Functional implications of CPVT1-associated RyR2 mutations in human cardiomyocytes

人类心肌细胞中 CPVT1 相关 RyR2 突变的功能意义

• 批准号: 10298481
• 负责人: MARTIN MORAD
• 金额: $ 51.83万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10298481
• 关键词: Adrenergic Agents; Adrenergic beta-Agonists; Adult; Affinity; Amino Acids; Arrhythmia; Back; Binding; Binding Sites; Biochemical; Biological Assay; CRISPR/Cas technology; Caffeine; Cardiac; Cardiac Myocytes; Cardiomyopathies; Catecholaminergic Polymorphic Ventricular Tachycardia; Cell model; Cells; Complement; Computer Simulation; Coupling; Data; Disease; Dissociation; Electrophysiology (science); Exhibits; FKBP1B gene; Functional disorder; Genes; Genetic; Heart; Heart Hypertrophy; Heart failure; Human; Human Engineering; Imaging Device; Impairment; Knock-in; Link; Maps; Mediating; Membrane; Missense Mutation; Modeling; Molecular; Mus; Muscle Cells; Mutagenesis; Mutate; Mutation; Myocardial dysfunction; Myocardium; Pathology; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphoric Monoester Hydrolases; Phosphotransferases; Protein Kinase; Proteins; Recombinants; Regulation; Research; Resolution; RyR1; Ryanodine; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcoplasmic Reticulum; Signal Pathway; Signal Transduction; Site; Specificity; Structural Models; Structure; Structure-Activity Relationship; System; Tacrolimus Binding Proteins; Tail; Testing; Total Internal Reflection Fluorescent; Transgenic Mice; alpha helix; base; confocal imaging; human stem cells; in vivo; induced pluripotent stem cell; insight; interdisciplinary approach; loss of function; loss of function mutation; molecular imaging; mouse model; mutant; novel; novel strategies; patch clamp; public health relevance; response; skeletal

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.

项目摘要/摘要： 心脏收缩性由Ca2+释放形式通过ryanodine接收器形式释放。 （RYR2），一种具有CA2+，MG2+，蛋白激酶，咖啡因和FKBP12.6的蛋白质。自从 许多RYR2错义突变与致命的心肌病有关，对 RYR2的调节机制对于治疗这些病理是必不可少的。两种异源策略 在HEK293细胞和转基因小鼠模型中，重组RYR2突变体的表达已用于 RYR2的研究结构/功能关系以及疾病连接的RYR2突变的功能后果。 尽管这些方法为RYR2调节机制提供了新的见解，但它们已经继承了 具有非心脏遗传背景的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，其中RyR2突变在人类诱导中引入 使用CRISPR/CAS9基因编辑的多能干细胞（HIPSC）衍生的心肌细胞（CMS）。突变体 然后将肌细胞培养在培养基中，该培养基在结构和功能上将其培养为成人心肌细胞 状态。使用这个人类的肌细胞平台，我们建议检查Ca2+的分子机制， 与CPVT1病理相关的RYR2的咖啡因和FKBP调节。具体我们的目的：1）比较 域特异性CPVT1相关的RYR2突变的Ca2+ - 信号后果 “成熟” hipsc-CMS，通过反刺激来营救其表型，并确定其药物 特异性； 2）表征突变RyR2 Ca2+和Cafeine的功能后果 结合位点，从近原子结构预测并确定其相互作用；和3）到 表征了功能丧失CPVT1相关的RYR2突变的机制，并识别 Ca2+泄漏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的多个突变线，携带不同的RYR2突变并检查其 CA2+信号异常。膜电流和野生型和突变hipsc-的细胞内CA2+信号 衍生的心肌细胞将在通过共聚焦/TIRF显微镜成像的斑块夹心肌细胞中进行量化 使用针对CA2+信号通路各种节点的一般编码的CA2+问题。我们还将使用 [3H] ryanodine结合测定法，以确定Ca2+，Cafeine和配件亲和力的可能改变 蛋白质。为了确保我们的HIPSC平台的可靠性，我们将比较CA2+信号异常 HIPSC-CMS中的诱变，在小鼠模型中ryR2突变的体内敲入。我们希望我们的小说 方法将使系统地表征CPVT1突变体的表型以及 非CPVT1突变体与人体心肌中的RyR2的原子结构有关，从而提供了一种新颖的 和RYR2调节研究的协同人类平台。