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Identifying an epicardial cell subset that drives cardiac repair

Zebrafish are an excellent genetic model organism. They are also an excellent model to study heart regeneration as they can repair their hearts after injury without scar formation.  Zebrafish repair heart injuries through a combination of cardiomyocyte proliferation and epicardium support. The epicardium is a layer of mesothelial tissue surrounding the heart that is a source of various cell types and extracellular matrix that facilitates heart repair. During zebrafish cardiac wound repair, epicardial cells are proposed to undergo an epithelial-mesenchymal transition (EMT) from progenitors to differentiated supporting cell types such as mural cells and fibroblasts. 

However, the EMT and specific epicardial progenitor cell type that responds to cardiac damage has not been fully characterized due to a lack of genetic tools for labeling and tracing epicardial subsets. In a recent Nature Communications study, researchers from Weill Cornell Medical College and the Icahn School of Medicine at Mount Sinai used single-cell RNA-sequencing in zebrafish to show that distinct epicardial subsets are involved in cardiac repair. They also identified a transiently activated epicardial progenitor cell subpopulation with an essential regenerative role. Thus, their study informs potential future cardiac injury repair treatments. 

Distinct epicardial subsets exist in the adult zebrafish 

To investigate the distribution of epicardial cells in uninjured adult zebrafish hearts, Xia et al used a nuclear EGFP reporter driven by the regulatory sequence of tcf21, a common epicardial marker. They first investigated cell populations of the ventricular wall using immunofluorescence in this model system. Two distinct epicardial subsets were observed: epithelial epicardial cells stained positive for aldh1a2 and were found in the outermost layer; mesenchymal epicardial cells were aldh1a2-negative and found in the interior compact muscle. This demonstrates heterogeneity within the epicardium. 

modRNAs confirm EMT 

To confirm the EMT during zebrafish heart regeneration, Xia et al created a Cre-modified RNA (modRNA) for transient epithelial expression of mCherry upon injection into the pericardial cavity of a transgenic zebrafish line. Their modRNA incorporated TriLink’s CleanCap® Reagent AG and N1- Methylpseudouridine-5’-Triphosphate. After injection into the epithelial layer, histological analysis showed that the mCherry+ epithelial cells entered the mesenchymal layer, verifying an EMT process. 

Single-cell RNA-seq reveals pro-regenerative subsets 

To interrogate epicardial subsets during heart regeneration, Xia et al performed single cell RNA-seq on live tcf21+ cells isolated from tcf21:nucEGFP zebrafish with partial ventricular amputation. Unbiased clustering of the RNA-seq results identified 12 clusters. Of these clusters, temporal dynamics and gene expression changes labeled clusters 5 and 9 as the main injury-induced pro-regenerative subsets. Notably, cluster 5 was found to contain almost all proliferating cells (identified by top2a, a G2/M phase marker), suggesting this cluster as the major driver to restore the epicardial population after injury. A subset of cluster 5 also exhibited relatively high levels of snail1a, indicative of EMT. 

ptx3 and col12a1b define a transient pro-regenerative epicardial subtype 

Further characterization of cluster 5 showed it was enriched with pro-regenerative ECM and related genes, including pentraxin 3 long a (ptx3a) and collagen type XII alpha 1b (col12a1b). ptx3a and col12a1b were undetectable in the epicardium of uninjured hearts. However, upon injury, both proteins were expressed throughout the tcf21+ epithelial layer. Their expression peaked at 3 days post amputation and was minimal by 14 days post injury. These results were seen in both the single-cell RNA-seq data and in staining data. Additional experiments showed that ptx3a+ col12a1b+ cells gave rise to mural cells, as well as the cells of the epithelial and mesenchymal epicardium, and so this cell population was named the activated epicardial progenitor cell (aEPC) subpopulation. Genetic ablation of this cell population prevented successful heart repair, demonstrating that aEPCs are indispensable for cardiac regeneration. This work identifies a cell population that could be critical for developing future cardiac repair therapies. 


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Article reference: Xia Y, Duca S, Perder B, et al. Activation of a transient progenitor state in the epicardium is required for zebrafish heart regeneration. Nat Commun. 2022 Dec 13;13(1):7704