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: 10570894
• 负责人: Aidan Mandy Fenix
• 金额: $ 1.12万
• 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 2023-03-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10570894
• 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; Knock-out; Laboratory Research; Lead; 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; Physical condensation; Physiology; Point Mutation; Protein Binding Domain; Proteins; RNA; RNA Binding; RNA Recognition Motif; RNA Splicing; RNA purification; Reaction; Recovery; Regenerative Medicine; Regulation; Reporter; Repression; Role; Scheme; Serine; Structure; Testing; Training; Universities; Washington; Work; biophysical properties; cardiac tissue engineering; career; collaborative environment; connectin; disease-causing mutation; early onset; experimental study; force sensor; genomic locus; heart function; human disease; human pluripotent stem cell; human stem cells; lens; live cell microscopy; mRNA Precursor; membrane assembly; monomer; mutant; new therapeutic target; novel; novel therapeutics; overexpression; segregation; stem cell model; stem cells; sudden cardiac death; superresolution microscopy; 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.

项目总结/摘要 肌肉特异性RNA剪接因子RBM 20的突变是最近发现的侵袭性扩张性肌萎缩症的原因。 心肌病（DCM），其特征在于严重的心律失常。然而，潜在的机制仍然是 不清楚，因此没有可用的治疗方法。我们最近发现了核RBM 20剪接的存在， 这是一个将来自不同染色体的多个共调控DNA基因座靠近的工厂。形成这种 三维染色质结构依赖于RBM 20焦点，该焦点由其主要剪接靶点成核， 编码巨蛋白肌联蛋白（TTN）的前mRNA。我的初步数据表明RBM 20病灶经历了 液-液相分离（LLPS），一种被认为有助于分离和调节 亚核隔间我的初步数据还表明RBM 20 LIPS在RBM 20 DCM中受到干扰 变种人因此，在该提议中测试的中心假设是RBM 20组装无膜 心脏内稳态功能所需的大分子凝聚物。我的具体目标是：（1） 确定RBM 20剪接工厂的生物物理特性和DCM突变的影响;和（2） 确定RBM 20功能是否需要RBM 20 LLPS，以及RBM 20突变如何驱动DCM 表型我将使用纯化的人RBM 20进行体外实验，并使用人RBM 20进行细胞实验。 人多能干细胞衍生的心肌细胞（HPSC-CM），以确定RBM 20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，和分子生物学，研究LLPS。将生物物理学和 由于RBM 20 LLPS与心脏功能的扰动引起的拓扑变化，我将描述三个- 三维工程心脏组织（3D-EHTs），心脏类器官模型。测试是否需要LLPS 对于RBM 20功能，我将进行靶基因的DNA荧光原位杂交和RT-qPCR，以测量 分别是拼接工厂装配和可选拼接。揭示RBM 20突变如何驱动DCM 表型，我将测量WT和突变体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RBM 20突变是如何驱动侵略性的 DCM影响RBM 20的生物物理性质、细胞动力学和功能。此外，这些实验 将揭示RBM 20 DCM突变体如何影响心脏生理学和驱动疾病，并有可能揭示 RBM 20 DCM的新治疗选择。更广泛地说，我们的工作将提高我们对LLPS如何的理解- 介导的区室化驱动人类健康以及LLPS的扰动如何促成疾病。这 该项目将在干细胞研究所的高度支持和协作环境中进行， 华盛顿大学的再生医学。在我的赞助商和共同赞助商的指导下， (Dr. Charles E. Murry和Nathan J Sniadecki博士），该项目将提供以下方面所需的培训： 我的职业目标是建立一个独立的研究实验室。