PROJECT SUMMARY/ABSTRACT Fibrosing cardiac diseases cause 25% of deaths in the United States. Fibrosis is a key pathological hallmark of nearly all cardiac diseases, including hypertensive heart disease, atrial fibrillation, and ischemic heart disease, which is caused by myocardial infarction (MI). Across these fibrosing cardiac diseases, cardiac fibroblasts are activated to deposit excessive extracellular matrix that causes organ dysfunction, including diastolic dysfunction, arrythmias, and heart failure with reduced ejection fraction. Despite the clinical burden of cardiac fibrosis, especially after MI, the molecular mechanisms driving cardiac fibroblasts to produce excessive scar tissue are unknown. Uncovering the pathophysiological mechanisms for cardiac fibrosis formation has the potential to reveal novel drug targets to reduce cardiac fibrosis and improve cardiac function. The Research Training Plan will investigate the molecular mechanisms for cardiac fibrosis formation after MI. Specifically, this project will test the hypothesis that thinning of the ventricle wall after MI increases wall stress to activate mechanotransduction pathways in cardiac fibroblasts to produce scar tissue. To investigate this hypothesis, this project will combine innovative computational and experimental approaches, such as advanced imaging, computational modeling, spatial transcriptomics, and animal models. Specific Aim 1 will spatially correlate tissue fibrosis and wall stress after MI in mice by using 2D histology, 3D whole-organ imaging, and finite element analysis. Specific Aim 2 will determine the spatial heterogeneity of cardiac fibroblast subpopulations after MI using publicly available mouse spatial transcriptomics datasets. Specific Aim 3 will functionally test if mechano- transduction pathways activate cardiac fibroblasts by using an in vitro fibroblast-seeded hydrogel stretch assay and an in vivo mouse model of MI. In summary, the proposed studies will investigate a possible pathophysiological mechanism for cardiac fibrosis formation after MI and may provide novel therapeutic targets to treat patients after MI. Importantly, through this project, the applicant, John Lu, will gain diverse expertise in cutting-edge experimental techniques, computational approaches, and scientific reasoning under the mentorship of global fibrosis expert, Dr. Michael Longaker, and leading cardiovascular biologist, Dr. Kristy Red-Horse. Through his training activities, John will also develop the professional and clinical skills to direct an independent laboratory as a future physician-scientist investigator. Furthermore, Stanford University offers an outstanding environment for innovative and collaborative research, with the necessary infrastructure and core facilities to ensure this project’s success. In summary, the strong mentoring environment and fellowship training plan will prepare John to be an independent physician-scientist investigator working at the frontiers o...