Limb Muscles Of Mice Research Articles

Mouse limb muscle is determined in the absence of the earliest myogenic factor myf-5.

myf-5 is the only member of the MyoD family of myogenic regulatory genes to be expressed in the mouse embryo prior to muscle cell differentiation. We have used the developing limb as a model in which to follow the formation of peripheral muscle, to address the question of whether myogenic precursor cells are already present in the limb bud before expression of myf-5. The lacZ reporter gene has been introduced into the myf-5 gene by homologous recombination so that its expression is under the control of the endogenous myf-5 locus. beta-Galactosidase (beta-gal) coloration provides a sensitive assay for myf-5+ cells. Embryos were generated from embryonic stem cells carrying this mutation, and the appearance of beta-gal+ (myf-5+) cells was followed during limb development in vivo. Limb buds, at a stage when they are beta-gal-, were cultured in vitro. After several days, beta-gal+ cells accumulated in the premuscle mass. We conclude that determined muscle precursor cells in the limb bud do not initially express any member of the MyoD family. Furthermore, myogenic precursor cells in the somite, which, according to the avian model, migrate from the ventral/lateral edge of the dermomyotome to form peripheral muscle masses, are also negative for these factors.

Quantitation of muscle precursor cell activity in skeletal muscle by Northern analysis of MyoD and myogenin expression: Application to dystrophic (mdx) mouse muscle

The regeneration of skeletal muscle is dependent upon proliferation and fusion of activated mononuclear muscle precursor cells. Early and specific markers of this population of activated cells are the transcription factors MyoD and myogenin. Northern analysis was used to determine levels of MyoD and myogenin mRNA in (i) muscles regenerating after experimental crush injury and (ii) in limb muscles of dystrophic mdx mice at various ages in comparison to controls. In crush-injured muscle, MyoD and myogenin mRNA increased at 24 h, peaked between 2 to 6 days, and returned to uninjured control levels by 15 days after injury. In both mdx and control mice, MyoD and myogenin mRNA levels were high in fetal muscles and decreased rapidly during the 2 weeks after birth. In mdx muscles, the mRNA levels increased significantly from about 21 days, remained high until around 40 days, and then decreased to a relatively constant yet elevated level when compared to control muscles. The elevated levels persisted to 420 days of age. The results show that this technique can be used to provide sensitive quantitative information on the size of the population of activated precursor cells in skeletal muscle. As such, it represents a novel and convenient means of measuring regenerative activity in vivo in whole muscles.

Actin and myosin genes are transcriptionally regulated during mouse skeletal muscle development

During primary and secondary myotube formation in utero and subsequent maturation of muscle fibers after birth there are complex changes in the pattern of contractile protein gene expression at the RNA and protein levels. In order to determine the degree of transcriptional regulation of actin and myosin genes we have carried out “nuclear run-on” experiments using nuclei prepared from the limb muscle of mice at 14.5, 15.5, 17.5, and 18.5 days in utero and at 10–12 and 12.5 days after birth. We show that transitions in the expression of these genes in vivo are regulated transcriptionally. Transcription of the sarcomeric α-actins changes from cardiac to predominantly skeletal actin over this time period; transcription of the β-actin gene is repressed. The myosin heavy chain and myosin light chain genes also undergo transcriptional transitions during muscle development. Notably, transcription from the MLC3 F promoter is activated after that of the MLC1 F promoter, which is part of the same gene. These results are discussed in the context of published RNA data.

The MyoD family of myogenic factors is regulated by electrical activity: isolation and characterization of a mouse Myf-5 cDNA

A full-length cDNA coding for a homolog of the human Myf-5 was isolated from a BC3H-1 mouse library and characterized. The clone codes for a protein of 255 amino acids that is 89%, 88% and 68% identical to the human, bovine and Xenopus myf-5, respectively. The mouse Myf-5 cDNA (mmyf-5), as well as sequences coding for MyoD, myogenin and Mrf-4, were used to probe Northern blots to analyze the effects of innervation on the expression of the MyoD family of myogenic factors. Mouse myf-5, MyoD and myogenin mRNAs levels were found to decline in hind limb muscles of mice between embryonic day 15 (E15) and the first postnatal week, a period that coincides with innervation. In contrast, Mrf-4 transcripts increase during this period and reach steady-state levels by 1-week after birth. To distinguish if the changes in myogenic factor expression are due to a developmental program or to innervation, mRNA levels were analyzed at different times after muscle denervation. Mmyf-5 transcripts begin to accumulate 2 days postdenervation; after 1 week levels are 7-fold higher than in innervated muscle. Mrf-4, MyoD and myogenin transcripts begin to accumulate as soon as 8h after denervation, and attain levels that are 8-, 15- and 40-fold higher than found in innervated skeletal muscle, respectively. The accumulation of these three mRNAs precedes the increase of nicotinic acetylcholine receptor alpha subunit transcripts, a gene that is transcriptionally regulated by MyoD-related factors in vitro. Using extracellular electrodes to directly stimulate in situ the soleus muscle of rats, we found that 'electrical activity' per se, in absence of the nerve, represses the increases of myogenic factor mRNAs associated with denervation.

The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy

Although murine X-linked muscular dystrophy (mdx) and Duchenne muscular dystrophy (DMD) are genetically homologous and both characterized by a complete absence of dystrophin, the limb muscles of adult mdx mice suffer neither the detectable weakness nor the progressive degeneration that are features of DMD. Here we show that the mdx mouse diaphragm exhibits a pattern of degeneration, fibrosis and severe functional deficit comparable to that of DMD limb muscle, although adult mice show no overt respiratory impairment. Progressive functional changes include reductions in strength (to 13.5% of control by two years of age), elasticity, twitch speed and fibre length. The collagen density rises to at least seven times that of control diaphragm and ten times that of mdx hind-limb muscle. By 1.5 years of age, similar but less severe histological changes emerge in the accessory muscles of respiration. On the basis of these findings, we propose that dystrophin deficiency alters the threshold for work-induced injury. Our data provide a quantitative framework for studying the pathogenesis of dystrophy and extend the application of the mdx mouse as an animal model.

Long term effect of low frequency chronic electrical stimulation on the fast hind limb muscles of dystrophic mice.

Low frequency chronic electrical stimulation can have a beneficial effect on dystrophic muscles. The present study was undertaken to assess the long term effect of such stimulation on the fast hind limb muscles of dystrophic mice. The relationship between the changes induced by stimulation and the initial condition of the dystrophic muscles, as well as other factors which might contribute to this relationship, were examined. The stimulation induced an increase in the force output of weak dystrophic muscles and a speeding of their time course of contraction and relaxation, as well as an increase in their fatigue resistance. In relatively strong dystrophic muscles, the stimulation induced similar changes in contractile speed and fatigue characteristics, but it led to a slight decrease in force output. Our results suggest that the stimulation promotes the growth and differentiation of the small regenerating fibres known to be present in the diseased muscles and, in addition, induces an increase in the mitochondrial content of the muscle fibres. Our results indicate that these effects are not permanent.

Great adaptability of brown adipose tissue mitochondria to extreme ambient temperatures in control and cold-acclimated gerbils as compared with mice

1. 1. The gerbil ( Gerbillus campestris) is a desert rodent able to tolerate high (38°C) and low (−20°C) ambient temperatures, probably due to both its low resting metabolic rate in hot environment and its high peak metabolic rate in cold. 2. 2. Measurement of mitochondrial state IV respiration and cytochrome-oxidase activity (COX) were made in interscapular brown adipose tissue (IBAT), liver and hind limb muscles of gerbils and mice of nearly equal body mass, acclimated for 4 weeks at cold ambient temperature (CA) or reared at thermoneutrality (TN). 3. 3. The most striking difference between these two animal species appears to be in IBAT mitochondria: in TN animals, the level of state IV respiration and COX activity was lower in gerbils than in mice, but the cold acclimation-induced increase in these parameters was greater in gerbils than in mice. 4. 4. Alternatively, in gerbils as in mice, cold acclimation induced a reduction in muscle mitochondrial COX activity. No important change due to cold acclimation was observed in liver mitochondria, either in gerbils or in mice. 5. 5. As compared with mice, the lower state IV respiration in IBAT mitochondria from TN gerbils may explain their low RMR, whereas the higher COX activity of IBAT mitochondria from CA gerbils may explain their higher PMR. 6. 6. As a result of this great adaptability of BAT mitochondria, the gerbil seemed to be able to live in a wide range of ambient temperatures in its natural habitat.

A multivariate study on enzymatic changes in limb muscles and heart muscle of dystrophic mice.

The present study was undertaken to elucidate further the enzymatic changes in dystrophic muscle using multivariate analysis. The activities of 14 kinds of enzymes, including 6 exopeptidases, 4 endopeptidases, beta-N-acetyl-D-glucosaminidase, phosphatase, esterase, and ribonuclease, were examined in forelimb and hindlimb muscles as well as in cardiac muscle of dystrophic mice and their controls. Two principal components identified from the enzymatic spectrum proved to be related especially to aminopeptidases and to serine proteinases, respectively. The enzymatic changes in forelimb muscle were very similar to those in hindlimb muscle when both were compared to those in cardiac muscle. The changes in aminopeptidases were unique to the limb muscles, whereas those of serine proteinases were unique to cardiac muscle of dystrophic mice. In the future, more attention should be focused on the role of exopeptidases in pathogenetic mechanisms of muscular dystrophy, because of the possibility that they play a major role in the initial stage of muscular dystrophy.

Age-related changes in oxidative capacity of the gastrocnemius muscle in normal and dystrophic ( [formula omitted]) mice

The hind limb muscles of dy 2J dy 2J mice appear more oxidative than normal hind limb muscles when assayed histochemically. In some cases, biochemical assay of oxidative capacity does not match histochemical data, and in some dy 2J mice, particularly old animals, there appears to be a decline in biochemically assayed oxidative enzymes. The current study cyto-biochemically assayed succinic dehdrogenase activity of the gastrocnemius muscle in dy 2J and normal mice. The assays were conducted over a maturation time course of 1 to 6 months of age. Additionally, the gastrocnemius muscle was divided into two distinct regions, a superficial region, containing largely glycolytic fast-twitch fibers (SGM), and a deeper region of mixed, largely oxidative fibers (DGM) in normal animals. Assay of the whole muscle and the two regions of each individual muscle showed that the dy 2J muscle increased in SDH activity with maturation and was significantly greater than the change observed in normal muscle during the same period. The increase in the whole muscle SDH activity was accounted for increases noted in the SGM. The dy 2J DGM, which showed marked morphologic degeneration, had neither an increase nor a decrease in measured SDH activity. Michaelis-Menton analysis of the enzymatic assay indicated that the V max, and not the K m , of the dystrophic muscle enzyme system was higher than normal, suggesting a change in quantity of enzyme present, and not altered function of the system. The observed increases occurred for 4 months of maturation and then began a decline, which was noted at 6 months. Coupled with the time course of gastrocnemius muscle degeneration (the glycolytic region was slow—very little by 4 months; the mixed was fast—highly degenerated by 1 to 2 months), the results of this study suggest that oxidative capacity in the dy 2J gastrocnemius muscle increased with maturation until it became obscured by inherant muscle wasting in a particular dy 2J muscle region. By comparison, analysis of the large glycolytic triceps muscle, a forelimb muscle which does not receive pseudomyotonia, indicated that this muscle did not increase in SDH activity in dystrophic animals, which showed the abnormal increase in the activity of the hind limb gastrocnemius. Thus the relationship of pseudomyotonia to increasing oxidative activity in the hind limb muscles of dystrophic animals was evident.

Sustained transmitter output by increased transmitter turnover in limb muscles of old mice

The ability of neuromuscular junctions in old animals to maintain tetanic output was tested in phasic and tonic limb muscles and the physiologic mechanism of maintenance was elucidated by analysis of the turnover of a false transmitter during prolonged tetani. Transmitter release during and after tetani was compared in limb muscles of young (8-9 month) and old (28-30 month) male CBF-1 mice. Amplitudes of end-plate potentials (epp's) in curarized preparations and of spontaneous miniature end-plate potentials (mepp's) were measured in vitro at 30 degrees C in soleus and extensor digitorum longus (edl) muscles. In both young and old soleus muscles, epp amplitude was maintained at about 45% of resting level during the latter part of trains of 1,200 stimuli at 10 Hz but recovered to about 90% control within a few seconds after stimulation ceased. In edl muscles of young mice, epp amplitudes during a 20 Hz train of 1,200 impulses steadily declined to about 20% of control and gradually recovered over 2 min after the tetanus. In old edl muscles, tetanic decay of the epp's was greater and recovery slower than in young muscles, but absolute epp amplitudes were invariably greater. During trains of 6,000 impulses at 10 Hz, plateau epp amplitude decayed to 40-50% in young soleus muscle and 30-40% control in old muscle, but recovery was similar and absolute epp amplitudes were greater in old soleus muscle. A false transmitter precursor, homocholine (HoCh), was used to investigate the mechanism of this prolonged output, and, therefore, the use of HoCh in this system was first validated.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevations of cathepsin B and cathepsin L activities in forelimb and hind limb muscles of genetically dystrophic mice

The combined activities of cathepsin B and cathepsin L were studied in the forelimb and hind limb muscles of dystrophic mice. The activities of these proteases in the forelimb and hind limb muscles of young and adult dystrophic mice were significantly higher than those in normal mice. However, clinical involvement of dystrophy appeared in the hind limbs but not in the forelimbs. We therefore suggest that the increase in protease activity begins at a very early age and that the clinical involvement is not linked with the increase in cathepsins B and L.

Isolation and sequence of cDNA clones coding for the precursor to the gamma subunit of mouse muscle nicotinic acetylcholine receptor.

cDNA libraries have been constructed in plasmid (pBR322) and bacteriophage lambda gammagt10) vectors with poly (A+) RNA isolated from the nonfusing mouse muscle cell line BC3H-1. The libraries were screened with a restriction fragment derived from a genomic clone coding for a human acetylcholine receptor gamma subunit. Several clones were obtained whose cDNA inserts possessed nucleotide and deduced amino acid sequence homology with acetylcholine receptor gamma subunits from Torpedo californica, chick, calf, and human. One isolate, lambda BMG419, has 88 nucleotides of 5'-untranslated sequence, an open reading frame of 1,557 nucleotides coding for the precursor to the mouse acetylcholine receptor gamma subunit, and 144 nucleotides of 3'-untranslated sequence. Alignment of the lambda BMG419-deduced amino acid sequence with homologs from other species predicts a precursor peptide of 519 amino acids and a mature protein of 497 amino acids, with nonglycosylated molecular weights of 58,744 and 56,424 daltons, respectively. Comparison of the deduced amino acid sequence of the mouse gamma subunit with Torpedo, chick, calf, and human sequences showed overall homologies of 54%, 67%, 90%, and 90%, respectively; however, significantly higher homologies were found in several putative functional domains. Radiolabeled lambda BMG419 has been used to identify homologous RNA species, one of approximately 2 kb and one of about 3.5 kb, in poly (A+) RNA prepared from BC3H-1 cells and denervated mouse limb muscle. gamma Subunit-coding RNA species are considerably more abundant in denervated than in innervated muscle, suggesting that neural regulation of the abundance of the gamma subunit is exerted through regulation of the amount of its mRNA.

Fatiguability and oxidative capacity of forelimb and hind limb muscles of dystrophic mice

Fatigue indices and succinic dehydrogenase (SDH) activities were determined in the extensor digitorum longus (EDL) and soleus muscles of the hind limb and the extensor carpi radialis longus of the forelimb in control and dystrophic mice aged 4 to 26 weeks. A good correlation was found between SDH activities and fatigue indices in muscles from normal mice. In the dystrophic (dy 2J) mice, however, this correlation was not present. The EDL muscles from 26-week-old dy 2J mice showed a much higher resistance to fatigue than age-matched controls but this was not accompanied by a significant change in SDH. The increased fatigue resistance in dy 2J EDL appeared between 8 and 12 weeks of age and was temporally correlated with the onset of fused bursts of spontaneous activity in the hind limb muscles. Nevertheless, there was no conclusive evidence for a link among this spontaneous activity, oxidative enzyme capacity, and fatigue resistance.

Content and synthesis of several abundant glycolytic enzymes in skeletal muscles of normal and dystrophic mice

1. 1. In both 2- and 3-month-old 129 ReJ mice, the catalytic activity levels of three enzymes involved in glycogen breakdown (phosphorylase, enolase, and aldolase) were found to be 35–50% lower in hind limb muscles of dystrophic mice as compared with normal mice. 2. 2. The reduced activities of these enzymes in the diseased tissue was directly due to corresponcling reductions in the number of enzyme molecules rather than being due to inactivation of the enzymes in the dystrophic muscle. 3. 3. Results of short term double isotope incorporation experiments conducted with muscle expiants in vitro suggested that the rates of synthesis of these enzymes, and of most other abundant cytosolic proteins, relative to each other, were similar in hind limb muscles of normal and dystrophic mice. 4. 4. The present work on murine muscular dystrophy is discussed in terms of our previous studies into the influence of avian muscular dystrophy on the content and synthesis of abundant glycolytic enzymes in chicken skeletal muscles.

Potassium contractures in mouse limb muscles.

The force of contractures produced by 14-400 mM-K+ (as methanesulphonate) was measured in whole mouse soleus (sol.), extensor digitorum longus (e.d.l.), and in bundles from these muscles. Frog semitendinosus muscles were used for comparison. Whole mouse muscles displayed biphasic contracture responses lasting more than 5 min when provoked by 150m M-K+. Contractures of bundles dissected from these muscles were monophasic and had a short duration. The time required for the muscle bundles to contract and relax to 1/2 maximum force (T) was an inverse function of [K+]. T was increased by lowering [K+] from 400 to 50 mM by a factor of 8.3 and 7.0 in proximal and distal portions of sol. and by a factor of 4.2 and 2.8 in proximal and distal portions of e.d.l., respectively. The force-[K+] relation was steeper for sol. than for e.d.l., and the proximal portions were more sensitive to K+ than the distal portions, particularly in e.d.l. The capability of the muscles to produce force in response to 400 mM-K+ after a 10 min exposure to 30 or 50 mM-K+ was high in sol., somewhat lower in proximal parts of e.d.l. and in frog semitendinosus, and lowest in distal parts of e.d.l. It was concluded that K contractures of mouse muscles are basically monophasic, and that biphasic contractures of whole muscles arise because of K+-diffusion delays, slow responses to intermediate [K+], and differences in responsiveness of the fibres contained in a particular muscle.

Slow potassium contractures in mouse limb muscles.

1. Mouse extensor digitorum longus and soleus muscles respond to a sudden maintained increase in external K ion concentration with a fast contracture which inactivates and is followed by a slow contracture. 2. The slow contracture could not be selectively eliminated by altering the tonicity, ionic strength, anionic composition or buffer system of the external solution and depended only on the increase on external potassium concentration. The slow contracture differed from the fast K contracture in its time course, temperature sensitivity, fibre type dependence, and inactivation kinetics. The fast and slow contractures were similarly altered by changes in external anion species, by changes in external divalent cations, and by the presence of 20 mM-caffeine. 3. The mechanism and functional significance of the slow contracture are obscure. The results suggest that its generation is not identical to that of the fast contracture, but may depend, in part, upon the normal activation processes.

Potassium contractures and mechanical activation in mammalian skeletal muscles.

Potassium (K-) contractures were recorded from slow-twitch (mouse soleus and fast-twitch (mouse extensor digitorum longus (EDL) and rat sternomastoid) muscles. The mouse limb muscles responded to a maintained increase in external potassium concentration with a rapid increase in tension (fast contracture) which inactivated and was followed by a slow contracture. Rat sternomastoid muscles responded with fast contractures only. The threshold potassium concentration for contraction was higher in fast-twitch muscles than in soleus muscles, at 22 and at 37 degrees C. After corrections had been made for the more rapid depolarization of soleus fibers, the threshold potential for soleus fiber contraction was 15mV closer to the resting membrane potential than the threshold for fast-twitch fiber contraction. The K-contracture results were confirmed by two microelectrode voltage-clamp experiments. Activation of fast twitch fibers required depolarizing pulses that were 15 to 20mV greater than the pulses required to activate soleus fibers. When the time courses of K-contractures were compared it was evident that inactivation with prolonged depolarization was much faster in the fast-twitch muscles than in the soleus muscles. The results suggest that the voltage dependence and kinetics of the process coupling T-tubule depolarization with calcium release from the sarcoplasmic reticulum may depend on fiber atype in mammalian skeletal muscle.

Level of nerve growth factor-like immunoreactivity in the lower limb muscles of muscular dystrophic mice

The level of nerve growth factor-like immunoreactivity in the lower limb muscles, the site where the dystrophic mice are effected, of both normal and dystrophic mice was studied by solid-phase radioimmunoassay. Nerve growth factor-like immunoreactivity levels of the homozygous dystrophic mice were about two times lower than those of the heterozygous dystrophic mice at 10–11 weeks and 7–8 weeks of age. The levels in 4–5 week old homozygous mice were too low for detection and remarkable differences between the homozygous and the heterozygous mice were observed at this age. These differences in the level of nerve growth factor-like immunoreactivity suggest that the factor may have some relation to the disease.

Distribution of histological changes on the limb muscles of dystrophic mice.

Distribution of histological changes on the limb muscles of dystrophic mice.

Some mechanical and electrical properties of distal hind limb muscles of genetically dystrophic mice (C57BL/6Jdy 2J/dy 2J)

Some mechanical and electrical properties of distal hind limb muscles of mice suffering from muscular dystrophy caused by the dy 2J allele have been defined. The dystrophic muscles were lighter and weaker than normal when subjected to either single or tetanic indirect stimulation. The twitch tetanus ratio was unchanged. The contraction time of the dystrophic muscles was prolonged, but time to half-relaxation was normal. All dystrophic muscle fibers generated an action potential when stimulated indirectly, and there was no myasthenialike response of the gross muscle EMG during high frequency stimulation. Resting membrane potentials were reduced in the dystrophic muscle fibers, and action potentials were slower and smaller in the dystrophic “fast-twitch” muscle fibers of the extensor digitorum longus muscle. Action potentials in the “slow twitch” muscle fibers of the soleus muscle were normal. Miniature end-plate potentials were present in all dystrophic muscle fibers examined. Many muscle fibers in both extensor digitorum longus and soleus muscles of dystrophic animals were sensitive to acetylcholine outside the end-plate region, and were also able to generate action potentials in the presence of 10 −6 m tetrodotoxin. Since no evidence of either functional or structural denervation was found, it is suggested that these are immature, regenerating muscle fibers. It is further suggested that the ability to regenerate in spite of the presence of the dystrophic gene is a major and possibly qualitative difference between murine dystrophy of the type dy 2J dy 2J , and dy dy in which regeneration of adult muscle is abortive. The implications of such a qualitative difference are discussed with reference to the current controversy concerning the etiology of the muscular dystrophies.