Site-specific promoter hypermethylation of GPX4 in heart failure with reduced ejection fraction (HFrEF): nonlinear dose-response with hepatic and coagulation markers.
Ma L, Li X, et al. • Molecular biology reports • 2026
GPX4 promoter methylation, particularly at site GPX4_FA3_CpG_5, is significantly elevated in HFrEF patients compared to healthy controls and exhibits nonlinear dose-response relationships with hepatic and coagulation markers, supporting GPX4 promoter methylation as a potential epigenetic marker for HFrEF.
Key Findings
Results
Methylation at GPX4_FA3_CpG_5 was significantly higher in HFrEF patients than in healthy controls.
The study enrolled 125 patients with HFrEF (LVEF < 50%) and 350 healthy controls.
The difference in methylation at GPX4_FA3_CpG_5 between groups was statistically significant (P = 0.019).
Methylation levels were quantified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).
The GPX4 promoter regions analyzed were designated FA3 and FA20, each containing multiple CpG sites.
Results
Methylation at GPX4_FA20_CpG_6 differed significantly between NYHA heart failure severity classes.
Stratified analysis by NYHA functional class revealed increased methylation at GPX4_FA20_CpG_6 in patients with class I/II heart failure compared to those with class III/IV disease (P = 0.017).
This suggests an inverse relationship between FA20_CpG_6 methylation level and heart failure severity as classified by NYHA.
The finding indicates site-specific methylation patterns may vary with disease stage.
Results
Restricted cubic spline modeling identified nonlinear, U-shaped or multiphasic associations between GPX4_FA3_CpG_5 methylation and several clinical indicators.
Nonlinear dose-response relationships were identified between GPX4_FA3_CpG_5 methylation and total bile acid (TBA), fibrinogen (FG), fibrin degradation products (FDP), and eosinophil percentage (EO%).
The relationships were described as 'U-shaped or multiphasic,' indicating complex, non-monotonic associations rather than simple linear correlations.
RCS (restricted cubic spline) models were applied specifically to assess potential nonlinear relationships between methylation levels and clinical variables.
Results
No significant associations were detected between GPX4 methylation and blood lipid or routine hematologic parameters.
Despite associations with coagulation and hepatic markers, GPX4 promoter methylation did not significantly correlate with blood lipid profiles.
Routine hematologic parameters were also not significantly associated with GPX4 methylation levels.
Statistical analyses included t-tests, Mann-Whitney U tests, and χ² tests for group comparisons.
Discussion
The authors propose that site-specific GPX4 promoter methylation may reflect dysregulation of GPX4-mediated antioxidant and ferroptosis-related pathways in cardiac dysfunction.
GPX4 encodes glutathione peroxidase 4, an enzyme involved in antioxidant defense and regulation of ferroptosis.
The authors suggest that epigenetic silencing via promoter methylation may impair GPX4 expression, contributing to oxidative stress and ferroptotic cell death in heart failure.
The nonlinear associations with hepatic markers (TBA) and coagulation markers (FG, FDP) are interpreted as indicating 'complex metabolic interplay' in HFrEF.
The authors conclude that 'these findings support GPX4 promoter methylation as a potential epigenetic marker for HFrEF.'
Methods
The study applied MALDI-TOF mass spectrometry to quantify CpG-specific methylation in peripheral blood genomic DNA from HFrEF patients and controls.
Peripheral blood genomic DNA was extracted from all 475 participants (125 HFrEF, 350 controls).
MALDI-TOF MS was used to quantify methylation levels at individual CpG sites within the GPX4 promoter FA3 and FA20 regions.
HFrEF was defined as LVEF < 50%.
Multiple statistical approaches were employed: t-tests, Mann-Whitney U tests, χ² tests, and restricted cubic spline (RCS) models.
What This Means
This research suggests that chemical modifications to the DNA that controls production of an important antioxidant protein called GPX4 may play a role in heart failure. Specifically, the study found that a type of modification called methylation was higher at a specific location in the GPX4 gene's control region (called FA3_CpG_5) in patients with heart failure with reduced pumping ability (HFrEF) compared to healthy individuals. Additionally, methylation at a different location (FA20_CpG_6) was actually higher in patients with milder heart failure than in those with more severe disease, suggesting these changes behave differently depending on where they occur and how advanced the disease is.
The study also found that the level of methylation at the FA3_CpG_5 site had complex, non-straight-line relationships with markers of liver function (total bile acids) and blood clotting (fibrinogen and fibrin degradation products), as well as a type of immune cell (eosinophils). These U-shaped or multi-phase relationships suggest that the interaction between GPX4 methylation and these body systems is not simple. Notably, GPX4 is a protein that protects cells from a form of cell death driven by oxidative damage called ferroptosis, and the authors propose that methylation-induced suppression of GPX4 may contribute to heart muscle cell damage.
This research matters because it points to a potentially measurable epigenetic marker in the blood that could help characterize heart failure biology. If GPX4 promoter methylation is confirmed in larger studies as a reliable marker, it could eventually inform understanding of how oxidative stress and ferroptosis contribute to heart failure progression, and possibly point toward new therapeutic targets related to antioxidant pathway regulation.
Ma L, Li X, Zhang D, Song L, Guo Y, Wang L, et al.. (2026). Site-specific promoter hypermethylation of GPX4 in heart failure with reduced ejection fraction (HFrEF): nonlinear dose-response with hepatic and coagulation markers.. Molecular biology reports. https://doi.org/10.1007/s11033-026-12328-2