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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., (© 2023. The Author(s).)

Cetaceans, early in their evolutionary history, had developed many physiological adaptations to secondarily return to the sea. Among these adaptations, changes in molecules that transport oxygen and that ultimately support large periods of acute tissue hypoxia probably represent one big step toward the conquest of aquatic environments. Myoglobin contributes to intracellular oxygen storage and transcellular diffusion of oxygen in muscle, and plays an important role in supplying oxygen in hypoxic or ischemic conditions. Here we looked for evidence of adaptive molecular evolution of myoglobin in the cetacean lineage, relative to their terrestrial counterparts. We performed a comparative analysis to examine the variation of the parameter ω (d N/d S) and infer past period of adaptive evolution during the cetacean transition from the terrestrial to the aquatic environment. We also analyzed the changes in amino acid properties. At the nucleotide level, the results showed significant differences in selective pressure between cetacean and non-cetacean myoglobin (ω value three times higher in cetaceans when compared to terrestrial mammals), and also among cetacean lineages according to their diving capacities. Interestingly, both families with long duration diving cetaceans present two parallel substitutions (on sites 4 and 12). Regarding the amino acid properties, our analysis identified four significant physicochemical amino acid changes among residues in myoglobin protein under positive destabilizing selection.

Respiratory proteins are responsible for transport and storage of oxygen. It is well established that specific requirements for oxygen during vertebrate ontogeny cause switches of hemoglobin chain expression. Here, we characterize the developmental profiles of zebrafish (Danio rerio) globins by means of quantitative real-time reverse transcription PCR. The total mRNA levels of the hemoglobin chains, including a newly identified embryonic α-chain, as well as myoglobin, neuroglobin, cytoglobin 1 and 2, and globin X were estimated. mRNAs of all globins were detectable in unfertilized eggs, suggesting maternal storage. Embryonic α- and β-hemoglobin mRNA peaked at hatching and the switch to adult hemoglobin expression occurred 16 dpf. Enhanced myoglobin mRNA levels were detected ~31 h post-fertilization (hpf), coinciding with the heart and the muscle development, while neuroglobin mRNA expression pattern correlates with the formation of the nervous system. Amounts of myoglobin and neuroglobin mRNA were similar within an order of magnitude throughout the ontogeny, tentatively supporting a respiratory role of neuroglobin. Cytoglobin 2 mRNA levels increased gradually, whereas cytoglobin 1 mRNA levels increased strongly after ~31 hpf, which is in agreement with a function in cell proliferation. Globin X mRNA level was highest in oocytes, but low in later stages. Together, these data suggest a specific role for each globin, which are also associated with certain events in fish development.

Variability profiles measured over a set of aligned sequences can be used to estimate evolutionary freedom to vary. Differences in variability profiles between clades can be used to identify shifts in function at the molecular level. We demonstrate such a shift between the alpha and beta subunits of hemoglobin. We also show that the variability profiles for myoglobin are different between whales and primates and speculate that the differences between the two clades may reflect a shift associated with the novel oxygen storage demands in the lineage leading to whales. We discuss the relationship between sequence variability and "evolutionary opportunity" and explore the utility of Maynard Smith's multidimensional evolutionary opportunity space metaphor for exploring functional constraints, genetic redundancy, and the context dependency of the genotype-phenotype map. This work has implications for quantitatively defining and comparing protein function. Supplementary data is available from bioinfo.mbb.yale. edu/align.

This report presents the first teleost myoglobin (Mb) genomic DNA sequence, several features of which are distinct from mammalian Mb genes. We have isolated and compared genomic clones of three closely related Antarctic icefish: Chionodraco rastrospinosus, which expresses Mb mRNA and functional polypeptide; Champsocephalus gunnari, which transcribes the Mb gene but does not produce polypeptide due to a 5-base pair (bp) frameshift insertion; and Chaenocephalus aceratus, which lacks both Mb protein and mRNA. The single-copy icefish Mb gene contains three introns: two at positions identical to those found in mammalian Mb and a novel intron located in the 5' untranslated region three nucleotides upstream from the initiator codon. All three introns are shorter than those found in mammalian Mb genes and exhibit a considerably higher A+T content. The entire Mb transcriptional unit is intact in C. aceratus, indicating that the failure to express this gene is not due to aberrations in the coding region, splice junctions, polyadenylation signals, or core promoter elements. The three icefish Mb sequences display an extreme degree of identity in the transcriptional unit and putative promoter region. In contrast, sequences 65 bp downstream from the polyadenylation site bear no homology among the three species, demonstrating that rapid sequence change has occurred in the 1 million years since the divergence of these species.

We determined the myoglobin cDNA sequence for seven Antarctic notothenioid fish species. These data identify mutations in the myoglobin gene for Champsocephalus gunnari and Pagetopsis macropterus, two icefish species that lack detectable quantities of the polypeptide but express myoglobin mRNA. a third species lacking myoglobin polypeptide, Chaenocephalus aceratus, is devoid of myoglobin mRNA and accordingly failed to produce myoglobin products on polymerase chain reaction (PCR) amplification. Myoglobin cDNA sequences were highly conserved among the species the express the protein, particularly in the coding region. Sequence variation among the myoglobin-expressing channichthyid species was 2.0% to 2.9% in the coding region and 2.6% to 3.3% over the entire cDNA. The same extent of variation, 1.6% to 3.2% in the coding sequence and 2.8% to 3.7% overall, was observed between the icefishes and more distantly related, red-blooded nototheniid species. The two species expressing mutant myoglobin mRNA, C. gunnari and P. macropterus, exhibited the highest degree of sequence variation among the fish myoglobins examined. Drift in the myoglobin sequence in these two species, and conservation of myoglobin cDNA among fishes from two distinct families, suggest that a selective pressure operates to maintain myoglobin in the species that express the protein.

To evaluate the relative importance of positive selection and neutral drift from the nucleotide base changes observed in the homologous alignment of genes, a theoretical equation of base changes is formulated by including both the influence of selection and the base substitutions due to mutations. Under the assumption that the average rate of base substitutions estimated from synonymous changes is the "true" mutation rate applicable at all positions, this method is applied to the vertebrate globin gene family, and evaluates the departures of base change rates from the "true" mutation rate at the first and second codon positions as a consequence of preferential selection for the conservation of important function. In addition to the strong effect of selection on the amino acid residues in the internal region mostly common to myoglobin and hemoglobin chains, the distinctive directions of selective parameter values are seen at sites on the globin surface, distinguishing the subunit contact residues of hemoglobins from the polar residues on the surface of myoglobins. Moreover, this effect of selection distinguishing between the myoglobin and hemoglobin chain genes becomes weaker in cold-blooded vertebrates, especially in fish, strongly suggesting the possibility that the clear distinction between these globins is a result of selection out of the changes regarded as neutral ones in an ancestor of vertebrates. Thus, the present method may also serve to investigate the homology of many other proteins from the aspect of molecular evolution, mainly focusing on the evolution of their biological functions.

Given that the analysis of a single gene does not necessarily provide unambiguous phylogenetic information, it is important to scrutinize as many genes as possible. Using the maximum likelihood method, particularly suitable for a total evidence approach, we evaluated the phylogenetic information provided by the 12S and 16S rRNA, cytochrome b, and myoglobin sequence data in order to resolve one of the most debated phylogenetic questions: the relationships among the major groups of cetaceans. Our analysis strongly supports the hypothesis that sperm whales are closer to baleen whales than to dolphins.

The intracellular localization of myoglobin mRNA in the skeletal muscles of normal subjects was examined by in situ hybridization using a biotin-labeled cDNA probe. By phase-contrast microscopy, myoglobin mRNA signals were demonstrated to be located preferentially on the A-band. Two different methods of tissue preparation, i.e., pre-embedding method and post-embedding method, were used for the electron microscopic study. With the pre-embedding method, only a few gold particles were found to be associated with cytoskeletal filaments in the intermyofibrillar space. With the post-embedding method, superior preservation of sections and higher signal intensities were obtained. Although most of the gold particles were localized on the A-band, some were seen in other regions; i.e., in the intermyofibrillar space, perinuclear space, or the I-band, where myoglobin is localized. These findings suggest that myoglobin is primarily synthesized on the A-band, where ribosomes predominantly exist, although myoglobin is also localized on the I-band. The predominant localization of myoglobin mRNA on the A-band may aid in the mRNA transcription and may be related to the regulation of myoglobin synthesis in skeletal muscle cells.

Quantitative microcomplement fixation tests employing rabbit antisera were done to compare immunologically 13 cetacean myoglobins and 15 mammalian lysozymes c of known amino acid sequence. In both cases there was a strong correlation between immunological distance (y) and percent sequence difference (x), as had been found for several other globular proteins. For myoglobin the relationship could be described by y = 10.5x and for lysozyme by y = 8.5x. The coefficients in both of these equations are appreciably higher than the values of 5.1-6.9 reported for three other vertebrate globular proteins (bird lysozyme c, mammalian ribonuclease, and mammalian serum albumin), and they imply that rabbit antisera to mammalian myoglobins and lysozymes are more sensitive to evolutionary substitutions. A strong inverse correlation (r = -0.95) was found when the slope of the line relating y to x for these five data sets was plotted against the percent sequence difference between the rabbit's own protein and the proteins immunized with. Specifically, the cetacean myoglobins on average differ in amino acid sequence from rabbit myoglobin by less than 13% and exhibit the steepest slope (10.5), while bird lysozyme sequences differ by nearly 40% from rabbit lysozyme and exhibit the shallowest slope (5.1).

The evolutionary relation of vertebrate myoglobin and the hemoglobin chains including the agnathan hemoglobin chain is investigated on the basis of a new view of amino acid changes that is developed by canonical discriminant analysis of amino acid residues at individual sites. In contrast to the clear discrimination of amino acid residues between myoglobin, hemoglobin alpha chain, and hemoglobin beta chain in warm-blood vertebrates, the three types of globins in the lower class of vertebrates show so much variation that they are not well discriminated. This is seen particularly at the sites that are ascertained in mammals to carry the amino acid residues participating in stabilizing the monomeric structure in myoglobin and the residues forming the subunit contacts in hemoglobin. At these sites, agnathan hemoglobin chains are evaluated to be intermediate between the myoglobin and hemoglobin chains of gnathostomes. The variation in the phylogenetically lower class of globins is also seen in the internal region; there the amino acid residues of myoglobin and hemoglobin chains in the phylogenetically higher class exhibit an example of parallel evolution at the molecular level. New quantities, the distance of sequence property between discriminated groups and the variation within each group, are derived from the values of discriminant functions along the peptide chain, and this set of quantities simply describes an overall feature of globins such that the distinction between the three types of globins has been clearer as the vertebrates have evolved to become jawed, landed, and warm-blooded. This result strongly suggests that the functional constraint on the amino acid sequence of a protein is changed by living conditions and that severe conditions constitute a driving force that creates a distinctive protein from a less-constrained protein.

Multivariate statistical analyses were applied to 16 physical and chemical properties of amino acids. Four of these properties; volume, polarity, isoelectric point (charge), and hydrophobicity were found to explain adequately 96% of the total variance of amino acid attributes. Using these four quantitative measures of amino acid properties, a structural discriminate function in the form of a weighted difference sum of squares equation was developed. The discriminate function is weighted by the location of each particular residue within a given tertiary structure and yields a numerical discriminate or difference value for the replacement of these residues by different amino acids. This resulting discriminate value represents an expression of the perturbation in the local positional environment of a protein when an amino acid substitution occurs. With the use of this structural discriminate function, a residue by residue comparison of the known mammalian myoglobin sequences was carried out in an attempt to elucidate the positions of possible deviations from the known tertiary structure of sperm whale myoglobin. Only 11 of the 153 residue positions in myoglobin demonstrated possible structural deviations. From this analysis, indices of difference were calculated for all amino acid exchanges between the various myoglobins. All comparisons yielded indices of difference that were considerably lower than would be expected if mutations had been fixed at random, even if the organization of the genetic code is taken into consideration. On the basis of these results, it is inferred that some form of selection has acted in the evolution of mammalian myoglobins to favor amino acid substitutions that are compatible with the retention of the original conformation of the protein.

Amino acid sequence determination of elephant myoglobin revealed the presence of the unusual substitution E7 His leads to Gln. Stereochemical analyses suggest that the most suitable residue which can functionally substitute for His at this position in vertebrate globins is Gln. Physiochemical studies imply that the slower rate of autooxidation of elephant myoglobin is the result of this substitution which may confer some selective advantage on the species. Comparative sequence data of paenungulate myoglobins suggest that the His leads to Gln mutation probably occurred in an ancestor of Elephantinae.

The complete amino acid sequence of the major component myoglobin from killer whale, Orcinus orca, was determined by automated Edman degradation. In this study residue 122 was found to be glutamic acid instead of glutamine as was originally reported (Castillo et al. 1977). This reassignment affects the phylogenetic relationship of killer whale myoglobin with the myoglobins from other closely related cetacean species and also affects studies concerned with the physical parameters of the protein.

We have examined the extensive amino acid sequence data now available for five protein families - the alpha crystallin A chain, myoglobin, alpha and beta hemoglobin, and the cytochromes c - with the goal of estimating the true spatial distribution of base substitutions within genes that code for proteins. In every case the commonly used Poisson density failed to even approximate the experimental pattern of base substitution. For the 87 species of beta hemoglobin examined, for example, the probability that the observed results were from a Poisson process was the minuscule 10(-44). Analogous results were obtained for the other functional families. All the data were reasonably, but not perfectly, described by the negative binomial density. In particular, most of the data were described by one of the very simple limiting forms of this density, the geometric density. The implications of this for evolutionary inference are discussed. It is evident that most estimates of total base substitutions between genes are badly in need of revision.