Remarkable heterogeneity in myosin heavy-chain composition of the human young masseter compared with young biceps brachii

Adult human jaw muscles differ from limb and trunk muscles in enzyme-histochemical fibre type composition. Recently, we showed that the human masseter and biceps differ in fibre type pattern already at childhood. The present study explored the myosin heavy-chain (MyHC) expression in the young masseter and biceps muscles by means of gel electrophoresis (GE) and immuno-histochemical (IHC) techniques. Plasticity in MyHC expression during life was evaluated by comparing the results with the previously reported data for adult muscles. In young masseter, GE identified MyHC-I, MyHC-IIa MyHC-IIx and small proportions of MyHC-fetal and MyHC-α cardiac. Western blots confirmed the presence of MyHC-I, MyHC-IIa and MyHC-IIx. IHC revealed in the masseter six isomyosins, MyHC-I, MyHC-IIa, MyHC-IIx, MyHC-fetal, MyHC α-cardiac and a previously not reported isoform, termed MyHC-IIx'. The majority of the masseter fibres co-expressed two to four isoforms. In the young biceps, both GE and IHC identified MyHC-I, MyHC-IIa and MyHC-IIx. MyHC-I predominated in both muscles. Young masseter showed more slow and less-fast and fetal MyHC than the adult and elderly masseter. These results provide evidence that the young masseter muscle is unique in MyHC composition, expressing MyHC-α cardiac and MyHC-fetal isoforms as well as hitherto unrecognized potential spliced isoforms of MyHC-fetal and MyHC-IIx. Differences in masseter MyHC expression between young adult and elderly suggest a shift from childhood to adulthood towards more fast contractile properties. Differences between masseter and biceps are proposed to reflect diverse evolutionary and developmental origins and confirm that the masseter and biceps present separate allotypes of muscle.     

Dystonia musculorum mutation and myosin heavy chain expression in skeletal and cardiac muscles.

This study evaluated the influence of dystonia musculorum (dt) mutation, characterized by spinocerebellar fibers degeneration, on cardiac and skeletal muscles: one respiratory (diaphragm, Dia), three masticatory (anterior temporalis, AT; masseter superficialis, MS; and anterior digastric, AD), one hindlimb (soleus, S), tongue (T), and one cardiac (ventricle, V). Body and muscle weight, muscle protein content, and myosin heavy chain (MHC) isoforms relative expression were then compared in dt mutant mice and in normal mice, according to sex. Male body and muscle weight was always greater than that of females, but there was no specific muscle difference in females. dt mutant mice showed a reduced whole body growth but no specific muscle atrophy, as well as a global decrease in muscle protein content that made muscles more fragile. dt mutation induced a global reduction of muscle protein concentration, whereas a general influence of sex could not be disclosed. Concerning MHC relative composition, all the muscles were fast-twitch: Dia, AT, MS, AD, S, and T expressed predominantly the fast type 2 MHC isoforms, whereas V contained only MHC alpha, also a fast MHC. Female muscles were slower than male muscles, except for S, which was faster. However, classification of muscles in terms of shortening velocity was very different in normal males and females. In other respects, dt mutant muscles were slower and consequently more fatigue resistant than normal, except for S, which became faster and less fatigue resistant. dt mutation exhibits then a specific effect on this continually active postural muscle. In the other muscles, global increased fatigue resistance could constitute an adaptive response to work requirements modifications linked to the muscle damage. It should be noted that a developmental MHC (neonatal) was present in female dt AD. Innervation, which influences muscle structure, is altered in dt mutant and could be another causal factor of the fast-to-slow MHC switches. It appears that dystonin, the dt gene product, is very important in maintaining the structural integrity of both cardiac and skeletal muscle and in its absence, the muscle becomes more fragile and is damaged by modified activity.     

Fiber content and myosin heavy chain composition of muscle spindles in aged human biceps brachii.

The present study investigated potential age-related changes in human muscle spindles with respect to the intrafusal fiber-type content and myosin heavy chain (MyHC) composition in biceps brachii muscle. The total number of intrafusal fibers per spindle decreased significantly with aging, due to a significant reduction in the number of nuclear chain fibers. Nuclear chain fibers in old spindles were short and some showed novel expression of MyHC alpha-cardiac. The expression of MyHC alpha-cardiac in bag1 and bag2 fibers was greatly decreased in the A region. The expression of slow MyHC was increased in nuclear bag1 fibers and that of fetal MyHC decreased in bag2 fibers whereas the patterns of distribution of the remaining MyHC isoforms were generally not affected by aging. We conclude that aging appears to have an important impact on muscle spindle composition. These changes in muscle spindle phenotype may reflect an age-related deterioration in sensory and motor innervation and are likely to have an impact in motor control in the elderly.     

Diversity of myosin heavy chain expression in satellite cells from mouse soleus and EDL muscles.

Postnatal myoblasts, the satellite cells, originating from slow and fast skeletal muscle fibres differentiate and fuse into myotubes expressing different phenotype of myosin heavy chain (MyHC) isoforms. Little is known, however, of factors which establish and maintain this phenotypic diversity. We used immunofluorescent labelling and Western blotting to examine the expression of slow and fast MyHC isoforms in myotubes formed in vitro from satellite cells isolated from mouse fast twitch extensor digitorum longus (EDL) and slow twitch soleus muscles. Satellite cells were cultured in serum-rich growth medium promoting myoblast proliferation until cross-striated and self-contracting myotubes were formed. We report that in both cultures myotubes expressed slow as well as fast MyHC isoforms, but the level of slow MyHC was higher in soleus culture than in EDL culture. Hence, the pattern of expression of slow and fast MyHC was characteristic of the muscle fibre type from which these cells derive. These results support the concept of phenotypic diversity among satellite cells in mature skeletal muscles and suggest that this diversity is generated in vitro irrespectively of serum mitogens.     

Neural control of the sequence of expression of myosin heavy chain isoforms in foetal mammalian muscles

The expression of myosin isoforms was studied during development of calf muscles in foetal and neonatal rats, using monoclonal antibodies against slow, embryonic and neonatal isoforms of myosin heavy chain (MHC). Primary myotubes had appeared in all prospective rat calf muscles by embryonic day 16 (E16). On both E16 and E17, primary myotubes in all muscles with the exception of soleus stained for slow, embryonic and neonatal MHC isoforms; soleus did not express neonatal MHC. In earlier stages of muscle formation staining for the neonatal isoform was absent or faint. Secondary myotubes were present in all muscles by E18, and these stained for both embryonic and neonatal MHCs, but not slow. In mixed muscles, primary myotubes destined to differentiate into fast muscle fibres began to lose expression of slow MHC, and primary myotubes destined to become slow muscle fibres began to lose expression of neonatal MHC. This pattern was further accentuated by E19, when many primary myotubes stained for only one of these two isoforms. Chronic paralysis or denervation from E15 or earlier did not disrupt the normal sequence of maturation of primary myotubes up until E18, but secondary myotubes did not form. By E19, however, most primary myotubes in aneural or paralyzed tibialis anterior muscles had lost expression of slow MHC and expressed only embryonic and neonatal MHCs. Similar changes occurred in other muscles, except for soleus which never expressed neonatal MHC, as in controls. Paralysis or denervation commencing later than E15 did not have these effects, even though it was initiated well before the period of change in expression of MHC isoforms. In this case, some secondary myotubes appeared in treated muscles. Paralysis initiated on E15, followed by recovery 2 days later so that animals were motile during the period of change in expression of MHC isoforms, was as effective as full paralysis. These experiments define a critical period (E15-17) during which foetuses must be active if slow muscle fibres are to differentiate during E19-20. We suggest that changes in expression of MHC isoforms in primary myotubes depend on different populations of myoblasts fusing with the myotubes, and that the normal sequence of appearance of these myoblasts has a stage-dependent reliance on active innervation of foetal muscles. A critical period of nerve-dependence for these myoblasts occurs several days before their action can be noted.     

Related expression of MyoD and Myf5 with myosin heavy chain isoform types in bovine adult skeletal muscles

Skeletal muscles are characterized as fast and slow muscles, according to the expression pattern of myosin heavy chain (MyHC) isoforms in the muscle fibers. To investigate the relationships between MyHC isoforms and myogenic regulatory factors (MRFs) including MyoD, Myf5, myogenin, and MRF4 in adult skeletal muscles, expressions of these MRFs in the ten muscles of three cows were analyzed by a semi-quantitative RT-PCR. The results showed that MyoD expression was significantly lower in the lingual muscles (TN), masseter (MS) and diaphragm (DP), which lack MyHC-2x (fast glycolytic) expression and abound with MyHC-slow (slow oxidative) and/or MyHC-2a (fast oxidative), than it was in the pectoralis (PP), psoas major (PM), longissimus thoracis (LT), spinnalis (SP), semitendinosus (ST), semimembranosus (SM), and biceps femoris (BF). In contrast, the Myf5 expression in TN, MS, and DP was significantly higher than in PM, LT, ST, SM, and BF. No significant difference was observed in myogenin and MRF4 expression among the muscles tested. The results suggest that MyoD and Myf5 influence the MyHC isoform expression, although the effects are not decisive in specifying the phenotypes of adult muscles.     

Eyestalk ablation has little effect on actin and myosin heavy chain gene expression in adult lobster skeletal muscles

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.     

Caenorhabditis elegans UNC-45 is a component of muscle thick filaments and colocalizes with myosin heavy chain B, but not myosin heavy chain A

In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B-dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.     

Regional differences in myosin heavy chain isoform expression and maximal shortening velocity of the rat vaginal wall smooth muscle

Contractility of the proximal and distal vaginal wall smooth muscle may play distinct roles in the female sexual response and pelvic support. The goal of this study was to determine whether differences in contractile characteristics of smooth muscle from these regions reside in differences in the expression of isoforms of myosin, the molecular motor for muscle contraction. Adult female Sprague-Dawley rats were killed on the day of estrus, and the vagina was dissected into proximal and distal segments. The Vmax at peak force was greater for tissue strips of the proximal vagina compared with that of distal (P < 0.01), although, at steady state, the Vmax for the muscle strips from the two regions was not different. Furthermore, at steady state, muscle stress was higher (P < 0.001) for distal vaginal strips (n = 5). Consistent with the high Vmax for the proximal vaginal strips, RT-PCR results revealed a higher %SM-B (P < 0.001) in the proximal vagina. A greater expression of SM-B protein (P < 0.001) was also detected by Western blotting (n = 4). Interestingly, there was no regional difference noted in SM-1/SM-2 isoforms (n = 6). The proximal vagina had a higher expression of myosin heavy chain protein (P < 0.01) and a greater percentage of smooth muscle bundles (P < 0.001). The results of this study are the first demonstration of a regional heterogeneity in Vmax and myosin isoform distribution in the vagina wall smooth muscle and confirm that the proximal vaginal smooth muscle exhibits phasic contractile characteristics compared with the distal vaginal smooth muscle, which is tonic.     

Differences in mitochondrial gene expression profiles, enzyme activities and myosin heavy chain types in yak versus bovine skeletal muscles

Hypoxia can affect energy metabolism. We examined gene expression and enzyme activity related to mitochondrial energy metabolism, as well as myosin heavy chain (MyHC) types in yaks (Bos grunniens) living at high altitudes. Real-time quantitative PCR assays indicated that the yak has significantly lower levels of carnitine palmitoyltransferase (CPT) mRNA in the biceps femoris and lower levels of uncoupling protein 3 (UCP3) mRNA in both biceps femoris and longissimus dorsi than in Yellow cattle. No significant differences between yak and Yellow cattle were observed in the activities of mitochondrial β-hydroxyacyl-CoA dehydrogenase, isocitrate dehydrogenase and cytochrome oxidase in the same muscles. Semi-quantitative RT-PCR analysis showed that the MyHC 1 mRNA levels in yak biceps femoris was lower than in Yellow cattle. We conclude that the yak has significantly lower mRNA levels of CPT, UCP3, and MyHC 1 in biceps femoris than in Yellow cattle, suggesting that the yak biceps femoris has lower fatty acid oxidation capacity and greater glycolytic metabolic potential.     

Structural organization and expression of the gene for bovine myosin I heavy chain.

Brush border myosin I heavy chain (MIHC), known previously as the brush border 110-kDa protein, contains an amino-terminal sequence which is highly homologous to the globular head domain of conventional myosin II heavy chain (MIIHC). The carboxyl-terminal sequence of MIHC completely diverges from that of MIIHC and functions as calmodulin-binding and membrane-interaction sites. In this investigation, we determined the structural organization of the bovine MIHC by isolating a set of genomic segments containing the whole MIHC gene. The bovine MIHC gene is 26 kilobase pairs long and consists of 28 exons. At the homologous amino-terminal portion of MIHC, many introns are located at positions equivalent to those of the rat MIIHC gene and the amoeba MIHC gene. At the carboxyl-terminal sequence of MIHC, the putative calmodulin-binding and membrane-interacting domains are specified by discrete sets of exons. These findings support the view that the amino-terminal head portions of MIHC and MIIHC evolved from a common ancestral origin and also that the MIHC protein was generated as a result of fusion of discrete genomic segments encoding different functional and structural protein domains. Analysis of tissue expression of the MIHC mRNA was also extended in this investigation, and the results indicated that this mRNA is expressed in some tissues other than the intestines.     

Molecular cloning and expression of cardiac-specific myosin heavy chain gene sequences in chick embryo

A recombinant DNA plasmid, pMHC8, that contains gene sequences for embryonic chick cardiac myosin heavy chain was constructed, identified and characterized. The identity of the clone was established by hybridization with labeled probes that afford screening of MHC²² with high specificity, by inhibition of MHC synthesis in the in vitro hybrid-arrested translation assay, and by tissue-specific hybridization of labeled pMHC8 DNA to MHC messenger RNA.The pMHC8 DNA probe is highly specific for chick heart muscle tissue, since it hybridized poorly to chick skeletal muscle RNA and did not detectably hybridize to adult rat heart RNA. Upon screening the embryonic chick heart cells in culture, no detectable level of MHC mRNA was observed in dividing myoblasts, but the mRNA appeared in differentiated cardiac myocytes paralleling morphogenetic changes in the embryonic cells.     

Fiber-type distribution and expression of myosin heavy chain isoforms in newborn heterozygous myostatin-knockout pigs

Objectives

To explore the effects of heterozygous myostatin-knockout (MSNT^+/?) on muscle characteristics, specifically fiber-type distribution and expression of myosin heavy chain isoforms in pigs.

Results

The fiber cross-sectional area of the semitendinosus and semimembranosus muscles were much larger in MSTN^+/? pigs at birth than in wild-type (WT) pigs. MSTN^+/? pigs had a higher proportion of fast-type fibers and lower succinate dehydrogenase activity in muscles than WT pigs. The myosin heavy chain IIB mRNA level in both two muscles was ~ threefold higher in MSTN^+/? pigs compared with WT pigs.

Conclusion

MSTN^+/? pigs exhibit a disproportionate increase in muscle mass and can have a higher body weight due to fiber hypertrophy, a change in the fiber-type distribution, and alteration of myosin heavy chain isoforms levels, leading to more fast glycolytic fibers.

     

The expression of myosin heavy chain isoforms in normal and hypertrophied chicken slow muscle

Hypertrophy was produced in the anterior latissimus dorsi (ALD) muscle of 5-wk-old chickens by application of a load to the humerus. After 4 wk, hypertrophied ALD muscles were greater than 2.5 times heavier than contralateral control ALD muscles. Two isomyosins are distinguishable in normal ALD muscles by their different electrophoretic mobilities. It is shown here that the faster migrating SM-1 isomyosin decreases in abundance with age and that the application of an overload enhances both the rate and extent of this process. Monoclonal antibodies were selected by an immunotransfer technique that were specific for the heavy chains associated with either SM-1 or SM-2, or cross-reacted with both isoforms. The cellular distribution of the SM-1 and SM-2 isomyosins was analyzed by immunofluorescent technique using these antibodies. Anti-SM-1 and anti-SM-2 antibodies reacted with separate populations of cells, whereas the third antibody reacted with all myocytes in the normal ALD muscle. These data suggest that there is an exclusive cellular distribution of myosin heavy chains associated with SM-1 and SM-2 proteins. Immunofluorescent analysis of hypertrophied muscle showed the anti-SM-2-specific antibody reacting with all myocytes, whereas the anti-SM-1-specific antibody reacted with none. This is consistent with the elimination of the SM-1 isoform in hypertrophied muscles.