Restoration of muscle contractile properties after partial and complete denervation

The subject of this experimental study was the isometric contractile properties of rat tibialis anterior muscle, number and average size of the motor units as well as type content and type-grouping of muscle fibres according to SDH activity in the same muscle after total and partial denervation (crushing the sciatic nerve and L4). It has been shown that in the process of reinnervation after total and partial denervation, quantitative differences with the general tendency in the dynamics of restoration of contractile properties of the whole muscle are found at different dynamics of restoration of electromyographic and muscle histochemical characteristics of motor units.     

Phospholipid distribution and stimulation of methylation during denervation and reinnervation in skeletal muscle

Denervation of the rat soleus and extensor digitorum longus muscles was induced by nerve crush. Functional signs of denervation were noted within 48 h with recovery beginning about the 12th day following denervation. There was also a marked decrease in muscle weight but only a small decrease in protein content per mg of muscle, subsequent to denervation. At 1, 2 and 3 weeks following nerve crush there was a relative decrease in muscle phosphatidylethanolamine (PE) and a corresponding increase in phosphatidylcholine (PC). The proportion of the other phospholipids did not significantly change. The levels of PC and PE returned to, or in some cases slightly overshot, control values at 4 and 5 weeks following nerve crush, i.e. during the period of reinnervation. Levels in non-denervated contralateral muscles did not significantly change. At 1 and 3 weeks following nerve crush a marked increase was observed in the activities of the enzymes PE-methyltransferases I and II, as measured by [³H]methyl group incorporation from S-adenosyl methionine into phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and PC. Increased activity of these methylases was seen in the contralateral control muscle, although less than in the denervated muscle. These enzymatic changes could be responsible for the changes in PE and PC distribution which we observed. Methylation of PE might also decrease the microviscosity of the membrane, thereby leading to other changes associated with denervation. Activation of this system might be another form of supersensitivity induced by denervation.     

Acetylcholinesterase activity and molecular forms during denervation and reinnervation in extensor digitorum longus muscle of the rat

The four principal molecular forms of acetylcholinesterase characteristic of the mammalian muscle (16.1 S., 12.5 S, 10.2 S, and 3.6. S) were identified by sucrose gradient sedimentation as the four activity peaks H, H₁, M and L.After denervation obtained by crushing the sciatic nerve five stages of the denervation-reinnervation process were examined. Days 7, 14, 22, 30, and 60 were chosen on the basis of previous electrophysiological and histochemical studies. The AChE activity showed an initial drop followed by recovery after nerve arrival at the muscle which was completed by day 60. Marked changes in the relative proportions of the four molecular forms were observed. The 16.1 S almost disappeared during the denervation period, reappeared after nerve arrival and was completely restored at day 60. Changes were also observed in the intermediate and lower forms and were tentatively related to processes of degradation, reaggregation and de novo synthesis.A comparison of the present data with those from parallel electrophysiological and histochemical studies suggests the presence and the functional role of molecular forms other than 16S in the neuromuscular junction.     

Ultrastructural changes in the connective tissue of skeletal muscles following denervation]

The ultrastructural changes found in the endomysium of rats after denervation of the diaphragm and m. plantaris were studied. Within the first week after crossing the peripheral nerve in the nedomysium there appeared an increased amount of neutrophils and monocytes as well as phagocytic material in the cytoplasm of histocytes. Activization of the cytoplasm of fibroblasts which manifested itself in the appearance of numerous vesicles and multiple free and bound ribosomes was detected by the end of the second and the beginning of the third weeks after denervation. At the same period eosinophils invaded the endomysium and became closely surrounded by numerous collagenic fibres. After reparation of neuromuscular synapses these changes disappeared. On the basis of these results and others founded in previous studies of denervated and reinnervated skeletal muscles the authors consider these changes in the endomysium appearing under the above experimental conditions to be manifestations of metabolic interrelations between the endomysium connective tissue and muscle fibres.     

Time course of changes in lipoprotein lipase activity in rat skeletal muscles during denervation-reinnervation.

The effects of denervation-reinnervation after sciatic nerve crush on the activity of extracellular and intracellular lipoprotein lipase (LPL) were examined in the soleus and red portion of gastrocnemius muscles. The activity of both LPL fractions was decreased in the two muscles within 24 h after the nerve crush and remained reduced for up to 2 wk. During the reinnervation period, LPL activity was still reduced in the soleus and started to increase only on the 40th day. In the red gastrocnemius, LPL activity increased progressively with reinnervation, exceeding control values on the 30th day post-crush. The LPL activity in the soleus from the contralateral to denervated hindlimb was also affected, being increased on the postoperation day and then gradually decreased during the following days. In conclusion, the time course of changes in muscle LPL activity after nerve crush confirmed the predominant role of nerve conduction in controlling muscle potential to take up free fatty acids derived from the plasma triacylglycerols. However, other factors, such as muscle fiber composition and the fiber transformation, should also be considered in this aspect of the denervation-reinnervation process. Moreover, it was found that denervation of muscles from one hindlimb may influence LPL activity in muscles from the contralateral leg.     

Influence of denervation and reinnervation on autolytic activity and on protein composition of skeletal muscle in rat.

Changes in the proteolytic activity and in the relative content of protein in soluble, myofibrillar and insoluble fractions were investigated following denervation and reinnervation of the soleus and tibialis anterior muscles of the rat. After denervation an increase of autolysis in the acid and neutral pH range, but not in the alkaline one, was found in both muscles. An increased autolysis at the acid and neutral pH range was also observed in both muscles after reinnervation, when the weight of the muscles increased. The results indicate the lack of inverse relationship between the changes of proteolytic activity and the decrease or increase of the amount of muscle protein in the course of muscle atrophy and regeneration.     

Satellite cells of the rat soleus muscle in the process of compensatory hypertrophy combined with denervation

Compensatory hypertrophy was induced in the rat soleus muscle by sectioning the tendon of the ipsilateral gastrocnemius and plantaris muscle. Seven days after tenotomy of synergistic muscles, when soleus hypertrophy attains about 40%, the number of satellite cells (expressed as percentage of all muscle nuclei found in the same cross-sections) as revealed by electron microscopy, was increased from 5.8+/-0.06% in the normal soleus muscle to 16.6+/-1.26%. After four days' denervation of the soleus muscle the percentage of satellite cells was increased to 7.2+/-0.62%. In experiments where hypertrophy of the soleus muscle was combined with denervation three days after tenotomy of synergists, and examined after another four days (during which time it loses, as has previously been shown, over 40% of its predenervation weight), the number of satellite cells was greatly increased to 29.9+/-3.42%. This increase is apparently due to two independent processes which take place during the first postoperative period: a) mitotic division of satellite cells during the early stages of compensatory hypertrophy and b) pinching off of muscle nuclei from rapidly atrophying muscle fibres due to subsequent denervation. Activation of satellite cells was mainly manifested by expansion of smooth and especially of rough endoplasmic reticulum, a rich Golgi complex, high pinocytotic activity, increased number of ribosomes and by nuclear changes. Concomitantly with the increased number of satellite cells, proliferation of fibroblasts, macrophages and mast cells could be observed.     

Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation.

To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41-44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.     

Degeneration of the afferent fibers simulating the effects of motor denervation on skeletal muscle

Degeneration of afferent nerve fibres was induced in rats in order to observe its effects on the properties of the extra-junctional membrane of soleus muscle fibres. In one approach, removal of dorsal root ganglia L4 and L5 was accomplished in preparations with intact or impulse-blocked (with tetrodotoxin containing cuffs around the sciatic nerve) efferent innervation. Spike resistance to tetrodotoxin developed in the inactive deafferented preparations earlier and to a greater extent than in control, that is only impulse-blocked, preparations. In another series of experiments, efferent denervation alone proved to be less effective than the association of efferent and afferent denervation. On the other hand, section of the afferent fibres central to the dorsal root ganglia was without effect. These results are consistent with the interpretation that products of nerve degeneration contribute together with inactivity to the development of the extrajunctional membrane changes observed in skeletal muscle after denervation.     

哺乳动物不活动化肌纤维的动作电位

用河豚毒素(TTX)慢性阻断大鼠坐骨神经的冲动传导,使后肢不活动,经过不同时间(最长7d)后离体观察了快肌伸趾长肌(EDL)和慢肌比目鱼肌(SOL)肌纤维终板区的诱发动作电位。我们发现在不活动期间动作电位超射和上升速率逐步下降,并从第4天起部分肌纤维能在含有1×10~(-7)g/ml TTX的溶液中被诱发产生动作电位(称抗TTX动作电位),待至第7天时全部SOL肌纤维和90％的EDL肌纤维都能被诱发出抗TTX动作电位。与去神经肌纤维相比,不仅抗TTX动作电位出现较晚,并且其超射和上升速率较低。在去掉TTX阻断使肌肉恢复活动后,动作电位超射和上升速率渐趋恢复,抗TTX动作电位逐渐消失。无论是动作电位的恢复还是抗TTX动作电位的消失,EDL肌纤维均快于SOL肌纤维。本文还讨论了不活动化使肌纤维动作电位变化以及快、慢肌差别的可能原因。     

Regulation of turnover and number of acetylcholine receptors at neuromuscular junctions

The number and metabolic stability of acetylcholine receptors (AChRs) at neuromuscular junctions of rat tibialis anterior (TA) and soleus (SOL) muscles were examined after denervation, paralysis by continuous application of tetrodotoxin to the nerve, or denervation and direct stimulation of the muscle through implanted electrodes. After 18 days of denervation AChR half-life declined from about 10 days to 2.3 days (TA) or 3.6 days (SOL) and after 18 days of nerve conduction block to 3.1 days (TA). In contrast, the total number of AChRs per endplate was unaffected by these treatments. Denervation for 33 days had no further effect on AChR half-life but reduced the total number of AChRs to about 54% (SOL) or 38% (TA) of normal. Direct stimulation of the 33-day denervated SOL from day 18 restored normal AChR stability and counteracted muscle atrophy but had no effect on the decline in AChR number. The results indicate that motoneurons control the stability of junctional AChRs through evoked muscle activity and the number of junctional AChRs through trophic factors.     

Unlike effects of denervation on the rate of entry of inorganic phosphate into rat slow and fast muscles.

The increased inorganic phosphate flow, characteristic of denervated gastrocnemius muscle is shown to be present in additional denervated fast muscles, i.e. the plantaris, tibialis anterior and extensor digitorum longus muscles. The response of the soleus, a slow muscle, to denervation is biphasic. After an initial decrease of the phosphate flow at the end of the first postoperative day, there is a secondary rise which has the same general characteristics as the rise observed in fast muscles i.e. an exponential or hyperbolic increase to an asymptotic value reached after thirty days. The denervated fast and slow muscles are not converging to an intermediate metabolic pattern. The changes in phosphate flow induced by denervation are reversible in the soleus as well as in the gastrocnemius muscles.     

Effect of reinnervation on collagen synthesis in rat skeletal muscle.

The effect of reinnervation on the activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), both enzymes of collagen biosynthesis, and on the concentration of hydroxyproline (Hyp) was studied in gastrocnemius, soleus, and tibialis anterior muscles of rat 19, 26, 40, and 61 days after crush denervation of the sciatic nerve. The GGT activity was elevated in denervated gastrocnemius and soleus muscles and the PH activity in gastrocnemius. Muscular Hyp concentration was increased in denervated tibialis anterior muscle. Both the PH and GGT activities and the Hyp concentration returned to the control level during the reinnervation period (19-61 days from the start of denervation). It seems that denervation atrophy of skeletal muscle is associated with an increased rate of muscular collagen biosynthesis and that during reinnervation collagen synthesis rate decreases despite accelerated muscular growth. The results thus suggest that innervation is a powerful suppressive regulator of muscular collagen biosynthesis.     

Calbindin D-28k-immunoreactivity in rat muscle spindles during postnatal maturation and after denervation

Summary Calbindin D-28k-immunoreactivity has been demonstrated in some of the intrafusal muscle fibres and in the capsule of adult rat muscle spindles. In this study, the immunocytochemical localization of calbindin D-28k in the muscle spindles of triceps surae muscle was studied during postnatal maturation and after denervation. In young rats calbindin D-28k-immunoreactivity was seen in a few intrafusal fibres, first at the age of 4 days. At the 7th day, three calbindin D-28k-immunoreactive fibres and one unlabelled fibre were seen in most muscle spindles, as in adult rats. The spindle capsule and perineurial sheath of nerves were first seen to exhibit calbindin D-28k immunoreactivity at the age of 14 days, and thereafter the localization of calbinding D-28k-like immunoreactivity was similar to that in adult rats. After denervation, calbindin D-28k-immunoreactivity remained in intrafusal muscle fibres and the spindle capsule for a long period. After two months of denervation, calbindin D-28k immunoreactivity could still be seen in the spindle capsule, but the intrafusal fibres were not labelled.The innervation is known to have trophic effects on the intrafusal fibres. The present findings suggest that the expression of calbindin D-28k-immunoreactivity in maturating muscle spindles may be induced by the developing innervation. The decrease of calbindin D-28k-immunoreactivity in intrafusal fibres after denervation may be due to the loss of trophic factors released by the nerves.     

Degradation rates of acetylcholine receptors can be modified in the postjunctional plasma membrane of the vertebrate neuromuscular junction

Denervation of vertebrate muscle causes an acceleration of acetylcholine receptor turnover at the neuromuscular junction. This acceleration reflects the composite behavior of two populations of receptors: "original receptors" present at the junction at the time of denervation, and "new receptors" inserted into the denervated junction to replace the original receptors as they are degraded (Levitt, T. A., and M. M. Salpeter, 1981, Nature (Lond.), 291:239-241). The present study examined the degradation rate of original receptors to determine whether reinnervation could reverse the effect of denervation. Sternomastoid muscles in adult mice were denervated by either cutting or crushing the nerve, and the nerves either allowed to regenerate or ligated to prevent regeneration. The original receptors were labeled with 125I-alpha-bungarotoxin at the time of denervation, and their degradation rate followed by gamma counting. We found that when the nerve was not allowed to regenerate, the degradation decreased from a t1/2 of approximately 8-10 d to one of approximately 3 d (as reported earlier for denervated original receptors) and remained at that half-life throughout the experiment (approximately 36 d). If the axons were allowed to regenerate (which occurred asynchronously between day 14 and day 30 after nerve cut and between day 7 and 13 after nerve crush), the accelerated degradation rate of the original receptors reverted to a t1/2 of approximately 8 d. Our data lead us to conclude that the effect of denervation on the degradation rate of original receptors can be reversed by reinnervating. The nerve can thus slow the degradation rate of receptors previously inserted into the postsynaptic membrane.     

Denervation and reinnervation of fast and slow muscles. A histochemical study in rats.

A histochemical study, using myosin-adenosine triphosphatase activity at pH 9.4, was conducted in soleus and plantaris muscles of adult rats, after bilateral crushing of the sciatic nerve at the sciatic notch. The changes in fiber diameter and per cent composition of type I and type II fibers plus muscle weights were evaluated along the course of denervation-reinnervation curve at 1, 2, 3, 4 and 6 weeks postnerve crush. The study revealed that in the early denervation phase (up to 2 weeks postcrush) both the slow and fast muscles, soleus and plantaris, resepctively, atrophied similarly in muscle mass. Soleus increased in the number of type II fibers, which may be attributed to "disuse" effect. During the same period, the type I fibers of soleus atrophied as much or slightly more than the type II fibers; whereas the type II fibers of plantaris atrophied significantly more than the type I fibers, reflecting that the process of denervation, in its early stages, may affect the two fiber types differentially in the slow and fast muscles. It was deduced that the type I fibers of plantaris may be essentially different in the slow (soleus) and fast (plantaris) muscles under study. The onset of reinnervation, as determined by the increase in muscle weight and fiber diameter of the major fiber type, occurred in soleus and plantaris at 2 and 3 weeks postcrush, respectively, which confirms the earlier hypotheses that the slow muscles are reinnervated sooner than the fast muscles. It is suggested that the reinnervation of muscle after crush injury may be specific to the muscle type or its predominant fiber type.     

The morphology of regeneration of skeletal muscles in the rat

Muscle regeneration was studied by light and electron microscopy in soleus muscles of rat. After segmental crushing the number of fibres increased in some muscles within 30 days, indicating that numerical hyperplasia can take place. Locally applied Ringer solution of 60-70 degrees C caused necrosis of myofibres but left satellite cells and blood supply largely intact. Following phagocytosis, four mechanisms of regeneration were seen. (1) Lost fibres were replaced by clusters of myotubes formed by satellite cells within persisting basal lamina tubes. These clusters displaced the surrounding endomysium and looked like longitudinally 'split' fibres. (2) Viable fibre fragments fused with satellite cells. (3) Satellite cells of surviving fibres proliferated and fused to myotubes localized beneath the basal lamina. (4) Thin new fibres occurred in the interstitium. Their origin remained unknown. After 6 months the mean size of the new myofibres was normal, but the scatter of diameters was increased, central nuclei, fibre 'spliting' and branching, and fibrosis were prominent. Staining for acetylcholinesterase revealed that many fibres were short and not innervated. The similarity with dystrophic muscles in man suggested, that the most prominent histological changes in myopathic muscles may be due to attempts of regeneration.     

Reduction of skeletal muscle atrophy by a proteasome inhibitor in a rat model of denervation

The ubiquitin-proteasome system is the primary proteolytic pathway implicated in skeletal muscle atrophy under catabolic conditions. Although several studies showed that proteasome inhibitors reduced proteolysis under catabolic conditions, few studies have demonstrated the ability of these inhibitors to preserve skeletal muscle mass and architecture in vivo. To explore this, we studied the effect of the proteasome inhibitor Velcade (also known as PS-341 and bortezomib) in denervated skeletal muscle in rats. Rats were given vehicle or Velcade (3 mg/kg po) daily for 7 days beginning immediately after induction of muscle atrophy by crushing the sciatic nerve. At the end of the study, the rats were euthanized and the soleus and extensor digitorum longus (EDL) muscles were harvested. In vehicle-treated rats, denervation caused a 33.5 +/- 2.8% and 16.2 +/- 2.7% decrease in the soleus and EDL muscle wet weights (% atrophy), respectively, compared to muscles from the contralateral (innervated) limb. Velcade significantly reduced denervation-induced atrophy to 17.1 +/- 3.3% in the soleus (P < 0.01), a 51.6% reduction in atrophy associated with denervation, with little effect on the EDL (9.8 +/- 3.2% atrophy). Histology showed a preservation of muscle mass and preservation of normal cellular architecture after Velcade treatment. Ubiquitin mRNA levels in denervated soleus muscle at the end of the study were significantly elevated 120 +/- 25% above sham control levels and were reduced to control levels by Velcade. In contrast, testosterone proprionate (3 mg/kg sc) did not alleviate denervation-induced skeletal muscle atrophy but did prevent castration-induced levator ani atrophy, while Velcade was without effect. These results show that proteasome inhibition attenuates denervation-induced muscle atrophy in vivo in soleus muscles. However, this mechanism may not be operative in all types of atrophy.     

Cytosolic androgen receptor in regenerating rat levator ani muscle.

The development of the cytosolic androgen receptor was studied after degeneration and regeneration of the rat levator ani muscle after a crush lesion. Muscle regeneration appears to recapitulate myogenesis in many respects. It therefore provides a model tissue in sufficiently in large quantity for investigating the ontogenesis of the androgen receptor. The receptor in the cytosol of the normal levator ani muscle has binding characteristics similar to those of the cytosolic receptor in other androgen-sensitive tissues. By day 3 after a crush lesion of the levator ani muscle, androgen binding decreased to 25% of control values. This decrease was followed by a 4-5 fold increase in hormone binding, which attained control values by day 7 after crush. Androgen binding remained stable at the control value up to day 60 after crushing. These results were correlated with the morphological development of the regenerating muscle after crushing. It is concluded that there is little, if any, androgen receptor present in the early myoblastic stages of regeneration; rather, synthesis of the receptor may occur after the fusion of myoblasts and during the differentiation of myotubes into cross-striated muscle fibres.     

Junctional form of acetylcholinesterase restored at nerve-free endplates.

Rat soleus muscles were ectopically innervated by implanting a foreign nerve in an endplate-free region of muscle and, 2–3 weeks later, cutting the original nerve. The junctional, 16 S form of acetylcholinesterase (AChE) and focal staining for AChE disappeared from the old endplate region within a few days after denervation. In muscles with an ectopic nerve, but not in paired control muscles, 16 S AChE and focal staining were restored in the old endplate region 1–2 weeks after denervation even though nerve fibers could not be detected in that region. These results suggest that the nerve exerts a local effect, specifying the site at which junctional AChE appears, and a nonlocal effect, perhaps mediated by muscle activity, regulating the amount of junctional AChE.