Functional implications of CPVT1-associated RyR2 mutations in human cardiomyocytes

项目摘要/摘要： 肌浆网钙离子释放通过兰尼定受体调节心肌收缩能力 RyR2是一种具有钙、镁、蛋白激酶、咖啡因和FKBP12.6多个调控结构域的蛋白质。自.以来 许多RyR2错义突变与致命性心肌病相关，详细了解 RyR2的调控机制对于这些疾病的治疗至关重要。异源的两种策略 重组RyR2突变体在HEK293细胞和转基因小鼠模型中的表达 研究RyR2的结构/功能关系以及与疾病相关的RyR2突变的功能后果。 尽管这些方法提供了对RyR2调控机制的新见解，但它们具有固有的 具有非心脏遗传背景的细胞的缺陷以及人和小鼠心脏的差异。我们有 从而建立了一个将RyR2突变引入人类诱导的替代研究平台 使用CRISPR/Cas9基因编辑的多能干细胞(HiPSCs)来源的心肌细胞(CMS)。突变型 然后在培养基中培养心肌细胞，使其在结构和功能上成熟为成年心肌细胞 州政府。利用这一人类心肌细胞平台，我们建议研究钙离子的分子机制， 咖啡因和FKBP对与CPVT1病理相关的RyR2的调节。具体来说，我们的目标是：1)比较 区域特异性CPVT1相关RyR2突变的CA2信号转导后果 成熟的hiPSC-CMS，通过反向诱变挽救其表型，并确定其药物 特异性；2)表征RyR2钙和咖啡因突变的功能后果 结合位置，从近原子结构预测并确定它们的相互作用；以及3) 描述CPVT1相关RyR2突变功能丧失的潜在机制并确定 钙渗漏突变与非渗漏突变的区别。为了实现这些目标，我们建议创建 我们更成熟的HiPSC-CMS的多个突变系携带不同的RyR2突变并检测它们的 CA2信号异常。野生型和突变型HiPSC的膜电流和细胞内钙信号 用共聚焦/TIRF显微镜成像的膜片钳心肌细胞将对衍生的心肌细胞进行量化 使用针对钙信号通路不同节点的遗传编码的钙探针。我们还将使用 [~3H]Ryanodine结合试验，以确定钙、咖啡因和附件亲和力的可能变化 蛋白质。为了确保我们的HiPSC-平台的可靠性，我们将比较 在小鼠模型中体内敲入RyR2突变的hiPSC-CMS的突变。我们希望我们的小说 该方法将使系统地表征CPVT1突变体的表型以及 非CPVT1突变体对RyR2原子结构的影响，从而提供了一种新的 以及研究RyR2调控的协同人体平台。

Project Summary/Abstract: Cardiac contractility is regulated by Ca2+ release form the sarcoplasmic reticulum through ryanodine receptor (RyR2), a protein with multiple regulatory domains for Ca2+, Mg2+, protein kinase, caffeine and FKBP12.6. Since a number of RyR2 missense mutations associate with lethal cardiomyopathies, a detailed understanding of regulatory mechanisms of RyR2 is essential for treatment of these pathologies. Two strategies of heterologous expression of recombinant RyR2 mutants in HEK293 cells and transgenic mouse models, have been used to study structure/function relationship of RyR2 and the functional consequences of disease-linked RyR2 mutations. Although these approaches have provided new insights into RyR2 regulatory mechanisms, they have inherent drawbacks of cells with non-cardiac genetic background and differences in human and mice hearts. We have therefore established an alternate research platform where RyR2 mutations are introduced in human induced pluripotent stem cells (hiPSCs)-derived cardiomyocytes (CMs) using CRISPR/Cas9 gene-editing. Mutant myocytes are then cultured in media that matures them structurally and functionally toward adult cardiomyocyte state. Using this human myocyte platform, we propose to examine molecular mechanisms underlying Ca2+, caffeine, and FKBP regulation of RyR2 associated with CPVT1 pathology. Specifically we aim: 1) To compare Ca2+-signaling consequences of domain specific CPVT1-associated RyR2 mutations expressed in “mature” hiPSC-CMs , rescue their phenotype by back-mutagenesis, and determine their drug specificity; 2) To characterize the functional consequence of mutating the RyR2 Ca2+ and caffeine binding sites, predicted from near atomic structure and determine their interaction; and 3) To characterize mechanisms underlying loss-of-function CPVT1-associated RyR2 mutations and identify the difference between Ca2+ leaky and non-leaky mutations. To accomplish these aims we propose to create multiple mutant lines of our more mature hiPSC-CMs carrying the different RyR2 mutations and examine their Ca2+ signaling aberrancies. Membrane currents and intracellular Ca2+ signals of wild type and mutant hiPSC- derived cardiomyocytes will be quantified in patch-clamped myocytes imaged by confocal/TIRF microscopy using genetically encoded Ca2+ probes targeted to various nodes of Ca2+ signaling pathway. We will also use [3H]ryanodine binding assay, to determine possible alterations in affinities of Ca2+, caffeine and accessory proteins. To assure the reliability of our hiPSC-platform, we will compare the Ca2+ signaling aberrancies of mutagenesis in hiPSC-CMs with in vivo knock-in of RyR2 mutations in mouse models. We hope that our novel approach will make it possible to systematically characterize the phenotype of the CPVT1 mutants, as well as non-CPVT1 mutants with implication to atomic structure of RyR2, in human myocardium, thus providing a novel and synergistic human platform for studies of RyR2 regulation.