Abstract
Redox modifications play an important role in many cellular processes, including cell death. Ischemic preconditioning (IPC) has been shown to involve redox signaling and protein S-nitrosylation (SNO) is greatly increased following myocardial IPC. Furthermore, inhibition of GSNO-reductase has also been shown to increase protein SNO and is cardioprotective. SNO is thought to provide cardioprotection, in part, by modulating enzyme activity. SNO may also provide cardioprotection by reducing cysteine oxidation during ischemia/reperfusion (IR) injury. In order to test the hypothesis that SNO provides cardioprotection by providing direct protection against cysteine oxidation following IR injury, we developed a method to measure protein oxidation using resin-assisted capture (Ox-RAC). This method is similar to the SNO-RAC method used in the quantification of SNO. Langendorff perfused hearts were subjected to various perfusion protocols (control, IPC, IR, IPC-IR) and homogenized. Each sample was divided into two equal aliquots, and subjected to the SNO-RAC/Ox-RAC procedure in order to simultaneously analyze SNO and oxidation. Using the SNO-RAC protocol, we identified 44 different proteins that were SNO with IPC. With the Ox-RAC protocol, we identified nearly 200 oxidized proteins following IR injury. Interestingly, IPC significantly reduced protein oxidation by more than 30% following ischemia and early reperfusion as determined via label free peptide analysis. Additionally, more than half of the proteins which showed significantly increased SNO with IPC, also showed a significant decrease in cysteine oxidation following IPC-IR at the same site when compared to IR alone. These results support the hypothesis that SNO provides cardioprotection by shielding cysteine residues from ROS-induced oxidation during IR injury. Therefore, the level of protein SNO plays a critical role in IR injury, where ROS production is increased.
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