Abstract Body (Do not enter title and authors here): Introduction and hypothesis: Cardiac fibroblasts and myofibroblasts drive post-myocardial infarction (MI) repair, remodeling, and fibrosis. TGF-β superfamily members exert pro-reparative, fibroblast activating actions, which are attributed primarily to Smad3 activation. The role of Smad1, another major receptor-regulated Smad, remains unknown. We hypothesized that Smad1 activation in infarct myofibroblasts may regulate repair and remodeling of the infarcted heart.

Results: In a mouse MI model, Smad1 was activated in a myofibroblast subpopulation. Although prevailing concepts suggest that Smad1 is activated predominantly by BMPs, Smad1 was phosphorylated not only by BMP4 and BMP7, but also by TGF-β1, -β2, and β3. TGF-β-induced Smad1 activation required both type I receptors: ALK1 and ALK5. Following MI, myofibroblast-specific Smad1 KO mice had exacerbated systolic dysfunction (ejection fraction 28d: p<0.01), increased LV dilation, and infarct expansion (scar/LV 28d: p<0.05), accompanied by accentuated fibrosis. In vitro, Smad1 loss markedly upregulated fibrosis-associated genes, increased fibroblast survival, and enhanced motility, as demonstrated by live single-cell algorithmic tracking. RNA-seq transcriptional profiling (in vitro/vivo) and bioinformatic analysis identified Smad3, p53, and WNT3A as key upstream regulators mediating Smad1 loss effects. In vitro, Smad1 interfered with nuclear translocation of activated pSmad3, directly interacted with p53, and upregulated Sfrp1. Pharmacological inhibition experiments demonstrated that Smad3 and p53 mediate in part the pro-fibrotic effects of Smad1 loss. Integrative scRNA-seq of infarct fibroblasts identified a distinct regulatory myofibroblast cluster, characterized by high Acvrl1 (ALK1) expression, and a transcriptional signature consistent with Smad1 activation, accompanied by an attenuated fibrogenic profile. This cluster expands during scar maturation, consistent with its role in restraining fibrosis.

Conclusions: We present the first in vivo evidence suggesting an anti-fibrotic role of fibroblast-specific Smad1 activation. TGF-β/ALK1-mediated Smad1 signaling promotes a fibrosis-restraining program by inhibiting Smad3 nuclear translocation, by suppressing WNT signaling and by interacting with p53. Smad1 favors a shift of a subpopulation of infarct myofibroblasts towards a protective regulatory phenotype. Cell-specific activation of Smad1 may hold therapeutic promise in fibrosis-associated conditions.