Human iPSC Model for Elucidating Crosstalk Signaling and Secretomes

扩张型心肌病（DCM）是一种严重且普遍的遗传性心脏缺陷，其特征在于心室腔扩大和收缩功能障碍。尽管DCM通常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表明非肌细胞可影响疾病的发病机制和进展。心肌细胞主动分泌多种蛋白质和囊泡到细胞外环境中，其内容物可以响应于应激和疾病而动态变化，这表明肌细胞和非肌细胞之间的串扰通信疾病状态的潜在途径。到目前为止，我们对心脏分泌组的理解是不完整的，这受到难以区分心脏分泌的蛋白质与患者血浆中其他器官分泌的蛋白质的阻碍。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。为了鉴定来自健康和患病心脏细胞的分泌蛋白的补体，我们首先提出从具有三种常见肌节突变的DCM患者产生人诱导多能干细胞衍生的心肌细胞（iPSC-CM）。为了阐明详细的分子机制，我们将使用患者特异性以及基因组编辑的同基因iPSC-CM进行结构、电生理学、发育、转录组学和机制分析。该等基因人类iPSC平台随后将用于系统地发现心脏细胞的（i）分泌蛋白和（ii）分泌外泌体，其使用能够定量数百种低丰度目标蛋白的大规模蛋白质组学平台。为了确认分泌蛋白的信号传导模式，我们将使用高通量平台对iPSC衍生的内皮细胞（iPSC-EC）和iPSC衍生的心脏成纤维细胞（iPSC-CF）进行详细的转录组学和功能分析，所述iPSC衍生的内皮细胞（iPSC-EC）和iPSC-CF与患病的iPSC-CM和健康的iPSC-CM共培养。我们预计，这些研究的成功完成将导致对DCM发病机制的新的机制见解，并帮助确定新的治疗靶点，可以阻止和逆转患病心脏中肌细胞和非肌细胞之间的疾病串扰信号传导。 在母体R 01 HL 141371的补充中，我们建议利用患者来源的人诱导多能干细胞（iPSC）平台研究唐氏综合征患者的CHD机制。我们假设21号染色体上心脏特异性、剂量敏感性三体基因的过度表达通过心脏串扰和肌细胞成熟受损导致心脏缺陷。目标1将产生40个唐氏综合征特异性iPSC系的生物储存库。为了研究细胞间串扰在唐氏综合征相关CHD发病机制中的作用，我们将设计类似于心肌细胞，内皮细胞和成纤维细胞的心脏组织组成的iPSC-心脏类器官，并确定源自唐氏综合征iPSC的心脏类器官的分子和功能表型。在目标2中，我们将使用泛组学方法研究唐氏综合征相关冠心病的机制。已确定的候选基因的机制将通过基因组编辑策略进一步研究。完成这一补充的目标可能会增加我们对唐氏综合征相关冠心病的理解，并扩大父母R 01奖的整体影响。在父母奖中，我们正在使用iPSC技术来确定体外遗传性心肌病的机制，并剖析心血管细胞类型之间的串扰在发病机制中的作用。唐氏综合征相关冠心病的潜在机制涉及复杂的细胞间通讯，导致发育和结构异常。因此，我们相信，利用唐氏综合征和非唐氏综合征CHD iPSC衍生的心肌细胞进行比较性体外和生物信息学分析可能会扩展我们对CHD的理解，并促进发现可能在其发病机制中至关重要的新基因和途径。

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.