New insights into skeletal muscle fibre types in the dog with particular focus towards hybrid myosin phenotypes

Electrophoresis, immunoblots, immunohistochemistry and image analysis methods were applied to characterise canine trunk and appendicular muscle fibres according to their myosin heavy chain (MyHC) composition and to determine, on a fibre-to-fibre basis, the correlation between contractile [MyHC (s), myofibrillar ATPase (mATPase) and sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) isoforms], metabolic [succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (GPDH) activities and glycogen and phospholamban (PLB) content] and morphological (cross-sectional area and capillary and nuclear densities) features of individual myofibres. An accurate delineation of MyHC-based fibre types was obtained with the developed immunohistochemical method, which showed high sensitivity and objectivity to delineate hybrid fibres with overwhelming dominance of one MyHC isoform. Phenotypic differences in contractile, metabolic and morphological properties seen between fibre types were related to MyHC content. All canine skeletal muscle fibre types had a relatively high histochemical SDH activity but significant differences existed in the order IIA>I>IIX. Mean GPDH was ranked according to fibre type such that I<IIA<IIX. Type IIA fibres were the smallest, type IIX fibres the largest and type I of intermediate size. Capillary and nuclear density decreased in the order IIA>I>IIX. Hybrid fibres, which represented nearly one third of the whole pool of skeletal muscle fibres analysed, had mean values intermediate between their respective pure phenotypes. Slow fibres expressed the slow SERCA isoform and PLB, whereas type II fibres expressed the fast SERCA isoform. Discrimination of myofibres according to their MyHC content was possible on the basis of their contractile, metabolic and morphological features. These intrafibre interrelationships suggest that myofibres of control dogs exhibit a high degree of co-ordination in their physiological, biochemical and morphological characteristics. This study demonstrates that canine skeletal muscle fibres have been misclassified in numerous previous studies and offers useful baseline data and new prospects for future work on muscle-fibre-typing in canine experimental studies.     

Immunohistochemical differences in myosin composition among intrafusal muscle fibres

Summary Mammalian intrafusal fibre types (nuclear chain, nuclear bag₁ and nuclear bag₂ fibres) are known to differ in their ultrastructure, intensity of the myofibrillar histochemical ATP-ase reaction, type of innervation and time course of contraction. The present study concerns the myosin composition of these intrafusal fibre types in the soleus muscle (mouse) and the extensor digitorum longus muscle (rat). We used an immunohistochemical method with three myosin antisera raised in rabbits: anti chicken pectoral myosin, anti chicken heart myosin (1) and anti chicken heart myosin (2) (=anti chicken heart myosin (1) adsorbed with muscle powder from soleus muscle of guinea pig). The results showed that three intrafusal fibre types differed in their myosin composition. A comparison of intrafusal fibre types with extrafusal fibre types for the histochemical myofibrillar ATP-ase reactivity and the reactivity with myosin antisera showed a resemblance of nuclear chain fibres with extrafusal type II fibres and a difference between nuclear bag₁ and nuclear bag₂ fibres and all other fibre types.     

Immunohistochemical analysis of the human psoas major muscle with regards to the body side and aging

The aim of our study was to explore the age related changes of the fibre type composition of the human psoas major muscle. Moreover, we wanted to compare the fibre type composition of the left and right muscle. Muscle samples were collected from 15 young and 15 old males. Type I, IIA and IIX muscle fibres were typed using myosin heavy chain identification. The serial transverse sections were analysed using a light microscope. Results of our study showed that the age-related atrophy affected all three fibre types. Type IIA fibres were affected most profoundly while type I fibres were affected most weakly. The percentage of the different fibre types did not change during aging. There were no differences in the fibre type composition between the left and right muscle. Human psoas major muscle undergoes normal aging changes with the atrophy of all three fibre types, whereas atrophy most profoundly affects type IIA fibres. No differences in the fibre type composition between the left and right muscle point to the equal engagement of both legs in normal everyday activities of human.     

Myosin-ATPase fibre typing of chemically skinned muscle fibres

Summary The efficacy of myosin (M)-ATPase fibre typing to differentiate fibre types in chemically (EGTA) skinned muscle fibres was investigated. Cryosections or single fibres from isolated bundles of chemically skinned rat extensor digitorum longus (EDL) and soleus (SOL) muscles were stained for M-ATPase activity. The results indicate that two major fibre types (type I and II, Brooke & Kaiser, 1970) can be indentified, as well as subgrouping of the type II fibres into types IIa and IIb. Thus, chemically skinning muscle fibres appears to have no detrimental effects on subsequent M-ATPase fibre typing.     

Nitric oxide synthase in human skeletal muscles related to defined fibre types

Skeletal muscle functions regulated by NO are now firmly established. However, the knowledge about the NO synthase (NOS) expression related to a defined fibre type in human skeletal muscles necessitates further clarification. To address this issue, we examined localization of NOS isoforms I, II and III, in human skeletal muscles employing immunocytochemical labeling with tyramide signal amplification complemented with enzyme histochemistry and Western blotting. The NOS immunoreactivity was related to fibre types of different classification systems: physiological classification into slow and fast, ATPase classification into I, IIA, IIAX, IIX, and physiological-metabolic classification into slow-oxidative (SO), fast-oxidative glycolytic (FOG) and fast-glycolytic (FG). We found a correlation of NOS I–III immunoreactivity to metabolic defined fibre types with strong expression in FOG fibres. This implies that NO as modulator of muscle function is involved in oxidative metabolism in connection with fast force development, which only occurs in FOG fibres. The NOS expression showed no correlation to ATPase fibre subtypes due to the metabolic heterogeneity of ATPase fibre types. Healthy and affected vastus medialis muscles after anterior cruciate ligament rupture revealed similar NOS expression level as shown by Western blotting with, however, different expression patterns related to the fibre types in affected muscles. This suggests an altered modulation of force development in the fibres of diseased muscles.     

A comparative histochemical study of intrinsic laryngeal muscles of ungulates and carnivores

The intrinsic laryngeal muscles of the horse, donkey, sheep, ox, pig, dog and cat were examined for myosin ATPase, following acid and alkali pre-incubation, SDH and M-alphaGPDH activities. In all laryngeal muscles two fibre types, betaR and alphaR, belonging to slow and fast-contracting, fatigue-resistant motor units (types S and FR) were present in different proportions. The alphaW fibre type, belonging to fast-contracting and fatigue-resistant motor units was absent (type FF). The alphaR fibres of the dog and the cat were subdivided into groups by the various degrees of acid stable myosin ATPase, oxidative and glycolytic activities. In the ox and pig laryngeal muscles, the same fibres showed an atypical myosin ATPase activity, as high as the fast-contracting fibres but acid-resistant like the slow-twitch fibres. The most uniform muscle was the CAD, which was formed of a higher percentage of slow-twitch fibres than the other laryngeal muscles of the same species. Also the VE muscle was very uniform in the dog, horse and donkey but the fast-twitch fibres were by far the most numerous, the highest in fact among all the laryngeal muscles. In the TA muscle of the cat, sheep and ox, the percentage of fast-twitch fibres was very high in the rostral portion decreasing gradually towards the caudal portion. Thus it was possible to separate histochemically the TA muscle in the rostral (pars ventricularis) and caudal (pars vocalis) portions which are related to the VE and the VO muscles of the dog, horse and donkey. In the VO muscle the slow-twitch fibres are more numerous than in the VE. The two portions of the TA were not detected by histochemical methods in the pig. However, this muscle has the highest percentage of fast-twitch fibres. The qualitative and quantitative data presented in this paper together with the data reported in the literature, enable us to correlate morphological and functional aspects of fibre composition among the species.     

Nitric oxide synthase II in rat skeletal muscles

Constitutive expression of nitric oxide synthase (NOS) II was found in rat hindlimb muscles by immunohistochemistry and western blotting during development from embryonic day 21 to the adult stage of 75 days. The immunohistochemical NOS II expression pattern was related to the physiological metabolic fibre types SO (slow-oxidative), FOG I, II (fast-oxidative glycolytic; I more glycolytic, II more oxidative) and FG (fast-glycolytic) and to the myosin-based fibre types I and IIA, IIB (IIX not separated) identified in serial sections by enzyme histochemistry and immunohistochemistry. In adult muscles only the small population of FOG II fibres, which is a part of both IIA and IIB fibre population, showed NOS II immunoreactivity. This is the reason that only weak NOS II expression in adult hindlimb muscles has been detected by western blotting. Hindlimb muscles of embryonic, neonatal and young rats of 8 days expressed more NOS II as compared with adult rat hindlimb muscles. This can be explained by the findings that before the age of 21 days fast fibres were metabolically undifferentiated, all of them were NOS II positive and contribute to the NOS II expression of the muscle. In muscles of diabetic rats the NOS II expression was elevated indicating an inhibition of glucose uptake into the muscle fibres of diabetic muscles. Our findings suggest that the NOS II may be designated both as constitutive and inducible.     

A histochemical analysis of mammalian oxidative skeletal muscle fibres using the enzymes of energetic metabolism

Summary Some histochemical parameters of the three main fibre types of rat vastus lateralis muscle were studied. Succinic dehydrogenase (SDH), creatine kinase (CK), sarcotubular ATPase (SR-ATPase) and mitochondrial ATPase activities were demonstrated in serial sections. The three fibre types, recognised by the distribution pattern of SDH activity, all show high CK activity. However, red Type I oxidative fibres when examined for ATPase and ATPase dependent CK activity, show distinct heterogeneity revealing sub-populations within the same homogeneous fibre type. Three distinct patterns were recognised in the red Type I fibres depending on the distribution of the final reaction product. The present histochemical evidence confirms the fact that subdivision of mammalian skeletal muscle into three fibre types is only approximate and probably more than three types exist.     

Relationship among fibre type, myosin ATPase activity and contractile properties

Summary At least two types of skeletal muscle myosin have been described which differ in ATPase activity and stability in alkaline or acidic media. Differences in ATPase characteristics distinguish Type I and Type II fibres histochemically. In this study, ATPase activity of myosin from muscles of several species with known histochemical and contractile properties has been determined to test the hypothesis that (1) myosin ATPase activity, (2) histochemical determination of fibre types and (3) maximum shortening velocity, all provide equivalent estimates of contractile properties in muscles of mixed fibre types. Maximum shortening velocity appears to be proportional to ATPase activity as expected from previous reports by Barany. However, both myosin ATPase and the maximum shortening velocity exhibit curvilinear relationships to the fraction of cross-sectional area occupied by Type II fibres. Therefore, we reject the hypothesis and conclude that histochemically determined myofibrillar ATPase does not accurately reflect the intrinsic ATPase activity or shortening velocity in muscles of mixed fibre types. Our data are consistent with the presence of more than two myosin isozymes or with a mixture of isozymes within single muscle fibres.     

The mATPase histochemical profile of rat type IIX fibres: correlation with myosin heavy chain immunolabelling

Summary In the present study we report a novel histochemical method which, by sequential pre-incubations in alkaline and acidic media, selectively differentiates muscle fibres expressing myosin heavy chain IIX, on the basis of a specific profile for myofibrillar actomyosin ATPase (mATPase) activity. The enzyme reactions were tested for specificity by means of anti-myosin heavy chain monoclonal antibodies, which were characterized on Western blots of muscle homogenates. Enzyme histochemical reactions with the traditional pH buffers were compared to those of the new method and, in conjunction with the immunoreactions, used to confirm the relationship between MyHC expression and the distinct profiles for mATPase. Imrnunohistochemical reactions demonstrated that the new method only differentiates those fibres expressing myosin heavy chain IIX. The method revealed a continuum in which the intermediate staining intensities corresponded to hybrid fibres expressing myosin heavy chain IIX in combination with either the IIA or IIB forms. Quantitative histochemistry and immunohistochemistry (by image analysis), used to examine the relationship between staining intensities for mATPase and amounts of myosin heavy chain IIX expression, revealed that the new method discriminates well between hybrid fibres expressing variable amounts of the IIX isoform (r² = 0.93).     

Comparison of red and white muscles by cytophotometry of their muscle fibre populations

Synopsis Samples from two red muscles (vastus intermedius and vastus medialis) and two white muscles (biceps femoris and gluteus medius) were taken from four pigs. Serial transverse sections were reacted for ATPase and NADH oxidative activity. Sections were mapped with a projection microscope so that the staining intensity of individual fibres for the two reactions could be measured with a simple microscope photometer. Transmission values at 600 nm were converted to units of 0–10 for the range from darkest to lightest staining fibres on each section to cancel variation in staining intensity between sections. The aim of the study was to use simple cytophotometry instead of subjective judgement in the categorization of different histochemical types of muscle fibres. Cytophotometry enabled clear resolution of the major fibre types (types I and II using the ATPase reaction), partial resolution of more variable characteristics (NADH oxidative activity in type I and II fibres) and no resolution of subtle subtypes (IA and IB with the NADH oxidative reaction). However, between the major fibre types, cytophotometry revealed variable numbers of fibres with transitional characteristics. There were more of these fibres in red muscles. With sections reacted for ATPase, transmission values for low magnification fields containing 100 to 200 fibres were correlated (r=–0.91) with the ratio of type I:II fibres.     

The effect of strength training on estimates of mitochondrial density and distribution throughout muscle fibres

The purpose of this study was to investigate the effect of strength training (12 weeks, 3 days/week, four lower-body exercises) of young individuals (mean age 23.6 years) on estimates of mitochondrial distribution throughout muscle fibres. A control group (mean age 21. 7 years) was followed simultaneously. Skeletal muscle biopsy samples were obtained from the vastus lateralis, pre- and post-training. The regional distribution of subsarcolemmal and intermyofibrillar mitochondrial populations was determined using quantitative histochemical staining of succinate dehydrogenase (SDH) in type I and II muscle fibres. Strength training resulted in significant increases of 26% and 28% in the cross-sectional area of type I and II fibres, respectively (P < 0.05). Overall SDH activity decreased by 13% with strength training (P < 0.05). The decrease in SDH activity with strength training between fibre types and between subsarcolemmal and intermyofibrillar regions of muscle fibres was not different. Fibre area and SDH activity was unchanged in the control group. We conclude that the muscle hypertrophy associated with strength training results in reduced density of regionally distributed mitochondria, as indicated by the reduction in the activity of SDH.     

Fibre type regionalization of forelimb muscles in two mammalian species, Galea musteloides (Rodentia, Caviidae) and Tupaia belangeri (Scandentia, Tupaiidae), with comments on postnatal myogenesis

To investigate structural differences between propulsory and antigravity muscles, the spatial distribution of slow (type I) and fast (type II) muscle fibres in forelimb muscles of two species of small mammals was studied, Galea musteloides and Tupaia belangeri. Serial sections through complete forelimbs were prepared. Following histochemical fibre typing, the forelimbs were reconstructed three-dimensionally using product design software. Most forelimb muscles of both species showed a homogenous distribution of type I fibres. In the supraspinatus and triceps brachii (capita longum et laterale) muscles, however, a segregation of fibre types into ”fast” superficial areas and ”slow” deep regions was observed. Slow regions contained at least 60% type I fibres and were positioned along intramuscular extensions of the tendons of insertion. The functional implications of fibre type regionalization are discussed. An analysis of intramuscular fibre type distribution during postnatal myogenesis revealed no significant differences in muscle fibre differentiation between altricial and precocial juveniles. Differences in locomotor ability probably arise from heterochronic development of connective tissue components (endo- and perimysium). Accepted: 10 June 1999     

The fibre type composition of the striated muscle of the oesophagus in ruminants and carnivores

The fibre type composition of the striated muscle layer of the oesophagus of the cow, sheep, donkey, dog and cat was examined with standard histochemical methods and immunohistochemical staining using type-specific antimyosin sera. The heavy chain and light chain composition of oesophageal myosin was also examined using electrophoretic peptide mapping and 2-dimensional gel electrophoresis respectively. In the ruminants and donkey the oesophagus was composed of fibre types I, IIA and IIC with immunohistochemical characteristics identical to those of the same fibre types found in control skeletal muscle. In the ruminants there was a gradient in the proportion of type I fibres from 1% (at the cervical end) to about 30% (at the caudal end). In the carnivores the oesophageal muscle was composed of a very small percentage of type I and IIC fibres, but the predominant type was very different histochemically and immunohistochemically from all the fibre types (I, IIA, IIB, IIC) present in the control muscles. This oesophageal fibre type ( IIoes ) had an acid- and alkaline-stable m-ATPase activity, a moderate histochemical Ca-Mg actomyosin ATPase activity, and reacted weakly with anti-IIA and anti-IIB myosin sera. Although the light chains of the IIoes myosin were the same as the light chains of a mixture of IIA and IIB myosins, their respective heavy chains gave different peptide maps. Greater differences were obtained between the heavy chains of IIoes and other striated muscle myosins. These observations lead us to conclude that this predominant fibre type of the carnivore oesophageal striated muscle is of the 'fast' type, and contains a distinct isoform of myosin similar but not identical to the other fast type myosins.     

Diameter changes of muscle fiber types of the inferior oblique muscle of the rabbit following denervation]

Changes in fibre diameters of extraocular muscles of the rabbit were studied at different times after denervation. The whole inferior oblique muscle hypertrophied, while some of the muscle fibres hypertrophied and others showed atrophy, depending on the fibre type. Fibre types have been determined by their histochemical enzyme profile. In the central layer of the muscle the phasic muscle fibres, which are rich in mitochondria, exhibited a transient hypertrophy being maximal 4-5 weeks after denervation and afterwards they atrophied; other phasic muscle fibres, which are poor in mitochondria, atrophied without having shown any sign of hypertrophy. Special, putatively slow tonic muscle fibres, which have low enzyme activities, underwent small long-lasting increases of their diameters. In the superficial layer of extraocular muscle there are two types of extremely thin muscle fibres rich in mitochondira. Both these fibre types hypertrophied to the greatest degree and for a very long time. Comparable changes in fibre diameters as described here for the muscle fibre types of an extraocular muscle are known from special muscle fibres in other vertebrate     

Effect of g.2728g > A and g.3996t > C polymorphisms at the leptin gene locus on microstructure and physicochemical properties of longissimus lumborum muscle of Polish Landrace pigs

The influence ofHindIII (g.2728G > A) and Bg/II (g.3996 T > C) polymorphisms at the leptin gene locus on muscle fibre characteristics and meat quality of longissimus lumborum muscle was studied in 146 barrows of the Polish Landrace breed. Leptin gene polymorphism was identified by PCR-RFLP. Fibre type percentage, fibre diameter and the following technological parameters ofmeat were also determined: pH45, pH24, L*a*b* colour, drip loss, water holding capacity, shear force and intramuscular fat content. Polymorphism was not detected in the locus studied in the Landrace pig herd analysed with the Bg/II restriction enzyme (g.3996 T > C). For the HindIII enzyme (g.2728G > A), there was a high frequency of GG homozygotes (0.78) and G allele (0.89), but the AA genotype was not present. Moreover, the genotypes ofleptin gene RFLP-HindIII polymorphism had no effect on intramuscular fat content and muscle fibre type percentage, but had a significant effect on muscle fibre size. Heterozygous GA fatteners had a significantly larger (P < 0.05) diameter of type IIB and type I fibres compared to homozygous GG fatteners. Generally, meat quality parameters were comparable among the examined genotypes except for water holding capacity (which was the lowest for the GG genotype) and colour lightness (L*) (which was the lightest for GA genotype). Moreover, regardless of genotype, large differences were observed between each animal in the distribution of intramuscular fat.     

The number of satellite cells in slow and fast fibres from human vastus lateralis muscle

The aim of this investigation was to study the distribution of satellite cells in slow (type I fibres) and fast (type II fibres) fibres from human vastus lateralis muscle. This muscle is characterised by a mixed fibre type composition and is considered as the site of choice for biopsies in research work and for clinical diagnosis. Biopsy samples were obtained from five healthy young volunteers and a total of 1,747 type I fibres and 1,760 type II fibres were assessed. Satellite cells and fibre type composition were studied on serial muscle cross-sections stained with specific monoclonal antibodies. From a total of 218 satellite cells, 116 satellite cells were found in contact with type I fibres (53.6±8% of the satellite cells associated to type I fibres) and 102 satellite cells in contact with type II fibres (46.4±8% of the satellite cells associated to type II fibres). There was no significant difference (P=0.4) between the percentages of satellite cells in contact with type I and with type II fibres. Additionally, there was no relationship between the mean number of satellite cells per fibre and the mean cross-sectional area of muscle fibres. In conclusion, our results show that there is no fibre type-specific distribution of satellite cells in a human skeletal muscle with mixed fibre type composition.     

Immunohistochemical demonstration of fibre type-specific isozymes of cytochrome c oxidase in human skeletal muscle

The immunohistochemical reaction of monoclonal as well as polyclonal antibodies against cytochrome c oxidase (COX) subunits with serial sections of normal human skeletal muscle was investigated. The stronger reactivity of polyclonal antibodies to COX subunits II-III and VIIbc with type I as compared to type II fibres, correlated well with the higher histochemical reactivity of NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase in type I fibres. In contrast an almost exclusive reaction of a monoclonal antibody against subunit IV with type I fibre and a preponderant reaction of a polyclonal antibody against subunits Vab with type II fibres was obtained. Antibodies against subunits I, Vb and VIc did not reveal a fibre-type-specific reactivity. The data indicate in human muscle the occurrence of fibre type-specific isozymes of cytochrome c oxidase differing in subunits IV and Va or Vb.     

Immunohistochemical demonstration of fibre type-specific isozymes of cytochrome c oxidase in human skeletal muscle

Summary The immunohistochemical reaction of monoclonal as well as polyclonal antibodies against cytochrome c oxidase (COX) subunits with serial sections of normal human skeletal muscle was investigated. The stronger reactivity of polyclonal antibodies to COX subunits II–III and VIIbc with type I as compared to type II fibres, correlated well with the higher histochemical reactivity of NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase in type I fibres. In contrast an almost exclusive reaction of a monoclonal antibody against subunit IV with type I fibre and a preponderan reaction of a polyclonal antibody against subunits Vab with type II fibres was obtained. Antibodies against subuntis I, Vb and VIc did not reveal a fibre-type-specific reactivity. The data indicate in human muscle the occurrence of fibre type-specific isozymes of cytochrome c oxidase differing in subunits IV and Va or Vb.     

Relationship between myosin heavy chain IId isoform and fibre types in soleus muscle of the rat after hindlimb suspension

The relationship between the myosin heavy chain (HC) IId isoform and histochemically defined fibre types was investigated in the rat soleus muscle after hindlimb suspension. After 4 weeks of suspension, right and left muscles were removed and fibre type composition and total fibre number were examined by histochemical myosin adenosine triphosphatase staining sections. Myosin HC isoforms were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. After the suspension, there was a significant decrease in the percentage of type I fibres and a concomitant increase in that of type IIa fibres. However, the total number of fibres was not affected by suspension. The synthesis of HC IId isoform, which was not found in the control, and the decrease in the ratio of slow type myosin heavy chain isoform (HC I) were observed after suspension. These results would may suggest that the change of fibre type composition was caused by a shift from type I to IIa fibres after suspension. Furthermore, it could be suggested that the synthesis of HC IId isoform occurred during the stage of type shift from type I to IIa fibres.