Epicardial Complement C3 Activation in Neonatal Cardiac Regeneration

The capacity for some neonatal mammals to regenerate damaged myocardium has a known dependency on immune signaling. By utilizing single cell RNA sequencing of the neonatal mouse heart two days following apical resection or a sham thoracotomy right after birth, we identify complement C3 signaling along an epicardial-to-fetal macrophage axis as playing a strong role in post-injury angiogenesis. Specifically, we identify differences in macrophage populations between surgical conditions, with major expansions in pro-angiogenic signaling fetal liver-derived macrophages post-injury. These results suggest a role for primordial macrophage lineages in the early injury response in the neonatal heart, which may in turn promote better recovery or regeneration. Overall design: Neonatal C57/BL6 mice of mixed-sex littermates underwent surgery at days P0-P1. 4 post-surgery (2 days) hearts per condition (sham and resected) were collected, perfused with PBS, and pooled. Ventricles were minced and 1200µL of dissociation media (RPMI 1640 media (Gibco 1187503), 0.04U/mL Dispase II in DMEM/F-12 (STEMCELL Technologies 07923), 2µg/mL Collagenase D (Roche 11088858001), 32U/mL DNase I (NEB M0303S)) were added. Samples were incubated (30min, 37°C) before deactivating the enzymes with cold 5% FBS in PBS. Samples were filtered through a 40µm cell strainer (Pluriselect 43-10040), centrifuged (300g, 5min, 4°C), resuspended in 2mL ACK lysing buffer (Gibco A10492-01), and incubated (5min, 37°C). Samples were centrifuged, washed (5% FBS in PBS), and recentrifuged. Live cells were enriched through dead cell removal (Miltenyi Biotech 130-090-101) and miniMACS separators. The suspension was washed and pelleted (400g, 5min, 4°C). Pellets were resuspended in 0.04% BSA in PBS at 1000 cells/µL and viability was determined through the LUNA-FL (Logos Biosystems). Cells were processed using the 10x Genomics Chromium pipeline and sequenced on an Illumina NextSeq 2000. Feature matrices were generated from raw FASTQ files via the Cell Ranger pipeline.