TRPV4通道介导心肌缺血再灌注损伤的机制研究

TRPV4 channel is a newly identified member of nonselective cation channels. Activation of the TRPV4 increases intracellular free Ca2+ concentration ([Ca2+]i), enhances production of reactive oxygen species (ROS), activates the inflammatory response, leads to cell apoptosis/death and probably involves in arrhythmia as well. However, whether TRPV4 channel involved in pathology of myocardial ischemic reperfusion injury (MIRI) has remained unknown. Recently, our preliminary studies found that: TRPV4 was expressed at low level in normal cardiomyocytes, however, its' expression level and function significant enhanced during MIRI; and TRPV4 agonist significant affect the reperfusion injury salvage kinase (RISK) expression and activity. Based on above results, we hypothesized that during MIRI, TRPV4 activation induced cardiomyocytes apoptosis/death and arrhythmia, which was through increasing in [Ca2+]i and production of ROS, inflammatory response and downregulation of RISK signaling pathway. Establishing the MIRI mice and cell models and using TRPV4-/- mice, TRPV4-siRNA, TRPV4 antagonits/agonists, we will explore molecular basis of MIRI mediated by TRPV4 channel. The results are expected to provide the new target of clinical treatment of MIRI.

TRPV4通道是新发现的一种非选择性阳离子通道，激活时可升高胞内钙离子（[Ca2+]i），促进氧自由基（ROS）释放，激发炎症反应，导致细胞凋亡坏死，还可诱发心律失常。然而TRPV4是否介导心肌缺血再灌注损伤（MIRI）的发生和发展，尚未见报道。最近我们预实验发现：正常心肌细胞有少量TRPV4表达，而在MIRI中，其表达及功能显著上调；另外，TRPV4激动剂可显著影响心肌细胞再灌注损伤挽救激酶（RISK）表达和活性。基于以上结果，我们推测在MIRI的发生和发展中，TRPV4上调，可通过升高[Ca2+]i、促进ROS释放、激发炎症反应以及下调RISK信号通路，导致心肌细胞凋亡坏死和心律失常。本研究拟建立MIRI小鼠及细胞模型，利用TRPV4-/-小鼠、TRPV4-siRNA及TRPV4阻断剂/激动剂，观察TRPV4如何参与MIRI的发生和发展，探索其分子机制，为临床防治MIRI提供新的靶点。

TRPV4通道是一种非选择性阳离子通道，可被缺血再灌注（I/R）激活，然而TRPV4是否参与心肌I/R损伤（MIRI）以及机制尚未见报道。因此本项目建立了在体、离体以及细胞MIRI模型，利用TRPV4-/-小鼠、TRPV4-siRNA及TRPV4阻断剂/激动剂， 研究了TRPV4在MIRI的作用及其机制。主要的结果如下：1）TRPV4在I/R后功能性表达升高；2）激动TRPV4可明显加重MIRI，主要表现为增加心梗面积、促进心肌细胞凋亡以及恶化心功能，同时可进一步增强I/R诱导的 [Ca2+]i升高、ROS释放增多、Δψm去极化、mPTP孔开放以及中性粒细胞浸润和炎症因子表达，而抑制TRPV4可显著减轻上述作用； 3）TRPV4阻断剂HC-067047治疗后可显著上调PI3K/Akt、ERK1/2和GSK-3活化（RISK信号通路），却对STAT3磷酸化水平（SAFE信号通路）影响不大，而RISK信号通路的阻断剂LY294002、wortmannin或U0126，均能废除HC-067047的心肌保护作用；4）HC-067047浓度依赖性减少H/R诱导的ROS和MDA释放、增强抗氧化酶活性、上调Akt磷酸化、促进Nrf2核转移以及ARE的基因表达，而Akt阻断剂LY294002可抑制HC-067047诱导的Nrf2核转移， LY294002以及Nrf2 siRNA均能显著抑制HC-067047的抗心肌氧化损伤的作用；5）静脉麻醉剂丙多酚可直接抑制心肌细胞上的TRPV4通道，减少I/R诱导的Ca2+内流，可完全逆转TRPV4激动剂GSK1016790A诱导的损伤，却无法进一步增加HC-067047的心肌保护作用。因此，本项目首次证实TRPV4介导了MIRI；激活TRPV4可通过[Ca 2+ ]i/ROS/Δψm/ mPTP以及炎症反应介导MIRI；阻断TRPV4可通过上调RISK信号通路减轻心肌细胞凋亡以及上调Akt/Nrf2/ARE信号通路增加内源性抗氧化酶活性减轻氧化应激损伤等分子机制减轻MIRI；此外丙多酚减轻MIRI损伤的作用也与抑制TRPV4，减轻细胞内钙超载相关。以上研究结果证实了TRPV4参与了MIRI，有望成为MIRI治疗的新靶点。