Dynamic transition of cardiac fibroblasts contributes critically to cardiac regeneration

Background Cardiovascular disease is the leading cause of death globally, with myocardial infarction (MI) often causing heart failure due to irreversible cardiomyocyte loss. While adult mammalian hearts have limited regenerative capacity, species like zebrafish, newts, and neonatal mice (within 7 days after birth)—can regenerate heart tissue through cardiomyocyte proliferation. Cardiac fibroblasts (CF), critical for ECM production, tissue repair, and angiogenesis, have been shown to be essential for cardiac regeneration in zebrafish, though their role in mammals remains unknown. This study aims to investigate the roles of CF in mammalian myocardial regeneration. Methods & Results We investigated the role of CF in heart regeneration using neonatal Col1a2-CreERT2; Rosa26RCAG-tdTomato;iDTR mice. By ablating Col1a2-expressing CF and subjecting the mice to apical resection (AR) and myocardial infarction (MI) at post-natal day 5, we observed impaired cardiac regeneration. CF ablation led to impaired cardiac regeneration and a paradoxical increase in myocardial fibrosis, as evidenced by a reduction in cardiomyocyte proliferation and excessive scar formation on Masson’s trichrome staining. Further analyses showed that CF depletion adversely affected survival, with mortality rates of 42% after AR and 55% after MI, compared to near 0% in controls. Using a lineage tracing approach, we have excluded the possibility that CF could transdifferentiate directly into cardiomyocytes in the neonatal mouse heart. To explore the potential mechanisms by which CF regulates cardiac regeneration, we performed bulk RNA sequencing on CF isolated from neonatal and adult mouse hearts. Differential expression analysis revealed that neonatal cardiac fibroblasts expressed higher levels of genes involved in cell cycle progression and cardiogenesis, highlighting their potential functions in promoting cardiac regeneration. Further insights were gained from single-cell RNA sequencing analysis on mouse cardiac cells (GSE153480), which revealed that in regenerative neonatal mouse hearts, CF populations transitioned from predominantly the FB4 (Col1a2+ S100a8+) cluster on day one post-injury to the FB3 (Col1a2+ CD36+) cluster by day three—populations that were scarcely present in non-regenerative hearts. These findings highlight a potential critical role for FB3 and FB4 in cardiac regenerative process. Conclusion Our findings demonstrates that CF, particularly those expressing Col1a2, are essential for heart regeneration in neonatal mice. Their ablation severely impairs regeneration and increases scarring in the neonatal mouse heart. While bulk RNA sequencing demonstrates enhanced cell cycle and cardiogenesis gene expression in neonatal CF, scRNA-Seq reveals dynamic shifts from FB4 to FB3 populations during regeneration, highlighting potential novel therapeutic targets for heart failure.