Arachidonic acid protects against diabetes-induced atrial fibrillation

Fig. 1. AA levels are significantly reduced in diabetic mice. A Metabolomic bubble plot of atrial tissues from diabetic and control mice reveals that the AA metabolic pathway exhibits the most prominent alterations. B Differential metabolite profile (DMP) indicates that the majority of metabolites within atrial metabolic pathways are downregulated in diabetic mice. C ELISA quantification shows that plasma AA levels are significantly decreased in diabetic (T2DM) mice (n = 10) compared to wild-type (WT) controls (n = 10). D ELISA analysis further confirms a marked reduction of AA levels in atrial tissues of T2DM mice (n = 5) relative to WT controls (n = 5). Data are presented as mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student’s t-test for (C) and (D). **P < 0.01, *** P < 0.001

Fig. 2. AA supplementation modulates islet function and metabolic parameters in diabetic mice. (A) Schematic diagram depicting the induction of diabetes and the AA supplementation protocol. _(B-C) ELISA quantification of AA levels in plasma (B) and atrial tissue (C) from DM and AA-treated diabetic (DM + AA) mice (n = 10 per group). (D) Fasting blood glucose levels in each group (n = 10 per group). (E1–E2) Body weight trajectories (E1) and corresponding quantification (E2) over time in each group (n = 10 per group). (F1–F2) Glucose tolerance test results displayed as time-course curves (F1) and area under the curve (AUC) analysis (F2) (n = 10 per group). (G1–G2) Insulin resistance test results presented as time-course curves (G1) and corresponding AUC (G2) (n = 3 per group). (H) Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index in each group (n = 5 per group). (I–L) Serum levels of alanine aminotransferase (ALT) (I), aspartate aminotransferase (AST) (J), total cholesterol (TC) (K), and triglycerides (TG) (L) in each group (n = 5 per group). Data are presented as mean ± SD. Statistical analysis was performed using a two-tailed unpaired t-test for (B, C), and one-way ANOVA…

Fig. 3. AA supplementation alleviates atrial remodeling in diabetic mice. (A1-A2) Representative cardiac echocardiography images and quantification of left atrial area in each group, demonstrating that AA supplementation attenuates atrial enlargement in diabetic mice (n = 4 per group). (B, C) Correlation analyses between left atrial area and AA levels in plasma (B) and atrial tissue (C), showing significant negative correlations. (D1-D3) Representative images and corresponding quantification of Masson’s trichrome staining (scale bar = 100 μm/20 µm) and wheat germ agglutinin (WGA) staining (scale bar = 20 μm) reveal that AA supplementation reduces atrial fibrosis and cardiomyocyte hypertrophy in diabetic mice (n = 4 per group). (E1-E2) Western blot analysis of atrial tissues showing reduced expression of atrial hypertrophy marker atrial natriuretic peptide (ANP) and fibrosis markers COL1A1 and COL3A1 following AA supplementation (n = 4 per group). (F1-F3) Results of intracardiac electrical stimulation indicate that AA supplementation significantly decreases both the incidence and duration of AF inducibility in diabetic mice. Data are presented as mean ± SD. Statistical analysis was…

Fig. 4. AA attenuates oxidative stress injury in the atria of diabetic mice. (A) Metabolomic heatmap of atrial tissues reveals a significant reduction inγ-GC levels in diabetic mice. (B1-B2) Detection of ROS in atrial tissues using the DCFH-DA probe demonstrates increased ROS production in diabetic mice, which is markedly attenuated by AA supplementation (scale bar = 50 μm, n = 4 per group). (C1-C2) JC-1 assay for mitochondrial membrane potential in isolated adult atrial cardiomyocytes indicates severe mitochondrial depolarization in diabetic mice, which is alleviated by AA treatment (scale bar = 100 μm, n = 5 per group). (D1-D2) MitoSOX™ Red staining shows elevated mitochondrial superoxide levels in diabetic mice, which are significantly reduced following AA supplementation (scale bar = 100 μm, n = 5 per group). (E1-E2) Western Blot analysis of atrial tissues shows decreased expression of CAT and SOD2 in diabetic mice, partially restored by AA treatment (n = 4 per group). (F1-F2) Western Blot analysis of IL-1β, IL-6, and tumor necrosis TNF-α reveals elevated expression in diabetic atria, which is attenuated by AA supplementation (n = 4 per group). Data are presented as mean ± SD.…

Fig. 5. AA attenuates atrial oxidative stress in diabetic mice via activation of the Nrf2/γ-GCS pathway. (A) RT–qPCR analysis shows significantly decreased γ-GCS mRNA levels in atrial tissues of diabetic mice, which are markedly restored by AA supplementation (n = 4 per group). (B) Western Blot analysis reveals reduced γ-GCS protein expression in diabetic atria, partially rescued by AA treatment (n = 4 per group). (C) Enzymatic activity assay shows significantly decreased γ-GCS activity in diabetic atrial tissues; although AA supplementation improves activity, the difference is not statistically significant (n = 5 per group). (D) GSH quantification shows a significant reduction in atrial GSH levels in diabetic mice, which is robustly restored by AA supplementation (n = 5 per group). (E) Western Blot analysis indicates decreased Nrf2 expression in diabetic atria, which is upregulated following AA treatment (n = 4 per group). (F) PCR electrophoresis of CUT&Tag assay products confirms direct binding of Nrf2 to the γ-GCS promoter region. (G) Dual-luciferase reporter assay demonstrates significantly increased relative luciferase activity (RLU) in the pGL3-basic–γ-GCS promoter construct, which…

Fig. 6. The protective effects of AA in diabetic miceare abrogated by γ-GCS inhibition. (A) Schematic illustration of the experimental design involving BSO, a γ-GCS inhibitor, used to construct the intervention model. (B1-B2) Body weight measurements in each group show that AA mitigates diabetes-induced weight gain, while BSO treatment partially reverses this effect (n = 5 per group). (C) Fasting blood glucose levels reveal that AA lowers glucose levels in diabetic mice, an effect that is partially blocked by BSO (n = 5 per group). (D1-D2) IPGTT results indicate that AA improves glucose tolerance in diabetic mice, whereas BSO attenuates this improvement (n = 3 per group). (E1-E2) ITT results show that AA ameliorates insulin resistance, an effect partially inhibited by BSO (n = 3 per group). (F) HOMA-IR index analysis confirms that AA reduces insulin resistance in diabetic mice, while BSO administration increases the index (n = 5 per group). (G1–G3) Representative images of Masson’s trichrome staining (scale bar = 100 μm) and WGA staining (scale bar = 100 μm) reveal that AA attenuates atrial fibrosis and cardiomyocyte hypertrophy, effects that are blocked by BSO (n = 5 per group). (H1-H3)…

Fig. 7. γ-GC exerts protective effects in diabetic mice. (A) Schematic representation of the experimental protocol for γ-GC intervention in diabetic mice. (B1-B2) Body weight measurements reveal that γ-GC administration alleviates diabetes-associated weight gain (n = 5 per group). (C) Fasting blood glucose levels are significantly reduced in γ-GC–treated diabetic mice compared to untreated controls (n = 5 per group). (D1-D2) IPGTT results demonstrate that γ-GC partially restores glucose tolerance in diabetic mice (n = 4 per group). (E1-E2) ITT results show that γ-GC improves insulin sensitivity in diabetic mice (n = 4 per group). (F) HOMA-IR index analysis indicates a significant reduction in insulin resistance following γ-GC supplementation (n = 5 per group). (G1-G3) Intracardiac electrophysiological stimulation reveals that γ-GC significantly reduces the inducibility of atrial fibrillation in diabetic mice. Data are presented as mean ± SD. Statistical analysis was performed using a two-tailed unpaired t-test. *P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 8. Decreased AA levels in patients with AF. A ELISA reveals that plasma AA levels are significantly lower in patients with AF compared to those with SR (n = 10 per group). B ELISA further demonstrate significantly reduced AA levels in atrial tissues of AF patients compared to SR controls (n = 10 per group). C, D Correlation analyses show significant negative associations between left atrial diameter and AA levels in plasma (C) and atrial tissue (D). E Schematic summary of the proposed mechanism: AA regulates the expression of γ-GC and GSH, modulates atrial oxidative stress injury via the γ-GC/GSH axis, and thereby exerts a protective effect against atrial remodeling and reduces AF susceptibility in diabetic mice. Data are presented as mean ± SD. Statistical analysis was performed using a two-tailed unpaired t-test. ** P < 0.01