Site-specific promoter hypermethylation of GPX4 in heart failure with reduced ejection fraction (HFrEF): nonlinear dose-response with hepatic and coagulation markers

Objective Heart failure with reduced ejection fraction (HFrEF) remains a major global health burden characterized by progressive cardiac dysfunction and high mortality. Emerging evidence suggests that epigenetic modifications, especially DNA methylation of antioxidant-related genes, may influence the molecular mechanisms underlying heart failure. This study aimed to investigate the methylation status of the glutathione peroxidase 4 (GPX4) promoter region in patients with HFrEF and to explore its associations with clinical and biochemical indicators. Methods A total of 125 patients with HFrEF (LVEF < 50%) and 350 healthy controls were enrolled. Peripheral blood genomic DNA was extracted, and CpG methylation levels within the GPX4 promoter (FA3 and FA20 regions) were quantified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Statistical analyses were performed using t-tests, Mann–Whitney U tests, and χ² tests. Restricted cubic spline (RCS) models were then applied to assess potential nonlinear dose–response relationships between methylation levels and clinical variables. Results Methylation at GPX4_FA3_CpG_5 was significantly higher in HFrEF patients than in healthy controls (P = 0.019). Stratified analysis by NYHA class revealed increased methylation at GPX4_FA20_CpG_6 in patients with class I/II heart failure compared with those with class III/IV disease (P = 0.017). RCS modeling identified nonlinear U-shaped or multiphasic associations between GPX4_FA3_CpG_5 methylation and several clinical indicators, including total bile acid (TBA), fibrinogen (FG), fibrin degradation products (FDP), and eosinophil percentage (EO%). No significant associations were detected between GPX4 methylation and blood lipid or routine hematologic parameters. Conclusion In HFrEF patients, GPX4 promoter methylation is closely associated with clinical and biochemical alterations. Site-specific changes suggest that dysregulation of the GPX4-mediated antioxidant network and ferroptosis-related pathways may contribute to cardiac dysfunction. The nonlinear dose–response relationships with hepatic and coagulation markers indicate complex metabolic interplay. These findings support GPX4 promoter methylation as a potential epigenetic marker for HFrEF.