Human iPSCs for Elucidating Intercellular Crosstalk Signaling in Dilated Cardiomyopathy

项目总结 LMNA相关扩张型心肌病(DCM)是最常见的遗传性心脏病之一， 以严重的收缩功能障碍和心室室扩大为特征。的主要特征 LMNADCM还涉及非心肌细胞功能障碍，包括心肌纤维化和内皮病变。 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，人类心脏分泌组的定义并不明确。为了克服这一挑战，我们建议利用 人类ipscs、基因组编辑技术和最先进的组学方法来识别和研究 串扰信号通路可能参与LMNA-DCM的发病机制。在目标1中，我们将全面 利用基于高通量适配子的蛋白质组学分析每种细胞类型的基线分泌组 方法，并进行跨井共培养试验，系统评价下游功能 蜂窝串扰的后果。在目标2中，我们将通过进一步调查来补充这些研究 不同LMNA-DCM/对照细胞在工程心脏组织中的细胞间通讯机制 文字成分。EHTS随后将通过单细胞RNA测序(scRNA-seq)进行分析，以 预测细胞-细胞串扰模式，并构建一个唯一和共享的配体受体对列表 条件。在目标3中，我们将对&gt；4,000种化合物进行大规模高通量筛选，使用 多细胞IPSC衍生的心脏器质(IPSC-CO)是从三系报告系分化而来的。已选择 候选者将通过蛋白质组学和定向功能得失研究进行验证和进一步研究。 我们预计，这些研究的成功完成将带来对DCM的新的机械论见解 发病机制，并帮助开发新的治疗策略，可以阻止和逆转异常串扰 病变心脏中不同类型的心肌细胞之间的信号传递。

PROJECT SUMMARY LMNA-related dilated cardiomyopathy (DCM) is among the most prevalent forms of inherited heart disease, characterized by severe systolic dysfunction and ventricular chamber enlargement. Major hallmarks of LMNADCM also involve features of non-myocyte dysfunction including myocardial fibrosis and endotheliopathy. However, precise mechanisms of intercellular communication in the heart remain unclear, in part because the human cardiac secretome to date has been poorly defined. To overcome this challenge, we propose to leverage human iPSCs, genome-editing technology, and state-of-the-art omics methods to identify and investigate crosstalk signaling pathways potentially involved in LMNA-DCM pathogenesis. In Aim 1, we will comprehensively profile the baseline secretomes of each cell type by employing high-throughput aptamer-based proteomics methods, and perform trans-well co-culture assays to systematically evaluate the downstream functional consequences of cellular crosstalk. In Aim 2, we will complement these studies with further investigation into intercellular communication mechanisms in engineered heart tissues (EHTs) of varying LMNA-DCM / control cell type compositions. The EHTs will be subsequently analyzed by single-cell RNA sequencing (scRNA-seq) to predict cell-cell crosstalk modalities and construct a list of unique and shared ligand receptor pairs across conditions. In Aim 3, we will perform large-scale high-throughput screening of >4,000 compounds using multicellular iPSC-derived cardiac organoid (iPSC-CO) differentiated from tri-lineage reporter lines. Selected candidates will be validated and further investigated using proteomics and targeted gain/loss-of function studies. We anticipate that the successful completion of these studies will lead to new mechanistic insight into DCM pathogenesis, and help develop novel therapeutic strategies that can impede and reverse aberrant crosstalk signaling between cardiac cell types in the diseased heart.