Characterisation of redox post-translational modifications upon acute antioxidant treatment in the diabetic heart, using quantitative mass spectrometry

Overproduction of reactive oxygen species (ROS) is a key determinant in the pathogenesis of Type 2 Diabetes (T2D) and cardiovascular disease (CVD) linked to obesity, hyperglycaemia, and loss of insulin sensitivity. Cellular redox levels directly reduce antioxidant capacity promoting protein alterations, manifesting as cardiac functional deficiencies. Current pre-clinical evidence reveals the potential for antioxidant therapies to reduce or attenuate ROS derived damage and dysfunction and restore redox balance. However, translation to clinical settings has been unsuccessful thus driving further investigation. We aimed to characterise modified cysteine-containing peptides in response to pathological levels of ROS and potential sites protected by acute antioxidant treatment. Using an 8-week rat model of T2D that combines a low-dose streptozotocin (STZ) and high-fat diet to model the T2D phenotype. Hearts are excised for Langendorff isolated ex vivo perfusion, a system enabling antioxidant intervention. This study featured two subsets, the first received 5-minute perfusion to define native redox balance; and the second received 40-minute perfusion with a global antioxidant, N-propionylglycine (MPG; 1mM). To determine how MPG was affording such protection, we performed quantitative redox proteomics using thiol-disulfide exchange to specifically identify and quantify cysteine-containing peptides using tandem mass spectrometry (MS/MS). In the presence of MPG, contractile performance was maintained in T2D myocardium upon ex vivo perfusion, contrary to our previous studies which showed a gradual decline in function. Percentage maintenance of rate pressure product of the T2D cohort increased by 13% with acute MPG treatment during ex vivo perfusion (84% vs 97%). Proteomics results had 23.48% of proteins in our native redox balance hearts change significantly by ANOVA and z-score. Increasing modified cystines were observed within cardiac antioxidant (e.g. Peroxidasin), mitochondrial, and contractile proteins. We have identified MPG treatment rescues reversibly modified cysteines in endogenous antioxidant mechanisms, mitochondrial proteins, and contractile proteins. Within this dataset 30.22% of proteins were observed significantly changing by ANOVA and z-score. Aforementioned peroxidasin was observed decreasing reversible cysteine modification at cysteine 264 upon MPG exposure, the same site reported in the native state as significantly increasing. Conversely, MPG showed little capacity to protect against redox changes to proteins regulating metabolic pathways. Metabolomics confirmed these observations with dysregulation of TCA intermediates unable to be salvaged by MPG-treatment. This suggests that redox changes in metabolic pathways occur during the development of the T2D phenotype (prior to ex vivo), while contractile dysfunction during ex vivo perfusion is specifically driven by redox alterations in the mitochondria and contractile apparatus.