Comparison of the sarcoplasmic and myofibrillar proteins of twitch and tonic fibres of frog muscle (Rana esculenta)

Sarcoplasmic and myofibrillar proteins of a frog mixed muscle (distal cruralis bundle) were investigated and compared to their fast twitch muscle homologues. Histochemical reactions revealed two populations of fibres in this muscle, differing from fast twitch fibres by the intensity of their myofibrillar ATPase reaction and by their mitochondrial NADH dehydrogenase activity. The distribution of parvalbumins and LDH isoenzymes in the whole muscle showed some features of tonic muscle type. Myosin light chains pattern of cruralis bundle fibres was characterized by the lower proportion of the LC3 subunit. These results confirmed the heterogeneity of this frog muscle and the presence of tonic or intermediate fibres with their typical sarcoplasmic and myofibrillar proteinic composition.     

A role for Insulin-like growth factor 2 in specification of the fast skeletal muscle fibre

Background  

Fibre type specification is a poorly understood process beginning in embryogenesis in which skeletal muscle myotubes switch myosin-type to establish fast, slow and mixed fibre muscle groups with distinct function. Growth factors are required to establish slow fibres; it is unknown how fast twitch fibres are specified. Igf-2 is an embryonically expressed growth factor with established in vitro roles in skeletal muscle. Its localisation and role in embryonic muscle differentiation had not been established.     

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.     

EFFECTS OF NEUROMUSCULAR BLOCKING AGENTS ON THE DIFFERENTIATION OF NERVE-MUSCLE CONNECTIONS IN SLOW AND FAST CHICK MUSCLES

The effects of neuromuscular blocking drugs on the development of slow and fast muscle fibres and their neuromuscular junctions was studied in chick embryos.
Treatment of embryos with the depolarizing neuromuscular blocking agent suxamethonium affected the development of muscle fibres of the slow anterior latissimus dorsi (ALD) muscle more than that of muscle fibres of the posterior latissimus dorsi (PLD). The differentiation of the presynaptic elements of the neuromuscular junction was delayed and this was particularly obvious in PLD. Normally the number of axon profiles at individual endplates is reduced by 18 days of incubation, but in suxamethonium treated embryos this reduction took place only at 21 days. During earlier stages of development the axon profiles from treated embryos were small with sparse synaptic vesicles. Nevertheless the subsynaptic site of endplates on ALD and PLD muscle fibres became specialized earlier than normal and to a greater extent. Treatment with hemicholinium (HC-3), a drug that reduces the synthesis of acetylcholine (ACh) in nerve terminals affected the development of PLD muscle fibres more than ALD muscle fibres. Although in HC-3 treated embryos nerve-muscle contacts were formed, the axon terminals look immature and remain small even in 18-day old embryos at both ALD and PLD muscle fibres. The reduction of the number of axon profiles normally seen at 18 days failed to take place in treated embryos. At 18 days of incubation many endplates on PLD muscle fibres showed little sign of postsynaptic specilization and resembled endplates usually seen at this stage on ALD muscle fibres.
It is concluded that while neuromuscular activity may be important for the reduction of the number of axon profiles at individual endplates, the specialization of the subsynaptic membrane is brought about by depolarizing effect of ACh.     

Fibre types in locomotory muscles of the cartilaginous fish Chimaera monstrosa

The cartilaginous fish Chimaera monstrosa swims slowly by means of pectoral fin movements, and fast by undulations of the tail. In order to compare the fibres in the corresponding muscles, they were studied by histochemistry and electron microscopy. Three fibre types were identified by microphotometry and morphometry. Most of the axial muscles are white fibres, containing little mitochondria and glycogen. Red fibres, with glycogen and about 5 % mitochondria constitute a thin sheet in the axial muscles, composed of one fibre layer only. Pink fibres, with intermediate amounts of glycogen and mitochondria are situated between these two types, but are often not covered by red fibres. Pectoral muscles contain numerous red and intermediate fibres, partially mixed, superficially, and white fibres deeper. Pectoral muscle red fibres contain about 25 % mitochondria, half of which are situated in subsarcolemmal accummulations. The sarcotubular system has T-tubules at the Z discs, and the terminal cisternae are partially divided by regularly spaced clefts.     

Passive electrical constants of skeletal muscular fibres studied in different muscles of normal and thiamine deficient rats (author's transl)]

10 Some effects of thiamine deficiency were studied in three skeletal rat muscles, having different proportions of "fast" and "slow" fibres: extensor longus digiti IV (a nearly pure fast muscle), soleus (having a predominant population of slow fibres) and diaphragm muscle (mixed fibre population). 20 Cross section area of fibres (fig. 2) is reduced in thiamine deficient animals, mostly for fast fibres having a glycolytic metabolism, the histochemical profile of which tends to become similar to that of slow fibres, in which oxydative metabolism is predominant, as shown by a marked increase in succinodehydrogenase activity. 30 Measurements of resting potential E, of membranes time constant tau and of fibre input resistance R were performed in normal and thiamine deficient muscles (table I). R and tau were obtained from square pulse analysis, using a double shifted sampling method permitting the use of a single microelectrode. E is not greatly affected by thiamine deficiency. tau changes appear not to be significant, except for fast fibres from extensor longus muscle, where tau is slightly reduced. R is increased in thiamine deficient animals (fig. 3). 40 Changes in R and tau do not exactly follow the predictions of cable theory, if one assumes that a purely dimensional factor is involved. Thus, the view that thiamine deficiency does not change basic passive electrical constants of fibres (membrane specific resistance and capacity, myoplasm resistivity) can be considered only as a first approximation. 50 R and tau values obtained in normal muscles are larger than data taken from other studies. The reasons for this discrepancy are discussed. It is suggested that diet differences may play a role.     

Contractile differentiation of foetal cattle muscles: intermuscular variability

off actile differentiation was studied in six foetal muscles exhibiting different contractile characteristics in adult cattle: the Masseter, Diaphragma, Biceps femoris, Longissimus thoracis, Semitendinosus and Cutaneus trunci. These muscles were excised from foetuses aged 60-260 days. Fibre types were identified by immunohistochemistry using three monoclonal antibodies raised against types 1, 2a, 2b (or 2x) and foetal myosin heavy chains. The different myosin isoforms were also separated by electrophoresis, identified by immunoblotting and quantified by ELISA. At least two generations of cells were observed in all the muscles studied. The primary, early differentiated one, gave rise to type II fibres in Cutaneus trunci and type I fibres in all remaining muscles. The secondary generation of cells differentiated later than the first generation of cells. Its pattern of differentiation was more complex in particular from 150 to 210 days. It formed slow fibres in slow adult muscles, fast fibres in fast adult muscles and both types in mixed muscles. Precocity of differentiation was muscle-type dependent and related to muscle function at birth.     

Histochemical and Ultrastructural Features of the Biceps Brachii of the African Chameleon (Chamaeleo senegalensis)

Abstract Characteristics of reptilian muscle fibres were investigated in the biceps brachii of the African chameleon, Chamaeleo senegalensis. Fibres were classified as slow and fast. These types of fibre were distinguished on the basis of histochemical staining for myofibrillar ATPase (mATPase). Fast fibres stained dark for mATPase while slow fibres stained light. The patterns of innervation of slow and fast fibres were investigated by staining nerve endings for acetylcholinesterase activity. Slow fibres have a pattern of multiple innervation, whereas fast fibres are associated with individual endplates. The organization of the myofibrils and the sarcoplasmic reticulum in slow muscle fibres from the chameleon biceps brachii was compared with that in fast fibres. Slow fibres lacked an M-line and the Z-lines were uneven. They had fibrils that were not clearly separated from each other and the sarcoplasmic reticulum was poorly developed. These features are in sharp contrast to those of fast fibres which had straight Z-lines, clear M-lines and well-developed sarcoplasmic reticulum.     

The membrane systems in different fibre types of the triceps surae muscle of cat

Summary The volume and surface area of mitochondria and sarcoplasmic reticulum in fast and slow twitch fibres of the cat triceps surae muscle were determined from thin sections. The width of the Z-line and the array of glycogen granules identified fast and slow twitch fibres.The relative volume occupied by mitochondria was largest in slow twitch gastrocnemius fibres. Fast twitch fibres showed the greatest scatter of mitochondrial content. This corresponds with the fact that motor units of the fast twitch type differ most with respect to resistance to fatigue.The relative volume of the sarcoplasmic reticulum was twice as large in fast as in slow twitch fibres. The volume fraction occupied by longitudinal tubules of the reticulum was the same in fast and slow twitch gastrocnemius fibres but was only half as large in the slow twitch soleus fibres. This difference may be related to post-tetanic potentiation: this property is present in all gastrocnemius fibres but is absent in soleus fibres.The specific tetanic force is 3 to 5 times smaller in slow twitch gastrocnemius than in slow twitch soleus fibres or fast gastrocnemius fibres. There was, however, no detectable morphological difference that might be related to this difference in force.Freeze fractures demonstrated directly that, in soleus fibres, terminal cisternae and longitudinal tubules of the reticulum were scarce as compared to gastrocnemius fibres. The plasma membranes of some gastrocnemius fibres displayed square arrays of 60-nm particles; these arrays were absent in other gastrocnemius fibres and in all soleus fibres. They probably characterize plasma membranes of fast twitch fibres.This study was supported by grants from the Danish Medical Research Council. I wish to thank Mrs. M. Bjærg for valuable technical help     

Further studies on single fibres of bovine muscles.

Young & Davey (1981) (Biochem. J. 195, 317-327) identified numbers of polymorphs of myofibrillar proteins by sodium dodecyl sulphate/polyacrylamide gel electrophoresis of single muscle fibres isolated from three bovine muscles. Fibres were classed according to the distribution of polymorphs. The study has now been extended to eight diverse bovine muscles. The previous distinction made between fast and slow fibres is valid without exception in the extended study. Within these classes, variations in myofibrillar expression are examined within and between fibres, muscles and animals. Two slow muscles are contrasted; masseter is homogeneous in fibre type, whereas diaphragm is subtly heterogeneous, possibly arising from greater physiological demands. Of the myofibrillar polymorphs, attention is concentrated on two variants of fast-muscle myosin heavy chain. Both are present in all fast and mixed muscles examined, except sternomandibularis, and each is respectively associated with certain unidentified proteins. Within a muscle the fast-muscle myosin light-chain expression is the same irrespective of the heavy-chain variant. Histochemical techniques demonstrated that the variants are respectively associated with types IIA and IIB as defined by other investigators.     

Co-ordinated expression of contractile and non-contractile features of control equine muscle fibre types characterised by immunostaining of myosin heavy chains

Combined methodologies of immunohistochemistry, histochemistry and photometric image analysis were applied: (1) to characterise control equine skeletal muscle fibres according to their myosin heavy chain (MyHC) composition and (2) to determine on a fibre-to-fibre basis the correlation between contractile [i.e. MyHC(s), myofibrillar ATPase (mATPase) and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms], metabolic [i.e. succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities, glycogen and phospholamban (PLB) contents], and morphological [i.e. cross-sectional area (CSA), capillary and nuclear densities] features of individual myofibres. An accurate delineation of MyHC-based fibre types was obtained with the immunohistochemical method developed. This protocol showed a high sensitivity and objectivity to delineate hybrid fibres with overwhelming dominance of one MyHC isoform and, furthermore, it allowed a semiquantitative delineation of fast hybrid fibres according to the predominant MyHC isoform expressed. The phenotypic differences in contractile, metabolic and morphological properties seen between fibre types were related to MyHC content. Slow fibres had the lowest mATPase activity (related to shortening velocity), the highest SDH activity (oxidative capacity), the lowest GPD activity (glycolytic metabolism) and glycogen content, the smallest CSA, the greatest capillary and nuclear densities, and expressed slow SERCA isoform and PLB, but not the fast SERCA isoform. The reverse pattern was true for pure IID/X fibres, and type IIA fibres had intermediate properties. Hybrid IIAD/X fibres had mean values intermediate to those of their respective pure phenotypes. Discrimination of fibres according to their MyHC content was possible on the basis of their contractile and non-contractile profiles. These intrafibre interdependencies suggest that, even when controlled by different mechanisms, myofibres of control horses exhibit a high degree of co-ordination in their physiological, biochemical and anatomical features.     

Electrophoretic analysis of proteins from single bovine muscle fibres.

A number of single fibres were isolated by dissection of four bovine masseter (ma) muscles, three rectus abdominis (ra) muscles and eight sternomandibularis (sm) muscles. By histochemical criteria these muscles contain respectively, solely slow fibres (often called type I), predominantly fast fibres (type II), and a mixture of fast and slow. The fibres were analysed by conventional sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the gels stained with Coomassie Blue. Irrespective of the muscle, every fibre could be classed into one of two broad groups based on the mobility of proteins in the range 135000-170000 daltons. When zones containing myosin heavy chain were cut from the single-fibre gel tracks and 'mapped' [Cleveland, Fischer, Kirschner & Laemmli (1977) J. Biol. Chem. 252, 1102-1106] with Staphylococcus proteinase, it was found that one group always contained fast myosin heavy chain, whereas the second group always contained the slow form. Moreover, a relatively fast-migrating alpha-tropomyosin was associated with the fast myosin group and a slow-migrating form with the slow myosin group. All fibres also contained beta-tropomyosin; the coexistence of alpha- and beta-tropomyosin is at variance with evidence that alpha-tropomyosin is restricted to fast fibres [Dhoot & Perry (1979) Nature (London) 278, 714-718]. Fast fibres containing the expected fast light chains and troponins I and C fast were identified in the three ra muscles, but in only four sm muscles. In three other sm muscles, all the fast fibres contained two troponins I and an additional myosin light chain that was more typical of myosin light chain 1 slow. The remaining sm muscle contained a fast fibre type that was similar to the first type, except that its myosin light chain 1 was more typical of the slow polymorph. Troponin T was bimorphic in all fast fibres from a ra muscles and in at least some fast fibres from one sm muscle. Peptide 'mapping' revealed two forms of fast myosin heavy chain distributed among fast fibres. Each form was associated with certain other proteins. Slow myosin heavy chain was unvarying in three slow fibre types identified. Troponin I polymorphs were the principal indicator of slow fibre types. The myofibrillar polymorphs identified presumably contribute to contraction properties, but beyond cud chewing involving ma muscle, nothing is known of the conditions that gave rise to the variable fibre composites in sm and ra muscles.     

Influence of neurons on the occurrence of fibre types and myosin light chains in cultured presumptive slow and fast myoblasts

Myoblasts from 9-day-old quail embryo slow anterior latissimus dorsi (ALD) and fast posterior and latissimus dorsi (PLD) muscles were co-cultured with neurons. The presence of neurons allowed ALD-derived muscle fibres to express characteristic features of a slow muscle (occurrence of alpha' and of beta' fibres and predominance of slow myosin light chains). On the contrary, PLD-derived fibres did not differentiate into normal fast fibres (occurrence of alpha'-like fibres and absence of LC3f). These results are compared with the differentiation of ALD and PLD myoblasts in aneural condition. It is suggested that neurons can modify some phenotypic expression of presumptive slow or fast myoblasts.     

The chloride conductance of intermediate fibres from frog muscles

Sheets of muscle fibres dissected from surface portions of frog ileofibularis and semitendinosus muscles were soaked in solutions with elevated K and Cl concentrations. The KCl-loaded muscles were then bathed in low [Cl-] solutions, whereby the membrane potential became transiently inside positive. The repolarization of the twitch fibres from the tonus bundle ("intermediate fibres") was faster than that of the fibres adjacent to it ("fast fibres") when the preceding exposure to high KCl was brief (7-15 min), and it was slower than that of the fast fibres when KCl was applied for 4 hours. Measurements of the voltage displacement at constant current and of the current in a point voltage clamp showed that inwardly rectifying K channels were present in the membranes of both types of fibres. The ionic conductance ratio, gK/gCl, was 191/523 in fast fibres and 335/230 in "intermediate" fibres. The different repolarization rates may thus be explained by differences in the chloride conductance of the fast and intermediate fibre membranes. The smaller diameter of the latter fibres may be another factor.     

Slowly contracting muscles power the rapid jumping of planthopper insects (Hemiptera, Issidae)

The planthopper insect Issus produces one of the fastest and most powerful jumps of any insect. The jump is powered by large muscles that are found in its thorax and that, in other insects, contribute to both flying and walking movements. These muscles were therefore analysed by transmission electron microscopy to determine whether they have the properties of fast-acting muscle used in flying or those of more slowly acting muscle used in walking. The muscle fibres are arranged in a parallel bundle that inserts onto an umbrella-shaped tendon. The individual fibres have a diameter of about 70 μm and are subdivided into myofibrils a few micrometres in diameter. No variation in ultrastructure was observed in various fibres taken from different parts of the muscle. The sarcomeres are about 15 μm long and the A bands about 10 μm long. The Z lines are poorly aligned within a myofibril. Mitochondrial profiles are sparse and are close to the Z lines. Each thick filament is surrounded by 10–12 thin filaments and the registration of these arrays of filaments is irregular. Synaptic boutons from the two excitatory motor neurons to the muscle fibres are characterised by accumulations of ~60 translucent 40-nm-diameter vesicle profiles per section, corresponding to an estimated 220 vesicles, within a 0.5-μm hemisphere at a presynaptic density. All ultrastructural features conform to those of slow muscle and thus suggest that the muscle is capable of slow sustained contractions in keeping with its known actions during jumping. A fast and powerful movement is thus generated by a slow muscle.     

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.     

Myofibrillar-protein isoforms and sarcoplasmic-reticulum Ca2+-transport activity of single human muscle fibres.

In this study the polymorphism of myofibrillar proteins and the Ca2+-uptake activity of sarcoplasmic reticulum were analysed in single fibres from human skeletal muscles. Two populations of histochemically identified type-I fibres were found differing in the number of light-chain isoforms of the constituent myosin, whereas the pattern of light chains of fast myosin of type-IIA and type-IIB fibres was indistinguishable. Regulatory proteins, troponin and tropomyosin, and other myofibrillar proteins, such as M- and C-proteins, showed specific isoforms in type-I and type-II fibres. Furthermore, tropomyosin presented different stoichiometries of the alpha- and beta-subunits between the two types of fibres. Sarcoplasmic-reticulum volume, as indicated by the maximum capacity for calcium oxalate accumulation, was almost identical in type-I and type-II fibres, whereas the rate of Ca2+ transport was twice as high in type-II as compared with type-I fibres. It is concluded that, in normal human muscle fibres, there is a tight segregation of fast and slow isoforms of myofibrillar proteins that is very well co-ordinated with the relaxing activity of the sarcoplasmic reticulum. These findings may thus represent a molecular correlation with the differences of the twitch-contraction time between fast and slow human motor units. This tight segregation is partially lost in the muscle fibres of elderly individuals.     

Ultrastructure and innervation of the swimbladder of Tetractenos glaber (Tetraodontidae)

Summary The general structure, ultrastructure and innervation of the swimbladder of the smooth toadfish, Tetractenos glaber, were examined with light-microscopic, fluorescence-histochemical, and transmission electron-microscopic techniques. The structure of the swimbladder is similar to that of other euphysoclists. Fluorescence histochemistry showed adrenergic fibres in both the secretory and resorptive areas of the swimbladder. Transmission electron microscopy revealed two morphologically distinct axon profiles type-I profiles containing many small, flattened vesicles; type-II profiles containing both large, granular vesicles and rounded, small clear vesicles in varying proportions.The gas-gland cells and surrounding muscularis mucosae are innervated by both type-I and type-II fibres. Type-I fibres also innervate pre-rete arteries. The rete- and gas-gland capillaries do not appear to be innervated. Arteries running to the resorptive area are innervated by type-I fibres. Both type-I and type-II profiles make contact with the muscularis mucosae in the resorptive area. Only type-I fibres innervate the radial dilator muscle in the oval sphincter region, whereas only type II fibres innervate the circular muscle of the oval sphincter.Type-I fibres took up -methyl-noradrenaline, and could not be found after pre-treatment with 6-hydroxydopamine. They are, therefore, assumed to be adrenergic. Type-II fibres were tentatively identified, by exclusion, as cholinergic.     

Quantitative histochemistry of three mouse hind-limb muscles: the relationship between calcium-stimulated myofibrillar ATPase and succinate dehydrogenase activities

Summary A quantitative modification of Meijer's calcium-lead capture method, for the demonstration of calcium-stimulated myofibrillar ATPase activity at physiological pH, is described. A range of myofibrillar ATPase activities has been found among fast muscle fibres in two mouse hind-limb muscles. The myofibrillar ATPase activity of fast muscle fibres is 1.5–3 times higher than the myofibrillar ATPase activity of slow muscle fibres.Myofibrillar ATPase activities and succinate dehydrogenase activities of individual muscle fibres have been determined in serial sections. Activities of the two enzymes are correlated positively in soleus (fast and slow fibres), and negatively in plantaris (almost all fast) and extensor digitorum longus muscle (all fast). However, this correlation is not significant among the oxidative fibres in the extensor digitorum longus. The fibres of the latter muscle cannot be classified satisfactorily into two sub-types.     

Effects of hypothyroidism on myosin heavy chain composition and fibre types of fast skeletal muscles in a small marsupial, Antechinus flavipes

Effects of drug-induced hypothyroidism on myosin heavy chain (MyHC) content and fibre types of fast skeletal muscles were studied in a small marsupial, Antechinus flavipes. SDS-PAGE of MyHCs from the tibialis anterior and gastrocnemius revealed four isoforms, 2B, 2X, 2A and slow, in that order of decreasing abundance. After 5 weeks treatment with methimazole, the functionally fastest 2B MyHC significantly decreased, while 2X, 2A and slow MyHCs increased. Immunohistochemistry using monospecific antibodies to each of the four MyHCs revealed decreased 2b and 2x fibres, and increased 2a and hybrid fibres co-expressing two or three MyHCs. In the normally homogeneously fast superficial regions of these muscles, evenly distributed slow-staining fibres appeared, resembling the distribution of slow primary myotubes in fast muscles during development. Hybrid fibres containing 2A and slow MyHCs were virtually absent. These results are more detailed but broadly similar to the earlier studies on eutherians. We hypothesize that hypothyroidism essentially reverses the effects of thyroid hormone on MyHC gene expression of muscle fibres during myogenesis, which differ according to the developmental origin of the fibre: it induces slow MyHC expression in 2b fibres derived from fast primary myotubes, and shifts fast MyHC expression in fibres of secondary origin towards 2A, but not slow, MyHC.