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

项目摘要 心肌病是一种改变心肌形态的广谱疾病。一 很大一部分以前无法解释的心肌病现在被认为是由于 在心脏中高度表达的基因突变。即使对于患有其他常见心脏病的患者 失败，如高血压或冠状动脉疾病，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，一种机械的 了解心肌病的遗传原因可以揭示心脏重塑的调节因子， 第一步是开发治疗方法来减弱这些过程。 对遗传性心肌病患者的研究显示， 编码肌节（心肌细胞的收缩装置）成分的基因。突变 这些肌节成分可损害心肌细胞正常收缩和舒张的能力， 最终导致病理性重塑和心力衰竭。 我们实验室和其他人最近进行的外显子组测序研究已经确定了一种新的 一种叫做ALPK 3的心肌病基因与大多数已知的心肌病基因不同， 作为肌节的组成部分，ALPK 3编码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 对心肌细胞生物学产生了深远的影响。我创造了人类同基因诱导多能干细胞 具有和不具有ALPK 3突变的衍生心肌细胞（hiPSC-CM）以询问分子途径 以及ALPK 3突变导致心肌病的机制。 这些研究是基于ALPK 3突变耗尽激酶活性和/或关键激酶活性的假设。 蛋白质-蛋白质相互作用导致细胞信号传导失调并最终导致收缩功能障碍。 我将以三个具体目标来阐述这一假设： 目标1.识别ALPK 3激酶活性和ALPK 3蛋白质-蛋白质相互作用的底物。 目标二。表征ALPK 3功能丧失突变对心肌细胞的影响 转录组和蛋白质组。 目标3。确定ALPK 3功能丧失突变对肌节动力学的功能影响。 总的来说，这些研究将提供新的遗传，机制和功能的见解的作用， ALPK 3在心脏生理学和疾病中的作用 !

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. !