Nitrite and Ischemic Preconditioning: A Common Mechanism of Protection Dependent on Myoglobin? (original) (raw)
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Proceedings of the National Academy of Sciences, 2008
The nitrite anion is reduced to nitric oxide (NO • ) as oxygen tension decreases. Whereas this pathway modulates hypoxic NO • signaling and mitochondrial respiration and limits myocardial infarction in mammalian species, the pathways to nitrite bioactivation remain uncertain. Studies suggest that hemoglobin and myoglobin may subserve a fundamental physiological function as hypoxia dependent nitrite reductases. Using myoglobin wild-type ( ؉/؉ ) and knockout ( ؊/؊ ) mice, we here test the central role of myoglobin as a functional nitrite reductase that regulates hypoxic NO • generation, controls cellular respiration, and therefore confirms a cytoprotective response to cardiac ischemia-reperfusion (I/R) injury. We find that myoglobin is responsible for nitrite-dependent NO • generation and cardiomyocyte protein iron-nitrosylation. Nitrite reduction to NO • by myoglobin dynamically inhibits cellular respiration and limits reactive oxygen species generation and mitochondrial enzyme oxidative inactivation after I/R injury. In isolated myoglobin ؉/؉ but not in myoglobin ؊/؊ hearts, nitrite treatment resulted in an improved recovery of postischemic left ventricular developed pressure of 29%. In vivo administration of nitrite reduced myocardial infarction by 61% in myoglobin ؉/؉ mice, whereas in myoglobin ؊/؊ mice nitrite had no protective effects. These data support an emerging paradigm that myoglobin and the heme globin family subserve a critical function as an intrinsic nitrite reductase that regulates responses to cellular hypoxia and reoxygenation. myoglobin knockout mice
Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver
Journal of Clinical Investigation, 2005
Nitrite represents a circulating and tissue storage form of NO whose bioactivation is mediated by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin, myoglobin, and tissue heme proteins. Because the rate of NO generation from nitrite is linearly dependent on reductions in oxygen and pH levels, we hypothesized that nitrite would be reduced to NO in ischemic tissue and exert NO-dependent protective effects. Solutions of sodium nitrite were administered in the setting of hepatic and cardiac ischemia-reperfusion (I/R) injury in mice. In hepatic I/R, nitrite exerted profound dose-dependent protective effects on cellular necrosis and apoptosis, with highly significant protective effects observed at near-physiological nitrite concentrations. In myocardial I/R injury, nitrite reduced cardiac infarct size by 67%. Consistent with hypoxia-dependent nitrite bioactivation, nitrite was reduced to NO, S-nitrosothiols, N-nitrosamines, and iron-nitrosylated heme proteins within 1-30 minutes of reperfusion. Nitrite-mediated protection of both the liver and the heart was dependent on NO generation and independent of eNOS and heme oxygenase-1 enzyme activities. These results suggest that nitrite is a biological storage reserve of NO subserving a critical function in tissue protection from ischemic injury. These studies reveal an unexpected and novel therapy for diseases such as myocardial infarction, organ preservation and transplantation, and shock states.
The role of vascular myoglobin in nitrite-mediated blood vessel relaxation
Cardiovascular Research, 2011
This work investigates the role of myoglobin in mediating the vascular relaxation induced by nitrite. Nitrite, previously considered an inert by-product of nitric oxide metabolism, is now believed to play an important role in several areas of pharmacology and physiology. Myoglobin can act as a nitrite reductase in the heart, where it is plentiful, but it is present at a far lower level in vascular smooth muscle-indeed, its existence in the vessel wall is controversial. Haem proteins have been postulated to be important in nitrite-induced vasodilation, but the specific role of myoglobin is unknown. The current study was designed to confirm the presence of myoglobin in murine aortic tissue and to test the hypothesis that vascular wall myoglobin is important for nitrite-induced vasodilation.
Circulating Nitrite Contributes to Cardioprotection by Remote Ischemic Preconditioning
Circulation Research, 2014
Rationale: Remote ischemic preconditioning (rIPC) with short episodes of ischemia/reperfusion (I/R) of an organ remote from the heart is a powerful approach to protect against myocardial I/R injury. The signal transduction pathways for the cross talk between the remote site and the heart remain unclear in detail. Objective: To elucidate the role of circulating nitrite in cardioprotection by rIPC. Methods and Results: Mice were subjected to 4 cycles of no-flow ischemia with subsequent reactive hyperemia within the femoral region and underwent in vivo myocardial I/R (30 minutes/5 minutes or 24 hours). The mouse experiments were conducted using genetic and pharmacological approaches. Shear stress–dependent stimulation of endothelial nitric oxide synthase within the femoral artery during reactive hyperemia yielded substantial release of nitric oxide, subsequently oxidized to nitrite and transferred humorally to the myocardium. Within the heart, reduction of nitrite to nitric oxide by ca...
Cardiovascular Drugs and Therapy, 2004
It is widely accepted that nitric oxide (NO) is a trigger and mediator of late ischaemic preconditioning (IP), however its role in classic (protection observed within 2-4 hours after the IP stimulus) IP is less certain. In addition, the contribution of cardiomyocyte nitric oxide synthase (NOS) activation to NO production in ischaemia is unknown. The aim of this study was therefore to investigate the role of NOS, NO, reactive oxygen species (ROS) and cGMP in IP in an isolated cardiomyocyte model. Methods: Adult rat cardiomyocytes were isolated by collagenase perfusion. Hypoxia was induced by covering pelleted cardiomyocytes with mineral oil. The IP protocol was one 10 min hypoxia/20 min reoxygenation cycle, followed by 2 hr sustained hypoxia. Non-IP cells were subjected to 2 hr sustained hypoxia only. The contribution of NO was investigated by NOS inhibition (L-NAME 50 µM) or by pretreatment of cells with a NO donor (SNP 100 µM), and that of ROS by inclusion of ROS scavengers (MPG and N-acetylcysteine) or pre-treatment with H 2 O 2. End-points were cellular cGMP content and cell viability as assessed by trypan blue exclusion (TBE) and cell morphology. Results: IP significantly improved myocyte viability (54% increase in TBE) at the end of sustained hypoxia. Treatment of cells with L-NAME and ROS scavengers during either the IP protocol or during sustained hypoxia had no effect on cell viability after 2 hr hypoxia, whereas viability of non-IP cells treated with L-NAME during sustained hypoxia improved significantly. cGMP levels were reduced in IP cells. Pre-treatment with SNP and H 2 O 2 did not mimic IP. Conclusions: IP conferred cardioprotection in isolated cardiomyocytes. Protection in this model was not due to activation of cardiomyocyte NOS or ROS production. However, NOS activation induced by sustained hypoxia, appeared to be harmful to non-IP cells.
Role of the anion nitrite in ischemia-reperfusion cytoprotection and therapeutics
Cardiovascular Research, 2007
The anion nitrite (NO 2 − ) constitutes a biochemical reservoir for nitric oxide (NO). Nitrite reduction to NO may be catalyzed by hemoglobin, myoglobin or other metal-containing enzymes and occurs at increasing rates under conditions of physiologic hypoxia or ischemia. A number of laboratories have now demonstrated in animal models the ability of nitrite to provide potent cytoprotection following focal ischemia-reperfusion (IR) injury of the heart, liver, brain, and kidney. While the mechanism of nitrite-mediated cytoprotection remains to be fully characterized, the release of nitritederived NO following IR appears to be central to this mechanism. The evidence of nitrite-mediated cytoprotection against IR injury in multiple animal models opens the door to potential therapeutic opportunities in human disease. Here we review the mechanisms for nitrite formation in blood and tissue, its metabolic equilibrium with NO, nitrate, and NO-modified proteins, the evidence supporting nitrite-mediated cytoprotection, and the potential mechanisms driving cytoprotection, and we explore the opportunities for the therapeutic application of nitrite for human disease.
Free Radical Biology and Medicine, 2009
Ischemia/reperfusion injury Hemoglobin-based oxygen carriers Nitric oxide Peroxynitrite Myocardial apoptosis Free radicals Ischemia/reperfusion (I/R) injury mainly caused by oxidative stress plays a major role in cardiac damage. The extent of the I/R injury is also an important factor that determines the function of a transplanted heart. This study first examined whether hemoglobin-based oxygen carriers (HBOCs) could protect isolated rat heart from I/R injury and then elucidated the underlying mechanism. Using the Langendorff model, isolated Sprague-Dawley rat hearts were arrested and stored at 4°C for 8 h and then reperfused for 2 h. Compared with St. Thomas' solution (STS) and rat self blood in STS, polymerized placenta hemoglobin (PolyPHb) in STS greatly improved heart contraction and decreased infarction size. The extent of myocardial apoptosis was also significantly decreased, which was related to reduced iNOS-derived nitric oxide production, increased protein ratio of Bcl-2/Bax, and reduced caspase-3 activity and cleavage level. Furthermore, PolyPHb in STS did not increase malondialdehyde, peroxynitrite, or mitochondrial hydrogen peroxide formation, but greatly elevated superoxide dismutase activity and preserved mitochondrial ATP synthesis, which served to maintain redox homeostasis in I/R heart. In conclusion, our results demonstrate that HBOCs protected isolated heart from I/R injury and this protection was associated with attenuation of NO-mediated myocardial apoptosis and restoration of the nitroso-redox balance.
Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics
Arteriosclerosis, Thrombosis, and Vascular Biology, 2006
The ability of oxyhemoglobin to inhibit nitric oxide (NO)-dependent activation of soluble guanylate cyclase and vasodilation provided some of the earliest experimental evidence that NO was the endothelium-derived relaxing factor (EDRF). The chemical behavior of this dioxygenation reaction, producing nearly diffusion limited and irreversible NO scavenging, presents a major paradox in vascular biology: The proximity of large amounts of oxyhemoglobin (10 mmol/L) to the endothelium should severely limit paracrine NO diffusion from endothelium to smooth muscle. However, several physical factors are now known to mitigate NO scavenging by red blood cell encapsulated hemoglobin. These include diffusional boundaries around the erythrocyte and a red blood cell free zone along the endothelium in laminar flowing blood, which reduce reaction rates between NO and red cell hemoglobin by 100-to 600-fold. Beyond these mechanisms that reduce NO scavenging by hemoglobin within the red cell, 2 additional mechanisms have been proposed suggesting that NO can be stored in the red blood cell either as nitrite or as an S-nitrosothiol (S-nitroso-hemoglobin). The latter controversial hypothesis contends that NO is stabilized, transported, and delivered by intra-molecular NO group transfers between the heme iron and -93 cysteine to form S-nitrosohemoglobin (SNO-Hb), followed by hypoxia-dependent delivery of the S-nitrosothiol in a process that links regional oxygen deficits with S-nitrosothiol-mediated vasodilation. Although this model has generated a field of research examining the potential endocrine properties of intravascular NO molecules, including S-nitrosothiols, nitrite, and nitrated lipids, a number of mechanistic elements of the theory have been challenged. Recent data from several groups suggest that the nitrite anion (NO 2 Ϫ ) may represent the major intravascular NO storage molecule whose transduction to NO is made possible through an allosterically controlled nitrite reductase reaction with the heme moiety of hemoglobin. As subsequently understood, the hypoxic generation of NO from nitrite is likely to prove important in many aspects of physiology, pathophysiology, and therapeutics. (Arterioscler Thromb Vasc Biol. 2006;26:697-705.) Key Words: nitric oxide Ⅲ nitrite Ⅲ hemoglobin Ⅲ vasodilation Ⅲ red blood cell N itric oxide (NO) is the endothelium-derived relaxing factor that modulates vascular tone by activating soluble guanylyl cyclase (sGC) in smooth muscle. 1-4 It is now appreciated that NO is produced in endothelial cells by the endothelial NO synthase enzyme and participates in many aspects of normal vascular physiology, including tonic vasodilation and inhibition of platelet activation and endothelial adhesion molecule expression. Endothelial dysfunction, characterized by reduced bioavailability of endothelial derived NO, is a central mechanistic feature of coronary artery disease and its risk factors, including diabetes, hypertension, smoking, and obesity. Excessive NO production from inducible NO synthase is a central mechanism of septic shock. Novel therapeutic strategies based on increasing or decreasing native concentrations of NO are undergoing investigation or have already translated into clinical practice.
Impact of mitochondrial nitrite reductase on hemodynamics and myocardial contractility
Scientific Reports
Inorganic nitrite (NO 2 −) can be reduced back to nitric oxide (NO) by several heme proteins called nitrite reductases (NR) which affect both the vascular tonus and hemodynamics. The objective of this study was to clarify the impact of several NRs on the regulation of hemodynamics, for which hemodynamic parameters such as heart rate, blood pressure, arterial stiffness, peripheral resistance and myocardial contractility were characterized by pulse wave analysis. We have demonstrated that NO 2 − reduced to NO in RBCs predominantly influences the heart rate, while myoglobin (Mb) and mitochondriaderived NO regulates arterial stiffness, peripheral resistance and myocardial contractility. Using ex vivo on-line NO-detection, we showed that Mb is the strongest NR occurring in heart, which operates sufficiently only at very low oxygen levels. In contrast, mitochondrial NR operates under both hypoxia and normoxia. Additional experiments with cardiomyocytes suggested that only mitochondria-derived generation of NO regulates cGMP levels mediating the contractility of cardiomyocytes. Our data suggest that a network of NRs is involved in NO 2 − mediated regulation of hemodynamics. Oxygen tension and hematocrit define the activity of specific NRs. Nitric oxide (NO) is a key player in the regulation of hemodynamic parameters such as peripheral resistance 1 , arterial pressure 2,3 , aortic stiffness, myocardial contractility 4,5 , heart rate, cardiac index 6,7 , and as a consequence tissue perfusion 8. Various isoforms of NO synthase (NOS) are the main sources of NO under normoxic conditions. However, NOS become inefficient under hypoxic conditions 9 , due to the fact that this enzyme family is oxygen dependent 10. Nitrite, the second major source of NO, becomes relevant exclusively under hypoxic conditions. Hypoxia has been shown to facilitate the reduction of nitrite to NO, a process catalyzed by so called nitrite reductases (NR) complementary to oxygen-dependent NO synthases 11. For this reason, nitrite administration has already been suggested for therapeutic treatment of diseases associated with impaired oxygen delivery 12,13. Both dietary nitrate and nitrate have also been shown to regulate blood pressure 14,15. Several reports suggest that hemodynamic effects of nitrite occur not only under hypoxic but also under normoxic conditions. In healthy volunteers under normoxic conditions circulating nitrite concentrations have been shown to correlate with systolic and diastolic blood pressure 16,17 and the administration of nitrite resulted in the dilation of conduit arteries 18. These data suggest that nitrite-mediated NO release may not only contribute to improve hemodynamics under hypoxic/ischemic conditions but also to physiological regulation of hemodynamics. There are three major sources of nitrite in the body (i) oxidation of NO formed by different NOS, (ii) absorption from food and (iii) the release from specific drugs, such as nitroglycerin. Hobbs et al. showed that beetroot bread, containing large amount of nitrate, which is reduced to nitrite in the gastrointestinal tract, increased endothelium-independent vasodilation and decreased diastolic blood pressure and arterial stiffness in healthy men 19. Several groups reported that diverse NRs regulate hemodynamics, from which two were found in red blood cells (RBC). Gladwin and co-authors suggested hemoglobin (Hb) in RBC as a key regulator of hemodynamics 20,21 , while Ahluwalia and coworkers presented evidence that blood pressure is regulated predominantly by xanthine oxidoreductase (XOR) which is also localized in RBCs 22. In contrast Rassaf 's group reported that Mb located in blood vessels, not Hb or XOR, is the major NR responsible for hemodynamic effects of nitrite 23,24. In addition, Zweier and coauthors suggested that cytoglobin is the key NR responsible for nitrite effects in aorta ring assays 25. In our previous studies we established the mitochondrial electron transport chain as a potent NR 26 , in particular,