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

Abstract

Abstract-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 Ca 2ϩ 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 Ca 2ϩ -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. (Circ Res. 2007;101:1155-1163.) Key Words: preconditioning Ⅲ S-nitrosylation Ⅲ cardioprotection I schemic preconditioning (IPC) is a cellular adaptive phenomenon whereby brief episodes of myocardial ischemia and reperfusion (I/R) render the heart resistant to subsequent prolonged ischemic injury. 1 Through activation of a complex cascade of signaling events, IPC has been shown to reduce arrhythmias, infarct size, and postischemic contractile dysfunction. [2][3][4][5] Nitric oxide (NO) has been shown to be an important signal in cardioprotection. 6 -8 In acute IPC, NO has been shown to mediate protection at least in part by activation of guanylyl cyclase, resulting in the production of cyclic guanosine monophosphate (cGMP) and the activation of protein kinase G, which in turn leads to the opening of the mitochondrial K ATP channel. 6 Recently, it has also been shown that protein kinase G results in activation of an endogenous mitochondrial protein kinase C that is involved in activation of the mitochondrial K ATP channel. 9,10 The opening of the mitochondrial K ATP channel is reported to reduce mitochondrial Ca 2ϩ loading 11,12 and also to lead to generation of reactive oxygen species, which activate signaling cascades in a feed-forward manner to elicit cardioprotection. 13,14 In addition to activating cGMP/protein kinase G-dependent signaling pathways, NO can directly modify sulfhydryl residues of proteins through S-nitrosylation, which ha...

Cardioprotective Pathways During Reperfusion: Focus on Redox Signaling and Other Modalities of Cell Signaling

“…This is in line with the observation that strategies to increase NO bioavailability (e.g., by nitrite infusion (157)) in reperfusion are very often protective (e.g., Refs. 169,[178][179][180] and is in line with the concept that when properly regulated ROS=RNS are not purely destructive (52), but rather engage an important cellular signaling function (see below, Redox signaling by ROS and RNS).…”

Redox- and Calmodulin-dependent S-Nitrosylation of the KCNQ1 Channel

“…verted to a nitrosothiol (1-3) and acts as a regulatory mechanism of various classes of proteins, including ion channels, such as the skeletal muscle type ryanodine receptor (ryanodine receptor type 1) channel (4,5), the N-methyl-D-aspartate receptor channel (6,7), the cardiac L-type Ca 2ϩ channel (8), and the cardiac Na ϩ channel (9). We have previously reported that NO derived from endothelial NO synthase activates ion currents through the cardiac slowly activating delayed rectifier potassium channel (I Ks ) composed of the pore-forming ␣-subunit KCNQ1 and the auxiliary ␤-subunit KCNE1.…”

A strategy for direct identification of protein S-nitrosylation sites by quadrupole time-of-flight mass spectrometry

“…They include the use of a SNOspecific antibody to detect in situ protein S-nitrosylation by immunohistochemistry [16], and the use of a biotinswitch method coupled with immunoblotting, 2D-gel electrophoresis, or fluorescence gel electrophoresis to detect changes in protein S-nitrosylation status under different biological conditions [17][18][19]. However, none of these methods identifies the specific S-nitrosylation sites within the proteins.…”