Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted murine heart

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1(+)) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1 alpha and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1(+) cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.