The Role of RBM20 Sequence and Expression in Dilated Cardiomyopathies

项目摘要 扩张型心肌病（DCM）是一种致命的疾病，其中异质性病因汇聚在 心力衰竭心律失常和猝死尽管选择性RNA剪接与DCM相关， 了解其致病性的特定分子介质。RBM 20是心肌特异性的 几种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)偶合器.我最近的研究表明，由RBM 20基因变异引起的扩张型心肌病， 具有高猝死率，这表明RBM 20对EC偶联的调节可能 会导致DCM患者致命的心律失常然而，只有一个关键的功能域的RBM 20已被 其特征在于，虽然其整体表达与DCM中的选择性剪接相关，但其程度 其直接控制人类心力衰竭中关键的与疟疾相关的基因的剪接的机制尚不清楚。 因此，我假设区域遗传变异和RBM 20表达减少导致了异常的 剪接钙处理基因，破坏人DCM中的EC偶联。 为了解决这个假设，我将进行三个实验目标：首先，我将确定序列- 我的初步数据高度预测RBM 20两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实现这一点， 诱导的多能干细胞衍生的心肌细胞（iPSC-CM），并测试所得的功能性。 通过评估RBM 20的剪接活性确定基因型。第二，为了鉴定所有RBM 20相关的剪接， 靶点相关的EC偶联，我将分析全球短读RNA测序（RNA-seq）数据，从人类 具有低与高RBM 20表达的心脏组织。我将继续直接操纵RBM 20表达， iPSC-CM，并使用长读段RNA-seq来表征这些靶标在以下条件下的全长同种型： RBM 20基因敲除最后，我将定义两个已知的异常剪接的机制， 疟疾相关的RBM 20剪接靶破坏EC偶联。为了做到这一点，我将使用靶向基因编辑 以校正其在RBM 20 +/-和RBM 20-/-iPSC-CM中的异常剪接同种型，并测量其作用。 操作EC偶联相关的表型，我以前已经建立在这些细胞系。 这些实验将为我今后的整体序列-功能研究提供广泛的基础 RBM 20成绩单长度之间的关系，以及我未来对测量和 调节RBM 20功能以改善猝死高危DCM患者的预后。在同一 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的来说，拟议的工作将产生一个创新的、多模式的 检查RBM 20在与DCM相关的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.