Effect of hind-limb suspension on young and adult skeletal muscle. II. Dystrophic mice

Disuse atrophy induced by limb immobilization reportedly protects dystrophic mouse muscle from histopathological changes. This study was conducted to determine whether disuse atrophy induced by hind-limb suspension (HS) limits the histopathology and contractile abnormalities typically observed in the dystrophic mouse. Two weeks of hind-limb suspension were applied to dystrophic mice (line 129B6F1) at two ages, 4 weeks (6 mice) and 12 weeks (8 mice). Thirty-one untreated dystrophics served as controls. In general, HS exaggerated the dystrophic signs, especially in the younger mice; it reduced animal weight, muscle weight, maximum tetanic and twitch tensions, and rates of tetanic and twitch tension development. HS further slowed the contractile properties of soleus (SOL) and extensor digitorum longus (EDL) muscles, and increased their fatigue resistance. HS reduced the size of type I and IIA fibers in the 6-week SOL and EDL, but not in the 14-week muscles. HS produced a preferential atrophy of SOL type I fibers, with a parallel increase in type IIA fibers. However, it did not alleviate the fiber size variability, degree of necrosis, central nucleation, inflammation, or muscle fibrosis in dystrophic muscles. These data demonstrate that disuse by hind-limb suspension does not prevent the histopathological deterioration or loss of muscle function in 6- and 14-week dystrophic mice.     

Effect of creatine supplementation on skeletal muscle of mdx mice

Dystrophic mice (mdx) and their controls (C57/Bl10) were fed for 1 month with a diet with or without creatine (Cr) enrichment. Cr supplementation reduced mass (by 19%, P < 0.01) and mean fiber surface (by 25%, P < 0.05) of fast-twitch mdx muscles. In both strains, tetanic tension increased slightly (9.2%) without reaching statistical significance (P = 0.08), and relaxation time increased by 16% (P < 0.001). However, Cr had no protective effect on the other hallmarks of dystrophy such as susceptibility to eccentric contractions; large numbers of centrally nucleated fibers in tibialis anterior; and elevated total calcium content, which increased by 85% (P = 0.008) in gastrocnemius mdx muscles. In conclusion, Cr may be a positive intervention for improving function of dystrophic muscle.     

Alteration of spinal cord IGF-I receptors and skeletal muscle IGF-I after hind-limb immobilization in the rat.

The effects of 4 weeks' hind-limb immobilization on the spinal cord insulin-like growth factor-I (IGF-I) receptors and skeletal muscle IGF-I level was investigated in rats. Quantitative receptor autoradiography using [125I]IGF-I as a ligand was performed to measure IGF-I receptors in cryosections from the lumbar region of the spinal cord. IGF-I receptor levels were significantly higher in all spinal cord laminae on the side ipsilateral to the immobilized limb than in the same spinal level of the controls. Using radioimmunoassay (RIA), IGF-I levels were significantly low in the soleus (SOL), but not the tibialis anterior (TIB) muscles, compared to the controls. The enhancement of the spinal cord IGF-I receptors after hind-limb immobilization may constitute part of the nervous system response to disuse.     

Full-length dystrophin cDNA transfer into skeletal muscle of adult mdx mice by electroporation

We showed that a LacZ expression plasmid (pCAG-lacZ) injection followed by electroporation increased the expression of the LacZ gene in the skeletal muscles of adult mdx mice up to ninefold higher as compared with simple intramuscular DNA injection. When full-length mouse dystrophin plasmid (pCAG-dys) and pCAG-lacZ were co-transfected by electroporation, 56% of dystrophin-positive fibers were stained for beta-galactosidase activity suggesting most of these myofibers are not revertants but transfected ones. Our data indicate that electroporation in vivo could introduce large full-length dystrophin cDNA into skeletal muscle of adult mdx mice.     

Effect of motor-response-deprivation on contingent negative variation (CNV). I: Normal adult data.

CNV's were obtained in S1-S2-MR paradigm from 22 normal subjects between the ages of 19 and 67 years (the mean age, 33.5+/-12.5). And then the effect of the presence or absence of a motor response on the CNV was studied. Results were as follows; (1) In the control session (S1-S2-MR), the mean CNV amplitude was 14+/-5 muV, and the mean response time was 291+/-76 msec. There was no significant difference with age, but the mean CNV amplitude for female was significantly higher than that for male. (2) Under the nonresponse condition (S1-S2) where the subject was instructed to attend to S1-S2 without a motor response, CNV amplitude significantly reduced for all subjects (7+/-5 muV). The mean voltage of the attentive-to-stimuli group was significantly higher than that of the inattentive group, and sex difference was not significant for the attentive group. However, CNV of the inattentive male disappeared completely, while the CNV of the inattentive female was significantly higher (8+/-5 muV). (3) Under the response condition (S1-S2-MR), CNV recovered almost to the magnitude level of the control condition (14+/-3 muV), in spite of the fact that the mean response time was significantly shortened between the control and response sessions. (4) We speculate that CNV changes may be dependent upon the dynamic balance of the excitatory-inhibitory activity.     

Effect of denervation on adenine nucleotides in skeletal muscle from normal and dystrophic mice

The effect of denervation on the adenine nucleotide content of fast- and slow-twitch skeletal muscle of the C57BL mouse was studied by high-performance liquid chromatography. From the adenine nucleotide content the energy charge, a measure of high-energy phosphate available to the cell, was calculated. The energy charge of the extensor digitorum longus muscle was significantly higher than that of the same muscle from dystrophic mice (C57BL/6J dy2j/dy2j) and on denervation decreased to the values found in the innervated muscle from dystrophic animals. Denervation of the muscle in dystrophic mice did not change the energy charge of that muscle. The energy charge of the soleus muscle from both normal and dystrophic mice was similar and did not change on denervation. It is proposed that in the dystrophic process a functional denervation of skeletal muscle occurs which preferentially affects fast-twitch muscle, leading to a reduction in the energy charge.     

Vasodilatation in hind-limb skeletal muscle evoked as part of the defence reaction in the rat

Electrical stimulation of the hypothalamic and mid-brain defence area of the rat's brain elicits a consistent cardiovascular pattern of response of which vasodilatation in the skeletal muscle is an integral component: the mechanisms mediating this vasodilatation were investigated. It is not sensitive to atropine in this species, but it was substantially affected, particularly its later stage (the prolonged tail-end of the response), by either beta-adrenoreceptor antagonists (propranolol, sotalol) or bilateral adrenalectomy. A major part of the hind-limb vasodilatation can therefore be attributed to the action of catecholamines released from the adrenal glands. The initial part of the vasodilatation, which still remained after beta-adrenoreceptor blockade and adrenalectomy, was abolished by intravenous injection of guanethidine and phentolamine and seems therefore due simply to withdrawal of vasoconstrictor tone. In confirmation, stimulation of the sympathetic outflow to the hind-quarters after phentolamine and guanethidine had been used to block vasoconstriction did not reveal a sympathetic vasodilator nerve supply to the hind-limb vasculature.     

Effective adenovirus-mediated gene expression in adult murine skeletal muscle.

We established an efficient method for obtaining expression of a foreign marker gene transferred in vitro into myoblasts and in vivo into adult mouse skeletal muscles using adenovirus vector. After infection of the C2 myoblasts with the adenovirus vector containing the beta-actin promoter with cytomegalovirus (CMV) enhancer (CAG promoter) AxCALacZ, significantly greater number of cells express beta-galactosidase when compared with the adenovirus vector expressing the lacZ gene under the control of the SR alpha viral terminal repeat promoter (AxSRLacZL) or the myosin heavy chain (MHC) IIB promoter (AxMHCLacZ). We also injected AxCALacZ into the skeletal muscles of 5- to 6-week-old C57BL/10 mice and determined that more than 60% of their muscle fibers expressed the lacZ gene 7 days after injection. The CAG promoter may have application in the development of gene therapy for Duchenne muscular dystrophy (DMD) using adenovirus vector.     

Mechanical properties of rat skeletal muscle after hind limb suspension

It is clear that there are events which occur in response to neuromuscular activity that are essential to maintain normal muscle properties. Two factors though to influence muscle contractile properties are tension and electrical activity. To study these variables the hind limbs of 10 postpubertal female Sprague-Dawley rats were prevented from supporting the weight of the body for 4 weeks, then in situ isometric properties were determined and compared with 10 age-matched control rats. The soleus (slow plantarflexor), the medial gastrocnemius (fast plantarflexor), and the tibialis anterior (fast dorsiflexor) were studied. The suspended soleus wet weight was 42% lower and the maximum isometric twitch (Pt) and tetanic (P0) tensions were 62 and 69% lower respectively, than in the control rats. The suspended medial gastrocnemius was 18% lower in wet weight and 14 and 9% lower in P0 and Pt than the controls. No differences in wet weight, P0 or Pt were observed in tibialis anterior. Mean contraction time was 22% shorter in the soleus, 9% in the medial gastrocnemius, and unchanged in the tibialis anterior following suspension. Further, the percent of P0 attained during a 330-ms tetanus at 20 Hz was 15% lower in the suspended soleus and unchanged in the other two muscles. The fatigue index (ratio of tension after 2 min of stimulation at 40 Hz for 330 ms once per second to the maximum tension developed during the test) was unaffected by suspension in the soleus and tibialis anterior but was reduced from 49% to 36% in the medial gastrocnemius. The maximum rate of shortening of all three muscles was unaffected by suspension. These results indicate that suspension of the hind limbs selectively affects the mass and force-generating capabilities of the plantarflexors, particularly the predominantly slow soleus. In contrast, the fatigability of only the fast plantarflexor was increased whereas the slow plantarflexor was unaffected. These results, considered in light of collaborative studies, suggest that the chronic force-time levels in a muscle have an important influence on tension- and speed-related properties but not necessarily the fatigability of that muscle. Further, a marked difference in the sensitivity of the contractile elements of slow and fast muscle to this influence was evident.     

Apoptosis in young and old denervated rat skeletal muscle

Introduction: We investigated the apoptotic response to different degrees of denervation in young and older rats randomized into control (C), partial (PD), and complete denervation (CD) of muscles innervated by the sciatic nerve. Methods: Muscle wet weight to body weight (MWW/BW), myosin heavy chain (MHC) isoforms, and fiber cross‐sectional area were determined in gastrocnemius and soleus muscles. Apoptotic responses were determined by changes in myonuclei and expression of Bcl‐2 and BAX. Results: PD and CD resulted in significant reductions in MWW/BW and FCSA in both young and older rats. Older controls had greater apoptotic responses than young controls. Apoptotic responses were greater in PD and CD than in C in both age groups. No statistical interaction between denervation and age group was seen. Conclusions: Older age was associated with increased level of apoptosis, but older muscle was not more vulnerable to the effect of denervation. Muscle Nerve 55 : 262–269, 2017     

Neural regulation of N-cadherin gene expression in developing and adult skeletal muscle.

Using monoclonal antibody and cDNA probes, we have studied N-cadherin gene expression in developing and adult chick skeletal muscle. N-cadherin was expressed by developing myotubes during the period of initial nerve-muscle contact but was downregulated within days of innervation. Treatment of embryos with d-tubocurare partially reversed this downregulation. In the adult, there were muscle fiber type differences in N-cadherin expression. N-cadherin was undetectable on normally innervated twitch fibers, while multiply innervated tonic muscle fibers expressed low but readily detectable levels of N-cadherin. Denervation led to the renewed expression of N-cadherin in twitch fibers as well as a marked increase in expression in tonic fibers. Levels of N-cadherin expressed by tonic fibers could also be modulated by animal housing conditions that favored either increased or decreased levels of daily motor activity. Increased motor activity was correlated with decreased levels of N-cadherin, while decreased motor activity correlated with increased levels of muscle N-cadherin. Results of in vitro studies using the calcium channel agonist ryanodine suggest that changes in intracellular calcium may be the initial signal linking neural stimulation with changes in muscle fiber expression of N-cadherin. Together, our results indicate that neural stimulation of chick skeletal muscle fibers dynamically and reversibly downregulates the expression of N-cadherin mRNA and protein. This pattern of regulation may be functionally important in limiting and/or promoting axon growth in innervated versus denervated muscle and may serve as a molecular model system for studies of the activity-dependent regulation of gene expression.     

Distinct subtypes of type I fibers of human skeletal muscle.

We have identified two distinct and consistent type I fiber subtypes in histochemically normal biopsies (vastus lateralis or biceps brachialis) from 12 healthy persons, both sexes, ages 15 to 42. The type I fibers were uniformly light with the regular ATPase (9.4) and uniformly dark with the acid-preincubated reverse ATPase reactions. However, the subtype IA fibers were stained darker than subtype IB fibers with the nicotinamide adenine nucleotide dehydrogenase-tetrazolium reductase, Oil red O, reduced nicotinamide adenine dinucleotide phosphate-tetrazolium reductase, nonspecific esterase, succinic-tetrazolium reductase, nicotinamide adenine dinucleotide-linked malic-tetrazolium reductase, and "nicotinamide adenine dinucleotide-linked lactic-tetrazolium reductase," and stained lighter with the phosphorylase, periodic acid-Schiff for glycogen, and menadione-mediated alpha-glycerophosphate-tetrazolium reductase reactions. The IB fibers were not as light (or as dark, depending on the stain) as any of the type II fibers. In one pathologic case, all the "hypotrophic type I muscle fibers with central nuclei" were subtype IB. In case of chronic infantile spinal atrophy the muscle showed some type-grouping and marked type I fiber predominance--the great majority of type I fibers were subtype IB, among which were islands (subtype-groups) of IA fibers. Subtyping of type I fibers may be informative in evaluating the pathologic process of certain human neuromuscular diseases.     

Ouabain binding sites in skeletal muscle from normal and dystrophic mice.

The specific binding of tritiated ouabain was used to estimate the density of Na+-K+-ATPase sites ("Na+-pump" sites) in segments of skeletal muscle from normal and dystrophic mice. Ouabain binding was approximately 4 times greater in red (soleus) muscle than in white (superficial gastrocnemius) muscle from normal animals. In dystrophic soleus muscles, ouabain binding was decreased by nearly one-half. Because Na+-K+-ATPase activity is associated with plasma membranes, these observations constitute further evidence for a sarcolemmal abnormality in dystrophic mice.     

Effects of prednisolone on skeletal muscle contractility in mdx mice

Current treatment for Duchenne muscular dystrophy (DMD) is chronic administration of the glucocorticoid prednisolone. Prednisolone improves muscle strength in boys with DMD, but the mechanism is unknown. The purpose of this study was to determine how prednisolone improves muscle strength by examining muscle contractility in dystrophic mice over time and in conjunction with eccentric injury. Mdx mice began receiving prednisolone (n = 23) or placebo (n = 16) at 5 weeks of age. Eight weeks of prednisolone increased specific force of the extensor digitorum longus muscle 26%, but other parameters of contractility were not affected. Prednisolone also improved the histological appearance of muscle by decreasing the number of centrally nucleated fibers. Prednisolone treatment did not affect force loss during eccentric contractions or recovery of force following injury. These data are of clinical relevance, because the increase in muscle strength in boys with DMD taking prednisolone does not appear to occur via the same mechanism in dystrophic mice. Muscle Nerve, 2009     

Nutritional rehabilitation of skeletal muscle in protein-deprived young rats

The effect of severe protein deprivation and subsequent nutritional rehabilitation on the fibre size and mitochondrial enzyme activity of the extensor digitorum longus (EDL) and soleus muscles of the young rat has been examined. Protein deprived rats showed atrophy of type 2 fibres predominantly, reduced histochemical activity of succinic dehydrogenase (SDH) and reduced biochemical activity of citrate synthase. Nutritional rehabilitation indicated by resumption of the original body weight resulted in complete restitution of the weight of the muscles and the size of type 1 and type 2 fibres, but not of the activity of SDH and citrate synthase. The results indicate that regarding size, type 2 fibres tend to be more influenced than type 1 fibres by the nutritional supply. The mitochondrial enzyme activity which is decreased by protein deprivation does not regain the normal levels as quickly as the muscle fibres resume their normal size.     

Effect of neural activity on skeletal muscle phosphoproteins

A number of phosphoproteins were found in the soluble fraction of rat skeletal muscle by two-dimensional polyacrylamide gel electrophoresis (PAGE). The pattern of some of these phosphoproteins differed in fast (extensor digitorum longus, EDL) or slow (soleus) muscles, was dependent on normal innervation, and was altered with denervation. In order to determine if the pattern was maintained by electrical activity or trophic factors, we compared the effect of electrical block by local neural application of tetrodotoxin with the effect of complete nerve section. Both methods produced similar alterations in phosphoproteins, indicating that the pattern is dependent on nerve activity, not trophic factors. Such phosphoproteins are possible mediators of neural activity on gene expression.