心脏兰尼碱受体功能缺失型突变致心律失常与心源性猝死的作用及机制研究

Sudden cardiac death (SCD) accounts for more than 544,000 deaths annually in our country, which is the highest mortality worldwide. Catecholaminergic polymorphic ventricular tachycardia (CPVT) plays an important role in SCD. It is well known that gain-of-function (GOF) mutations in cardiac ryanodine receptor (RyR2) can cause catecholaminergic polymorphic ventricular tachycardia (CPVT). The arrhythmogenic mechanism, diagnosis and treatment of RyR2 GOF-associated CPVT have also been well established. On the other hand, although loss-of-function (LOF) RyR2 mutations have also been associated with ventricular arrhythmias and sudden death, very little is known about the mechanism, diagnosis, and treatment of RyR2 LOF-associated arrhythmias. As a consequence, patients with LOF RyR2 mutations are likely to be misdiagnosed and mismanaged, which may lead to sudden death. Therefore, systematic and detailed investigation of LOF RyR2 mutations is urgently needed. The Overall Goals of this project are to demonstrate the existence, understand the mechanism, and develop the diagnosis and treatment of a new disease entity associated with RyR2 LOF. To achieve these goals, we will focus on 3 specific Objectives: ① to determine whether LOF RyR2 mutations are linked to cardiac arrhythmias distinct from CPVT, ② to understand the arrhythmogenic mechanism of LOF RyR2 mutations, and ③ to develop an effective diagnostic test and treatment for RyR2 LOF associated arrhythmias. By discovering new disease mechanism and developing effective diagnosis and treatment, we expect to advance health-related knowledge and health outcomes.

在我国心源性猝死发病率居全球之首。儿茶酚胺敏感性室性心动过速（CPVT）是引起猝死的重要因素。目前对RyR2功能获得型突变和CPVT的研究已十分成熟。但针对RyR2功能缺失型突变（RyR2 LOF）的研究则十分缺乏。目前在与猝死有关的3例RyR2 LOF报道中，患者都呈现出了与典型的CPVT不同的表型。由于缺少有效的筛选手段，这类患者常面临着误诊和猝死的风险。因此目前急需系统而深入的研究RyR2 LOF突变与心律失常的关系，并揭示其致心律失常的机制。基于以上论据和前期结果，本课题假设：RyR2 LOF可引起与典型的CPVT不同的一类新型心律失常。课题将①确立RyR2 LOF与心律失常、猝死的关系；②阐明RyR2 LOF致心律失常的机制；③初步建立针对RyR2 LOF有效的诊断和治疗策略。本研究将提高对RyR2 LOF有关疾病发生发展的认识，为恶性心律失常和心源猝死的防治带来突破。

RyR2是肌浆网上重要的钙离子释放通道，其功能失调可以导致心脏功能出现严重障碍，而RyR2的突变是引起RyR2功能失调的主要原因。目前已经有超过300个RyR2致病突变被发现，其中RyR2功能获得型（Gain-of-function，GOF）突变引起的儿茶酚胺敏感性室性心动过速（Catecholaminergic Polymorphic Ventricular Tachycardia, CPVT）一直被认为是引起心源性猝死的重要原因。CPVT的发病机制目前已经较为清楚，运动负荷测试（Exercise Stress Test， EST）是诊断CPVT的有效手段。但申请人课题组发现RyR2功能缺失型（Loss-of-function，LOF）突变也可引起携带者出现心源性猝死，且这类患者对EST测试不敏感，发病机制不明确，且目前尚无明确的诊断方法和治疗手段。因此申请人课题组将这种未知疾病首次命名为钙释放缺乏综合征（calcium release deficiency syndrome, CRDS），并对其开展了系统性的研究。我们的实验结果显示1）RyR2-LOF携带患者有病因不明的心源性猝死症状，且临床表型与RyR2-GOF引起的CPVT迥异。通过对多个RyR2-LOF突变携带家族遗传谱系进行基因关联分析, 我们发现RyR2-LOF与CRDS显著相关。提示CRDS是一种全新的疾病。2）课题组构建了RyR2-D4646A点突变模型小鼠，并展开了深入研究。我们发现RyR2-D4646A小鼠对EST和CPVT不敏感，但是通过给予LBLPS程序性电刺激来模拟CRDS患者心源性猝死前的异常心电活动，可以诱发RyR2-D4646A小鼠出现CRDS；3）通过共聚焦钙成像和膜片钳技术我们发现RyR2-LOF诱发CRDS的机制主要是由于RyR2钙离子释放减弱从而引起的多种离子通道代偿性重构，且与增加早后除极（Early afterdepolarizations, EADs）易感性有关；4）广谱抗心律失常药物奎尼丁和氟卡尼可以有效抑制RyR2-D4646A小鼠的CRDS发生频率和发作时间；5）我们证实LBLPS电刺激可诱发临床上RyR2-LOF突变携带患者出现心律失常。综上所述，我们首次鉴定出了一种新的心律失常、揭示了RyR2-LOF突变致心律失常的机制，并开发了首个RyR2-LOF相关心