Functional implications of CPVT1-associated RyR2 mutations in human cardiomyocytes

项目摘要/摘要： 心肌收缩力通过兰尼碱受体从肌浆网释放 Ca2+ 来调节 (RyR2)，一种具有 Ca2、Mg2、蛋白激酶、咖啡因和 FKBP12.6 多个调节域的蛋白质。自从 许多 RyR2 错义突变与致命性心肌病相关，详细了解 RyR2 的调节机制对于治疗这些病理学至关重要。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已被用于 研究 RyR2 的结构/功能关系以及疾病相关 RyR2 突变的功能后果。 尽管这些方法为 RyR2 调控机制提供了新的见解，但它们具有固有的缺陷 非心脏遗传背景细胞的缺点以及人类和小鼠心脏的差异。我们有 因此建立了一个替代研究平台，将 RyR2 突变引入人类诱导的 使用 CRISPR/Cas9 基因编辑技术对多能干细胞 (hiPSC) 衍生的心肌细胞 (CM) 进行研究。突变体 然后将心肌细胞在培养基中培养，使它们在结构和功能上成熟为成年心肌细胞 状态。使用这个人类心肌细胞平台，我们建议检查 Ca2 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 域特异性 CPVT1 相关 RyR2 突变的 Ca2 信号传导后果 “成熟”的 hiPSC-CM，通过反向突变挽救其表型，并确定其药物 特异性； 2) 表征 RyR2 Ca2 和咖啡因突变的功能后果 从近原子结构预测结合位点并确定它们的相互作用； 3) 至 表征功能丧失 CPVT1 相关 RyR2 突变的潜在机制并确定 Ca2 泄漏突变和非泄漏突变之间的差异。为了实现这些目标，我们建议创建 我们更成熟的 hiPSC-CM 的多个突变系携带不同的 RyR2 突变，并检查它们的 Ca2 信号传导异常。野生型和突变型 hiPSC- 的膜电流和细胞内 Ca2信号 衍生的心肌细胞将在通过共聚焦/TIRF 显微镜成像的膜片钳心肌细胞中进行定量 使用针对 Ca2 信号通路各个节点的基因编码 Ca2 探针。我们还将使用 [3H]ryanodine 结合测定，以确定 Ca2、咖啡因和辅助物的亲和力可能发生的变化 蛋白质。为了确保我们的 hiPSC 平台的可靠性，我们将比较 Ca2 信号传导异常 在小鼠模型中通过体内 RyR2 突变敲入 hiPSC-CM 中的诱变。我们希望我们的小说 该方法将使系统地表征 CPVT1 突变体的表型成为可能，以及 非 CPVT1 突变体与人心肌中 RyR2 的原子结构有关，从而提供了一种新的 用于 RyR2 调控研究的协同人类平台。

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.