Multiomics reveals epigenetic control of fibroblast activity after myocardial infarction and a key role for RUNX transcription factors

ABSTRACT Background After myocardial infarction (MI), cardiac fibroblasts proliferate and undergo a sequential differentiation process. They first transition into cardiac myofibroblasts, a transient and highly contractile state, and ultimately into matrifibrocytes, a more stable state that partially resembles chondrocytes. These dynamic transitions are essential for infarct healing and scar formation. While insufficient fibroblast activation can compromise infarct integrity, excessive activation promotes pathological fibrosis that impairs cardiac function. Despite its clinical importance, the transcriptional and epigenetic regulation of these transitions remain poorly understood. Elucidating underlying mechanisms is critical for developing strategies to fine-tune fibroblast activity during cardiac repair. Methods We performed bulk RNAseq, ATACseq, CUT&Tag, CUT&RUN, EMseq, and Hi-C on cardiac fibroblasts from uninjured and post-MI mouse hearts. In parallel, we conducted single-nucleus multiomic (snRNAseq and snATACseq) profiling across multiple time points after MI. Subsequent integrated analysis explored epigenetic mechanisms regulating cardiac fibroblast gene expression and activity. Using an improved computational strategy, we constructed gene regulatory networks to identify key transcription factors and biological processes regulated by these transcription factors. To assess the role of Runx1 specifically, we used tamoxifen-inducible, fibroblast-specific Runx1 knockout mice to evaluate transcriptional, epigenetic, and functional outcomes with the same genomic tools and additional complementary assays. Results Cardiac fibroblasts undergo extensive chromatin remodeling after MI, which is highly correlated with changes in transcriptomic profiles. In contrast, the role of DNA methylation is relatively minor. Gene regulatory network analysis identified Runx1 as a central regulator of cardiac fibroblast proliferation and matrifibrocyte differentiation. In vitro and in vivo validation confirmed Runx1 as a key modulator of transcriptional and epigenetic changes in cardiac fibroblasts. Runx1 KO reduced cardiac fibroblast proliferation, disrupted the myofibroblast-to-matrifibrocyte transition, and affected macrophage cytokine expression through altered cardiac fibroblast–macrophage communication. Fibroblast-specific Runx1 knockout mice showed improved post-MI survival and reduced cardiac dilatation, especially in males. Simultaneous Runx2 deletion further enhanced the effects of Runx1 knockout. Conclusions Cardiac fibroblast activation and differentiation after MI are regulated by dynamic epigenetic changes. Runx1 plays a pivotal role in modulating cardiac fibroblast activities, and its deletion improves cardiac repair by mitigating maladaptive fibroblast responses. By illuminating the centrality of Runx1 in post-MI repair, this study identifies an actionable pathway for therapeutically steering fibroblast responses.