Abstract 4371228: Single-cell multiomic analysis identifies a gene regulatory network governing cardiac fibroblast activities after myocardial infarction and reveals the critical role of RUNX transcription factors

Background: After myocardial infarction (MI), cardiac fibroblasts (CFs) undergo stepwise differentiation into cardiac myofibroblasts (CMFs) and matrifibrocytes, supporting tissue repair. While inadequate CF activation can compromise infarct stability, excessive activation may lead to pathological fibrosis that impairs cardiomyocyte function. Although previous studies have examined transcriptional and epigenetic regulation of this process, its complexity has limited a full understanding of the underlying mechanisms, knowledge that is crucial for developing strategies to fine-tune CF activity during healing. Hypothesis: We hypothesize that CF differentiation is driven by coordinated epigenetic remodeling involving key transcription factors (TFs), including members of the RUNX family. Methods: We performed bulk RNA-seq, ATAC-seq, Cut&Tag, Cut&Run, and Hi-C on CFs from uninjured and post-MI mouse hearts. In parallel, we conducted single-nucleus multiomic profiling (RNA-seq and ATAC-seq) across multiple time points after MI. Using a novel integrative strategy, we constructed a gene regulatory network (GRN) to identify key TFs and regulatory pathways. To assess the role of Runx1 , we used tamoxifen-inducible, CF-specific Runx1 knockout (KO) mice and evaluated transcriptional, epigenetic, and functional outcomes with the same genomic tools and complementary assays. Results: CFs undergo extensive epigenetic remodeling after MI. GRN analysis identified Runx1 as a central regulator of CF proliferation and differentiation. In vitro and in vivo validation confirmed Runx1 as a key modulator of transcriptional and epigenetic changes in CFs. Runx1 KO reduced CF proliferation, disrupted the CMF-to-matrifibrocyte transition, and altered cytokine expression involved in CF–macrophage communication. CF-specific Runx1 KO mice showed improved post-MI survival and reduced cardiac dilatation, especially in males. Combined Runx1/Runx2 deletion further enhanced these effects. Conclusion: Post-MI CF differentiation is regulated by dynamic epigenetic changes involving RUNX transcription factors. Runx1 plays a pivotal role in modulating CF activity and identity, and its deletion improves cardiac repair by mitigating maladaptive fibroblast responses.