Enzyme histochemistry of the mesocoxal muscles of Periplaneta americana

Histochemical techniques have been employed to characterize enzymatic activity in the mesocoxal muscles of the cockroach, Periplaneta americana. Through our studies of the enzymes myosin-ATPase, NADH reductase, succinic dehydrogenase (SDH), and lactic dehydrogenase (LDH), we were able to classify fibers within these muscles according to criteria established for muscle fibers of vertebrates. Many of the mesocoxal muscles possess two different and distinct populations of fibers, whereas the remaining muscles are homogeneous with respect to their constituent fibers. The data presented here indicate biochemical heterogeneity for muscles of differing structural and functional features and possible neurotrophic influences upon oxidative enzymes and myosin-ATPase isozymes.     

Characteristics of paravertebral muscles – fibre type distribution pattern in the pika, Ochotona rufescens (Mammalia: Lagomorpha)

The evolution of the locomotor apparatus in vertebrates is marked by major reorganizations in trunk's musculature. The hypothesized functions of mammalian back muscles in the literature are discussed under consideration of the distribution and proportion of oxidative, type‐I‐fibres, oxidative‐glycolytic, type‐IIa‐fibres and glycolytic, type‐IIb‐fibres in paravertebral muscles of a small mammal. The fibre type distribution was examined from a complete series of histological sections maintaining topographical relationships between the muscles as well as within the muscle, in order to establish the overall distribution pattern. The deep and short muscles showed the highest percentage of oxidative fibres. The larger, superficial paravertebral muscles contained the highest percentage of glycolytic fibres. Two muscles were intermediate in their proportion of fibre types. All epaxial muscles together can be interpreted as an antigravity muscle–complex counteracting enduringly against the rebound tendency caused by gravitation, comparable with antigravity muscles in limbs. A gradient from deep to superficial, or a clear regionalization of oxidative muscle fibres in central deep regions around a large intramuscular tendon was found in the m. spinalis and the m. quadratus lumborum, respectively. Concepts of the function of human back muscles as those of A. Bergmark (1989: Acta Orthop. Scand. 230 , 1) or S.G.T. Gibbons & M.J. Comerford (2001: Orthop. Division Rev. March/April, 21) were exposed to be more general within mammals. Functional specializations of different muscles and muscle parts are discussed under the consideration of evolutionary reorganization of the paravertebral musculature in tetrapods. Along the cranio‐caudal axis, the percentage of oxidative fibres was decreased in caudal direction within the same muscles, whereas the proportion of glycolytic fibres was increased. Therefore, classifications of muscles as ‘glycolytic’ or ‘oxidative’ based on biopsies or analyses of single cross‐sections may result in wrong interpretations. Changes in the proportions of the fibre type distribution pattern were mostly due to oxidative and glycolytic fibre types, whereas the percentage of oxidative‐glycolytic fibres had only minor influence. A significant positive correlation between the cross‐sectional area of the single fibre and its percentage in the area investigated were observed for oxidative fibres, whereby the size was positive correlated to the proportion of the oxidative fibres.     

Altered fibre types in gastrocnemius muscle of high wheel-running selected mice with mini-muscle phenotypes

Selective breeding of mice for high voluntary wheel running has favoured characteristics that facilitate sustained, aerobically supported activity, including a "mini-muscle" phenotype with markedly reduced hind limb muscle mass, increased mass-specific activities of oxidative enzymes, decreased % myosin heavy chain IIb, and, in the medial gastrocnemius, reduced twitch speed, reduced mass-specific isotonic power, and increased fatigue resistance. To evaluate whether selection has altered fibre type expression in mice with either "mini" or normal muscle phenotypes, we examined fibre types of red and white gastrocnemius. In both the medial and lateral gastrocnemius, the mini-phenotype increased activities of oxidative enzymes and decreased activities of glycolytic enzymes. In red muscle samples, the mini-phenotype markedly changed fibre types, with the % type I and type IIA fibres and the surface area of type IIA fibres increasing; in addition, mice from selected lines in general had an increased % type IIA fibres and larger type I fibres as compared with mice from control lines. White muscle samples from mini-mice showed dramatic structural alterations, with an atypical distribution of extremely small, unidentifiable fibres surrounded by larger, more oxidative fibres than normally present in white muscle. The increased proportion of oxidative fibres and these atypical small fibres together may explain the reduced mass and increased mitochondrial enzyme activities in mini-muscles. These and previous results demonstrate that extension of selective breeding beyond the time when the response of the selected trait (i.e. distance run) has levelled off can still modify the mechanistic underpinnings of this behaviour.     

Distribution of isoenzymes of the glycogenolytic cascade in different types of muscle fibre.

Distinct isoenzyme patterns of the glycogenolytic enzymes exist in different fibre types. Fast twitch glycolytic and slow twitch oxidative fibres differ in the proportion of the two isoenzymes of cyclic AMP dependent protein kinase and in the type of phosphorylase kinase that is present. Slow twitch oxidative fibres and cardiac fibres resemble one another in these two respects, but differ in that the type I phosphorylase of cardiac muscle is absent in slow twitch oxidative fibres. In all examples, the functional differences between the isoenzymes seem to be related to the regulatory rather than the catalytic behavior of the molecules. In the case of cyclic AMP dependent protein kinase and phosphorylase kinase, it is a regulatory subunit that appears to be affected [16,23], while in the case of phosphorylase, the type I isoenzyme is known to have a five to eight-fold Ka for the allosteric activator 5' AMP [6]. However, the precise physiological significance of these differences remains to be elucidated.     

Distribution of serotonin-containing neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of serotonin (5HT)-containing neurons in the central nervous system of the snail Helix pomatia has been determined in whole-mount preparations by use of immunocytochemical and in vivo 5,6-dihydroxy-tryptamine labelling. 5HT-immunoreactive neuronal somata occur in all but the buccal and pleural ganglia. Immunoreactive fibres are present throughout the central nervous system. The 5HT-immunoreactive neuronal somata characteristically appear in groups, located mainly in the cerebral, pedal, visceral and right parietal ganglia. The majority of 5HT-immunoreactive neurons is located in the pedal ganglia. Additionally a dense network of 5HT-immunoreactive varicose fibres is found in the neural sheath of the central nervous system including all the nerves and ganglia. The number and distribution of 5HT-immunoreactive neurons correlates with that demonstrated by 5,6-dihydroxytryptamine labelling method.     

Skeletal muscle characteristics of reindeer (Rangifer tarandus L.)

Fibre type composition, fibre areas, capillaries, enzyme activities and intramuscular substrates were analysed on skeletal muscle samples from reindeer. The muscles contained 10-20% Type I fibres and a higher percentage of Type IIB (40-60%) than Type IIA fibres (20-40%). All fibre types revealed medium or dark staining intensity for oxidative capacity. Glycolytic capacity was greatest in Type IIB fibres. All fibres stained for glycogen, while Type I and IIA fibres stained for lipids. The mean number of capillaries in contact with fibres of each type, relative to fibre type area was high in all muscle types. The metabolic profile of reindeer muscle indicates that energy, to a great extent, is produced through oxidative pathways.     

Fibres of intermediate type 1C and 2C are found continuously in mdx soleus muscle up to 52 weeks

The mdx mutant is a murine homologous model of Duchenne muscular dystrophy (DMD). Fibre types determined by the myosin-ATPase technique in soleus muscles were compared in C57BL/10 control and mdx mice from 3 to 52 weeks of age. The control strain continuously presented 70% of type 2 fibres whereas the mdx strain showed an increase to 80% at 6 weeks and a subsequent decline. In mdx muscles, necrosis which begins at 3 weeks of age did not affect specifically one type of fibre. Type grouping was never observed when muscle regeneration occurred. Fibres of intermediate type (1C and 2C) were found continuously up to 52 weeks of age in the mdx mutant. The foci of small immature regenerating fibres were of type 2C but never 1C. A few mature fibres were either of type 2C or 1C. We suggest that the presence of intermediate type fibres could result from the co-expression of type 1 and 2 myosin heavy chains, indicating a transition from type 2 to type 1 in regenerating fibres.     

Development of chequerboard pattern in human foetal skeletal muscle.

The study was undertaken to race the histochemical and electron microscopic development patterns of human myogenesis from the 9th to the 26th week of foetal life. Particular attention was paid to the possibility of appearance of metabolic or structural differences between individual skeletal muscle fibres in early periods of myogenesis. The 9th week of foetal life is the period when primitive myotubes are formed. Irregular distribution of the ATPase (pH 9-4) activity observed at this time is due to differences in the structure of fusing myoblasts. The early myotubes show a high activity of the oxidative enzymes and lack of phosphorylase. Conversion of immature muscle cells into structurally mature fibres begins between the 20th and the 24th week. The latter fibres exhibit a uniform activity of all the studied enzymes and thus resemble the intermediate type of fibres of mature muscles. From about the 26th week on the typical mosaic pattern of the enzymatic activity is observed. All the differences in enzymatic activity which appear in fibers prior to their full morphologicaal maturity result from differences in developmental stages of the fibres at the given moment. The present study also suggests that there are no morphological or metabolic differences between individual humanskeletal muscle fibres prior to their metabolic differentiation into types which occurs after their innervation.     

Post-larval growth in the lateral white muscle of the eel, Anguilla anguilla

The post-larval growth of lateral white muscle was studied in eels at different stages of post-larval development (glass, yellow and silver eels) by means of histochemical methods for myosin-ATPase (mATPase) activity, immunohistochemistry (for myosin isoforms) and electron microscopy.
Morphological, histo- and immunohistochemical data reveal a uniform appearance of white muscle in glass eels, whereas in following stages the typical mosaic appearance is present. Small-diameter fibres show a more acid-labile mATPase activity than large fibres and react with anti-F, anti-FHC and anti-S sera, but not with anti-SHC serum. In the silver stage, the small fibres tend to decrease in number as the size of the eels increases.
Electron microscopy reveals the presence of satellite cells at every stage: in glass eels there are also 'activated' elements showing scarce myofilaments in their cytoplasm; in yellow eels very small fibres are present, enveloped within the basal lamina of well-differentiated muscle fibres; in silver eels there are no fibres showing signs of immaturity.
Presumably the post-larval development of white muscle involves in juvenile eels a substantial recruitment of fibres from the satellite cell population; later the hyperplasia decreases or ceases and hypertrophy remains the only mechanism for muscle growth.     

Histochemical demonstration of enzymatic heterogeneity within the mesocoxal and metacoxal muscles of Periplaneta americana

Enzymatic activity in the mesocoxal and metacoxal muscles of the cockroach, Periplaneta americana, has been characterized using histochemical techniques. Our interpretation of the histochemical stains for myosin-ATPase (pre-incubated at pH 9.4, 10.4 and 4.3). NADH reductase, succinic dehydrogenase (SDH), and lactic dehydrogenase (LDH) has enabled us to classify fibres within these muscles according to established fibre typing strategies. The results reaffirm the biochemical heterogeneity reported for the mesocoxal muscles and allow us to confirm the expected heterogenity in the homologous metacoxal muscles. These techniques have proven to be an effective tool for assessing the biochemical properties of muscle fibres.     

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.     

Differentiation of metacoxal muscles in Periplaneta americana (L.) (Dictyoptera : Blattidae)

Flight muscles of the cockroach, Periplaneta americana (Dictyoptera : Blattidae) in different development stages (10 mm and 30 mm nymphs, and adult) are investigated for histochemical activity and by electron microscope. The 177 C muscle of 10 mm nymph shows low succinic dehydrogenase (SDH) and myosin-ATPase activities (+). Each myofibril is surrounded by an extensive network of sarcoplasmic reticulum. Regarding myofilament array, one thick filament is surrounded by 10–12 thin filaments. At the stage of 30 mm nymph, SDH and myosin-ATPase activities increase (+ +). Except for an increase in the number of mitochondria, electron microscopic features are similar to those in the 10 mm nymph. In the adult stage, both SDH and myosin-ATPase activities are highest. The distribution of sarcoplasmic reticulum and T-tubules is fundamentally unchanged, whereas the myofilament array is drastically changed, so that 6 thin filaments surround a thick one.     

Metabolic variability within individual fibres of the cat tibialis posterior and diaphragm muscles

Summary Variance in succinate dehydrogenase activity along the transverse and longitudinal axes of fibres from the cat tibialis posterior and diaphragm muscles was determined in order to estimate the three-dimensional distribution of mitochondria within single fibres. The variance (coefficient of variation) in succinate dehydrogenase activity along the transverse fibre axis was greatest in type IIB fibres from both muscles. Intracellular compartmentalization (i.e. subsarcolemmal vs central core differences in succinate dehydrogenase activity) was observed only in type II fibres from the tibialis posterior; the succinate dehydrogenase activity of the subsarcolemmal region was significantly greater than that of the central core. The extent of succinate dehydrogenase variance along the longitudinal fibre axis was dependent on the total length of the fibre segment analyzed, the muscle, and fibre type. The coefficient variation for short fibre segments, i.e. 40 m, was significantly lower than that for much longer fibre segments (840 m). Significant differences in the coefficient variation for 840 m fibre segments were observed between the diaphragm and tibialis posterior muscles. The longitudinal variance in succinate dehydrogenase activity was higher in diaphragm muscle fibres. The succinate dehydrogenase variance along the longitudinal axis of type II fibres was significantly greater than that of the type I fibre population. These results indicate that mitochondria are heterogeneously distributed within muscle fibres. Possible functional implications of such intrafibre metabolic variance are discussed.     

Redox balance following magnetic stimulation training in the quadriceps of patients with severe COPD

In severe COPD patients, oxidative stress, which is involved in their peripheral muscle dysfunction, increases in response to exercise. In this study, muscle oxidative stress was explored after quadriceps magnetic stimulation training. A randomized controlled study was conducted on very severe COPD patients, who underwent quadriceps magnetic stimulation training for 8 weeks. A control group was also studied. In both groups, vastus lateralis specimens were obtained before and after the 8-week period. Muscle protein carbonylation and nitration and antioxidant enzymes were determined using immunoblotting and proportions and sizes of type I and II fibres using immunohistochemistry. Compared to controls, magnetic stimulation muscle training did not modify redox balance, whilst inducing a significant increase in type I fibre sizes. In severe COPD patients, it is concluded that quadriceps magnetic stimulation training was a well-tolerated therapeutic intervention, which did not enhance muscle oxidative stress, while increasing the size of slow-twitch fibres.     

Localization of lactate dehydrogenase isozymes in human muscle tissues by the mixed aggregation immunocytochemical technique.

Lactate dehydrogenase (LDH) isozyme composition and localization was determined in sections of skeletal, heart and smooth muscle by the mixed aggregation immunocytochemical method using first antibody directed against purified human LDH-A4 (M4) or LDH-B4 (H4) followed by the enzymes LDH-A4 and LDH-B4, respectively. An even distribution of the two monomers in all fibres was seen with heart muscle and smooth muscle. Heart muscle had a low concentration of A-monomers and a high concentration of B-monomers, whereas the smooth muscle had equal concentrations of the two monomers. In contrast, skeletal muscle from m. quadriceps femoris was found to be composed of two muscle fibre types, one containing mainly A-, the other mainly B-monomers. On the basis of succinate dehydrogenase activity it was shown that the red (type 1) fibres contain mainly B-monomers and the white (type 2) fibres mainly A-monomers of LDH.     

Extreme endurance training: evidence of capillary and mitochondria compartmentalization in human skeletal muscle.

Biopsies from the medial gastrocnemius muscle of three experienced endurance runners who had completed an ultramarathon run (160 km) the previous day were assessed for their oxidative characteristics (fibre types, capillarization and mitochondria content). Also, a regional comparison was made for fibres located centrally (completely surrounded by other fibres) versus fibres located peripherally (next to the interfascicular space) and the capillarization of these peripheral fibres was determined. Subsarcolemmal mitochondria were abundant and 'compartmentalized' close to the capillaries. The number of capillaries around fibres ranged from 5.8 to 8.5 and 5.7 to 8.5, and the number of capillaries.mm-2 ranged from 665 to 810 and 727 to 762, for type I (slow twitch) and type II (fast twitch) fibres, respectively. Central fibres contained a greater number of capillaries and more capillaries.mm-2 than their peripheral counterparts. Peripheral fibres contained more capillaries.micron-1 between fibres than at the interfascicular space. Type I fibres were more distributed (63%-78%) and larger than type II fibres. An abundance of subsarcolemmal mitochondria located close to the capillaries, efficient capillary proliferation between fibres where sharing can occur and greater relative distribution and size of type I fibres are, collectively, efficient characteristics of extreme endurance training.     

Activities of malate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and fructose-1,6-diphosphatase with regard to metabolic subpopulations of fast- and slow-twitch fibres in rabbit muscles

Summary Activities of malate dehydrogenase (MDH), 3-hydroxyacyl-CoA dehydrogenase (HAD) and fructose-1,6-diphosphatase (FDPase) were determined in single fibres dissected from freeze-dried rabbit psoas and soleus muscles. Slow-twitch fibres as determined by qualitative ATPase reaction represent a rather uniform population with regard to HAD and MDH activities. In these fibres the two enzymes are in constant proportions. FDPase is found at extremely low activities in slow-twitch fibres and because of its relatively high activity in fast-twitch fibres of soleus and psoas muscle it might be used as a marker enzyme. Fast-twitch fibres in psoas muscle represent a heterogeneous population with regard to activities of MDH as well as of HAD. The two enzyme activities are not proportional in fasttwitch psoas fibres. These findings suggest the existence of metabolic sub-populations of fast-twitch fibres having a wide range of aerobic oxidative capacities and having differences in their capacity to oxidizing fatty acids.     

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.     

Biological durability and oxidative potential of man-made vitreous fibres as compared to crocidolite asbestos fibres

In this study we investigated relationships between redox properties and biodurability of crocidolite asbestos fibres and three different man-made vitreous fibres (MMVF): traditional stone wool fibres (MMVF 21), glass fibres (MMVF 11) and refractory ceramic fibres (RCF). Each fibre type was incubated up to 22 weeks in four different incubation media: gamble solution (GS) pH 5.0 and pH 7.4, representing blood plasma without proteins, and surfactant-like solution (SLS) pH 5.0 and pH 7.4. During incubation time aliquots of incubation mixtures were removed and analysed in a biochemical model reaction, mimicking activated phagocytes. In addition, changes of fibre morphology and chemical composition were examined using SEM- and EDX-technology. In the presence of crocidolite asbestos fibres and MMVF 21 the formation of OH*-radicals according to the Haber-Weiss sequence could be demonstrated, whereas MMVF 11 and RCF showed no reactivity. Crocidolite asbestos fibres exhibited a significant higher activity compared with the stone wool fibres at the onset of incubation. The oxidative capacities of these fibre types were shown to depend on both specific surface area and iron content. The oxidative potentials of crocidolite asbestos fibres as well as MMVF 21 were not constant during incubation over several weeks in each incubation medium. The reactivities showed sinoidal curves including reactivities much higher than those at the onset of incubation time. These irregular changes of oxidative capacity may be explained by changes of the redox state of fibre surface-complexed iron. Furthermore our results showed clear differences between incubation of fibres in GS and SLS, respectively, indicating that phospholipids play an important part in fibre dissolution behaviour and oxidative reactivity. In conclusion we suggest, that biodurability testing procedures should not exclusively concentrate on dissolution rates of fibres. They should include fibre characteristics concerning known pathogenic mechanisms to evaluate the real toxic potential of the fibre type looking at. Secondly we suggest, that phospholipids should be constituents of incubation liquids used for standardised fibre biodurability test procedures thus representing more realistic incubation conditions.     

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.