Abstract Body: Rationale: Type-2 diabetes (T2D) greatly increases the risk for developing heart failure. Recent evidence indicates that the cardiac expression and function of the sodium-glucose cotransporter 1 (SGLT1) is elevated in disease states, including T2D, leading to structural and functional remodeling of the heart. The mechanisms underlying SGLT1 activation in the diabetic heart are currently unknown.
Objective: To investigate if SGLT1 undergoes the O-linked attachment of β-N-acetylglucosamine (O-GlcNAcylation), a post-translational modification that is exacerbated in T2D, and its effect on SGLT1 activity in diabetic hearts.
Methods/Results: SGLT1 co-immunoprecipitates with O-GlcNAc in heart homogenates and HL-1 cardiomyocyte lysates. Enhancing global protein O-GlcNAcylation in HL-1 cells via si-RNA-mediated silencing of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, resulted in increased amounts of O-GlcNAc that precipitate with SGLT1. SGLT1 function, measured as the SGLT1-mediated Na⁺ influx, was significantly increased in HL-1 cells with silenced OGA. These data suggest that SGLT1 undergoes O-GlcNAcylation and this modification results in SGLT1 activation. Using rats that express human amylin in the pancreatic β-cells (HIP rats) as a model of late-onset T2D and their wild-type littermates as controls, we found that a larger fraction of SGLT1 co-immunoprecipitates with O-GlcNAc in hearts from T2D rats compared to controls. The SGLT1-mediated Na⁺ influx was larger in myocytes from T2D vs. control rats. Increasing total O-GlcNAcylation with Thiamet-G in control myocytes resulted in larger Na⁺ influx. In reverse experiments, inhibition of O-GlcNAcylation with 6-diazo-5-oxo-L-norleucine crystalline reduced SGLT1-mediated Na⁺ uptake in myocytes from T2D rats. Inhibition of O-GlcNAcylation in T2D myocytes reduced Ca²⁺ spark frequency and this effect was significantly less pronounced with SGLT1 blocked. These results suggest that O-GlcNAcylation affects myocyte Na⁺ regulation by activating SGLT1, which contributes to myocyte Na⁺ overload and its downstream effects on sarcoplasmic reticulum Ca²⁺ leak in T2D.
Conclusion: Exacerbated O-GlcNAcylation underlies SGLT1 activation in diabetic hearts. Preventing O-GlcNAcylation may limit myocyte Na⁺ overload and the frequency of pro-arrhythmogenic Ca²⁺ sparks in myocytes from T2D rats.