Hypertrophic cardiomyopathy-induced paracrine signaling and stromal activation

项目总结/摘要 肥厚型心肌病（HCM）影响1：500的人口，是心脏病突发的主要原因。 年轻人的死亡。HCM的临床表现包括左室壁增厚、舒张功能不全、心功能不全、心功能不全和心功能不全。 功能障碍和纤维化。纤维化导致的组织重塑在终末期取代了30 - 50%的心肌 HCM是患者结局的关键决定因素。许多肌节蛋白的突变， 心肌收缩力已被确定为HCM的原因，其中约30%位于β-肌球蛋白重 链（MYH 7），但仍不清楚心肌细胞收缩力的内在变化如何导致纤维化。 重塑虽然先前的研究已经提供了对纤维化的重要见解，但实验研究的局限性仍然存在。 模型，例如有限的患者样本，离体研究人类心肌细胞的能力有限，以及物种 心血管生物学的变化，使得难以确定间质纤维化之前的机制， activation.人类诱导性多能干细胞（hiPSC）和CRISPR/Cas9技术的进展 允许研究人员建模和研究遗传性心脏病，与健康的同基因背景相比， 体外虽然hiPSC-CM为患病心肌细胞的功能变化提供了重要的见解， 需要多细胞3D模型来研究纤维化中的病理重塑。心脏微组织（CMT） 平台提供了一种独特的工具来研究心肌细胞对基质细胞和微环境的影响。 该提议的总体假设是MYH 7变体hiPSC-CM致病性地激活基质细胞， 细胞通过旁分泌信号传导，导致纤维化表型。该提案将确定目标， 在以下具体目的中减弱纤维化表型。目标1.为了模拟MYH 7变体中的基质活化， HCM的hiPSC-CM体外模型。目标二。确定致病性MYH 7的关键旁分泌因子信号传导 导致基质细胞纤维化反应的变异。目标3：靶向间质中的旁分泌因子受体， 细胞以减少MYH 7变体CMT中的纤维化发展。基质活化的体外模型将是 通过定量胶原沉积、硬度、基因表达和 收缩性将鉴定来自MYH 7变体hiPSC-CM的导致这些变化的旁分泌信号传导 使用条件培养基实验、磷酸化蛋白质组学和RNA测序的组合。关键 信号通路将被小分子抑制剂靶向，基质激活的减弱将 通过量化胶原沉积、硬度、基因表达和收缩性来证实。的 这项研究的结果将为HCM的疾病病理学提供新的见解， 治疗靶点，以减轻这种病理，从而改善临床结果。

PROJECT SUMMARY/ABSTRACT Hypertrophic cardiomyopathy (HCM) affects 1:500 of the population and is the leading cause of sudden cardiac death in young people. Clinical presentation of HCM includes thickening of the left ventricular wall, diastolic dysfunction, and fibrosis. Tissue remodeling from fibrosis replaces 30 to 50% of the myocardium in end-stage HCM and is a key determinant in patient outcome. Mutations in numerous sarcomeric proteins that regulate cardiac contractility have been identified as causes of HCM, about 30% of which are in located β-myosin heavy chain (MYH7), but it remains unclear how the intrinsic changes in contractility of cardiomyocytes lead to fibrotic remodeling. While previous studies have provided important insight into fibrosis, limitations in experimental models, such as limited patient samples, limited ability to study human cardiomyocytes ex vivo, and species variances in cardiovascular biology, have made it difficult to determine a mechanism of fibrosis preceding stromal activation. Advancements in human induced pluripotent stem cell (hiPSC) and CRISPR/Cas9 technology have allowed investigators to model and study inherited cardiac diseases compared to a healthy isogenic background in vitro. While hiPSC-CMs have provided important insight into functional changes in diseased cardiomyocytes, a multicellular 3D model is needed to study the pathological remodeling in fibrosis. Cardiac microtissue (CMT) platforms offer a unique tool to study the effects of cardiomyocytes on stromal cells and the microenvironment. The overall hypothesis of this proposal is that MYH7-variant hiPSC-CMs pathogenically activate stromal cells through paracrine signaling, leading to a fibrotic phenotype. This proposal will determine targets to attenuate a fibrotic phenotype in the following Specific Aims. Aim 1. To model stromal activation in MYH7-variant hiPSC-CM in vitro models of HCM. Aim 2. To determine key paracrine factor signaling from pathogenic MYH7 variants that leads to a fibrotic response in stromal cells. Aim 3. To target paracrine factor receptors in stromal cells to decrease fibrotic development in MYH7-variant CMTs. The in vitro model of stromal activation will be characterized and validated with the quantification of collagen deposition, stiffness, gene expression, and contractility. The paracrine signaling from MYH7-variant hiPSC-CM leading to these changes will be identified using a combination of conditioned media experiments, phosphoproteomics, and RNA-sequencing. Key signaling pathways will be targeted with small-molecule inhibitors, and the attenuation of stromal activation will be confirmed through the quantification of collagen deposition, stiffness, gene expression, and contractility. The results of this study will provide new insights into the disease pathology of HCM and will provide potential therapeutic targets to attenuate this pathology, and thus improve clinical outcomes.