Myoglobin Protects Breast Cancer Cells Due to Its ROS and NO Scavenging Properties.

Myoglobin (MB) is an oxygen-binding protein usually found in cardiac myocytes and skeletal muscle fibers. It may function as a temporary storage and transport protein for O2 but could also have scavenging capacity for reactive oxygen and nitrogen species. In addition, MB has recently been identified as a hallmark in luminal breast cancer and was shown to be robustly induced under hypoxia. Cellular responses to hypoxia are regulated by the transcription factor hypoxia-inducible factor (HIF). For exploring the function of MB in breast cancer, we employed the human cell line MDA-MB-468. Cells were grown in monolayer or as 3D multicellular spheroids, which mimic the in vivo avascular tumor architecture and physiology with a heterogeneous cell population of proliferating cells in the rim and non-cycling or necrotic cells in the core region. This central necrosis was increased after MB knockdown, indicating a role for MB in hypoxic tumor regions. In addition, MB knockdown caused higher levels of HIF-1α protein after treatment with NO, which also plays an important role in cancer cell survival. MB knockdown also led to higher reactive oxygen species (ROS) levels in the cells after treatment with H2O2. To further explore the role of MB in cell survival, we performed RNA-Seq after MB knockdown and NO treatment. 1029 differentially expressed genes (DEGs), including 45 potential HIF-1 target genes, were annotated in regulatory pathways that modulate cellular function and maintenance, cell death and survival, and carbohydrate metabolism. Of these target genes, TMEFF1, TREX2, GLUT-1, MKNK-1, and RAB8B were significantly altered. Consistently, a decreased expression of GLUT-1, MKNK-1, and RAB8B after MB knockdown was confirmed by qPCR. All three genes of interest are often up regulated in cancer and correlate with a poor clinical outcome. Thus, our data indicate that myoglobin might influence the survival of breast cancer cells, possibly due to its ROS and NO scavenging properties and could be a valuable target for cancer therapy.

Metabolic physiology and skeletal muscle phenotypes in male and female myoglobin knockout mice.

Myoglobin (Mb) is a regulator of O2 bioavailability in type I muscle and heart, at least when tissue O2 levels drop. Mb also plays a role in regulating cellular nitric oxide (NO) pools. Robust binding of long-chain fatty acids and long-chain acylcarnitines to Mb, and enhanced glucose metabolism in hearts of Mb knockout (KO) mice, suggest additional roles in muscle intermediary metabolism and fuel selection. To evaluate this hypothesis, we measured energy expenditure (EE), respiratory exchange ratio (RER), body weight gain and adiposity, glucose tolerance, and insulin sensitivity in Mb knockout (Mb-/-) and wild-type (WT) mice challenged with a high-fat diet (HFD, 45% of calories). In males (n = 10/genotype) and females (n = 9/genotype) tested at 5-6, 11-12, and 17-18 wk, there were no genotype effects on RER, EE, or food intake. RER and EE during cold (10°C, 72 h), and glucose and insulin tolerance, were not different compared with within-sex WT controls. At ∼18 and ∼19 wk of age, female Mb-/- adiposity was ∼42%-48% higher versus WT females (P = 0.1). Transcriptomics analyses (whole gastrocnemius, soleus) revealed few consistent changes, with the notable exception of a 20% drop in soleus transferrin receptor (Tfrc) mRNA. Capillarity indices were significantly increased in Mb-/-, specifically in Mb-rich soleus and deep gastrocnemius. The results indicate that Mb loss does not have a major impact on whole body glucose homeostasis, EE, RER, or response to a cold challenge in mice. However, the greater adiposity in female Mb-/- mice indicates a sex-specific effect of Mb KO on fat storage and feed efficiency.NEW & NOTEWORTHY The roles of myoglobin remain to be elaborated. We address sexual dimorphism in terms of outcomes in response to the loss of myoglobin in knockout mice and perform, for the first time, a series of comprehensive metabolic studies under conditions in which fat is mobilized (high-fat diet, cold). The results highlight that myoglobin is not necessary and sufficient for maintaining oxidative metabolism and point to alternative roles for this protein in muscle and heart.

On the potential role of globins in brown adipose tissue: a novel conceptual model and studies in myoglobin knockout mice.

Myoglobin (Mb) regulates O2 bioavailability in muscle and heart as the partial pressure of O2 (Po2) drops with increased tissue workload. Globin proteins also modulate cellular NO pools, "scavenging" NO at higher Po2 and converting NO2- to NO as Po2 falls. Myoglobin binding of fatty acids may also signal a role in fat metabolism. Interestingly, Mb is expressed in brown adipose tissue (BAT), but its function is unknown. Herein, we present a new conceptual model that proposes links between BAT thermogenic activation, concurrently reduced Po2, and NO pools regulated by deoxy/oxy-globin toggling and xanthine oxidoreductase (XOR). We describe the effect of Mb knockout (Mb-/-) on BAT phenotype [lipid droplets, mitochondrial markers uncoupling protein 1 (UCP1) and cytochrome C oxidase 4 (Cox4), transcriptomics] in male and female mice fed a high-fat diet (HFD, 45% of energy, ∼13 wk), and examine Mb expression during brown adipocyte differentiation. Interscapular BAT weights did not differ by genotype, but there was a higher prevalence of mid-large sized droplets in Mb-/-. COX4 protein expression was significantly reduced in Mb-/- BAT, and a suite of metabolic/NO/stress/hypoxia transcripts were lower. All of these Mb-/--associated differences were most apparent in females. The new conceptual model, and results derived from Mb-/- mice, suggest a role for Mb in BAT metabolic regulation, in part through sexually dimorphic systems and NO signaling. This possibility requires further validation in light of significant mouse-to-mouse variability of BAT Mb mRNA and protein abundances in wild-type mice and lower expression relative to muscle and heart.NEW & NOTEWORTHY Myoglobin confers the distinct red color to muscle and heart, serving as an oxygen-binding protein in oxidative fibers. Less attention has been paid to brown fat, a thermogenic tissue that also expresses myoglobin. In a mouse knockout model lacking myoglobin, brown fat had larger fat droplets and lower markers of mitochondrial oxidative metabolism, especially in females. Gene expression patterns suggest a role for myoglobin as an oxygen/nitric oxide-sensor that regulates cellular metabolic and signaling pathways.

The role of myoglobin in the evolution of mammalian diving capacity - The August Krogh principle applied in molecular and evolutionary physiology.

After the Devonian tetrapod land invasion, groups of terrestrial air-breathing and endothermic mammals repeatedly went back to live in the sea, relying on air intake at the surface for extended breath-hold dives to forage underwater, often at great depths and even in the coldest oceans. Studies on the physiological mechanisms behind prolonged breath-hold diving have a long history, including August Krogh's estimates of the maximal dive duration of the blue whale. Yet the molecular underpinnings of such extreme physiological adaptations are only beginning to be understood. The present review focuses on the molecular properties of the respiratory protein myoglobin that has repeatedly evolved an elevated net positive surface charge in several distantly related groups of diving mammals. This has enabled substantial increases of maximal myoglobin concentration in muscle cells, and hence muscle oxygen storage capacity and maximal dive duration. Using myoglobin net surface charge as a marker has allowed unprecedented insights into the evolution of mammal diving capacity and into the general mechanisms of adaptive protein evolution. From these findings it is argued, in an extension of the August Krogh principle, that for a large number of problems in molecular and evolutionary physiology there will be some protein of choice, or a few such proteins, on which it can be most conveniently studied.

Myoglobin clearance with continuous veno-venous hemodialysis using high cutoff dialyzer versus continuous veno-venous hemodiafiltration using high-flux dialyzer: a prospective randomized controlled trial.

BACKGROUND: Myoglobin clearance in acute kidney injury requiring renal replacement therapy is important because myoglobin has direct renal toxic effects. Clinical data comparing different modalities of renal replacement therapy addressing myoglobin clearance are limited. This study aimed to compare two renal replacement modalities regarding myoglobin clearance. METHODS: In this prospective, randomized, single-blinded, single-center trial, 70 critically ill patients requiring renal replacement therapy were randomized 1:1 into an intervention arm using continuous veno-venous hemodialysis with high cutoff dialyzer and a control arm using continuous veno-venous hemodiafiltration postdilution with high-flux dialyzer. Regional citrate anticoagulation was used in both groups to maintain the extracorporeal circuit. The concentrations of myoglobin, urea, creatinine, ß2-microglobulin, interleukin-6 and albumin were measured before and after the dialyzer at 1 h, 6 h, 12 h, 24 h and 48 h after initiating continuous renal replacement therapy. RESULTS: Thirty-three patients were allocated to the control arm (CVVHDF with high-flux dialyzer) and 35 patients to the intervention arm (CVVHD with high cutoff dialyzer). Myoglobin clearance, as a primary endpoint, was significantly better in the intervention arm than in the control arm throughout the whole study period. The clearance values for urea and creatinine were higher in the control arm. There was no measurable albumin clearance in both arms. The clearance data for ß2-microglobulin and interleukin-6 were non-inferior in the intervention arm compared to those for the control arm. Dialyzer lifespan was 57.0 [38.0, 72.0] hours in the control arm and 70.0 [56.75, 72.0] hours in the intervention arm (p = 0.029). CONCLUSIONS: Myoglobin clearance using continuous veno-venous hemodialysis with high cutoff dialyzer and regional citrate anticoagulation is better than that with continuous veno-venous hemodiafiltration with regional citrate anticoagulation. TRIAL REGISTRATION: German Clinical Trials Registry (DRKS00012407); date of registration 23/05/2017. https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00012407 .

Myoglobin induces mitochondrial fusion, thereby inhibiting breast cancer cell proliferation.

Myoglobin is a monomeric heme protein expressed ubiquitously in skeletal and cardiac muscle and is traditionally considered to function as an oxygen reservoir for mitochondria during hypoxia. It is now well established that low concentrations of myoglobin are aberrantly expressed in a significant proportion of breast cancer tumors. Despite being expressed only at low levels in these tumors, myoglobin is associated with attenuated tumor growth and a better prognosis in breast cancer patients, but the mechanism of this myoglobin-mediated protection against further cancer growth remains unclear. Herein, we report a signaling pathway by which myoglobin regulates mitochondrial dynamics and thereby decreases cell proliferation. We demonstrate in vitro that expression of human myoglobin in MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells induces mitochondrial hyperfusion by up-regulating mitofusins 1 and 2, the predominant catalysts of mitochondrial fusion. This hyperfusion causes cell cycle arrest and subsequent inhibition of cell proliferation. Mechanistically, increased mitofusin expression was due to myoglobin-dependent free-radical production, leading to the oxidation and degradation of the E3 ubiquitin ligase parkin. We recapitulated this pathway in a murine model in which myoglobin-expressing xenografts exhibited decreased tumor volume with increased mitofusin, markers of cell cycle arrest, and decreased parkin expression. Furthermore, in human triple-negative breast tumor tissues, mitofusin and myoglobin levels were positively correlated. Collectively, these results elucidate a new function for myoglobin as a modulator of mitochondrial dynamics and reveal a novel pathway by which myoglobin decreases breast cancer cell proliferation and tumor growth by up-regulating mitofusin levels.

Onset of Accelerated Muscle Deoxygenation During the 20-m Shuttle Run Test in Boys.

PURPOSE: This study aimed to compare the relative exercise intensity at which the onset of accelerated muscle deoxygenation occurs during the 20-m shuttle run test (20mSRT) between boys and men and to examine whether the timing of the onset of acceleration appearance is related to 20mSRT performance in boys. METHODS: Twenty-four boys performed the 20mSRT, during which concentration changes in oxygenated and deoxygenated hemoglobin and myoglobin (ΔOxy-Hb and ΔDeoxy-Hb, respectively) in the m. vastus lateralis were monitored using a portable near-infrared spectroscopy device. The boys' data were compared with those of 29 men in a previous study. RESULTS: An onset of accelerated decrease in Δ[Oxy-Hb - Deoxy-Hb] was found in 11 of the 24 boys (45.8%) and 20 of the 29 men (69.0%) and was found at a higher relative exercise intensity in the boys than in the men. The number of laps at which the onset of acceleration occurred correlated with total laps in the boys (r = .87). CONCLUSIONS: These findings demonstrate that the onset of accelerated muscle deoxygenation during the 20mSRT occurs at a higher relative exercise intensity in boys than in men. Our findings also show that the timing of the onset of acceleration appearance is associated with 20mSRT performance in boys.

[Myoglobin: still a useful biomarker in 2017?] / Y a-t-il un intérêt au dosage de la myoglobine en 2017 ?

The clinical biologist plays a role as a consultant for the relevant use of biological examination. Advisory activities of the medical laboratory may help physician in diagnosis or therapeutic algorithm, avoiding redundant ordering or useless tests. In this context, we performed a review of literature about the clinically interest of myoglobin assays. The indications of myoglobin's assays appear fairly limited. It is no longer mentioned in the European guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. In patients with rhabdomyolysis myoglobin is neither a diagnostic nor a prognostic criterion. Its interest in predicting the occurrence of acute renal failure is also discussed. The most recent clinico-biological score (such as the McMahon score) do not integrate it. In this context, we decided to stop performing myoglobin assay.

The Role of Hemoproteins: Hemoglobin, Myoglobin and Neuroglobin in Endogenous Thiosulfate Production Processes.

Thiosulfate formation and biodegradation processes link aerobic and anaerobic metabolism of cysteine. In these reactions, sulfite formed from thiosulfate is oxidized to sulfate while hydrogen sulfide is transformed into thiosulfate. These processes occurring mostly in mitochondria are described as a canonical hydrogen sulfide oxidation pathway. In this review, we discuss the current state of knowledge on the interactions between hydrogen sulfide and hemoglobin, myoglobin and neuroglobin and postulate that thiosulfate is a metabolically important product of this processes. Hydrogen sulfide oxidation by ferric hemoglobin, myoglobin and neuroglobin has been defined as a non-canonical hydrogen sulfide oxidation pathway. Until recently, it appeared that the goal of thiosulfate production was to delay irreversible oxidation of hydrogen sulfide to sulfate excreted in urine; while thiosulfate itself was only an intermediate, transient metabolite on the hydrogen sulfide oxidation pathway. In the light of data presented in this paper, it seems that thiosulfate is a molecule that plays a prominent role in the human body. Thus, we hope that all these findings will encourage further studies on the role of hemoproteins in the formation of this undoubtedly fascinating molecule and on the mechanisms responsible for its biological activity in the human body.

Functional roles of globin proteins in hypoxia-tolerant ectothermic vertebrates.

Globins are heme-containing proteins ubiquitously expressed in vertebrates, where they serve a broad range of biological functions, directly or indirectly related to the tight control of oxygen levels and its toxic products in vivo. Perhaps the most investigated of all proteins, hemoglobin and myoglobin are primarily involved in oxygen transport and storage, but also in facilitating arterial vasodilation, suppressing mitochondrial respiration, and preventing tissue oxidative damage via accessory redox enzymatic activities during hypoxia. By contrast, the more recently discovered neuroglobin and cytoglobin do not seem to function as reversible oxygen carriers and are instead involved in redox activities, although their exact biological roles remain to be clarified. In this context, hypoxia-tolerant ectotherms, such as freshwater turtles and members of the carp family that survive winter in extreme hypoxia, have proven as excellent models to appreciate the diversity of biological functions of globin proteins. Unraveling physiological roles of globin proteins in these extreme animals will clarify an important part of the adaptive mechanisms for surviving extreme fluctuations of oxygen availability that are prohibitive to mammals.

The expression of VEGF, myoglobin and CRP2 proteins regulating endometrial remodeling in the porcine endometrial tissues during follicular and luteal phase.

Endometrial remodeling is important for successful embryo development and implantation in pigs. Therefore, this study investigated change of proteins regulating endometrial remodeling on follicular and luteal phase in porcine endometrial tissues. The endometrial tissue samples were collected from porcine uterus during follicular and luteal phase, vascular endothelial growth factor (VEGF), myoglobin and cysteine-rich protein 2 (CRP2) proteins were expressed by immnofluorescence, immunoblotting, and determined by 2-DE and MALDI-TOF/MS. We found that VEGF, myoglobin and CRP2 were strongly localized in endometrial tissues during luteal phase, but not follicular phase. The protein levels of VEGF, myoglobin and CRP2 in endometrial tissues were higher than luteal phase (P < 0.05). These results may provide understanding of intrauterine environment during estrous cycle in pigs, and will be used in animal reproduction for developing specific biomarkers in the future.

Platelet-activating factor mediates the cytotoxicity induced by W7FW14F apomyoglobin amyloid aggregates in neuroblastoma cells.

W7FW14F apomyoglobin (W7FW14F ApoMb) amyloid aggregates induce cytotoxicity in SH-SY5Y human neuroblastoma cells through a mechanism not fully elucidated. Amyloid neurotoxicity process involves calcium dyshomeostasis and reactive oxygen species (ROS) production. Another key mediator of the amyloid neurotoxicity is Platelet-Activating Factor (PAF), an inflammatory phospholipid implicated in neurodegenerative diseases. Here, with the aim at evaluating the possible involvement of PAF signaling in the W7FW14F ApoMb-induced cytotoxicity, we show that the presence of CV3899, a PAF receptor (PAF-R) antagonist, prevented the detrimental effect of W7FW14F ApoMb aggregates on SH-SY5Y cell viability. Noticeably, we found that the activation of PAF signaling, following treatment with W7FW14F ApoMb, involves a decreased expression of the PAF acetylhydroase II (PAF-AH II). Interestingly, the reduced PAF-AH II expression was associated with a decreased acetylhydrolase (AH) activity and to an increased sphingosine-transacetylase activity (TA(S)) with production of N-acetylsphingosine (C2-ceramide), a well known mediator of neuronal caspase-dependent apoptosis. These findings suggest that an altered PAF catabolism takes part to the molecular events leading to W7FW14F ApoMb amyloid aggregates-induced cell death.

Cardiac myoglobin deficit has evolved repeatedly in teleost fishes.

Myoglobin (Mb) is the classic vertebrate oxygen-binding protein present in aerobic striated muscles. It functions principally in oxygen delivery and provides muscle with its characteristic red colour. Members of the Antarctic icefish family (Channichthyidae) are widely thought to be extraordinary for lacking cardiac Mb expression, a fact that has been attributed to their low metabolic rate and unusual evolutionary history. Here, we report that cardiac Mb deficit, associated with pale heart colour, has evolved repeatedly during teleost evolution. This trait affects both gill- and air-breathing species from temperate to tropical habitats across a full range of salinities. Cardiac Mb deficit results from total pseudogenization in three-spined stickleback and is associated with a massive reduction in mRNA level in two species that evidently retain functional Mb. The results suggest that near or complete absence of Mb-assisted oxygen delivery to heart muscle is a common facet of teleost biodiversity, even affecting lineages with notable oxygen demands. We suggest that Mb deficit may affect how different teleost species deal with increased tissue oxygen demands arising under climate change.

Myoglobin functions in the heart.

The physiological role of myoglobin (Mb) within the heart depends on its oxygenation state. The myocardium exhibits a broad oxygen partial pressure (pO2) spectrum with a transmural gradient from the epicardial to the subendocardial layer, ranging from arterial values to an average of 19.3 mm Hg down to 0 mm Hg. The function of Mb as an O2 storage depot is well appreciated, especially during systolic compression. In addition, Mb controls myocardial nitric oxide (NO) homeostasis and thus modulates mitochondrial respiration under physiological and pathological conditions. We recently discovered the role of Mb as a myocardial O2 sensor; in its oxygenated state Mb scavenges NO, protecting the heart from the deleterious effects of excessive NO. Under hypoxia, however, deoxygenated Mb changes its role from an NO scavenger to an NO producer. The NO produced protects the cell from short phases of hypoxia and from myocardial ischemia/reperfusion injury. In this review we summarize the traditional and novel aspects of Mb and its (patho)physiological role in the heart.

Molecular evolution tracks macroevolutionary transitions in Cetacea.

Cetacea (whales, dolphins, and porpoises) is a model group for investigating the molecular signature of macroevolutionary transitions. Recent research has begun to reveal the molecular underpinnings of the remarkable anatomical and behavioral transformation in this clade. This shift from terrestrial to aquatic environments is arguably the best-understood major morphological transition in vertebrate evolution. The ancestral body plan and physiology were extensively modified and, in many cases, these crucial changes are recorded in cetacean genomes. Recent studies have highlighted cetaceans as central to understanding adaptive molecular convergence and pseudogene formation. Here, we review current research in cetacean molecular evolution and the potential of Cetacea as a model for the study of other macroevolutionary transitions from a genomic perspective.

Regulation of cellular gas exchange, oxygen sensing, and metabolic control.

Cells must continuously monitor and couple their metabolic requirements for ATP utilization with their ability to take up O2 for mitochondrial respiration. When O2 uptake and delivery move out of homeostasis, cells have elaborate and diverse sensing and response systems to compensate. In this review, we explore the biophysics of O2 and gas diffusion in the cell, how intracellular O2 is regulated, how intracellular O2 levels are sensed and how sensing systems impact mitochondrial respiration and shifts in metabolic pathways. Particular attention is paid to how O2 affects the redox state of the cell, as well as the NO, H2S, and CO concentrations. We also explore how these agents can affect various aspects of gas exchange and activate acute signaling pathways that promote survival. Two kinds of challenges to gas exchange are also discussed in detail: when insufficient O2 is available for respiration (hypoxia) and when metabolic requirements test the limits of gas exchange (exercising skeletal muscle). This review also focuses on responses to acute hypoxia in the context of the original "unifying theory of hypoxia tolerance" as expressed by Hochachka and colleagues. It includes discourse on the regulation of mitochondrial electron transport, metabolic suppression, shifts in metabolic pathways, and recruitment of cell survival pathways preventing collapse of membrane potential and nuclear apoptosis. Regarding exercise, the issues discussed relate to the O2 sensitivity of metabolic rate, O2 kinetics in exercise, and influences of available O2 on glycolysis and lactate production.

Heme binding site in apomyoglobin may be effectively targeted with small molecules to control aggregation.

A number of ligands with affinities for the heme binding site of apomyoglobin were tested to control amorphous and fibrillar aggregation in the protein. Several techniques, including fluorescence, dynamic light scattering, transmission electron microscopy, dot blot analysis combined with viability studies were employed for structural characterization and cytotoxicity assessment of the intermediate and final protein structures formed during the aggregation process. Of the small molecules investigated, chrysin and Nile red with high structural similarities to heme were chosen for further studies. Only fibril formation was found to be prevented by Nile red, while chrysin, with a greater structural flexibility, was able to prevent both types of aggregate formation. The two ligands were found to influence aggregation at different stages of intermediate structure formation, an ability determined by their degrees of similarities with heme. Based on structural characterization and toxicity studies, it is concluded that ligands similar in structure to heme may be effective in influencing various stages of aggregate formation and toxicity potencies of the protein structures. Since metalloproteins constitute more than thirty percent of all known proteins, it is concluded that the present strategy may be of general significance.

Functional differentiation of myoglobin isoforms in hypoxia-tolerant carp indicates tissue-specific protective roles.

Because of a recent whole genome duplication, the hypoxia-tolerant common carp and goldfish are the only vertebrates known to possess two myoglobin (Mb) paralogs. One of these, Mb1, occurs in oxidative muscle but also in several other tissues, including capillary endothelial cells, whereas the other, Mb2, is a unique isoform specific to brain neurons. To help understand the functional roles of these diverged isoforms in the tolerance to severe hypoxia in the carp, we have compared their O(2) equilibria, carbon monoxide (CO) and O(2) binding kinetics, thiol S-nitrosation, nitrite reductase activities, and peroxidase activities. Mb1 has O(2) affinity and nitrite reductase activity comparable to most vertebrate muscle Mbs, consistent with established roles for Mbs in O(2) storage/delivery and in maintaining nitric oxide (NO) homeostasis during hypoxia. Both Mb1 and Mb2 can be S-nitrosated to similar extent, but without oxygenation-linked allosteric control. When compared with Mb1, Mb2 displays faster O(2) and CO kinetics, a lower O(2) affinity, and is slower at converting nitrite into NO. Mb2 is therefore unlikely to be primarily involved in either O(2) supply to mitochondria or the generation of NO from nitrite during hypoxia. However, Mb2 proved to be significantly faster at eliminating H(2)O(2,) a major in vivo reactive oxygen species (ROS), suggesting that this diverged Mb isoform may have a specific protective role against H(2)O(2) in the carp brain. This property might be of particular significance during reoxygenation following extended periods of hypoxia, when production of H(2)O(2) and other ROS is highest.