Functional implications of RyR2 mutations in human cardiomyocytes

RyR2 突变对人类心肌细胞的功能影响

• 批准号: 10009812
• 负责人: MARTIN MORAD
• 金额: $ 60.73万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10009812
• 关键词: Affinity; Amino Acids; Arrhythmia; Back; Binding Sites; Biological Models; Biopsy; C-terminal; CRISPR/Cas technology; Caffeine; Calcium Signaling; Calmodulin; Cardiac; Cardiac Myocytes; Cardiomyopathies; Catecholaminergic Polymorphic Ventricular Tachycardia; Catecholamines; Cell Line; Cell model; Cells; Characteristics; Coupling; Cryoelectron Microscopy; Data; Disease; Electrophysiology (science); Evaluation; Exertion; Exhibits; FKBP1B gene; Failure; Fluorescent Probes; Functional disorder; Genes; Genetic; Genetic Engineering; Heart; Heart Hypertrophy; Heart failure; Human; Ion Channel; Link; Membrane; Missense Mutation; Modeling; Molecular; Mus; Muscle Cells; Mutagenesis; Mutate; Mutation; Myocardial; Myocardial Contraction; N-terminal; Pathologic; Pathology; Patients; Pharmacotherapy; Phenotype; Play; Prevention; Protein Kinase; Protein phosphatase; Proteins; Recombinants; Regulation; Reporting; Research; Resolution; Role; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Signaling Protein; Site; Stem cells; Structural Models; Structure; Structure-Activity Relationship; System; Time; Total Internal Reflection Fluorescent; Transgenic Mice; base; comparative; confocal imaging; experimental study; heart dimension/size; induced pluripotent stem cell; insight; mouse model; mutant; novel; novel strategies; patch clamp; public health relevance; receptor; stem cell differentiation

Project Summary/Abstract: Cardiac contractility is regulated by transient release of Ca2+ form the sarcoplasmic reticulum through type-2 ryanodine receptor (RyR2), a tetrameric ~5000 amino acids protein with multiple regulatory domains for Ca2+, Mg2+, protein kinase and phosphatase, and RyR2-stabilizing protein, FKBP12.6. Since a number of RyR2 missense mutations have been reported to associate with lethal cardiomyopathies, a better understanding of regulatory mechanisms of RyR2 is essential for prevention and treatment of these pathologies. Two major research strategies, heterologous cell expression in HEK293 cell lines carrying RyR2 mutations and transgenic mouse models expressing mutant RyR2, have been thus far used to study structure/function relationship of RyR2 and its pathological consequences. These approaches have advanced the understanding of RyR2 regulatory mechanisms, but suffer from inherent drawbacks of cells with non-cardiac genetic backgrounds or size and electrophysiological differences between human and mice. Thus, functional consequences of RyR2 mutagenesis remain to be fully explored in human myocardial model system. In this proposal, we aim to establish a new research platform where RyR2 mutagenesis is carried out in cardiomyocytes derived from human-induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 gene editing directed to Ca2+ and caffeine binding sites associated with cardiac pathology. Toward this end, we set three specific aims: (1) Establish the RyR2 mutagenesis in hiPSC-derived cardiomyocyte as a reliable platform to reproduce the calcium signaling aberrancies associated with the arrhythmia-linked mutations, by comparing the Ca2+ signaling aberrancies of gene-edited F2483I-RyR2 mutation carrying myocytes with cells derived directly from patient biopsies harboring the same mutations, and previously characterized by us, (2) Characterize functional consequences of mutating the potential Ca2+ and caffeine binding site of RyR2, recently identified in the high resolution cryo-electron microscopy studies, (3) Characterize the Ca2+ signaling phenotypes of RyR2 mutations associated with cardiac pathology in the three structurally distinct domains of RyR2. The membrane currents and global and focal 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 engineered Ca2+ fluorescent probes targeted to various nodes of Ca2+ signaling proteins. This novel approach may enable us to systematically characterize the phenotype of the mutant RyR2 in cells with more relevant genetic background of human cardiac cells in time-effective manner, leading hopefully to better understanding of molecular mechanism of RyR2 regulation and cardiac excitation-contraction coupling based on the near-atomic structural model of RyRs.

项目摘要/摘要： 心肌收缩能力是通过2型肌浆网瞬间释放钙离子来调节的 Ryanodine受体(RyR2)是一种四聚体~5000氨基酸的蛋白质，具有多个钙离子调节域， 镁离子，蛋白激酶和磷酸酶，RyR2稳定蛋白，FKBP12.6。由于许多RyR2 据报道，错义突变与致命性心肌病有关，更好地了解 RyR2的调控机制对于这些疾病的预防和治疗至关重要。两大 携带RyR2突变和转基因的HEK293细胞中异源细胞表达的研究策略 到目前为止，表达突变体RyR2的小鼠模型已被用于研究RyR2的结构/功能关系 RyR2及其病理后果。这些方法促进了对RyR2的理解 调节机制，但受累于具有非心脏遗传背景或 人和小鼠的大小和电生理差异。因此，RyR2的功能后果 在人类心肌模型系统中，诱变作用仍有待充分探索。在这项建议中，我们的目标是 建立RyR2基因突变心肌细胞的新研究平台 利用CRISPR/Cas9基因编辑定向钙离子从人诱导多能干细胞(HiPSCs)中分离出 以及与心脏病理有关的咖啡因结合部位。为此，我们制定了三个具体目标： (1)在hPSC来源的心肌细胞中建立RyR2突变作为一个可靠的平台来复制 通过比较钙离子与心律失常相关突变的钙信号异常 基因编辑的F2483I-RyR2突变携带心肌细胞与直接来源细胞的信号异常 具有相同突变的患者活检组织，以及之前由我们确定的特征，(2)特征为功能性 RyR2潜在的钙和咖啡因结合位点突变的后果，最近在高 分辨冷冻电子显微镜研究，(3)RyR2突变的钙信号表型 在RyR2的三个结构不同的结构域中与心脏病理相关。膜电流 野生型和突变型HiPSC来源的心肌细胞的全局和局部细胞内钙信号将 利用基因工程钙离子在共聚焦/TIRF显微镜下对膜片钳心肌细胞进行定量研究 针对钙信号蛋白不同节点的荧光探针。这种新颖的方法可能使我们能够 在具有更多相关遗传背景的细胞中系统地表征突变的RyR2的表型 以时间有效的方式对人类心脏细胞进行研究，有望更好地理解分子 基于近原子结构的RyR2调控和心肌兴奋收缩偶联机制 RYR模型。