Abstract 13440: S-Nitrosylation Occupancy is Increased With Ischemic Preconditioning
Thiol-based redox modifications play an important role in many different cellular processes and are vital for proper myocardial function. Myocardial ischemic preconditioning (IPC) is a redox sensitive process that provides cardioprotective effects to a large extent, via oxidative signaling. Protein S-nitrosylation (SNO) is a reversible, thiol-based modification that increases with IPC and provides protective effects by modulating enzyme activity and reducing the irreversible cysteine oxidation that occurs with ischemia-reperfusion (IR) injury. In order to fully examine the role of SNO in cardioprotection, a measure of cysteine occupancy is necessary, as the occupancy for a given cysteine residue should correlate with a change in activity and/or protection. Therefore, we developed a novel technique using cysteine-reactive Tandem Mass Tags (cysTMT) to determine SNO occupancy. Following differential cysTMT labeling, proteins were subjected to tryptic digestion, and the resulting peptides were analyzed via liquid chromatography-tandem mass spectrometry. Our results indicate that the occupancy for IPC-induced protein SNO ranges from 1% to more than 48%. The majority of proteins showed SNO occupancy in the range of 1-10%. However, there was a subset of proteins which showed a large change in SNO occupancy with IPC compared to baseline. This subset included α-ketoglutarate dehydrogenase (C477, not detected at baseline to 31.1%), annexin A6 (C114, 6.9 to 21.3%), hexokinse-2 (Cys256, not detected at baseline to 48%), the mitochondrial phosphate carrier protein (C131, 2.1 to 27.4%), and short-chain specific acyl-CoA dehydrogenase (C109, 4.5 to 32.7%). Large changes in occupancy were also observed for many proteins upon treatment with the cardioprotective/S-nitrosylating agent GSNO, including aconitate hydratase (C385, 4.9 to 20.3%), the adenine nucleotide translocase (C129, 4.3 to 22.7%), creatine kinase (C397, 3.3 to 33.6%), fructose bisphosphate aldolase A (C290, 2.5 to 37.9%), and the sarcoplasmic reticulum Ca2+ ATPase (C560, 4.1 to 21.4%). These results suggest that both IPC and GSNO increase SNO occupancy, and this increase in SNO serves to modulate enzyme activity and protect against irreversible cysteine oxidation.
- © 2011 by American Heart Association, Inc.