Project Summary Myocardial infarction (MI) induces the massive death of cardiomyocytes. Quiescent cardiac fibroblasts (CFs) are rapidly activated after MI. They proliferate and differentiate into cardiac myofibroblasts (CMFs), mediating cardiac fibrosis. We recently found that in more stabilized infarct scar CMFs further differentiate into matrifibrocytes, a state resembling partially differentiated chondrocytes, which indicated an advanced level of fibrosis. The post-MI fibrotic response in the heart stabilizes the infarcted myocardium, which may prevent cardiac rupture but also reduce ventricle wall compliance and conductivity, and even interfere with regeneration efforts. Understanding the mechanisms regulating post-injury CF activities will help the development of treatment strategies that enhance the beneficial effect of CFs and inhibit their deleterious effects. We found that in response to MI the expression of runt-related transcription factor 1 (Runx1) in CFs increased significantly during both myofibroblast and matrifibrocyte differentiation. Our preliminary study showed that Runx1 promoted CF proliferation and inhibited their differentiation into myofibroblasts in vitro. In addition, we identified Ccn2 as a potential target of Runx1. Ccn2 plays multiple roles in the regulation of cell proliferation and differentiation through interacting with different cofactors. Thus, we hypothesize that Runx1 functions to balance the proliferation and myofibroblast differentiation of CFs during the acute phase of MI and promote matrifibrocyte differentiation in more stabilized infarct scars through direct targeting Ccn2, which is critical for maintaining tissue integrity but also promotes adverse remodeling. In Aim #1, CF-specific tamoxifen-inducible Runx1 knockout (KO) mice and wild type mice will be subjected to MI injury. Immunohistochemical staining, RNA-seq, and functional assay will be employed to study the function of Runx1 in the proliferation, myofibroblast differentiation, and matrifibrocyte differentiation of CFs after MI. In Aim #2, the mechanism through which Runx1 regulates the expression of Ccn2 will be studied using CUT&RUN, ATAC-seq, Hi-C, and co-IP coupled to mass spectrometry. The unbiased approach will also identify other direct and indirect targets of Runx1. In Aim #3, CF-specific tamoxifen-inducible Ccn2 KO mice will be employed to compare the effect of Ccn2 KO and Runx1 KO on CF proliferation and differentiation after MI. The requirement of Ccn2 in the regulation of CF proliferation and differentiation by Runx1 will also be studied. The synergistic effect between Ccn2 and its cofactors, HB-EGF and TGFβ3, on cardiac fibroblast proliferation and differentiation will be studied as well.