Molecular Mechanisms of RyR2-triggered Arrhythmias

描述(申请人提供)：兰尼定受体(RyRs)是肌浆网的钙释放通道，提供诱导心肌细胞收缩所需的大部分钙。在它们的细胞内环境中，RyRs受各种胞浆和腔因子的调节，因此它们的输出信号(Ca~(2+))诱导精细分级的细胞收缩，而不会触发可能导致异常电活动(室性心律失常)的细胞过程，而室性心律失常是猝死(SD)的主要原因。RyR功能障碍的重要性最近被强调，心脏RyR基因点突变(HRYR2)与儿茶酚胺能多形性室性心动过速(CPVT)相关，CPVT是一种心律失常综合征，其特征是在心脏明显正常的个体中发生肾上腺素介导性室性心动过速。绝大多数CPVT突变定位于RyR2蛋白的三个位点(热点)，影响RyR功能的不同方面，然而，RyR2蛋白突变与快速性心律失常发生的分子机制尚不完全清楚。我们的一般假设是，CPVT突变导致多种形式的RyR2功能障碍，表型的严重程度取决于受影响的结构域在控制钙释放方面的等级。为了验证这一假说，我们将使用单个RyR2通道、野生型小鼠的分离的心室肌细胞和全心以及敲入小鼠的CPVT模型来：(1)确定CPVT突变引起的三个“热点”中每一个是否出现了不同的RyR2功能障碍模式；(2)确定由三个“热点”中的每一个突变引起的可能多样化的RyR2功能障碍是否会聚成异常电活动的优势细胞机制；以及(3)确定CPVT的敲入小鼠模型是否发展出类似的表型，并且对2-肾上腺素能刺激和2-受体阻滞剂做出同样的反应。我们将使用一系列最先进的技术，包括通过激光光解“笼式”钙离子进行单通道活动的动力学分析，使用激光扫描共聚焦显微镜进行高速钙离子成像，以及记录整个心脏跳动中的异常电活动。因此，拟议的实验设计将结合分子、细胞和全心脏研究，以前所未有的综合生理学水平阐明RyR引发的快速性心律失常的分子机制。与公共健康相关：离子通道的突变可能会导致心动过速和猝死。离子通道是负责在心脏中产生电和钙信号的蛋白质。该项目研究一种名为钙释放通道(兰尼定受体)的重要离子通道的突变是如何导致室性心律失常和猝死的。这项研究的成功完成将使我们能够使治疗方法合理化，从而实现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.