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

Summary 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 fibres (Samaha et al. 1970). Gross correspondence (>85%) existed between IIA and IIB (Brooke and Kaiser 1970) and and 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 and fibres on the other hand as well as between IIB fibres and and 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.     

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.     

Inter- and intraspecies comparisons of fibre type distribution and of succinate dehydrogenase activity in type I, IIA and IIB fibres of mammalian diaphragms

Fibre types in the costal region of the diaphragm muscle of several mammalian species with widely different respiratory rates were examined microphotometrically for succinate dehydrogenase (SDH) activity. Mean activities indicated no significant (p greater than 0.05) difference between the type I and IIA fibres for any of the species examined. SDH activities in type IIB fibres were significantly lower (p less than 0.05) than either the type I or type IIA fibres in the cat, guinea pig, rat and rabbit whereas in the mouse no difference was found. The dog had no classical type 1B fibres. Analysis of the distribution of SDH activities by fibre type indicated a wide scattering of scores with no distinct separation between fibre types. Large differences in SDH activity were noted between species. Mean SDH activities were highest in the mouse and rat, intermediate in the rabbit and guinea pig and lowest in the cat and dog. These data suggest an association between respiratory rate and aerobic oxidative potential of the various fibre types in diaphragms of the species examined.     

Inter- and intraspecies comparisons of fibre type distribution and of succinate dehydrogenase activity in type I,IIA and IIB fibres of mammalian diaphragms

Summary Fibre types in the costal region of the diaphragm muscle of several mammalian species with widely different respiratory rates were examined microphotometrically for succinate dehydrogenase (SDH) activity. Mean activities indicated no significant (p>0.05) difference between the type I and IIA fibres for any of the species examined. SDH activities in type IIB fibres were significantly lower (p<0.05) than either the type I or type IIA fibres in the cat, guinea pig, rat and rabbit whereas in the mouse no difference was found. The dog had no classical type 1B fibres. Analysis of the distribution of SDH activities by fibre type indicated a wide scattering of scores with no distinct separation between fibre types. Large differences in SDH activity were noted between species. Mean SDH activities were highest in the mouse and rat, intermediate in the rabbit and guinea pig and lowest in the cat and dog. These data suggest an association between respiratory rate and aerobic oxidative potential of the various fibre types in diaphragms of the species examined.     

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.     

Purification and characterization of myosins from human and rabbit skeletal muscles by using specific monoclonal antibodies

By using immunoaffinity column chromatography slow (I) and fast (IIA, IIB) myosins were isolated from human (vastus lateralis) and rabbit (tibialis anterior, psoas and conoidal bundle) skeletal muscles. The peptide pattern revealed that slow (I) and fast (IIA, IIB) myosin heavy chains are quite distinct, as are those from pure slow (conoidal bundle) and fast (psoas) rabbit skeletal muscles. Unlike Billeter et al. (1981) the authors observed that fast human myosins were always associated with a small amount of slow myosin light chains. The fast myosins (IIA, IIB) from rabbit tibialis anterior muscle did not appear very distinct and contained only fast myosin light chains. These myosins were different from the IIB myosin from the psoas muscle. Ten per cent of the fibres revealed histochemically as fast IIA also reacted with an anti-slow myosin antibody. The classical histochemical techniques appear inadequate to demonstrate the existing differences among fibre types, but the monoclonal antibodies hold promise.     

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.     

Lactate dehydrogenase isozymes in type I,IIA and IIB fibres of rabbit skeletal muscles

Summary Lactate dehydrogenase (LDH) isozyme patterns were analysed by polyacrylamide (PAA) slab gel electrophoresis in extracts prepared from various rabbit skeletal muscles of defined fibre composition and by PAA microelectrophoresis of microdissected, histochemically typed single muscle fibres. The results obtained by electrophoresis of whole muscle extracts generally agreed with the data obtained from single fibre electrophoresis, i.e. the LDH isozyme pattern corresponded to that of the predominant fibre type. Type I Fibres from soleus and semitendinosus muscles were characterized by a unique pattern of all 5 LDH isozymes with a predominance of LDH-1, 2 and 3. The major fraction (80%) of the type II fibres from extensor digitorum longus and tibialis anterior muscles contained only LDH-5 (M₄). About 20% of the type II fibres contained in addition to LDH-5 small amounts of LDH-4 and LDH-3. The fraction of fibres containing LDH-5, LDH-4, and LDH-3 was similar (ca. 20%) in the histochemically defined IIA and IIB subpopulations In view of the fact that the major fractions of rabbit IIB fibres display low and of IIA fibres high aerobic oxidative capacities (Reichmann and Pette 1982), these data indicate that the expression of the H-subunit of LDH is not correlated with the aerobic-oxidative capacity of the fibre. It also appears not to be correlated with the presence of different myosin isoforms in IIA and IIB fibres.     

The multiplicity of combinations of myosin light chains and heavy chains in histochemically typed single fibres. Rabbit tibialis anterior muscle.

1. Combined histochemical and biochemical single-fibre analyses [Staron & Pette (1987) Biochem. J. 243, 687-693], were used to investigate the rabbit tibialis-anterior fibre population. 2. This muscle is composed of four histochemically defined fibre types (I, IIC, IIA and IIB). 3. Type I fibres contain slow myosin light chains LC1s and LC2 and the slow myosin heavy chain HCI, and types IIA and IIB contain the fast myosin light chains LC1f, LC2f and LC3f and the fast heavy chains HCIIa and HCIIb respectively. 4. A small fraction of fibres (IIAB), histochemically intermediate between types IIA and IIB, contain the fast light myosin chains but display a coexistence of HCIIa and HCIIb. 5. Similarly to the soleus muscle, C fibres in the tibialis anterior muscle contain both fast and slow myosin light chains and heavy chains. The IIC fibres show a predominance of the fast forms and the IC fibres (histochemically intermediate between types I and IIC) a predominance of the slow forms. 6. A total of 60 theoretical isomyosins can be derived from these findings on the distribution of fast and slow myosin light and heavy chains in the fibres of rabbit tibialis anterior muscle.     

Fine structure of atrial natriuretic peptide (ANP)-granules in the atrial cardiocytes in the hamster, guinea pig, rabbit, cat and dog.

In the hamster, guinea pig, rabbit, dog and cat, the right and left atria and ventricles were examined by immunohistochemistry, and the right auricular cardiocytes were studied by transmission electron microscopy. Moreover, ANP-granules in the cardiocytes were analyzed by ultrastructural morphometry. Immunohistochemically, the most intensely ANP-reactive cardiocytes were localized in the right auricle, particularly more prominent in the hamster and guinea pig than in the rabbit, dog and cat. The immunoreaction in the dog and cat was weaker than that in the rabbit. ANP-immunoreactivity was not detected in the ventricular myocardium of any of all species examined, but was occasionally observed in the subendocardium of the ventricular septum. Ultrastructurally, ANP-granules were localized principally in the perinuclear region associated with the Golgi apparatus and scattered throughout the sarcoplasmic layers. The Golgi apparatus of the cardiocytes was better developed in the hamster and guinea pig than in the rabbit, dog and cat. It was poorly-developed in the dog and cat. By ultrastructural morphometry, the number of granules was greatest in the hamster followed by the guinea pig, rabbit and dog or cat, in this order. On the other hand, the diameter of granules was largest in the guinea pig and reduced via the hamster to the rabbit. The diameter was significantly smaller in the dog than in the rabbit. The diameter of granules of the cat was lay between the rabbit and dog.     

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.     

Monospecific antibodies against the three mammalian fast limb myosin heavy chains

Skeletal muscle fibres in mammalian limb muscles are of four types: slow, 2A, 2X, and 2B, each characterized by a distinct myosin heavy chain (MyHC) isoform. Existing monoclonal antibodies (mabs) against fast MyHCs lack fibre-type specificity across species and could not positively identify 2X fibres. In this work, mabs were raised against each of the fast MyHCs. These mabs were shown to be monospecific by Western blots and immunohistochemistry in the rat. The advantages of using these mabs for identifying the three fast fibre types and hybrid fibres expressing multiple isoforms were illustrated using rat tibialis anterior muscle. Immunohistochemical analyses confirmed the monospecificity of these mabs in the following additional species: mouse, guinea pig, rabbit, cat, and baboon. 2B fibres were absent in limb muscles of the cat and baboon. These mabs constitute a set of powerful tools for studying muscle fibre types and their transformations.     

Fibre composition and enzyme activities in five different muscles from the Svalbard reindeer

beta R fibres (type I) constitute less than 10% of the semimembranosus and longissimus dorsi muscles and about twice as much of the gluteobiceps and flexor hallucis. Except for longissimus dorsi, 50% or more consist of alpha W (type IIB) fibres--in semimembranous, as much as 70%. Despite the comparatively large content of alpha W fibres, both the oxidative capacity and the capacity to metabolize fatty acids is high. Furthermore, unexpectedly small differences in oxidative capacity between the three fibre types beta R, alpha R and alpha W (I, IIA and IIB) are revealed by histochemical staining. These results indicate a tendency to bring the three fibre types closer together as regards metabolic activities, as an adaptation to the relatively tranquil life of this animal. However, the large content of alpha W fibres does not accord well with this way of life, as they guarantee quick movements. The comparatively high oxidative capacity of the alpha W fibres in the Svalbard reindeer and the fact that during starvation it is primarily alpha W fibres that contribute to the energy supply by protein degradation may nevertheless account for their abundant occurrence.     

Speciesabhängigkeit der Monoaminoxydase-Aktivität in verschiedenem Lebensalter

Summary The monoamine oxidase activity in ten species (man, dog, cat, rabbit, guinea pig, rat, hamster, mouse, chicken, goose) was histochemically studied in the myocardium, liver, kidney and psoas muscle in newborn and older individuals.An age-dependent increase of monoamine oxidase activity is established in the liver and kidney of man, dog, cat, guinea pig and hamster. In the psoas muscle of the rat the monoamine oxidase activity is consistently weak. In the myocardium only the rat shows an increase with age.     

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.     

No classical type IIB fibres in dog skeletal muscle

Summary To analyse the fibre type composition of adult dog skeletal muscle, enzyme histochemistry, immunohistochemistry for type I, IIA and IIB myosins, and peptide mapping of myosin heavy chains isolated from typed single fibres were combined. Subdivision of type II fibres into two main classes according to the activity of the m-ATPase after acidic and alkaline preincubation proved to be rather difficult and was only consistently achieved after a very careful adjustment of the systems used. One of these sub-classes of type II fibres stained more strongly for m-ATPase activity after acidic and alkaline preincubation, was oxidative-glycolytic and showed a strong reaction with an anti-type IIA myosin. The other one, however, although unreactive with anti-IIA myosin, was also oxidative-glycolytic, and only showed a faint reaction with an anti-type IIB myosin. Peptide mapping of the myosin heavy chains of typed single fibres revealed two populations of heavy chains among the type II fibre group. Thus, in dog muscle, we are confronted with the presence of two main classes of type II fibres, both oxidative-glycolytic, but differing in the structure of their myosin heavy chains. In contrast to some reports in the literature, no classical type IIB fibres could be detected.     

Histochemical comparison of oxidative enzymes in adrenal glands of mammals

Summary Succinic dehydrogenase, five DPN-linked dehydrogenases-lactic dehydrogenase, malic dehydrogenase, glutamic dehydrogenase, -glycerophosphate dehydrogenase, -hydroxybutyric dehydrogenase, two TPN linked dehydrogenases — glucose-6-phosphate dehydrogenase, isocitric dehydrogenase and 3-ol steroid dehydrogenase were studied in mouse, rat, guinea pig, rabbit, dog, cat, cow, monkey and human adrenal glands. Histochemical studies were made of a characteristic distribution of different level of enzyme activity. In mammals adrenal glands, glucose-6-phosphate dehydrogenase showed the highest activity and its localization was divided into the following two groups: 1) High enzymatic activity was demonstrated in the zona fasciculata and reticularis of the rat, guinea pig, rabbit, cat and 2) high enzymatic activity was demonstrated in the zona glomerulosa and reticularis of the dog, cow and monkey. A precise relationship between the localization and endocrinological function remains abscure.     

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.     

EXERCISE MYOPATHY: CHANGES IN MYOFIBRILS OF FAST-TWITCH MUSCLE FIBRES

The purpose of the present study was to determine the relationships between the changes of myofibrils in fast-twitch oxidative-glycolytic (type IIA) fibres and fast-twitch glycolytic (type IIB) muscle fibres, protein synthesis and degradation rate in exercise-induced myopathic skeletal muscle. Exhaustive exercise was used to induce myopathy in Wistar rats. Intensity of glycogenolysis in muscle fibres during exercise, protein synthesis rate, degradation rate and structural changes of myofibrils were measured using morphological and biochemical methods. Myofibril cross sectional area (CSA) in type IIA fibres decreased 33% and type IIB fibres 44%. Protein degradation rate increased in both type IIA and IIB fibres, 63% and 69% respectively in comparison with the control group. According to the intensity of glycogenolysis, fast oxidative-glycolytic fibres are recruited more frequently during overtraining. Myofibrils in both types of fast-twitch myopathic muscle fibres are significantly thinner as the result of more intensive protein degradation. Regeneration capacity according to the presence of satellite cells is higher in type IIA fibres than in type IIB fibres in myopathic muscle.