Junctophilin-4依赖性Ca2+信号介导CD4+ JP4+ T细胞迁移在大鼠急性心肌梗死转归的作用研究

Acute myocardial infarction arises inflammation causes heart remodeling. Regulatory T cells (Treg) dissipate inflammation and promote myocardium healing, though the underlining mechanisms need further unveiling. T cell receptor (TCR) activation elevates store-operated Ca2+ entry to the endoplasmic reticulum by STIM1. A recent study proved that junctophilin 4（JP4）directly interacts to STIM1 upon TCR activation, suggesting its involvement in AMI stimulated Treg migration. And the underlining mechanisms need further explorations. Previously, we discovered the presence of CD4+JP4+ lymph node structures at the endocardium in a rat AMI model by ligating coronary anterior descending branch. Meanwhile, CD4+JP4+ infiltration into the myocardium was also found increased. Hence, in the current study, we would focus on the distribution of Foxp3+CD4+JP4+ T cells in myocardium, peripheral blood and thymus. Also, the correlation of heart function and Foxp3+CD4+JP4+ T cells, as well as, the downstream dynamic signaling pathways of TCR activation need further verification. In vitro, Jp4 knock-down using shRNA will be constructed in CD4+ T cells from rat peripheral blood among sham, AMI model and AMI+300 μg/kg GSNO interference. Based on Jp4 knock-down, chemotactic migration and Ca2+ transient will be tested to determine the essential role of JP4 in CD4+ T cell modulated AMI prognosis. In the present study, we hope to preliminarily clarify the regulating mechanisms of JP4 mediated Ca2+ signaling stimulated by AMI activated TCR, and the contribution to T cell migration in AMI. We also hope to provide more experimental evidence of CD4+JP4+ T as a potential diagnostic marker in AMI.

急性心肌梗死（AMI）时炎症可导致心肌重构。调节T细胞在梗死组织调控炎症向消退转化而促进愈合，但始发机制不明。T细胞受体被激活促进连接蛋白junctophilin-4（JP4）上调，由此调控内质网STIM1操纵胞内Ca2+平衡。申请人前期研究发现AMI大鼠心内膜出现多个CD4+JP4+ T细胞集落，伴CD4+JP4+ T细胞浸润增加，推测与JP4参与CD4+ T细胞迁移相关，亟待深入研究予以阐明。本项目拟进一步考察上述细胞是否表达Foxp3，在梗死心肌及外周分布差异，与心功能相关性及分子机制，评估JP4在CD4+ T细胞调控AMI转归的不可或缺性。体外考察Jp4沉默对CD4+ T细胞趋化迁移、Ca2+瞬变的影响，论证AMI时CD4+JP4+ T细胞表达增加的生命现象本质。阐明JP4依赖Ca2+信号在AMI调控CD4+ T细胞迁移的机制，项目的实施可望为AMI诊治潜在靶点提供新颖的科学依据。

急性心肌损伤（AMI）病理机制的研究提示，T细胞在AMI急性期发挥重要作用。然而，T细胞如何在损伤信号刺激下向心肌迁移、分化成具有抑制炎症作用的调节T细胞（Treg），成为亟待阐明的科学问题。T细胞受体激活促进连接蛋白junctophilin-4（JP4）上调，由此调控内质网STIM1操纵胞内Ca2+穿梭。本项目前期研究发现AMI大鼠心内膜出现多个CD4+JP4+ T细胞集落，伴CD4+JP4+ T细胞浸润增加，推测与JP4参与CD4+ T细胞迁移调控相关，亟待深入研究予以阐明。本项目进一步发现，Treg主要标志物Foxp3在上述CD4+JP4+ T细胞表达，胞浆与胞核均有分布。S-亚硝基谷胱甘肽（GSNO）预给药能促进Foxp3+ JP4+ T细胞向梗死心肌迁移，并伴随左心室M1型巨噬细胞浸润减少、促炎因子减少和炎症抑制性细胞因子增加，心功能增强等心肌修复效应。据此推测GSNO通过S-亚硝基化蛋白修饰促进JP4相关的Treg分化/迁移信号通路激活，相关蛋白修饰位点值得后续研究深入探索。体外构建Jp4沉默T细胞，给予GSNO未能提高其趋化迁移、向Treg分化能力，进一步证明JP4表达对T细胞迁移和发挥炎症抑制效应的关键作用。临床急性冠脉综合征患者急性期外周血T细胞的表型检测与转录组学测序分析，均初步验证了JP4在缺血性心肌损伤急性期的特征表达。本项目论证了AMI时CD4+JP4+ T细胞表达显著变化的生命现象本质，阐明JP4依赖Ca2+信号在AMI调控CD4+ T细胞迁移、向Treg分化的调节机制，相关研究成果可望为揭示AMI诊治新靶点提供更多科学依据。