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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

基本信息

批准号：
10475205
负责人：
MARTIN MORAD
金额：
$ 50.68万
依托单位：
UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
依托单位国家：
美国
项目类别：
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/10475205
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 induced pluripotent stem cell derived cardiomyocytes 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

项目摘要

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.

项目概要/摘要：肌浆网Ca ~（2+）释放通过Ryanodine受体调节心肌收缩（RyR 2），一种具有Ca 2+、Mg 2+、蛋白激酶、咖啡因和FKBP 12.6的多个调节结构域的蛋白。以来许多RyR 2错义突变与致死性心肌病有关，详细了解RyR 2错义突变的机制， RyR 2的调节机制对于治疗这些病理是必不可少的。两种异源策略重组RyR 2突变体在HEK 293细胞和转基因小鼠模型中的表达，已被用于研究RyR 2的结构/功能关系以及与疾病相关的RyR 2突变的功能后果。尽管这些方法为RyR 2调节机制提供了新的见解，但它们具有固有的局限性。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RyR 2突变被引入人类诱导使用CRISPR/Cas9基因编辑的多能干细胞（hiPSC）衍生的心肌细胞（CM）。突变体然后将肌细胞培养在培养基中，使其在结构和功能上向成年心肌细胞成熟状态使用这个人类肌细胞平台，我们建议检查Ca 2+的分子机制，咖啡因和FKBP调节与CPVT 1病理相关的RyR 2。具体来说，我们的目标是：1）比较 Ca 2+信号传导的结果，结构域特异性CPVT 1相关的RyR 2突变表达于 “成熟的”hiPSC-CM，通过回复诱变挽救它们的表型，并确定它们的药物特异性; 2）表征突变RyR 2 Ca 2+和咖啡因的功能后果结合位点，从近原子结构预测，并确定它们的相互作用;和3）表征功能丧失CPVT 1相关RyR 2突变的潜在机制，并鉴定 Ca 2+渗漏和非渗漏突变之间的差异。为了实现这些目标，我们建议创建携带不同RyR 2突变的我们更成熟的hiPSC-CM的多个突变系，并检查它们的 Ca 2+信号异常。野生型和突变型hiPSC-1的膜电流和细胞内Ca 2+信号将在通过共聚焦/TIRF显微镜成像的膜片钳肌细胞中定量衍生的心肌细胞使用遗传编码的Ca 2+探针靶向Ca 2+信号通路的各个节点。我们还将使用 [3 H]ryanodine结合试验，以确定Ca 2+、咖啡因和辅料亲和力的可能改变 proteins.为了确保我们的hiPSC平台的可靠性，我们将比较HiPSC的Ca 2+信号传导异常。在小鼠模型中用RyR 2突变的体内敲入在hiPSC-CM中进行诱变。我们希望我们的小说这种方法将使系统地表征CPVT 1突变体的表型成为可能，非CPVT 1突变体与RyR 2的原子结构的含义，在人类心肌，从而提供了一种新的以及用于RyR 2调节研究的协同人类平台。