Myoglobin causes oxidative stress,increase of NO production and dysfunction of kidney's mitochondria

Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis. Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferryl-myoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial dysfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions release from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology.     

Molecular cloning,characterization and expression of myoglobin in Tibetan antelope (Pantholops hodgsonii), a species with hypoxic tolerance

The Tibetan antelope (Pantholops hodgsonii) is a hypoxia-tolerant species that lives at an altitude of 4000–5000 m above sea level on the Qinghai–Tibetan plateau. Myoglobin is an oxygen-binding cytoplasmic hemoprotein that is abundantly expressed in oxidative skeletal and cardiac myocytes. Numerous studies have implicated that hypoxia regulates myoglobin expression to allow adaptation to conditions of hypoxic stress. Few studies have yet looked at the effect of myoglobin on the adaptation to severe environmental stress on TA. To investigate how the Tibetan antelope (TA) has adapted to a high altitude environment at the molecular level, we cloned and analyzed the myoglobin gene from TA, compared the expression of myoglobin mRNA and protein in cardiac and skeletal muscle between TA and low altitude sheep. The results indicated that the full-length myoglobin cDNA is composed of 1154 bp with a 111 bp 5′ untranslated region (UTR), a 578 bp 3′ UTR and a 465 bp open reading frame (ORF) encoding a polypeptide of 154 amino acid residues with a predicted molecular weight of 17.05 kD. The TA myoglobin cDNA sequence and the deduced amino acid sequence were highly homologous with that of other species. When comparing the myoglobin sequence from TA with the Ovis aries myoglobin sequence, variations were observed at codons 21 (GGT → GAT) and 78 (GAA → AAG), and these variations lead to changes in the corresponding amino acids, i.e., Gly → Asp and Glu → Lys, respectively. But these amino acid substitutions are unlikely to effect the ability of binding oxygen because their location is less important, which is revealed by the secondary structure and 3D structure of TA myoglobin elaborated by homology modeling. However, the results of myoglobin expression in cardiac and skeletal muscles showed that they were both significantly higher than that in plain sheep not only in mRNA but also protein level. We speculated that the higher expression of myoglobin in TA cardiac and skeletal muscles improves their ability to obtain and store oxygen under hypoxic conditions. This study indicated that TA didn't improve the ability of carrying oxygen by changing the molecular structure of myoglobin, but through increasing the expression of myoglobin in cardiac and skeletal muscles.     

Chimeras vs X inactivation mosaics: significance of differences in pigment distribution

Cell cultures of cardiac, pectoral, and thigh muscle of chick embryos synthesized myoglobin, as measured by incorporation of radioactive lysine detected by radioimmunoprecipitation. Liver and skin cultures, although active in protein synthesis, failed to demonstrate myoglobin synthesis. Puromycin inhibited myoglobin synthesis by the cell cultures. The electrophoretic characteristics of the myoglobin antigen synthesized by thigh and pectoral muscle were identical. Myglobin synthesizing progenitor cells attached to plastic dishes in 1 hr, but not completely in 0.5 hr. Cells, unattached at 0.5 hr, were enriched in myoglobin synthesizing cells. Incorporation of lysine-U-¹⁴C into myoglobin was maximal in confluent cultures and its increase paralleled the increase of cell fusion in the cultures. The ability of pectoral, white muscle to synthesize myoglobin in a manner equivalent to that of cardiac tissue was unexpected because of its failure to synthesize myoglobin in vivo and may indicate that factors in the whole organism may regulate the expression of this muscle cell's capabilities.     

In silico analysis of Myoglobin in Channa striata

Myoglobin is a cytoplasmic hemoprotein, expressed solely in cardiac myocytes and oxidative skeletal muscle fibers, that reversibly binds O₂ by its heme residue. Myoglobin is an essential oxygen-storage hemoprotein capable of facilitating oxygen transport and modulating nitric oxide homeostasis within cardiac and skeletal myocytes. Functionally, myoglobin is well accepted as an O₂- storage protein in muscle, capable of releasing O₂ during periods of hypoxia or anoxia. There is no evidence available regarding active sites, ligand binding sites, antigenic determinants and the ASA value of myoglobin in Channa striata. We further document the predicted active sites in the structural model with solvent exposed ASA residues. During this study, the model was built by CPH program and validated through PROCHECK, Verify 3D, ERRAT and ProSA for reliability. The active sites were predicted in the model with further ASA analysis of active site residues. The discussed information thus provides the predicted active sites, ligand binding sites, antigenic determinants and ASA values of myoglobin model in Channa striata.     

Embryonic synthesis of myoglobin in vivo estimated by radioimmunoassay

Chick embryos in ovo incorporated radioactivity from lysine-U-¹⁴C into myoglobin, as measured by an immunoprecipitation technique. The most consistent results were obtained by injection of the precursor into the yolk sac fluid.Incorporation, or apparent myoblobin synthesis, occurred in cardiac and skeletal muscle but not in liver, although incorporation of amino acid into total soluble proteins was equivalent in all tissues studied. Synthesis was highest in cardiac muscle and appeared there first in younger embryos. Myoglobin synthesis was detectable in the heart of embryos as early as 6 days of age and rose with age thereafter. Myoglobin synthesis appeared later and at lower levels in skeletal muscle.In vitro at neutral pH, tissue extracts of liver and muscle possessed only slight properties of myoglobin degradation.Using nonradioactive precipitin techniques, sensitive to 5–10 μg/ml, myoglobin was detected in embryonic heart muscle by week 2 of life and rose in content thereafter. Two of 8 embryos had trace amounts in thigh muscle near the time of hatching, and no embryos possessed measurable amounts of myoglobin in liver tissue or in pectoral skeletal muscle. Adult birds possessed equivalent amounts of myoglobin in heart and thigh muscle while pectoral muscle and liver tissue had no detectable myoglobin content.     

Physical conditioning: Effect on the myoglobin concentration in skeletal and cardiac muscle of bar-headed geese

• 1.1. A 12 week program of treadmill exercise (0.7 m/sec, 30 min per day, five days per week), significantly increased the myoglobin concentration of the femorotibialis medius muscle in bar-headed geese as compared to nonexercised controls.
• 2.2. The myoglobin concentration differed among various muscles within a bird. The highest myoglobin concentrations were found in the primary flight muscle, the pectoralis major, and in cardiac muscle.
• 3.3. By physically conditioning their muscles, bar-headed geese may improve the oxygen flow to mitochondria and, thereby, enhance their ability to exercise under conditions of low oxygen partial pressures.

     

Amino Acid Sequence, Spectral, Oxygen-Binding, and Autoxidation Properties of Indoleamine Dioxygenase-Like Myoglobin from the Gastropod Mollusc Turbo cornutus

Myoglobin was isolated from the radular muscle of the archaeogastropod mollusc Turbo cornutus (Turbinidae). This myoglobin is a monomer carrying one protoheme group; the molecular mass was estimated by SDS–PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA-derived amino acid sequence of 375 residues was determined, of which 327 residues were identified directly by chemical sequencing of internal peptides. The amino acid sequence of Turbo myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 36% identity with the myoglobin from Sulculus diversicolor (Haliotiidae) and 27% identity with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. Thus, the Turbo myoglobin can be counted among the myoglobins which evolved from the same ancestor as that of indoleamine 2,3-dioxygenase. The absorbance ratio of γ to CT maximum (γ/CT) of Turbo metmyoglobin was 17.8, indicating that this myoglobin probably possesses a histidine residue near the sixth coordination position of heme iron. The Turbo myoglobin binds oxygen reversibly. Its oxygen equilibrium properties are similar to those of Sulculus myoglobin, giving P ₅₀ = 3.5 mm Hg at pH 7.4 and 20°C. The pH dependence of autoxidation of Turbo oxymyoglobin was quite different from that of mammalian myoglobin, suggesting a unique protein folding around the heme cavity of Turbo myoglobin. A kinetic analysis of autoxidation indicates that the amino acid residue with pK _a = 5.4 is involved in the reaction. The autoxidation reaction was enhanced markedly at pH 7.6, but not at pH 5.5 and 6.3 in the presence of tryptophan. We suggest that a noncatalytic binding site for tryptophan, in which several dissociation groups with pK _a ≥ 7.6 are involved, remains in Turbo myoglobin as a relic of molecular evolution.     

藏羚羊与藏绵羊心肌、骨骼肌中肌红蛋白含量和乳酸脱氢酶活性的比较

为了探讨藏羚羊(Pantholops hodgsonii)对低氧环境的适应机制。以生活在同海拔(4 300 m)的藏绵羊(Tibetan Sheep)为对照,用分光光度法测定2种动物心肌、骨骼肌中肌红蛋白(myoglobin,Mb)含量、乳酸(lactic acid,LD)含量及乳酸脱氢酶(lactate dehydrogenase,LDH)活力。结果显示,藏羚羊心肌和骨骼肌中Mb含量明显高于藏绵羊(P0.05),但心肌和骨骼肌的Mb含量无差别(P0.05),而藏绵羊心肌Mb含量明显高于骨骼肌(P0.05);藏羚羊心肌和骨骼肌中LD含量及LDH活力明显低于藏绵羊(P0.05),且2种动物心肌中的LDH活力均明显低于其骨骼肌(P0.01)。结果表明,藏羚羊尽管生活在高寒缺氧地区,其心肌和骨骼肌细胞仍然能得到丰富的氧供应,并非处于缺氧状态,这可能是通过增加心肌和骨骼肌中Mb的含量,提高其在低氧环境获取和储存氧的能力,从而提高有氧获能水平。与之相反,藏绵羊尽管也生活在高寒缺氧地区,但其心肌和骨骼肌中Mb含量相对于藏羚羊较低,且LD含量和LDH活力较高,说明其心肌和骨骼肌细胞内氧供不如藏羚羊丰富,提示藏绵羊可能主要以糖酵解获能。我们推测这种差异可能与两种动物不同的运动习性密切相关,且认为藏羚羊较高的Mb含量可能是其适应高原缺氧条件的分子基础之一。     

The synthetic polyphenol tert-butyl-bisphenol inhibits myoglobin-induced dysfunction in cultured kidney epithelial cells

Abstract

Rhabdomyolysis caused by severe burn releases extracellular myoglobin (Mb) that accumulates in the kidney and urine (maximum [Mb] ～ 50 μM) (termed myoglobinuria). Extracellular Mb can be a pro-oxidant. This study cultured Madin-Darby-canine-kidney-Type-II (MDCK II) cells in the presence of Mb and tested whether supplementation with a synthetic tert-butyl-polyphenol (tert-butyl-bisphenol; t-BP) protects these renal cells from dysfunction. In the absence of t-BP, cells exposed to 0–100 μM Mb for 24 h showed a dose-dependent decrease in ATP and the total thiol (TSH) redox status without loss of viability. Gene expression of superoxide dismutases-1/2, haemoxygenase-1 and tumour necrosis factor increased and receptor-mediated endocytosis of transferrin and monolayer permeability decreased significantly. Supplementation with t-BP before Mb-insult maintained ATP and the TSH redox status, diminished antioxidant/pro-inflammatory gene responses, enhanced monolayer permissiveness and restored transferrin uptake. Overall, bolstering the total antioxidant capacity of the kidney may protect against oxidative stress induced by experimental myoglobinuria.     

Optical measurements of intracellular oxygen concentration of rat heart in vitro

The optical characteristics of hemoglobin-free perfused rat heart have been examined in detail. Ethyl hydrogen peroxide is found to convert myoglobin into “ferryl compound” in the perfused heart, as is also seen in vitro. After pretreatment with ethyl hydrogen peroxide, a typical mitochondrial absorption spectrum, similar to that of isolated rat heart mitochondria, is obtained in perfused heart. The overall absorption spectrum of the heart obtained by the aerobic to anaerobic transition is a superposition of the mitochondrial spectrum on that of myoglobin. By comparing these spectra, it is found that measurement of cytochrome a + a₃ at 605–620 nm is possible in spite of the absorbance change due to the oxygenation-deoxygenation of myoglobin, whereas the wavelength pairs for cytochrome c at 550-540 nm, cytochrome b at 562–575 nm and cytochrome a + a₃ at 445–450 nm can not be used in the heart because of interference from the absorption change of myoglobin. The partial pressure of O₂ (P₅₀) which is required for half maximal deoxygenation (or oxygenation) of myoglobin in perfused heart is found to be 2.4 mm Hg at room temperature and the Hill constant, n, is 1.1; these values are similar to those of myoglobin purified from rat heart. The steady-state O₂ titration has been performed by using absorbancy changes of myoglobin and cytochrome a + a₃ as intracellular O₂ indicators. In the perfused heart, the percentage change of oxygenation-deoxygenation of myoglobin parallels the oxidation-reduction of cytochrome a + a₃, while the mixture of purified myoglobin and isolated mitochondria shows a deviation, reflecting the difference of O₂ affinities between myoglobin and cytochrome a + a₃. The results indicate that there may be an O₂ gradient between cytosolic and mitochondrial compartments in the hemoglobin-free perfused heart. The absorption changes of myoglobin and of cytochrome a + a₃ can be measured in a single contraction-relaxation cycle. A triple beam method was introduced to eliminate the effect of light scattering changes in these measurements. The results demonstrated that myoglobin is more oxygenated during the systolic and diastolic periods and deoxygenated in the resting period, whereas cytochrome a + a₃ is more reduced in systole and diastole and oxidized in the resting state. Changing the perfusion conditions greatly alters the time course of the events which occur during the contraction-relaxation cycle of the perfused heart.     

Hypoxia-induced left ventricular dysfunction in myoglobin-deficient mice

Myoglobin-deficient mice are viable and have preserved cardiac function due to their ability to mount a complex compensatory response involving increased vascularization and the induction of the hypoxia gene program (hypoxia-inducible factor-1alpha, endothelial PAS, heat shock protein27, etc.). To further define and explore functional roles for myoglobin, we challenged age- and gender-matched wild-type and myoglobin-null mice to chronic hypoxia (10% oxygen for 1 day to 3 wk). We observed a 30% reduction in cardiac systolic function in the myoglobin mutant mice exposed to chronic hypoxia with no changes observed in the wild-type control hearts. The cardiac dysfunction observed in the hypoxic myoglobin-null mice was reversible with reexposure to normoxic conditions and could be prevented with treatment of an inhibitor of nitric oxide (NO) synthases. These results support the conclusion that hypoxia-induced cardiac dysfunction in myoglobin-null mice occurs via a NO-mediated mechanism. Utilizing enzymatic assays for NO synthases and immunohistochemical analyses, we observed a marked induction of inducible NO synthase in the hypoxic myoglobin mutant ventricle compared with the wild-type hypoxic control ventricle. These new data establish that myoglobin is an important cytoplasmic cardiac hemoprotein that functions in regulating NO homeostasis within cardiomyocytes.     

Molecular characterisation and expression of Atlantic cod (Gadus morhua) myoglobin from two populations held at two different acclimation temperatures

Much previous research has demonstrated the plasticity of myoglobin concentrations in both cardiac and skeletal myocytes in response to hypoxia and training. No study has yet looked at the effect of thermal acclimation on myoglobin in fish. Atlantic cod (Gadus morhua) from two different populations, i.e. the North Sea and the North East Arctic, were acclimated to 10 and 4 degrees C. Both the myoglobin mRNA and myoglobin protein in cod hearts increased significantly by up to 3.7 and 2.3 fold respectively as a result of acclimation to 4 degrees C. These increments were largest in the Arctic population, which in earlier studies have been shown to possess cold compensated metabolic demands at low temperatures. These metabolic demands associated with higher mitochondrial capacities may have driven the increase in cardiac myoglobin concentrations, in order to support diffusive oxygen supply. At the same time the increase in myoglobin levels may serve further functions during cold acclimation, for example, protection of the cell against reactive oxygen species, and scavenging nitric oxide, thereby contributing to the regulation of mitochondrial volume density.     

Clinical and analytical performance of automated immunochemiluminometric assays for determination of cardiac markers in serum

Three new immunochemiluminometric assays for quantitation of cardiac markers, i.e. creatine kinase isoenzyme MB (CK-MB), myoglobin and cardiac troponin I (cTnI), were evaluated with the Sanofi Access analyser. The complete profile requires 20 min to perform, the method being suitable in true stat situations. In patients with early myocardial infarction (median time of sample collection: 210 min from onset, range 30–450; n = 44), the diagnostic sensitivity of Access cTnI was 66%, compared with 80% for myoglobin, and 43% for CK-MB. For comparison, cTnI, with an automated immunofluorimetric assay was also measured (sensitivity, 45%; p < 0.05 vs. Access cTnI). Our data confirmed myoglobin as the first biochemical marker to appear elevated after infarction. However, cTnI may be a more sensitive marker for early detection of cardiac damage than initially thought, when determined by an ultrasensitive method such as an immunochemiluminometric assay. © 1998 John Wiley & Sons, Ltd.     

Absence of water at the sixth co-ordination site in ferric Aplysia myoglobin

The acidic ferric form of hemoglobin and myoglobin carries a water molecule as the sixth ligand of the iron atom. On the other hand, Aplysia met myoglobin shows a pentaco-ordinated active site below the pK of the acid-alkaline transition (7.5). This finding rationalizes some peculiar properties of Aplysia myoglobin as compared with sperm whale myoglobin.     

Myocardial oxygenation in isolated hearts predicted by an anatomically realistic microvascular transport model

An anatomically realistic model for oxygen transport in cardiac tissue is introduced for analyzing data measured from isolated perfused guinea pig hearts. The model is constructed to match the microvascular anatomy of cardiac tissue based on available morphometric data. Transport in the three-dimensional system (divided into distinct microvascular, interstitial, and parenchymal spaces) is simulated. The model is used to interpret experimental data on mean cardiac tissue myoglobin saturation and to reveal differences in tissue oxygenation between buffer-perfused and red blood cell-perfused isolated hearts. Interpretation of measured mean myoglobin saturation is strongly dependent on the oxygen content of the perfusate (e.g., red blood cell-containing vs. cell-free perfusate). Model calculations match experimental values of mean tissue myoglobin saturation, measured mean myoglobin, and venous oxygen tension and can be used to predict distributions of intracellular oxygen tension. Calculations reveal that approximately 20% of the tissue is hypoxic with an oxygen tension of <0.5 mmHg when the buffer is equilibrated with 95% oxygen to give an arterial oxygen tension of over 600 mmHg. The addition of red blood cells to give a hematocrit of only 5% prevents tissue hypoxia. It is incorrect to assume that the usual buffer-perfused Langendorff heart preparation is adequately oxygenated for flows in the range of < or =10 ml. min-1. ml tissue-1.     

Pulmonary vein isolation of symptomatic refractory paroxysmal and persistent atrial fibrillation

Aim. To investigate long-term outcome and to determine predictors of successful pulmonary vein isolation (PVI) in patients with symptomatic paroxysmal or persistent atrial fibrillation (AF) who are refractory or intolerant to antiarrhythmic drugs. Background. The treatment of AF has traditionally been pharmacological aimed at rate or rhythm control. However, rhythm control remains difficult to establish. PVI is reported to be effective in selected patient groups. Methods. Ninety-nine consecutive patients with a mean age of 54±10 years who had paroxysmal or persistent AF were treated in the University Medical Center Groningen. All patients underwent PVI by the same electrophysiologist. Successful PVI was defined as absence of AF on Holter or electrocardiogram (ECG), and no symptoms of AF. Results. After six months of follow-up, 60 (61%) patients were free of AF episodes, both on 96-hour Holter monitoring and on ECGs, and had no symptoms related to AF. Thirty-nine of these 60 patients (65%) were no longer treated with any class I or III antiarrhythmic drugs. Independent determinants of successful PVI were paroxysmal AF (OR 18 [3.5–93], p=0.001), and left pulmonary vein ablation time >55 minutes (OR 15 [2.7–81], p=0.002). Left atrial (parasternal view 42±6 vs. 40±5 mm, p<0.05 and apical view 61±9 vs. 58±8 mm, p<0.05) and right atrial (59±7 vs. 56±5 mm, p<0.05) sizes decreased significantly in the successfully treated patients after six months of follow-up. Conclusion. Independent determinants of a successful outcome after PVI are paroxysmal AF and a longer left atrial ablation time. (Neth Heart J 2009;17:366–72.)     

Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.     

An unusual ‘bow tie’ image in pacemaker implantation

Upper venous system anatomic variations may cause difficulties during cardiac pacemaker implantation. Persistent left superior vena cava (PLSVC) and absent right superior vena cava could be an arrhythmogenic source of atrial arrhythmias and cardiac conduction disease. We represent dual-chamber pacemaker implantation in a patient with a very rare upper venous system anomaly, paroxysmal atrial fibrillation, sick sinus syndrome, that cause unusual fluoroscopic image.