Hypertrophic cardiomyopathy-induced paracrine signaling and stromal activation

项目摘要/摘要 肥厚型心肌病(HCM)影响1：500的人口，是导致心脏骤停的主要原因 年轻人的死亡。肥厚性心肌病的临床表现包括左室壁增厚、舒张期 功能障碍和纤维化。纤维化引起的组织重塑在终末期取代了30%到50%的心肌 HCM，是患者预后的关键决定因素。调节多种肌瘤蛋白的突变 心肌收缩功能已被确认为肥厚性心肌梗死的原因，其中约30%位于β-肌球蛋白重建区。 链(MYH7)，但尚不清楚心肌细胞收缩性能的内在变化如何导致纤维化 改建。虽然之前的研究已经为纤维化提供了重要的见解，但实验的局限性 模型，如有限的患者样本，有限的体外研究人类心肌细胞的能力，以及物种 心血管生物学的差异，使得确定间质前纤维化的机制变得困难 激活。人类诱导多能干细胞(HiPSC)和CRISPR/Cas9技术的进展 允许研究人员将遗传性心脏病与健康的等位基因背景进行对比建模和研究 在试管中。虽然HiPSC-CMS为了解病变心肌细胞的功能变化提供了重要的见解， 为了研究纤维化的病理重塑，需要建立一个多细胞三维模型。心脏微组织(CMT) 这些平台为研究心肌细胞对基质细胞和微环境的影响提供了独特的工具。 这一提议的总体假设是MYH7变异的HiPSC-CMS致病地激活基质 细胞通过旁分泌信号，导致纤维化表型。这项提案将确定以下目标 在以下特定的目的中减轻纤维化的表型。目的1.建立MYH7基因突变的间质激活模型 HIPSC-CM体外建立人肥厚性心肌病模型。目的2.确定致病MYH7的关键旁分泌因子信号转导途径 导致间质细胞纤维化反应的变体。目的3.靶向间质中的旁分泌因子受体 细胞减少MYH7变异型CMTs的纤维化发展。基质激活的体外模型将是 通过量化胶原沉积、僵硬、基因表达和 伸缩性。将确定导致这些变化的来自MYH7变异型HiPSC-CM的旁分泌信号 使用条件培养实验、磷酸蛋白质组学和RNA测序相结合的方法。钥匙 信号通路将以小分子抑制剂为靶点，基质激活的减弱将 通过胶原沉积、硬度、基因表达和收缩能力的量化得到确认。这个 这项研究的结果将对肥厚性心肌炎的疾病病理提供新的见解，并将提供潜在的 治疗的目标是减轻这种病理，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.