PROJECT SUMMARY Cardiac fibrosis contributes to the progression of heart failure. Currently, there is no FDA-approved drug specifically targeting cardiac fibrosis due to the lack of understanding of the underlying mechanism and a reliable drug discovery platform. Here, we will use a novel multiplexing methodology of creating a "cell village" by pooling multiple patients' induced pluripotent stem cell (iPSC) lines in a dish to map the genetic basis of interindividual differences in response to a profibrotic cocktail. In Aim 1, we will co-culture 100 iPSC lines from 60 healthy donors and 40 dilated cardiomyopathy (DCM) patients in 10 distinct "cell villages," where each "cell village" contains ten independent patient-specific iPSC lines. Next, we will differentiate each "cell village" into iPSC-derived cardiomyocytes (iPSC-CMs), endothelial cells (iPSC-ECs), and cardiac fibroblasts (iPSC-CFs), and then generate 3D engineered heart tissues (EHTs). Finally, we will perform a single-cell multiomics sequencing analysis of the "cell villages" to understand the impact of internal genetic predisposition and external profibrotic stimuli on EHTs. In Aim 2, we will perform high throughput screening to identify novel small molecules for treating cardiac fibrosis using novel iPSC-CF reporter lines. The antifibrotic effects of the drug hits will be validated in primary CFs and EHTs. We will perform RNA- and ATAC- sequencing to determine the downstream signaling pathways of the newly identified compounds. In Aim 3, we will validate the therapeutic effects of antifibrotic compounds in mice models of dilated cardiomyopathy. The cardiac function will be measured by echocardiography, and fibrosis severity will be assessed by histology at multiple time points. We will also perform single-nuclei multiome and spatial transcriptomics to delineate the impact of therapeutic drug treatment in each cell type cluster and cell-cell interactions in the heart based on the 10x Genomics platform. Collectively, we expect to identify new signaling pathways and novel compounds for patients with cardiac fibrosis and heart failure.