RYANODINE RECEPTORS, Ca2+-TRIGGERED ARRHYTHMIAS AND HCM

心脏RyR基因(RYR2)突变与儿茶酚胺能多形性室性心动过速(CPVT)有关，CPVT是一种心律失常综合征，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，最近又与肥厚性心肌病(HCM)有关，HCM是心脏骤停的主要原因，心脏质量过大会导致收缩、松弛、传导和节律异常。 因此，RyR2基因突变可能导致钙离子动态平衡紊乱，这很可能是快速性心律失常(CPVT)和/或病理性结构重构(HCM)的关键事件。然而，RvR2蛋白突变与CPVT或HCM发展之间的分子机制仍不完全清楚。我们的一般假设是，RyR2起源的CPVT和HCM表型是由多种通道功能障碍机制引起的，其严重程度与受影响区域在控制钙释放方面的等级相称。我们认为，CPVT突变在正常情况下是沉默的，但在应激条件下(Padreneric Exhibition)使RyR2通道进入灾难性的钙释放，导致快速性心律失常；另一方面，HCM突变由于RyR2通道的结构性激活而引发慢性和隐匿性的钙释放，导致病理性心脏重构。为了验证这些假说，我们将使用来自野生型小鼠的单个RyR2通道、分离的心室肌细胞和全心以及敲入的HCM和CPVT模型来：(1)确定三个CPVT结构功能域(CPVT-I、CPVT-II和CPVT-III)和新发现的HCM结构域的突变是否会出现不同的RyR2功能障碍模式；(2)确定由HCM和CPVT突变引起的各种RyR2功能障碍是否汇聚为异常电活动的单一或优势细胞机制，这是心脏骤停的主要原因；(3)确定CPVT和HCM敲入小鼠模型的表型是否相似，对β-肾上腺素能刺激和其他加重因素的反应是否相同。我们将使用一系列最先进的技术，包括通过激光光解“笼状”钙离子来动态分析单通道活动，使用激光扫描共聚焦显微镜进行高速钙离子成像，记录心脏跳动中的异常电活动，以及超声心动图。因此，拟议的实验设计将结合分子、细胞、整个心脏和完整的动物研究，以前所未有的综合生理学水平阐明RyR2引发的快速性心律失常的分子机制。

Mutations in the cardiac RyR gene {RYR2) 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, and have very recently been associated with Hypertrophic Cardiomyopathy (HCM), a major cause of sudden cardiac arrest where excessive cardiac mass leads to abnormalities in contraction, relaxation, conduction and rhythm. Hence, RyR2 mutations may lead to deranged Ca2+ homeostasis, quite possibly the pivotal event for the initiation of tachyarrhythmias (CPVT) and/or pathological structural remodeling (HCM). However, the molecular mechanisms that link a mutation in the RvR2 protein and the development of CPVT or HCM remain incompletelv understood. Our general hypothesis is that RyR2-originated CPVT and HCM phenotypes arise from multiple mechanisms of channel dysfunction, the severity of which is commensurate with the hierarchy of the affected domain in the control of Ca2+ release. We propose that CPVT mutations are normally silent but throw RyR2 channels into catastrophic Ca2+ release under conditions of stress (Padrenergic stimulation), leading to tachyarrhythmias; HCM mutations, on the other hand, elicit chronic and insidious Ca2+ release due to constitutive activation of RyR2 channels, leading to pathological cardiac remodeling. To test these hypotheses, we will use single RyR2 channels, isolated ventricular myocytes and whole hearts from wild-type mice and knock-in mouse models of HCM and CPVT to: (1) determine whether distinct patterns of RyR2 dysfunction emerge from mutations in each of the three CPVT structure-function domains (CPVT-I, CPVT-II, and CPVT-III) and the newly-identified HCM domain; (2) determine whether the presumably diverse RyR2 dysfunctions elicited by HCM and CPVT mutations converge into a single or preponderant cellular mechanism of aberrant electrical activity, a major cause of sudden cardiac arrest; (3) determine if the knock-in mouse models of CPVT and HCM develop similar phenotype and respond equally to p-adrenergic stimulation and other exacerbating factors. 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, recording of aberrant electrical activity in beating hearts, and echocardiography. The proposed experimental design will therefore combine molecular, cellular, whole heart and intact animal studies to elucidate the molecular mechanisms of RyR2-initiated tachyarrhythmias with an unprecedented level of integrative physiology.