Human iPSCs for Elucidating Intercellular Crosstalk Signaling in Dilated Cardiomyopathy

项目摘要 LMNA相关的扩张型心肌病（DCM）是遗传性心脏病中最常见的形式， 以严重的收缩功能障碍和心室腔扩大为特征。主要特点 LMNADCM还涉及非肌细胞功能障碍的特征，包括心肌纤维化和内皮病变。 然而，心脏中细胞间通讯的精确机制仍不清楚，部分原因是 迄今为止，人类心脏分泌物组的定义很差。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 可能参与LMNA-DCM发病机制的串扰信号通路。目标1：全面 通过使用基于高通量适体的蛋白质组学来描绘每种细胞类型的基线分泌组 方法，并进行跨孔共培养测定，以系统地评估下游功能性 蜂窝串扰的后果。在目标2中，我们将通过进一步调查补充这些研究， 不同LMNA-DCM /对照细胞工程化心脏组织（EHT）中细胞间通讯机制 类型组成。随后将通过单细胞RNA测序（scRNA-seq）分析EHT， 预测细胞间的串扰模式，并构建跨细胞的独特和共享的配体受体对的列表。 条件在目标3中，我们将使用以下方法对> 4，000种化合物进行大规模高通量筛选： 多细胞iPSC衍生的心脏类器官（iPSC-CO）从三谱系报告细胞系分化。选择 将使用蛋白质组学和靶向功能获得/丧失研究来验证和进一步研究候选物。 我们期望这些研究的成功完成将导致对DCM的新的机制见解 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 疾病心脏中心脏细胞类型之间的信号传导。

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.