Junhui Sun

Deoxymyoglobin is a Nitrite Reductase That Generates Nitric Oxide and Regulates Mitochondrial Respiration

Previous studies have revealed a novel interaction between deoxyhemoglobin and nitrite to generate nitric oxide (NO) in blood. It has been proposed that nitrite acts as an endocrine reservoir of NO and contributes to hypoxic vasodilation and signaling. Here, we characterize the nitrite reductase activity of deoxymyoglobin, which reduces nitrite approximately 36 times faster than deoxyhemoglobin because of its lower heme redox potential. We hypothesize that physiologically this reaction releases NO in proximity to mitochondria and regulates respiration through cytochrome c oxidase. Spectrophotometric and chemiluminescent measurements show that the deoxymyoglobin-nitrite reaction produces NO in a second order reaction that is dependent on deoxymyoglobin, nitrite and proton concentration, with a bimolecular rate constant of 12.4 mol/L-1s-1 (pH 7.4, 37°C). Because the IC50 for NO-dependent inhibition of mitochondrial respiration is approximately 100 nmol/L at physiological oxygen tensions (5 to 10 &mgr;mol/L); we tested whether the myoglobin-dependent reduction of nitrite could inhibit respiration. Indeed, the addition of deoxymyoglobin and nitrite to isolated rat heart and liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. The addition of nitrite to rat heart homogenate containing both myoglobin and mitochondria resulted in NO generation and inhibition of respiration; these effects were blocked by myoglobin oxidation with ferricyanide but not by the xanthine oxidoreductase inhibitor allopurinol. These data expand on the paradigm that heme-globins conserve and generate NO via nitrite reduction along physiological oxygen gradients, and further demonstrate that NO generation from nitrite reduction can escape heme autocapture to regulate NO-dependent signaling.

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

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 &agr;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, &agr;-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase &agr;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.

Hypercontractile Female Hearts Exhibit Increased S-Nitrosylation of the L-Type Ca2+ Channel α1 Subunit and Reduced Ischemia/Reperfusion Injury

Mechanisms underlying gender differences in cardiovascular disease are poorly understood. We found previously that, under hypercontractile conditions, female hearts exhibit significantly less ischemia/reperfusion injury than males. Here we show that male wild-type (WT) mouse hearts pretreated with 10 nmol/L isoproterenol before ischemia exhibited increased injury versus female hearts, but this relative protection in females was absent in eNOS−/− and nNOS−/− hearts. In isoproterenol-treated female versus male hearts, there was also more endothelial NO synthase (eNOS) associated with cardiomyocyte caveolin-3, and more neuronal NOS (nNOS) translocation to caveolin-3 during ischemia/reperfusion. S-nitrosothiol (SNO) formation was increased in isoproterenol-treated ischemic/reperfused hearts in all mouse genotypes, but only in WT mice was SNO content significantly higher in females than males. Using the biotin switch method, we identified the L-type Ca2+ channel &agr;1 subunit as the predominant S-nitrosylated protein in membrane fractions, and following isoproterenol and ischemia/reperfusion male/female differences in SNO were seen only in WT hearts, but not in constitutive NOS−/− genotypes. The isoproterenol-induced increase in L-type Ca2+ current (ICa) was smaller in females versus in males, but NOS blockade increased ICa in females. This gender difference in ICa in isoproterenol-treated myocytes (and abolition on NOS inhibition) was mirrored exactly in Ca2+ transients and SR Ca2+ contents. In conclusion, these data suggest that eNOS and nNOS both play roles in the gender differences observed in ischemia/reperfusion injury under adrenergic stimulation, and also demonstrate increased S-nitrosylation of the L-type Ca2+ channels in female cardiomyocytes.

Protein S-Nitrosylation and Cardioprotection

Nitric oxide (NO) plays an important role in the regulation of cardiovascular function. In addition to the classic NO activation of the cGMP-dependent pathway, NO can also regulate cell function through protein S-nitrosylation, a redox dependent, thiol-based, reversible posttranslational protein modification that involves attachment of an NO moiety to a nucleophilic protein sulfhydryl group. There are emerging data suggesting that S-nitrosylation of proteins plays an important role in cardioprotection. Protein S-nitrosylation not only leads to changes in protein structure and function but also prevents these thiol(s) from further irreversible oxidative/nitrosative modification. A better understanding of the mechanism regulating protein S-nitrosylation and its role in cardioprotection will provide us new therapeutic opportunities and targets for interventions in cardiovascular diseases.

Simultaneous measurement of protein oxidation and S-nitrosylation during preconditioning and ischemia/reperfusion injury with resin-assisted capture.

Rationale: Redox modifications play an important role in many cellular processes, including cell death. Ischemic preconditioning (IPC) has been shown to involve redox signaling. Protein S-nitrosylation (SNO) is increased following myocardial IPC, and SNO is thought to provide cardioprotection, in part, by reducing cysteine oxidation during ischemia/reperfusion (IR) injury. Objective: To test the hypothesis that SNO provides cardioprotection, in part, by shielding against cysteine oxidation following IR injury. Methods and Results: We developed a new method to measure protein oxidation using resin-assisted capture (Ox-RAC), which 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, IPC/reperfusion) and homogenized. Each sample was divided into 2 equal aliquots, and the SNO-RAC/Ox-RAC procedure was performed to simultaneously analyze SNO and oxidation. We identified 31 different SNO proteins with IPC, 27 of which showed increased SNO compared to baseline. Of the proteins that showed significantly increased SNO with IPC, 76% showed decreased oxidation or no oxidation following ischemia and early reperfusion (IPC-IR) at the same site when compared to IR alone; for non-SNO proteins, oxidation was reduced by only 50%. We further demonstrated that IPC-induced protein SNO is quickly reversible. Conclusions: These results support the hypothesis that IPC-induced protein SNO provides cardioprotection by shielding cysteine residues from reactive oxygen species–induced oxidation during IR injury. Therefore, the level of protein SNO plays a critical role in IR injury, where ROS production is increased.

The NAD-dependent deacetylase SIRT2 is required for programmed necrosis

Although initially viewed as unregulated, increasing evidence suggests that cellular necrosis often proceeds through a specific molecular program. In particular, death ligands such as tumour necrosis factor (TNF)-α activate necrosis by stimulating the formation of a complex containing receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3). Relatively little is known regarding how this complex formation is regulated. Here, we show that the NAD-dependent deacetylase SIRT2 binds constitutively to RIP3 and that deletion or knockdown of SIRT2 prevents formation of the RIP1–RIP3 complex in mice. Furthermore, genetic or pharmacological inhibition of SIRT2 blocks cellular necrosis induced by TNF-α. We further demonstrate that RIP1 is a critical target of SIRT2-dependent deacetylation. Using gain- and loss-of-function mutants, we demonstrate that acetylation of RIP1 lysine 530 modulates RIP1–RIP3 complex formation and TNF-α-stimulated necrosis. In the setting of ischaemia-reperfusion injury, RIP1 is deacetylated in a SIRT2-dependent fashion. Furthermore, the hearts of Sirt2−/− mice, or wild-type mice treated with a specific pharmacological inhibitor of SIRT2, show marked protection from ischaemic injury. Taken together, these results implicate SIRT2 as an important regulator of programmed necrosis and indicate that inhibitors of this deacetylase may constitute a novel approach to protect against necrotic injuries, including ischaemic stroke and myocardial infarction.

Estrogen Receptor-β Activation Results in S-Nitrosylation of Proteins Involved in Cardioprotection

Background— It has been shown that the activation of estrogen receptor-&bgr; (ER-&bgr;) plays an important cardioprotective role against ischemia/reperfusion injury. However, the mechanism for this protection is not clear. We hypothesize that estrogen protects by ER-&bgr; activation, which leads to S-nitrosylation (SNO) of key cardioprotective proteins. Methods and Results— We treated ovariectomized C57BL/6J mice with the ER-&bgr; selective agonist 2,2-bis(4-hydroxyphenyl)-proprionitrile (DPN), 17&bgr;-estradiol (E2), or vehicle using Alzet minipumps for 2 weeks. Isolated hearts were Langendorff perfused and subjected to ischemia and reperfusion. Compared with vehicle-treated hearts, DPN- and E2-treated hearts had significantly better postischemic functional recovery and decreased infarct size. To test the specificity of DPN, we treated ER-&bgr;–knockout mice with DPN. However, no cardioprotective effect of DPN was found in ER-&bgr;–knockout mice, indicating that the DPN-induced cardioprotection occurs through the activation of ER-&bgr;. Using DyLight-maleimide fluors and a modified biotin switch method, we used a 2-dimensional DyLight fluorescence difference gel electrophoresis proteomic method to quantify differences in SNO of proteins. DPN- and E2-treated hearts showed an increase in SNO of a number of proteins. Interestingly, many of these proteins also had been shown to have increased SNO in preconditioned hearts. In addition, the DPN-induced cardioprotection and increased SNO were abolished by treatment with a nitric oxide synthase inhibitor. Conclusion— The activation of ER-&bgr; by DPN treatment leads to increased protein SNO and cardioprotection against ischemia/reperfusion injury, suggesting that long-term estrogen exposure protects hearts largely via activation of ER-&bgr; and nitric oxide/SNO signaling.

Characterization of potential S-nitrosylation sites in the myocardium

S-nitrosylation (SNO) is a reversible protein modification that has the ability to alter the activity of target proteins. However, only a small number of SNO proteins have been found in the myocardium, and even fewer specific sites of SNO have been identified. Therefore, this study aims to characterize potential SNO sites in the myocardium. We utilized a modified version of the SNO-resin-assisted capture technique in tandem with mass spectrometry. In brief, a modified biotin switch was performed using perfused mouse heart homogenates incubated with or without the S-nitrosylating agent S-nitrosoglutathione. Our modified SNO-resin-assisted capture protocol identified 116 unique SNO-modified proteins under basal conditions, and these represent the constitutive SNO proteome. These constitutive SNO proteins are likely to be physiologically relevant targets, since nitric oxide has been shown to play an important role in the regulation of normal cardiovascular physiology. Following S-nitrosoglutathione treatment, we identified 951 unique SNO proteins, many of which contained multiple SNO sites. These proteins show the potential for SNO. This study provides novel information regarding the constitutive SNO proteome of the myocardium, as well as potential myocardial SNO sites, and yields additional information on the SNO sites for many key proteins involved in myocardial contraction, metabolism, and cellular signaling.

Estrogen Receptor Activation and Cardioprotection in Ischemia Reperfusion Injury

Premenopausal females have a comparably lower incidence of cardiovascular disease than their male counterparts. Although estrogen and activation of estrogen receptors (ERs) have been found to contribute to female protection, the complex mechanisms involved are unclear. Besides altering gene transcription, estrogen could elicit its cardioprotective effect via ER-mediated nongenomic signaling pathways. In addition to the two classic nuclear ER isoforms, ERα and ERβ, a G-protein coupled ER (GPR30 or GPER) has been found to be expressed in cardiomyocytes and plays an acute cardioprotective role in ischemia reperfusion injury. By using isoform-specific ER knockout mouse models and/or their specific modulators, the mechanisms of the different ERs involved in cardioprotection have been explored. In this review, we will focus on the signaling pathways leading to cardioprotection in ischemia reperfusion injury after ER activation and discuss the possibility and promise of specific ER modulators to treat ischemic heart diseases.

Regulation of myocyte contraction via neuronal nitric oxide synthase: role of ryanodine receptor S-nitrosylation

The sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor, RyR2) has been proposed to be an end target of neuronal nitric oxide synthase (NOS1) signalling. The purpose of this study is to investigate the mechanism of NOS1 modulation of RyR2 activity and the corresponding effect on myocyte function. Myocytes were isolated from NOS1 knockout (NOS1−/−) and wild‐type mice. NOS1−/− myocytes displayed a decreased fractional SR Ca2+ release, NOS1 knockout also led to reduced RyR2 S‐nitrosylation levels. RyR2 channels from NOS1−/− hearts had decreased RyR2 open probability. Additionally, knockout of NOS1 led to a decrease in [3H]ryanodine binding, Ca2+ spark frequency (CaSpF) and a rightward shift in the SR Ca2+ leak/load relationship. Similar effects were observed with acute inhibition of NOS1. These data are indicative of decreased RyR2 activity in myocytes with NOS1 knockout or acute inhibition. Interestingly, the NO donor and nitrosylating agent SNAP reversed the depressed RyR2 open probability, the reduced CaSpF, and caused a leftward shift in the leak/load relationship in NOS1−/− myocytes. SNAP also normalized Ca2+ transient and cell shortening amplitudes and SR fractional release in myocytes with NOS1 knockout or acute inhibition. Furthermore, SNAP was able to normalize the RyR2 S‐nitrosylation levels. These data suggest that NOS1 signalling increases RyR2 activity via S‐nitrosylation, which contributes to the NOS1‐induced positive inotropic effect. Thus, RyR2 is an important end target of NOS1.