钙库超载诱导钙释放（SOICR）：防控心肌缺血再灌注心律失常的新靶点

The mechanism of malignant arrhythmias induced by myocardial ischemia reperfusion (I/R) remains unclear. There is compelling evidence showing that intracellular calcium overload and spontaneous calcium release from the sarcoplasmic reticulum (SR) via the calcium release channel (Ryanodine receptor, RyR2) are critically involved. Our previous work has shown that dysfunction of RyR2 due to genetic mutations leads to a common phenotype of enhanced spontaneous calcium release under conditions of SR calcium overload, a process also known as Store Overload Induced Calcium Release (SOICR). And pathological basis for imbalanced calcium homeostasis during myocardial I/R is considered to be existed. Based on these observations, we hypothesize that dysfunction of RyR2 induced by I/R injury can trigger SOICR and consequently arrhythmias. In this project, we propose to employ fluorescence probes, single cell calcium imaging, and gene knock-in technology to explore the following 3 specific aims. Firstly, the role of SOICR in the development of I/R-induced arrhythmias will be determined in multilevel from single cell to whole animal. Secondly, we will define the molecular mechanisms by which I/R induces the occurrence of SOICR. Finally, we will test the possibility that suppressing SOICR may prevent I/R related triggered activities and arrhythmias. This study will provide a better understanding of the role of SOICR in myocardial I/R injury and its underlying mechanism, and may lead to new therapeutic targets for the treatment and prevention of I/R arrhythmias.

心肌缺血再灌注（I/R）诱发恶性心律失常的机制尚不明确，可能与细胞内钙超载和细胞钙库-肌浆网（SR）钙释放通道（RyR2）介导的自发性钙释放有关。本课题组前期研究发现：RyR2基因突变致其通道功能异常活跃，在合并SR钙超载时出现自发性钙释放，即钙库超载诱导钙释放（SOICR）。而在心肌I/R过程中存在致钙稳态失衡的病理基础。基于这些观察，我们推测：心肌I/R损伤诱导RyR2功能障碍致其异常开放引起SOICR，进而触发心律失常。本项目拟采用荧光蛋白转染、钙离子成像和转基因动物等技术，从单细胞到整体动物多层次研究SOICR在I/R心律失常发生中的作用，并深入RyR2单通道水平探究I/R诱发SOICR的分子机制，进一步探索通过抑制SOICR防治I/R相关触发活动及心律失常的可能性。本研究将有助于阐明SOICR在I/R心律失常发生过程中的作用及其分子基础，为探寻其防治靶点提供新的思路。

心肌缺血再灌注（I/R）诱发恶性心律失常的机制尚不明确，可能与细胞内钙超载和细胞钙库-肌浆网（SR）钙释放通道（RyR2）介导的自发性钙释放（SCR）有关。我们前期研究发现：RyR2基因突变致其通道功能异常活跃，在合并SR钙超载时出现自发性钙释放，即钙库超载诱导钙释放（SOICR）。而在心肌I/R过程中存在致钙稳态失衡的病理基础。因此，我们推测：心肌I/R损伤诱导RyR2功能障碍致其异常开放引起SOICR进而触发心律失常。本实验主要从两个方面来探索心肌I/R时心律失常的发生机制：1.构建单细胞（HEK293细胞系和大鼠心肌细胞）缺血再灌注损伤模型，应用钙离子荧光技术记录胞内钙离子的实时变化并寻找有效的抑制剂。2.观察在体局部缺血再灌注模型心律失常的发生情况及应用RyR2抑制剂、L型钙通道阻滞剂后的效果。结果显示：1.在I/R时，SCR发生情况：单个心肌细胞SCR发生率升高；钙库总量：与对照组相比，缺血时减少，复灌时恢复至对照组水平；钙瞬变振幅：与对照组相比，缺血时降低，复灌时增加；进一步应用RyR2抑制剂（卡维地洛）、L型钙通道阻滞剂（硝苯地平）和对照组（美托洛尔）后表现为：⑴对钙库的影响：各药物组与I/R相比无显著差异，硝苯地平组较卡维地洛组、美托洛尔组有所下降⑵对钙瞬变振幅的影响：硝苯地平组心肌细胞钙瞬变振幅均明显低于其他各组；其他各组间钙瞬变振幅无显著差异⑶对SCR发生率的影响：卡维地洛和硝苯地平都可显著降低起搏心肌细胞再灌注后 SCR 的发生率。2.观察在体缺血再灌注模型发现：常见的I/R心律失常类型为-室早（PVC）、室速(VT)、室颤（VF），进一步应用RyR2抑制剂和L型钙通道阻滞剂后得出：卡维地洛组明显抑制室颤的发生，3.我们前期的研究显示：卡维地洛可通过直接抑制RyR2开放时间从而抑制RyR2功能达到减少SOICR发生的效果。基于以上实验结果，我们推论：ryanodine 受体可能参与缺血再灌注心律失常的发生，可能与缺血再灌注导致的RyR2 功能发生改变有关。我们预期，研究结果可能为缺血再灌注性心律失常的药物治疗提供新的理论基础和思路。