Clycerolphosphate oxidase and succinate dehydrogenase activities in IIA and IIB fibres of mouse and rabbit tibialis anterior muscles

Summary Quantitative microphotometric measurements of two mitochondrial flavoproteins, glycerolphosphate oxidase (GP-OX) and succinate dehydrogenase (SDH), were performed on serial sections of mouse and rabbit tibialis anterior (TA) muscles in order to study the distribution of these two enzymes and their activity ratios in IIA and IIB fibres. The measurements showed a large scatter of the two enzyme activities in these two myosin-based fibre types. In rabbit TA, IIA and IIB fibres have similar GP-OX activities, whereas generally IIA fibres have higher SDH activities than IIB fibres. An inverse distribution of the two enzymes exists in mouse muscle. Generally, IIA fibres of mouse TA display low SDH and IIB fibres high SDH activities. The mean activity of GP-OX is slightly higher in IIA than in IIB fibres of mouse TA. Since measurements of both enzymes were taken in the same fibres, the ratio of their activities in each fibre could be evaluated. The SDH/ GP-OX activity ratios vary significantly between the two fibre populations both in rabbit and in mouse. The ratio is high in IIA and low in IIB fibres of rabbit TA, whereas it is low in IIA and high in IIB fibres of mouse TA.     

Glycerolphosphate oxidase and succinate dehydrogenase activities in IIA and IIB fibres of mouse and rabbit tibialis anterior muscles

Quantitative microphotometric measurements of two mitochondrial flavoproteins, glycerolphosphate oxidase (GP-OX) and succinate dehydrogenase (SDH), were performed on serial sections of mouse and rabbit tibialis anterior (TA) muscles in order to study the distribution of these two enzymes and their activity ratios in IIA and IIB fibres. The measurements showed a large scatter of the two enzyme activities in these two myosin-based fibre types. In rabbit TA, IIA and IIB fibres have similar GP-OX activities, whereas generally IIA fibres have higher SDH activities than IIB fibres. An inverse distribution of the two enzymes exists in mouse muscle. Generally, IIA fibres of mouse TA display low SDH and IIB fibres high SDH activities. The mean activity of GP-OX is slightly higher in IIA than in IIB fibres of mouse TA. Since measurements of both enzymes were taken in the same fibres, the ratio of their activities in each fibre could be evaluated. The SDH/GP-OX activity ratios vary significantly between the two fibre populations both in rabbit and in mouse. The ratio is high in IIA and low in IIB fibres of rabbit TA, whereas it is low in IIA and high in IIB fibres of mouse TA.     

The effect of glucocorticoids on the myosin heavy chain isoforms' turnover in skeletal muscle

The purpose of this study was to find the effect of dexamethasone on the myosin heavy chain (MyHC) isoforms' composition in different skeletal muscles and glycolytic (G) fibres in relation with their synthesis rate and degradation of MyHC isoforms by alkaline proteinases. Eighteen-week-old male rats of the Wistar strain were treated with dexamethasone (100 microg/100 g bwt) during 10 days. The forelimb strength decreased from 9.52 to 6.19 N (P<0.001) and hindlimb strength from 15.54 to 8.55 N (P<0.001). Daily motor activity decreased (total activity from 933 to 559 and ambulatory activity from 482 to 226 movements/h, P<0.001). The degradation rate of muscle contractile proteins increased from 2.0 to 5.9% per day (P<0.001), as well as the myosin heavy chain IIB isoform degradation with alkaline proteinase in fast-twitch (F-T) muscles (12 +/- 0.9%; P<0.05) and glycolytic muscle fibres (15 +/- 1.1%; P<0.001). The synthesis rate of MyHC type II isoforms decreased in Pla muscles (P<0.05) and MyHC IIA (P<0.05) and IIB in EDL muscle and G fibres (P<0.001). The relative content of MyHC IIB isoform decreased in F-T muscles (P<0.001) and in G fibres (P<0.01), and the relative content of IIA and IID isoforms increased simultaneously. Dexamethasone decreased the MyHC IIB isoform synthesis rate and increased the sensibility of MyHC IIB isoform to alkaline proteinase, which in its turn led to the decrease of MyHC IIB isoform relative content in F-T muscles with low oxidative potential and G muscle fibres.     

Heterogeneity and distribution of fast myosin heavy chains in some adult vertebrate skeletal muscles.

Three monoclonal antibodies, LM5, F2 and F39 raised to chicken fast skeletal muscle myosin, specific for myosin heavy chain (MHC) subunit, were used to study the composition and distribution of this protein in some vertebrate skeletal muscles. These antibodies in immunohistochemical investigations did not react with the majority of the type I fibres in most muscles. Antibodies LM5 and F39 stained all the type II fibres in all the adult chicken skeletal muscles studied. Antibody F2 also stained all the type II fibres in most chicken skeletal muscles tested except in gastrocnemius in which a proportion of both the type IIA and IIB fibres either did not stain or stained only weakly. Antibody F2 unlike LM5 and F39 stained most of the type IIIB fibres in anterior latissimus dorsi (ALD) and IB fibres in red strip of chicken Pectoralis muscle. Antibodies LM5 and F2 in the rat diaphragm reacted with all the type IIA and IIB fibres, while antibody F39 stained only the type IIB fibres darkly with most IIA fibres being either not stained or only weakly stained. In the rat extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, antibody LM5 stained all the IIA and IIB fibres. Antibody F2 in these muscles stained all the type IIA fibres but only a proportion of the IIB fibres. The remaining IIB fibres were either unstained or only weakly positive. Antibody F39 in rat EDL and TA muscles did not only distinguish subgroups of IIB fibres (dark, intermediate and negative or very weak) but also of the IIA fibres. These three antibodies used together therefore detected a great deal of heterogeneity in the myosin heavy chain composition and muscle fibre types of several skeletal muscles.     

Heterogeneity and distribution of fast myosin heavy chains in some adult vertebrate skeletal muscles

Summary Three monoclonal antibodies, LM5, F2 and F39 raised to chicken fast skeletal muscle myosin, specific for myosin heavy chain (MHC) subunit, were used to study the composition and distribution of this protein in some vertebrate skeletal muscles. These antibodies in immunohistochemical investigations did not react with the majority of the type I fibres in most muscles. Antibodies LM5 and F39 stained all the type II fibres in all the adult chicken skeletal muscles studied. Antibody F2 also stained all the type II fibres in most chicken skeletal muscles tested except in gastrocnemius in which a proportion of both the type IIA and IIB fibres either did not stain or stained only weakly. Antibody F2 unlike LM5 and F39 stained most of the type IIIB fibres in anterior latissimus dorsi (ALD) and IB fibres in red strip of chicken Pectoralis muscle. Antibodies LM5 and F2 in the rat diaphragm reacted with all the type IIA and IIB fibres, while antibody F39 stained only the type IIB fibres darkly with most IIA fibres being either not stained or only weakly stained. In the rat extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, antibody LM5 stained all the IIA and IIB fibres. Antibody F2 in these muscles stained all the type IIA fibres but only a proportion of the IIB fibres. The remaining IIB fibres were either unstained or only weakly positive. Antibody F39 in rat EDL and TA muscles did not only distinguish subgroups of IIB fibres (dark, intermediate and negative or very weak) but also of the IIA fibres. These three antibodies used together therefore detected a great deal of heterogeneity in the myosin heavy chain composition and muscle fibre types of several skeletal muscles.     

BDNF rescues myosin heavy chain IIB muscle fibers after neonatal nerve injury

Neonatal sciatic nerve injury is known to result in an extensive loss of lumbar motor neurons as well as the disappearance of their respective muscle fibers in the hindlimb musculature. The loss of motor neurons and muscle fibers can be prevented by immediate administration of target-derived neurotrophic factors to the site of injury. In the present study, we investigated the role of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the survival and maturation of a subset of motor neurons innervating the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. We have shown that combined administration of CNTF and BDNF prevented the loss of motor units after neonatal nerve injury and contributed to the maintenance of muscle mass. Importantly, this combined neurotrophin regimen also prevented the disappearance of muscle fibers that express myosin heavy chain IIB (MyHC IIB) in both EDL and TA muscles 3 mo after neonatal sciatic nerve crush. In parallel studies, we observed a higher level of BDNF in EDL muscle during the critical period of development when motor neurons are highly susceptible to target removal. Given our previous findings that combined administration of CNTF with neurotrophin-3 (NT-3) or neurotrophin-4/5 (NT-4/5) did not result in the rescue of MyHC IIB fibers in EDL, the present results show the importance of muscle-derived BDNF in the survival and maturation of a subpopulation of motor neurons and of MyHC IIB muscle fibers during neonatal development of the neuromuscular system. motor neurons; neuromuscular development; neurotrophins     

Nonmuscle myosin IIB, a sarcomeric component in the extraocular muscles

Extraocular muscles (EOMs) are categorized as skeletal muscles; however, emerging evidence indicates that their gene expression profile, metabolic characteristics and functional properties are significantly different from the prototypical members of this muscle class. Gene expression profiling of developing and adult EOM suggest that many myofilament and cytoskeletal proteins have unique expression patterns in EOMs, including the maintained expression of embryonic and fetal isoforms of myosin heavy chains (MyHC), the presence of a unique EOM specific MyHC and mixtures of both cardiac and skeletal muscle isoforms of thick and thin filament accessory proteins. We demonstrate that nonmuscle myosin IIB (nmMyH IIB) is a sarcomeric component in ∼ 20% of the global layer fibers in adult rat EOMs. Comparisons of the myofibrillar distribution of nmMyHC IIB with sarcomeric MyHCs indicate that nmMyH IIB co-exists with slow MyHC isoforms. In longitudinal sections of adult rat EOM, nmMyHC IIB appears to be restricted to the A-bands. Although nmMyHC IIB has been previously identified as a component of skeletal and cardiac sarcomeres at the level of the Z-line, the novel distribution of this protein within the A band in EOMs is further evidence of both the EOMs complexity and unconventional phenotype.     

Expression of Myosin heavy chain isoforms in rat soleus muscle spindles after 19 days of hypergravity.

The aim of this study was to determine whether a period of 19 days in hypergravity was long enough to induce changes in the expression of myosin heavy chain (MyHC) isoforms in the muscle spindles. The soleus muscle of 10 male Wistar rats (control: CONT, n=5; hypergravity: HG, n=5) was frozen, cut into serial sections, and labeled with antibodies against MyHCs: I, IIA, IIA + IIX + IIB, slow-tonic, and alpha-cardiac. Forty CONT and 45 HG spindles were analyzed. The results from HG spindles compared to CONT showed that there was no change in the cross-sectional area of intrafusal fibers. However, along the entire length of B1 fibers, the expression of both MyHC I and alpha-cardiac was increased significantly, whereas the labeling against MyHC IIA and MyHC slow-tonic was decreased. In B2 fibers, the labeling against MyHC IIA (region A), slow-tonic (region A), and fast myosins (regions A-C) was statistically decreased. In chain fibers, the labeling against both MyHC IIA and fast MyHC was reduced significantly. We conclude that hypergravity has a real impact on the MyHC content in the muscle spindles and induces some inverse changes of those observed in hypogravity for MyHCs I, alpha-cardiac, and slow-tonic.     

Immunohistochemical Analysis of the Effects of Cross-innervation of Murine Thyroarytenoid and Sternohyoid Muscles

This work uses cross-innervation of respiratory muscles of different developmental origins to probe myogenic and neurogenic mechanisms regulating their fiber types. The thyroarytenoid (TA) originates from the sixth branchial arch, whereas the sternohyoid (SH) is derived from somitic mesoderm. Immunohistochemical analysis using highly specific monoclonal antibodies to myosin heavy chain (MyHC) isoforms reveals that normal rat SH comprises slow, 2a, 2x, and 2b fibers, as in limb fast muscles, whereas the external division of the TA has only 2b/eo fibers coexpressing 2B and extraocular (EO) MyHCs. Twelve weeks after cross-innervation with the recurrent laryngeal nerve, the SH retained slow and 2a fibers, greatly increased the proportion of 2x fibers, and their 2b fibers failed to express EO MyHC. In the cross-innervated TA, the SH nerve failed to induce slow and 2A MyHC expression and failed to suppress EO MyHC expression in 2b/eo fibers. However, 2x fibers amounting to 4.2% appeared de novo in the external division of the TA. We conclude that although MyHC gene expression in these muscles can be modulated by neural activity, the patterns of response to altered innervation are largely myogenically determined, thus supporting the idea that SH and TA differ in muscle allotype. (J Histochem Cytochem 58:1057–1065, 2010)     

Training affects myosin heavy chain phenotype in the trapezius muscle of women

The aim of this investigation was to determine whether 10 weeks of three different types of training can alter the myosin heavy chain (MyHC) composition of the trapezius muscle. Twenty-one women were randomly assigned to three training groups that performed strength (n=9), endurance (n=7) or coordination training (n=5). Pre and post biopsies were taken from the upper part of the descending trapezius muscle and were analysed for MyHC isoform content using 5% gel electrophoresis. In addition, we have studied the expression of embryonic and neonatal MyHCs using double-immunofluorescence staining. In the strength-trained group, there was a significant increase in the amount of MyHC IIA and a significant decrease in the amount of MyHC IIB and MyHC I. In the endurance group, there was a significant decrease in the amount of MyHC IIB. MyHC composition in the coordination group was not altered. Following the training period, myotubes and individual small-sized muscle fibres were observed in the strength and endurance trained groups. These structures were stained with the markers for early myogenesis (MyHC embryonic and neonatal). These data suggest that specific shifts in MyHC isoforms occur in the trapezius muscle following strength and endurance training. The presence of small-sized muscle fibres expressing the developmental isoforms of MyHC suggests that strength and endurance training induced the formation of new muscle fibres. Accepted: 31 March 1999     

Direct correlation of parvalbumin levels with myosin isoforms and succinate dehydrogenase activity on frozen sections of rodent muscle

Parvalbumin (PV) is a soluble Ca++ binding protein which is particularly concentrated in fast muscles of rodents. We have developed a new protocol to fix frozen sections of muscle by formaldehyde vapor, which enabled us to immunochemically stain serial frozen sections for PV. Fiber types were defined on the basis of myosin ATPase stability, and of isomyosins identified by a variety of antibodies because ATPase stability alone yielded ambiguous results in the mouse. Slow Type I fibers in mouse and rat were devoid of PV and had intermediate to high SDH levels. Fast fiber subtypes IIA, IIB, and IIX-like were defined in the mouse on the basis of the similarity of their myosin heavy chain immunoreactivity to these types in the rat. The soleus muscle was usually PV negative, but a small population of strongly PV-positive IIX-like fibers was present in the mouse. In mouse fast muscle, small diameter IIA fibers were PV negative with high SDH activity. In both mouse and rat, PV reactivities of IIB and IIX fibers were higher than those of IIA and I, whereas SDH levels of IIA, IIX, and I fibers were higher than those of IIB. Thus, PV content correlated with the type of myosin ATPase but not with SDH levels. The method described for immunocytochemistry of PV may be applicable to other highly soluble proteins.     

Fiber composition and oxidative capacity of hamster skeletal muscle.

The hamster is a valuable biological model for physiological investigation. Despite the obvious importance of the integration of cardiorespiratory and muscular system function, little information is available regarding hamster muscle fiber type and oxidative capacity, both of which are key determinants of muscle function. The purpose of this investigation was to measure immunohistochemically the relative composition and size of muscle fibers composed of types I, IIA, IIX, and IIB fibers in hamster skeletal muscle. The oxidative capacity of each muscle was also assessed by measuring citrate synthase activity. Twenty-eight hindlimb, respiratory, and facial muscles or muscle parts from adult (144-147 g bw) male Syrian golden hamsters (n=3) were dissected bilaterally, weighed, and frozen for immunohistochemical and biochemical analysis. Combining data from all 28 muscles analyzed, type I fibers made up 5% of the muscle mass, type IIA fibers 16%, type IIX fibers 39%, and type IIB fibers 40%. Mean fiber cross-sectional area across muscles was 1665 +/- 328 microm(2) for type I fibers, 1900 +/- 417 microm(2) for type IIA fibers, 3230 +/- 784 microm(2) for type IIX fibers, and 4171 +/- 864 microm(2) for type IIB fibers. Citrate synthase activity was most closely related to the population of type IIA fibers (r=0.68, p<0.0001) and was in the rank order of type IIA > I > IIX > IIB. These data demonstrate that hamster skeletal muscle is predominantly composed of type IIB and IIX fibers.     

Fiber types in rat laryngeal muscles and their transformations after denervation and reinnervation.

The intrinsic laryngeal muscles cricothyroid (CT) and thyroarythenoid (TA) differ in myosin expression. CT expresses limb myosin heavy chains (MyHCs) and TA expresses an MyHC found in extraocular (EO) muscles, in addition to limb isoforms. We used immunohistochemical (IHC) analyses with highly specific monoclonal antibodies (MAbs) against various MyHCs to study muscle fiber types in rat CT and TA and to investigate whether nerves to laryngeal muscles control MyHC expression. CT was found to have the full complement of limb fiber types. TA had three major fiber types: 2b/eo, co-expressing 2B and EO MyHCs, 2x/2b, co-expressing 2X and 2B MyHCs, and 2x, expressing 2X MyHC. Type 2a and slow fibers were absent. TA consisted of two divisions: the external division (TA-X), which is homogeneously 2b/eo, and the vocalis division (TA-V), composed principally of 2x and 2b/eo fibers with a minority of 2x/2b fibers. TA-V had two compartments that differ in fiber type composition. At 4 weeks after cutting and re-uniting the recurrent laryngeal nerve (RLN), many 2b/eo fibers in the TA-X began to express 2X MyHC, while EO and 2B MyHC expression in these fibers progressively declined. By 12 weeks, up to 16.5% of fibers in the TA-X were of type 2x. These findings suggest that nerve fibers originally innervating 2x fibers in TA-V and other muscles have randomly cross-innervated 2b/eo fibers in the TA-X and converted them into 2x fibers. We conclude that CT and TA are distinct muscle allotypes and that laryngeal muscle fibers are subject to neural regulation.     

Fiber type and myosin heavy chain compositions of adult pretarsal orbicularis oculi muscle

Summary Pretarsal orbicularis oculi muscle (POOM) is an important structure of eyelid movement in human. The aim of this study was to investigate fiber histomorphology and myosin heavy chain (MyHC) isoform composition of adult POOM, and to clarify their age-related changes. Eyelid specimens from 58 subjects (age range, 21 to 91 years) were collected during upper blepharoplasty procedures. Serial cross sections of POOM were ATPase-stained and examined under miscroscope. Quantitative measures of muscle fiber size and fiber type distribution were obtained in 35 subjects with adequate fiber cross sections. Relative MyHC isoform contents of POOM were retrieved by gel electrophoresis in all 58 subjects. Examination of the histochemical staining revealed an abundance of type II fiber ( >85%) in human POOM, with more type IIX than IIA fibers. Decreased mean area of all fibers and type IIA fibers were noted in the old group when compared to the young. As for MyHC analysis, the relative content of MyHC isoforms exhibited an order of IIX > IIA > I, and the relative MyHC IIA content showed a negative correlation with age. Comparing with previous studies of limb or masticatory muscles, adult POOM exhibits a unique fiber and MyHC composition, as well as a different aging pattern.     

A comparative microphotometric study of succinate dehydrogenase activity levels in type I,IIA and IIB fibres of mammalian and human muscles

Summary Activity levels of succinate dehydrogenase (SDH) were determined kinetically by means of comparative microphotometric measurements in situ. Activities were correlated with fibre types classified histochemically according to Brooke and Kaiser (1970). Analyses of tibialis anterior muscles in the mouse, rat, guinea pig, rabbit, cat and the human showed pronounced variations in the activity profiles of type I, type IIA and IIB fibres of these muscles. Large scattering of enzyme activity existed in the three fibre populations. Overlaps of varying extent were found for the SDH profiles between the different muscles. Type I fibres reveal species diffeences in aerobic oxidative capacity. Whereas the majority of the IIB fibres in rabbit muscle tended to be low in SDH activity, the main fraction of this fibre population was characterized by high activities in mouse muscle. Similarly, the IIA fibre populations revealed opposite properties in mouse and rabbit muscles. These extremes as well as intermediate activity patterns indicate that no general scheme exists according to which the histochemically assessable myosin ATPase is correlated with the aerobic oxidative capacity of muscle fibres in various mammalian muscles.     

A comparison of two ATPase based schemes for histochemical muscle fibre typing in various mammals

A comparative study was performed on the fibre populations in tibialis anterior muscles of mouse, rat, guinea pig, rabbit, cat and dog using the two different methods of histochemical staining for myofibrillar ATPase after acid (Brooke and Kaiser 1970) or alkaline preincubations (Guth and Samaha 1970). For all species a complete correspondence existed between type I (Brooke and Kaiser 1970) and beta fibres (Samaha et al. 1970). Gross correspondence (greater than 85%) existed between IIA and IIB (Brooke and Kaiser 1970) and alpha beta and alpha fibres (Samaha et al. 1970) respectively in mouse, guinea pig, rabbit, cat and dog. In the case of mouse and dog, this high degree of correspondence was based on the assumption that mouse tibialis anterior contains no type I and the dog no type IIB fibres. For the rat, a pronounced overlap existed between IIA fibres on the one hand and alpha beta and alpha fibres on the other hand as well as between IIB fibres and alpha beta and alpha fibres. These observations lead to the conclusion that the two classification schemes are not interchangeable for all species and that the two terminologies should be used only in relation with the methods from which they were derived.     

Differential effects of short-term β agonist and growth hormone treatments on expression of myosin heavy chain IIB and associated metabolic genes in sheep muscle

Growth hormone (GH) and β agonists increase muscle mass, but the mechanisms for this response are unclear and the magnitude of response is thought to vary with age of animal. To investigate the mechanisms driving the muscle response to these agents, we examined the effects of short-term (6 day) administration of GH or cimaterol (a β2-adrenergic agonist, BA) on skeletal muscle phenotype in both young (day 60) and mature (day 120) lambs. Expression of myosin heavy chain (MyHC) isoforms were measured in Longissimus dorsi (LD), Semitendinosus (ST) and Supraspinatus (SS) muscles as markers of fibre type and metabolic enzyme activities were measured in LD. To investigate potential mechanisms regulating the changes in fibre type/metabolism, expression or activity of a number of signalling molecules were examined in LD. There were no effects of GH administration on MyHC isoform expression at either the mRNA or protein level in any of the muscles. However, BA treatment induced a proportional change in MyHC mRNA expression at both ages, with the %MyHCI and/or IIA mRNA being significantly decreased in all three muscles and %MyHCIIX/IIB mRNA significantly increased in the LD and ST. BA treatment induced de novo expression of MyHCIIB mRNA in LD, the fastest isoform not normally expressed in sheep LD, as well as increasing expression in the other two muscles. In the LD, the increased expression of the fastest MyHC isoforms (IIX and IIB) was associated with a decrease in isocitrate dehydrogenase activity, but no change in lactate dehydrogenase activity, indicating a reduced capacity for oxidative metabolism. In both young and mature lambs, changes in expression of metabolic regulatory factors were observed that might induce these changes in muscle metabolism/fibre type. In particular, BA treatment decreased PPAR-γ coactivator-1β mRNA and increased receptor-interacting protein 140 mRNA. The results suggest that the two agents work via different mechanisms or over different timescales, with only BA inducing changes in muscle mass and transitions to a faster, less oxidative fibre type after a 6-day treatment.     

Distribution of sarcoplasmic reticulum Ca-ATPase and of calsequestrin in rabbit and rat skeletal muscle fibers

Muscle fibers in rabbit extensor digitorum longus (EDL), tibialis anterior (TA) and soleus, and rat soleus, were examined immunohistochemically for two proteins of the sarcoplasmic reticulum. Ca-ATPase and calsequestrin (CaS). Fibers were typed with the histochemical reaction for actomyosin ATPase. In the rabbit EDL and TA, type I fibers clearly reacted less for Ca-ATPase and CaS than type II fibers, but the difference was less with CaS than with Ca-ATPase. Although the differences were relatively small, IIB fibers consistently presented greater amounts of Ca-ATPase than IIA fibers. No type II subgroups could be recognized after incubation with anti-CaS. These findings confirm results from previous immunochemical measurements on whole muscles containing different proportions of IIA and IIB fibers (Leberer and Pette 1986). Type IIA and IIC in the rabbit and rat soleus reacted stronger for Ca-ATPase and for CaS than type I fibers. Small differences in Ca-ATPase, but not in CaS, were recognized within the type I fiber population. Therefore, type I fibers in the rabbit and rat soleus are not a homogeneous population.