TRPV4通道调节T细胞分化介导自身免疫性心肌炎的发病机制研究

Myocarditis is a common cause of sudden death in young people under the age 40, which can develop into chronic dilated cardiomyopathy and heart failure, but the treatment for myocarditis is very scarce. CD4+Th17 cells have been proved to be an important initiator of the immune responses in myocarditis. Inhibition of Th17 cell differentiation can alleviate the myocardium inflammation. TRPV4 channel is a non-selective cation channel, which can mediate the Ca2+ influx and take part in the inflammation reponse. But There is no report about the role of TRPV4 in T cell differentiation. So we isolated NaiveT cells from human peripheral blood and gave the stimulation for the directional differentiation of Th17 cell. We found that the expression of TRPV4 channel mRNA increased significantly in this process, while TRPV4 channel blocker can inhibit the Ca2+ influx of T cells and apparently reduced the proliferation proportion of Th17 cells. These results suggest that TRPV4 channel may take part in the pathogenesis of myocarditis by regulating the differentiation of Th17 cells. Therefore, this project intends to establish a mouse model of autoimmune myocarditis, using TRPV4 specific agonist / antagonist /siRNA, to explore the molecular mechanism of TRPV4 channel in the pathogenesis of myocariditis, which may provide new ideas for the drug development for myocarditis and other Th17 cell-mediated autoimmune diseases.

心肌炎是40岁以下青年人群猝死的常见原因，并可迁延为扩张型心肌病及慢性心衰，但治疗药物十分匮乏。已知CD4+ Th17细胞是心肌炎中免疫反应的重要启动者，抑制Th17细胞的分化可减轻心肌组织炎症反应。TRPV4通道是非选择性阳离子通道，可介导Ca2+内流，参与炎症反应，但其能否调节T细胞分化尚未见相关报道。我们分离了人外周血的Naive T细胞，给予刺激诱导其向Th17细胞分化时，发现TRPV4通道mRNA的表达明显上升，加入TRPV4通道阻断剂可抑制T细胞的Ca2+内流，并明显降低增殖分化的Th17细胞比例，以上结果提示TRPV4通道可能通过影响Th17细胞分化参与心肌炎的发病。因此，本课题拟建立自身免疫性心肌炎小鼠模型，利用TRPV4通道特异性激动剂/阻断剂/siRNA，阐明TRPV4通道参与心肌炎发病的分子机制，为心肌炎及其他Th17细胞介导的自身免疫性疾病的治疗药物开发提供新思路。

心肌炎是40岁以下青年人群猝死的常见原因，并可迁延为扩张型心肌病及慢性心衰，但治疗药物十分匮乏。TRPV4通道通道是非选择性阳离子通道，可介导Ca2+内流，参与炎症反应，但其在心肌炎发病中的作用尚未见相关报道。本课题计划以EAM小鼠为模型，观察TRPV4通道通道的表达，给予TRPV4通道特异性激动剂和阻断剂，观察其对心肌炎症反应及T细胞亚群的影响；并通过体外实验观察激活或阻断TRPV4通道通道对Th17细胞分化的STAT3信号通路的影响，进一步阐明TRPV4通道通道的作用机制。该项目基本按计划执行，我们检测了TRPV4通道在EAM小鼠的表达，发现其在EAM模型中表达呈现上升趋势，生物信息学分析提示TRPV4通道在髓系细胞中表达较高，而在T细胞中表达较低。在EAM小鼠体内给予TRPV4通道抑制剂GSK2193874，心脏IL-17水平无明显变化，小鼠脾脏Th17细胞比例不变，但髓系细胞有明显变化。我们分离了正常小鼠的CD4+细胞及Naive T细胞，在体外分别给予TRPV4通道激动剂和抑制剂，发现TRPV4通道对CD4+ T细胞的钙内流、活化、增殖及Th17细胞的分化无明显作用。所以，我们将实验计划调整后有新的发现：（1）TRPV4通道抑制剂对小鼠CD4+ T细胞功能的直接影响不大，但在体内和体外明显调控髓系细胞的比例，并间接影响T细胞的功能。（2）在新冠疫情期间由于实验无法开展，我们通过生物信息学的方法筛选并发现了在心肌炎进展为扩张型心肌病过程中可能起重要作用的分子：CCL5和DPT，随后在人体心脏标本中进行了验证，为心肌炎的诊断和治疗提供了新的靶点。总之，本研究首次证实，TRPV4通道主要在EAM小鼠的髓系细胞上高表达，抑制TRPV4通道可调控髓系细胞的分化和比例，间接影响T细胞的功能，从而减轻EAM炎症；同时，我们首次筛选并验证了，CCL5与DPT可能成为心肌炎向扩张型心肌病的关键调控分子，为心肌炎及扩张型心肌病治疗药物开发提供新靶点。