Introduction

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Resistance (hyposensitivity) to NDMRs has been reported after upper and lower motor neuron lesions (Hogue et al., 1990), burn trauma (Kim et al., 1988), chronic administration of NDMRs with (Dodson et al., 1995) or without (Hogue et al., 1992) muscle paralysis and conditions that predispose to muscle disuse (Gronert et al., 1984). These have been the subject of a comprehensive review (Martyn et al., 1992). In most, if not all, of these conditions, the resistance to NDMRs is associated with muscle atrophy and up-regulation of AChRs on muscle membranes (Kim et al., 1988; Hogue et al., 1990; Martyn et al., 1992; Fung et al., 1995). Although the exact mechanism(s) of the resistance to NDMRs has not yet been elucidated, the resistance is usually attributed to the changes in muscle mass and expression of AChRs. However, resistance to NDMRs has also been reported in the absence of overt muscle fiber atrophy (Waud et al., 1985) or increased membrane AChRs (Marathe et al., 1989), whereas increased membrane AChRs may exist without resistance to NDMRs (Ward and Martyn, 1993). Hence, there is disparity as to whether the changes in muscle mass (atrophy) and membrane AChRs are causally related to the observed resistance to NDMRs.

Disuse or immobilization of muscles caused by application of plaster casts, pain, restriction in bed or denervation is a common feature of most conditions associated with resistance to NDMRs. Intuitively, the muscle atrophy produced by immobilization would be expected to increase sensitivity to NDMRs. However, independent studies have shown that muscle disuse atrophy per se produces resistance to NDMRs, increased sensitivity to agonists (acetylcholine and succinylcholine), proliferation of AChRs or sprouting of nerve terminals (Fischbach and Robbins, 1971; Waud et al., 1985; Fung et al., 1995; Yanez and Martyn, 1996). The increased sensitivity to agonists and resistance to NDMRs are reported to revert to normal within 3 to 7 weeks of termination of immobilization (Martyn et al., 1992; Fung et al., 1995). However, it is not clear whether the changes in sensitivity to NDMRs, AChRs and muscle morphology persist indefinitely. It is also not clear whether the changes in AChRs and muscle morphology after disuse correlate with the changes in sensitivity to NDMRs, neither is it clear whether the proliferation of AChRs after disuse is associated with changes in the subunit composition of AChRs as after denervation (Witzemann et al., 1991). In mature innervated muscle, AChRs are composed of , ,  and  subunits. With denervation, AChRs consisting of , ,  and  subunits are also expressed (Witzemann et al., 1991) .

Therefore, in this study we investigated (1) the effects of chronic disuse on the morphology of muscle fibers and of the neuromuscular junction, membrane expression of AChRs, the levels of the mRNAs that encode for subunits of the AChR, and the sensitivity to the NDMR, dTC, (2) whether these morphologic and pharmacologic changes persist with continued disuse and (3) whether the associations between these morphologic and pharmacologic changes support a causal relationship between them as well as a role for disuse in their etiology. Preliminary data from this study have been reported in abstract form (Ibebunjo et al., 1996a).

	Methods

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Animals

Adult male Sprague-Dawley rats (200-250 g) were used for this study. The study was approved by the Subcommittee on Research Animal Care at the Massachusetts General Hospital. The animals were housed three per cage under a 12-hr light-dark cycle with food and water available ad libitum. The rats were randomly allocated to the two (immobilization or sham immobilization) treatment groups.

Surgical Preparations

After 1 week of acclimatization, the rats were anesthetized with pentobarbital (60 to 70 mg kg¹ i.p.) and one hind limb was immobilized by pinning the knee at 90° flexion and ankle at 90° dorsiflexion. The contralateral unimmobilized leg, and a separate group of animals in which one hind limb was sham-operated served as controls. In the sham-immobilized control group, the sham-immobilized limb was subjected to the same manipulations, including boring a hole through the joints, but a pin was not inserted to immobilize the joint. At 7, 14 or 28 days after immobilization, the rats were again anesthetized and an endotracheal tube inserted for mechanical ventilation with air at 70 to 80 breaths per minute using a tidal volume of 10 ml kg¹ (Rodent Ventilator model 683; Harvard Apparatus Inc., South Natick, MA) to maintain arterial blood carbon dioxide tension at 30 to 40 mm Hg. Anesthesia was maintained with intermittent doses of pentobarbital given i.p. Lack of the withdrawal response to toe clamping was taken to indicate an adequate depth of anesthesia. Rectal temperature was monitored and maintained at 37-38°C with a heating lamp. The jugular vein was cannulated for drug administration.

Muscle Sensitivity to dTC

With the rat in dorsal recumbency, the tendon of insertion of the tibialis muscle on both sides was surgically exposed and each attached to a Grass FT03 force displacement transducer (Grass Instruments Co, Quincy, MA). The sciatic nerves on both sides were exposed in the thigh for indirect nerve stimulation of the muscles. After stabilizing the knee rigidly in a clamp, a base-line tension of approximately 10 and 50 g, which yielded maximal evoked twitch tensions, was applied to the immobilized and control tibialis muscles, respectively. The atrophic muscle often could not contract against tensions greater than ~10 g. Supramaximal electrical stimuli of 2 msec duration were applied to the sciatic nerve at 2 Hz for 2 sec every 12 sec (train-of-four pattern) using a Grass S88 stimulator and SIU5 stimulus isolation units. The evoked twitch of the tibialis muscle was recorded on a Type 7500 Linearcorder (Western Graphtec, Irvine, CA) that was calibrated in grams of force.

After stabilization of evoked contractile responses and studies of the changes in muscle contractility produced by disuse (Ibebunjo and Martyn, 1996), indirect train-of-four stimulation was continued for 30 to 60 min after which the sensitivity of the two tibialis muscles to dTC was investigated by the cumulative dose-response method (Hogue et al., 1992). Bolus doses of dTC were given intravenously in increments of 20 µg kg¹ until the first twitch, T1, in the train-of-four was 5% of base-line tension (that is, 95% depression) in both muscles. Each incremental dose was given only when the previous dose had produced maximal effect as indicated by three equal consecutive T1 twitches in both muscles or increasing T1 response in either muscle. The interval between doses was 60 to 120 sec. After the last dose of dTC, evoked twitch was allowed to recover to base-line levels. When T1 first recovered to base-line levels, as well as when the ratio of the fourth to the first twitch (the T4/T1 ratio) first reached unity, the ability of the muscle to sustain contractions at 50 Hz for 5 sec was tested. In five (four immobilized and one sham-immobilized) rats of the day 28 group, 1 ml of venous blood samples was collected when T1 first recovered to 50% of base-line in each muscle. The concentration of dTC in plasma was estimated by reverse-phase high-performance liquid chromatography as described previously (Annan et al., 1990). The lower detection limit of the assay was 10 ng of dTC ml¹ plasma. The mean plasma concentration of dTC when T1 recovered to 50% of base-line on each side was calculated.

The percent depression of T1 relative to base-line was transformed to logit scale and plotted against the logarithm of the cumulative dose by linear regression analysis to determine the ED₅₀ and ED₉₅ of dTC (µg kg¹) for each muscle. The time (minutes) taken for spontaneous recovery of T1 to 50% of base-line, the T4/T1 ratio at recovery of T1 to 50% of base-line, tetanic (50 Hz) fade when T1 first recovered to 100% of base-line as well as tetanic fade when the T4/T1 ratio first equaled unity were determined. Tetanic fade was calculated as follows: [100(tension at start  tension at end of 5 sec of 50 Hz stimulation)/(tension at start)].

Muscle Morphology

At the end of the dose-response study, the rats were euthanized by anesthetic overdose. Both tibialis muscles were dissected out, weighed, rapidly frozen in isopentane precooled in liquid nitrogen and stored at 70°C for muscle histology, AChR assay and RNA extraction.

Fiber and motor endplate size. The diameter of muscle fibers and surface area of motor endplates were determined as reported previously (Karnovsky and Roots, 1964; Robbins et al., 1980). Muscle tissue was dissected at 20°C in a cryostat into 2- to 3-mm thin strips along the muscle length and partially fixed at 4°C in 4% (v/v) glutaraldehyde in buffer containing 0.1 mM dTC to prevent muscle contracture. The specimen was then dissociated into single fibers, incubated at 37°C for 90 min in an acetylthiocholine iodide medium (Karnovsky and Roots, 1964) and fiber segments bearing an endplate were isolated and mounted on microscope slides (Aqua-Mount, Lerner Laboratories, Pittsburgh, PA). The surface area of the cholinesterase-stained endplates on single muscle fibers was measured by tracing the smallest smooth perimeter around each endplate presented en face (Robbins et al., 1980). The diameter of the corresponding fiber was measured, and the ratio of motor endplate surface area to fiber diameter was calculated.

Intramuscular connective tissue. Serial cryostat sections, 10 µm thick, were cut, air-dried and stained with Sirius Red F3BA (Aldrich Chemical Co., St. Louis, MO) as described previously but omitting the rehydration step (Sweat et al., 1964). This stained muscle fibers pale yellow and collagen dark red. The section was scanned, fields devoid of blood vessels other than capillaries were selected and the relative areas occupied by muscle fibers and connective tissue were estimated by isodata thresholding and expressed as a percentage of total muscle area.

Morphometry. Fiber diameter, endplate surface area and intramuscular connective tissue content were measured on live video images captured and processed using a Nikon Diaphot Microscope (Nikon Corporation, Tokyo, Japan) linked to a CCD Video Camera System VI-470 (Optronics Engineering, Goleta, CA) and the image analysis software, MetaMorph (Universal Imaging Corporation, West Chester, PA).

AChR Assay

The amount of AChRs on the muscle membrane was quantitated by ¹²⁵I-BTX binding as described previously (Ward and Martyn, 1993). The frozen muscles were thawed and homogenized at 4°C for 1 min in 4 volumes of 0.01 M potassium phosphate buffer, pH 7.4, containing 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mg ml¹, bacitracin, 2 mM benzamidine hydrochloride and 0.02% (w/v) sodium azide. The homogenate was centrifuged at 20,000 × g for 30 min at 4°C. The precipitates were resuspended and homogenized for 1 min in the same buffer containing an additional 2% (v/v) Triton X-100 (Sigma Chemical, St. Louis, MO), a detergent that extracts AChRs. The extraction procedure was continued overnight, on a shaker, at 4°C. The solution was centrifuged at 20,000 × g for 50 min at 4°C, and the supernatant recovered and stored at 70°C. Triplicate samples of crude muscle extract were incubated with 2.5 nM ¹²⁵I-BTX (specific activity ~16.8 µCi µg¹, DuPont NEN, Wilmington, DE) in the 2% Triton X-100 buffer for 90 min at room temperature. Excess ¹²⁵I-BTX was separated from toxin-bound AChR complexes by vacuum filtration with polyethylenimine-pretreated Whatman GF/B glass fiber filters. Nonspecific binding of ¹²⁵I-BTX was detected by preincubating extracts with excess unlabeled 1 µM BTX. The protein concentration of muscle extract was assayed according to the method of Hartree (Hartree, 1972). The concentration of AChRs was calculated from the molecular weight and specific activity of BTX and counts per minute, and expressed as femtomoles per milligram of protein.

AChR Subunit mRNA Expression

Total muscle RNA was extracted by the acid guanidinium isothiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). The extracted RNA was purified of contaminating DNA with RNase-free DNase (Boehringer-Mannheim, Indianapolis, IN) (Kienzle et al., 1996). The concentration and purity of the RNA was determined spectrophotometrically by measuring the absorbance at 260 and 280 nm: an A260/A280 ratio in the range 1.7 to 2.0 was considered acceptable. The structural integrity of the RNA was verified by electrophoresis on 2% agarose gels followed by ethidium bromide staining to visualize 18S and 28S ribosomal RNA.

The levels of expression of the mRNA encoding for the , , ,  and  subunits of the AChR were quantitated by RT-PCR with radio-labeled oligonucleotides. cDNA was prepared from 1 µg total RNA with the GeneAmp RNA PCR Kit using 2.5 µM oligo-d(T)₁₆ as primer and 2.5 U µl¹ of Murine Leukemia Virus RT in a final reaction volume of 20 µl as specified by the manufacturer (Roche Molecular Systems Inc., Branchburg, NJ). A portion (1 µl) of the cDNA from the RT reaction was then amplified by PCR in a total volume of 20 µl with AChR-subunit specific primers (table 1). Both unlabeled dCTP and ³²P-dCTP were incorporated in the PCR reaction mixture, each at 0.05 mM, to achieve a final dCTP molar concentration of 0.1 mM. The conditions for the PCR reactions are summarized in table 1. Electrophoresis of 15 µl of the PCR reaction product was performed on 5% Tris-Boric acid-EDTA Ready Gels (Bio-Rad Laboratories, Melville, NY). The levels of -actin mRNA were used to control for intersample variations in the amount of RNA used for the RT-PCR reactions (Witzemann et al., 1991). The gels were dried and exposed to X-ray films (DuPont Lighting Plus, Wilmington, DE). The autoradiographs were scanned with a ScanMaker E6 scanner and AdobePhotoshop 3.0 (Adobe Systems, Mountain View, CA), and the intensity of the bands was quantitated densitometrically by NIH Image 1.47 software (NTIS, Springfield, VA).

Statistical Analysis

The sample sizes for the dTC dose-response studies were 9, 11 and 9 at days 7, 14 and 28, respectively, for the immobilization group, and 6 at each time point for the sham immobilization group. Because muscle tissue samples obtained from each animal were limited, tissues from one half of the rats at each time point (n = 4-6) were used for muscle histology, whereas the other half (n = 4-6) was used to study changes in membrane AChRs and the AChR subunits.

One-way analysis of variance for repeated measurements and the Scheffe (post hoc) test were used to investigate significant differences among the day 7, 14 and 28 groups. At each time point, the paired t test was used to compare the immobilized or sham-immobilized tibialis muscles versus their respective contralateral controls. Linear and stepwise regression analyses were used to investigate the associations between muscle sensitivity to dTC and muscle weight, fiber diameter, the ratio of endplate surface area to fiber diameter or membrane AChR content. Differences were assumed to be significant if the P value was .05. Values are expressed as mean ± S.E.M.

	Results

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Effects on Body Weight

Before anesthesia and surgery, the rats that subsequently had one hind limb immobilized for 7, 14 and 28 days gained weight at 7.2 ± 0.5, 7.9 ± 0.4 and 6.2 ± 0.3 g day¹, respectively. Corresponding values after unilateral hind limb immobilization were 2.1 ± 0.8, 3.2 ± 0.7 and 3.4 ± 0.5 g day¹, respectively (P < .01 compared with corresponding presurgery rates). In sham-operated rats, the presurgery growth rates were 7.9 ± 0.7, 7.0 ± 1.0 and 6.1 ± 0.7 for the day 7, 14 and 28 groups, respectively. Corresponding values after sham immobilization were 4.1 ± 1.2, 4.3 ± 0.7 and 3.9 ± 0.5 g day¹ (P > .05 vs. presurgery rates). The growth rates were not different between sham-immobilized and immobilized rats (P > .05).

Muscle Sensitivity to dTC

Dose-response curves. The dose of dTC that produced 50% depression of T1 (ED₅₀) in immobilized and contralateral control tibialis muscles is presented in figure 1. Immobilization for 7, 14 and 28 days increased the ED₅₀ of dTC 3.0-, 3.2- and 2.1-fold, respectively (P < .05 vs. corresponding controls). The ED₅₀ of dTC in sham-operated tibialis muscles did not differ from their contralateral control (P > .05). The ED₅₀ of dTC in contralateral control tibialis muscle was smaller at day 28 than at days 7 or 14 (28.6 ± 2.1 vs. 48.7 ± 6.7 or 43.0 ± 6.2 µg kg¹, respectively; F = 0.04, P > .05; fig. 1). A similar trend was evident in the sham-immobilized group (28 ± 3 vs. 38 ± 8 or 39 ± 7 at day 7, 14 and 28, respectively; P > .05). The reasons for this discrepancy are not clear but might reflect differences in sensitivity among batches of rats or the effects of age. Nonetheless, our conclusion that the immobilized muscle is more resistant to dTC than control muscle remains valid because the rats were randomly allocated to each group and comparisons were of paired muscles (the immobilized or sham-immobilized muscle versus their contralateral controls) from rats within the same group.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   The effects of disuse on the effective dose (ED50) of dTC. The ED50 of dTC was greater in tibialis muscles immobilized for 7, 14 or 28 days than in contralateral control tibialis muscles (P < .01, paired t test). The ED50 of dTC in sham-immobilized tibialis muscles did not differ from their contralateral controls (data not shown). Values are mean ± S.E.M., n = 9-11 (* P < .05; *** P < .001;  P < .0001 vs. Control)

Recovery characteristics. The relative resistance of the immobilized tibialis muscle to dTC was also reflected in several recovery parameters. Spontaneous recovery of T1 to 50% of base-line was 2.93-, 2.91- and 2.30-fold faster in immobilized than in contralateral control tibialis muscles at days 7, 14 and 28, respectively (fig. 2a). The recovery index (time for recovery of T1 from 25 to 75% of base-line) was also faster in immobilized than in control tibialis muscles (fig. 2b). The T4/T1 ratio at 50% recovery of T1 in control tibialis muscles was 52, 36 and 65% of that in tibialis muscles immobilized for 7, 14 and 28 days, respectively (fig. 2c). When T1 first recovered to base-line tension, tetanic fade did not differ between control and immobilized muscles. However, when the T4/T1 ratio first equaled unity, tetanic fade was greater in control than in immobilized tibialis muscles (fig. 2d). Sham-immobilized tibialis muscles did not differ from their contralateral controls in ED₅₀ or recovery parameters of dTC (data not shown).

View larger version (35K): [in this window] [in a new window]   Fig. 2.   The effect of chronic immobilization on the recovery of the tibialis muscle from dTC-induced paralysis. Immobilization produced resistance to dTC as indicated by the (a) shorter time for T1 to recover to 50% of base-line, (b) faster recovery of T1 from 25 to 75% of base-line (recovery index), (c) greater T4/T1 ratio at recovery of T1 to 50% of base-line and (d) lesser tetanic fade when the T4/T1 ratio first reached unity. Values are mean ± S.E.M., n = 9-11 (* P < .05; ** P < .01; *** P < .001;  P < .0001 vs. Control).

Plasma concentrations of dTC. The mean plasma concentration of dTC when T1 first recovered to 50% of base-line in the immobilized and contralateral control tibialis muscles were 682 ± 99 and 291 ± 97 ng ml¹, respectively (P < .05, paired t test). That is, plasma dTC concentration at 50% T1 depression was 2.3-fold greater in immobilized than in control tibialis muscles, in close agreement with the 2.1-fold increase in the ED₅₀ at day 28. The plasma dTC concentrations when T1 first recovered to 50% of base-line in the one sham-immobilized and contralateral control tibialis muscle studied at day 28 were 299 and 178 ng ml¹, respectively.

Muscle Morphology

Muscle weight. Pinning the knee and ankle joints at 90° flexion and 90° dorsiflexion, respectively, immobilized the tibialis muscle in partial flexion. Under these conditions, control tibialis muscles weighed 618 ± 27, 713 ± 19 and 741 ± 30 mg at days 7, 14 and 28, respectively. Corresponding values for immobilized tibialis muscles were 427 ± 24, 391 ± 15 and 451 ± 35 mg, representing 68%, 55% and 61% the weight of contralateral controls, respectively (P < .0001). Sham operation did not alter muscle weight relative to contralateral controls (P > .05). During the 28 days of immobilization, immobilized tibialis muscles did not gain weight (P > .05), whereas contralateral control tibialis muscles did (P < .01).

Muscle fiber and endplate size. The mean diameter of fibers (unclassified as to the type) in control tibialis muscles was 73 ± 2, 81 ± 4 and 75 ± 4 µm at days 7, 14 and 28, respectively. After 7, 14 and 28 days of immobilization, fibers in the tibialis muscle atrophied to 80%, 71% and 78% of control (P < .01), respectively. The mean surface area of motor endplates in control tibialis muscles was 1116 ± 41, 1119 ± 67 and 1006 ± 89 µm² at days 7, 14 and 28, respectively, and did not differ from that for tibialis muscles immobilized for 7 (1096 ± 102 µm²), 14 (1092 ± 85 µm²) or 28 (1280 ± 77 µm²) days (P > .05). Consequently, the ratio of motor endplate surface area to fiber diameter increased 1.25- (P > .05), 1.39- (P < .05) and 1.66-fold (P < .05) after immobilization for 7, 14 and 28 days, respectively (fig. 3).

View larger version (50K): [in this window] [in a new window]   Fig. 3.   The ratio of the surface area of motor endplates to the diameter of the corresponding fiber in immobilized and control tibialis muscles. The ratio of endplate size to fiber size increased after 7, 14 and 28 days of immobilization. Values are mean ± S.E.M., n = 4-6 (* P < .05 vs. Control).

Intramuscular connective tissue. The content of connective tissue varied widely between muscle samples. In control tibialis muscles, connective tissue represented 3.2 ± 0.5%, 2.8 ± 0.7% and 2.5 ± 0.7% of muscle cross-sectional area at days 7, 14 and 28, respectively. Corresponding values for immobilized tibialis muscles were 3.58 ± 0.71, 4.83 ± 1.35 and 2.93 ± 1.01, representing an insignificant increase in intramuscular connective tissue (P > .05, immobilized vs. contralateral control muscles).

Muscle Membrane AChR

The levels of BTX binding sites on the membrane of control tibialis muscles at days 7, 14 and 28 were 19 ± 4, 24 ± 6 and 18 ± 2 fmol mg protein¹, respectively. Corresponding values for immobilized tibialis muscles were 112 ± 31, 150 ± 45 and 21 ± 2 fmol mg protein¹, representing a 6.0- (P < .05), 6.3- (P > .05) and 1.2-fold (P > .05) increase (fig. 4). The level of BTX binding sites in sham-operated tibialis muscles did not differ from controls (data not shown). Nonspecific binding of ¹²⁵I-BTX in control tibialis muscles represented 30 to 50% of total binding, and did not differ between immobilized versus control muscles (P > .05) or among muscles from rats in the day 7, 14 and 28 groups (P > .05).

View larger version (34K): [in this window] [in a new window]   Fig. 4.   The levels of AChRs on tibialis muscle membrane after chronic immobilization. Membrane AChR content was determined by 125I-BTX binding after 7, 14 or 28 days of immobilization and compared with values for the contralateral control muscles. The up-regulation of membrane AChR seen at day 7 of immobilization was normalized by day 28. Values are mean ± S.E.M., n = 4-6 (* P < .05 vs. Control).

Expression of mRNA of AChR Subunits

In control tibialis muscles, only the transcripts encoding for the  and  subunits of AChR were detectable after 20 cycles of PCR amplification of the RT product. The  and  subunits were clearly detectable after 25 cycles, whereas the  subunit was not. Immobilization of the tibialis muscle for 7 days increased the mRNAs encoding for the , , ,  and  subunits 7.8-, 1.6-, 3.4-, 3.5- and 9.6-fold, respectively, relative to contralateral control tibialis muscles (P < .05) (fig. 5). By day 14 of immobilization, the up-regulation had decreased to 2.8-, 1.1-, 3.9-, 2.5- and 6.5-fold of control, respectively, with only the levels of the  and  subunits being significantly greater than controls (P < .05). By day 28 of immobilization there were no differences between immobilized and control muscles in the levels of mRNA encoding for any of the five AChR subunits (fig. 5). Sham-immobilized tibialis muscles did not differ from their contralateral controls in the levels of the transcripts encoding for any of the five subunits of the AChR (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Levels of AChR-subunit mRNAs after chronic immobilization. Seven days of immobilization produced significant up-regulation of the mRNAs encoding for the , , ,  and  subunits of the AChR. By day 14 of continued immobilization, only the levels of the  and  subunit mRNAs were greater than controls. The level of expression of all subunits returned to normal by day 28 of immobilization. Values are mean ± S.E.M., n = 4-6 (* P  .05; ** P < .01; *** P < .001;  P < .0001 vs. Control).

Muscle Sensitivity versus Morphology

Linear regression analysis revealed a positive correlation between the ED₅₀ of dTC versus (a) muscle membrane AChRs (R² = 0.51; P < .0001) or (b) the ratio of motor endplate size to fiber size (R² = 0.30; P < .001) (fig. 6, a and b). The ED₅₀ of dTC correlated inversely with (a) tibialis muscle weight (R² = 0.512; P < .0001) and (b) mean muscle fiber diameter (R² = 0.43; P < .0001) (fig. 7, a and b). Stepwise regression analysis indicated that 64.8% of the variability in the ED₅₀ of dTC could be explained by changes in membrane AChRs (48.5%), fiber diameter (11.1%), tibialis muscle weight (2.7%) and the ratio of endplate size to fiber size (2.5%). On the other hand, 41.4% of the variability in the time for spontaneous recovery of T1 to 50% of base-line could be explained by the changes in muscle weight (18.7%), fiber diameter (14.5%), the ratio of endplate to fiber size (5.7%) and membrane AChRs (2.5%). Muscle sensitivity to dTC did not correlate with intramuscular connective tissue content.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Regression plot of the ED50 of dTC versus (a) muscle membrane AChR content or (b) the ratio of endplate size to fiber diameter in tibialis muscles immobilized (filled symbols), sham-immobilized (open symbols) or unimmobilized (gray symbols) for days 7 (circles), 14 (squares) and 28 (triangles). There was a positive association among these variables.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Regression plot of the ED50 of dTC versus (a) the weight of the tibialis muscle or (b) mean muscle fiber diameter in tibialis muscles immobilized (filled symbols), sham-immobilized (open symbols) or unimmobilized (gray symbols) for 7 (circles), 14 (squares) or 28 (triangles) days. There was an inverse association between the ED50 and these morphologic variables.

	Discussion

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The salient findings from this study are that (1) disuse caused resistance to dTC, the magnitude of which decreased with duration of disuse; (2) the fiber atrophy that follows disuse is not accompanied by a corresponding decrease in the size of the neuromuscular junction, hence disused fibers in effect have larger endplates relative to fiber size; (3) the up-regulation of membrane AChRs induced by chronic disuse is transient and is preceded by a more transient increase in the expression of transcripts encoding all five (including the ) subunits of the AChR; and (4) the resistance to NDMRs produced by disuse can be explained in part by the changes in membrane AChRs, muscle mass, fiber size and the ratio of endplate size to fiber size. Although surgical trauma to muscles and the associated inflammation have been shown to increase sensitivity to agonist drugs, an indirect pharmacologic evidence of AChR proliferation (Katz and Miledi, 1964; Jones and Vrbova, 1974), it is unlikely that they or anesthesia contributed to the present findings because similar effects were not seen after sham immobilization. The present findings can, therefore, be attributed solely to the muscle disuse that followed immobilization.

The present results confirm previous reports that limb immobilization (Gronert, 1981; Gronert et al., 1984; Waud et al., 1985; Fung et al., 1995) and clinical conditions that predispose to disuse atrophy (Martyn et al., 1980, 1992; Dodson et al., 1995) produce resistance to NDMRs. As in the guinea pig (Waud et al., 1985), the resistance was evident by day 7, peaked soon after and was followed by a drop toward control levels by day 28. In the dog, onset of resistance to the NDMR, metocurine, was evident by the fourth day of immobilization produced by plaster casting, and was still present by day 21; removal of the cast at day 21 resulted in gradual return of muscle sensitivity to control levels during another 2 weeks (Fung et al., 1995). It is not clear whether the magnitude of resistance in those dogs would have been sustained had immobilization been allowed to continue beyond the third week. It would appear from the present in vivo and a previous in vitro study (Waud et al., 1985), however, that resistance might begin to wane with continued immobilization. Indirect evidence supports this concept. Fischbach and Robbins (1971), with use of iontophoretic application of ACh to estimate the proliferation of AChRs along fibers in disused rat soleus muscles, showed that the spread of ACh sensitivity which starts 3 to 5 days after disuse returns to control states by 3 weeks despite persistence of disuse. This finding is supported by the present study which provides direct evidence that the sensitivity of disused muscles to ACh antagonists (i.e., NDMRs) begins to return toward normal levels during continued disuse. Also consistent with this concept is the finding that even in denervation, the number of junctional and extrajunctional AChRs decreases with time despite persistence of the denervated state (Andreose et al., 1995).

The resistance to NDMRs that followed muscle disuse was associated with proliferation of AChRs on the muscle at 7 and 14 days but not at 28 days. In turn, the up-regulation of AChRs was associated with an increase in the expression of the transcripts encoding for the subunits of the AChR. The mature AChR is a pentameric protein composed of , , , and  subunits. In embryonic muscles as well as in conditions in which there is deprivation of neural influence (e.g., denervation), the immature AChRs, comprising the , , , and  subunits, are synthesized and distributed both junctionally and extrajunctionally. These immature AChRs differ from mature receptors in several respects: notably their metabolic half-life, mean channel open time, and sensitivity to ACh agonists and antagonists; the immature AChRs are more sensitive to agonists and less sensitive to antagonists such as dTC (Martyn et al., 1992; Steinbach and Chen, 1995). After denervation, expression of the mRNAs coding for all the subunits of the AChR including the  subunit increases, which thus indicates that some of the newly synthesized AChRs are of the immature type (Witzemann et al., 1991). However, these changes in gene expression after denervation do not persist indefinitely; the increased gene expression of AChR subunits and membrane AChRs return to normal (innervated) levels containing predominantly the adult-type AChRs after 1 to 2 months of continued denervation (Adams et al., 1995). The present study demonstrates, probably for the first time, that the up-regulation of AChRs seen after muscle disuse alone is associated with increased expression of all five subunits of the AChR including the  subunit, and that these changes return to normal within 1 month of muscle disuse. Thus, the proliferation of AChRs after disuse is probably a gene-mediated phenomenon akin to that in denervation. However, expression of  subunit mRNA (and, possibly, the membrane expression of immature AChRs) after disuse is somewhat surprising, because there was no physical disruption of the nerve-muscle contact. Nevertheless, the up-regulation of AChRs and its subunit transcripts after immobilization support the hypothesis that both neural factors and electrical activity influence the quantity and quality of AChRs expressed on muscle membranes (Schuetze and Role, 1987; Goldman et al., 1988). It should be emphasized that the immobilization model of disuse studied is probably more reflective of clinical states (e.g., plaster casting, bed-ridden) than other models, such as spinal isolation, which also cause damage to the nerve.

Unlike the changes in AChRs and its transcripts which were transient, the changes in muscle structure (notably fiber atrophy and the increase in the ratio of endplate size to fiber size) persisted throughout the period of disuse. These morphometric findings are consistent with previous reports (Fischbach and Robbins, 1971; Sieck and Prakash, 1995). However, the present findings differ slightly from previous reports (Fischbach and Robbins, 1971; Jozsa et al., 1988) with respect to the rate of onset and magnitude of fiber atrophy and proliferation of connective tissue in muscles after immobilization. The relevance of changes in fiber size or endplate size relative to fiber size to resistance to NDMRs derives from previous findings that muscle sensitivity to NDMRs correlates with these morphologic variables (Ibebunjo et al., 1996b). In the goat, mean fiber size increased in the order: laryngeal < diaphragm < masseter < limbs < abdominal wall muscles, but motor endplate size did not differ among these muscle groups. Hence, the ratio of endplate size to fiber size increased in the reverse order; that is, the ratio was greatest in laryngeal and least in abdominal muscles (Ibebunjo et al., 1996c). The sensitivity (duration of blockade) of these muscles to the NDMR, vecuronium, increased in the same order as fiber size and inversely as the ratio of endplate size to fiber size (Ibebunjo et al., 1996b). Similarly, changes in intramuscular connective tissue content were investigated because the connective tissue might represent the unknown "tissue factor" that was hypothesized to regulate the rates of onset and offset of paralysis by limiting diffusion of NDMRs to and from AChRs at the neuromuscular junction (Armstrong and Lester, 1979). Our results suggest that the intramuscular connective tissue is not the "tissue factor" limiting recovery from blockade (Armstrong and Lester, 1979) .

The present results suggest that the changes in membrane AChRs and fiber size are important predictors (explaining ~49 and 11%, respectively) of the variability in the ED₅₀ of dTC during muscle disuse. In contrast, muscle mass and fiber size may be better predictors of the duration of action of dTC than membrane AChRs or relative endplate size. These results thus support the views that up-regulation of membrane AChRs is an important mechanism for the resistance to NDMRs (Martyn et al., 1992), and that the potency and duration of action of NDMRs might be regulated by slightly different factors (Ibebunjo et al., 1996b). Apparently, taken singly, neither the changes in membrane AChRs, fiber size, muscle mass nor relative endplate size adequately explain the time course of the resistance to dTC, especially at day 28 of disuse. However, these discrepancies can be resolved, and the time course of the resistance explained, if these factors are considered to act in concert as follows: In the early stages (first 2-3 days) of disuse, before fiber atrophy manifests, resistance to NDMRs may result from the sheer numerical increase in membrane AChRs as well as the expression of immature AChRs which are relatively resistant to NDMRs (Martyn et al., 1992; Steinbach and Chen, 1995). These effects of changes in AChRs are later compounded by fiber atrophy and the increase in relative endplate size both of which further enhance resistance to dTC (Ibebunjo et al., 1996b) to the maximum observed at 7 to 14 days of disuse. Thereafter, the gradual decline in the surface expression of membrane AChRs and probably the cessation of synthesis of immature AChRs (as suggested by the decreased expression of mRNAs of all, including the , subunits of the AChR) result in the decline in resistance to dTC toward normal levels at day 28. However, sensitivity does not completely return to normal at day 28 because of persistence of fiber atrophy, the increased relative endplate size and possibly persistence of a small population of -subunit-containing AChRs. That the changes in membrane AChRs, fiber size and relative endplate size together accounted for only ~60% of the variability in the ED₅₀ of dTC produced by chronic disuse suggests that other factors (such as changes in the input resistance of fibers, acetylcholinesterase activity, the quantal content of the endplate potentials and/or the ratio of - to -containing AChRs) probably contribute to the changes in sensitivity to dTC during chronic disuse. These deserve further investigation.

In conclusion, this study with dTC as the prototypical drug confirmed previous findings that muscle disuse produces resistance to NDMRs (Gronert et al., 1984; Waud et al., 1985; Fung et al., 1995). Our findings of decreased resistance to dTC, decreased membrane AChRs and decreased expression of subunits of the AChR confirm previous indirect evidence, obtained from iontophoretic studies (Fischbach and Robbins, 1971), that immobilization-induced up-regulation of AChRs begins to wane in the presence of continued immobilization. Furthermore, this study documents that transcriptionally mediated up-regulation of AChRs can occur even in the absence of nerve disruption. Another important and possibly novel finding is the expression of the mRNA of the  subunit of AChR in the presence of an intact nerve and neuromuscular junction. Disuse also produced fiber atrophy and increased endplate size relative to fiber size, both of which persisted throughout the period of disuse. The results suggest that the magnitude and time course of the resistance to dTC during chronic muscle disuse may be explained, at least in part, by the additive effects produced by qualitative and quantitative changes in AChRs, fiber size and endplate size relative to fiber size, although, when taken singly, neither of these biochemical or structural changes alone adequately explain the resistance to dTC produced by chronic disuse.