广东省大学生创新创业训练计划项目”1项。