Translational Control of Cardiac Excitability

项目摘要/摘要 心脏节律性跳动背后的离子通道的表达必须精确 协调完成它们的生理作用，保护心脏免受心律失常的影响。多个方面的方式 这项任务已经完成，人们对此仍知之甚少。我们的初步发现提出了一种新的方法 哪个离子通道的表达是协调的。这一提议的中心假设是，一个“微观-- 相互作用的信使核糖核酸的“翻译组”编码功能相关的蛋白质，如编码 脑室动作电位。这些离子通道以共翻译方式组装成大分子 控制高阶心脏兴奋性的复合体。我们将使用以下范围来验证此假设 人心肌、人心肌细胞等实验制剂 诱导多能干细胞(IPSC-CM)、动物模型和HEK293细胞。我们将调查联合- 对编码心室离子通道的转录本的调节和相互作用，并确定这是否 利用RNA组装预测心肌细胞内稳定的大分子蛋白质复合体 免疫共沉淀实验，蛋白质免疫共沉淀，膜片钳电生理学和 超分辨率显微镜。使用RNA-seq，我们将识别微翻译组中的其他转录本， 包括那些编码RNA结合蛋白的基因，这些蛋白将转录本捆绑在一起，并测试它们的作用 使用RNAi。我们将检验这样一种假设，即信使核糖核酸的加工机制，如无意义- 介导的衰变和miRNA调节，协调控制动作电位的成分 微翻译组，用于改变疾病状态，完成正常的生理功能。这些实验 将揭示定量调节心脏兴奋性的关键平衡的机制， 它的扰动会引发灾难性的室性心律失常。

Project Summary/Abstract The expression of ion channels underlying the rhythmic beating of the heart must be precisely coordinated to fulfill their physiological roles and protect the heart from arrhythmia. Many aspects of how this task is accomplished remain poorly understood. Our preliminary findings suggest a novel way in which ion channel expression is coordinated. The central hypothesis of this proposal is that a “micro- translatome” of interacting mRNA species encodes functionally related proteins, such as those encoding the ventricular action potential. These ion channels assemble co-translationally into macromolecular complexes that govern higher-order cardiac excitability. We will test this hypothesis using a range of experimental preparations including human ventricular myocardium, cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CM's), animal models and HEK293 cells. We will probe the co- regulation and interaction of transcripts encoding ventricular ion channels, and determine whether such assemblies predict stable macromolecular protein complexes within cardiomyocytes using RNA immunoprecipitation experiments, protein co-immunoprecipitation, patch- clamp electrophysiology and super-resolution microscopy. Using RNA-seq, we will identify other transcripts in the micro-translatome, including those encoding RNA binding proteins that tether the transcripts together, and test their roles using RNAi. We will test the hypothesis that mechanisms of mRNA processing, such as nonsense- mediated decay and miRNA regulation, coordinately control the components of the action potential micro-translatome to modify the disease state and fulfill normal, physiological roles. These experiments will uncover mechanisms that quantitatively regulate the critical balance of cardiac excitability, the perturbation of which triggers catastrophic ventricular arrhythmias.