Junctophilin-2 对心房肌细胞钙泄漏和房颤发生的影响及机制研究

Atrial cardiomyocyte calcium homeostasis abnormality, which is reported mainly due to the increased sarcoplasmic reticulum (SR) Ca2+ leak (Ca2+ spark or Ca2+ wave) from “leaky” Ca2+ release channel ryanodine receptor (RyR2), is one of the most important mechanisms in atrial fibrillation (AF) initiation and progression. Previous studies showed the role of junctophilin-2 (JPH2), a membrane-coupling protein, in stabilizing RyR2, and calcium dependent neutral proteases calpain-I is activated as the cytosolic calcium increases, leading to cytoplasmic or membrane protein proteolysis. Our previous works have documented a down-regulated expression of JPH2 in both mRNA and protein levels in the atrial myocardium of persistent AF patients with an increased Ca2+ spark frequency. Thus, we hypothesized that JPH2 degradation, induced by activated calpain-I, can modulate RyR2 gating and SR Ca2+ leak leading to AF..This study is designed to elucidate whether calpain-I is involved in the degradation of JPH2 proteins, and furthermore, to explore the mechanism of JPH2 on RyR2 gating instability (single RyR2 channels open probability [RyR2 Po], Ca2+ spark or Ca2+ wave), subsequently the effect of JPH2-mediated regulation of RyR2 on arrthymogenic trigger activity (delayed after depolarization [DAD]) and the AF incidence, with stimulated emission depletion microscopy (STED), single-particle cryo-EM, single channel recordings, whole-cell patch clamp as well as laser confocal Ca2+ imaging techniques in the established rapid atrial pacing rabbit AF model and isolated rabbit atrial myocytes. The results of this research will help understand the mechanism of AF and provide new vision on the optimized therapeutic options based on the mechanistic insights.

心房肌细胞钙稳态异常是房颤发生的重要机制之一。肌浆网钙通道RyR2稳定性下降导致钙泄漏是钙稳态异常的重要原因。我们前期研究发现，房颤患者心房肌JPH2基因及蛋白表达水平均明显降低，伴胞内钙火花等异常钙释放事件发生频率明显增多。提示JPH2可能参与调节RyR2关闭状态的稳定性，而其本身可被钙蛋白酶calpain-I酶解。本项目在业已建立的快速起搏心房兔房颤模型上，采用受激发射损耗显微技术、单颗粒冷冻电子显微技术、全细胞膜片钳、激光共聚焦及平面脂质膜单通道技术等手段，从整体、细胞及超微结构水平，进一步探讨calpain-I对JPH2表达变化的影响，重点研究JPH2对RyR2蛋白空间构象、单通道开放频率、肌浆网异常钙释放事件以及细胞延迟后除极（DAD）等异常电触发活动的调控机制，明确JPH2在稳定RyR2通道、影响钙泄漏中的作用及可能机制。本研究有助于阐明房颤发生的可能机制，为其治疗提供新思路。

心房肌细胞钙稳态异常是房颤发生的重要机制之一。肌浆网钙通道RyR2稳定性下降导致钙泄漏是钙稳态异常的重要原因。我们前期研究发现，房颤患者心房肌JPH2基因及蛋白表达水平均明显降低，伴胞内钙火花等异常钙释放事件发生频率明显增多。提示JPH2可能参与调节RyR2关闭状态的稳定性，而其本身可被钙蛋白酶calpain-I酶解。本项目在业已建立的快速起搏心房兔房颤模型上，采用受激发射损耗显微技术、单颗粒冷冻电子显微技术、全细胞膜片钳、激光共聚焦及平面脂质膜单通道技术等手段，从整体、细胞及超微结构水平，进一步探讨calpain-I对JPH2表达变化的影响。. 研究发现:1.在房颤患者及多种房颤实验模型中，随着外界刺激持续存在，心房肌组织中JP2蛋白表达水平逐渐降低，而calpain-1表达明显上调且其酶活性显著激活；2.P2-OE可以通过改善心房肌细胞钙稳态异常、AERP缩短等电重构，以及心房扩大等结构重构，降低CAPN1-OE小鼠的房颤诱发率及发作持续时间，但JP2-OE的保护作用具有时间局限性，无法提供长期抗心律失常的保护作用；3.利用MDL28170抑制calpain酶活性，可以调控JP2至正常水平，并明显改善小鼠心房肌细胞钙稳态异常、电重构及心房扩大、纤维化增多等病理改变，可实现持久有效的抗房颤保护作用。本研究通过阐明calpain-I对JPH2表达变化对房颤发生的影像和可能机制，为未来房颤的防治提供了理论基础和全新思路。