The Role of RBM20 Sequence and Expression in Dilated Cardiomyopathies

项目总结 扩张型心肌病(DCM)是一种不同病因汇聚在一起的致命疾病。 心力衰竭、心律失常和猝死。尽管替代的RNA剪接与DCM有关，但几乎没有 了解其致病性的特定分子介体。RBM20是一种心肌特异的 包括兴奋收缩关键基因在内的几个重要心肌细胞基因的剪接调控 (EC)耦合。我最近表明，由RBM20的遗传变异引起的DCM尤其是 致心律失常，猝死率高，提示RBM20‘S对EC偶联的调节可能 导致扩张型心肌病的致命性心律失常。然而，RBM20只有一个关键的功能结构域是 虽然它的总体表达与DCM中的选择性剪接有关，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，目前尚不清楚。 因此，我假设RBM20的区域遗传变异和表达降低导致了异常 钙处理基因的剪接，扰乱人类扩张性心肌病的EC偶联。 为了解决这一假设，我将承担三个实验目标：第一，我将确定序列- 我的初步数据高度预测了RBM20两个区域的变异体的功能关系 功能域。我将使用这些区域的高通量饱和基因编辑来实现这一点 诱导多能干细胞来源的心肌细胞(IPSC-CM)及其功能检测 基因分型通过评估RBM20‘S的剪接活性。第二，识别所有与RBM20相关的剪接 与EC偶联相关的靶，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肌组织。我将继续直接操作RBM20表达式 IPSC-CMS和Long Read RNA-seq来表征这些靶子在该条件下的全长亚型 RBM20击倒。最后，我将定义两个已知的异常剪接的机制 心律失常相关的RBM20剪接靶点破坏EC偶联。为了做到这一点，我将使用定向基因编辑 纠正其在RBM20+/-和RBM20-/-iPSC-CMS中的异常剪接异构体并测量其影响 我之前在这些细胞系中建立的对EC偶联相关表型的操纵。 这些实验将为我今后研究全局序列函数提供广泛的基础 整个RBM20成绩单的长度之间的关系，以及我未来对测量和 调节RBM20功能以改善猝死高危DCM患者的预后。同时 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 对未来的调查至关重要。总而言之，拟议的工作将产生一种创新的、多模式的 探讨RBM20在扩张型心肌病心律失常和猝死中的作用。

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.