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PubMed1996

Molecular cloning of the rat NADH:ubiquinone oxidoreductase subunit and its up-regulation in the facial muscle after denervation: detected by means of differential display.

The differential display method was applied to identify gene expression which is especially up-regulated in the rat denervated skeletal muscle. Total RNA from normal and denervated facial muscles was isolated, amplified by PCR using certain primers, and separated by electrophoresis on polyacrylamide gel. PCR products which were apparently higher in the operated than in the control side were cut out from the gel and subsequently sequenced. One of the cDNA fragments obtained in the present study showed about 80% identity in nucleotide sequence and about 84% identity in amino acid sequence to one of the NADH: ubiquinone oxidoreductase (complex I) subunits from the bovine heart mitochondria (813). Complex I, the first and largest enzyme of the mitochondrial respiratory chain, transfers electrons from NADH to ubiquinone-10 (UQ-10). B13 is a 13 kDa subunit; deficiency of which causes incomplete or incorrect assembly of a functional complex I, resulting in one of the types of human mitochondrial myopathy. Northern blot analysis and in situ hybridization histochemistry confirmed that rat B13 mRNA expression was up-regulated after denervation and was distributed throughout whole muscle cell body. These findings suggest that up-regulation of the rat B13 mRNA expression after denervation plays an important role in the effective electron transfer from NADH to UQ-10 in skeletal muscle cells, resulting in restriction of lipid peroxidation and biological tissue damage.

Amino Acid Sequence↗
PubMed2000

Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles.

The mitotic activity in muscles of growing rats and the effect of denervation were studied by means of continuous infusion of 5-bromo-2-deoxyuridine (BRDU). Denervated muscles after 10 weeks contained 20 to 60% fewer muscle nuclei than normal; BRDU labeled about 25% of the nuclei of normal soleus and extensor digitorum longus (EDL) and of denervated EDL muscles but only 5% in the denervated soleus muscle. Labeled nuclei persisted in denervated but not in normal muscles. After the main growth period, the turnover of myonuclei was at most 1 to 2% per week. The behavior of connective tissue nuclei was similar to that in muscle fibers. Infusion of BRDU had no effect on contractile properties. It is suggested that the exceptionally rapid atrophy of the denervated rat soleus associated with loss of satellite cells was due to loss of myonuclei and differentiation and fusion of satellite cells. The cause may possibly be that the phase of postdenervation fibrillation is shorter than in other muscles.

Animals↗
PubMed1982

The potentiating action of acetylcholine on the chemosensitivity of denervated skeletal muscle.

The acetylcholine sensitivity of soleus and extensor digitorum longus muscles of young rats was studied in vitro by measuring the isometric tension and membrane depolarisation developed in response to bath application of acetylcholine. Contractures of denervated muscles to acetylcholine gradually increased when the same concentration of acetylcholine was applied at 10 min intervals up to 60 min incubation whilst contractures evoked by potassium sulphate did not change significantly. The twitch tension of the muscles elicited in response to direct electrical stimulation did not increase and direct electrical stimulation failed to increase the acetylcholine sensitivity. Increased responses in vitro to acetylcholine were observed only in muscles denervated between 2 and 10 days previously with an optimum increase in sensitivity at 4-6 days. The increases in sensitivity were due to an action of acetylcholine since muscles left untreated exhibited no increases. Responses to carbamylcholine chloride, which is not hydrolysed by acetylcholinesterase, also increased. Membrane depolarisations in response to acetylcholine increased in a similar manner to the contracture responses in both soleus and extensor digitorum longus muscles, whilst the resting membrane potentials did not change. Denervated muscles which had been treated with acetylcholine bound more 125I-labelled alpha-bungarotoxin than muscles which had not been treated with acetylcholine. Denervated soleus muscles which had been previously blocked with unlabelled alpha-bungarotoxin responded to applications of acetylcholine with a gradually increasing depolarisation response and subsequently bound more [125I]alpha-bungarotoxin than muscles left without acetylcholine treatment. The possibility is discussed that acetylcholine acts upon the surface membranes of denervated muscles to increase the number of active acetylcholine receptors.

Acetylcholine↗
PubMed1983

Responses to electrical stimulation of denervated human muscle fibres recorded with single fibre EMG.

Denervated muscle fibres were stimulated electrically with needle electrodes introduced close to a recording single fibre electrode. The denervated muscle fibre could be driven with rates up to 100 Hz. The jitter was large at threshold but low at suprathreshold stimulus strength. There was evidence of discrete low threshold sites along the denervated muscle fibre, seen as stepwise latency change on smoothly changing stimulus strength, hepatic activation from other fibres and also as extra-discharges originating from such sites.

Action Potentials↗
PubMed1999

Tau expression in denervated rat muscles.

We studied whether denervation affects the expression of tau, in particular phosphorylated tau, and how it is degraded in rat soleus muscles. Immunoblot analysis showed a high molecular weight, approximately 110 kDa (big tau), in normal muscle. Tau levels increased significantly in denervated muscles treated with chloroquine (a lysosomotrophic agent) and in untreated ones, as compared to levels of similarly treated contralateral, innervated muscles. Most of the tau in the innervated and denervated muscles was phosphorylated. Immunohistochemically, tau and beta-tubulin colocated in the sarcoplasm of innervated, saline-treated (intact) muscle, but the staining intensities were very weak. Both proteins, however, were expressed extensively in these areas in the denervated muscles from saline-treated rats. In the denervated muscle of chloroquine-treated rats there were numerous autophagic vacuoles in the sarcoplasm, and phosphorylated-tau accumulation was marked within these vacuoles, indicative that tau first was taken into autophagic, vacuoles by nonselective autophagy then degraded via the lysosomal as well as the nonlysosomal calpain system. Our findings suggest that phosphorylated big tau accumulates with beta-tubulin in denervated muscular atrophy, possibly in order to maintain or preserve the integrity of the muscle fiber during progressive atrophy or regeneration.

Animals↗
PubMed1985

Change in fiber type in partially-denervated soleus muscle of the rat.

In 30% or less partially denervated muscle, the reinnervation of denervated muscle fiber may give rise to a change in motor unit size or number of muscle fibers innervated by a single motor neuron. This study was designed to evaluate changes in fiber type and contractibility of partially denervated rat soleus muscle. Partial denervation (by 30% or less) of the soleus nerve does not cause a decrease in the number of muscle fibers. A histochemical study was performed on frozen sections of the muscle. The total number of muscle fibers, atrophied fibers and type II fibers were counted. In the muscle 4 weeks after partial denervation, the number of type II fibers was fewer with a decrease of about 40% which was not significant. The twitch time to peak and half-relaxation time were not changed. The number of type II fibers was significantly decreased (p less than 0.01) after 8 weeks. There was a prolongation of contraction time. The decrease of type II fibers was extensive involving not only the denervated area but also the rest of the muscle area. The transformation of fiber type observed in partially denervated muscle may be attributed to a possible diminution of neurotrophic substances in intact motor neurons.

Animals↗
PubMed1995

Up-regulation of ferritin heavy chain mRNA expression in the rat skeletal muscle after denervation: detected by means of differential display.

The differential display method was applied to identify gene expression, which is especially up-regulated in denervated skeletal muscle. Total RNA from normal and denervated rat facial muscles (muscles zygomaticus, levator nasolabialis and caninus) was isolated, amplified by polymerase chain reaction (PCR) using certain primers and separated by electrophoresis on a polyacrylamide gel. PCR products, the amounts of which were significantly higher in the operated side than in the control side, were cut out from the gel and sequenced. One of the cDNA fragments obtained in the present study showed 100% identity in nucleotide sequence to the rat ferritin heavy chain (FHC) mRNA. Northern blot analysis and in situ hybridization histochemistry confirmed that FHC mRNA expression was up-regulated after denervation and was distributed throughout whole muscle cell bodies. The biological damage attributed to superoxide and hydrogen peroxide is dependent on the presence of intracellular free iron. Intracellularly, most of the iron that is not metabolized is sequestered in ferritin as a crystalline core of ferric irons (Fe3+). These findings suggest that alterations in the ferritin subunit composition after denervation play an important role in iron metabolism in skeletal muscle cells, resulting in restriction of the biological tissue damage caused by reactive oxygen species.

Animals↗
PubMed1991

Experimental study on the influence of tension, immobilisation and denervation on muscle transplantation.

To observe the effects of tension and immobilisation on denervated muscle, rats' denervated soleus muscles were transplanted orthotopically at different tensions: high, neutral, moderate and low tension with immobilisation. Results were evaluated by measuring muscle mass, twitch and tetanic tension, and histologically by HE staining. The results showed better preservation of muscle weight in the high tension group. Atrophy and loss of function were rapid during the first 4 weeks and were greatest in the low tension group. Rate of atrophy was slowed and relatively stabilised at the eighth postoperative week. We conclude that increased tension with immobilisation produced slower initial atrophy and loss of function in denervated muscle flaps. Although slower, the atrophy continued during the prolonged immobilisation and there was no significant difference compared with neutral tension at a later period. Moreover, immobilisation produced atrophy and affected function regardless of the amount of tension. Increased tension decelerates and decreased tension accelerates denervation atrophy of muscle. Thus denervation, immobilisation and reduced tension are all detrimental in muscle transplantation.

Animals↗
PubMed1998

Enlargement of motoneuron peripheral field following partial denervation with or without dorsal rhizotomy.

In partially denervated skeletal muscle, spared motor fibres sprout, enlarging motor unit size. Neuritogenesis and sprouting are known to depend on the synaptic input to the neurons. This suggests that spared motoneuron reaction to partial muscle denervation might be controlled by primary sensory neurons which directly or indirectly project to motoneurons. In two groups of rats, different surgical procedures were carried out: partial denervation of the extensor digitorum longus muscle without or with homolateral dorsal rhizotomy. Spared motoneuron peripheral field was evaluated by nerve-evoked tension measures. Following partial muscle denervation, spared motoneurons enlarged their projection peripheral field five to six times, innervating most of the denervated portion of the muscle. When dorsal rhizotomy was carried out together with partial denervation, the enlargement of the motoneuron's peripheral field occurred later; however, the peripheral field size was the same or greater than that found in partially denervated muscles without dorsal rhizotomy in the long term. Excitatory postsynaptic potential recordings at neuromuscular junctions consistently showed that innervation of denervated muscle cells by spared motoneurons was impaired when the dorsal roots were cut. Finally, in both groups of operated rats an increase in motor unit number occurred early after surgery, anticipating a process normally occurring in the same age range. These findings are consistent with the idea that sensory input trans-synaptically controls motoneuron peripheral field size.

Animals↗
PubMed1992

The importance of frequency and amount of electrical stimulation for contractile properties of denervated rat muscles.

Soleus (SOL) and extensor digitorum longus (EDL) muscles were denervated and directly stimulated for 23-69 days through implanted electrodes employing three different patterns. The stimulation was delivered in impulse trains where the pulse frequency differed (20, 75, and 150 Hz), while the train duration (0.3 s) and train repetition rate (1 min-1) were identical. Consequently, the number of pulses varied such that higher frequency was combined with a higher amount of stimulation. In both SOL and EDL the high-frequency pattern resulted in shorter twitch time-to-peak, greater post-tetanic potentiation, and greater tetanic force than the low frequency. Isotonic shortening velocity was increased to the same extent by all the patterns in SOL whereas in EDL fast intrinsic shortening velocity was maintained by the low-frequency pattern while it was decreased by the high-frequency pattern. We attribute this unexpected effect on the EDL to the larger number of pulses in the high-frequency pattern. By combining the present findings with previous data on directly stimulated rat muscles we conclude: in SOL the twitch duration is influenced by both the frequency and the amount of impulse activity, higher frequencies and smaller amounts leading to faster twitches. The EDL twitch duration is similarly dependent on the amount of activity, but the role of frequency is more unclear. In both SOL and EDL the isotonic shortening velocity is reduced by increasing amounts of activity and there is no evidence that impulse frequency plays a role. In EDL force output is strongly influenced by the impulse frequency, low frequencies resulting in low force outputs irrespective of the amount of activity.

Animals↗
PubMed1997

Developmental regulation of interstitial cell density in bullfrog skeletal muscle.

Denervation of skeletal muscle results in striking connective tissue remodelling in junctional areas of muscle. Since extracellular matrix molecules mediate axonal growth and synaptic differentiation, it is likely that the interstitial cells and matrix molecules that accumulate near synaptic sites after denervation influence the regrowth and regeneration of synaptic connections. The experiments presented here addressed the question of whether the junctional connective tissue in developing bullfrog skeletal muscle was also specialized in its cellular and molecular composition. Denervation responses of muscle, such as extrajunctional sensitivity to acetylcholine, often reproduce the characteristics of developing muscle during synaptogenesis. In developing muscle, the distribution of interstitial cells was nonuniform during the period of muscle fibre birth and synaptogenesis. Interstitial cells were concentrated near synaptic sites as in denervated adult muscle. Unlike denervated adult muscle, there were no junctional accumulations of fibronectin or tenascin, matrix molecules produced by interstitial cells, in developing muscles. These results demonstrate that the junctional connective tissue in developing muscle is identified by a high density of interstitial cells that may play a role in the identification and formation of synaptic sites. Further, the junctional matrix environment of developing muscle is distinct from the matrix remodelling that occurs in response to denervation, suggesting that the matrix production by interstitial cells during development is regulated differently from that after denervation of the neuromuscular junction.

Aging↗
PubMed2004

Effects of denervation on cell cycle control in laryngeal muscle.

Denervation of skeletal muscle is thought to lead to an accelerated proliferation of myogenic stem cells known as satellite cells. The transition of these cells from a quiescent to a proliferative state is thought to require satellite cells to enter the cell cycle and replicate. Little is known about the expression of genes associated with cell cycle control, and so the objective of this study was to examine the effects of denervation and reinnervation of the posterior cricoarytenoid (PCA) muscle on key cell cycle genes. Female Sprague-Dawley rats were assigned to control, denervated, or reinnervated groups. Animals were killed at 7, 14, and 30 days after ligation of the recurrent laryngeal nerve. The PCA muscle was then analyzed for changes in the messenger RNA levels of key genes associated with cell cycle control, differentiation, and proliferation. Cyclin D1 is a key gene responsible for initiating progression of the cell cycle from G1 to S phase. Interestingly, neither denervation nor reinnervation affected the expression of this gene. In contrast, we found large increases in key cell cycle inhibitors (p21 and the growth arrest and DNA destruction 45 [GADD45] gene) in both the denervated and reinnervated groups. We interpret the increases in these cell cycle inhibitors to reflect (1) an inhibition of satellite cell proliferation and/or (2) a special form of apoptosis that results in the loss of myonuclei known to occur under atrophic conditions. To our knowledge, this is the first study to examine the effects of denervation on cell cycle regulation.

Analysis of Variance↗
PubMed1980

Membrane and biochemical alterations after denervation and during reinnervation of mouse skeletal muscle.

Denervation of the extensor digitorum longus (EDL) muscle of the mouse by either nerve crush or nerve section produced: a reduction of the resting membrane potential (Em), alterations in the properties of muscle fibre action potentials and the development of tetrodotoxin (TTX)-resistant action potentials. These changes in membrane electrical properties were accompanied by an increase in the endocytic activity of the muscle and an increase in the activities of the lysosomal enzymes cathepsin D and N-acetylglucosaminidase (NAGA). Reinnervation of muscle was indicated at 9 days after nerve crush by the presence of miniature end-plate potentials. The recovery of membrane electrical properties, beginning with the onset of reinnervation, were not temporally related. The Em increased in two stages: an early rapid repolarization and a later slower repolarization. The muscle fibers were sensitive to the blocking action of TTX by 12 days after nerve crush, whereas the rate of rise (dV/dt) of the action potential did not approach values of innervated muscles until 21 days. Reinnervation resulted in a decrease in endocytosis and a decrease in the activities of cathepsin D and NAGA toward innervated values by 21 days after nerve crush. The results suggest that membrane alterations after denervation and during reinnervation may occur by endo- and exocytosis of membrane constituents and that the lysosomal system may play a role in the breakdown and/or recycling of these structures.

Acetylglucosaminidase↗
PubMed1988

Insulin resistance in denervated skeletal muscle. Inability of insulin to stimulate dephosphorylation of glycogen synthase in denervated rat epitrochlearis muscles.

We have investigated the effects of insulin and motor denervation on the phosphorylation of glycogen synthase in skeletal muscle. Rat epitrochlearis muscles were denervated in vivo 3 days before the contralateral and denervated muscles were incubated in vitro with 32Pi to label sites in glycogen synthase. The 32P-labeled synthase was rapidly immunoprecipitated from extracts under conditions which prevented changes in the phosphorylation state of the enzyme. When 32P-labeled synthase from contralateral muscles was cleaved with CNBr, essentially all of the 32P was recovered in two fragments, denoted CB-1 and CB-2. Incubating these muscles with insulin decreased the 32P content of each fragment by approximately 25%, indicating that the hormone stimulated dephosphorylation of at least two sites. Peptide mapping by reverse phase high performance liquid chromatography was performed to resolve phosphorylation sites more completely. The results suggest that the enzyme was phosphorylated in sites 1a, 1b, 2, 3(a+b+c), and 5. Insulin stimulated dephosphorylation of sites in peptides presumed to contain sites 1b, 2, and 3(a+b+c). Synthase from denervated muscles appeared to contain the same amount of phosphate as enzyme from contralateral muscles, and denervation did not detectably affect the distribution of 32P within the subunit. However, denervation abolished the effect of insulin on decreasing the 32P content of synthase. The results indicate that the insulin resistance induced by denervation involves a loss in the ability of insulin to stimulate dephosphorylation of glycogen synthase.

Animals↗