Hypoxia Stimulates Neonatal Hypoxia-inducible Factor 1 alpha Dependent Macrophage Secretion Of Cardiomyocyte Mitogens

Background: The neonatal murine heart can regenerate after injury. This regenerative capacity is dependent on macrophages and lost shortly after birth. How macrophages facilitate regeneration remains vague, and the neonatal myeloid response to hypoxia as well as the role of myeloid Hypoxia-Inducible Factors (HIFs) in cardiac regeneration are unknown. We hypothesized that post-ischemic regulation of regenerative programs are confined to neonatal cardiac macrophages and are lost in the adult. We also hypothesized hypoxia is a stimulus for neonatal macrophages to secrete cardiomyocyte mitogens in a HIF dependent manner. Methods: Single-cell sequencing was performed on cardiac myeloid cells post myocardial infarction (MI) in wildtype neonatal and adult mice to explore differences in regenerative vs reparative responses to injury. Neonatal cardiomyocytes harvested from wildtype mice were co-cultured with supernatant from hypoxia stimulated neonatal bone marrow-derived macrophages from myeloid HIF-1 α expressing vs HIF-1 α genetically depleted mice. Immunofluorescent staining with antibodies to Troponin T, Ki67, and DAPI was performed to quantify cardiomyocyte proliferation. Results: High-resolution clustering identified 3 distinct macrophage populations present in neonatal and adult mice post MI. Gene Ontology identified significantly upregulated genes in biological pathways associated with regeneration and HIF-1α stabilization in Timd4+Mrc1+ macrophages in the neonate but not the adult. Immunofluorescent staining of neonatal cardiomyocytes at steady state revealed an average of 6.0% Ki67+ cardiomyocytes compared to 35.4% Ki67+ cardiomyocytes exposed to supernatant from hypoxia stimulated HIF-1 α + macrophages (N=10/group; SEM + 1.96; p value <0.0001) and 10.1% Ki67+ cardiomyocytes exposed to supernatant from hypoxia stimulated HIF-1 α - macrophages (N=10/group; SEM + 1.54; p value 0.0162). Conclusions: We have newly identified enrichment of regenerative and HIF-1α stabilization pathways specific to neonatal resident cardiac macrophages after ischemic injury. We also newly conclude hypoxia is a stimulus for neonatal macrophages to secrete HIF-1 α dependent mitogenic factors that induce cardiomyocyte proliferation.