ERBB4 coordinates fibroblast and myeloid cell function during neonatal cardiac regeneration

BACKGROUNDNeuregulin-1 (NRG1) is indispensable for scarless regeneration of the injured neonatal mammalian heart, by stimulating cardiomyocyte proliferation through ERBB4 tyrosine kinase receptors. The role of ERBB4 signaling in fibroblasts and myeloid cells in this process remains poorly understood. HYPOTHESISWe hypothesized that fibroblast- or myeloid-specific Erbb4 deletion impairs neonatal cardiac regeneration following myocardial infarction (MI). METHODSWild type (WT), fibroblast-specific (FB-Erbb4-KO) and myeloid-specific (M-Erbb4-KO) mice underwent LAD ligation at postnatal day 1 (total n=200). Hearts were harvested at 4, 7, 10, and 21 days post-injury (dpi) for histological analyses, bulk RNA sequencing, and targeted qPCR of the apical LV region. Cardiomyocyte cell-cycle activity was quantified by pH3 and Aurora B kinase staining. Scar size was quantified by Massons trichrome staining. RESULTSFB-Erbb4-KO and M-Erbb4-KO showed similar levels of cardiomyocyte cell cycle activity post-MI compared to WT controls. However, there were significant differences in the dynamics of myocardial scar size, capillary density and myocardial gene expression. In FB-Erbb4-KO mice, infarct scar size at 4 dpi was comparable to WT. Although scar size decreased 6-fold in both WT and KO mice by 7 dpi, FB-Erbb4-KO mice retained a significantly larger infarct scar than WT controls. At 10 dpi, KO mice had a larger scar size, lower myocardial capillary density and increased expression of Col1a1 mRNA levels in the ventricular apex. At 21 dpi, regeneration was nearly complete in both WT and FB-Erbb4-KO mice, although the limited residual scar in KO mice was significantly larger compared to WT. In M-Erbb4-KO mice, infarct scar size at 4 dpi was 2-fold larger compared to WT. Infarct size remained larger at 7 dpi and 10 dpi while Mmp2 and Mmp9 mRNA expression was upregulated. At 21 dpi, regeneration was nearly complete in both WT and KO mice, with no significant size difference between the remaining scars. CONCLUSIONFibroblast-specific ERBB4 deletion modestly impaired scar resolution, whereas myeloid-specific ERBB4 deletion resulted in a larger initial injury response but did not impair the ultimate regenerative outcome. These findings indicate that ERBB4 in fibroblasts and myeloid cells coordinates regenerative microenvironment dynamics beyond cardiomyocyte proliferation rather than being an indispensable switch for neonatal cardiac regeneration.