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The Role of RBM20 Sequence and Expression in Dilated Cardiomyopathies

RBM20 序列和表达在扩张型心肌病中的作用

基本信息

批准号：
10670204
负责人：
Victoria Parikh
金额：
$ 14.5万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-05 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10670204
关键词：
Action Potentials Address Alternative Splicing Antisense Oligonucleotides Arrhythmia Calcium Cardiac Cardiac Myocytes Cardiomyopathies Cell Line Cells Characteristics Clinical Computational Biology Coupling Data Diagnostic Dilated Cardiomyopathy Disease Electrophysiology (science)Etiology Event Exons Frequencies Funding Future Genes Genetic Genetic Predisposition to Disease Genetic Transcription Genetic Variation Genomics Genotype Heart Heart failure Human Investigation Knowledge L-Type Calcium Channels Label Lead Length Measurement Measures Mediator Modeling Molecular Molecular and Cellular Biology Mus Myocardium National Heart, Lung, and Blood Institute Pathogenicity Patients Phenotype Phosphorylation Population Process Prognosis Protein Isoforms RNA RNA Splicing Regulation Rodent Role RyR2 Sarcoplasmic Reticulum Site Sorting Sudden Death Testing Time Tissues Training Transcript Variant Work arrhythmogenic cardiomyopathy biobank end stage disease experimental study genetic variant high risk human data improved individualized medicine induced pluripotent stem cell induced pluripotent stem cell derived cardiomyocytes innovation knock-down multimodality novel patch clamp prevent skills therapeutic target transcriptome sequencing

项目摘要

PROJECT SUMMARY Dilated cardiomyopathy (DCM) is a deadly disease in which heterogeneous etiologies converge on heart failure, arrhythmias, and sudden death. Although alternative RNA splicing is associated with DCM, little is understood about specific molecular mediators of its pathogenicity. RBM20 is a cardiac muscle-specific splicing regulator of several important cardiomyocyte genes including those critical to excitation-contraction (EC) coupling. I have recently shown that DCM caused by genetic variants in RBM20 is particularly arrhythmogenic, with high rates of sudden death, suggesting that RBM20’s regulation of EC coupling may contribute to deadly arrhythmias in DCM. However, only one critical functional domain of RBM20 has been characterized, and while its overall expression has been associated with alternative splicing in DCM, the extent to which it directly controls splicing of key arrhythmia-associated genes in human heart failure is not known. Therefore, I hypothesize that regional genetic variation and decreased expression of RBM20 lead to aberrant splicing of calcium handling genes, disrupting EC coupling in human DCM. To address this hypothesis, I will undertake three experimental aims: First, I will determine sequence- function relationships of variants in two regions of RBM20 highly predicted by my preliminary data to represent functional domains. I will accomplish this using high throughput saturation gene editing of these regions in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) and test the functionality of resultant genotypes by assessment of RBM20’s splicing activity. Second, to identify all RBM20-associated splicing targets relevant to EC coupling, I will analyze global short read RNA-sequencing (RNA-seq) data from human cardiac tissue with low vs. high RBM20 expression. I will go on to directly manipulate RBM20 expression in iPSC-CMs and use long read RNA-seq to characterize full length isoforms of those targets under the condition of RBM20 knockdown. Lastly, I will define the mechanism by which the aberrant splicing of two known arrhythmia-associated RBM20 splicing targets disrupts EC coupling. To do this, I will use targeted gene editing to correct their aberrant splice isoforms in RBM20+/- and RBM20-/- iPSC-CMs and measure the effect of this manipulation on EC coupling-related phenotypes that I have previously established in these cell lines. These experiments will provide a broad basis for my future studies of global sequence-function relationships across the length of the RBM20 transcript, as well as my future investigation of measurement and modulation of RBM20 function to improve prognosis in DCM patients at high risk of sudden death. At the same time, this project will develop my skills in computational biology, gene editing, and single cell electrophysiology critical to this future investigation. Taken together, the proposed work will produce an innovative, multimodality examination of the role of RBM20 in arrhythmia and sudden death associated with DCM.

项目概要扩张型心肌病 (DCM) 是一种致命的疾病，其病因多种多样心力衰竭、心律失常和猝死。尽管选择性 RNA 剪接与 DCM 相关，但很少有研究表明了解其致病性的特定分子介质。 RBM20 是一种心肌特异性几个重要心肌细胞基因的剪接调节因子，包括对兴奋收缩至关重要的基因（EC）耦合。我最近发现，由 RBM20 基因变异引起的 DCM 尤其严重致心律失常，猝死率高，表明 RBM20 对 EC 耦合的调节可能导致 DCM 中致命的心律失常。然而，RBM20 只有一个关键功能域已被虽然其总体表达与 DCM 中的选择性剪接相关，但其程度它直接控制人类心力衰竭中关键心律失常相关基因的剪接尚不清楚。因此，我假设区域遗传变异和 RBM20 表达减少导致异常钙处理基因的剪接，破坏人类 DCM 中的 EC 偶联。为了解决这个假设，我将实现三个实验目标：首先，我将确定序列- 我的初步数据高度预测了 RBM20 两个区域中变体的功能关系功能域。我将使用这些区域的高通量饱和基因编辑来完成此任务诱导多能干细胞来源的心肌细胞（iPSC-CM）并测试所得产物的功能通过评估 RBM20 的剪接活性来确定基因型。其次，识别所有RBM20相关剪接与 EC 偶联相关的目标，我将分析来自人类的全球短读 RNA 测序 (RNA-seq) 数据 RBM20 低表达与高表达的心脏组织。我将继续直接操作 RBM20 表达 iPSC-CM 并使用长读长 RNA-seq 来表征条件下这些靶标的全长亚型 RBM20 敲低。最后，我将定义两个已知的异常拼接的机制心律失常相关的 RBM20 剪接靶标会破坏 EC 耦合。为此，我将使用靶向基因编辑纠正 RBM20+/- 和 RBM20-/- iPSC-CM 中的异常剪接亚型并测量其效果对我之前在这些细胞系中建立的 EC 偶联相关表型进行操作。这些实验将为我今后研究全局序列函数提供广泛的基础 RBM20 转录本长度上的关系，以及我未来对测量和调节 RBM20 功能可改善猝死高风险 DCM 患者的预后。同时到时候，这个项目将培养我在计算生物学、基因编辑和单细胞电生理学方面的技能对未来的调查至关重要。总而言之，拟议的工作将产生一个创新的、多模式的检查 RBM20 在 DCM 相关心律失常和猝死中的作用。