Published online before print October 4, 2007, doi: 10.1161/CIRCRESAHA.107.155879
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Submitted on May 9, 2007
Revised on August 29, 2007
Accepted on September 20, 2007
Preconditioning Results in S-Nitrosylation of Proteins Involved in Regulation of Mitochondrial Energetics and Calcium Transport
Junhui Sun ;
From the Vascular Medicine Branch (J.S., E.M.), and Proteomics Core Facility (M.M., R.-F.S.), National Heart Lung and Blood Institute, NIH, Bethesda; and Department of Pathology (C.S.), Johns Hopkins University, Baltimore, Md.
* To whom correspondence should be addressed. E-mail: murphy1{at}niehs.nih.gov.
Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC). Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion; (2) IPC; or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca2+ channel 
1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca2+-ATPase, -ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase 1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart.
Key words: preconditioning • S-nitrosylation • cardioprotection
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