Abstract 18108: Critical Cysteine Oxidation is Reduced During Ischemia-Reperfusion Injury via S-nitrosothiol Formation
Myocardial ischemic preconditioning (IPC) yields an increase in protein S-nitrosothiol (SNO) formation. Furthermore, inhibition of GSNO-reductase has been shown to increase protein SNO and is cardioprotective. SNO is a reversible protein modification that has been proposed to reduce cysteine oxidation during ischemia-reperfusion (IR) injury. To test this hypothesis, we utilized two-dimensional DyLight fluorescence difference gel electrophoresis (2D DIGE) with mass spectrometry to concomitantly examine protein SNO and oxidation in a model of IR injury. A modified biotin switch was performed using homogenates from hearts subjected to IR (60′ perfusion, 20′ ischemia, 5′ reperfusion) and IPC-IR (20′ perfusion, 4 cycles IPC, 20′ ischemia, 5′ reperfusion). Each sample was divided into two equal aliquots in order to analyze SNO and oxidation. After blocking free thiols with NEM, SNO thiols were reduced with ascorbic acid (AA) and labeled with DyLight maleimide for detection. For oxidation, free and AA-reduced thiols were blocked with NEM, and the remaining oxidized thiols were reduced with DTT and labeled. Four samples (IRSNO, IROx, IR-IPCSNO, IR-IPCOx), each labeled with a unique DyLight fluor, were then combined and run via 2D DIGE. Analysis revealed an increase in protein SNO with IPC-IR, compared to IR alone. SNO proteins included GAPDH, electron transfer flavoprotein, creatine kinase, 3-ketoacyl-CoA thiolase, malate dehydrogenase and α-ketoglutarate dehydrogenase (α-KGDH). Consistent with the hypothesis, the majority of these SNO proteins showed decreased cysteine oxidation. Interestingly, α-KGDH produced 83% less reactive oxygen species (ROS) when pretreated with the S-nitrosylating agent GSNO in an in vitro assay using purified enzyme (1.9±0.6 vs. 11.0±1.5 nmol/L/min). Further experiments showed that oxidation of α-KGDH with H2O2 in the absence of GSNO increased ROS production by 66% (18.2±1.4 nmol/L/min), whereas GSNO pretreatment consistently reduced ROS production by α-KGDH despite oxidation with H2O2 (2.7±0.4 nmol/L/min). Therefore, these results suggest that SNO provides cardioprotection in a two-fold manner: 1) SNO blocks critical cysteine residues, preventing further oxidation, and 2) SNO decreases the production of ROS.
- © 2010 by American Heart Association, Inc.