RYANODINE RECEPTORS, Ca2+-TRIGGERED ARRHYTHMIAS AND HCM

心脏RyR基因（RYR 2）中的突变与儿茶酚胺能多态性室性心动过速（CPVT）相关，CPVT是一种致心律失常综合征，其特征在于在具有明显正常心脏的个体中发展肾上腺素能介导的室性心动过速，并且最近与肥厚性心肌病（HCM）相关，HCM是心脏骤停的主要原因，其中过度的心脏质量导致收缩异常，放松、传导和节奏。 因此，RyR 2突变可能导致Ca 2+稳态紊乱，很可能是引发快速性心律失常（CPVT）和/或病理性结构重塑（HCM）的关键事件。然而，将RvR 2蛋白突变与CPVT或HCM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，RyR 2起源的CPVT和HCM表型由通道功能障碍的多种机制引起，其严重程度与控制Ca 2+释放的受影响区域的层次结构相称。我们认为，CPVT突变通常是沉默的，但在应激条件下（肾上腺素能刺激），RyR 2通道进入灾难性的Ca 2+释放，导致快速性心律失常;另一方面，HCM突变，由于RyR 2通道的组成性激活，引起慢性和阴险的Ca 2+释放，导致病理性心脏重塑。为了验证这些假设，我们将使用单个RyR 2通道、分离的心室肌细胞和来自野生型小鼠和HCM和CPVT敲入小鼠模型的整个心脏来：（1）确定RyR 2功能障碍的不同模式是否出现于三个CPVT结构-功能域中的每一个中的突变（CPVT-I、CPVT-II和CPVT-III）和新鉴定的HCM结构域;（二）确定由HCM和CPVT突变引起的推测不同的RyR 2功能障碍是否收敛为单一或优势的异常细胞机制，电活动，心脏骤停的主要原因;（3）确定CPVT和HCM的基因敲入小鼠模型是否产生相似的表型，并对β-肾上腺素能刺激和其他加重因素产生同等的反应。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，记录跳动的心脏异常电活动，和超声心动图。因此，拟议的实验设计将结合联合收割机的分子，细胞，整个心脏和完整的动物研究，以阐明RyR 2引发的快速性心律失常的分子机制，具有前所未有的综合生理学水平。

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.