Investigating Mechanisms of RBM20 Liquid-liquid Phase Separation Driving Cardiomyocyte Physiology and Dilated Cardiomyopathy

RBM20液-液相分离驱动心肌细胞生理学和扩张型心肌病的机制研究

• 批准号: 10540300
• 负责人: Aidan Mandy Fenix
• 金额: $ 6.98万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-16 至 2024-02-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10540300
• 关键词: 3-Dimensional; Affect; Alternative Splicing; Arginine; Arrhythmia; Automobile Driving; Behavior; Binding Sites; Biochemical; Biological Models; Biological Phenomena; Biology; Biophysics; Calcium; Cardiac; Cardiac Myocytes; Cells; Characteristics; Chromatin; Chromatin Structure; Chromosomes; DNA; DNA Repair; DNA Sequence Alteration; Data; Dilated Cardiomyopathy; Disease; Disease model; Electrophysiology (science); Engineering; Fluorescence Recovery After Photobleaching; Fluorescent in Situ Hybridization; Genes; Goals; Health; Heart; Heart Diseases; Heart failure; Homeostasis; Human; Image; Imaging Techniques; In Vitro; Inherited; Institutes; Knock-out; Laboratory Research; Lead; Light; Liquid substance; Magnetism; Measures; Mediating; Membrane; Mentorship; Microscopy; Modeling; Molecular Biology; Muscle; Mutation; Myocardial dysfunction; N-terminal; Nuclear; Nuclear RNA; Nuclear Structure; Organelles; Organoids; Pathogenicity; Performance; Phase; Phenotype; Photobleaching; Physiology; Point Mutation; Protein Binding Domain; Proteins; RNA; RNA Binding; RNA Recognition Motif; RNA Splicing; Reaction; Recovery; Regenerative Medicine; Regulation; Reporter; Repression; Resolution; Role; Scheme; Serine; Structure; Testing; Training; Universities; Washington; Work; biophysical properties; cardiac tissue engineering; career; collaborative environment; connectin; disease-causing mutation; early onset; exhaustion; experimental study; force sensor; genomic locus; heart function; human disease; human pluripotent stem cell; human stem cells; lens; live cell microscopy; mRNA Precursor; mutant; new therapeutic target; novel; novel therapeutics; overexpression; segregation; stem cell model; stem cells; sudden cardiac death; supportive environment; tool

PROJECT SUMMARY/ABSTRACT Mutations in the muscle-specific RNA splicing factor RBM20 are a recently identified cause of aggressive dilated cardiomyopathy (DCM) characterized by severe arrhythmias. However, the underlying mechanisms are still unclear, and thus no therapies are available. We recently uncovered the existence of a nuclear RBM20 splicing factory that brings into proximity multiple co-regulated DNA loci from different chromosomes. Formation of this three-dimensional chromatin structure relies on RBM20 foci that are nucleated by its main splicing target, the pre-mRNA encoding for the giant protein titin (TTN). My preliminary data suggests that RBM20 foci undergo liquid-liquid phase separation (LLPS), a mechanism thought to contribute to the segregation and regulation of sub-nuclear compartments. My preliminary data also suggests RBM20 LLPS is perturbed in RBM20 DCM mutants. Thus, the central hypothesis tested in this proposal is that RBM20 assembles membrane-less macromolecular condensates required for homeostatic function in the heart. My specific aims are to: (1) determine the biophysical properties of the RBM20 splicing factory and impact of DCM mutations; and (2) determine whether RBM20 LLPS is required for RBM20 function, and how RBM20 mutations drive DCM phenotypes. I will perform in vitro experiments using purified human RBM20, and cellular experiments using human pluripotent stem cell-derived cardiomyocytes (HPSC-CMs), to determine the mechanisms of RBM20 LLPS and impact of DCM mutations. I will leverage a combination of super-resolution microscopy, photo- bleaching and photo-conversion microscopy, and molecular biology, to study LLPS. To correlate biophysical and topological changes due to perturbation of RBM20 LLPS with cardiac function, I will characterize three- dimensional engineered heart tissues (3D-EHTs), a cardiac organoid model. To test whether LLPS is required for RBM20 function, I will perform DNA fluorescent in situ hybridization and RT-qPCR of target genes, to measure splice factory assembly and alternative splicing, respectively. To reveal how RBM20 mutations drive DCM phenotypes, I will measure electrophysiological and contractile performance of WT and mutant 3D-EHTs. Collectively, these experiments will exhaustively characterize how RBM20 mutations, which drive aggressive DCM, affect the biophysical properties, cellular dynamics, and function of RBM20. In addition, these experiments will reveal how RBM20 DCM mutants affect cardiac physiology and drive disease, with the potential to reveal new therapeutic options for RBM20 DCM. More broadly, our work will enhance our understanding of how LLPS- mediated compartmentalization drives human health and how perturbation of LLPS contributes to disease. This project will take place in the highly supportive and collaborative environment of the Institute for Stem Cell and Regenerative Medicine at the University of Washington. With the mentorship of my Sponsor and Co-Sponsor (Dr. Charles E. Murry and Dr. Nathan J Sniadecki, respectively), this project will provide the training required for my career goal of establishing an independent research laboratory.

项目概要/摘要 肌肉特异性 RNA 剪接因子 RBM20 的突变是最近发现的导致侵袭性扩张的原因 以严重心律失常为特征的心肌病（DCM）。但其根本机制仍是 不清楚，因此没有可用的治疗方法。我们最近发现了核 RBM20 剪接的存在 使来自不同染色体的多个共同调控的 DNA 基因座接近的工厂。这个的形成 三维染色质结构依赖于 RBM20 焦点，该焦点由其主要剪接目标（即 编码巨蛋白肌联蛋白 (TTN) 的前 mRNA。我的初步数据表明 RBM20 焦点经历 液-液相分离（LLPS），一种被认为有助于分离和调节的机制 亚核区室。我的初步数据还表明 RBM20 LLPS 在 RBM20 DCM 中受到干扰 突变体。因此，该提案测试的中心假设是 RBM20 组装无膜 心脏稳态功能所需的大分子缩合物。我的具体目标是：（1） 确定 RBM20 剪接工厂的生物物理特性以及 DCM 突变的影响；和（2） 确定 RBM20 LLPS 是否是 RBM20 功能所必需的，以及 RBM20 突变如何驱动 DCM 表型。我将使用纯化的人 RBM20 进行体外实验，并使用 人多能干细胞来源的心肌细胞 (HPSC-CM)，以确定 RBM20 的机制 LLPS 和 DCM 突变的影响。我将利用超分辨率显微镜、照片- 漂白和光转换显微镜以及分子生物学，以研究 LLPS。将生物物理和 由于 RBM20 LLPS 与心脏功能的扰动导致的拓扑变化，我将描述三个特征： 三维工程心脏组织（3D-EHT），一种心脏类器官模型。测试是否需要 LLPS 对于RBM20功能，我会对目标基因进行DNA荧光原位杂交和RT-qPCR，以测量 分别是拼接工厂组装和替代拼接。揭示 RBM20 突变如何驱动 DCM 表型，我将测量 WT 和突变 3D-EHT 的电生理和收缩性能。 总的来说，这些实验将详尽地描述 RBM20 突变如何驱动攻击性 DCM，影响 RBM20 的生物物理特性、细胞动力学和功能。此外，这些实验 将揭示 RBM20 DCM 突变体如何影响心脏生理和驱动疾病，并有可能揭示 RBM20 DCM 的新治疗选择。更广泛地说，我们的工作将增强我们对 LLPS 如何 介导的区室化驱动人类健康以及 LLPS 的扰动如何导致疾病。这 项目将在干细胞研究所的高度支持和协作环境中进行 华盛顿大学再生医学。在我的赞助商和共同赞助商的指导下 （分别为 Charles E. Murry 博士和 Nathan J Sniadecki 博士），该项目将提供以下人员所需的培训： 我的职业目标是建立一个独立的研究实验室。