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(Circulation Research. 2007;101:1155.)
© 2007 American Heart Association, Inc.

Cellular Biology

Preconditioning Results in S-Nitrosylation of Proteins Involved in Regulation of Mitochondrial Energetics and Calcium Transport

Junhui Sun, Meghan Morgan, Rong-Fong Shen, Charles Steenbergen, Elizabeth Murphy

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.

Correspondence to Dr Elizabeth Murphy, Senior Scientist, NHLBI, NIH, Vascular Medicine Branch, Room 7N112, Building 10, Magnuson CC, 10 Center Dr, Bethesda, MD 20892. E-mail murphy1{at}mail.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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