Regulation and Dysregulation of Cardiac EC coupling by Calmodulin

项目摘要/摘要 钙释放通道兰尼定受体(RyR2)的遗传和获得性缺陷是一系列 致命的心脏疾病，从心律失常到心力衰竭(即兰氏病)。尽管RyR2是 被认为是治疗这些疾病的合理目标，有效的治疗基于 RyR2功能缺乏规范化。这在一定程度上是由于RyR2调控的复杂性。在……里面 在这个提议中，我们将检验这样一个假设，即智能工程蛋白与AAV介导的结合 基因转移为合理设计治疗兰氏病提供了一种策略。基于 钙调素(CaM)在调节RyR2中的作用，我们提出了一种新的心脏基因治疗方法 使用RyR2特异性和多靶点的凸轮，即治疗性凸轮(TCAM)。这一策略是 根据我们最初的研究提出的概念，RyR2功能的各种调节因子收敛于 难治性控制RyR2活性动态和钙信号稳定性的共同机制 心跳周期。遗传和获得性RyR2缺陷，包括RyR2、CASQ2和CaM突变，改变RyR2 不耐药导致钙循环紊乱，钙过早异常释放，继而发生心律失常。 治疗性凸轮(TCAM)旨在重置RyR2难治性，无论潜在病因如何，都将 为兰氏病提供一般的治疗策略。为了实现这一目标，我们将使用多尺度研究 (从分子到整个动物)结合了蛋白质生物化学(Davis)实验室和细胞学的专业知识 生理学(Gyorke)实验室使用新型和尖端的蛋白质输送方法，用于工程凸轮和 最先进的多隔室和多细胞成像工具，用于测试其功能效果。 此外，使用遗传小鼠模型和临床相关的犬心脏制剂进行的研究 失败(HF)模型将为基于去同步化的新的治疗策略提供“原则证明” 钙调素介导的钙信号不应性减缓钙的异常释放。我们建议：1)测试 假设不同遗传形式的CPVT通过共同的机制损害RyR2的功能： 缩短钙信号的不稳定；和2)工程“治疗性”(T)凸轮治疗 自闭症。

Project Summary/Abstract Genetic and acquired defects in Ca release channels, ryanodine receptors (RyR2s), underlie a spectrum of lethal cardiac disorders ranging from arrhythmias to heart failure (i.e. ryanopathies). Although RyR2 is considered to be a logical target for the treatment of these disorders, effective therapies based on normalization of RyR2 function are lacking. This is in part due to the complex nature of RyR2 regulation. In this proposal, we will test the hypothesis that intelligently-engineered proteins coupled with AAV-mediated gene transfer provide a strategy for the rational design of therapies to treat ryanopathies. Based on calmodulin's (CaM) role in regulating RyR2, we propose a novel cardiac gene therapy approach against ryanopathies using RyR2-specific and multi-target CaMs, i.e therapeutic CaMs (TCaMs). This strategy is based on the concept suggested by our initial studies that various regulators of RyR2 function converge on a common mechanism of refractoriness that controls RyR2 activity dynamics and Ca signaling stability during the cardiac cycle. Genetic and acquired RyR2 defects, including mutations in RyR2, CASQ2 and CaM, alter RyR2 refractoriness resulting in disturbed Ca cycling, premature aberrant Ca release and consequent arrhythmias. Therapeutic CaMs (TCaMs) designed to reset RyR2 refractoriness, regardless of the underlying etiology, will provide a general treatment strategy for ryanopathies. To accomplish this goal, we will use multi-scale studies (from molecule to whole animal) that combine the expertise of a protein biochemistry (Davis) lab and a cellular physiology (Gyorke) lab using novel and cutting edge protein delivery approaches for engineered CaMs and state-of-the art multi-compartmental and multicellular imaging tools for testing their functional effects. Additionally, studies using genetic mouse models and preparations from a clinically relevant canine heart failure (HF) model will provide a “proof-of-principle” for new therapeutic strategies based on desynchronization of aberrant Ca release by CaM-mediated slowing of Ca signaling refractoriness. We propose to: 1) Test the hypothesis that different genetic forms of CPVT impair RyR2 function through a common mechanism: shortening Ca signaling refractoriness; and 2) Engineer “therapeutic”(T)CaMs for the treatment of ryanopathies.