Distinct transcriptional and functional features of regenerative mouse neonatal cardiac macrophages

Abstract

Background The myocardium of neonatal mice is able to regenerate after myocardial infarction (MI), whereas in adults the formation of scar predominantly occurs following heart injury. Macrophages are involved in the fibrotic response in adult mouse hearts, but also required for successful regeneration in neonates. Recent work has demonstrated that macrophages directly contribute collagen to scar formation following MI. Furthermore, neonatal and adult cardiac macrophages have divergent transcriptional responses to injury. Here, we describe differential transcriptomes and signalling pathways of these functionally distinct neonatal resident cardiac macrophages. Methods Hearts from neonatal P1, P7 and adult CD1 mice (n=3 per group) were digested with collagenase to produce a single cell suspension. Macrophages were isolated by FACS and identified as Ly6G, F4/80+, LyChi/lo cells. Macrophage whole transcriptomes were measured by Illumina RNA-sequencing. Transcript abundance was quantified from raw reads by Salmon and analysis of differentially expressed (DE) genes was carried out with DESeq2. Gene Ontology (GO) enrichment analysis of DE genes was performed with PANTHER. Genes were ranked according to p-value for differential expression, then these ranked genes were used for Gene Set Enrichment Analysis (GSEA) to detect enriched gene sets from the Molecular Signatures Database. Results RNA-sequencing of transcriptomes from neonatal P1, P7 and adult mouse macrophages from hearts highlighted distinct gene expression profiles. The greatest differences were between P1 vs. adult (4,494 differentially expressed (DE) genes at p<0.05) and P7 vs. adult (3,347 DE genes), whereas P1 and P7 macrophages were relatively similar (478 DE genes). A set of 171 genes was found to be DE in P1 vs. P7 and adult macrophages. This P1-specific gene set was highly enriched for GO terms including matrix disassembly (29-fold enrichment, p<0.05) and regulation of chemokine production (12-fold enrichment, p<0.05). GSEA analysis highlighted key functional pathways that were differentially regulated in P1 macrophages, including oxidative phosphorylation and glycolysis, the E2F transcription factors and cell cycle regulators Myc and p53. Conclusions We highlight key genes and pathways distinct to resident neonatal cardiac macrophages to determine the basis for the regenerative capacity of these cells. Interestingly, while genes associated with extracellular matrix were previously shown to be altered after MI in neonatal macrophages, similar differences were also observed here in the basal state of resident neonatal macrophages. We also identified transcriptional and cell cycle regulators linked to the programming and regenerative capacity of these macrophages. The functional differences found in neonatal macrophages might represent potential targets for novel therapeutics. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Novo Nordisk Fonden the Tripartite Immunometabolism Consortium (NNF15CC0018486), British Heart Foundation Centre of Research Excellence Awards (RE/13/1/30181)