Objective  To investigate the molecular mechanisms of immune cells (ICs)- and senescence-related biomarkers in atrial fibrillation (AF).

Methods  In this study, immune cell-related genes (ICRGs) were obtained using weighted gene co-expression network analysis (WGCNA) based on the GSE2240 dataset. Subsequently, the intersection of ICRGs, senescence-related genes (SRGs), and differentially expressed genes (DEGs) derived from differential expression analysis between AF and sinus rhythm (SR) groups was obtained to screen candidate genes. Biomarkers were further identified using the least absolute shrinkage and selection operator (LASSO) algorithm and validated in the GSE115571 dataset. Gene set enrichment analysis (GSEA), GeneMANIA network construction, molecular regulatory network construction, and molecular docking were performed to investigate the molecular mechanisms of the biomarkers in AF. A total of 8 male Sprague-Dawley rats of 87 weeks old and weighing 632-656 g were randomly assigned to the AF group and the sinus rhythm (SR) group (n = 4 per group). An AF rat model was established. The mRNA expression levels of myosin light chain kinase (MYLK) and insulin-like growth factor binding protein 2 (IGFBP2) in rat myocardial tissue were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR).

Results  Two biomarkers, MYLK and IGFBP2, were identified. GSEA revealed that MYLK was significantly enriched in the olfactory transduction pathway, whereas IGFBP2 was significantly enriched in the extracellular matrix-receptor interaction pathway. The GeneMANIA network demonstrated functional similarity between these biomarkers and 20 other genes. In addition, MYLK was regulated by 28 transcription factors (TFs) and 41 microRNAs (miRNAs), whereas IGFBP2 was regulated by 63 TFs and 4 miRNAs, including TAF1 and MIRT020526. Drug prediction analysis indicated that only MYLK had potential interactions with 3 drugs, among which Tozasertib exhibited the strongest binding affinity to MYLK, with a binding energy of －7.8 kcal/mol. RT-qPCR results showed that IGFBP2 mRNA expression was upregulated and MYLK mRNA expression was downregulated in myocardial tissue of rats in the AF group compared with the SR group (both P < 0.05).

Conclusion  In this study, MYLK and IGFBP2 are identified as AF-related biomarkers, and their potential molecular mechanisms are elucidated, providing new theoretical insights into AF research.