Amelioration of Cardiac Fibrosis Using Diverse Cell Village Human Cardiac Organoids

Cardiac fibrosis, the excessive buildup of fibrous connective tissue in the heart resulting in impaired function and structure, is a prominent hallmark of dilated cardiomyopathy (DCM), and significantly contributes to the pathogenesis of this inherited heart disease. DCM is characterized by systolic dysfunction and the enlargement of ventricular chambers, often linked to mutations in myocyte-specific genes that impair contractile function. However, beyond myocyte dysfunction, non-myocyte elements play a crucial role, particularly in the form of cardiac fibrosis and endotheliopathy. Notably, the extent of fibrosis correlates with the progression of DCM and serves as a critical predictor of adverse patient outcomes, including the development of heart failure. This strong association suggests a possible causal relationship between cardiomyocyte (CM), and endothelial cell (EC) dysfunction which results in the dysfunctional activation of fibroblasts (Fb) from a quiescent state turning into ECM-secreting myofibroblasts. To discover a way to combat fibroblast activation, we will take two hits from a screen of 5,000 FDA-approved compounds that was previously performed in the lab, with the goal of validating them in a complex human 3D iPSC cardiac organoid (iPSC-CO) initially, followed by further validation in a mouse model of LMNA DCM. iPSC-CO will be generated from 20 sex and racially diverse iPSC lines (10 healthy, 10 DCM) currently available to our lab, and pooled into 2 cell villages before differentiation into CM, EC, and Fb. Using whole genome sequencing in tandem with single nucleus RNAseq and ATACseq, we will profile the transcriptome and the chromatin state of each cell within the organoid, enabling us to assign a cell line identity to the exact cell line origin. In addition, these organoids will be physiologically profiled using measurements of fibrosis and stiffness through AFM contraction kinematics. These two hits will also be tested in an animal model of DCM, the LMNAH222P mouse. The cardiac tissue of these mice naturally becomes excessively fibrotic by 20 weeks of age, so treatments will begin 1 month before, and will be monitored using echocardiography and tail pressure cuff. At the end of the experiment these animals will be used for 10x multiomics analysis as well as histological and immunological analysis of fibrosis. Here, our research aims to uncover effective strategies to mitigate the detrimental effects of cardiac fibrosis in diseases like DCM. By validating and describing the anti- fibrotic mechanism of two hits from prior compound screen, CGS15943 an adenosine receptor antagonist and AM404 a TRPV1 agonist, and correlating their efficacy in the LMNA DCM mouse model, we aim to provide a therapeutic for those affected by DCM. Using advanced techniques like whole genome sequencing, single nucleus RNAseq and ATACseq, and pairing it with physiological profiling, our study will reveal the interplay between three cardiac cell types, CM, EC, and Fb potentially leading to new treatments and knowledge to better combat excessive cardiac fibrosis. Project Number: 1F32HL174081-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Peter James Zushin | Institution: STANFORD UNIVERSITY, STANFORD, CA | Award Amount: $79,756 | Activity Code: F32 | Study Section: Special Emphasis Panel[ZRG1 F10A-R (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F32HL17408101A1