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Effects of membrane potential on just detectable movement in rat skeletal muscle: effects of denervation.

The potential, Vt, at which a brief test depolarization first elicited movement was determined using two-microelectrode point voltage clamp. We expected that inactivation of excitation-contraction coupling at conditioning potentials between -60 and 0 mV would shift Vt to more positive potentials, and that fibers would become inactivatable with less conditioning depolarization in EDL than soleus. The curve relating Vt to conditioning potential had a negative slope (which was insensitive to addition of 1 mM cobalt or replacement of calcium with 20 mM CaEGTA) between -60 and -35 mV and a steep positive slope with further depolarization Unexpectedly, fibers became inactivatable with less conditioning depolarization in soleus than in EDL when Vt was measured with 50 msec test pulses. However, the positive shift in Vt became less steep as test pulse duration lengthened in soleus fibers. When Vt obtained with test pulses approaching rheobase (10 msec in EDL and 500 msec in soleus) was compared, EDL fibers became inactive with less conditioning depolarization than soleus fibers. The increase in Vt became steeper with 1 mM cobalt or 20 mM CaEGTA and was shifted to more positive potentials by denervation in soleus fibers. We conclude that inactivation (i) does not strongly influence threshold contractions at conditioning potentials between -60 and -40 mV and (ii) influences Vt between -40 and 0 mV in a manner that depends on test pulse duration.

Animals↗

Similarity of acetylcholine receptors of denervated, innervated and embryonic chicken muscles. 1. Molecular species and their purification.

Acetylcholine receptors were purified to homogeneity from chicken embryonic, adult innervated and denervated muscles, by bio-specific chromatographies using immobilised alpha-neurotoxin and lentil lectin. A minimum specific activity for the pure receptor was estimated to be 6000 nmol alpha-toxin binding sites/g protein. For analysis, the receptors were radio-iodinated or tritiated to high specific radioactivity with succinimidyl-[2,3-3H]propionate. All of the iodinated protein present in the purified receptor preparation reacted with antibody against the pure acetylcholine receptor from Torpedo marmorata electric organ. In the case of all three muscle types used the same oligomeric forms were obtained. The principal form has a sedimentation coefficient of about 9 S, while a minor species (approximately 5S) was also appreciable in crude preparations of embryonic and denervated muscles. Immunization of rabbits with the homogenous receptor from chicken denervated muscle produced muscle weakness characteristic of experimental autoimmune myasthenia gravis. These antisera were equally reactive towards the receptor --125I-alpha-bungarotoxin complexes from chick innervated and denervated muscles. Likewise, the electrophoretic mobilities of the receptors (9-S form) from all three muscle types were identical, as were the isoelectric points of their complexes with 125I-alpha-bungarotoxin. Collectively, these findings and associated ones on subunit structure denote that the 9-S receptor molecules from junctional and extra-junctional area and embryonic stage of chicken muscle are indistinguishable by all criteria yet applied to them.

Animals↗

Extension and magnitude of denervation in skeletal muscle from ageing mice.

In this work we hypothesized that denervation in flexor digitorum brevis (FDB) muscle from ageing mice is more extensive than predicted by standard functional and structural assays used in the past. In addition, we asked whether denervation is a fully or partially developed process. Despite the reported alteration in skeletal muscle innervation, the quantification of the extension and magnitude of denervation in ageing rodents has remained elusive. To address these two questions we utilized a combination of electrophysiological and immunohistochemical assays directed to detecting the expression of tetrodotoxin (TTX)-resistant sodium channels (Na(v)1.5) in FDB muscles from young-adult and senescent mice. Sodium current density measured with the macropatch cell-attached technique did not show significant differences between FDB fibres from young and old mice. The TTX dose-response curve, using the whole cell voltage-clamp technique, showed three populations of fibres in senescent mice, one similar to fibres from young mice (TTX sensitive), another one similar to fibres from experimentally denervated muscle (TTX resistant), and a third group intermediate between these two. Partially and fully denervated fibres added up to approximately 50% of the total number of fibres tested, a number that concurs with the percentage of fibres positive for the Na(v)1.5 channel by specific immunostaining.

Aging↗

Differential expression of tenascin after denervation, damage or paralysis of mouse soleus muscle.

The expression of the extracellular matrix molecule tenascin was studied by immunocytochemistry and Western blotting in soleus muscles of adult mice after nerve damage (denervation), muscle injury (induced by enforced running or freezing) and functional block of synaptic transmission (botulinum toxin). Enhanced expression of tenascin in the extracellular spaces around focally damaged muscle fibres was found already 10 h after onset of running on a motor-driven treadmill which causes muscle injury in soleus muscle. Tenascin expression reached a peak at 2-3 days post-exercise, after which it declined gradually and became undetectable by two weeks after injury. Similarly, cryo-damage of soleus muscles in situ led to upregulation of tenascin. Chronic muscle denervation after sciatic nerve transection caused a persistent (studied up to 31 days) expression of tenascin at denervated endplates and in intramuscular nerve branches but not in other tissue compartments. Local application of botulinum toxin Type A, which results in muscle inactivity but not in tissue degeneration, however, did not induce tenascin expression 12 h to 12 days post-injection. Expression of tenascin after denervation and muscle damage, but its absence after paralysis, were verified by SDS-PAGE and Western blot analysis. Independent of the type of injury (muscle, nerve or both) the known major isoforms of mouse tenascin, as judged by M(r) comparison, were re-expressed, with no preponderance of individual M(r) forms. These results show that tenascin expression in adult muscles is induced by both axon and muscle fibre damage but not by muscle inactivity. In contrast, NCAM, in accordance with previous observations, showed enhanced expression both as a result of inactivity and in association with tissue repair.

Animals↗

Cyclic adenosine 3',5'-monophosphate regulates GLUT4 and GLUT1 glucose transporter expression and stimulates transcriptional activity of the GLUT1 promoter in muscle cells.

We have previously reported that innervation-dependent basal contractile activity regulates in an inverse manner the expression of GLUT1 and GLUT4 glucose transporters in skeletal muscle. Based on the facts that muscle innervation decreases and muscle denervation increases cAMP levels, we investigated whether cAMP might mediate the effects of innervation/denervation on glucose transporter expression. Treatment of L6E9 myotubes with 8-bromo-cAMP, forskolin, or monobutyryl-8-bromo-cAMP led to a marked decrease in GLUT4 protein levels; 8-bromo-cAMP also diminished GLUT4 messenger RNA (mRNA), suggesting pretranslational repression. In contrast, L6E9 myoblasts and myotubes responded to 8-bromo-cAMP or forskolin by increasing the cell content of GLUT1 protein. Induction of GLUT1 protein was a consequence of the activation of different mechanisms in myoblast and myotube cells; whereas 8-bromo-cAMP treatment caused a substantial increase in GLUT1 mRNA in myoblasts, no change in GLUT1 mRNA was detected in myotubes. The increase in GLUT1 mRNA in L6E9 myoblasts induced by 8-bromo-cAMP was the result of transcriptional activation, as concluded from transfection analysis of 2.1 kilobases of the rat GLUT1 gene promoter fused to the bacterial chloramphenicol acetyltransferase gene. Furthermore, the stimulatory effect of 8-bromo-cAMP on the transcriptional activity of the GLUT1 promoter required a 33-bp sequence lying 5' upstream of the transcription start site. In all, cAMP inversely regulates GLUT4 and GLUT1 glucose transporter expression in muscle cells. Furthermore, our results suggest that down-regulation of GLUT4 expression and up-regulation of GLUT1 expression in muscle associated with denervation are partly attributable to cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗

Myotrophic effects on denervated fast-twitch muscles of mice: correlation of physiologic, biochemical, and morphologic findings.

Certain morphological, biochemical, and physiological parameters were assessed in fast-twitch muscles of 6-week-old mice with unilateral hindlimb denervation for 4 weeks. Some of the mice received daily injections (i.p.) of nerve extract throughout the period of denervation. Values from treated and untreated denervated muscles were compared with each other and with those from contralateral, innervated controls. The cross-sectional areas of denervated types IIA and IIATy muscle fibers were 45% and 28% greater, respectively, in muscles of treated than of untreated mice, which resulted in greater maximal tetanic tension. Injection with nerve extract did not influence the postdenervation reduction of phosphorylation of myosin light chain 2-fast nor the loss of posttetanic twitch potentiation, two parameters thought to be related. Denervation produced a significant decrease in relative content of cytosolic parvalbumin; however, this change was completely prevented by administration of nerve extract. This latter finding correlated with the amelioration of greater than 50% of the postdenervation prolongation of half-relaxation time of the twitch in treated than in untreated muscles. More than half of the prolongation of time-to-peak of the twitch was also prevented in denervated muscles of treated than of untreated mice.

Animals↗

Denervation syndromes of the shoulder girdle: MR imaging with electrophysiologic correlation.

OBJECTIVE: To investigate the use of MR imaging in the characterization of denervated muscle of the shoulder correlated with electrophysiologic studies. DESIGN AND PATIENTS: We studied with MR imaging five patients who presented with shoulder weakness and pain and who underwent electrophysiologic studies. On MR imaging the distribution of muscle edema and fatty infiltration was recorded, as was the presence of masses impinging on a regional nerve. RESULTS: Acute/subacute denervation was best seen on T2-weighted fast spin-echo images with fat saturation, showing increased SI related to neurogenic edema. Chronic denervation was best seen on T1-weighted spin-echo images, demonstrating loss of muscle bulk and diffuse areas of increased signal intensity within the muscle. Three patients showed MR imaging and electrophysiologic findings of Parsonage Turner syndrome. One patient demonstrated an arteriovenous malformation within the spinoglenoid notch, impinging on the suprascapular nerve with associated atrophy of the infraspinatus muscle. The fifth patient demonstrated fatty atrophy of the teres minor muscle caused by compression by a cyst of the axillary nerve and electrophysiologic findings of an incomplete axillary nerve block. CONCLUSION: MR imaging is useful in detecting and characterizing denervation atrophy and neurogenic edema in shoulder muscles. MR imaging can provide additional information to electrophysiologic studies by estimating the age (acute/chronic) and identifying morphologic causes for shoulder pain and atrophy.

Adult↗

Neural control of embryonic acetylcholine receptor and skeletal muscle.

The manner by which motor neurons exert control over the distribution and number of acetylcholine receptors, and muscle development was investigated in the superior oblique muscle of white Peking duck embryos. Clusters of receptors in the normally developing muscle first appeared on day 10 of incubation as determined with I125 alpha-bungarotoxin autoradiography. The initial appearance of receptor clusters coincided with the arrival of motor nerve fibers in the muscle. Clusters of receptors also appeared in normal fashion in muscles made aneural by destruction of motor neurons on day 7. But after day 14 these clusters had disappeared and no new clusters were seen thereafter in the aneural muscle. Receptor clusters persisted throughout development in muscle in which neuromuscular transmission was blocked with either curare or botulinum toxin and in muscles denervated on day 10.5, i.e., shortly after the initial nerve-muscle contact but prior to the onset of muscle activity. A progressive increase in the total number of receptors and in the total amount of protein occurred during the course of normal development. However, the specific activity of the receptor protein declined sharply following innervation on day 10. The total number of receptors and the specific activity of the receptor was affected depending on whether the motor neurons were destroyed before or after innervation and following chronic blockade of neuromuscular transmission. The half-life of the receptor protein was similar in normal, aneural, and paralyzed muscles (26, 25, 26 h, respectively). Measurements of total protein indicated that essentially no muscle growth occurred in the complete absence of innervation. Paralyzed muscles continued to develop but at a slower pace.

Animals↗

The expression of ion channel mRNAs in skeletal muscles from patients with myotonic muscular dystrophy.

We investigated gene expression patterns of ion channels including the apamin-sensitive small-conductance Ca(2+)-activated K(+) (SK3) channel, the adult isoform of the skeletal muscle Na(+) channel (SkM1), the fetal isoform of skeletal muscle Na(+) channel (H1), and the Cl(-) channel (ClC-1) by using the semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) for muscle samples from patients with adult onset myotonic dystrophy (DM), amyotrophic lateral sclerosis, and polymyositis. Patients with DM showed a significant increase in SK3 mRNA but not in mRNAs for other ion channels. The increased expression of SK3 gene in DM did not correlate with H1, the marker of muscle denervation, or the percentage of type 2C fiber, the marker of muscle regeneration.

Adult↗

Patterns of motoneuron dysfunction and recovery.

Electrophysiological studies have been carried out on five patients with neuropathies of different etiologies. In each patient serial estimates were made of the numbers of functioning motor units in various muscles. It was found that the intensity of the neuropathic process and the rate of recovery differed in a consistent way among the motoneuron pools investigated. The lesion was more severe in extensor digitorum brevis neurons than in thenar neurons, while the hypothenar ones were least affected. A stage of partial synaptic failure has been recognized in which a motoneuron appears to be no longer able to excite a muscle fiber, but still capable of maintaining certain trophic activities. By comparing the number of functioning motor units with the size of the maximum evoked muscle response it has been possible to detect the adoption of denervated muscle fibers by axonal sprouts from 'healthy' surviving neurons (collateral reinnervation). Lastly, in some muscles it appears that the adopted muscle fibers may subsequently be recaptured by the original motoneurons following recovery of the latter from the neurotoxic insult.

Adult↗

Oral administration of creatine monohydrate retards progression of motor neuron disease in the wobbler mouse.

BACKGROUND AND AIMS: Creatine has a neuroprotective effect in mutant superoxide dismutase (G93A) transgenic mice, an animal model of motor neuron disease (MND). Treatment with creatine monohydrate enhances muscle strength in patients with neuromuscular disorders. The purpose of our study was to determine whether administration of creatine monohydrate can attenuate progressive disease in wobbler mice. METHODS: After an initial diagnosis of disease at the age of 3-4 weeks, creatine monohydrate (5 or 50 mg/kg, po) or vehicle was given to wobbler mice daily for 4 weeks in a blinded fashion. We compared symptomatic and neuropathological assessments among the three groups. RESULTS: Creatine levels in biceps muscles were increased by approximately 20% following administration of higher-dose creatine monohydrate. In comparison with vehicle, treatment with higher doses of creatine monohydrate potentiated grip strength, attenuated forelimb contracture and increased the weight of biceps muscles. Mice treated with higher doses of creatine monohydrate showed retarded denervation muscle atrophy in the biceps muscles and reduced degeneration of the spinal motor neurons. Thus, oral administration of creatine monohydrate delayed the progression of disease in wobbler mice. CONCLUSION: Our results indicate that this molecule may have therapeutic potential in human motor neuropathy or MND.

Administration, Oral↗

Denervation-induced changes in myosin heavy chain expression in the rat diaphragm muscle.

Unilateral denervation (Dnv) of the rat diaphragm muscle (Diam) markedly alters expression of myosin heavy chain (MHC) isoforms. After 2 wk of Diam Dnv, MHC content per half-sarcomere decreases in fibers expressing MHC(2X) and MHC(2B). We hypothesized that changes in MHC protein expression parallel changes in MHC mRNA expression. Relative MHC isoform mRNA levels were determined by Northern analysis after 1, 3, 7, and 14 days of Dnv of the rat Diam. MHC protein expression was determined by SDS-PAGE. Changes in MHC isoform protein and mRNA expression were not concurrent. Expression of MHC(Slow) and MHC(2X) mRNA isoforms decreased dramatically by 3 days of Dnv, whereas that of MHC(2A) and MHC(2B) did not change. Expression of all MHC protein isoforms decreased by 3 days of Dnv. We observed a differential effect of rat Diam Dnv on MHC isoform protein and mRNA expression. The time course of the changes in MHC isoform mRNA and protein expression suggests a predominant effect of altered protein turnover rates on MHC protein expression instead of altered transcription after Dnv.

Animals↗

Effect of denervation on regenerating muscle plasma membrane integrity: freeze-fracture and dystrophin immunostaining analyses.

We investigated time sequentially, the densities of intramembranous assembly "orthogonal arrays" of regenerating rat extensor digitorum longus muscle after bupivacaine-induced muscle injury. There was no evidence of orthogonal arrays at the early stage, but the densities of orthogonal arrays increased with the maturation of the innervated regenerating myofibers. In contrast, the orthogonal arrays were scarcely observed at any time point examined in denervated regenerating muscles. Therefore, the neural factor may have an important effect on the appearance of orthogonal arrays. Moreover, we studied the immunostainability of Duchenne muscular dystrophy gene product "dystrophin" in the regenerating muscle at the same time points. Positive immunostaining was observed in both innervated and denervated regenerating myofibers even from the early stage of regeneration. On the basis of these data, the relationship between orthogonal array and dystrophin are discussed.

Animals↗

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.

Amino Acid Sequence↗

Pharmacologic denervation of frown muscles enhances baseline expression of happiness and decreases baseline expression of anger, sadness, and fear.

BACKGROUND: Individuals with intense frowning are commonly perceived as expressing negative emotions. Anger, fear, and sadness are associated with corrugator ("frown") muscle activity. OBJECTIVE: We sought to study how faces were perceived by others after denervation of frown muscles with localized botulinum toxin injections for treatment of facial frown lines. METHODS: Facial photographs were taken from volunteers before and after botulinum toxin injection. These photographs were shown to viewers who were naive to the procedure and asked to rate the expressed intensity of anger, sadness, fear, and happiness. As reference for this task we used a standard set of pictures of facial affect displaying different intensity levels for each emotion tested. RESULTS: Of 40 viewers, 39 were able to discriminate different intensity levels (0%, 25%, 50%, 75%, and 100%) of emotional affect in the control task. According to their ratings faces with denervated frown muscle activity expressed relatively less anger (-40%), fear (-49%), sadness (-10%), and more happiness (+71%). CONCLUSION: Frown muscle activity is essential for both negative and positive emotional expressions. Temporary denervation using botulinum toxin enhances the facial expression of positive emotion resulting in a shift rather than a loss of facial affect.

Anti-Dyskinesia Agents↗

Denervation of newborn rat muscle does not block the appearance of adult fast myosin heavy chain.

Several observations, both in vivo and in vitro, have indicated that the development and maturation of mammalian skeletal muscle fibres is influenced by nerve-muscle interactions. Morphological maturation of newly regenerated adult mouse muscle fibers in an organotypic nerve-muscle culture system depends on the presence of spinal cord neurones. Sciatic nerve transection in newborn rats has been shown to modify the development of the histochemical and contractile properties of the denervated muscles. In addition, neural influences are important for the appearance of certain of the myosin small subunits. It has been proposed that the nerve also controls the changes in myosin heavy chain isozymes appearing during development. One such transition occurs in rat muscle where the neonatal form of myosin heavy chain is replaced by the adult form during the second post-natal week. Here we demonstrate that innervation of the rat gastrocnemius muscle (a fast-contracting muscle in the adult) is not required for the appearance of the adult form of myosin heavy chain.

Animals↗

Innervation is necessary for the development of fast contraction kinetics of singing muscles in a katydid.

The twitch duration of mesothoracic wing muscles of the male katydid Neoconocephalus robustus (Insecta; Orthoptera; Tettigoniidae) decreases rapidly within the first 5 days of adulthood, to about half of its value in newly molted adults. To determine if this change is dependent upon neural input, male mesothoracic first tergocoxal muscles were unilaterally denervated on the second day of adulthood. The contraction kinetics of the denervated and contralateral innervated muscles were tested four days later. The development of rapid contraction kinetics was slowed or stopped in the denervated muscles, while the contralateral innervated muscles did become faster. Mesothoracic wing muscles of females do not develop faster contraction kinetics. When the female mesothoracic first tergocoxal muscle is denervated, there is no difference in twitch duration after 4 days between the innervated and contralateral denervated muscles. Therefore, denervation in newly molted adult male katydids interrupts a developmental program for the acquisition of adult contraction kinetics.

Animals↗

Effects of simultaneous denervation and tenotomy on the ultrastructure of a rat slow twitch muscle.

Simultaneous denervation and tenotomy causes atrophy of rat slow twitch soleus muscle. Apparently, the alterations are similar to those seen in denervated or tenotomized slow muscle. However, the extent of disorganization of myofibrils appears lower than that expected after a moderate period of time in only denervated or tenotomized muscle. This study indicates that after an initial proliferation of lysosomes, alterations of sarcotubular system and mitochondria proceed. Nevertheless, 45 days after denervation and tenotomy many mitochondria have a normal aspect.

Animals↗