Metformin reprograms cardiac fibroblast heterogeneity and metabolome to attenuate post-inflammatory fibrosis in experimental autoimmune myocarditis
M Stefanska, K Sarad, M Kot, M Szyszka, M Blatkiewicz, M RucinskiAbstract
Background/Introduction
Dilated cardiomyopathy (DCM) evolves from autoimmune myocarditis via TGF-β1/Smad-driven post-inflammatory fibrosis. Experimental autoimmune myocarditis (EAM) models acute inflammation transitioning to chronic fibrosis. Metformin activates AMPK to inhibit TGF-β/myofibroblast differentiation and restore mitochondrial function via metabolic reprogramming. The effect of metformin in established EAM fibrosis and cardiac metabolome remain undefined.
Purpose
The aim of this study was to investigate whether therapeutic metformin administration from day 17 attenuates chronic EAM fibrosis by day 40, to elucidate transcriptomic mechanisms via single-nuclei RNA sequencing and characterize cardiac metabolomic reprogramming.
Methods
6–8-week-old male BALB/c mice (n=8–10/group) were immunized with αMyHC peptide in CFA to induce EAM. Metformin (250 mg/kg/day) or PBS (control) was administered via nasogastric tube from day 17 to the study endpoint (day 40). Hearts were harvested for untargeted metabolomics, single-nucleus RNA sequencing (~200,000 nuclei/sample, >99% purity via FACS; 10x Genomics GEM-X 3' v4; NovaSeq X, targeting 50,000 reads/nucleus), and Seurat analysis (RStudio).
Results
After quality control, 47,416 nuclei were retained for analysis, identifying 11 distinct cardiac cell populations (Fig. 1A). Metformin attenuated fibrotic remodelling and restored cell-type proportions in experimental autoimmune myocarditis (EAM) hearts, significantly reducing fibroblast, leukocyte, and microvascular endothelial cell fractions. Subclustering of 10,516 fibroblasts yielded four transcriptionally distinct subsets (FB1–FB4; Fig. 1B). Notably, the dominant profibrotic FB1 expanded during EAM but contracted with administration of metformin. In contrast, FB2 exhibited a metabolic profile enriched for mitochondrial ATP synthesis (GO:0046034), oxidative phosphorylation (GO:0006119), cardiac muscle tissue development (GO:0048738), and cardiac muscle contraction (GO:0060048) (Fig. 1C). Metformin preferentially reactivated this protective module in both FB2 and ventricular cardiomyocytes (Fig. 1D), consistent with AMPK-mediated mitochondrial restoration. Untargeted heart tissue metabolomics at day 40 (Fig. 1E) confirmed metformin accumulation alongside the significant upregulation of serotonin, butyrate/isobutyrate, and gamma-glutamylasparagine; TCA suppression (e.g., α-ketoglutarate, malate, succinate), purine depletion (xanthine), aspartate reduction, itaconate elevation, and glycosylceramide upregulation.
Conclusions
Therapeutic metformin mitigates EAM fibrosis by reprogramming fibroblast heterogeneity, restoring AMPK-driven cardioprotective programs, and inducing distinct cardiac metabolomic signatures, notably TCA suppression and itaconate elevation. These findings collectively support the repurposing of metformin for post-inflammatory cardiomyopathies.ESC2026_AbstractFigure