Characteristics of acetylcholine-activated channels of innervated and chronically denervated skeletal muscles

Characteristics of the ACh-activated channels before and after denervation of the frog interosseal muscle were studied using the patch clamp technique. Acetylcholine sensitivity was increased on extrajunctional portions of the muscle 7, 42, and 73 days after sectioning of the sciatic nerve. Nonjunctional regions of the innervated muscle appeared to contain one type of ACh channels having a conductance of 28 pS and a mean channel lifetime of 3.8 ms at -90 mV. The denervated muscles contained two classes of channels with conductance of 18 and 28 pS which were present as early as 7 days postdenervation and remained for 93 days. The channel open times of the innervated muscles increased with membrane hyperolarization. The open times of the channels present at 42 days postdenervation showed longer lifetimes than those of innervated muscles and were 10.8 ms and 9.6 ms at -90 mV. These channels also showed less voltage dependence than the control fibers.     

Phosphorylase kinase isozymes and phosphorylase in denervated skeletal muscles

The effects of motor denervation on levels of phosphorylase kinase isozymes and phosphorylase were investigated in rat epitrochlearis, hemidiaphragm, and soleus muscles. Amounts of the proteins were measured after quantitative immunoprecipitation and found to be decreased by as much as 70% 2 weeks after denervation. Unexpectedly, denervation had little, if any, effect on the relative proportions of the two phosphorylase kinase isozymes. Phosphorylase and phosphorylase kinase were decreased by essentially the same extent after denervation, and the effects of denervation were comparable in all three muscles. The decreases in these enzymes explain, at least in part, the marked alterations in glycogen metabolism that occur after motor denervation.     

Mechanical and electrical properties of denervated rat skeletal muscles

Mechanical activity (twitch and tetanus) and electrical activity (single and repetitive action potentials) were recorded in vitro (34 degrees C) in control and denervated (3 to 14 days) soleus and extensor digitorum longus muscles of the rat. After denervation tetanic tension (100 to 200 Hz, 500 ms duration) was decreased in both types of muscles. Denervation reduced significantly the rates of rise and fall and the amplitude of the action potential in both types of muscle fibers. In denervated fibers with very low resting membrane potential no action potentials could be recorded: in these fibers only a slow response without overshoot was detected. Hyperpolarization of denervated fibers to -90 mV prior to application of the depolarizing pulse increased their excitability. Action potential amplitudes were well maintained during tetanic stimulation (200 Hz, 40 to 90 ms) in innervated fibers. Depolarization of the innervated fibers with cathodic current before the tetanic pulse hindered the generation of repetitive action potentials at 200 Hz. A proportion of denervated fibers stimulated at 100 to 200 Hz generated only one action potential or gave rise to an incomplete train. Hyperpolarization of the denervated fibers resulted in an improvement in the ability to generate a train of action potentials at 100 to 200 Hz. A group of denervated fibers exhibited well maintained action potentials during tetanus. We suggest that failure in the repetitive electrical activity of denervated fibers could be the reason for the reduced tension of tetanus. Depolarization of the fibers and/or the increment in the electrical time constant of the sarcolemma are suggested for the decrease in the electrical excitability of denervated fibers.     

Reinnervation of denervated skeletal muscles by grafted dorsal root ganglion.

We examined whether or not the cervical dorsal root ganglion (DRG) of the rat, when isografted and connected to the distal stump of the severed common peroneal nerve, could survive, project axons to the denervated leg muscles, and exert beneficial influences to delay the degeneration of the denervated muscles. Rats in which the muscles were similarly denervated but no DRG was grafted served as the control. After a postoperative period of 72 to 286 days, histological study showed that nerve cells at the superficial part of the grafted DRG survived. Indirect electrical stimulation via the distal stump of the common peroneal nerve produced no contraction of the muscles, indicating that no functional neuromuscular contacts had been reestablished. Direct stimulation of the denervated muscles did elicit contraction, and the isometric twitch and tetanic tensions were significantly much higher in the experimental rats with a grafted DRG than in the control rats. Cholinesterase-silver staining indicated the presence of axons in the denervated muscles, but the axons did not terminate on endplates. Compared with the control muscles, the experimental muscles had significantly more axons, and had atrophied less as indicated by muscle wet weight and histological appearance. These results indicate that the sensory axons can delay the weakening and atrophy of muscles after denervation. We suggest that the sensory axons may exert certain trophic influence on the denervated muscle fibers, though the actual mechanism is unknown.     

Decreased acetylcholine receptor content in denervated skeletal muscles infused with nerve extract

The influence of a concentrated extract of soluble substances from the sciatic nerve upon the acetylcholine receptor (AChR) content in the soleus muscle of adult rats was examined by in vivo infusions. Internal and membrane-inserted AChR were quantitated by the specific binding of 125I-alpha-bungarotoxin (a-BuTX). Interestingly, the nerve extract had no apparent effect unless the soleus muscle was also denervated at the start of the infusion. Then, after 66 hr, substantially less (60-80%) binding of 125I-a-BuTX to AChR was observed compared to denervated solei that did not receive an infusion of nerve extract. However, the concentration of protein in the nerve extract had to exceed 5 mg/ml before this effect was evident. Infusions of phosphate-buffered saline, bovine serum albumin, rat liver extract, or human transferrin had no striking effect upon AChR. The prevention of the characteristic denervation-induced increase in non-junctional AChR by an active component in the nerve extract may be due to a trophic signal for decreased synthesis of AChR, but it is also possible that the degradation of AChR was increased.     

Reinnervation is followed by necrosis in previously denervated skeletal muscles of dystrophic hamsters

Hind leg muscles of dystrophic hamsters were continually denervated by multiple crushes of the sciatic nerve to as long as 93 days of age. In these muscles, the prevalence of centronucleated fibers which is a cumulative index of prior necrosis, remained very low. In control dystrophic muscles the prevalence of centronucleated fibers increased steadily to approximately 80% where it leveled off. By omitting further crushes in other groups of animals, previously denervated muscles became adequately reinnervated. In the reinnervated muscles the prevalence of centronucleated fibers steadily increased throughout the necrotic phase of dystrophy at a rate that was comparable to corresponding stages of the natural history of the disease. These experiments indicated that continued denervation was effective in negating skeletal muscle fiber necrosis throughout the necrotic phase and that the electromechanical activity of muscle fibers which allows muscle fiber necrosis was not a time-locked factor.     

Sodium influx during action potential in innervated and denervated rat skeletal muscles.

Resting Na(+) influx (J(i)(Na)) was measured in innervated and denervated (1-6 days) rat extensor digitorum longus muscle in the absence and presence of 2 micromol/L tetrodotoxin (TTX).The mean value of Na(+) permeability (P(Na)) in innervated muscles was 49.6 +/- 2.6 pm.s(-1). At the second day postdenervation, it decreased by about 45%. This was followed, between the second and fourth days, by a sharp rise, which by the sixth day reached a steady value approximately 2.5 times greater than that of innervated muscles. This, most likely, generated the 30% increase in internal [Na(+)] concentration ([Na(+)](I)) observed at this time. Tetrodotoxin reduced P(Na) of both innervated and denervated muscles by about 25%. In 6-day denervated muscles, virtually all the TTX effect on P(Na) represents the blockage of TTX-resistant Na(+) channels. Denervation produced a depolarization of about 20 mV by the sixth day. The extra J(i)(Na) per action potential (AP) decreased monotonically with time after denervation from 20.0 +/- 3.8 in innervated to 11.1 +/- 1.0 nmol.g(-1).AP(-1) in 6-day denervated muscles. The overshoot of the AP decreased from 15 +/- 1 in innervated to 7 +/- 1 mV in 6-day denervated muscles. Likewise, the maximum rate of rise (+dV/dt), an expression of the inward Na(+) current, fell from 305 +/- 14 in innervated to 188 +/- 18 V.s(-1) in 6-day denervated muscles. The estimated 6-day denervated/innervated ratio of peak Na(+) conductance (g(Na)) was 0.67. The changes in AP parameters promoted by denervation were substantially reduced when both innervated and denervated fibers were hyperpolarized to -90 mV. These results suggest that the depolarization, mainly due to the increase in P(Na) /P(K) ratio, increases Na(+) inactivation and consequently reduces peak g(Na), in spite of the absolute increment in resting TTX-sensitive P(Na). This, in addition to the moderate reduction in the inward driving force on Na(+), decreases the inward Na(+) current and the extra J(i)(Na) per AP.     

An electrophysiological study of the effects of D-tubocurarine, atropine, and alpha-bungarotoxin on the cholinergic receptor in innervated and chronically denervated mammalian skeletal muscles

An electrophysiological study of the action of d-tubocurarine (d-TC), atropine, and α-bungarotoxin (BuTX) was made on the innervated and chronically denervated diaphragm and soleus muscles of the rat and mouse. All three drugs were able to block endplate potentials of innervated muscles as well as acetylcholine (ACh) sensitivity of chronically denervated muscles. The effects of atropine and d-TC were fully reversed upon washing with Ringer's solution, white the effects of BuTX were only partially reversed. The reversibility of BuTX was more evident at extrajunctional areas in chronically denervated muscles than at the innervated endplate region, but in both cases only a fraction of the normal response could be detected after intensive washing for at least 2 hr. The blockade of ACh sensitivity in chronically denervated muscles required a concentration of d-TC 10-fold higher than that necessary to block the endplate potentials of innervated muscles. BuTX and d-TC did not affect the ionic permeabilities of the muscle fiber during an action potential while atropine decreased both Na⁺ and K⁺ conductances, the magnitude of the effect being dependent on the frequency of stimulation. At the endplate region, d-TC was much more effective than atropine in protecting against the irreversible effect of BuTX. In the chronically denervated preparation, however, neither of the two drugs effectively protected against BuTX. It is concluded that in terms of their reactivities to cholinolytic agents, the extrajunctional receptors induced by chronic denervation of skeletal muscles are qualitatively similar to those found at endplate regions of normal muscles, but that they exhibit differences in their quantitative interaction with different cholinolytic agents. The data further indicate that atropine interacts with the ionic conductance modulator unit associated with the cholinergic receptor, rather than with the ACh receptor itself.     

Inactivity-induced motor nerve terminal sprouting in amphibian skeletal muscles chronically blocked by alpha-bungarotoxin

There is a positive correlation between contractile inactivity and the initiation of motor neuron sprouting. However, the exact mechanism responsible for this neuronal growth remains obscure. In a previous study (M. M. Wines and M.S. Letinsky, 1988, J. Neurosci. 8: 3909-3919) we investigated this phenomenon by inducing chronic contractile inactivity of an amphibian muscle by exposure to formamide and found that motor neuron sprouting occurs in the presence of normal pressynaptic transmitter release and propagated muscle fiber action potentials. The present study investigates motor neuron sprouting in response to inactivity produced when neuromuscular transmission is blocked by chronic exposure to alpha-bungarotoxin (alpha-BTX). The alpha-BTX-induced muscle paralysis was maintained for 1-63 days by repetitive application of the toxin to the cutaneous pectoris muscle of adult Rana pipiens. During the chronic alpha-BTX treatment end-plate potentials were reduced below threshold, which therefore removed both muscle fiber action potentials and contractile activity. Our findings showed only terminal sprouting. Also, higher sprouting frequencies (up to 100% of the observed terminals) were observed after chronic alpha-BTX treatment, compared to the sprouting response induced by formamide treatment. In view of our earlier formamide results, these observations suggest that the inhibition of the postsynaptic acetylcholine response, and consequently inhibition of muscle fiber electrical and contractile activity, produces a stronger stimulus to motor neuron sprouting than the presence of contractile inactivity alone coupled with normal synaptic transmission and muscle electrical activity.     

Satellite cells in innervated and denervated muscles treated with clenbuterol.

The sympathomimetic agent, clenbuterol, induces a muscle-specific hypertrophy in both normal and catabolic muscle. Drug-induced hypertrophy is not generally associated with an increase in DNA content, thus the role of satellite cells in the response of soleus muscles from weanling rats is questioned. Following simultaneous sciatic section and administration of clenbuterol, responses are similar in innervated and denervated muscles after 4 days. Increased protein accretion in treated muscles is associated with evidence of satellite cell activation, but with little evidence of division. It is speculated that satellite cell production of growth factors may play an important role in the hypertrophic action of clenbuterol, and the clinical implications of the findings are discussed.     

Parvalbumin in cross-reinnervated and denervated muscles

The extensor digitorum longus (EDL) muscle was cross-reinnervated by the soleus (SOL) nerve, leading to the well-known transformation toward a slow muscle. Nine weeks after the operation, the quantitative analysis of the Ca2+-binding protein, parvalbumin (PV), using high-performance liquid chromatography, showed a threefold reduction of PV in the cross-reinnervated EDL muscle. Denervation of the EDL muscle, which leads to an increase of the half-relaxation time, resulted in a 20% decrease of the PV concentration within 4 days. This significant lower PV level was detectable prior to any change of the myofibrillar adenosine triphosphatase (ATPase). Normal PV concentrations were reached after 9 weeks following self-reinnervation of the EDL muscle. The experiments support the view that PV is involved in the relaxation of rat fast skeletal muscles and that its expression is dependent on nerve-muscle interaction. Since PV changes preceded histochemical changes after denervation, this protein may be a sensitive marker for early stages of neuromuscular disturbances.     

Functional compensation in partially denervated muscles

In patients with various types of chronic motor denervation, the numbers of surviving motor units have been compared with the twitch tensions developed by the same muscle (extensor digitorum brevis). It was found that functional compensation in partially denervated muscles was often marked; in most patients abnormally small twitches occurred only when fewer than 10% of motor axons remained. The factors responsible for this compensation are considered. The twitch speeds of partially denervated muscles differed markedly, even among patients with the same disorder; there was evidence to suggest that the twitches of some motor units might become slower than those found in normal muscles.     

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 β‐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 β‐tubulin in denervated muscular atrophy, possibly in order to maintain or preserve the integrity of the muscle fiber during progressive atrophy or regeneration. © 1999 John Wiley & Sons, Inc. Muscle Nerve 22: 61–70, 1999     

Effect of reinnervation on the degradation rate of junctional acetylcholine receptors synthesized in denervated skeletal muscles

Two populations of ACh receptors (AChRs) with different degradation rates have been shown to coexist in the postsynaptic membrane after denervation of the neuromuscular junction (NMJ). One population, consisting of the slowly degrading original AChRs inserted into the plasma membrane prior to denervation, has a degradation half-life (t1/2) of approximately 8 d. This degradation rate accelerates after denervation (to a t1/2 approximately 3 d), but can be decelerated back to the predenervation rate by reinnervation. The second population, the rapidly degrading new AChRs, which replace the degrading original AChRs at the NMJ after denervation, resembles embryonic AChRs, with a t1/2 of approximately 1 d. In the present study, we report that the degradation rate of these new junctional AChRs is unaltered for 3-6 half-lives after reinnervation. We further report that a small amount (less than 10%) of slowly degrading AChRs (t1/2 approximately 3 d) may also be synthesized in denervated muscle. We suggest that, unlike its effect on the original, slowly degrading AChRs, reinnervation does not modulate the degradation rate of the rapidly degrading new junctional AChRs. It merely regulates the ratio of rapidly to slowly degrading AChRs being synthesized and inserted at the NMJ.