Abstract Text: Macrophages facilitate neonatal murine cardiac regeneration. How macrophages orchestrate regeneration remains vague and the neonatal myeloid response to hypoxia as well as the role of myeloid Hypoxia-Inducible Factors (HIFs) in 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 mitogens in a HIF-dependent manner. To explore differences in regenerative vs reparative responses to injury, single-cell sequencing was performed on cardiac myeloid cells post myocardial infarction (MI) in wildtype neonatal and adult mice. The neonatal macrophage response to hypoxia was investigated by co-culturing supernatant from hypoxia stimulated neonatal bone marrow-derived macrophages from myeloid HIF-1a expressing vs HIF-1a genetically depleted mice with neonatal cardiomyocytes harvested from wildtype mice. Immunofluorescent staining was performed to quantify cardiomyocyte proliferation. Neutralizing antibodies were utilized to identify mitogens. Gene Ontology identified significantly upregulated genes in biological pathways associated with regeneration and HIF-1a stabilization in C1qa+TLF+ macrophages in the neonate but not the adult. Immunofluorescent staining of neonatal cardiomyocytes revealed a significant increase in mitotically active cardiomyocytes exposed to supernatant from hypoxia stimulated HIF-1a+ macrophages compared to HIF-1a- macrophages. IGF-1 was identified as a mitogen secreted from hypoxic macrophages. In summary, we have newly identified enrichment of regenerative and HIF-1a stabilization pathways specific to C1qa+TLF+ neonatal cardiac macrophages after ischemic injury. We also newly conclude hypoxia is a stimulus for neonatal macrophages to secrete HIF-1a dependent mitogenic factors such as IGF-1.
