Oral Presentation 28th Annual Lorne Proteomics Symposium 2023

Acute treatment with antioxidant N-propionylglycine attenuates mitochondrial cysteine redox post-translational modifications and restores endogenous antioxidants in the diabetic heart, identified using quantitative mass spectrometry. (#5)

Molly K Talbot 1 2 , Alexander W Rookyard 2 3 , Desmond K Li 3 4 , Stuart J Cordwell 2 3 5 , Melanie Y White 1 2
  1. School of Medical Science, University of Sydney, Camperdown, NSW, Australia
  2. Charles Perkins Centre, University of Sydney, Camperdown, NSW, Australia
  3. School of Life and Environmental Sciences, University of Sydney, Camperdown, NSW, Australia
  4. Heart Research Institute , Sydney , NSW, Australia
  5. Sydney Mass Spectrometry , University of Sydney, Camperdown, NSW, Australia

Excessive production of reactive oxygen species (ROS) is a powerful shared mechanism in the pathogenesis of both cardiovascular disease (CVD) and Type 2 Diabetes (T2D). These disease states are common complications of metabolic syndrome as defined by hyperglycaemia, insulin insensitivity and obesity. The altered metabolic states promote a pathological increase in ROS, driving decreased cellular antioxidant capacity and promoting contractile dysfunction through protein oxidation. Clinically the cumulative effect is witnessed through increased risk of mortality following a single, acute cardiac event in T2D cohorts (Sarwar et al., 2010). Cysteine-containing proteins are at risk of oxidation, with damage limited by protecting these sites with antioxidant therapies. Using a rat model of T2D that combines low-dose streptozotocin (STZ) and high-fat diet, we performed longitudinal in-vivo echocardiography imaging at 0- (pre-diet), 4- (pre-STZ), and 8-week (pre-termination) time points to assess the development of cardiac dysfunction. T2D cohorts showed depressed left ventricular performance, as indicated by the reversal of the E/A ratio. Langendorff ex vivo perfusion was conducted on two groups of T2D hearts, the first perfused for 5 mins to clear the blood and define the degree of native protein oxidation, and the second, in the presence of an acute dose of antioxidant N-propionylglycine (MPG). With the addition of MPG, the ex-vivo contractile function was maintained in the T2D setting. Thiol-disulfide exchange and differential alkylation permitted examination of the T2D redox-proteome to identify potential sites afforded protection by MPG treatment. Redox-sensitive peptides were identified and quantified with isobaric labelling and liquid-chromatography tandem mass spectrometry (MS/MS), showing redox-sensitive sites within the contractile filament and mitochondria. Parallel metabolomics showed MPG rescued endogenous antioxidant glutathione in T2D cardiac tissue. This study demonstrated that restoring the redox imbalance by MPG improves cardiac contractile functionality in T2D conditions, identifying both essential sites of cysteine modifications and increased provision of endogenous antioxidants.

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