Cardiomyocyte phenotype and mechanotransduction in Filamin C gene variants causing arrhythmogenic cardiomyopathy

导致致心律失常性心肌病的Filamin C基因变异的心肌细胞表型和机械转导

基本信息

批准号：
10333325
负责人：
Luisa Mestroni
金额：
$ 51.03万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10333325
关键词：
Actins Address Adhesions Arrhythmia Arrhythmogenic Right Ventricular Dysplasia Biomechanics CRISPR/Cas technology Cardiac Cardiac Myocytes Cardiomyopathies Cells Complex Cytoskeleton Data Desmosomes Development Disease Electrophysiology (science)Experimental Models Failure Foundations Freezing Functional disorder Gene Expression Gene Mutation Genes Genetic Genetic Transcription Heart Heart failure Human Impairment Integrin Binding Integrins Intercellular Junctions Interdisciplinary Study Knowledge Laboratories Lead Life Link Mechanics Modeling Molecular Morbidity - disease rate Mutation Myocardial Pathway interactions Patients Phenotype Property Proteins Research Role Sarcomeres Series Signal Pathway Spectrum Analysis Stress Striated Muscles TGFB1 gene Technology Tissues Transforming Growth Factor beta Variant Ventricular Arrhythmia Viscosity Work arrhythmogenic cardiomyopathy base cardiac muscle disease citrate carrier clinical phenotype crosslink design experience experimental study fibrogenesis filamin functional disability genetic variant genome editing high risk induced pluripotent stem cell induced pluripotent stem cell derived cardiomyocytes innovation interdisciplinary approach lipid biosynthesis loss of function mechanotransduction mortality multidisciplinary mutant novel novel therapeutics personalized medicine sudden cardiac death therapeutic development

项目摘要

Cardiomyocyte phenotype and mechanotransduction in Filamin C gene variants causing arrhythmogenic cardiomyopathy Project Summary For over two decades, our laboratories have investigated the genetic basis of cardiomyopathies, heart muscle diseases that are a major cause of morbidity and mortality in the world. Recently, we discovered a novel cardiomyopathy disease gene, filamin C (FLNC), and noted that truncating loss-of-function variants (FLNCtv) in FLNC lead to arrhythmogenic cardiomyopathy (ACM), characterized by a high risk of life-threatening ventricular arrhythmias and progression to heart failure. However, FLNC function is still poorly understood and significant knowledge gaps preclude therapeutic development. Notably, (i) the cellular localization and interactions of FLNC, in particular at the cell-cell junction, are incompletely resolved, (ii) the spectrum of molecular networks involved in filaminopathies is largely unknown, (iii) the biomechanical properties of cardiomyocytes with mutant FLNC are also unknown, (iv) the role of FLNC in sarcomere function is not completely elucidated, and (v) finally, the mechanism by which FLNC variants cause different clinical phenotypes is unknown. This proposal aims to determine mechanisms of myocardial failure and cardiac arrhythmia in FLNCtv. Our overarching hypothesis is that FLNCtv perturb mechanotransduction machinery due to disruption of the sarcomeric cytoskeleton, resulting in stress signaling pathway activation (integrins/hippo pathway) which in turn triggers fibrogenesis and adipogenesis, ultimately providing the substrate for arrhythmia. To address these gaps, we have generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from FLNCtv patients and from CRISPR/Cas9-edited lines, collected frozen explanted hearts from FLNCtv patients, and gathered a multidisciplinary research team experienced in experimental modeling of cardiomyopathies. Based on a series of proof-of-concept experiments and preliminary data, we propose three Specific Aims: Aim 1. Determine the phenotype and mechanisms of functional impairment and electrical dysfunction in FLNCtv. We will determine the mechanisms of structural and functional alterations, changes in electrophysiological function, and dysregulation of the interactome at the sarcomere-cytoskeletal-desmosomal interface in hiPSC-CMs. Aim 2. Identify the mechanisms of altered biomechanics in FLNCtv human hearts and hiPSC-CMs. We will determine the mechanisms of altered biomechanics by single cell spectroscopy and myofibrillar mechanics of mutant FLNC hiPSC-CMs and explanted hearts of FLNCtv patients. Aim 3. Define the mechanism of gene expression dysregulation in FLNCtv cardiomyopathy. Using cardiac tissue from FLNCtv patients and FLNC hiPSC-CM models, we will assess role of altered mechanosignaling (Hippo/YAP, TGFβ, Wnt), discover novel transcriptional changes in FLNCtv heart tissue and hiPSC-CMs models, and provide the mechanistic link with structural, contractile and electrophysiological alterations. The elucidation of molecular networks activated in FLNCtv will provide the mechanistic link with the structural, contractile and electrophysiological alterations, and lay the foundation for targeted rescue experiments.

Filamin C 基因变异引起的心肌细胞表型和机械转导致心律失常性心肌病项目概要二十多年来，我们的实验室研究了心肌病、心肌病的遗传基础最近，我们发现了一种新的疾病，这些疾病是世界上发病和死亡的主要原因。心肌病疾病基因 filamin C (FLNC)，并指出截短功能丧失变异 (FLNCtv) FLNC 会导致致心律失常性心肌病 (ACM)，其特点是危及生命的高风险然而，人们对 FLNC 的功能仍知之甚少。显着的知识差距阻碍了治疗的发展，值得注意的是，(i) 细胞定位和 FLNC 的相互作用，特别是在细胞与细胞连接处的相互作用，尚未完全解决，(ii) 丝蛋白病涉及的分子网络在很大程度上是未知的，(iii) 丝蛋白病的生物力学特性具有突变 FLNC 的心肌细胞也是未知的，(iv) FLNC 在肌节功能中的作用尚不清楚完全阐明，以及（v）最后，FLNC变异导致不同临床表型的机制该提案旨在确定心肌衰竭和心律失常的机制。在 FLNCtv 中，我们的总体假设是 FLNCtv 扰乱机械传导机制是由于肌节细胞骨架破坏，导致应激信号通路激活（整合素/河马途径），进而触发纤维生成和脂肪生成，最终为心律失常提供基础。为了解决这些空白，我们生成了人类诱导多能干细胞衍生的心肌细胞（hiPSC-CM）来自 FLNCtv 患者和 CRISPR/Cas9 编辑的细胞系，收集了冷冻外植心脏 FLNCtv 患者，并聚集了一支在实验建模方面经验丰富的多学科研究团队基于一系列概念验证实验和初步数据，我们提出了三种。具体目标：目标 1. 确定功能障碍和电障碍的表型和机制我们将确定 FLNCtv 的结构和功能改变、变化的机制。电生理功能以及肌节-细胞骨架-桥粒相互作用组的失调 hiPSC-CM 中的界面目标 2. 确定 FLNCtv 人类心脏和生物力学改变的机制。我们将通过单细胞光谱学确定生物力学改变的机制突变 FLNC hiPSC-CM 和 FLNCtv 患者移植心脏的肌原纤维力学。目标 3。使用来自 FLNCtv 的心脏组织研究 FLNCtv 心肌病中基因表达失调的机制。患者和 FLNC hiPSC-CM 模型，我们将评估改变的机械信号传导（Hippo/YAP、TGFβ、 Wnt），发现 FLNCtv 心脏组织和 hiPSC-CM 模型中新的转录变化，并提供与结构、收缩和电生理改变的机械联系。 FLNCtv 中激活的网络将提供与结构、收缩和电生理改变，为针对性抢救实验奠定基础。