Translational Control of Cardiac Excitability

项目概要/摘要 作为心脏节律性跳动基础的离子通道的表达必须精确地 协调以履行其生理作用并保护心脏免受心律失常。许多方面如何 这一任务的完成仍然知之甚少。我们的初步发现提出了一种新的方法 其离子通道表达是协调的。这一建议的核心假设是，一个"微观- 相互作用的mRNA种类的"翻译组"编码功能相关的蛋白质，例如编码 心室动作电位这些离子通道协同组装成大分子 控制高级心脏兴奋性的复合物。我们将使用一系列的 包括人心室心肌、来源于人的心肌细胞的实验制剂 诱导多能干细胞（iPSC-CM）、动物模型和HEK293细胞。我们会调查- 调控和相互作用的转录编码心室离子通道，并确定是否这样 利用RNA组装预测心肌细胞内稳定的大分子蛋白质复合物 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，我们将鉴定微翻译组中的其他转录物， 包括那些编码RNA结合蛋白的基因，这些蛋白将转录本连接在一起， 使用RNAi。我们将检验mRNA加工机制的假设，如无意义- 介导的衰变和miRNA调节，协调控制动作电位的组分 微转译来改变疾病状态并履行正常的生理作用。这些实验 将揭示定量调节心脏兴奋性临界平衡的机制， 其扰动触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.