Human iPSCs for Elucidating Stress-mediated Paracrine Signaling in Dilated Cardiomyopathy

项目总结 扩张型心肌病(DCM)是遗传性心脏病最常见的形式之一，其特征是 收缩功能障碍和心室室扩大。尽管DCM经常与基因突变有关 损害收缩功能的肌细胞特异性基因，病理特征也包括非心肌细胞 功能障碍，包括心脏纤维化和内皮病变。纤维化尤其与血管病变的程度有关。 DCM进展，是患者预后不良(例如，心力衰竭)的重要指标，提示 心肌细胞(CM)功能障碍与成纤维细胞的异常激活可能存在因果关系。潜力 两种细胞类型之间的病理性串扰信号似乎越来越可信，因为患病或 与对照CMS相比，逆境下的CMS产生了明显不同的分泌模式。 然而，心脏细胞间通讯的确切机制仍不清楚，部分原因是 到目前为止，由于难以区分蛋白质，人类心脏分泌组的定义一直很模糊。 由心脏相对于病人血浆中的其他器官分泌。 在这里，我将利用IPSC衍生的工程化心脏组织(IPSC-EHT)、基因组编辑技术和切割- 边缘蛋白质组学验证应激诱导的CM分泌体信号促进成纤维细胞的假说 DCM发病机制中的活化和纤维化。为了实现这一点，我将首先产生IPSC来源的心肌细胞 (IPSC-CMS)来自携带三种常见肉瘤基因突变的DCM患者，以及基因组- 编辑的等基因线。IPSC-CMS将用于创建3D IPSC-EHTS，它将启用增强型CM 成熟以及细胞对电刺激和/或增加的机械反应的检查 装填。然后，来自健康和疾病IPSC-EHTS的分泌蛋白质和外切体将被 使用高通量蛋白质组学平台，在定义的条件下全面分析。为了澄清 潜在串扰信号的机制和下游效应，IPSC来源的心脏的激活 成纤维细胞(IPSC-CFs)将通过条件培养液处理和共培养试验进行检测。 拟议研究的成功完成将带来对扩张型心肌病发病机制的新见解， 并帮助确定可以干扰扩张型心肌病病理信号的新的治疗靶点。

PROJECT SUMMARY Dilated cardiomyopathy (DCM) is among the most common forms of inherited heart disease, characterized by systolic dysfunction and ventricular chamber enlargement. Although DCM is often associated with mutations in myocyte-specific genes that impair contractile function, pathological hallmarks also include non-myocyte dysfunction, including cardiac fibrosis and endotheliopathy. Fibrosis in particular correlates with the extent of DCM progression and is an important indicator of adverse patient outcomes (e.g., heart failure), suggesting that cardiomyocyte (CM) dysfunction and aberrant activation of fibroblasts could be causally coupled. Potential pathological crosstalk signaling between the two cell types seems increasingly plausible given that diseased or stressed CMs have been shown to produce remarkably distinct secretory profiles compared to control CMs. However, precise mechanisms of intercellular communication in the heart remain unclear, in part because the human cardiac secretome to date has been poorly defined, hampered by the difficulty of distinguishing proteins secreted by the heart versus other organs in patient plasma. Here, I will leverage iPSC-derived engineered heart tissue (iPSC-EHT), genome-editing technology, and cutting- edge proteomics to test the hypothesis that stress-induced CM secretome signaling promotes fibroblast activation and fibrosis in DCM pathogenesis. To achieve this, I will first generate iPSC-derived cardiomyocytes (iPSC-CMs) from DCM patients that carry mutations in three common sarcomeric genes, along with genome- edited isogenic lines. The iPSC-CMs will be used to create 3D iPSC-EHTs, which will enable enhanced CM maturation as well as examination of cellular responses to electrical stimulation and/or increased mechanical load. The secreted proteins and exosomes from healthy versus diseased iPSC-EHTs will then be comprehensively profiled under defined conditions using high-throughput proteomics platforms. To elucidate mechanisms and downstream effects of potential crosstalk signaling, activation of iPSC-derived cardiac fibroblasts (iPSC-CFs) will be examined by treatment with conditioned media and by co-culture assays. Successful completion of the proposed studies will lead to new mechanistic insights into DCM pathogenesis, and help identify novel therapeutic targets that can disrupt pathological signaling in DCM.