Differential effects of muscle fibre length and insulin on muscle-specific mRNA content in isolated mature muscle fibres during long-term culture

The aims of this study were (1) to determine the relationship between muscle fibre cross-sectional area and cytoplasmic density of myonuclei in high- and low-oxidative Xenopus muscle fibres and (2) to test whether insulin and long-term high fibre length caused an increase in the number of myonuclei and in the expression of α-skeletal actin and of myogenic regulatory factors (myogenin and MyoD) in these muscle fibres. In high- and low-oxidative muscle fibres from freshly frozen iliofibularis muscles, the number of myonuclei per millimetre fibre length was proportional to muscle fibre cross-sectional area. The in vivo myonuclear density thus seemed to be strictly regulated, suggesting that the induction of hypertrophy required the activation of satellite cells. The effects of muscle fibre length and insulin on myonuclear density and myonuclear mRNA content were investigated on high-oxidative single muscle fibres cultured for 4–5 days. Muscle fibres were kept at a low length (~15% below passive slack length) in culture medium with a high insulin concentration (~6 nmol/l: “high insulin medium”) or without insulin, and at a high length (~5% above passive slack length) in high insulin medium. High fibre length and high insulin medium did not change the myonuclear density of isolated muscle fibres during culture. High insulin increased the myonuclear α-skeletal actin mRNA content, whereas fibre length had no effect on α-skeletal actin mRNA content. After culture at high fibre length in high insulin medium, the myonuclear myogenin mRNA content was 2.5-fold higher than that of fibres cultured at low length in high insulin medium or in medium without insulin. Myonuclear MyoD mRNA content was not affected by fibre length or insulin. These in vitro experiments indicate that high muscle fibre length and insulin enhance muscle gene expression but that other critical factors are required to induce adaptation of muscle fibre size and performance.This work was partially supported by a research grant from the Haak Bastiaanse Kuneman Stichting.     

Cellular adaptation of the trapezius muscle in strength-trained athletes

 The aim of this study was to elucidate the cellular events that occur in the trapezius muscle following several years of strength training. In muscle biopsies from ten elite power lifters (PL) and six control subjects (C), several parameters were studied: cross-sectional area of muscle fibres, myosin heavy chain composition (MHC) and capillary supply [capillaries around fibres (CAF) and CAF/fibre area]. A method was also developed for counting the number of myonuclei and satellite cell nuclei. The proportion of fibres expressing MHC IIA, the cross-sectional area of each fibre type and the number of myonuclei, satellite cells and fibres expressing markers for early myogenesis were significantly higher in PL than in C (P<0.05). A significant correlation between the myonuclear number and the cross-sectional area was observed. Since myonuclei in mature muscle fibres are not able to divide, we suggest that the incorporation of satellite cell nuclei into muscle fibres resulted in the maintenance of a constant nuclear to cytoplasmic ratio. The presence of small diameter fibres expressing markers for early myogenesis indicates the formation of new muscle fibres. Accepted: 17 November 1998     

Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation.

To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41-44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.     

Fibre type specificity of haem oxygenase-1 induction in rat skeletal muscle.

The expression of the inducible haem oxygenase (HO-1) gene was examined in different skeletal muscles. Rats were treated with haemin and a time course of HO-1 mRNA expression was determined in soleus and extensor digitorum longus (EDL) muscles. Fibre type composition and tissue myoglobin content were also measured. We found that HO-1 mRNA expression markedly increased in soleus (type I fibres) muscle but was only slightly affected in EDL (type II fibres). HO-1 expression directly correlated with both percentage of red fibres and tissue myoglobin. These data demonstrate that HO-1 gene expression follows a fibre type-specific pattern which might indicate an important role for this protein in the maintenance of skeletal muscle function.     

Changes in FGF and FGF receptor expression in low-frequency-stimulated rat muscles and rat satellite cell cultures

This study compares effects of chronic electrical stimulation on the expression levels of FGF-1, FGF-2 and their receptors (FGFRI, FGFR4) in rat tibialis anterior (TA) muscle of hypothyroid rat, as well as in satellite cell cultures derived from normal rat TA and soleus (SOL) muscles. In 5-day (5-d)-stimulated hypothyroid TA muscle, FGF-1 and FGF-2 mRNA levels were threefold elevated over control. FGFR1 and FGFR4 mRNAs were twofold and 1.5-fold elevated, respectively. In longer stimulated muscles, FGF-1 and FGFR4 mRNAs returned to basal levels, whereas FGF-2 mRNA remained elevated. FGFR1 mRNA decreased to control levels in 10-d stimulated muscles, but increased again after 20 days of stimulation. SOL- and TA-derived satellite cell cultures were stimulated for 5 days. At this time point, changes in myosin heavy chain isoforms were detectable consisting of increases in MHCI mRNA and decreases in MHCIIb and MHCIId mRNA. The comparison between 5-d-stimulated hypothyroid TA muscle and 5-d-stimulated TA- and SOL-derived satellite cell cultures revealed differences in the expression of FGF-1 and FGF-2, but similar expression levels of FGFR1 and FGFR4. Even though FGF-1 and FGF-2 mRNAs were elevated in the satellite cell cultures, their increases were less pronounced than in the stimulated hypothyroid muscle. Taking into consideration that skeletal muscle contains muscle fibres and various non-muscle tissues, e.g. blood vessels, these results suggest that the latter contribute to the observed increases in FGF-1 and FGF-2 expression in stimulated muscle.     

Optimality in the encoding/decoding relations of motoneurones and muscle units

Experimental data indicate the existence of a matching between many motoneurone characteristics and those of the corresponding innervated muscle fibres. This association between the properties of motoneurones and muscle fibres is quite important for the adequate and efficient functioning of the motor system. We present a view of motoneurones and muscle fibres as encoders and decoders of signals. An analysis of some of the encoding/decoding relations performed by motoneurones and muscle fibres in functionally relevant situations indicates that they are optimal and we indicate in the paper what performance measures are optimised. Besides its relevance in the study of motor control, the encoding/decoding framework should also be useful in the task of model validation.     

Mef2s are required for thick filament formation in nascent muscle fibres

During skeletal muscle differentiation, the actomyosin motor is assembled into myofibrils, multiprotein machines that generate and transmit force to cell ends. How expression of muscle proteins is coordinated to build the myofibril is unknown. Here we show that zebrafish Mef2d and Mef2c proteins are required redundantly for assembly of myosin-containing thick filaments in nascent muscle fibres, but not for the earlier steps of skeletal muscle fibre differentiation, elongation, fusion or thin filament gene expression. mef2d mRNA and protein is present in myoblasts, whereas mef2c expression commences in muscle fibres. Knockdown of both Mef2s with antisense morpholino oligonucleotides or in mutant fish blocks muscle function and prevents sarcomere assembly. Cell transplantation and heat-shock-driven rescue reveal a cell-autonomous requirement for Mef2 within fibres. In nascent fibres, Mef2 drives expression of genes encoding thick, but not thin, filament proteins. Among genes analysed, myosin heavy and light chains and myosin-binding protein C require Mef2 for normal expression, whereas actin, tropomyosin and troponin do not. Our findings show that Mef2 controls skeletal muscle formation after terminal differentiation and define a new maturation step in vertebrate skeletal muscle development at which thick filament gene expression is controlled.     

Development of immunohistochemical characteristics of intrafusal fibres in normal and de-efferented rat muscle spindles

Intrafusal muscle fibres in adult muscle spindles differ in their myosin composition. After selective motor denervation intrafusal muscle fibres develop mature ultrastructural characteristics. In order to evaluate the role of fusimotor innervation on the maturation of the myosin composition of intrafusal muscle fibres we have examined with immunohistochemical techniques i) the postnatal development of muscle spindles in new-born rats and in 7-21 day old rats; ii) muscle spindles in the EDL of 21-day-old rats de-efferented at birth. For the characterization of myosins in intrafusal fibres we used three myosin antisera: antipectoral myosin, antiheart myosin and antiheart myosin adsorbed with muscle powder from the soleus muscle of guinea pig. We show in this study that during development intrafusal fibres change immunoreactivity and that in the absence of motor innervation bag fibres do not fully develop the myosin characteristics of control spindles. We conclude that the maturation of bag1 and bag2 fibres apparently requires next to the inductive influence of sensory axon terminals the presence and activity of fusimotor axons.     

Dipeptidyl peptidases in the soleus muscle of the rat before and after treatment with 5-hydroxytryptamine

A moderate peptidase activity against L-lysyl-L-proline-4-methoxy-beta-napththylamide was detected histochemically in unfixed sections of soleus muscle fibres of inbred male Wistar rats using two variants of the semipermeable membrane technique. One variant involved simultaneous coupling with tetrazotised 3,3'-dimethoxybenzidine, the other post-coupling. The activity at pH 6 increased approximately three-fold in many fibres showing signs of insult in rats that had been given a single low dose of 5-hydroxytryptamine (10 mg/kg body weight) 48-72 h earlier. The hydroxytryptamine treatment was found to induce a selective myopathy. Some of the increased peptidase activity within insulted muscle fibres appeared to arise from invading mononuclear cells, but the majority seemed endogenous to muscle fibres. The peptidase activity persisted in some fibres 21-28 days after 5-hydroxytryptamine administration, by which time the whole muscle appeared histologically normal. The variation of the activity of the peptidase with pH in the presence of various inhibitors was investigated in both control and insulted muscle fibres. From its sensitivity and behaviour towards Zn2+, Hg2+, Cu2+, puromycin, benzethonium chloride and phenylmethylsulphonyl fluoride and its indifference towards Co2+, Cd2+, Mn2+ and o-phenanthroline, it is concluded that the activity can be attributed to a mixture of at least two peptidases, dipeptidyl peptidase II and an unidentified neutral dipeptidyl peptidase. The possible role of the peptidase(s) in muscle regeneration in discussed.     

Development of immunohistochemical characteristics of intrafusal fibres in normal and de-efferented rat muscle spindles

Summary Intrafusal muscle fibres in adult muscle spindles differ in their myosin composition. After selective motor denervation intrafusal muscle fibres develop mature ultrastructural characteristics. In order to evaluate the role of fusimotor innervation on the maturation of the myosin composition of intrafusal muscle fibres we have examined with immunohistochemical techniques i) the postnatal development of muscle spindles in new-born rats and in 7–21 day old rats; ii) muscle spindles in the EDL of 21-day-old rats de-efferented at birth. For the characterization of myosins in intrafusal fibres we used three myosin antisera: antipectoral myosin, antiheart myosin and antiheart myosin adsorbed with muscle powder from the soleus muscle of guinea pig. We show in this study that during development intrafusal fibres change immunoreactivity and that in the absence of motor innervation bag fibres do not fully develop the myosin characteristics of control spindles. We conclude that the maturation of bag₁ and bag₂ fibres apparently requires next to the inductive influence of sensory axon terminals the presence and activity of fusimotor axons.     

Localisation and quantification of dehydrogenase activities in single muscle fibres of mdx gastrocnemius

The kinetics of succinate (SDH) and lactate (LDH) dehydrogenases were determined in single muscle fibres in unfixed sections of the gastrocnemius of dystrophic mdx mice (with an X-linked genetic disorder lacking a cytoskeletal protein, dystrophin) and age-matched C57BL/10 control mice. Quantitative gel substrate-film techniques and a real-time image analysis system were used. Three main fibre types were observed in regenerated mdx gastrocnemius and in corresponding controls: small fibres (S) with high SDH and LDH initial reaction velocities and activities, large fibres (L) with low activities of these dehydrogenases and intermediate-sized fibres (I) with intermediate enzyme activities. The small and intermediate fibres in both mdx and control muscles exhibited respectively high and moderate subsarcolemmal SDH and LDH activities attributable to accumulated mitochondria. The ratios of the initial velocities of the intrinsic enzyme reactions in the sarcoplasm, excluding the subsarcolemmal regions, of mdx muscle fibres compared to those in control fibres were 0.958 (S), 1.09 (I) and 0.959 (L) for SDH, and 1.03 (S), 1.06 (I) and 1.07 (L) for LDH. A parameter a, a measure of the diffusion of LDH out of muscle sections during incubation on gel substrate films, was found to be 0.981 and 1.00 in mdx and control muscles, respectively. Thus there are no significant differences in the activities and microenvironments of the enzymes between regenerated mdx muscle fibres and normal control muscle fibres. These data suggest that dystrophin deficiency in mdx muscles has no effects on the interactions of LDH with cytoskeletal proteins or on SDH activities in mitochondria whose number and morphology differ in mdx muscle fibres compared to those in normal controls. SDH and LDH activities were also found in the mitochondria clustered on two longitudinally directed poles of each central nucleus in regenerated mdx muscle fibres. They were proportional to the activities in the sarcoplasm excluding the subsarcolemmal regions. Accepted: 12 October 1999     

Effect of glucocorticoids on the turnover rate of actin and myosin heavy and light chains on different types of skeletal muscle fibres

The catabolic action of glucocorticoids on the molecular level of the two main muscular proteins, myosin and actin, was found to depend on the type of muscle fibres. The synthesis rate of actin and myosin heavy chain was decreased in all types of muscle fibres, and in myosin light chain only in the slow-twitch red fibres. The turnover rate of actin and myosin heavy chain was also found decreased in all types of muscle fibres. The myosin light chains turned over more rapidly in dexamethasone-treated than in the control rats in all types of muscle fibres except in the case of the slow-twitch red ones as was shown by single and double isotope methods. Dexamethasone treatment enhanced the urinary 3-methylhistidine excretion in rats by 60%.     

Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy

The aim of this study was to investigate the function of the Hippo pathway member Yes-associated protein (Yap, gene name Yap1) in skeletal muscle fibres in vivo. Specifically we bred an inducible, skeletal muscle fibre-specific knock-in mouse model (MCK-tTA-hYAP1 S127A) to test whether the over expression of constitutively active Yap (hYAP1 S127A) is sufficient to drive muscle hypertrophy or stimulate changes in fibre type composition. Unexpectedly, after 5–7 weeks of constitutive hYAP1 S127A over expression, mice suddenly and rapidly lost 20–25% body weight and suffered from gait impairments and kyphosis. Skeletal muscles atrophied by 34–40% and the muscle fibre cross sectional area decreased by ≈40% when compared to control mice. Histological analysis revealed evidence of skeletal muscle degeneration and regeneration, necrotic fibres and a NADH-TR staining resembling centronuclear myopathy. In agreement with the histology, mRNA expression of markers of regenerative myogenesis (embryonic myosin heavy chain, Myf5, myogenin, Pax7) and muscle protein degradation (atrogin-1, MuRF1) were significantly elevated in muscles from transgenic mice versus control. No significant changes in fibre type composition were detected using ATPase staining. The phenotype was largely reversible, as a cessation of hYAP1 S127A expression rescued body and muscle weight, restored muscle morphology and prevented further pathological progression. To conclude, high Yap activity in muscle fibres does not induce fibre hypertrophy nor fibre type changes but instead results in a reversible atrophy and deterioration.     

Age-related changes and nutritional regulation of myosin heavy-chain composition in longissimus dorsi of commercial pigs

The objective of this study is to investigate the age-related changes of and the effects of dietary conjugated linoleic acid (CLA) on muscle-fibre types in commercial pigs. We divided 25 crossbred male pigs into five age groups (7, 30, 60, 100 and 180 days) and 30 finishing pigs into two dietary groups (one fed a CLA-enriched diet and the other fed a control diet for 30 days). We analysed the composition (%) of myosin heavy-chain (MyHC) mRNA according to the absolute copies of each MyHC (I, IIa, IIb and IIx) mRNA, and the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) in the longissimus muscle. From days 7 to 180, the MyHC I mRNA abundance and SDH and MDH activities presented a decreasing trend, the MyHC IIb mRNA abundance presented a steady trend and the MyHC IIa and IIx mRNA abundances presented an increasing trend. On day 30, MyHC I and IIb mRNA abundances were at their lowest (P < 0.05), and the MyHC IIa and IIx mRNA abundances were at their highest (P < 0.05). In the CLA group, the MyHC I mRNA abundance and the activities of SDH and MDH were improved in the longissimus muscle, whereas pressure loss, drip loss and average back fat depth significantly decreased (P < 0.01) and shear force significantly increased (P < 0.01). Loin eye area, feed conversion rate and meat colour showed some tendency to be improved. These results indicated that more oxidative fibres might convert to glycolytic fibres with increasing age or weight, and that the early developmental stage might be a key stage for this conversion. During the finishing stage, the proportion of oxidative fibres might be increased by dietary CLA supplementation, which may contribute to the water-holding capacity of meat. The results would provide an important basis for the application of muscle-fibre types in the improvement of pork quality.     

Regeneration following orthotopic transplantation of entire plantaris muscle in Sprague Dawley rats

Regeneration of rat plantaris muscle has been studied without predeneration. The original muscle fibres were completely degenerated within one week, but were completely regenerated at the end of experiment. The weight of regenerate showed 51% recovery. The breadth, length and total area of muscle and number of muscle fibres was less by 42, 37, 26 and 29% respectively, compared to control muscle at the end of experiment. The diameter of regenerated fibres was almost normalized at the end of experiment. The number and diameter of nuclei in the regenerate was more than that of control which coincides with the increase in DNA and RNA contents. The protein contents after an initial loss started increasing and continued to do so till the end of the experiment. The better regeneration of the plantaris muscle seems to be due to the presence of supporting gastrocnemius muscle.     

Effect of disuse on the ultrastructure of the achilles tendon in rats

We examined the influence exerted, through disuse of the hindlimb, on the collagen fibres of the achilles tendon in rats. With disuse the body mass decreased by 28%, and the mass of soleus muscle decreased by 20%. A decrease in the surface area and diameter was observed in the experimental group when compared to the control group. A histogram of the collagen fibres showed a decrease of the thick fibres in the experimental group. The maximum surface area of collagen fibres in the experimental group was seen to be only 43% of that of the control group. These results showed a decrease in the thickness of the collagen fibres of the achilles tendon through disuse. This seemed to suggest that resistance to tension is decreased by disuse.     

A Neuro-Mechanical Model of a Single Leg Joint Highlighting the Basic Physiological Role of Fast and Slow Muscle Fibres of an Insect Muscle System

In legged animals, the muscle system has a dual function: to produce forces and torques necessary to move the limbs in a systematic way, and to maintain the body in a static position. These two functions are performed by the contribution of specialized motor units, i.e. motoneurons driving sets of specialized muscle fibres. With reference to their overall contraction and metabolic properties they are called fast and slow muscle fibres and can be found ubiquitously in skeletal muscles. Both fibre types are active during stepping, but only the slow ones maintain the posture of the body. From these findings, the general hypothesis on a functional segregation between both fibre types and their neuronal control has arisen. Earlier muscle models did not fully take this aspect into account. They either focused on certain aspects of muscular function or were developed to describe specific behaviours only. By contrast, our neuro-mechanical model is more general as it allows functionally to differentiate between static and dynamic aspects of movement control. It does so by including both muscle fibre types and separate motoneuron drives. Our model helps to gain a deeper insight into how the nervous system might combine neuronal control of locomotion and posture. It predicts that (1) positioning the leg at a specific retraction angle in steady state is most likely due to the extent of recruitment of slow muscle fibres and not to the force developed in the individual fibres of the antagonistic muscles; (2) the fast muscle fibres of antagonistic muscles contract alternately during stepping, while co-contraction of the slow muscle fibres takes place during steady state; (3) there are several possible ways of transition between movement and steady state of the leg achieved by varying the time course of recruitment of the fibres in the participating muscles.     

Neural control of gene expression in the skeletal muscle fibre: the nature of the lesion in the muscular protein-synthesizing machinery following denervation

Experiments are reported showing that following 8 days of denervation the function of the protein-synthesizing machinery, operating in the rat gastrocnemius fibres, is altered, probably as a consequence of decreased amounts of ribosomes and actively translated mRNA. In addition, the data obtained show that the amount per muscle and the availability per ribosome of the soluble factors involved in the process of protein synthesis are markedly decreased, thus suggesting that the amounts of ribosomes, mRNA and soluble factors are regulated in a concerted fashion when muscular protein synthesis is decreased after denervation.     

Two different forms of beta myosin heavy chain are expressed in human striated muscle

Summary We have found evidence for two beta-like myosin heavy chains in humans, one cardiac and one skeletal. The cDNA sequences of the cardiac beta myosin heavy chain cDNA clone pHMC3 and the skeletal beta-like myosin heavy chain cDNA clone pSMHCZ, were compared to each other. It was found that the 3 untranslated regions as well as 482 nucleotides specifying the carboxyl coding region, were 100% homologous. Further examination revealed that the skeletal clone pSMHCZ diverges from the human cardiac beta myosin heavy chain cDNA clone pHMC3 at the 5 end. We present evidence in this report which indicates that the cardiac beta myosin heavy chain mRNA is expressed in skeletal muscle tissues. The human cardiac beta myosin heavy chain cDNA clone, pHMC3, which codes for a portion of the light meromyosin section of the myosin heavy chain, was used as a probe for S1 nuclease mapping studies with RNA derived from cardiac tissue, smooth muscle and skeletal muscle tissues consisting of fast-twitch, slow-twitch and mixed fast- and slow-twitch muscle fibres. Two probes were used to examine the expression of the mRNA. One probe (406 nucleotides) constitutes the 3 untranslated region and a portion of the coding region of the beta cardiac myosin heavy chain cDNA clone, which is 100% homologous to pSMHCZ, the skeletal cDNA clone. The other constitutes the majority of the coding region (1017 nucleotides) of the cardiac clone pHMC3 in which the first 216 nucleotides from the labelled end are 100% homologous to the skeletal clone pSMHCZ. In the soleus muscle, which is rich in slow-twitch type I muscle fibres, the expression of the cardiac beta myosin heavy chain mRNA was very prominent. In gastrocnemius muscle, a mixed fibre muscle, the expression of this mRNA was detected to a lesser degree than that for the soleus muscle. In vastus lateralis and vastus medialis, which consist of predominantly type II, fast-twitch fibres, there were trace amounts of the cardiac beta myosin heavy chain mRNA. When expression of this mRNA was tested in smooth muscle tissue none could be detected.