Exploring molecular markers of atrial fibrillation fibrosis and drug targets of paricalcitol based on network pharmacology, single-cell transcriptomics, and molecular docking

Atrial fibrillation (AF) is closely associated with cardiac fibrosis, where fibroblasts play a central role. However, systematic research into the key regulatory factors to fibroblasts in AF remains lacking. The single-cell dataset (GSE238242) and bulk transcriptomic datasets (GSE41177, GSE79768) were acquired from Gene Expression Omnibus (GEO). Single-cell data underwent quality control, normalization, and clustering using Seurat, and cell communication was analyzed with CellChat. High-dimensional WGCNA (hdWGCNA) was performed on fibroblast populations to identify key modules. Differentially expressed genes (DEGs) from bulk data were intersected with hdWGCNA hub genes to pinpoint biomarkers. Functional enrichment was assessed using the gene set enrichment analysis (GSEA). Drug prediction via DSigDB and molecular docking with AutoDock were conducted to identify potential therapeutics. In vitro validation was performed using TGF‑β1‑treated H9c2 cells. Candidate gene expression was confirmed by qRT‑PCR and Western blot, and functional impact on proliferation was assessed via CCK‑8 assay following siRNA‑mediated knockdown. Single-cell analysis identified ten major cell clusters in AF, with fibroblasts exhibiting active communication via pathways like EFNA5-EPHA3. Integrated hdWGCNA and transcriptomic differential expression analysis pinpointed COL15A1 and SCN7A as fibroblast-associated biomarkers, which were enriched in fibrosis-related pathways. In vitro validation confirmed their upregulation in TGF-β1-induced fibrotic H9c2 cells, and siRNA-mediated knockdown significantly suppressed cell proliferation. Drug prediction and molecular docking indicated that Paricalcitol formed a stable complex with COL15A1 . This study identified COL15A1 and SCN7A as fibroblast-associated molecular markers in AF, highlighting their potential value in understanding fibrotic mechanisms and informing future therapeutic strategies.