Human iPSC Model for Elucidating Crosstalk Signaling and Secretomes

扩张型心肌病（DCM）是一种严重而普遍的遗传性心脏缺陷，以心室增大和收缩功能障碍为特征。虽然DCM通常与与收缩性和其他肌细胞特异性功能相关的基因突变有关，但患者的纤维化和内皮功能障碍提示非肌细胞可影响疾病的发病机制和进展。心肌细胞积极分泌多种蛋白质和囊泡到细胞外环境中，其含量可以动态变化以响应应激和疾病，这表明肌细胞和非肌细胞之间的疾病状态之间存在潜在的串扰沟通途径。到目前为止，我们对心脏分泌组的理解是不完整的，因为很难区分患者血浆中心脏和其他器官分泌的蛋白质。为了克服这一挑战，我们建议利用尖端的iPSC技术、基因组编辑技术和蛋白质组学技术来发现和验证心脏分泌组及其在DCM发病机制中调节的串扰信号通路。为了鉴定健康和患病心脏细胞分泌蛋白的补体，我们首先提出从三种常见的肌体突变的DCM患者中产生人诱导多能干细胞来源的心肌细胞（iPSC-CMs）。为了阐明详细的分子机制，我们将使用患者特异性和基因组编辑的等基因iPSC-CMs进行结构、电生理、发育、转录组和机制分析。该等基因人类iPSC平台随后将用于系统地发现(i)分泌蛋白和（ii）分泌心肌细胞外泌体，使用大规模蛋白质组学平台，能够定量数百种低丰度的感兴趣蛋白。为了确认分泌蛋白的信号传导方式，我们将使用高通量平台对ipsc衍生的内皮细胞（iPSC-ECs）和ipsc衍生的心脏成纤维细胞（iPSC-CFs）与患病和健康iPSC-CMs共培养进行详细的转录组学和功能分析。我们预计这些研究的成功完成将导致对DCM发病机制的新认识，并有助于确定新的治疗靶点，这些靶点可以阻止和恢复患病心脏中肌细胞和非肌细胞之间的疾病串扰信号。

PROJECT SUMMARY Dilated cardiomyopathy (DCM) is a severe and prevalent inherited cardiac defect, characterized by ventricular chamber enlargement and systolic dysfunction. Although DCM is commonly associated with mutations in genes associated with contractility and other myocyte-specific functions, fibrotic and endothelial dysfunctions in patients suggest non-myocytes can influence disease pathogenesis and progression. Cardiac myocytes actively secrete a diverse array of proteins and vesicles into the extracellular milieu, the contents of which can change dynamically in response to stress and disease, suggesting a potential avenue of crosstalk communicating disease status between myocytes and non-myocytes. Thus far, our understanding of the cardiac secretomes is incomplete, hampered by difficulty of differentiating proteins secreted by the heart vs. other organs in patient plasma. To overcome this challenge, we propose to leverage cutting-edge iPSC technology, genome-editing technology, and proteomics technology to discover and validate cardiac secretomes and the crosstalk signaling pathways they regulate in the context of DCM pathogenesis. To identify the complement of secreted proteins from healthy and diseased cardiac cells, we first propose to generate human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from DCM patients with three common sarcomeric mutations. To clarify the detailed molecular mechanisms, we will conduct structural, electrophysiological, developmental, transcriptomic, and mechanistic analyses using patient- specific as well as genome-edited isogenic iPSC-CMs. This isogenic human iPSC platform will then be used to systematically discover the (i) secreted proteins and (ii) secreted exosomes of cardiac cells using large- scale proteomics platforms capable of quantifying hundreds of low-abundance proteins of interest. To confirm the signaling modality of secreted proteins, we will perform detailed transcriptomic and functional analysis of iPSC-derived endothelial cells (iPSC-ECs) and iPSC-derived cardiac fibroblasts (iPSC-CFs) co-cultured with diseased vs. healthy iPSC-CMs using high-throughput platforms. We anticipate that the successful completion of these studies will lead to new mechanistic insights into DCM pathogenesis, and help identify novel therapeutic targets that can impede and revert disease crosstalk signaling between myocytes and non- myocytes in the diseased heart. In a Supplement to the Parent R01 HL141371, we propose to leverage patient-derived human induced pluripotent stem cell (iPSC) platform towards studying mechanisms of CHD in people with Down syndrome. We hypothesize overexpression of cardiac-specific, dosage-sensitive trisomy genes on chromosome 21 leads to heart defects through impaired cardiac crosstalk and myocyte maturation. Aim 1 will generate a biorepository of 40 Down syndromes-pecific iPSC lines. To investigate the role of intercellular crosstalk in the pathogenesis of Down syndrome-related CHD, we will engineer iPSC-cardiac organoids resembling the heart tissue composition of cardiomyocytes, endothelial cells, and fibroblasts and determine molecular and functional phenotypes of cardiac organoids derived from the Down syndrome iPSCs. In Aim 2, we will investigate the mechanism of Down syndrome-related CHD using a pan-omic approach. The mechanisms of identified gene candidates will be further investigated through genome editing strategy. Completing the aims of this supplement will likely increase our understanding of Down syndrome-related CHD as well as broaden the overall impact of the parent R01 award. In the parent award, we are using iPSC technology to identify mechanisms of genetic cardiomyopathy in vitro and dissecting the role of crosstalk between cardiovascular cell types in pathogenesis. Mechanism underlying Down syndrome-related CHD involves complex intercellular communication leading to developmental and structural anomalies. Hence, we are confident that a comparative in vitro and bioinformatics analysis utilizing both Down syndrome and non-Down syndrome CHD iPSC-derived cardiomyocytes will likely extend our understanding of CHD as well as facilitate the discovery of novel genes and pathways that may be critical in its pathogenesis.