Poster Presentation 26th Annual Lorne Proteomics Symposium 2021

Quantitative mass spectrometry of cysteine redox post translational modifications in the diabetic heart (#98)

Alexander Rookyard 1 2 , Desmond Li 3 , Stuart Cordwell 1 2 4 5 , Melanie White 2 4
  1. School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia
  2. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia
  3. Heart Research Institute , Sydney, NSW, Australia
  4. Discipline of Pathology, School of Medical Sciences, University of Sydney, Sydney, NSW, Australia
  5. Sydney Mass Spectrometry, The University of Sydney, Sydney, NSW, Australia

The global rate of diabetes mellitus is projected to grow to 700 million by 2045, of which type 2 diabetes mellitus (T2DM) accounts for ~90% of incidences. T2DM develops in response to caloric excess and hyperlipidaemia which leads to peripheral insulin resistance, pancreatic insufficiency, and eventual hyperglycaemia. In addition to this, T2DM is an independent risk factor for cardiovascular disease (CVD). Increased oxidative stress via reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been implicated with the aetiology of the diabetic heart, however, the targets of ROS/RNS are poorly defined. Reactive Cys can readily undergo redox post translation modifications (PTMs), however, with the prolonged exposure anticipated in the diabetic setting, irreversible PTM of Cys (Cys-SO2H and Cys-SO3H) are predicted to be a feature. We utilised our pre-clinical rodent model of T2DM (high fat diet and streptozotocin), to excise the hearts for Langendorff ex-vivo perfusion, where a progressive loss of function was observed over time. This was functional depression was prevented when perfused with the antioxidant intervention with N-2-mercaptopropoinylglycine (MPG). Enrichment of irreversible Cys PTMs using strong cation exchange followed by hydrophilic interaction chromatography and identification by LC-MS/MS permitted identification of close to 700 sites, of which 242 were not identified in the control hearts and 90 were unique to the diabetic phenotype. Modified Cys-SO3H were increased, localised to the mitochondria and contractile filament. This correlative increase was further confirmed by quantitation of 160 sites by PRM-MS, of which 20 sites were modified in the combined HFD-STZ setting. Krebs cycle enzyme malate dehydrogenase (MDH) was shown to be highly modified by irreversible oxidation in the diabetic hearts. In the heart, MDH interacts with the malate-aspartate shuttle (MAS), which is the primary pathway responsible for the transfer of reducing equivalents between the cytosol and mitochondria, making it an attractive target for increased redox modifications.