Cell-type specific roles of ERBB4 in regenerating neonatal hearts after injury

Abstract Background Scarless regeneration of the neonatal mammalian heart is a remarkable but poorly understood process. Previous studies have shown that paracrine actions of neuregulin-1 (NRG1) are indispensable to stimulate cardiomyocyte proliferation in the injured neonatal heart, and are mediated by activation of the ERBB4 receptor. How NRG1 influences other aspects of neonatal cardiac regeneration, especially in other cell types, remains unclear. Purpose To study temporal evolution of the infarct size during 21 days after myocardial infarction (MI) in neonatal cell-specific Erbb4 knock-out (KO) and wild type (WT) mice. We hypothesized that scarless healing would be altered in mice lacking the ERBB4 receptor in fibroblasts or myeloid cells. Methods Fibroblast-specific Erbb4 KO mice (FB-Erbb4-KO, Erbb4F/+ Col1a2-Cre+) and myeloid-specific Erbb4 KO mice (M-Erbb4-KO, Erbb4F/F-Cre+) were generated. MI was induced in one-day old KO and WT littermates of both sexes by permanent ligation of the LAD. Animals were euthanized at 4, 7, 10, and 21 days post-MI. For infarct scar quantification, transverse sections at four distinct levels between the ligation site and the apex were stained with Masson’s Trichrome. Cardiac tissue was processed for transcriptomic analysis (including qPCR and bulk RNA seq). Results No significant differences in the initial infarct scar size were observed between WT and FB-Erbb4-KO mice 4 days post-MI. By day 7, scar size had decreased ~6-fold in both groups, with a trend toward larger scars in FB-Erbb4-KO mice. At day 10 post-MI, infarct scar size in the FB-Erbb4-KO group was significantly larger compared to WT. At 21 days post-MI, the infarct size had almost completely disappeared in the WT group, but remained clearly present in the FB-Erbb4-KO group. Transcriptome analysis showed significant upregulation in the expression of TNFa and Col1a1 at 10 days after MI. In contrast, in the M-Erbb4-KO mice, the initial infarct scar size at day 4 and 7 was significantly larger compared to WT. During this early post-MI stage, gene expression IL-6 and IL-1β was increased in the KO group. From day 10 post-MI on, scar sizes were the same within the groups, coinciding with upregulation in gene expression of MMP’s in the KO group. Conclusion Fibroblast-specific deletion of ERBB4 impairs post-MI scar resolution in neonatal mice. Although early scar formation is comparable to WT, deficits in scar regression become more pronounced towards day 21 post-MI. These findings indicate that effective neonatal cardiac regeneration requires intact NRG1/ERBB4 signalling in fibroblasts. In contrast, myeloid-specific ERBB4 deletion leads to a larger early infarct scar but does not affect the final resolution phase, indicating that myeloid ERBB4 is dispensable for long-term regenerative healing. Together, these findings identify fibroblast—but not myeloid—ERBB4 signalling as a critical determinant of scarless cardiac repair in the neonatal heart.