Human iPSCs for Elucidating Intercellular Crosstalk Signaling in DCM - Diversity Supplement

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Summary of Parent R01: 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. 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. Proposed Supplement: There are 62.1 million Hispanics living in the United States, according to data from the 2020 United States Census. In the Hispanic population, heart diseases have been relatively understudied. In this proposed diversity supplement, we will extend the scope of the parent R01 to include patients from Hispanic ethnicity to investigate LMNA-related dilated cardiomyopathy (LMNA-DCM. Specifically, in this proposal, we will include 11 individuals (10 healthy, 1 diseased) from the Hispanic community to expand the diversity of the parent R01. This proposal will first characterize healthy and diseased Hispanic heart cells derived from induced pluripotent stem cells and introduce LMNA-DCM variants using CRISPR genome-editing technology to 9 healthy Hispanic cell lines to validate functional changes between healthy and diseased cells. Then in extended Aim 2, we will generate 3D Cardiac Organoids using the cell types generated in Aim 1 to capture cell-cell signaling pathways by way of transcriptomic analysis and gene expression using single-cell RNA sequencing.