Effects of eccentric treadmill running on mouse soleus: degeneration/regeneration studied with Myf-5 and MyoD probes.

AIM: The aim of this report is to show that eccentric exercise under well-controlled conditions is an alternative model, to chemical and mechanical analyses, and analyse the process of degeneration/regeneration in mouse soleus. METHODS: For this, mice were submitted to a single bout of eccentric exercise on a treadmill down a 14 degrees decline for 150 min and the soleus muscle was analysed at different times following exercise by histology and in situ hybridization in comparison with cardiotoxin-injured muscles. RESULTS: We analyse the regenerative process by detection of the accumulation of transcripts coding for the two myogenic regulatory factors, Myf-5 and MyoD, which are good markers of the activated satellite cells. From 24 h post-exercise (P-E), clusters of mononucleated Myf-5/MyoD-positive cells were detected. Their number increased up to 96 h P-E when young MyoD-positive myotubes with central nuclei began to appear. From 96 to 168 h P-E the number of myotubes increased, about 10-fold, the new myotubes representing 58% of the muscle cells (168 h P-E). CONCLUSION: These results show that this protocol of eccentric exercise is able to induce a drastic degeneration/regeneration process in the soleus muscle. This offers the opportunity to perform biochemical and molecular analyses of a process of regeneration without muscle environment defects. The advantages of this model are discussed in the context of fundamental and therapeutical perspectives.

[High-performance society and doping]. / Société de la performance et dopage.

Doping is not limited to high-level athletes. Likewise it is not limited to the field of sports activities. The doping phenomenon observed in sports actually reveals an underlying question concerning the notion of sports itself, and more widely, the society's conception of sports. In a high-performance society, which is also a high-risk society, doping behavior is observed in a large number of persons who may or may not participate in sports activities. The motivation is the search for individual success or profit. The fight against doping must therefore focus on individual responsibility and prevention in order to preserve athlete's health and maintain the ethical and educational value of sports activities.

Expression and neural control of myogenic regulatory factor genes during regeneration of mouse soleus.

Given the importance of the myogenic regulatory factors (MRFs) for myoblast differentiation during development, the aims of this work were to clarify the spatial and temporal expression pattern of the four MRF mRNAs during soleus regeneration in mouse after cardiotoxin injury, using in situ hybridization, and to investigate the influence of innervation on the expression of each MRF during a complete degeneration/regeneration process. For this, we performed cardiotoxin injury-induced regeneration experiments on denervated soleus muscle. Myf-5, MyoD, and MRF4 mRNAs were detected in satellite cell-derived myoblasts in the first stages of muscle regeneration analyzed (2--3 days P-I). The Myf-5 transcript level dramatically decreased in young multinucleated myotubes, whereas MyoD and MRF4 transcripts were expressed persistently throughout the regeneration process. Myogenin mRNA was transiently expressed in forming myotubes. These results are discussed with regard to the potential relationships between MyoD and MRF4 in the satellite cell differentiation pathway. Muscle denervation precociously (at 8 days P-I) upregulated both the Myf-5 and the MRF4 mRNA levels, whereas the increase of both MyoD and myogenin mRNA levels was observed later, in the late stages of regeneration (30 days P-I). This significant accumulation of each differentially upregulated MRF during soleus regeneration after denervation suggests that each myogenic factor might have a distinct role in the regulatory control of muscle gene expression. This role is discussed in relation to the expression of the nerve-regulated genes, such as the nAChR subunit gene family. (J Histochem Cytochem 49:887-899, 2001)

Neural and hormonal control of expression of myogenic regulatory factor genes during regeneration of Xenopus fast muscles: myogenin and MRF4 mRNA accumulation are neurally regulated oppositely.

With the aim to investigate the influence of both innervation and thyroid hormone, on the expression of the MRFs during muscle regeneration, we performed cardiotoxin injury-induced regeneration experiments on fast muscles of adult Xenopus laevis subjected to different experimental conditions, including denervation and T3 treatment, and analyzed the accumulation of the four myogenic regulatory factors (MRFs) using RT-PCR and in situ hybridization. We show here that manipulation of hormone levels or innervation resulted in differential alterations of MRF expression. Denervation and T3 treatment transiently down-regulated Myf-5 mRNA levels at the beginning of the regeneration process. Myf-5 was the only myogenic factor subject to thyroid hormone influence. Muscle denervation persistently reduces the levels of MRF4 transcripts as early as the first stages of regeneration, whereas the levels of myogenin mRNA were increased in the late stages of regeneration. This suggests that MRF4 expression may be induced by innervation and hence may be involved in mediating transcriptional responses to innervation and that myogenin expression may compensate for the down-regulation of MRF4 gene. This switch in MRF gene expression following denervation could have important consequences for the ability of Xenopus regenerating muscles to recover function after denervation.

Expression of the proteoglycan SPOCK during mouse embryo development.

SPOCK is a modular proteoglycan, with homology with proteins involved in cell adhesion processes and neurogenesis. We have previously shown that SPOCK transcripts predominate in the adult mouse brain. Here, we report its expression during mouse embryonic development by in situ hybridization, and immunocytochemistry. SPOCK is actively expressed at the onset of neurogenesis during periods of neuron migration and axonal outgrowth. At a later developmental stage, its expression is particularly prevalent within developing synaptic fields. In the peripheral nervous system, SPOCK expression is also developmentally regulated particularly in dorsal root ganglion neurons.

Long-term denervation modulates differentially the accumulation of myogenin and MRF4 mRNA in adult Xenopus muscle.

In adult Xenopus laevis, we analyzed, using reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization, the influence of long-term muscle denervation on the accumulation of MRF4 and myogenin transcripts. The brachial muscle was denervated by cutting the brachial nerve and was examined after 4 months. MRF4 mRNA levels decreased about two-fold in denervated muscle as compared with contralateral muscle. Myogenin mRNA levels, by contrast, were induced about five-fold by denervation. This report shows that muscle denervation persistently reduces the levels of MRF4 transcripts suggesting that MRF4 expression may be induced by innervation and hence may be involved in mediating transcriptional responses to innervation. The up-regulation of myogenin by denervation suggests that myogenin expression may compensate for the down-regulation of MRF4 gene.

Expression of myogenic regulatory factors during muscle development of Xenopus: myogenin mRNA accumulation is limited strictly to secondary myogenesis.

To clarify the acquisition of the adult muscle pattern in Xenopus laevis, in situ hybridization and reverse transcriptase-polymerase chain reaction were used to correlate the time course of gene expression for myogenic regulatory factors (Myf-5, MyoD, and myogenin) with the expression of contractile protein (myosin heavy chain; MHC) genes during hindlimb formation compared with their expression in dorsal body muscles. After the precocious expression of Myf-5 and MyoD mRNA in limb bud (stage 50), myogenin mRNA strongly accumulated later at paddle stages (stages 52/53) concomitantly with the accumulation of both the larval and the adult MHC mRNAs. In dorsal body muscles, as early as stage 52, myogenin transcripts accumulated in a few small, secondary myofibers expressing the adult MHC mRNA that were located along the dorsomedial edge, but they were never detected in the large, primary myofibers of the body expressing the larval MHC mRNA. During metamorphosis, the areas expressing both the adult MHC and the myogenin transcripts gradually expanded from the dorsomedial edge to the ventral side of the dorsal body muscles, accounting for the progression of the secondary "adult" myogenesis described previously (Nishikawa and Hayashi [1994] Dev. Biol. 165:86-94). This work shows that, in Xenopus, the accumulation of myogenin mRNA is restricted to secondary myogenesis, including the formation of new muscles in developing limbs as well as in dorsal muscles during body remodeling. This shows that myogenin is not required for primary myogenesis, and it suggests a crucial role for myogenin in the terminal differentiation program, including myoblast fusion and the activation of adult-type muscle genes.

Localization of Myf-5, MRF4 and alpha cardiac actin mRNAs in regenerating Xenopus skeletal muscle.

We have analysed the spatial and temporal expression patterns of Myf-5, MRF4 and alpha cardiac actin mRNAs during muscle regeneration following cardiotoxin injury in adult Xenopus laevis using in situ hybridization. Myf-5 transcripts began to be detected in the activated satellite cells as early as the beginning of the regeneration process, then dramatically decreased in young plurinucleated myotubes. MRF4 mRNA was detected later, just before the young myotube stage, and was strongly expressed during the different stages of the maturation of myotubes. Like Myf-5, alpha cardiac actin mRNA began to accumulate early in activated satellite cells. These results, which contribute to an overview of the expression of the genes coding for myogenic bHLH proteins during muscle regeneration, are discussed in relation to the expression of these factors during development.

Analysis of muscle adaptations to terrestrial stepping in the Urodelan amphibian Pleurodeles waltlii.

The changes of myosin isoform pattern and of its associated light chains in relation to the myosin ATPase profile were analysed in different muscles of the hypothyroidian amphibian Pleurodeles waltlii submitted to terrestrial stepping, using electrophoretic and histochemical techniques. These changes were specific to the muscle type but appeared globally characterized by a type-IIB to type-IIA/I fibre transition associated with a transition from fast to intermediate and/or slow myosin isoforms. These results are similar to the effects of endurance training on locomotor muscles of mammals. The diaphragm of experimental animals was also characterized by a complete disappearance of the larval myosin isoforms which were detected in the diaphragm of control animals. The myosin pattern of ventricular muscle did not change following terrestrial stepping. This work indicates that thyroid hormone does not regulate the muscle adaptations that occur following terrestrial stepping and suggests a more complex mechanism of regulation in which innervation could be implicated.

Analysis of MyoD, myogenin, and muscle-specific gene mRNAs in regenerating Xenopus skeletal muscle.

We have analyzed in adult Xenopus laevis, using in situ hybridization, the spatial and temporal expression patterns of MyoD, myogenin, and alpha-skeletal actin and fast myosin heavy chain mRNAs during muscle regeneration following cardiotoxin injury. MyoD transcripts could be detected in the satellite cells as early as the first stage of regeneration and were expressed persistently throughout the regeneration process. Myogenin mRNAs were transiently expressed in forming myotubes. alpha-Skeletal actin and fast myosin heavy chain mRNAs were detected precociously, before the young myotube stage. This work has shown, for the first time, the presence of myogenin transcripts during Xenopus myogenesis.

Myosin isoforms and their light chains from the ventricular muscle of the urodelan amphibian Pleurodeles waltlii: comparison with myosin from skeletal muscles.

Myosin extracted from ventricular muscle of the urodelan amphibian Pleurodeles waltlii was analyzed in comparison with myosin extracted from skeletal muscles by native, one-dimensional SDS gel electrophoresis and two-dimensional gel electrophoresis. Two myosin isoforms were detected in ventricular muscle using pyrophosphate gel electrophoresis. These isomyosins contained two types of light chain subunits, LC1v and LC2v. Two-dimensional gel electrophoresis showed that LC1v comigrated with the slow light chain LC1s, whereas LC2v was characterized by a specific mobility, distinct from LC2s and LC2f. Diaphragm muscle was characterized by the coexistence of larval and adult myosin isoforms.

Cloning and expression of a thyroid hormone receptor alpha 1 in the perennibranchiate amphibian Proteus anguinus.

We demonstrated the presence of thyroid hormone receptor alpha mRNAs in tissues of the perennibranchiate amphibian Proteus anguinus, which is insensitive to thyroid hormone. From P. anguinus muscle we cloned and sequenced the 3' coding and untranslated region of a cDNA corresponding to a thyroid hormone receptor alpha 1. Using cDNA-PCR and in situ hybridization, we showed a tissue-specific expression of thyroid hormone receptor alpha genes, which was not upregulated by thyroid hormone as opposed to that observed in the TH-sensitive species, Xenopus laevis.

The emergence of an adult muscle phenotype in urodelan amphibians: an immunohistochemical study.

Electrophoretic techniques adapted for the analysis of muscles of lower invertebrates reveal four myosin heavy chain isoforms in the dorsalis trunci of Pleurodeles waltlii: two fast (MHC-IIA, MHC-IIB), and one slow (MHC-I) in the adult and one isoform (MHC-La) in the larvae. Polyclonal antibodies were prepared against the larval (anti-MHC-La) and one of the fast myosin (MHC-IIA) isoforms and their specificity was confirmed by western blot analysis. An immunohistochemical analysis was then carried out on frozen sections of the dorsalis trunci of P. waltlii at different stages of development. From stage 44 it was possible to demonstrate the presence of MHC-IIA in the small diameter fibers at the periphery of the muscle; the number and diameter of these fibers increased from stage 44 to stage 56 when anatomical metamorphosis had finished. By stage 56 these fibers could also be readily identified using standard histochemical techniques as type IIA fibers. We conclude that fast IIA myosin is expressed well before the final adult muscle phenotype has been established and its expression is therefore independent of thyroid hormone.

Morphological and biochemical analysis of regeneration after cardiotoxin injury of Xenopus laevis fast muscles.

The anterior brachial muscle of Xenopus laevis forelimb was characterized as a fast-type muscle composed of type II fibers exclusively. Larval and adult muscles showed three distinct isomyosins composed by two different heavy chains, HCl and HCf, respectively, associated with the same fast light chains. Muscle regeneration was examined after degeneration of the myofibers by injection of cardiotoxin, a snake toxin. 24 h after the injury no myofibers and no myosin were detected. New myosins of larval and adult fast types started to be synthesized two weeks after the injury, during a stage of proliferation of mononucleated cells. 1 month after the injury, the regenerated muscles which showed structural differences with the normal muscle contained only fast isomyosins. The precocious larval to fast heavy chain transition observed in regenerating muscles of the adult X. laevis without any thyroid hormone influence shows that the myogenic program in adult muscle regeneration is regulated by factors that are different from those regulating normal development.

Pituitary-thyroid axis controls the final differentiation of the dorsal skeletal muscle in urodelan amphibians.

A histoenzymological study of the ATPase activity of myosin in the dorsal axis muscle (dorsalis trunci) was carried out on two species of urodelan amphibians: Pleurodeles waltlii, a euthyroid species with spontaneous metamorphosis and Ambystoma mexicanum, a neotenic hypothyroid species. P. waltlii and A. mexicanum underwent an operation after which cytological analysis of the remaining pituitary were carried out in parallel. The muscle phenotype of urodelan amphibians varies according to the thyroid status of the species. In euthyroid adults, IIA fibers are dominant whereas in hypothyroid adults, IIC fibers are dominant. The number of type IIB (fast) and type I fibers (slow) are similar in both species. Physiological or experimental modulation of the concentration of circulating thyroid hormones results in a modification of the muscle fiber type profile pertaining to the considered species. We found that pituitary (TSH) plays a dominant role in the maturation of type IIC fibers in both species. Moreover, it seems to modulate the development of IIA fibers in P. waltlii and that of IIB fibers in A. mexicanum. Its action is thus species specific. Through partial or total hypophysectomy experiments, we have been able to demonstrate the influence of the hypophysothyroidian axis on the appearance of the adult muscle phenotype during metamorphosis.

A new approach of urodele amphibian limb regeneration: study of myosin isoforms and their control by thyroid hormone.

In P. waltlii, an urodele amphibian species which undergoes spontaneous metamorphosis, study of native myosin in pyrophosphate gels at various stages of normal development demonstrates a complete larval to fast myosin isoforms transition, which occurs more precociously in forelimb muscles than in the dorsal and ventral muscles. In the neotenic species A. mexicanum, forelimb muscles development also presents a complete myosin isoforms transition which is in contrast with the partial myosin isoforms transition observed in the dorsal muscle. In metamorphosed or neotenic animals of both species aged 1 year, forelimb regeneration is characterized by a complete transition from larval to fast myosin isoforms, that occurs earlier and more rapidly than in normal forelimb development. When forelimb regeneration is studied in P. waltlii aged 4 years, the adult fast and slow isomyosins are expressed very early in the regeneration process. In experimental hypothyroidian P. waltlii, the larval to fast isoforms transition in regenerating forelimb muscles is slightly delayed. Experimental hyperthyroidism accelerates the disappearance of larval isomyosins in regenerating forelimb muscles, both in P. waltlii and A. mexicanum aged 1 year. This work demonstrates that changes in myosin isoform pattern during forelimb regeneration in adult urodele amphibians are different from changes occurring in the normal forelimb development. They take place without any thyroid hormone influence, as opposed to normal development, and appear to be age-dependent.

White muscle differentiation in the eel (Anguilla anguilla L.): changes in the myosin isoforms pattern and ATPase profile during post-metamorphic development.

Myosin isoforms and their light and heavy chains subunits were studied in the white lateral muscle of the eel during the post metamorphic development, in relation with the myosin ATPase profile. At elver stage VI A1 the myosin isoforms pattern was characterized by at least two isoforms, FM3 and FM2. The fast isomyosin type 1 (FM1) appeared during subsequent development. It increased progressively in correlation with the increase in the level of the light chain LC3f. FM1 became predominant at stage VI A4. At the elver stage VI A1, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed at least two heavy chains, namely type II-1 and II-2. The type II-1 heavy chain disappeared in the yellow eel white muscle, and V8-protease peptide map showed the appearance of a minor heavy chain type II-3 as early as stage VI B. Comparison of myosin heavy chains and myosin isoforms patterns showed the comigration of different myosin isoforms during white muscle development. The myosin ATPase profile was characterized by a uniform pattern as far as stage VI A4. A mosaic aspect in white muscle was observed as early as stage VI B, showing the appearance of small acid labile fibers. This observation suggests that the type II-3 heavy chain is specific to the small fibers.

Myosin structure in the eel (Anguilla anguilla L.). Demonstration of three heavy chains in adult lateral muscle.

Myosin extracts from central white fibers and peripheral red fibers of the lateral muscle of eel (Anguilla anguilla) were analysed by electrophoresis under non-dissociating conditions, which demonstrated a polymorphism of myosin isoforms. The light and heavy subunit content of the isomyosins was established using SDS-PAGE and two-dimensional electrophoresis. In the central white muscle, 3 myosin isoforms FM3, FM2, FM1, were characterized by 3 types of fast light chain and one fast heavy chain HCf; the existence of a fourth isomyosin is discussed. In the peripheral red muscle, two myosin isoforms were found, SM1 and SM2, each characterized by a specific heavy chain, HCs1 or HCs2, and containing the same slow light chain content. This work demonstrates for the first time the existence of 3 heavy chains in the skeletal muscle of a fish.

Myosin structure and thyroidian control of myosin synthesis in urodelan amphibian skeletal muscle.

Electrophoretic analysis in non-dissociating conditions reveals three types of myosin in adult urodelan amphibian skeletal muscles: 3 isoforms of fast myosin (FM), one isoform of intermediate myosin (IM) and one or two isoforms of slow myosin (SM). Each type is characterized by a specific heavy chain HCf (FM), HCi (IM) and HCs (SM), respectively. In all urodelan species, as in mammals, fast isomyosins associate HCf and the three fast light chains LC1f, LC2f, and LC3f. In most urodelan species the intermediate myosin contains LC1f and LC2f and can be considered as an homodimer of the alkali LC1f. However, in Euproctus asper, IM is characterized by the association of both slow and fast LC with HCi. Slow myosin is a hybrid molecule associating HCs with slow and fast LC. During metamorphosis, a myosin isoenzymic transition occurs consisting in the replacement of three larval myosins (LM) characterized by a specific heavy chain (HCI), by the adult isomyosins with lower electrophoretic mobilities. At the same time there is a change in the ATPase myofibrillar pattern, with the larval fiber types being replaced by adult fibers of types I, IIA and IIB. In the neotenic and perennibranchiate species, which do not undergo spontaneous metamorphosis, sexually mature larval animals present a change in the myosin isoenzymic profile, but no complete transition. The coexistence of larval and adult isomyosins and the persistence of transitional fibers of type IIC in the skeletal muscle are demonstrated. Experimental hypo- and hyperthyroidism indicate that thyroid hormone stimulates the regression of the larval isomyosins, possibly through indirect pathways. In contrast, the appearance and the persistence of the adult isomyosins seem to be independent of thyroid hormone. Thus, the control of the isoenzymic transition in the skeletal muscle of urodelan amphibians appears to imply indirect mechanisms, operating differently on each of the two phases of the complete transition.

Thyroidal status and myosin isoenzymic pattern in the skeletal dorsal muscle of urodelan amphibians--the perennibranchiate Proteus anguinus.

In the perennibranchiate Proteus anguinus, larval myosin isoforms were shown to coexist for life with the adult isomyosins that appeared at the end of the larval stage. Analysis of the myofibrillar ATPase profile also revealed that a high percentage of immature fibers persisted in adults. A long-term treatment with large amounts of T3 had no effect on juvenile individuals. Applied to subadult animals it promoted a regression of larval myosin isoforms and a reduction in the percentage of immature fiber types. The regulative effect of T3 in the myosin isoenzymic transition may be delayed and depends on metabolic conditions, which suggests it is indirect.