Platelet bioenergetic screen in sickle cell patients reveals mitochondrial complex V inhibition, which contributes to platelet activation.

Bioenergetic dysfunction, although central to the pathogenesis of numerous diseases, remains uncharacterized in many patient populations because of the invasiveness of obtaining tissue for mitochondrial studies. Although platelets are an accessible source of mitochondria, the role of bioenergetics in regulating platelet function remains unclear. Herein, we validate extracellular flux analysis in human platelets and use this technique to screen for mitochondrial dysfunction in sickle cell disease (SCD) patients, a population with aberrant platelet activation of an unknown mechanism and in which mitochondrial function has never been assessed. We identify a bioenergetic alteration in SCD patients characterized by deficient complex V activity, leading to decreased mitochondrial respiration, membrane hyperpolarization, and augmented oxidant production compared with healthy subjects. This dysfunction correlates with platelet activation and hemolysis in vivo and can be recapitulated in vitro by exposing healthy platelets to hemoglobin or a complex V inhibitor. Further, reproduction of this dysfunction in vitro activates healthy platelets, an effect prevented by attenuation of mitochondrial hyperpolarization or by scavenging mitochondrial oxidants. These data identify bioenergetic dysfunction in SCD patients for the first time and establish mitochondrial hyperpolarization and oxidant generation as potential pathogenic mechanism in SCD as well as a modulator of healthy platelet function.

Human neuroglobin functions as a redox-regulated nitrite reductase.

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

Nitrite activates protein kinase A in normoxia to mediate mitochondrial fusion and tolerance to ischaemia/reperfusion.

AIMS: Nitrite (NO2(-)), a dietary constituent and nitric oxide (NO) oxidation product, mediates cardioprotection after ischaemia/reperfusion (I/R) in a number of animal models when administered during ischaemia or as a pre-conditioning agent hours to days prior to the ischaemic episode. When present during ischaemia, the reduction of nitrite to bioactive NO by deoxygenated haem proteins accounts for its protective effects. However, the mechanism of nitrite-induced pre-conditioning, a normoxic response which does not appear to require reduction of nitrite to NO, remains unexplored. METHODS AND RESULTS: Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocytes, we demonstrate that a transient (30 min) normoxic nitrite treatment significantly attenuates cell death after a hypoxic episode initiated 1 h later. Mechanistically, this protection depends on the activation of protein kinase A, which phosphorylates and inhibits dynamin-related protein 1, the predominant regulator of mitochondrial fission. This results morphologically, in the promotion of mitochondrial fusion and functionally in the augmentation of mitochondrial membrane potential and superoxide production. We identify AMP kinase (AMPK) as a downstream target of the mitochondrial reactive oxygen species (ROS) generated and show that its oxidation and subsequent phosphorylation are essential for cytoprotection, as scavenging of ROS prevents AMPK activation and inhibits nitrite-mediated protection after H/R. The protein kinase A-dependent protection mediated by nitrite is reproduced in an intact isolated rat heart model of I/R. CONCLUSIONS: These data are the first to demonstrate nitrite-dependent normoxic modulation of both mitochondrial morphology and function and reveal a novel signalling pathway responsible for nitrite-mediated cardioprotection.

Oxygen regulates tissue nitrite metabolism.

AIMS: Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO(2)(-)) is now accepted as an endocrine reservoir of NO that elicits biological responses in major organs. While it is known that tissue nitrite is derived from NO oxidation and the diet, little is known about how nitrite is metabolized by tissue, particularly at intermediate oxygen tensions. We investigated the rates and mechanisms of tissue nitrite metabolism over a range of oxygen concentrations. RESULTS: We show that the rate of nitrite consumption differs in each organ. Further, oxygen regulates the rate and products of nitrite metabolism. In anoxia, nitrite is reduced to NO, with significant formation of iron-nitrosyl proteins and S-nitrosothiols. This hypoxic nitrite metabolism is mediated by different nitrite reductases in each tissue. In contrast, low concentrations (∼3.5 µM) of oxygen increase the rate of nitrite consumption by shifting nitrite metabolism to oxidative pathways, yielding nitrate. While cytochrome P(450) and myoglobin contribute in the liver and heart, respectively, mitochondrial cytochrome c oxidase plays a significant role in nitrite oxidation, which is inhibited by cyanide. Using cyanide to prevent artifactual nitrite decay, we measure metabolism of oral and intraperitoneally administered nitrite in mice. INNOVATION: These data provide insight into the fate of nitrite in tissue, the enzymes involved in nitrite metabolism, and the role of oxygen in regulating these processes. CONCLUSION: We demonstrate that even at low concentrations, oxygen is a potent regulator of the rate and products of tissue nitrite metabolism.

Nitrite activates AMP kinase to stimulate mitochondrial biogenesis independent of soluble guanylate cyclase.

Nitrite, a dietary constituent and endogenous signaling molecule, mediates a number of physiological responses including modulation of ischemia/reperfusion injury, glucose tolerance, and vascular remodeling. Although the exact molecular mechanisms underlying nitrite's actions are unknown, the current paradigm suggests that these effects depend on the hypoxic reduction of nitrite to nitric oxide (NO). Mitochondrial biogenesis is a fundamental mechanism of cellular adaptation and repair. However, the effect of nitrite on mitochondrial number has not been explored. Herein, we report that nitrite stimulates mitochondrial biogenesis through a mechanism distinct from that of NO. We demonstrate that nitrite significantly increases cellular mitochondrial number by augmenting the activity of adenylate kinase, resulting in AMP kinase phosphorylation, downstream activation of sirtuin-1, and deacetylation of PGC1α, the master regulator of mitochondrial biogenesis. Unlike NO, nitrite-mediated biogenesis does not require the activation of soluble guanylate cyclase and results in the synthesis of more functionally efficient mitochondria. Further, we provide evidence that nitrite mediates biogenesis in vivo. In a rat model of carotid injury, 2 weeks of continuous oral nitrite treatment postinjury prevented the hyperproliferative response of smooth muscle cells. This protection was accompanied by a nitrite-dependent upregulation of PGC1α and increased mitochondrial number in the injured artery. These data are the first to demonstrate that nitrite mediates differential signaling compared to NO. They show that nitrite is a versatile regulator of mitochondrial function and number both in vivo and in vitro and suggest that nitrite-mediated biogenesis may play a protective role in the setting of vascular injury.

Nitrite protects against morbidity and mortality associated with TNF- or LPS-induced shock in a soluble guanylate cyclase-dependent manner.

Nitrite (NO(2)(-)), previously viewed as a physiologically inert metabolite and biomarker of the endogenous vasodilator NO, was recently identified as an important biological NO reservoir in vasculature and tissues, where it contributes to hypoxic signaling, vasodilation, and cytoprotection after ischemia-reperfusion injury. Reduction of nitrite to NO may occur enzymatically at low pH and oxygen tension by deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, mitochondrial complexes, or NO synthase (NOS). We show that nitrite treatment, in sharp contrast with the worsening effect of NOS inhibition, significantly attenuates hypothermia, mitochondrial damage, oxidative stress and dysfunction, tissue infarction, and mortality in a mouse shock model induced by a lethal tumor necrosis factor challenge. Mechanistically, nitrite-dependent protection was not associated with inhibition of mitochondrial complex I activity, as previously demonstrated for ischemia-reperfusion, but was largely abolished in mice deficient for the soluble guanylate cyclase (sGC) alpha1 subunit, one of the principal intracellular NO receptors and signal transducers in the cardiovasculature. Nitrite could also provide protection against toxicity induced by Gram-negative lipopolysaccharide, although higher doses were required. In conclusion, we show that nitrite can protect against toxicity in shock via sGC-dependent signaling, which may include hypoxic vasodilation necessary to maintain microcirculation and organ function, and cardioprotection.