Role of S-nitrosylation in regulating cardiac function and disease

我们进行了研究，以检查一氧化氮在心脏保护中的作用。 一氧化氮是缺血预适应（IPC）中重要的信号信使。因此，我们研究了蛋白质S-亚硝基化是否发生在IPC心脏和S-亚硝基谷胱甘肽（GSNO）是否eleventh类似的作用，S-亚硝基化和心脏保护。在无血流缺血和再灌注20分钟之前，以Langendorff模式灌注来自C57 BL/6 J小鼠的心脏，并进行以下条件：（1）对照灌注;（2）IPC;或（3）0.1 mmol/L GSNO处理。与对照组相比，IPC和GSNO可明显改善缺血后左室发展压的恢复，缩小梗死面积。IPC和GSNO均显著增加心肌细胞膜组分中S-亚硝基硫醇含量和L-型Ca ~（2+）通道α 1亚基的S-亚硝基化水平。我们确定了几个候选的S-亚硝基化蛋白质组学分析后的生物素开关方法，包括心肌肌浆网Ca 2 +-ATP酶，α-酮戊二酸脱氢酶，和线粒体F1-ATP酶α 1亚基。GSNO处理后，这些酶的活性以浓度依赖性方式发生变化。我们进一步开发了一种2D DyLight荧光差异凝胶电泳蛋白质组学方法，该方法使用DyLight荧光和改良的生物素开关方法来鉴定S-亚硝基化蛋白质。IPC和GSNO对S-亚硝基化的修饰方式和心肌缺血再灌注损伤的保护作用相似，提示S-亚硝基化可能在心肌保护中起重要作用。 已经提出，SNO可以提供心脏保护，部分通过减少缺血再灌注（IR）损伤期间的半胱氨酸氧化。为了验证这一假设，我们开发了一种新的方法，使用树脂辅助捕获（RAC）来测量氧化，类似于S-亚硝基化定量中使用的SNO-RAC方法。 对Langendorff灌注的心脏进行各种灌注方案（对照、IPC、IR、IPC-IR）并匀浆。将每份样品分成两等份，并进行改良的生物素转换，以同时分析SNO和氧化。使用两个独立的措施，我们确定了44个不同的蛋白质，显示增加的S-亚硝基化与IPC。这些蛋白质中的大多数（40）也表现出减少半胱氨酸氧化后IR。我们确定了53个半胱氨酸残基，从这40个蛋白质，显示在同一网站的S-亚硝基化的氧化减少。这些蛋白质包括甘油醛-3-磷酸脱氢酶、电子转移黄素蛋白和电压依赖性阴离子选择性通道蛋白2。此外，在体外测定中，纯化的α-酮戊二酸脱氢酶的氧化挑战使活性氧（ROS）的产生增加超过60%。有趣的是，预处理与S-亚硝基化剂S-亚硝基谷胱甘肽防止氧化诱导的ROS产生的增加。这些结果表明，SNO通过两种不同的机制产生心脏保护作用：（1）通过直接阻断ROS诱导的半胱氨酸残基氧化，和（2）通过减少ROS的产生。

We performed studies to examine the role of nitric oxide in cardioprotection. 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 alpha1 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, alpha-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase alpha1 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. It has been proposed that SNO may provide cardioprotection, in part, by reducing cysteine oxidation during ischemia-reperfusion (IR) injury. To test this hypothesis, we developed a new method to measure oxidation using resin assisted capture (RAC), similar to the SNO-RAC methods used in the quantification of S-nitrosylation. 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 a modified biotin switch was performed in order to simultaneously analyze SNO and oxidation. Using two independent measures, we identified 44 different proteins which showed increased S-nitrosylation with IPC. The majority (40) of these proteins also showed a decrease in cysteine oxidation following IR. We identified 53 cysteine residues from these 40 proteins that showed a decrease in oxidation at the same site of S-nitrosylation. These proteins included glyceraldehyde-3-phosphate dehydrogenase, electron transfer flavoprotein , and voltage-dependent anion-selective channel protein 2. Further, in an in vitro assay, oxidative challenge of purified alpha-ketoglutarate dehydrogenase increased the production of reactive oxygen species (ROS) by more than 60%. Interestingly, pretreatment with the S-nitrosylating agent S-nitrosoglutathione prevented the oxidation-induced increase in ROS production. These results suggest that SNO yields cardioprotective effects through two distinct mechanisms: (1) by directly blocking against the ROS-induced oxidation of cysteine residues, and (2) by reducing the production of ROS.