Characterizing the mechanism of loss-of-function mutations in ALPK3 in the pathogenesis of cardiomyopathy

项目摘要 心肌病是一系列改变心肌形态的疾病。一个 以前无法解释的心肌病的很大一部分现在被理解为 在心脏中高表达的基因突变。即使是有其他常见心脏原因的患者也是如此 高血压或冠状动脉疾病等衰竭，心肌病相关基因的遗传变异 可单独或共同影响心脏形态并影响疾病严重程度。因此，一个机械主义者 了解心肌病的遗传原因可以揭示心脏重构的调节因素，这是一个关键的 开发治疗方法以减弱这些过程的第一步。 对遗传型心肌病患者的研究发现， 编码肌节成分的基因，肌节是心肌细胞的收缩装置。基因突变 这些肌节成分会削弱心肌细胞正常收缩和放松的能力， 最终导致病理性重构和心力衰竭。 最近由我们的实验室和其他实验室进行的外显子组测序研究发现了一种新的 导致心肌病的基因称为ALPK3。与大多数已知的心肌病基因不同，心肌病基因编码 ALPK3是肌节的组成部分，编码一种定位于细胞核的肌肉特异性激酶。 因此，了解这种激酶的功能有可能揭示来自细胞核的新信号 这对心肌细胞生物学产生了深远的影响。我创造了人类同源诱导的多能干细胞 携带和不携带ALPK3突变的来源心肌细胞(HiPSC-CMS)询问分子通路 以及ALPK3突变导致心肌病的机制。 这些研究的前提是ALPK3突变耗尽了激酶活性和/或关键的 蛋白质-蛋白质相互作用导致细胞信号失控，最终导致收缩功能障碍。 我将以三个具体目标来解释这一假设： 目的1.鉴定ALPK3活性底物及ALPK3蛋白与蛋白质的相互作用。 目的2.研究ALPK3功能缺失突变对心肌细胞的影响 转录组和蛋白质组。 目的3.确定ALPK3功能缺失突变对肌节动力学的功能影响。 总而言之，这些研究将提供新的遗传学、机械学和功能方面的洞察力，以了解 ALPK3在心脏生理和疾病中的作用 好了！

PROJECT ABSTRACT Cardiomyopathies are a broad spectrum of diseases that alter the morphology of the heart muscle. A significant portion of previously unexplained cardiomyopathies are now understood to be the result of mutations in genes that are highly expressed in the heart. Even for patients with other common causes of heart failure such as hypertension or coronary artery disease, genetic variants in cardiomyopathy-associated genes can individually or collectively affect cardiac morphology and influence disease severity. Thus, a mechanistic understanding of the genetic causes of cardiomyopathy can reveal regulators of cardiac remodeling, a critical first step for the development of therapeutics to attenuate these processes. Studies of patients with inherited forms of cardiomyopathy have revealed monogenic mutations in genes that encode components of the sarcomere – the contractile apparatus of a cardiomyocyte. Mutations in these sarcomeric components can impair the ability of the cardiomyocyte to contract and relax normally, and ultimately result in pathologic remodeling and heart failure. Recent exome sequencing studies, conducted by our lab and others, have identified a novel cardiomyopathy-causing gene called ALPK3. Unlike most known cardiomyopathy genes, which encode components of the sarcomere, ALPK3 encodes a muscle-specific kinase that is localized to the nucleus. Therefore, understanding the function of this kinase has the potential to reveal novel signals from the nucleus that profoundly impact cardiomyocyte biology. I have created human isogenic induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) with and without ALPK3 mutations to interrogate the molecular pathways in which this kinase participates, and the mechanisms by which ALPK3 mutations result in cardiomyopathy. These studies are predicated on the hypothesis that ALPK3 mutations deplete kinase activity and/or critical protein-protein interactions that lead to dysregulated cellular signaling and culminate in contractile dysfunction. I will address this hypothesis with three specific aims: Aim 1. Identify substrates of ALPK3 kinase activity and ALPK3 protein-protein interactions. Aim 2. Characterize the effects of ALPK3 loss-of-function mutations on the cardiomyocyte transcriptome and proteome. Aim 3. Determine the functional effect of ALPK3 loss-of-function mutations on sarcomere dynamics. Collectively, these studies will provide new genetic, mechanistic, and functional insights into the role of ALPK3 in cardiac physiology and disease. !