Molecular Mechanisms of RyR2-triggered Arrhythmias

描述（由申请人提供）：Ryanodine受体（RyR）是肌浆网的Ca 2+释放通道，提供诱导心肌细胞收缩所需的大部分Ca 2+。在其细胞内环境中，RyR受多种胞质和腔因子调节，使得其输出信号（Ca 2+）诱导精细分级的细胞收缩，而不引发可能导致异常电活动（室性心律失常）的细胞过程，异常电活动是猝死（SD）的主要原因。RyR功能障碍的重要性最近已被强调，心脏RyR基因（hRYR 2）的点突变与儿茶酚胺能多态性室性心动过速（CPVT），一种以肾上腺素能介导的室性心动过速在具有明显正常心脏的个体中的发展为特征的致心律失常综合征相关。绝大多数CPVT突变已定位于影响RyR功能的不同方面的RyR 2蛋白的三个位点（“热点”），然而，将RyR 2蛋白中的突变与快速性心律失常的发展联系起来的分子机制仍不完全清楚。我们的一般假设是，CPVT突变导致多种形式的RyR 2功能障碍，表型的严重程度由受影响的结构域在控制Ca 2+释放的层次结构决定。为了验证这一假设，我们将使用单个RyR 2通道、分离的心室肌细胞和来自CPVT野生型小鼠和敲入小鼠模型的整个心脏来：（1）确定是否由CPVT突变改变的三个“热点”中的每一个产生不同的RyR 2功能障碍模式;（2）确定由三个“热点”中的每一个中的突变引起的可能不同的RyR 2功能障碍是否汇聚成异常电活动的优势细胞机制;和（3）确定CPVT的基因敲入小鼠模型是否产生相似的表型并对β 2-肾上腺素能刺激和β 2-阻断剂同等地应答。我们将使用一系列最先进的技术，包括单通道活动的动力学分析，通过激光光解的“笼”Ca 2+，高速Ca 2+成像与激光扫描共聚焦显微镜，并记录异常的电活动在整个，跳动的心脏。因此，拟议的实验设计将结合联合收割机的分子，细胞和整个心脏的研究，以阐明RyR引发的快速性心律失常的分子机制与前所未有的水平的综合生理学。 公共卫生相关性：离子通道（负责在心脏中产生电信号和钙信号的蛋白质）的突变可能导致心动过速和猝死。本项目研究钙释放通道（兰尼碱受体）中一种重要离子通道的突变如何导致室性心律失常和猝死。这项研究的成功完成将使我们能够合理化的治疗方法，为这些疾病的最佳治疗。

DESCRIPTION (provided by applicant): Ryanodine receptors (RyRs) are the Ca2+ release channels of sarcoplasmic reticulum that provide the majority of Ca2+ necessary to induce contraction of cardiac cells. In their intracellular environment, RyRs are regulated by a variety of cytosolic and luminal factors so that their output signal (Ca2+) induces finely graded cell contraction without igniting cellular processes that may lead to aberrant electrical activity (ventricular arrhythmias), the main cause of sudden death (SD). The importance of RyR dysfunction has been recently highlighted with the demonstration that point mutations in the cardiac RyR gene (hRYR2) are associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia in individuals with an apparently normal heart. The vast majority of CPVT mutations have been localized to three loci ("hot spots") of the RyR2 protein that affect different aspects of RyR function, however, the molecular mechanism that links a mutation in the RyR2 protein and the development of tachyarrhythmias remains incompletely understood. Our general hypothesis is that CPVT mutations cause multiple forms of RyR2 dysfunction, with the severity of the phenotype determined by the hierarchy of the affected domain in the control of Ca2+ release. To test this hypothesis, we will use single RyR2 channels, isolated ventricular myocytes and whole hearts from wild-type mice and knock-in mouse models of CPVT to: (1) determine whether distinct patterns of RyR2 dysfunction emerge from each of the three "hot spots" altered by CPVT mutations; (2) determine whether the presumably diverse RyR2 dysfunctions elicited by mutations in each of the three "hot spots" converge into a preponderant cellular mechanism of aberrant electrical activity; and (3) determine if the knock-in mouse models of CPVT develop similar phenotype and respond equally to 2-adrenergic stimulation and 2-blockers. We will use an array of state-of-the-art techniques including kinetic analysis of single channel activity by laser photolysis of "caged" Ca2+, high-speed Ca2+ imaging with laser scanning confocal microscopy, and recording of aberrant electrical activity in whole, beating hearts. The proposed experimental design will therefore combine molecular, cellular, and whole heart studies to elucidate the molecular mechanisms of RyR-initiated tachyarrhythmias with an unprecedented level of integrative physiology. PUBLIC HEALTH RELEVANCE: Mutations in ion channels, the proteins that are responsible for generating electrical and calcium signals in the heart, can cause tachycardia and sudden death. This project studies how mutations in one important ion channel called the calcium release channel (ryanodine receptor) cause ventricular arrhythmia and sudden death. Successful completion of this study will allow us to rationalize a therapeutic approach for the optimal treatment of these disorders.