Major Myosin Heavy Chain Isoforms Research Articles

Fiber-type phenotype of the jaw-closing muscles in Gorilla gorilla, Pan troglodytes, and Pan paniscus: A test of the Frequent Recruitment Hypothesis

Skeletal muscle fiber types are important determinants of the contractile properties of muscle fibers, such as fatigue resistance and shortening velocity. Yet little is known about how jaw-adductor fiber types correlate with feeding behavior in primates. Compared with chimpanzees and bonobos, gorillas spend a greater percentage of their daily time feeding and shift to herbaceous vegetation when fruits are scarce. We thus used the African apes to test the hypothesis that chewing with unusually high frequency is correlated with the expression in the jaw adductors of a high proportion of type 1 (slow, fatigue-resistant) fibers at the expense of other fiber types (the Frequent Recruitment Hypothesis). We used immunohistochemistry to determine the presence and distribution of the four major myosin heavy chain (MHC) isoforms in the anterior superficial masseter (ASM), superficial anterior temporalis, and deep anterior temporalis of four Gorilla gorilla, two Pan paniscus, and four Pan troglodytes. Serial sections were stained against slow (MHC-1/-α-cardiac) and fast (MHC-2/-M) fibers. Fibers were counted and scored for staining intensity, and fiber cross-sectional areas (CSAs) were measured and used to estimate percentage of CSA of each MHC isoform. Hybrid fibers accounted for nearly 100% of fiber types in the masseter and temporalis of all three species, resulting in three main hybrid phenotypes. As predicted, the gorilla ASM and deep anterior temporalis comprised a greater percentage of CSA of the slower, fatigue-resistant hybrid fiber type, significantly so for the ASM (p = 0.015). Finally, the results suggest that fiber phenotype of the chewing muscles contributes to behavioral flexibility in ways that would go undetected in paleontological studies relying solely on morphology of the bony masticatory apparatus.

A novel MYH2 mutation in family members presenting with congenital myopathy, ophthalmoplegia and facial weakness.

Myosin heavy chain (MyHC) is a major structural component of the striated muscle contractile apparatus. In adult human limb skeletal muscle, there are three major MyHC isoforms, slow/beta cardiac MyHC, MyHC IIa and MHC IIx, which are important for the functional characteristics of different muscle fiber types. Hereditary myosin myopathies have emerged as an important group of diseases with variable clinical and morphological expression dependent on the mutated isoform, and also the type and location of the mutation. Myosin myopathy with external ophthalmoplegia is associated with mutations in MYH2, encoding for MyHC IIa that is mainly expressed in type 2A muscle fibers and is inherited in dominant as well as recessive manner. We present a family with myopathy with early onset proximal muscle weakness, facial muscle involvement and ophthalmoplegia. Muscle biopsy demonstrated lack of type 2A muscle fibers and genetic work up demonstrated that the disease was caused by a novel recessive MYH2 mutation: c.1009-1G>A resulting in skipping of exon 12, which is predicted to result in a frame shift and introducing at premature stop codon at position 347 (p.Ser337Leufs*11).

Myosin Heavy Chain-2b Transcripts and Isoform Are Expressed in Human Laryngeal Muscles

Three fast myosin heavy chain (MyHC) isoforms, i.e. MyHC-2a, -2x and -2b, are expressed in skeletal muscles of smaller mammals. In contrast, only MyHC-2a and -2x have been revealed in humans so far. The expression of MyHC isoforms is known to be wider in the functionally more specialized laryngeal muscles. Though mRNA transcripts of the MyHC-2b gene were found to be expressed in certain human skeletal and laryngeal muscles, the corresponding isoform has not been demonstrated in these muscles. To our knowledge, we are the first to demonstrate not only the expression of MyHC-2b transcripts using an in situ hybridization technique but also the corresponding protein, i.e. the MyHC-2b isoform, in some human laryngeal muscles by immunohistochemistry but not by polyacrylamide gel electrophoresis. Using a set of antibodies specific to MyHC isoforms, we demonstrated that MyHC-2b was always co-expressed with the major MyHC isoforms, not only with the fast ones (MyHC-2a and -2x) but with the slow isoform (MyHC-1) as well.

Identification of myosin heavy chain isoforms in porcine longissimus dorsi muscle by electrophoresis and mass spectrometry

Myosin heavy chain (MHC) isoforms have been considered as makers for muscle fiber types in relation to meat quality, whereas MHC isoforms in porcine skeletal muscle have not been fully identified. The improved technique of SDS-PAGE and 2DE were used to separate porcine MHC isoforms. Western blotting with monoclonal antibodies including BA-F8 (anti-MHC slow/I), SC-71 (anti-MHC 2a and 2x), 10F5 (anti-MHC 2b), and BF-35 (anti-MHC slow/I and 2a) and MS were used to confirm MHC migration rate and identify MHC isoforms from separated bands and spots. Up to 45% w/v of glycerol, 8% w/v of acrylamide content, and 25 h of electrophoretic time at 70 V allowed a clear separation of MHC isoforms. Major MHC isoforms such as slow, 2a, 2x, and 2b were clearly separated by SDS-PAGE. A total of 23 MHC spots were separated and identified by 2DE and MS. Therefore, four MHC isoforms such as slow/I, 2a, 2x, and 2b could be identified by the improved SDS-PAGE technique, 2DE and MS. Therefore, these techniques allow more accurate and accessible analysis in muscle fiber typing and in relationship between MHC isoforms, muscle fiber characteristics, and pork quality.

Electrophoretic Mobility of Cardiac Myosin Heavy Chain Isoforms Revisited: Application of MALDI TOF/TOF Analysis

The expression of two cardiac myosin heavy chain (MyHC) isoforms in response to the thyroid status was studied in left ventricles (LVs) of Lewis rats. Major MyHC isoform in euthyroid and hyperthyroid LVs had a higher mobility on SDS-PAGE, whereas hypothyroid LVs predominantly contained a MyHC isoform with a lower mobility corresponding to that of the control soleus muscle. By comparing the MyHC profiles obtained under altered thyroid states together with the control soleus, we concluded that MyHCα was represented by the lower band with higher mobility and MyHCβ by the upper band. The identity of these two bands in SDS-PAGE gels was confirmed by western blot and mass spectrometry. Thus, in contrast to the literature data, we found that the MyHCα possessed a higher mobility rate than the MyHCβ isoform. Our data highlighted the importance of the careful identification of the MyHCα and MyHCβ isoforms analyzed by the SDS-PAGE.

Characterization of the Human Skeletal Muscle Proteome by One-dimensional Gel Electrophoresis and HPLC-ESI-MS/MS

Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.

Myosin heavy chain–based fiber types in the adult human cricopharyngeus muscle

The cricopharyngeus (CP) muscle is a major component of the upper sphincter of the esophagus. Its physiology is complex; a variety of reflexes maintain CP sustained contraction except during swallowing, when it relaxes to allow a food bolus to pass into the esophagus. In order to understand CP function, we previously studied the normal adult human CP and found that it has an unusual layered structure, with a slow inner and fast outer layer. In addition, a majority of its muscle fibers express unusual myosin heavy chain (MHC) isoforms (slow-tonic, alpha-cardiac, neonatal, and embryonic) as well as the major MHC isoforms (types I, IIa, and IIx). In this study, autopsied adult human CP muscles were studied with immunocytochemical techniques to determine the patterns of MHC coexpression in CP muscle fibers. The results show that CP fibers were hybrids expressing from two to six MHC isoforms. Ten different combinations of MHC isoforms were identified in CP fibers, with the most common (54%) containing three MHC isoforms. The variety of hybrid CP fiber types suggests that the CP is capable of a wide range of contraction characteristics. Determination of MHC expression patterns of the CP muscle fibers is critical for evaluating the contractile properties of the sphincter.

Intrinsic Properties of the Adult Human Mylohyoid Muscle: Neural Organization, Fiber-Type Distribution, and Myosin Heavy Chain Expression

The mylohyoid (MH) muscle plays a critical role in chewing, swallowing, respiration, and phonation. The present study was designed to test the hypothesis that the functional properties of the MH are reflected by its intrinsic specializations, including the neural organization, fiber-type distribution, and myosin heavy chain (MHC) expression. Adult human MH muscles were investigated to determine the nerve supply pattern using Sihler's stain, banding pattern and types of motor endplates using acetylcholinesterase (AChE) staining and silver impregnation, and muscle fiber type and MHC composition using immunocytochemical and immunoblotting techniques. The adult human MH was found to have the following neuromuscular specializations. First, the muscle was innervated by several branches of the MH nerve derived from the mandibular division of the trigeminal nerve. Each of the nerve branches supplied a distinct region of the muscle, forming a segmental innervation pattern. Second, the MH had a single motor endplate band which was located in the middle of the muscle length. Both en plaque and en grappe types of motor endplates were identified on the MH muscle fibers. Finally, the adult human MH fibers expressed unusual MHC isoforms (i.e., slow-tonic, alpha-cardiac, embryonic, and neonatal) which coexisted with the major MHC isoforms (i.e., slow type I, fast type IIa, and fast type IIx), thus forming various major/unusual (or m/u) MHC hybrid fiber types. The m/u hybrid fibers (84% of the total fiber population) were the predominant fiber types in the adult MH muscle. Determination of the neuromuscular specializations of the MH is helpful for better understanding of the muscle functions and for development of strategies to treat MH-related upper airway disorders.

ACaenorhabditis elegans model of the myosin heavy chain IIa E706R mutation

Mutations in myosin heavy chain (MyHC) genes recently have been shown to be associated with various forms of congenital myopathies: myosin myopathies. The MyHC IIa E706K mutation is associated with congenital joint contractures, early-onset muscle weakness, and progressive course with moderate to severe muscle weakness later in life. To study the pathogenicity of this MyHC mutation, we investigated the effect of the corresponding mutation (E710K) in the major MyHC isoform (MyHC B) of the body wall muscle of the nematode Caenorhabditis elegans. Worms with null mutations in the MyHC B gene (unc-54) are severely paralyzed and depleted of thick filaments in the body wall muscle sarcomeres. unc-54 null mutants with extrachromosomal arrays of a gene construct including the entire wild-type unc-54 gene were partially rescued as determined by a motility assay and by morphological analysis of the body wall muscle. Analysis of unc-54 null mutants with extrachromosomal arrays of the unc-54 gene with the E710K mutation were severely paralyzed but showed formation of thick filaments in the body wall muscle. We conclude that the MyHC E706K (E710K in C. elegans) mutation is pathogenic and that the effect is primarily functional rather than structural because thick filaments are formed. The C. elegans model may be useful to study suspected pathogenic mutations in MyHC genes associated with human muscle diseases.

Adult human pharyngeal muscle fibers express some unusual myosin-heavy chain isoforms

Problem: Fiber type and myosin-heavy chain (MHC) composition in the human pharyngeal constrictor (PC) and upper esophageal sphincter (UES) muscles were analyzed to better understand the muscle functions. Methods: Twelve adult human PC and UES muscles obtained from autopsies were studied using immunohistochemical and immunoblotting techniques. For comparison, masseter and biceps brachii muscles of the same subjects served as controls. Serial cross-sections were incubated with a panel of isoform-specific anti-MHC monoclonal antibodies. Results: The human PC and UES muscles were composed of 2 histochemically defined fiber layers, a slow inner layer (SIL) (78% type I) and a fast outer layer (FOL) (59% type II). The adult PC and UES fibers expressed unusual MHC isoforms (ie, slow-tonic, alpha-cardiac, embryonic, and neonatal) that were also identified in the masseter, but not in the biceps brachii. These unusual MHC isoforms demonstrated by immunocytochemistry were confirmed by electrophoretic immunoblotting. The slow-tonic and alpha-cardiac fibers were concentrated mainly in the SIL, whereas the developmental MHC-containing fibers were located primarily in the FOL. The unusual MHC isoforms were coexpressed with the major MHC isoforms (ie, slow type I and fast type II), thus forming various major/unusual (m/u) MHC hybrid fiber types. The slow-tonic and alpha-cardiac MHC isoforms coexisted mainly with type I MHC, whereas the embryonic and neonatal MHC isoforms were coexpressed primarily with type IIa MHC. At least 8 m/u MHC hybrid fiber types were identified in these muscles. Conclusion: The multiplicity of MHC isoforms in the PC and UES fibers is believed to be related to embryonic origin, innervation, and unique functional requirements. Significance: Age-related changes in the fiber type and MHC composition in the human PCs and UES may be associated with pathogeneses of dysphagia and obstructive sleep apnea that are commonly seen in the elderly. Support: None reported.

Adult human mylohyoid muscle fibers express slow-tonic, alpha-cardiac, and developmental myosin heavy-chain isoforms.

Some adult cranial muscles have been reported to contain unusual myosin heavy-chain (MHC) isoforms (i.e., slow-tonic, alpha-cardiac, embryonic, and neonatal), which exhibit distinct contractile properties. In this study, adult human mylohyoid (MH) muscles obtained from autopsies were investigated to detect the unusual MHC isoforms. For comparison, the biceps brachii and masseter muscles of the same subjects were also examined. Serial cross-sections from the muscles studied were incubated with a panel of isoform-specific anti-MHC monoclonal antibodies that distinguish major and unusual MHC isoforms. On average, the slow type I and fast type II MHC-containing fibers in the MH muscle accounted for 54% and 46% of the fibers, respectively. In contrast to limb and trunk muscles, the adult human MH muscle was characterized by a large proportion of hybrid fibers (85%) and a small percentage of pure fibers (15%; P < 0.01). Of the fast fiber types, the proportion of the type IIa MHC-containing fibers (92%) was much greater than that of the type IIx MHC-containing fibers (8%; P < 0.01). Our data demonstrated that the adult human MH fibers expressed the unusual MHC isoforms that were also identified in the masseter, but not in the biceps brachii. These isoforms were demonstrated by immunocytochemistry and confirmed by electrophoretic immunoblotting. Fiber-to-fiber comparisons showed that the unusual MHC isoforms were coexpressed with the major MHC isoforms (i.e., MHCI, IIa, and IIx), thus forming various major/unusual (or m/u) MHC hybrid fiber types. Interestingly, the unusual MHC isoforms were expressed in a fiber type-specific manner. The slow-tonic and alpha-cardiac MHC isoforms were coexpressed predominantly with slow type I MHC isoform, whereas the developmental MHC isoforms (i.e., embryonic and neonatal) coexisted primarily with fast type IIa MHC isoform. There were no MH fibers that expressed exclusively unusual MHC isoforms. Approximately 81% of the slow type I MHC-containing fibers expressed slow-tonic and alpha-cardiac MHC isoforms, whereas 80% of the fast type IIa MHC-containing fibers expressed neonatal MHC isoform. The m/u hybrid fibers (82% of the total fiber population) were found to constitute the predominant fiber types in the adult human MH muscle. At least seven m/u MHC hybrid fiber types were identified in the adult human MH muscle. The most common m/u hybrid fiber types were found to be the MHCI/slow-tonic/alpha-cardiac and MHCIIa/neonatal, which accounted for 39% and 33% of the total fiber population, respectively. The multiplicity of MHC isoforms in the adult MH fibers is believed to be related to embryonic origin, innervation pattern, and unique functional requirements.

Endogenous expression and localization of myostatin and its relation to myosin heavy chain distribution in C2C12 skeletal muscle cells.

Myostatin is a negative regulator of skeletal muscle growth. We have previously reported that recombinant myostatin protein inhibits DNA and protein synthesis in C2C12 cells. Our objective was to assess if C2C12 cells express myostatin, determine its sub-cellular localization and the developmental stage of C2C12 cells in which myostatin mRNA and protein are expressed. To study the endogenous expression of myostatin, C2C12 myoblasts were allowed to progress to myotubes, and changes in the levels of endogenous myostatin mRNA expression were determined by RT-PCR. The myostatin protein and the two major myosin heavy chain (MHC) isoforms (MHC-I and -II) were determined by Western blot. Confirmation of the relative MHC expression patterns was obtained by a modified polyacrylamide gel electropheretic (PAGE) procedure. Imunofluorescence staining was employed to localize the site of myostatin expression and the relative distribution of the MHC isoforms. Co-expression of these proteins was studied using a dual staining approach. Expression of myostatin mRNA was found in myotubes but not in myoblasts. Myostatin protein was seen in most but not all, of the nuclei of polynucleated fibers expressing MHC-II, and myostatin was detected in the cytoplasm of myotube. The localization of myostatin protein in myotube nuclei was confirmed by Western blot of isolated nuclear and cytoplasmic fractions. Incubation of C2C12 myotubes with graded doses of dexamethasone dose-dependently increased the intensity of nuclear myostatin immunostaining and also resulted in the appearance of cytoplasmic expression. In conclusion, myostatin was expressed mostly in C2C12 myotubes nuclei expressing MHC-II. Its predominant nuclear localization suggests that it may play a role in transcriptional regulation.

Myosin isoforms in mammalian skeletal muscle

Skeletal muscles of different mammalian species contain four major myosin heavy-chain (MHC) isoforms: the "slow" or beta-MHC and the three "fast" IIa-, IIx-, and IIb-MHCs; and three major myosin light-chain (MLC) isoforms, the "slow" MLC1s and the two "fast" MLC1f and MLC3f. The differential distribution of the MHCs defines four major fiber types containing a single MHC isoform and a number of intermediate hybrid fiber populations containing both beta/slow- and IIa-MHC, IIa- and IIx-MHC, or IIx- and IIb-MHC. The IIa-, IIx-, and IIb-MHCs were first detected in neonatal muscles, and their expression in developing and adult muscle is regulated by neural, hormonal, and mechanical factors. The transcriptional mechanisms responsible for the fiber type-specific regulation of MHC and MLC gene expression are not known and are presently being explored by in vivo transfection experiments. The functional role of MHC isoforms has been in part clarified by correlated biochemical-physiological studies on single skinned fibers: these studies, in agreement with results from in vitro motility assays, indicate that both MHC and MLC isoforms determine the maximum velocity of shortening of skeletal muscle fibers.

Metabolic and contractile protein expression in developing rat diaphragm muscle

Progressive changes in myosin isozyme expression and in energy-generating enzyme activities were followed in the diaphragm and, for comparison, in axial and appendicular muscles of rats from 18 d gestation to maturity. Native myosins were characterized by pyrophosphate gel electrophoresis. Myosin heavy-chain (MHC) isozymes were measured with ELISA using monoclonal antibodies and were localized by immunocytochemistry. RNA transcripts for the MHCs were demonstrated on Northern blots and by RNase protection assays. Quantitative activities of malate dehydrogenase (MDH), beta-hydroxyacyl CoA dehydrogenase (beta OAC), 1-phosphofructokinase (PFK), lactate dehydrogenase (LDH), creatine kinase (CK), and adenylokinase (AK) were measured in muscle homogenates and in individual fibers by fluorometric pyridine nucleotide-dependent assays. Compared to limb muscles, expression of neonatal myosin in the diaphragm is precocious. Neonatal MHC mRNA is prominent in the diaphragm at 19 d gestation, and neonatal myosin is the major MHC isoform present at birth. Slow and fast IIa MHCs are also present at birth. Transcripts for IIa MHC are detectable in the diaphragm at 21 d gestation and are upregulated at birth. Comparable signal for IIa MHC mRNA is not found in the gastrocnemius until 10 d postpartum. Adult fast IIb MHC mRNA was detected only as a faint signal at 30-40 d in the diaphragm and then disappeared. Results indicate that a separate phenotype, the IIx type, matures late in diaphragmatic development. The activities of enzymes representing all of the major energy pathways are higher in the fetal diaphragm than in the fetal hindlimb muscles. For example, beta OAC had sixfold higher activity in the diaphragm than in the extensor digitorum longus (EDL) muscle at birth, activity in the diaphragm than in the extensor digitorum longus (EDL) muscle at birth.

The unc-45 gene of Caenorhabditis elegans is an essential muscle-affecting gene with maternal expression.

We have isolated three novel alleles of the unc-45 locus in C. elegans, that are recessive lethals. Two of these alleles, when homozygous, result in a nearly total loss of muscle contraction with a concomitant arrest of development and a displacement of muscle cells. The third allele is similar, but showed maternal rescue by a wild-type allele. All previously identified unc-45 alleles were temperature sensitive and, although they produced paralysis of adult animals, all were homozygous viable. Prior genetic studies with these temperature sensitive alleles had suggested that at least one function of the unc-45 gene product was to interact with the major myosin heavy chain isoform, MHC B, of body wall muscles. Our observations of the lethal alleles suggest that the unc-45 product normally interacts with additional muscle components in both the body wall and pharyngeal muscles. In particular, we suggest that the unc-45 product might interact with all four myosin heavy chains: MHC B; MHC A; and the pharyngeal isoforms, MHC C and MHC D. Maternal rescue of the lethality of the third allele shows that the unc-45 gene product is present in the oocytes, although it may not be necessary until late in development when myofilaments begin to assemble.