Masticatory Myosin Research Articles

388. Hypersensitivity of MYH16 Fibers (M Fibers) to Myodegeneration in Canine Muscular Dystrophy

Golden Retriever Muscular Dystrophy (GRMD) dogs frequently develop severe restriction to mouth opening, a condition known as trismus, as one of their earliest physical signs of the disease. Trismus is generally due to fibrosis and/or tonic contraction of the muscles of mastication, the temporalis and the masseter muscle. These muscles are primarily composed of unique myofibers exhibiting a masticatory myosin heavy chain encoded by the MYH 16 gene (M fibers). M fibers have been shown to produce the highest specific force among mammalian myofibers, based on their atypical crossbridge kinetics. Here, we show for a first time a morphological analysis of M fibers for the purposes of their classification, distribution and proportion in the masticatory muscles in normal and GRMD dogs based on immunofluorescence staining by a highly specific anti-MYH16 antibody. Our data demonstrate that in both temporalis and masseter muscles, the predominant M fibers are larger in size when compared to any other striated myofiber. A comparative histological analysis of these and other muscles in GRMD dogs demonstrates a significant relative increase in the proportion of injured, hypercontracted M fibers, a highly significant relative abundance of regenerating fibers expressing the embryonic myosin isoform encoded by the MYH3 gene, and prominent fibrosis when compared to the other affected striated muscles. These results suggest that the higher specific force developed by M fibers, due to the MYH16 encoded MyHC isoform, accelerates the fibers’ rate of degeneration in response to myonecrotizing sarcolemmal injury in the absence of dystrophin. Trismus is not seen in Duchenne MD, suggesting that the deletional frameshift in the human MYH16 gene (Nature 428: 373-4, 415-8) dramatically reduces the relative rate of sarcolemmal injury. These observations may offer novel insights into the physiological role of dystrophin in striated muscles, and provide an especially sensitive test for use in experimental therapeutics in the canine models for DMD.

Insights into hominin phenotypic and dietary evolution from ancient DNA sequence data

Nuclear genome sequence data from Neandertals, Denisovans, and archaic anatomically modern humans can be used to complement our understanding of hominin evolutionary biology and ecology through i) direct inference of archaic hominin phenotypes, ii) indirect inference of those phenotypes by identifying the effects of previously-introgressed alleles still present among modern humans, or iii) determining the evolutionary timing of relevant hominin-specific genetic changes. Here we review and reanalyze published Neandertal and Denisovan genome sequence data to illustrate an example of the third approach. Specifically, we infer the timing of five human gene presence/absence changes that may be related to particular hominin-specific dietary changes and discuss these results in the context of our broader reconstructions of hominin evolutionary ecology. We show that pseudogenizing (gene loss) mutations in the TAS2R62 and TAS2R64 bitter taste receptor genes and the MYH16 masticatory myosin gene occurred after the hominin-chimpanzee divergence but before the divergence of the human and Neandertal/Denisovan lineages. The absence of a functional MYH16 protein may explain our relatively reduced jaw muscles; this gene loss may have followed the adoption of cooking behavior. In contrast, salivary amylase gene (AMY1) duplications were not observed in the Neandertal and Denisovan genomes, suggesting a relatively recent origin for the AMY1 copy number gains that are observed in modern humans. Thus, if earlier hominins were consuming large quantities of starch-rich underground storage organs, as previously hypothesized, then they were likely doing so without the digestive benefits of increased salivary amylase production. Our most surprising result was the observation of a heterozygous mutation in the first codon of the TAS2R38 bitter taste receptor gene in the Neandertal individual, which likely would have resulted in a non-functional protein and inter-individual PTC (phenylthiocarbamide) taste sensitivity variation, as also observed in both humans and chimpanzees.

Patterns of tropomyosin and troponin-T isoform expression in jaw-closing muscles of mammals and reptiles that express masticatory myosin

We recently reported that masticatory ('superfast') myosin is expressed in jaw-closing muscles of some rodent species. Most mammalian limb muscle fibers express tropomyosin-β (Tm-β), along with fast-type or slow-type tropomyosin-β (Tm-β), but jaw-closing muscle fibers in members of Carnivora express a unique isoform of Tm [Tm-masticatory (Tm-M)] and little or no Tm-β. The goal of this study was to determine patterns of Tm and troponin-T (TnT) isoform expression in the jaw-closing muscles of rodents and other vertebrate species that express masticatory myosin, and compare the results to those from members of Carnivora. Comparisons of electrophoretic mobility, immunoblotting and mass spectrometry were used to probe the Tm and fast-type TnT isoform composition of jaw-closing and limb muscles of six species of Carnivora, eight species of Rodentia, five species of Marsupialia, big brown bat, long-tailed macaque and six species of Reptilia. Extensive heterogeneity exists in Tm and TnT isoform expression in jaw-closing muscles between phylogenetic groups, but there are fairly consistent patterns within each group. We propose that the differences in Tm and TnT isoform expression patterns between phylogenetic groups, which share the expression of masticatory myosin, may impart fundamental differences in thin-filament-mediated muscle activation to accommodate markedly different feeding styles that may require high force generation in some species (e.g. many members of Carnivora) and high speed in others (e.g. Rodentia).

Regulation of Jaw-specific Isoforms of Myosin-binding Protein-C and Tropomyosin in Regenerating Cat Temporalis Muscle Innervated by Limb Fast and Slow Motor Nerves

Cat jaw-closing muscles are a distinct muscle allotype characterized by the expression of masticatory-specific myofibrillar proteins. Transplantation studies showed that expression of masticatory myosin heavy chain (m-MyHC) is promoted by fast motor nerves, but suppressed by slow motor nerves. We investigated whether masticatory myosin-binding protein-C (m-MBP-C) and masticatory tropomyosin (m-Tm) are similarly regulated. Temporalis muscle strips were transplanted into limb muscle beds to allow innervation by fast or slow muscle nerve during regeneration. Regenerated muscles were examined postoperatively up to 168 days by peroxidase IHC using monoclonal antibodies to m-MyHC, m-MBP-C, and m-Tm. Regenerates in both muscle beds expressed fetal and slow MyHCs, m-MyHC, m-MBP-C, and m-Tm during the first 4 weeks. Longer-term regenerates innervated by fast nerve suppressed fetal and slow MyHCs, retaining m-MyHC, m-MBP-C, and m-Tm, whereas fibers innervated by slow nerve suppressed fetal MyHCs and the three masticatory-specific proteins, induced slow MyHC, and showed immunohistochemical characteristics of jaw-slow fibers. We concluded that expression of m-MBP-C and m-Tm is coregulated by m-MyHC and that neural impulses to limb slow muscle are capable of suppressing masticatory-specific proteins and to channel gene expression along the jaw-slow phenotype unique to jaw-closing muscle.

The myosin light chain 1 isoform associated with masticatory myosin heavy chain in mammals and reptiles is embryonic/atrial MLC1

We recently reported that masticatory myosin heavy chain (MHC-M) is expressed as the exclusive or predominant MHC isoform in masseter and temporalis muscles of several rodent species, contrary to the prevailing dogma that rodents express almost exclusively MHC isoforms that are typically found in fast limb muscles and not masticatory myosin. We also reported that the same rodent species express the embryonic/atrial isoform of myosin light chain 1 (MLC1E/A) in jaw-closing muscles and not a unique masticatory MLC1 isoform that others have reported as being expressed in jaw-closing muscles of carnivores that express MHC-M. The objective of this study was to test the hypothesis that MLC1E/A is consistently expressed in jaw-closing muscles whenever MHC-M is expressed as the predominant or exclusive MHC isoform. Jaw-closing muscles, fast and slow limb muscles, and cardiac atria and ventricles of 19 species (six Carnivora species, one Primates species, one Chiroptera species, five marsupial species, an alligator and five turtle species) were analyzed using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA sequencing. Gel electrophoresis and immunoblotting indicate that MHC-M is the exclusive or predominant MHC isoform in the jaw-closing muscles of each of the studied species. The results from all of the approaches collectively show that MLC1E/A is exclusively or predominantly expressed in jaw-closing muscles of the same species. We conclude that MLC1E/A is the exclusive or predominant MLC1 isoform that is expressed in jaw-closing muscles of vertebrates that express MHC-M, and that a unique masticatory isoform of MLC1 probably does not exist.

Tropomyosin and Troponin-T Isoform Differences in Jaw-Closing Muscles of Rodentia and Carnivora that Express Masticatory Myosin

We recently reported that, contrary to prevailing dogma, masticatory (“superfast”) myosin is expressed in jaw-closing muscles of some members of Rodentia (Reiser et al., J. Exp. Biol. 212:2511-2519, 2009). Whereas virtually all mammalian limb muscle fibers express tropomyosin-β (Tm-β), along with fast-type or slow-type Tm-α, jaw-closing muscle fibers that express masticatory myosin in members of Carnivora express a unique isoform of Tm-α and do not express Tm-β (Rowlerson et al., Biochem. Biophys. Res. Commun. 113:519-525, 1983; Hoh et al., Proc. Aust. Physiol. Pharmacol. Soc. 20:192P, 1989). The goal of this study was to examine thin filament protein isoform composition in jaw-closing muscles of rodents that express masticatory myosin and compare the results to those from members of Carnivora. Tm or troponin-T (TnT) specific antibodies and immunoblotting were used to probe homogenates of limb and jaw-closing muscles of six species of Rodentia and three species of Carnivora. The results verify the almost exclusive expression of Tm-α, presumably the unique isoform reported earlier, in the jaw-closing muscles of Carnivora and reveal that members of Rodentia express both Tm-α and Tm-β in jaw-closing muscles, similar to limb muscles in the same species. The results also indicate that the same complement of TnT isoforms are expressed in jaw-closing and limb muscles of Carnivora, but differ markedly between the two muscle groups in Rodentia. Fast-type TnC and TnI appear to be expressed in jaw-closing muscles of both orders. It is postulated that the differences in Tm and TnT isoform expression patterns between Carnivora and Rodentia may impart fundamental differences in calcium-sensitivity of force generation to accommodate markedly different feeding styles, with shared expression of masticatory myosin, between these two animal orders. Supported by the National Science Foundation.

Protruding masticatory (superfast) myosin heads from staggered thick filaments of dog jaw muscle revealed by X-ray diffraction

To characterize the structure of jaw muscle fibres expressing masticatory (superfast) myosin, X-ray diffraction patterns of glycerinated fibres of dog masseter were compared with those of dog tibialis anterior in the relaxed state. Meridional reflections of masseter fibres were laterally broad, indicating that myosin filaments are staggered along the filament axis. Compared with tibialis anterior fibres, the peak of the first myosin layer line of masseter fibres was lower in intensity and shifted towards the meridian, while lattice spacings were larger at a similar sarcomere length. These suggest that the myosin heads of masticatory fibres are mobile, and tend to protrude from the filament shaft towards actin filaments. Lowering temperature or treating with N-phenylmaleimide shifted the peak of the first myosin layer line of tibialis anterior fibres towards the meridian and the resulting profile resembled that of masseter fibres. This suggests that the protruding mobile heads in the non-treated masticatory fibres are in the ATP-bound state. The increased population of weakly binding cross-bridges may contribute towards the high specific force of masticatory fibres during contraction. Electron micrographs confirmed the staggered alignment of thick filaments along the filament axis within sarcomeres of masticatory fibres, a feature that may confer efficient force development over a wide range of the sarcomere lengths.

Masticatory (`superfast') myosin heavy chain and embryonic/atrial myosin light chain 1 in rodent jaw-closing muscles

Masticatory myosin is widely expressed among several vertebrate classes. Generally, the expression of masticatory myosin has been associated with high bite force for a carnivorous feeding style (including capturing/restraining live prey), breaking down tough plant material and defensive biting in different species. Masticatory myosin expression in the largest mammalian order, Rodentia, has not been reported. Several members of Rodentia consume large numbers of tree nuts that are encased in very hard shells, presumably requiring large forces to access the nutmeat. We, therefore, tested whether some rodent species express masticatory myosin in jaw-closing muscles. Myosin isoform expression in six Sciuridae species was examined, using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA analysis. The results indicate that masticatory myosin is expressed in some Sciuridae species but not in other closely related species with similar diets but having different nut-opening strategies. We also discovered that the myosin light chain 1 isoform associated with masticatory myosin heavy chain, in the same four Sciuridae species, is the embryonic/atrial isoform. We conclude that rodent speciation did not completely eliminate masticatory myosin and that its persistent expression in some rodent species might be related to not only diet but also to feeding style.

Masticatory myosin unveiled: first determination of contractile parameters of muscle fibers from carnivore jaw muscles

Masticatory myosin heavy chain (M MyHC) is a myosin subunit isoform with expression restricted to muscles derived from the first branchial arch, such as jaw-closer muscles, with pronounced interspecies variability. Only sparse information is available on the contractile properties of muscle fibers expressing M MyHC (M fibers). In this study, we characterized M fibers isolated from the jaw-closer muscles (temporalis and masseter) of two species of domestic carnivores, the cat and the dog, compared with fibers expressing slow or fast (2A, 2X, and 2B) isoforms. In each fiber, during maximally calcium-activated contractions at 12 degrees C, we determined isometric-specific tension (P(o)), unloaded shortening velocity (v(o)) with the slack test protocol, and the rate constant of tension redevelopment (K(TR)) after a fast shortening-relengthening cycle. At the end of the mechanical experiment, we identified MyHC isoform composition of each fiber with gel electrophoresis. Electrophoretic migration rate of M MyHC was similar in both species. We found that in both species the kinetic parameters v(o) and K(TR) of M fibers were similar to those of 2A fibers, whereas P(o) values were significantly greater than in any other fiber types. The similarity between 2A and M fibers and the greater tension development of M fibers were confirmed also in mechanical experiments performed at 24 degrees C. Myosin concentration was determined in single fibers and found not different in M fibers compared with slow and fast fibers, suggesting that the higher tension developed by M fibers does not find an explanation in a greater number of force generators. The specific mechanical characteristics of M fibers might be attributed to a diversity in cross-bridge kinetics.

Autoantibodies in Canine Masticatory Muscle Myositis Recognize a Novel Myosin Binding Protein-C Family Member

Inflammatory myopathies are a group of autoimmune diseases that affect muscles. In humans, the most common inflammatory myopathies are polymyositis, dermatomyositis, and inclusion body myositis. Autoantibodies may be found in humans with inflammatory myopathies, and these play an important role in diagnosis and disease classification. However, these Abs are typically not muscle specific. Spontaneously occurring canine inflammatory myopathies may be good parallel disorders and provide insights into human myositis. In dogs with inflammatory myopathy, muscle-specific autoantibodies have been found, especially in masticatory muscle myositis. We have identified the major Ag recognized by the autoantibodies in canine masticatory muscle myositis. This Ag is a novel member of the myosin binding protein-C family, which we call masticatory myosin binding protein-C (mMyBP-C). mMyBP-C is localized not only within the masticatory muscle fibers, but also at or near their cell surface, perhaps making it accessible as an immunogen. The gene for mMyBP-C also exists in humans, and mMyBP-C could potentially play a role in certain human inflammatory myopathies. Understanding the role of mMyBP-C in this canine inflammatory myopathy may advance our knowledge of mechanisms of autoimmune inflammatory muscle diseases, not only in dogs, but also in humans.

Cross-bridge kinetics of fast and slow fibres of cat jaw and limb muscles: correlations with myosin subunit composition

Mechanical properties of the jaw-closing muscles of the cat are poorly understood. These muscles are known to differ in myosin and fibre type compositions from limb muscles. This work aims to correlate mechanical properties of single fibres in cat jaw and limb muscles with their myosin subunit compositions. The stiffness minimum frequency, f(min), which reflects isometric cross-bridge kinetics, was measured in Ca(2+)-activated glycerinated fast and slow fibres from cat jaw and limb muscles for temperatures ranging between 15 and 30 degrees C by mechanical perturbation analysis. At 15 degrees C, f(min) was 0.5 Hz for limb-slow fibres, 4-6 Hz for jaw-slow fibres, and 10-13 Hz for limb-fast and jaw-fast fibres. The activation energy for f(min) obtained from the slope of the Arrhenius plot for limb-slow fibres was 30-40% higher than values for the other three types of fibres. SDS-PAGE and western blotting using highly specific antibodies verified that limb-fast fibres contained IIA or IIX myosin heavy chain (MyHC). Jaw-fast fibres expressed masticatory MyHC while both jaw-fast and jaw-slow fibres expressed masticatory myosin light chains (MLCs). The nucleotide sequences of the 3' ends of the slow MyHC cDNAs isolated from cat masseter and soleus cDNA libraries showed identical coding and 3'-untranslated regions, suggesting that jaw-slow and limb-slow fibres express the same slow MyHC gene. We conclude that the isometric cross-bridge cycling kinetics of jaw-fast and limb-fast fibres detected by f(min) are indistinguishable in spite of differences in MyHC and light chain compositions. However, jaw-slow fibres, in which the same slow MyHCs are found in combination with MLCs of the jaw type, show enhanced cross-bridge cycling kinetics and reduced activation energy for cross-bridge detachment.

Structure and Function of Masticatory (Superfast) Myosin

Masticatory (superfast) myosin is specifically expressed in jaw-closing muscles and is found in carnivorous lower vertebrates and several orders of mammals. In humans, this myosin is not expressed in those whose masticatory myosin heavy chain gene is defective due to a frame-shift deletion. This myosin has a very ancient phylogenetic origin, and is considered as a distinct subclass (labeled as II M) of vertebrate striated myosins, its heavy chain exhibiting a less than 70% sequence homology to other known striated muscle myosin heavy chains.ATPase activity of masticatory myosin is higher than that of fast myosin, and fibers expressing masticatory myosin exhibit a higher maximal tension than fast or slow fibers and intermediate shortening velocity between those of fast and slow limb fibers. These characteristics are considered very appropriate in carnivores for catching and grasping prey, and in folivores for the mastication of tough vegetable matter.Regarding the structural aspect, myosin heads of masticatory fibers are reported to be mobile and protrude more from the thick filaments toward actin by X-ray diffraction experiments at the synchrotron facility. Electron micrographs of masticatory fibres showed that the sarcomere structure of fibers is disordered and the M-line is barely discernible. These structural characteristics may explain the unique contracting properties.

Structure and Function of Masticatory (Superfast) Myosin

Structure and Function of Masticatory (Superfast) Myosin

1P170 咀嚼筋ミオシンの力学的特性(筋肉 : 筋蛋白・収縮)

1P170 咀嚼筋ミオシンの力学的特性(筋肉 : 筋蛋白・収縮)

A distinct subclass of mammalian striated myosins: structure and molecular evolution of "superfast" or masticatory myosin heavy chain.

"Superfast" or masticatory myosin is the molecular motor in the powerful and specialized jaw-closing muscles of carnivores, folivores, and frugivores. This myosin presumably underpins the unusual high force and moderate shortening velocity of muscle fibers expressing it. Here, we report the cloning and sequencing of the cDNA encoding the full-length masticatory myosin heavy chain (MyHC) from cat temporalis muscle. This was obtained by immunoscreening a cDNA expression library and RACE-PCR (rapid amplification of cDNA ends-PCR). Sequence comparisons at the DNA and amino acid levels show that masticatory MyHC has less than 70% homology to known striated MyHCs, compared with 87-96% between other mammalian fast isoforms themselves. Nucleotide substitution rates at the nonsynonymous sites between masticatory MyHC and other mammalian striated MyHCs are considerably higher than between these striated MyHCs themselves. Phylogenetic analysis revealed that masticatory MyHC diverged from invertebrate MyHC before the avian cardiac MyHC subclass and the mammalian fast/developmental and slow/cardiac MyHC subclasses. Masticatory MyHC is thus a distinct new subclass of vertebrate striated myosins. The early divergence from invertebrate MyHC, combined with immunochemical evidence of its expression in reptilian and shark jaw-closing muscles, suggests that masticatory MyHC evolved in early gnathostomes, driven by benefits derived from powerful jaw closure. During the mammalian radiation, some taxa continued to express it, while others adapted to new types of food and eating habits by replacing masticatory MyHC with more appropriate isoforms normally found in limb and cardiac muscles.

'Superfast' or masticatory myosin and the evolution of jaw-closing muscles of vertebrates.

There are four fibre types in mammalian limb muscles, each expressing a different myosin isoform that finely tunes fibre mechanics and energetics for locomotion. Functional demands on jaw-closer muscles are complex and varied, and jaw muscles show considerable phylogenetic plasticity, with a repertoire for myosin expression that includes limb, developmental, alpha-cardiac and masticatory myosins. Masticatory myosin is a phylogenetically ancient motor with distinct light chains and heavy chains. It confers high maximal muscle force and power. It is highly jaw-specific in expression and is found in several orders of eutherian and marsupial mammals including carnivores, chiropterans, primates, dasyurids and diprotodonts. In exceptional species among these orders, masticatory myosin is replaced by some other isoform. Masticatory myosin is also found in reptiles and fish. It is postulated that masticatory myosin diverged early during gnathostome evolution and is expressed in primitive mammals. During mammalian evolution, mastication of food became important, and in some taxa jaw closers replaced masticatory myosin with alpha-cardiac, developmental, slow or fast limb myosins to adapt to the variety of diets and eating habits. This occurred early in some taxa (rodents, ungulates) and later in others (macropods, lesser panda, humans). The cellular basis for the uniqueness of jaw-closing muscles lies in their developmental origin.