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 致心律失常性心肌病 项目摘要 二十多年来，我们的实验室一直在研究心肌病的遗传基础，即心肌 疾病是世界上发病率和死亡率的主要原因。最近，我们发现了一部小说 心肌病基因细丝C(Flnc)，并注意到截断功能丧失变异体(Flnctv) 导致致心律失常的心肌病(ACM)，其特征是危及生命的高风险 室性心律失常和进展为心力衰竭。然而，人们对FLNC的功能仍知之甚少 重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-细胞交界处的相互作用没有完全分辨，(Ii)光谱 参与丝氨酸病的分子网络在很大程度上是未知的，(Iii) 具有突变的FlnC的心肌细胞也未知，(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 是未知的。这项建议旨在确定心肌衰竭和心律失常的机制。 在FLNCtv中。我们的主要假设是，FLNCtv扰动机械转导机械是由于 破坏肌节细胞骨架，导致应激信号通路激活(整合素/河马 途径)，进而触发纤维化和脂肪生成，最终为心律失常提供底物。 为了解决这些差距，我们创造了人类诱导的多能干细胞来源的心肌细胞。 (HiPSC-CMS)来自FLNCtv患者和CRISPR/Cas9编辑的株系，从 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。 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.确定功能损害和电损伤的表型和机制 FLNCtv的功能障碍。我们将确定结构和功能改变的机制，改变 肌节-细胞骨架-桥粒相互作用体的电生理功能和失调 在HiPSC-CMS中的接口。目的2.确定FLNCtv人心脏和心脏生物力学改变的机制 HiPSC-CMS。我们将通过单细胞光谱分析来确定生物力学改变的机制 突变的FlnC-hiPSC-CMS和FlNCtv患者移植心脏的肌原纤维力学。目标3.定义 FlnCtv心肌病基因表达异常的机制。利用来自FLNCtv的心脏组织 患者和FNC HiPSC-CM模型，我们将评估机械信号变化(HIPPO/YAP，转化生长因子β， 在FlnCtv心脏组织和HiPSC-CMS模型中发现新的转录变化，并提供 与结构、收缩和电生理改变的机械性联系。关于分子的阐明 在FLNCtv中激活的网络将提供与结构、收缩和 电生理改变，为有针对性的抢救实验奠定基础。

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.