ELECTROMYOGRAPHY—Uses and Limitations

A normal muscle at rest emits no detectable electric current, but in action, in diseases of the muscle and in denervation it emits electric impulses characteristic of these states. The impulses can be amplified and studied through the sonic and oscilloscopic patterns they create. These patterns are sufficiently different so that simple atrophy of disuse can be distinguished from the denervation that may be associated with it. Since denervation can be localized to individual muscles and thence to the nerves controlling them, electromyography serves much the same function as myelography, with comparable accuracy and with greater safety and simplicity. It aids in the diagnosis of several muscular diseases of children and adults.Because electromyographic changes due to injury do not appear until 18 to 21 days later, a study made soon after injury can either disclose or rule out preexisting lesions. Then a later study indicating denervation is objective evidence that any disability is due to the injury in question.     

Effects of Electrical Stimulation on Molecular Forms of Butyrylcholinesterase in Denervated Fast and Slow Latissimus Dorsi Muscles of Newly Hatched Chicken

The effects of denervation and direct electrical stimulation upon the activity and the molecular form distribution of butyrylcholinesterase (BuChE) were studied in fast-twitch posterior latissimus dorsi (PLD) and in slow-tonic anterior latissimus dorsi (ALD) muscles of newly hatched chicken. In PLD muscle, denervation performed at day 2 substantially reduced the rate of rapid decrease of BuChE specific activity which takes place during normal development, whereas in the case of ALD muscle little change was observed. Moreover, the asymmetric forms which were dramatically reduced in denervated PLD muscle were virtually absent in denervated ALD muscle at day 14. Denervated PLD and ALD muscles were stimulated from day 4 to day 14 of age. Two patterns of stimulation were applied, either 5-Hz frequency (slow rhythm) or 40-Hz frequency (fast rhythm). Both patterns of stimulation provided the same number of impulses per day (about 61,000). In PLD muscle, electrical stimulation almost totally prevented the postdenervation loss in asymmetric forms and led to a decrease in BuChE specific activity. In ALD muscle, electrical stimulation partially prevented the asymmetric form loss which occurs after denervation. This study emphasizes the role of evoked muscle activity in the regulation of BuChE asymmetric forms in the fast PLD muscle and the differential response of denervated slow and fast muscles to electrical stimulation.     

Influence of Electroporation Conditions on Transfection of Muscle Fibers in Vivo

This study is a survey of in vivo experiments on transfection of laboratory mouse muscle fibers by electoporation using an original device generating electric impulses. Transfection efficiency proved to depend on DNA dose and the number of electric impulses. It can be increased significantly by electroporation at varying pulse burst polarity. At both direct electrode application to muscles and electroporation through the skin, the muscle fiber transfection was more efficient under electroporation conditions much milder than those usually reported. The use of electroporation method for gene therapy of Duchenne muscular dystrophy is discussed.     

Dependence of the efficacy of transfecting muscle fibers in vivo on electroporation conditions

This study is a survey of in vivo experiments on transfection of laboratory mouse muscle fibers by electroporation using an original device generating electric impulses. Transfection efficiency proved to depend on DNA dose and the number of electric impulses. It can be increased significantly by electroporation at varying pulse burst polarity. At both direct electrode application to muscles and electroporation through the skin, the muscle fiber transfection was more efficient under electroporation conditions much milder than those usually reported. The use of electroporation method for gene therapy of Duchenne muscular dystrophy is discussed.     

Impulse Activity Evokes Precocious Sprouting of Nociceptive Nerves into Denervated Skin

We have studied the sprouting of intact high-threshold mechanosensory nerves into adjacent denervated trunk skin in adult rats behaviorally, histologically, and electrophysiologically. In the anesthetized animal, stimulation of high-threshold endings in back skin by localized pinching elicits a bilateral reflex excitation of the underlying skeletal muscle, the cutaneous trunci muscle (CTM), visible as a twitch-like puckering of the skin. The reflex was also evoked by electrical excitation of Aδ and of C fibers in the dorsal cutaneous nerves (DCNs), with characteristic latencies of 7–20 msec and 40–60 msec, respectively; excitation of low-threshold (Aα) fibers was ineffective. After cutting selected DCNs, the deprived skin became insensible, but pinch responsiveness gradually recovered over the following 2 weeks. Regeneration of cut axons was not responsible for this recovery; when neighboring intact DCNs were cut, however, all responses were abolished in the recovered skin that had been initially denervated. By 3–5 days after denervation, axons in the dermis were all histologically absent or degenerating; when pinch sensitivity was restored to such skin, silver-stainable axons reappeared in the formerly empty Schwann tubes. During the work we noticed that the periodic examination by pinching, used to follow the time course of recovery of function in individual animals, led to an earlier development of this recovery than in animals that were examined only once at a specified time after denervation, This apparent acceleration in the redevelopment of pinch sensitivity was correlated with the appearance of axons in the recovered skin, and was shown to be due to the impulse activity evoked in the remaining nerves by the periodic pinching; it did not occur when the nerves were blocked by tetrodotoxin (TTX), and it was mimicked by a brief (10-min) period of electrical excitation of the Aδ fibers in a remaining nerve carried out at the time when the denervation of skin was done. The time course of the phenomenon suggested that the principal effect of the impulses was to shorten the latency to the onset of sprouting in the activated Aδ axons; that is, they induced precocious sprouting. The impulses needed to be conducted centrally for the effect to occur, and precocious sprouting failed to occur if the impulses were allowed to proceed only distally toward the skin.     

Increased histaminase activity in the atrophic muscle of denervated frog.

Sciatic denervation for 1 month in the frog Rana hexadactyla resulted in progressive atrophy of the gastrocnemius muscle without any change in the total DNA content of the whole muscle. Histamine content of the muscle decreased; glutamic and acid content increased and histidine level remained unaltered on denervation. Histaminase activity localized in the muscle decreased; glutamic acid content increased and histidine level remained unaltered on denervation. Histaminase activity localized in the muscle mitochondria increased on denervation. The histidine-degrading enzymes, histidine ammonia lyase, urocanate hydratase and imidazol-5yl lactate dehydrogenase, are localized in the sarcoplasm of the muscle and their activities are not altered on denervation. The histidine decarboxylase activity localized in the mitochondria is not altered on denervation. The reduction in the histamine content of the atrophied muscle may be due to increased mitochondrial histaminase activity but not due to increased decarboxylation of histidine. The loss of 'trophic influence' due to denervation may be manifested in the impairment of mitochondrial histaminase activity.     

Reciprocal neural regulation of extrajunctional acetylcholinesterase and collagen Q in rat muscles--the role of calcineurin signaling

There are two main differences regarding acetylcholinesterase (AChE) expression in the extrajunctional regions of fast and slow rat muscles: (1) the activity of AChE catalytic subunits (G1 form) is much higher in fast than in slow muscles, and (2) the activity of the asymmetric forms of AChE (A(8) and A(12)) is quite high extrajunctionally in slow muscles but virtually absent in fast muscles. The latter is due to the absence of the expression of AChE-associated collagen Q (ColQ) in the extrajunctional regions of fast muscle fibers, in contrast to its ample expression in slow muscles. We showed that both differences are caused by different neural activation patterns of fast vs. slow muscle fibers, which determine the respective levels of mRNA of both proteins. Whereas the changes in AChE mRNA levels in fast and slow muscles, as well as the levels of ColQ mRNA levels in slow muscles, observed in response to exposing either slow or fast muscles to different muscle activation patterns, are completely reversible, the extrajunctional suppression of ColQ expression in fast muscle fibers seems to be irreversible. Calcineurin signaling pathway in muscles is activated by high-average sarcoplasmic calcium concentration resulting from tonic low-frequency muscle fiber activation pattern, typical for slow muscle fibers, but is inactive in fast muscle fibers, which are activated by infrequent high-frequency bursts of neural impulses. Application to rats of two inhibitors of calcineurin (tacrolimus-FK506 and cyclosporin A) demonstrated that the mRNA levels of both the AChE catalytic subunit and ColQ in the extrajunctional regions of the soleus muscle are regulated by the calcineurin signaling pathway, but in a reciprocal way. Under the conditions of low calcineurin activity, AChE expression is enhanced and that of ColQ is suppressed, and vice versa. Our results also indicated that different, calcineurin-independent regulatory pathways are responsible for the reduction of AChE expression during muscle denervation, and for maintaining high ColQ expression in the neuromuscular junctions of fast muscle fibers.     

Proteomic analysis of rat laryngeal muscle following denervation

Laryngeal muscle atrophy induced by nerve injury is a major factor contributing to the disabling symptoms associated with laryngeal paralysis. Alterations of global proteins in rat laryngeal muscle following denervation were, therefore, studied using proteomic techniques. Twenty-eight adult Sprague-Dawley rats were divided into normal control and denervated groups. The thyroarytenoid (TA) muscle was excised 60 days after right recurrent laryngeal nerve was resected. Protein separation and identification were preformed using 2-DE and MALDI-MS with database search. Forty-four proteins were found to have significant alteration in expression level after denervation. The majority of these proteins (57%), most of them associated with energy metabolism, cellular proliferation and differentiation, signal transduction and stress reaction, were decreased levels of expression in denervated TA muscle. The remaining 43% of the proteins, most of them involved with protein degradation, immunoreactivity, injury repair, contraction, and microtubular formation, were found to have increased levels of expression. The protein modification sites by phosphorylation were detected in 22% of the identified proteins that presented multiple-spot patterns on 2-D gel. Significant changes in protein expression in denervated laryngeal muscle may provide potential therapeutic strategies for the treatment of laryngeal paralysis.     

Ciliary neurotrophic factor (CNTF) affects the excitable and contractile properties of innervated skeletal muscles

The well-established trophic role of CNTF upon neurons led to performing clinical trials in patients of neurodegenerative diseases. However, trials were suspended due to side effects such as severe weight loss, hyperalgesia, coughing, muscle cramps and pain. So far it is not known how CNTF triggers the problems related to skeletal muscle cramps and pain. CNTF has also been described as a myotrophic factor for denervated skeletal muscles, but the possibility that it affects innervated muscles has also been considered. Since a myotrophic factor could be a valuable tool for treatment of several muscle diseases, we studied the effects of low doses of CNTF delivered systemically by an osmotic pump, over the electrical and mechanical properties of innervated and denervated fast and slow muscles. CNTF induced spontaneous electrical discharges and slowed twitches in innervated muscles, but did not prevent the changes induced by denervation. We postulate that the spontaneous discharges induced by CNTF in innervated muscles may be the cause of the cramps, coughing, and muscle ache reported by patients. At low doses, CNTF does not exert its myotrophic role over denervated muscles but clearly affects the excitable and contractile properties of innervated muscles.     

Evidence for active chloride accumulation in normal and denervated rat lumbrical muscle

Intracellular Cl- activity (aiCl) was measured with Cl(-)-sensitive microelectrodes in normal and denervated rat lumbrical muscle. In normal muscle bathed in normal Krebs solution, aiCl lay close to that predicted by the Nernst equation. The addition of 9-anthracene carboxylic acid, which blocks Cl- conductance, caused aiCl to increase far above that predicted by a passive distribution. Furosemide (10 microM) reversibly blocked this accumulation. After muscle denervation, aiCl progressively increased for 1-2 wk. The rise occurred in two stages. The initial stage (1-3 d after denervation) reflected passive Cl- accumulation owing to membrane depolarization. At later times, aiCl continued to increase, with no further change in membrane potential, which suggests an active uptake mechanism. This rise approximately coincided with the natural reduction in membrane conductance to Cl- that occurs several days after denervation. Na+ replacement, K+ replacement, and furosemide each reversibly blocked the active Cl- accumulation in denervated muscle. Quantitative estimates suggested that there was little difference between Cl- flux rates in normal and denervated muscles. The results can be explained by assuming that, in normal muscle, an active accumulation mechanism operates, but that Cl- lies close to equilibrium owing to the high membrane conductance to Cl-. The rise in aiCl after denervation can be accounted for by the membrane depolarization, the reduction in membrane Cl- conductance, and the nearly unaltered action of an inwardly directed Cl- "pump."     

Modification of motoneuron size after partial denervation in neonatal rats

Our previous studies have shown that partial denervation of extensor digitorum longus muscle (EDL) in the rat at 3 days of age causes an increase in the activity of the intact motoneurons. The originally phasic pattern of activity of EDL became tonic after partial denervation. These modifications of motoneuron activity were associated with the change in the phenotype of the muscle from fast to slow contracting and with a conversion of the muscle fibres from a fast to a slow type. The present study investigates whether the size of the cell body of the active EDL motoneurons change in parallel with the altered muscular activity. The study involved partial denervation of rat EDL muscle by section of the L4 spinal nerve at 3 days of age. Then the remaining motoneurons from L5 spinal nerve supplying the EDL muscle were retrogradly labelled with horseradish peroxidase two months later. The results show a reduction in motoneuron size in parallel with an increase in activity of the motoneurons after partial denervation of EDL muscle.     

Proteasome-dependent Activation of Mammalian Target of Rapamycin Complex 1 (mTORC1) Is Essential for Autophagy Suppression and Muscle Remodeling Following Denervation

Drastic protein degradation occurs during muscle atrophy induced by denervation, fasting, immobility, and various systemic diseases. Although the ubiquitin-proteasome system is highly up-regulated in denervated muscles, the involvement of autophagy and protein synthesis has been controversial. Here, we report that autophagy is rather suppressed in denervated muscles even under autophagy-inducible starvation conditions. This is due to a constitutive activation of mammalian target of rapamycin complex 1 (mTORC1). We further reveal that denervation-induced mTORC1 activation is dependent on the proteasome, which is likely mediated by amino acids generated from proteasomal degradation. Protein synthesis and ribosome biogenesis are paradoxically increased in denervated muscles in an mTORC1-dependent manner, and mTORC1 activation plays an anabolic role against denervation-induced muscle atrophy. These results suggest that denervation induces not only muscle degradation but also adaptive muscle response in a proteasome- and mTORC1-dependent manner.     

Neural Regulation of Muscle Glucose 6-Phosphate Dehydrogenase: Effect of Batrachotoxin and Tetrodotoxin

Abstract: We tested the hypothesis that glucose 6-phosphate dehydrogenase (G6PD) activity in the rat skeletal muscle is regulated by putative axonally derived neurotrophic factors. This was accomplished by comparing the effects of nerve section and subperineural injection of batrachotoxin (BTX) or tetrodotoxin (TTX) on G6PD in rat extensor digitorum longus (EDL) muscle. BTX, an agent known to block nerve impulse conduction and axonal transport, increased G6PD activity to 155% and 163% of control by days 2 and 4 after injection. Denervation of the EDL muscle by section of the peroneal nerve 10–20 mm from its entrance to the muscle caused G6PD activity to increase to 170% of control by day 1 and to 200% and 180% of control by days 2 and 4, respectively. The increase in enzyme activity after denervation and after subperineural injection of BTX was due in part to muscle inactivity resulting from blockade of nerve impulses. This conclusion is based upon the observation that subperineural injection of TTX at an identical site in the peroneal nerve caused a small but significant (30%) increase in G6PD activity after 4 days. Choline acetyltransferase (CAT) activity was assessed as a measure of the efficacy of blockade of slow axonal transport. Decreases in CAT activity following denervation or injection of BTX or TTX were parallel to increases in G6PD activity observed under these conditions. These results argue for a role of axonal transport in neural regulation of muscle G6PD, with a small contribution by neuromuscular activity.     

Reciprocal neural regulation of extrajunctional acetylcholinesterase and collagen Q in rat muscles—The role of calcineurin signaling

There are two main differences regarding acetylcholinesterase (AChE) expression in the extrajunctional regions of fast and slow rat muscles: (1) the activity of AChE catalytic subunits (G1 form) is much higher in fast than in slow muscles, and (2) the activity of the asymmetric forms of AChE (A₈ and A₁₂) is quite high extrajunctionally in slow muscles but virtually absent in fast muscles. The latter is due to the absence of the expression of AChE-associated collagen Q (ColQ) in the extrajunctional regions of fast muscle fibers, in contrast to its ample expression in slow muscles. We showed that both differences are caused by different neural activation patterns of fast vs. slow muscle fibers, which determine the respective levels of mRNA of both proteins. Whereas the changes in AChE mRNA levels in fast and slow muscles, as well as the levels of ColQ mRNA levels in slow muscles, observed in response to exposing either slow or fast muscles to different muscle activation patterns, are completely reversible, the extrajunctional suppression of ColQ expression in fast muscle fibers seems to be irreversible. Calcineurin signaling pathway in muscles is activated by high-average sarcoplasmic calcium concentration resulting from tonic low-frequency muscle fiber activation pattern, typical for slow muscle fibers, but is inactive in fast muscle fibers, which are activated by infrequent high-frequency bursts of neural impulses. Application to rats of two inhibitors of calcineurin (tacrolimus-FK506 and cyclosporin A) demonstrated that the mRNA levels of both the AChE catalytic subunit and ColQ in the extrajunctional regions of the soleus muscle are regulated by the calcineurin signaling pathway, but in a reciprocal way. Under the conditions of low calcineurin activity, AChE expression is enhanced and that of ColQ is suppressed, and vice versa. Our results also indicated that different, calcineurin-independent regulatory pathways are responsible for the reduction of AChE expression during muscle denervation, and for maintaining high ColQ expression in the neuromuscular junctions of fast muscle fibers.     

Acetylcholine--inhibition of transmitter release from adrenergic nerve terminals mediated by muscarinic receptors.

The evidence is reviewed for the presence of muscarinic receptors on the sympathetic nerves to blood vessels. Activation of these receptors by acetylcholine in doses that are too small to affect the smooth muscle cells directly inhibits the release of norepinephrine evoked by electric impulses or potassium ions. This inhibitory action of acetylcholine is prevented by muscarinic blocking agents and is probably due to hyperpolarization of the adrenergic nerve terminals.     

The TWEAK-Fn14 system: breaking the silence of cytokine-induced skeletal muscle wasting

The occurrence of skeletal muscle atrophy, a devastating complication of a large number of disease states and inactivity/disuse conditions, provides a never ending quest to identify novel targets for its therapy. Proinflammatory cytokines are considered the mediators of muscle wasting in chronic diseases; however, their role in disuse atrophy has just begun to be elucidated. An inflammatory cytokine, tumor necrosis factor (TNF)- like weak inducer of apoptosis (TWEAK), has recently been identified as a potent inducer of skeletal muscle wasting. TWEAK activates various proteolytic pathways and stimulates the degradation of myofibril protein both in vitro and in vivo. Moreover, TWEAK mediates the loss of skeletal muscle mass and function in response to denervation, a model of disuse atrophy. Adult skeletal muscle express very low to minimal levels of TWEAK receptor, Fn14. Specific catabolic conditions such as denervation, immobilization, or unloading rapidly increase the expression of Fn14 in skeletal muscle which in turn stimulates the TWEAK activation of various catabolic pathways leading to muscle atrophy. In this article, we have discussed the emerging roles and the mechanisms of action of TWEAK-Fn14 system in skeletal muscle with particular reference to different models of muscle atrophy and injury and its potential to be used as a therapeutic target for prevention of muscle loss.     

Functional Recovery of Denervated Skeletal Muscle with Sensory or Mixed Nerve Protection: A Pilot Study

Functional recovery is usually poor following peripheral nerve injury when reinnervation is delayed. Early innervation by sensory nerve has been indicated to prevent atrophy of the denervated muscle. It is hypothesized that early protection with sensory axons is adequate to improve functional recovery of skeletal muscle following prolonged denervation of mixed nerve injury. In this study, four groups of rats received surgical denervation of the tibial nerve. The proximal and distal stumps of the tibial nerve were ligated in all animals except for those in the immediate repair group. The experimental groups underwent denervation with nerve protection of peroneal nerve (mixed protection) or sural nerve (sensory protection). The experimental and unprotected groups had a stage II surgery in which the trimmed proximal and distal tibial nerve stumps were sutured together. After 3 months of recovery, electrophysiological, histological and morphometric parameters were assessed. It was detected that the significant muscle atrophy and a good preserved structure of the muscle were observed in the unprotected and protective experimental groups, respectively. Significantly fewer numbers of regenerated myelinated axons were observed in the sensory-protected group. Enhanced recovery in the mixed protection group was indicated by the results of the muscle contraction force tests, regenerated myelinated fiber, and the results of the histological analysis. Our results suggest that early axons protection by mixed nerve may complement sensory axons which are required for promoting functional recovery of the denervated muscle natively innervated by mixed nerve.