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

细丝蛋白C基因变异引起的心肌细胞表型和机械转导 致心肌病 项目摘要 二十多年来，我们的实验室研究了心肌病的遗传基础， 这些疾病是世界上发病率和死亡率的主要原因。最近我们发现了一本小说 心肌病基因细丝蛋白C（FLNC），并指出，截短功能丧失变体（FLNCtv） 在FLNC中，导致以危及生命高风险为特征的致瘤性心肌病（ACM 室性心律失常并进展为心力衰竭。然而，FLNC功能仍然知之甚少， 巨大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-细胞连接处，不完全解析，（ii）FLNC的光谱 参与细丝蛋白病的分子网络在很大程度上是未知的，（iii） 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不是已知的。 完全阐明，和（v）最后，FLNC变异体引起不同临床表型的机制 不明该提案旨在确定心肌衰竭和心律失常的机制 在FLNCtv。我们的总体假设是FLNCtv干扰机械转导机制， 肌节细胞骨架的破坏，导致应激信号通路激活（整合素/河马 这反过来触发纤维形成和脂肪形成，最终为心律失常提供底物。 为了解决这些差距，我们已经产生了人类诱导多能干细胞衍生的心肌细胞， 从FLNCtv患者和来自CRISPR/Cas9编辑的细胞系的hiPSC-CM中收集冷冻的心脏， FLNCtv患者，并聚集了一个多学科的研究团队，在实验建模经验丰富， 心肌病基于一系列概念验证实验和初步数据，我们提出了三个 具体目标：目标1。确定功能障碍和电刺激的表型和机制 FLNCtv中的功能障碍。我们将确定结构和功能改变的机制， 肌节-细胞骨架-桥粒相互作用组的电生理功能和失调 hiPSC-CM中的接口。目标2.确定FLNCtv人类心脏生物力学改变的机制， hiPSC-CM。我们将通过单细胞光谱学来确定生物力学改变的机制， 图1示出了突变FLNC hiPSC-CM和FLNC tv患者的心脏的肌原纤维力学。目标3：定义 FLNCtv心肌病基因表达失调的机制使用FLNCtv的心脏组织 患者和FLNC hiPSC-CM模型，我们将评估改变的机械信号传导（Hippo/雅普，TGFβ， Wnt），在FLNCtv心脏组织和hiPSC-CM模型中发现了新的转录变化，并提供了 与结构、收缩和电生理改变的机械联系。分子的阐明 FLNCtv中激活的网络将提供与结构性、收缩性和 电生理改变，并为有针对性的救援实验奠定基础。

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.