The Impact of Normobaric Hypoxia and Intermittent Hypoxic Training on Cardiac Biomarkers in Endurance Athletes: A Pilot Study.

This study explores the effects of normobaric hypoxia and intermittent hypoxic training (IHT) on the physiological condition of the cardiac muscle in swimmers. Hypoxia has been reported to elicit both beneficial and adverse changes in the cardiovascular system, but its impact on the myocardium during acute exercise and altitude/hypoxic training remains less understood. We aimed to determine how a single bout of intense interval exercise and a four-week period of high-intensity endurance training under normobaric hypoxia affect cardiac marker activity in swimmers. Sixteen young male swimmers were divided into two groups: one undergoing training in hypoxia and the other in normoxia. Cardiac markers, including troponin I and T (cTnI and cTnT), heart-type fatty acid-binding protein (H-FABP), creatine kinase-MB isoenzyme (CK-MB), and myoglobin (Mb), were analyzed to assess the myocardium's response. We found no significant differences in the physiological response of the cardiac muscle to intense physical exertion between hypoxia and normoxia. Four weeks of IHT did not alter the resting levels of cTnT, cTnI, and H-FABP, but it resulted in a noteworthy decrease in the resting concentration of CK-MB, suggesting enhanced cardiac muscle adaptation to exercise. In contrast, a reduction in resting Mb levels was observed in the control group training in normoxia. These findings suggest that IHT at moderate altitudes does not adversely affect cardiac muscle condition and may support cardiac muscle adaptation, affirming the safety and efficacy of IHT as a training method for athletes.

Dietary Hemin Remodels Gut Microbiota and Mediates Tissue Inflammation and Injury in the Small Intestine.

SCOPE: Epidemiological studies have linked excessive red and processed meat intake to gut disorders. Under laboratory conditions, high heme content is considered the primary health risk factor for red meat. However, heme in meat is present in myoglobin, which is an indigestible protein, suggesting the different functions between myoglobin and heme. This study aims to explore how dietary myoglobin and heme affect gut health and microbiota differently. METHODS AND RESULTS: Histological and biochemical assessments as well as 16S rRNA sequencing are performed. Moderate myoglobin intake (equivalent to the recommended intake of 150 g meat per day for human) has beneficial effects on the duodenal barrier. However, a too high myoglobin diet (equivalent to intake of 3000 g meat per day for human) triggers duodenum injury and alters the microbial community. The hemin diet destroys intestinal tissue and ileal microbiota more significantly. The in vitro experiments further confirm that free heme exhibits high toxicity to beneficial gut bacteria while myoglobin promotes the growth and metabolism of Limosilactobacillus reuteri. CONCLUSION: Moderate intake of myoglobin or hemin is beneficial to intestinal health and microbiota, but too high amounts lead to tissue inflammation and injury in the small intestine by reshaping ileal microbiota.

Not All Binding Sites Are Equal: Site Determination and Folding State Analysis of Gas-Phase Protein-Metallodrug Adducts.

Modern approaches in metallodrug research focus on compounds that bind protein targets rather than DNA. However, the identification of protein targets and binding sites is challenging. Using intact mass spectrometry and proteomics, we investigated the binding of the antimetastatic agent RAPTA-C to the model proteins ubiquitin, cytochrome c, lysozyme, and myoglobin. Binding to cytochrome c and lysozyme was negligible. However, ubiquitin bound up to three Ru moieties, two of which were localized at Met1 and His68 as [Ru(cym)], and [Ru(cym)] or [Ru(cym)(PTA)] adducts, respectively. Myoglobin bound up to four [Ru(cym)(PTA)] moieties and five sites were identified at His24, His36, His64, His81/82 and His113. Collision-induced unfolding (CIU) studies via ion-mobility mass spectrometry allowed measuring protein folding as a function of collisional activation. CIU of protein-RAPTA-C adducts showed binding of [Ru(cym)] to Met1 caused a significant compaction of ubiquitin, likely from N-terminal S-Ru-N chelation, while binding of [Ru(cym)(PTA)] to His residues of ubiquitin or myoglobin induced a smaller effect. Interestingly, the folded state of ubiquitin formed by His functionalization was more stable than Met1 metalation. The data suggests that selective metalation of amino acids at different positions on the protein impacts the conformation and potentially the biological activity of anticancer compounds.

Endurance training increases mitochondrial myoglobin and enhances its interaction with complex IV in rat plantaris muscle.

AIM: Endurance exercise training is known to increase mitochondrial respiration in skeletal muscle. However, the molecular mechanisms behind this are not fully understood. Myoglobin (Mb) is a member of the globin family, which is highly expressed in skeletal and cardiac muscles. We recently found that Mb localizes inside mitochondria in skeletal muscle and interacts with cytochrome c oxidase subunit IV (COXIV), a subunit of mitochondrial complex IV, which regulates respiration by augmenting complex IV activity. In the present study, we investigated the effect of endurance training on Mb-COXIV interaction within mitochondria in rat skeletal muscle. METHODS: Eight-week-old male Wistar rats were subjected to 6-week treadmill running training. Forty-eight hours after the last training session, the plantaris muscle was removed under anesthesia and used for biochemical analysis. RESULTS: The endurance training increased mitochondrial content in the skeletal muscle. It also augmented complex IV-dependent oxygen consumption and complex IV activity in isolated mitochondria from skeletal muscle. Furthermore, endurance training increased Mb expression at the whole muscle level. Importantly, mitochondrial Mb content and Mb-COXIV binding were increased by endurance training. CONCLUSION: These findings suggest that an increase in mitochondrial Mb and the concomitant enhancement of Mb interaction with COXIV may contribute to the endurance training-induced upregulation of mitochondrial respiration by augmenting complex IV activity.

Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Paraspinal muscle oxygenation and mechanical efficiency are reduced in individuals with chronic low back pain.

This study aimed to compare the systemic and local metabolic responses during a 5-min trunk extension exercise in individuals with chronic low back pain (CLBP) and in healthy individuals. Thirteen active participants with CLBP paired with 13 healthy participants performed a standardised 5-min trunk extension exercise on an isokinetic dynamometer set in continuous passive motion mode. During exercise, we used near-infrared spectroscopy to measure tissue oxygenation (TOI) and total haemoglobin-myoglobin (THb). We used a gas exchange analyser to measure breath-by-breath oxygen consumption (V̇O2) and carbon dioxide produced (V̇CO2). We also calculated mechanical efficiency. We assessed the intensity of low back pain sensation before and after exercise by using a visual analogue scale. In participants with CLBP, low back pain increased following exercise (+ 1.5 units; p < 0.001) and THb decreased during exercise (- 4.0 units; p = 0.043). Paraspinal muscle oxygenation (65.0 and 71.0%, respectively; p = 0.009) and mechanical efficiency (4.7 and 5.3%, respectively; p = 0.034) were both lower in participants with CLBP compared with healthy participants. The increase in pain sensation was related to the decrease in tissue oxygenation (R2 = - 0.420; p = 0.036). Decreases in total haemoglobin-myoglobin and mechanical efficiency could involve fatigability in exercise-soliciting paraspinal muscles and, therefore, exacerbate inabilities in daily life. Given the positive correlation between tissue oxygenation and exercise-induced pain exacerbation, muscle oxygenation may be related to persisting and crippling low back pain.

Regulating the Heme Active Site by Covalent Modifications: Two Case Studies of Myoglobin.

Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46 C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H2 O2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins.

The interaction of perfluorooctane sulfonate with hemoproteins and its relevance to molecular toxicology.

Perfluorooctane sulfonate (PFOS), a representative of perfluorinated compounds in industrial and commercial products, has posed a great threat to animals and humans via environmental exposure and dietary consumption. Herein, we investigated the effects of PFOS binding on the redox state and stability of two hemoproteins (hemoglobin (Hb) and myoglobin (Mb)). Fluorescence spectroscopy, circular dichroism and UV-vis absorption spectroscopy demonstrated that PFOS could induce the conformational changes of proteins along with the exposure of heme cavity and generation of hemichrome, which resulted in the increased release of free hemin. After that, free hemin liberated from hemoproteins led to reactive oxygen species formation, lipid peroxidation, cell membrane damage and loss of cell viability in vascular endothelial cells, while neither Hb nor Mb did show cytotoxicity. Chemical inhibitors of ferroptosis effectively mitigated hemin-caused toxicity, identifying the hemin-dependent ferroptotic cell death mechanisms. These data demonstrated that PFOS posed a potential threat of toxicity through a mechanism which involved its binding to hemoproteins, decreased oxygen transporting capacity, and increased hemin release. Altogether, our findings elucidate the binding mechanisms of PFOS with two hemoproteins, as well as possible risks on vascular endothelial cells, which would have important implications for the human and environmental toxicity of PFOS.

Sex Differences in Test-Retest Reliability of Near-Infrared Spectroscopy During Postocclusive Reactive Hyperemia of the Vastus Lateralis.

ABSTRACT: Shoemaker, ME, Smith, CM, Gillen, ZM, and Cramer, JT. Sex differences in test-retest reliability of near-infrared spectroscopy during postocclusive reactive hyperemia of the vastus lateralis. J Strength Cond Res 38(2): e40-e48, 2024-The purpose of this study was to determine test-retest reliability for vascular reactivity measures and ranges for normalization of near-infrared spectroscopy (NIRS) variables from the vastus lateralis using postocclusive reactive hyperemia (PORH) procedure in male subjects, female subjects, and combined. Concentrations of oxygenated hemoglobin (Hb) + myoglobin (Mb) (O 2 Hb) and deoxygenated Hb + Mb (HHb) to derive total Hb + Mb (THb), difference in Hb + Mb signal (Hbdiff), and muscle tissue oxygen saturation (StO 2 ) from the vastus lateralis were measured during the PORH in 12 male subjects (age: 23.17 ± 1.77 years; stature: 180.88 ± 4.59 cm; and mass: 81.47 ± 9.68 kg) and 10 female subjects (age: 23.80 ± 2.07 years; stature: 165.95 ± 4.92 cm; and mass: 70.93 ± 10.55 kg) on 2 separate days. Adipose tissue thickness at the NIRS site was measured with ultrasonography. There were no significant differences between the mean values from visit 1 to visit 2 ( p > 0.076-0.985). In the composite sample, intraclass correlation coefficient (ICC) and coefficient of variation (CV) ranged from 0.35 to 0.91 and 4.74 to 39.18%, respectively. In male subjects, ICC and CV values ranged from 0.57 to 0.89 and 2.44 to 28.55%, respectively. In female subjects, ICC and CV values ranged from -0.05 to 0.75 and 7.83 to 61.19%, respectively. Although NIRS variables were overall reliable during PORH, when separated by sex, reliability in male subjects generally increased, whereas female subjects were not reliable, suggesting adipose tissue thickness may be a contributing factor. Understanding sex differences in reliability is important when using this technique for normalization or examining vascular reactivity during athletic performance. With greater utilization of NIRS monitoring in athletes to examine training adaptations, it is important for practitioners to understand the capabilities and potential limitations of the tool.

Cardiac nitric oxide scavenging: role of myoglobin and mitochondria.

Vascular production of nitric oxide (NO) regulates vascular tone. However, highly permeable NO entering the cardiomyocyte would profoundly impact metabolism and signalling without scavenging mechanisms. The purpose of this study was to establish mechanisms of cardiac NO scavenging. Quantitative optical studies of normoxic working hearts demonstrated that micromolar NO concentrations did not alter mitochondria redox state or respiration despite detecting NO oxidation of oxymyoglobin to metmyoglobin. These data are consistent with proposals that the myoglobin/myoglobin reductase (Mb/MbR) system is the major NO scavenging site. However, kinetic studies in intact hearts reveal a minor role (∼9%) for the Mb/MbR system in NO scavenging. In vitro, oxygenated mitochondria studies confirm that micromolar concentrations of NO bind cytochrome oxidase (COX) and inhibit respiration. Mitochondria had a very high capacity for NO scavenging, importantly, independent of NO binding to COX. NO is also known to quickly react with reactive oxygen species (ROS) in vitro. Stimulation of NO scavenging with antimycin and its inhibition by substrate depletion are consistent with NO interacting with ROS generated in Complex I or III under aerobic conditions. Extrapolating these in vitro data to the intact heart supports the hypothesis that mitochondria are a major site of cardiac NO scavenging. KEY POINTS: Cardiomyocyte scavenging of vascular nitric oxide (NO) is critical in maintaining normal cardiac function. Myoglobin redox cycling via myoglobin reductase has been proposed as a major NO scavenging site in the heart. Non-invasive optical spectroscopy was used to monitor the effect of NO on mitochondria and myoglobin redox state in intact beating heart and isolated mitochondria. These non-invasive studies reveal myoglobin/myoglobin reductase plays a minor role in cardiac NO scavenging. A high capacity for NO scavenging by heart mitochondria was demonstrated, independent of cytochrome oxidase binding but dependent on oxygen and high redox potentials consistent with generation of reactive oxygen species.

Construction of a myoglobin scaffold-based biocatalyst for the biodegradation of sulfadiazine and sulfathiazole.

Sulfonamide antibiotics, a family of broad-spectrum antibiotic drugs, are increasingly used in aquaculture and are frequently detected in aquatic environments. This poses a potential threat to organisms and may cause the evolution of antimicrobial resistance. Therefore, it is important to develop an environmentally friendly and efficient biocatalyst to degrade sulfonamides (SAs) such as sulfadiazine (SD) and sulfathiazole (ST). Here, we realized the direct and efficient degradation of SD and ST using a hydrogen peroxide-dependent artificial catalytic system based on myoglobin (Mb). The arrangements of amino acids at positions 29, 43, 64, and 68 were found to influence catalytic activity. An L29H/H64D/V68I myoglobin mutant showed the best catalytic efficiency (i.e., kcat/Km = 720.42 M-1 s-1) against SD. Next, mutant H64D/V68I showed the best degradation rate against SD (i.e., 91.45 ± 0.16%). Moreover, L29H/H64D/V68I Mb was found to efficiently catalyze ST oxidation (kcat/Km = 670.08 M-1 s-1), while H64D/V68I had the best degradation rate against ST (i.e., 99.45 ± 0.23%). Our results demonstrate that SAs can be efficiently degraded by artificial peroxygenases constructed using a myoglobin scaffold. This therefore provides a simple and economical method for the biodegradation of SD and ST.

Generation and validation of a myoglobin knockout zebrafish model.

Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.

Protective effect of thymoquinone on glycation of human myoglobin induced by d-ribose.

Nonenzymatic glycation and the subsequent accumulation of advanced glycation end-products (AGEs) in proteins are factors underlying long-term pathogenesis in diabetes. The study of protein glycation is crucial for elucidating their relationship with diabetes mellitus and related disorders. This study explores the interaction between d-ribose and human myoglobin (HMb), as well as the protective effect of thymoquinone (TQ) on glycation. A time-dependent in-vitro glycation study was performed to investigate the mechanism of d-ribose-induced structural interference of HMb in the absence and presence of TQ. Spectroscopic and proteomic analysis indicated that the presence of TQ significantly reduced the total amount of AGEs while maintaining structural characteristics of HMb. 14 glycated sites on HMb were further identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) after incubation with d-ribose for 12 h, predominantly interacting with lysine residues. TQ was found to disrupt this interaction, reducing the glycated sites from 14 to 12 sites and the percentage of glycated peptides from 26.50 % to 12.97 %. Additionally, there was a significant decrease in the degree of glycation at the same sites. In summary, our findings suggest that TQ has the potential to act as an anti-glycation agent and provide a comprehensive understanding underlying the inhibition mechanism of glycation.

Enhancing Supplemental Effects of Acute Natural Antioxidant Derived from Yeast Fermentation and Vitamin C on Sports Performance in Triathlon Athletes: A Randomized, Double-Blinded, Placebo-Controlled, Crossover Trial.

This study investigated the acute effects of natural antioxidants, derived from yeast fermentation containing glutathione and dietary vitamin C supplementation, on metabolic function, skeletal muscle oxygenation, cardiac function, and antioxidant function during submaximal exercise in middle-aged triathlon athletes. Twelve participants (aged 49.42 ± 5.9 years) completed 90 min submaximal cycling trials corresponding to 70% maximal oxygen uptake with either vitamin C and glutathione (VitC+Glu), vitamin C (VitC), glutathione (Glu) supplementation, or placebo. Metabolic function (minute ventilation, oxygen uptake, carbon dioxide output [VCO2], respiratory exchange ratio [RER], oxygen pulse [O2pulse], carbohydrate oxidation, fat oxidation, and energy expenditure), skeletal muscle oxygenation (oxidized hemoglobin and myoglobin in skeletal muscle tissue, total hemoglobin and myoglobin in skeletal muscle tissue [tHb]), cardiac function (heart rate [HR], stroke volume [SV], cardiac output, end-diastolic volume, end-systolic volume, and ejection fraction), and antioxidant function parameters (blood lactate, superoxide dismutase, catalase, glutathione peroxidases, glutathione [GSH], diacron reactive oxygen metabolite [dROM], and biological antioxidant potential [BAP]) were measured during submaximal exercise and recovery. VCO2, RER, HR, blood lactate after exercise, and dROM were significantly lower, and O2pulse, tHb, and BAP were significantly higher for VitC+Glu than for the other trials (p < 0.05). In conclusion, combined vitamin C and glutathione supplementation was more effective in improving metabolic function, skeletal oxygenation, cardiac function, and antioxidant function during prolonged submaximal exercise in middle-aged triathletes.

Generation of myoglobin (MB)-knockout human embryonic stem cell (hESC) line (KAIMRCe002-A-1S) using CRISPR/Cas9 technology.

Myoglobin (MB) is a cytoplasmic hemoprotein that is predominantly expressed in the heart and oxidative myofibers of skeletal muscle. It has been demonstrated that MB binds to oxygen and promotes its diffusion for energy production in the mitochondria. Recently, MB was found to be expressed in different forms of malignant tumors and cancer cell lines. Further studies using gene disruption technology will enhance the understanding of MB's role in human cardiovascular biology and cancers. Here, we describe the generation of a homozygous MB knockout in human embryonic stem cells (hESC-MB-/-) via CRISPR/Cas9 to study MB function in human biology and diseases.

The mechanism of Leuconostoc mesenteroides subsp. IMAU:80679 in improving meat color: Myoglobin oxidation inhibition and myoglobin derivatives formation based on multi enzyme-like activities.

The Leuconostoc mesenteroides subsp. IMAU:80679 (LM) was chosen for its superior capability in enhancing redness, and was incubated in a broth system containing metmyoglobin (MetMb) to investigate its mechanisms for color improvement. The a* value of LM group reached its highest level of 52.75 ± 1.04 at 24 h, significantly higher than control of 19.75 ± 0.6 (p < 0.05). The addition of LM could inhibit myoglobin oxidation to some extent. Meanwhile, higher content of nitrosylmyoglobin (NOMb) and Zn-protoporphyrin (Znpp) were observed in LM samples during the whole incubation period. Furthermore, enzymatic activity and encoded genes related to MetMb reduction and pigment formation were determined to explain its possible mechanism on color enhancement. Finally, by extracting crude enzymes and adding them to meat batters, the redness of crude enzyme group was comparable to that achieved with 20 ppm nitrite, providing a potential method on compensating for nitrite/nitrate substitution in meat products.

Do ultrasound form spontaneously nitrous pigments in nitrite-free pork meat batter?

The effects of ultrasound (US) on myoglobin modification, nitrous pigment formation, color, and total and free sulfhydryl content in nitrite-free pork meat batter were assessed. Five treatments were elaborated: Control (without US); TUS10'12 and TUS20'12 (sonication at 25 kHz, at 12 °C for 10 and 20 min, respectively); TUS10'18 and TUS20'18 (sonication at 25 kHz, at 18 °C for 10 and 20 min, respectively). Sonication for 20 min at 12 °C increased OxyMb and DeoxyMb pigments while reducing MetMb levels. This US condition also yielded higher red color indices and lower yellow color indices. Moreover, TUS20'12 exhibited enhanced nitrous pigment formation and decreased FerrylMb and free sulfhydryl (SH) values, indicating reduced oxidation in OxyMb and DeoxyMb pigments. In conclusion, the findings demonstrate that US can impart a cured color to nitrite-free meat products.

Nitrite Reductase Activity of Ferrous Nitrobindins: A Comparative Study.

Nitrobindins (Nbs) are all-ß-barrel heme proteins spanning from bacteria to Homo sapiens. They inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and promoting peroxynitrite isomerization to NO3-. Here, the nitrite reductase activity of Nb(II) from Mycobacterium tuberculosis (Mt-Nb(II)), Arabidopsis thaliana (At-Nb(II)), Danio rerio (Dr-Nb(II)), and Homo sapiens (Hs-Nb(II)) is reported. This activity is crucial for the in vivo production of NO, and thus for the regulation of blood pressure, being of the utmost importance for the blood supply to poorly oxygenated tissues, such as the eye retina. At pH 7.3 and 20.0 °C, the values of the second-order rate constants (i.e., kon) for the reduction of NO2- to NO and the concomitant formation of nitrosylated Mt-Nb(II), At-Nb(II), Dr-Nb(II), and Hs-Nb(II) (Nb(II)-NO) were 7.6 M-1 s-1, 9.3 M-1 s-1, 1.4 × 101 M-1 s-1, and 5.8 M-1 s-1, respectively. The values of kon increased linearly with decreasing pH, thus indicating that the NO2--based conversion of Nb(II) to Nb(II)-NO requires the involvement of one proton. These results represent the first evidence for the NO2 reductase activity of Nbs(II), strongly supporting the view that Nbs are involved in NO metabolism. Interestingly, the nitrite reductase reactivity of all-ß-barrel Nbs and of all-α-helical globins (e.g., myoglobin) was very similar despite the very different three-dimensional fold; however, differences between all-α-helical globins and all-ß-barrel Nbs suggest that nitrite reductase activity appears to be controlled by distal steric barriers, even though a more complex regulatory mechanism can be also envisaged.

TOM complex-independent transport pathway of myoglobin into mitochondria in C2C12 myotubes.

Recently, we found that myoglobin (Mb) localizes in both the cytosol and mitochondrial intermembrane space in rodent skeletal muscle. Most proteins of the intermembrane space pass through the outer mitochondrial membrane via the translocase of the outer membrane (TOM) complex. However, whether the TOM complex imports Mb remains unknown. The purpose of this study was to investigate the involvement of the TOM complex in Mb import into the mitochondria. A proteinase K protection assay of mitochondria from C2C12 myotubes confirmed that Mb integrated into the mitochondria. An immunoprecipitation assay verified the interaction of Mb and TOM complex receptors (Tom20, Tom70) in isolated mitochondria. The assay showed a clear interaction of Mb with Tom20 and Tom70. A knockdown experiment using siRNA for TOM complex receptors (Tom20, Tom70) and TOM complex channel (Tom40) did not alter the amount of Mb expression in the mitochondrial fraction. These results suggested that Mb does not necessarily require the TOM complex for mitochondrial import of Mb. Although the physiological role of Mb interactions with TOM complex receptors remains unclear, further studies are needed to clarify how Mb enters the mitochondria independently of the TOM complex.

Low myoglobin concentration in skeletal muscle of elite cyclists is associated with low mRNA expression levels.

Myoglobin is essential for oxygen transport to the muscle fibers. However, measurements of myoglobin (Mb) protein concentrations within individual human muscle fibers are scarce. Recent observations have revealed surprisingly low Mb concentrations in elite cyclists, however it remains unclear whether this relates to Mb translation, transcription and/or myonuclear content. The aim was to compare Mb concentration, Mb messenger RNA (mRNA) expression levels and myonuclear content within muscle fibers of these elite cyclists with those of physically-active controls. Muscle biopsies were obtained from m. vastus lateralis in 29 cyclists and 20 physically-active subjects. Mb concentration was determined by peroxidase staining for both type I and type II fibers, Mb mRNA expression level was determined by quantitative PCR and myonuclear domain size (MDS) was obtained by immunofluorescence staining. Average Mb concentrations (mean ± SD: 0.38 ± 0.04 mM vs. 0.48 ± 0.19 mM; P = 0.014) and Mb mRNA expression levels (0.067 ± 0.019 vs. 0.088 ± 0.027; P = 0.002) were lower in cyclists compared to controls. In contrast, MDS and total RNA per mg muscle were not different between groups. Interestingly, in cyclists compared to controls, Mb concentration was only lower for type I fibers (P < 0.001), but not for type II fibers (P > 0.05). In conclusion, the lower Mb concentration in muscle fibers of elite cyclists is partly explained by lower Mb mRNA expression levels per myonucleus and not by a lower myonuclear content. It remains to be determined whether cyclists may benefit from strategies that upregulate Mb mRNA expression levels, particularly in type I fibers, to enhance their oxygen supply.