Muscle differentiation in blackspot seabream (Pagellus bogaraveo, Brunnich): histochemical and immunohistochemical study of the fibre types.

The aim of this work was to gain insights into the mechanism of muscle differentiation and growth in Pagellus bogaraveo, by studying muscle fibre phenotypes identified by immunohistochemistry. At hatching, several layers of deep fast-white fibres were covered by a superficial fibre monolayer. At 5 days, slow-red fibres appeared near the lateral line nerve. At 40 days, the intermediate-pink muscle became visible, and in the slow-red and fast-white muscle layers transitions from larval myosin isoforms to the isoforms typical of adult muscle occurred. Between 70 and 100 days, small fibres with a distinct ATPase profile appeared throughout the fast-white muscle, marking the onset of "mosaic" hyperplasia. The myosin of the original superficial monolayer fibres underwent two myosin transformations, before being slowly replaced by an adult slow-red isoform. In juveniles and adults, the slow-red muscle layer could be resolved into two distinct types. The analysis of fibre phenotypes indicated that post-larval muscle growth occurred by two distinct stages of hyperplasia. This study offers a basis for further comparative and experimental studies with this economically relevant species, namely for identifying factors influencing its muscle growth dynamics and disclosing underlying mechanisms.

Selective regulation of myofiber differentiation by energy status during postnatal development.

The role of energy status in postnatal regulation of porcine skeletal muscle development has been determined in littermate animals kept for 3-4 wk on a high (H) or low (L) energy intake (H = 2L), at a thermally neutral [26 degrees C (26H and 26L, respectively)] or low [10 degrees C (10H and 10L, respectively)] environmental temperature. A variety of skeletal muscles was assessed at 7 wk of age for changes in myofiber hypertrophy and differentiation. In contrast with findings in adult humans and rats, there was no selective preservation of type I slow-oxidative fiber size during energy restriction. However, differentiation between mature skeletal myosin heavy-chain isoforms was markedly affected by energy status, and in rhomboideus there were particularly striking effects of both nutrition and temperature: proportions of type I fibers from the four groups 26H, 26L, 10H, and 10L were 34 +/- 2, 50 +/- 4, 73 +/- 2, and 72 +/- 3 (P < 0.005 for diet at 26 degrees C; P < 0.001 for temperature). These changes may have been induced by alterations in both thyroid status and contractile activity. They support the hypothesis of a key role for rhomboideus muscle in thermoregulation and demonstrate the plasticity of skeletal muscle differentiation to environmental change during postnatal life.

Myosin expression in the jaw-closing muscles of the domestic cat and American opossum.

Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), glycerol SDS-PAGE, two-dimensional electrophoresis, and protein immunoblotting techniques were used to identify myosin heavy chain (MHC) and light chain (MLC) isoforms in limb and masticatory muscles of the cat and American opossum. The fibre types in which these isoforms are expressed were identified by histochemistry and immunohistochemistry. Antibodies specific for the type IIM MHC isoform characteristic of cat jaw-closing muscles and the type I MHC isoform were produced and characterized. The IIM antibody stained the majority of fibres found in the jaw-closing muscles of both species. These IIM-containing fibres characteristically had a histochemical ATPase that remained active after both acid and alkali pre-incubations. A minority of type I fibres was also present in cat jaw-closing muscles, and these reacted positively with antibody specific for type I MHC. It was confirmed that the vast majority of fibres in the cat jaw-closing muscles contained only the characteristic masticatory MHC (IIM) and masticatory MLCs (LC1m and LC2m). These muscles did not contain either the type II fibre isoforms of limb muscles or the atrial cardiac (alpha-cardiac) MHC. The type IIM MHC could also be identified in jaw-closing muscles of the opossum. Two-dimensional gel electrophoresis was used to identify the MLC composition of single, histochemically defined, type I fibres in the cat soleus and deep masseter. The type I fibres of limb muscle contained the usual slow MLCs, but type I fibres from the jaw-closing muscles contained only the masticatory light chains.

Fibre type classification and myosin isoforms in the human masseter muscle.

Human masseter muscle is highly unusual since it contains relatively large numbers of fibres with variable myofibrillar ATPase staining as well as fibres that express neonatal and alpha-cardiac myosin heavy chain (MHC). These findings however, have not been organised together into a fibre type classification scheme. Biopsies from the anterior superficial area of masseter were collected from a large sample of healthy young adults. Biopsies were sectioned and stained for myofibrillar ATPase reactivity and the presence of MHC isoforms as detected by a series of antibodies. The MHC composition of the same biopsies was also analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). A series of rectus abdominis muscle biopsies were analysed similarly to serve as a control for type I, IIA and IIB fibres and isoforms. From the histochemical, immunohistochemical and biochemical experiments we found the masseter to contain type I, IM, IIC, IIA and IIB fibres as previously classified, but in addition there were type neonatal, alpha-cardiac, and 'other' (three or more myosins including neonatal and alpha-cardiac). The percentage of each fibre type was highly variable in masseter biopsies, but generally type I fibres were most common, and the proportion of IIB, neonatal, alpha-cardiac and 'other' fibres was low. Even in biopsies that contained relatively large amounts of these last three fibre types, the amount of neonatal and/or alpha-cardiac MHC detected on SDS-PAGE was limited, suggesting that these MHCs are a minor component in the fibres in which they are expressed.

Fish muscle structure: fibre types in flatfish and mullet fin muscles using histochemistry and antimyosin antibody labelling.

In studies of the myosin crossbridge interaction with actin in vertebrate muscles, the muscles of bony fish have the unique advantage for ultrastructural work that the A-band has a simple 'crystalline' lattice of myosin filaments. However, the anatomy and physiology of these fish muscles is relatively poorly understood compared with the rabbit, chicken or frog muscles conventionally used for crossbridge studies. Here the fibre types in fish fin muscles have been characterized to allow sensible selection of single fish fibres for ultrastructural studies. The fibre type compositions of the fin muscles of mullet, plaice, sole and turbot were examined by histochemistry and immunohistochemistry using polyclonal antibodies raised against various myosin isoforms: fish slow, fish fast, mammalian fast (type IIA) and chicken tonic myosins. In the mullet, fin muscles were composed of variable proportions of fast and slow fibres. In the three flatfish, the fin muscle showed a zonal arrangement with slow fibres, binding anti-slow myosin antibody, next to the skin (alpha region). The bulk of the muscle, distal to the skin, was a typical fast muscle both histochemically and in its reaction with antibodies (delta region). Between these two regions there may be one (sole) or two (turbot, plaice) intermediate zones (beta and gamma regions) comparable to the pink/intermediate layer of myotomal muscle. In the plaice fin muscle, two kinds of slow fibre could be distinguished immunohistochemically.

Myosin isoform transitions during development of extra-ocular and masticatory muscles in the fetal rat.

The late fetal development of rat extra-ocular and masticatory muscles was examined by myosin immunohistochemistry. The pattern of slow and neonatal myosin isoform expression in primary and secondary myotubes in these muscles was generally similar to that seen by others in limb muscles. We observed a consistent difference between the Sprague-Dawley and Wistar rats in the degree of maturity reached by all muscles studied at a particular age. In both strains, extra-ocular muscles were also about one day in advance of the masticatory muscles. Thus, secondary myotubes were first seen at E17 in Wistar extraocular muscles, at E18 in Sprague-Dawley extra-ocular muscles and Wistar masticatory muscles, and at E19 in Sprague-Dawley masticatory muscles. There was a strikingly early and complete type differentiation of primary myotubes in extraocular muscles, and tonic myosin first appeared before birth in presumptive extrafusal tonic fibres in the orbital layer of the oculorotatory muscles. Throughout the late fetal period, retractor bulbi was composed of fast myotubes only, but these myotubes were not arranged in classical clusters. In the masticatory muscles at E17/E18 some slow primary myotubes started to express tonic myosin, and these presumptive spindle bag2 fibres were located only in regions of the muscles known to contain spindles in the adult. Presumptive bag1 fibres appeared about a day later (initially without tonic myosin), and in the region of the spindle cluster in anterior deep masseter extrafusal secondary myotube production appeared to be suppressed.

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

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

Quantitative analysis of histochemical and immunohistochemical reactions in skeletal muscle fibres of Rana and Xenopus.

Intensities of histochemical and immunohistochemical reactions in muscle fibres of Rana and Xenopus have been estimated microphotometrically, and the data from serial sections statically analysed. Quantitative validities of reactions and measurements have also been assessed against independent published evidence. It is concluded that NADH-tetrazolium reductase overestimates tonic-fibre aerobic capacities and the actomyosin ATPase reaction overestimates their contraction speeds. However, it appears that succinate dehydrogenase, despite being a near-equilibrium enzyme of particulate distribution, indicates the relative aerobic capacities of fibres with acceptable accuracy when lightly reacted. Capacities for aerobic and anaerobic metabolism are positively correlated over all types of fibre (r typically approximately 0.6 for 200 fibres), perhaps as an adaptation to environmental hypoxia. Multivariate clusters (indicating fibre types) have been sought, using Ward's method with optimizing procedures (iterative relocation and multivariate-normal modelling). Cluster analysis confirms the subjective identifications of two 'slow/tonic' types in Xenopus (labelled T5 and S4) but of only one (T5) in Rana. Division of the 'fast family' twitch fibres into three types (F1-F3) in both genera, with metabolic capacity related inversely to apparent shortening velocity, is highly supportable by objective criteria. However, statistically significant subdivisions also present themselves. Rana F2 and Xenopus F1 clusters can be bisected according to metabolic capacity; and Xenopus F2 fibres fall into three subtypes reflecting different isomyosin contents. In the different types of twitch fibre, ratios of myofibrillar ATP consumption rate to aerobic capacity increase up to 30-fold with contraction speed, but anaerobic/aerobic ratios do so only 5-fold.

Changes in the concentration of the calcium-binding parvalbumin in cross-reinnervated rat muscles. Comparison of biochemical with physiological and histochemical parameters.

The fast extensor digitorum longus (EDL) and the slow soleus (SOL) muscles were cross-reinnervated in both directions in the rat. During the following transformation of muscle type properties, the expression of the Ca2+-binding parvalbumin (parvalbumin, Mr = 12,000) was investigated. The combined biochemical, histochemical, and physiological results demonstrated that the amount of parvalbumin decreased in the fast to slow (X-EDL) and increased in the slow to fast (X-SOL) transformation. Alterations of parvalbumin-mRNA levels were similar to changes found at the protein level, indicating a tight transcriptional regulation of the parvalbumin expression. The close correlation, however, between parvalbumin and relaxation speed found in normal muscles had changed after cross-reinnervation. After the altered nervous input, a slow contracting/slow relaxing muscle may even contain more parvalbumin than a fast contracting/fast relaxing one. The expression of parvalbumin may depend on the nerve-muscle interaction, and parvalbumin may thus be used as a sensitive marker for early stages of muscular transformation and neurological disorders.

Correlation of parvalbumin concentration with relaxation speed in mammalian muscles.

The physiological role of the Ca2+-binding protein parvalbumin in skeletal muscle has been investigated by measuring the parvalbumin content by HPLC in a variety of mammalian muscles, including man, and comparing the results with the respective muscle relaxation properties and fiber type compositions. The parvalbumin concentrations were highest in the skeletal muscles of the smallest animal investigated (mouse, gastrocnemius: 4.9 g/kg), which has the highest relaxation speed, and lowest in the larger animals (horse, deep gluteal muscle: less than or equal to 0.001 g/kg) and man (vastus, triceps: less than or equal to 0.001 g/kg), which have much lower relaxation speeds. Analysis of three type-homogeneous muscles of the guinea pig revealed highest parvalbumin concentrations (0.25 g/kg) in sartorius (type IIB) and lowest concentrations (less than or equal to 0.007 g/kg) in soleus (type I), consistent with the different half-relaxation times of fast and slow muscles. Denervation of the rat extensor digitorum longus, which increases the half-relaxation time from 9.4 to 19 msec, resulted in a 20% decrease of the parvalbumin content. Given this close correlation between parvalbumin content and relaxation speed in a variety of muscles and species, we suggest that parvalbumin is involved directly in the relaxation process in fast muscles.