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

• 批准号: 10463600
• 负责人: Victoria Parikh
• 金额: $ 14.5万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 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/10463600
• 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; Label; Lead; Length; Measurement; Measures; Mediator of activation protein; 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; Sarcoplasmic Reticulum; Site; Sudden Death; Testing; Time; Tissues; Training; Transcript; Variant; Work; arrhythmogenic cardiomyopathy; base; 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

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.

项目摘要 扩张的心肌病（DCM）是一种致命疾病，其中异质病因融合了 尽管替代的RNA剪接与DCM相关，但很少 了解其致病性的特定分子介体。 RBM20是一种心脏肌肉特异性 几个重要心肌细胞基因的剪接调节剂，包括至关重要的兴奋性征收基因 （EC）耦合。我最近表明，RBM20中遗传变异引起的DCM尤其是 心律失常，猝死率很高，表明RBM20的EC耦合调节可能 在DCM中导致致命心律不齐。但是，RBM20的一个关键功能域是 表征，虽然其总体表达与DCM中的替代剪接有关 它直接控制人体心力衰竭中与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的表达改善导致异常 钙处理基因的剪接，破坏人类DCM中的EC耦合。 为了解决这一假设，我将实现三个实验目的：首先，我将确定序列 - 我的初步数据高度预测的RBM20区域的变体的功能关系以表示 功能域。我将使用这些区域的高吞吐量满意度基因编辑来完成此操作。 诱导多能干细胞衍生的心肌细胞（IPSC-CM）并测试所得的功能 通过评估RBM20的剪接活动的基因型。第二，确定所有与RBM20相关的剪接 与EC耦合相关的目标，我将分析来自人类的全局简短读取RNA-Servisting（RNA-Seq）数据 具有低RBM20表达的心脏组织。我将继续直接操纵RBM20的表达 IPSC-CMS并使用长读RNA-Seq来表征条件下这些靶标的全长同工型 RBM20敲除的。最后，我将定义两个已知的异常剪接的机制 心律不齐相关的RBM20剪接靶标会破坏EC耦合。为此，我将使用目标基因编辑 纠正RBM20 +/-和RBM20 - / - IPSC-CMS中的异常剪接同工型 我以前在这些细胞系中已经建立的与EC耦合相关的表型操纵。 这些实验将为我对全球序列功能的未来研究提供广泛的基础 跨RBM20成绩单的关系以及我未来的测量和投资 RBM20功能的调节以改善高死亡风险的DCM患者的预后。同样 时间，这个项目将发展我在计算生物学，基因编辑和单细胞电生理学方面的技能 对于未来的投资至关重要。综上所述，拟议的工作将产生创新的多模式 检查RBM20在心律不齐和与DCM相关的猝死的作用。