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

Abstract

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)-1α 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.

Epicardial Contribution to the Developing and Injured Heart: Exploring the Cellular Composition of the Epicardium

“…Since a tamoxifen-inducible Wt1 reporter line was used to obtain the scRNA seq data, the contribution of Wt1 + -derived cells to the identified populations could be analyzed. In this set-up no contribution of traced cells to Wt1 − populations was observed within the 5 days post-MI ( 119 ). In a subset of the Wt1 + population there was a high expression of Tmbs4x , the gene coding for Tß4 which can induce cardiomyogenesis in vivo and induce cardiac repair when given prior to injury ( 99 , 120 ).…”

“…When comparing transcriptional changes between a model for mitochondrial cardiomyopathy and healthy postnatal mouse hearts the epicardium displayed no heterogeneity, although this may be due to the disease model used ( 164 ). Recently, the epicardial layer and subepicardial mesenchyme (or epicardial stromal cells – epiSCs) were subjected to single cell sequencing 5 days post-MI by Hesse et al ( 119 ). This approach provided a higher resolution by focusing on the epicardial layer specifically.…”

“…The function of the identified subpopulations was not assessed in mice, nor whether these populations resulted from differential progression, as observed during development. Nevertheless, a high degree of heterogeneity was detected in stromal and ECM producing cells ( 119 ) (see below). Strikingly, there was some conservation between these data and functionally heterogenous epicardial subpopulations in zebrafish as described by Weinberger et al ( 119 , 151 ).…”

“…Nevertheless, a high degree of heterogeneity was detected in stromal and ECM producing cells ( 119 ) (see below). Strikingly, there was some conservation between these data and functionally heterogenous epicardial subpopulations in zebrafish as described by Weinberger et al ( 119 , 151 ). Since a tamoxifen-inducible Wt1 reporter line was used to obtain the scRNA seq data, the contribution of Wt1 + -derived cells to the identified populations could be analyzed.…”

“…Therefore, considering the marginal cellular contribution to the injured heart it could be relevant to promote epicardial proliferation and migration via external stimuli. One approach to stimulate cardiac repair via the epicardium is by treating mice with thymosin ß4 (Tß4), a peptide secreted by endothelial cells and the epicardium during development and after injury (117)(118)(119). Importantly, treatment post-MI resulted in an increase in proliferating EPDCs and neovascularization of the injured heart (120).…”