Translational Control of Cardiac Excitability

心脏兴奋性的转化控制

基本信息

批准号：
9187729
负责人：
Gail A Robertson
金额：
$ 63.82万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-15 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9187729
关键词：
Action Potentials Affect Animal Model Antibodies Arrhythmia Basic Science Biological Process Cardiac Cardiac Myocytes Cells Co-Immunoprecipitations Complex Data Disease Electrophysiology (science)Elements Equilibrium Fluorescence Resonance Energy Transfer Heart Human Immunoprecipitation Ion Channel Link Macromolecular Complexes Membrane Messenger RNA MicroRNAs Microscopy Modeling Multiprotein Complexes Mutation Myocardium Nonsense-Mediated Decay Patients Physiological Preparation Process Proteins RNA RNA Interference RNA-Binding Proteins Regulation Resolution Role Shapes Specific qualifier value Testing Therapeutic Intervention Time Transcript Translations Ventricular Ventricular Arrhythmia abstracting base biophysical properties disease phenotype heart rhythm human RNA sequencing induced pluripotent stem cell knock-down mRNA Stability novel patch clamp polypeptide prevent research study small hairpin RNA stoichiometry transcriptome sequencing

项目摘要

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.

项目摘要/摘要离子引导的表达是心脏的节奏跳动必须是正确的协调以履行生理角色并保护心脏免受心律不齐。该任务是对我们的初步发现的良好理解。哪个离子通道表达是协调的。相互作用的mRNA物种的翻译组编码功能相关的蛋白质，例如编码这些离子通道的室性动作电位组装成大分子控制高阶心脏兴奋性的复合物。实验制剂包括人类心肌，源自人类的心肌细胞软性干细胞（IPSC-CM），动物模型和HEK293细胞。编码心室离子通道的转录本的调节和相互作用，并确定是否这样组件预测使用RNA在心肌细胞内稳定的大分子蛋白复合物免疫沉淀实验，蛋白质共免疫沉淀，斑块电生理学和超分辨率显微镜。将那些编码RNA结合蛋白编码转录本的人组合在一起，并测试其角色使用RNAi。介导的衰减和miRNA调节，协调控制动作电位的组成部分微型转运剂可以改变疾病状态并履行正常的生理作用将发现定量调节心脏兴奋性临界平衡的机制扰动会触发灾难性的心室心律不齐。