Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Molecular Identifiers and localization of mural epicardial cell clusters in the infarcted heart identified by scRNA-Seq and RNAscope

R. Zalfen1, J. Hesse1, C. Owenier1, Z. Ding1, J. Schrader1

1Institut für Molekulare Kardiologie, Universitätsklinikum Düsseldorf, Düsseldorf;

Background: The epicardium of the adult heart, a quiescent cellular monolayer, becomes activated after myocardial infarction (MI) by upregulating embryonic genes. Epicardial cells form a multi-cell layer termed epicardial stromal cells (EpiSCs) and secrete paracrine factors that can stimulate cardiomyocyte growth and angiogenesis. Therefore, EpiSCs play an important role in cardiac healing/repair. Still, little is known about cell heterogeneity and molecular identifiers within the epicardial layer of the post MI heart. The present study defined the localization of EpiSC populations within the activated epicardium by RNA in situ hybridization, which were identified by scRNA-Seq. Methods: After performing single cell RNA sequencing of EpiSCs, which were isolated 5 days after myocardial infarction (50 min ischemia followed by reperfusion), marker genes for each of the 11 found clusters were selected. RNA in situ hybridization of gene transcripts for the EpiSC populations was performed using the RNAscope 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics, Hayward, USA) on cryo-sections of the mouse heart 5 days after myocardial infarction and sham operation. Results: To characterize the specific location of EpiSC populations in the infarcted heart, RNA in situ hybridization of gene transcripts for selected EpiSC populations was performed.  In the outer part of the epicardium Msln expression (EpiSC-7) could be observed, which was consistent with the epithelial signature of EpiSC-7. Besides, cells which expressed Wt1 (EpiSC-7, 1, 8), Tgfb2 (EpiSC-8) and Cd44 (EpiSC-4) were also localized on the outer layer. Cluster EpiSC-6 could be defined by Sfrp2 expression, as well as Gatm, which was expressed in EpiSC-1 was found homogenously distributed throughout the epicardium. Gatm also labelled activated cardiac fibroblasts (aCSCs) in the myocardium. Similarly, Top2a (EpiSC-9), Dkk3 (EpiSC-10) and Pcsk6 (EpiSC-3) were homogenously dispersed throughout the epicardium and in the case of Pcsk6 also in the myocardium. Interestingly, Ifit3 and Cxcl10 (EpiSC-11) were found heterogeneously distributed in the epicardium. Conclusion: This study demonstrates that major EpiSC populations identified by scRNA-Seq are localized within distinct areas of the activated post-MI epicardium. The spacial distribution of cluster-specific identifiers point to a cellular hierarchy within this highly heterogeneous epicardial cell population.

https://dgk.org/kongress_programme/jt2022/aV32.html