Single-Cell RNA Sequencing Reveals Potential for Endothelial-to-Mesenchymal Transition in Tetralogy of Fallot

Background: Tetralogy of Fallot (TOF) is a very common cyanotic congenital heart disease. Endothelial-tomesenchymal transition (EndoMT) is recognized as a physiological mechanism involved in embryonic heart development and endothelial formation. However, there is still a gap in the reports related to the mechanism of EndoMT development in TOF. Methods: First, transcriptomic data of single cell nuclei of TOF and Donor were obtained based on the Gene Expression Omnibus (GEO) database, and the data were normalized and clustered by dimensionality reduction using the Seurat package. Subsequently, differentially expressed genes (DEGs) between TOF and Donor were screened using the "FindMarkers" function, and the gene sets of interest were enriched. Finally, to characterize the dynamics of EndoMT occurrence in TOF, we performed pseudotime cell trajectory inference as well as utilized SCENIC analysis to probe the gene regulatory networks (GRNs) dominated by transcription factors (TFs) in endothelial cells. Results: We identified a total of six cell clusters based on single-cell nuclear transcriptome data from TOF and Donor. We found that 611 genes with up-regulated expression within TOF showed conversion to mesenchyme. By subdividing endothelial cell subtypes, endothelial cells 2 were shown to be involved in cell adhesion, migration and extracellular matrix processes. Pseudo-time and SCENIC analyses showed that endothelial cell 2 has EndoMT potential. In addition, ERG and TEAD1 are TFs that play key regulatory roles in this subtype, and both of their target genes are also highly expressed in TOF. This demonstrates that ERG and TEAD1 effectively promote the EndoMT process. Conclusion: Our study reveals the molecular mechanisms underlying the development of EndoMT in TOF, which demonstrates that manipulating the endothelial-to-mesenchymal transition may offer unprecedented therapeutic potential for the treatment of TOF.