INTRODUCTION

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THE TWO PRIMARY SITES OF muscle protein breakdown are the lysosome and the cytoplasm. Although muscle atrophy (i.e., wasting) results from either denervation (i.e., nerve severing) or unweighting, the cellular mechanisms of protein loss in these models appear to be fundamentally different. With denervation atrophy, the lysosome seems to be recruited for protein destruction (17). In vitro incubation of denervated muscles with lysosome inhibitors including chloroquine (19), methylamine (14), or leucine methyl ester (8, 9) caused decreased protein degradation relative to control muscles (17). Furthermore, intramuscular administration of chloroquine lowers in vivo protein degradation of denervated muscle (17). In contrast, accelerated proteolysis resulting from unweighting atrophy is related to greater protein breakdown in the cytosol rather than an increased response of the lysosome (17).

When muscles are subjected to both unweighting and denervation, the effect of nerve severing predominates and causes more severe atrophy (15). Similar results have been obtained in comparing denervated and immobilized muscles (4). Therefore, maintaining an intact nerve supply, with muscle unweighting or immobilization, offers some protective effects. We hypothesize that this effect of intact nerves is a consequence of one or more neurotrophic factors. This premise is based on reports that injection of sciatic nerve extract into denervated extensor digitorum longus (EDL) muscles results in smaller decreases in muscle weight, protein content, and fiber cross-sectional area (3, 4).

To evaluate our hypothesis, we compared the effects of proximal (short nerve stump) and distal (long nerve stump) denervation in contralateral soleus muscles at 24-96 h after severing the tibial or sciatic nerves, respectively. We reasoned that the long nerve stump might retain neurotrophic influence longer than with proximal denervation. Accordingly, severing the sciatic nerve does not cause significant atrophy until 48 h after denervation (7). Besides analyzing total muscle protein content, we monitored the time course of responses of in vitro protein degradation and lysosomal latency to link the influence of neurotrophic substances to breakdown of muscle protein. To verify the denervation surgical technique, we also analyzed protein content of the EDL, which would not be affected by severing the tibial nerve.

	MATERIALS AND METHODS

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Animals and experimental treatment. Procedures were approved by the University of Arizona Animal Care and Use Committee. Juvenile female Sprague-Dawley rats (~100 g; Sasco, Omaha, NE) were used and maintained on food and water ad libitum. In other studies, we have found no difference in food intake between control and denervated rats (15). Rats were anesthetized with xylazine (8 mg/kg) plus ketamine (63 mg/kg) during surgery. We selected the soleus as the primary muscle of interest for consistency with previous studies from this laboratory. One hindlimb was denervated proximally and the other distally to the soleus so that contralateral limbs could be compared directly, as described previously (5). For distal denervation (long nerve stump), a 3-mm section of the sciatic nerve was removed above the femoral trochanter in the sciatic notch, thereby affecting all hindlimb muscles on that side. Proximal denervation (short nerve stump) of the soleus consisted of excising a 6- to 10-mm section of the three branches of the tibial nerve where it passes between the two heads of the gastrocnemius. This procedure left the EDL innervated, providing a control that was not expected to atrophy in the proximally denervated leg. Measurements were made 24, 48, 72, or 96 h after denervation.

At the end of the experimental period, animals were killed by cervical dislocation while under anesthesia. The soleus or EDL muscles from both hindlimbs were rapidly dissected and weighed. These tissues were analyzed for total protein, in vitro protein degradation, or lysosomal latency.

Total protein. After excision, soleus and EDL muscles were homogenized in 10% trichloroacetic acid (TCA) and centrifuged for 5 min at 3,000 g. The pellets were washed once with 10% TCA and then once with ethanol:ether (1:1). After the protein pellets were solubilized in 2 ml of 0.5 N NaOH, total protein was assayed by the Biuret procedure (11).

In vitro protein degradation. Protein degradation was measured as the release of tyrosine from muscle protein in the presence of cycloheximide, an inhibitor of protein synthesis (6). Soleus muscles were placed into a 25-ml flask containing 3 ml of oxygenated (95% O₂-5% CO₂) Krebs-Ringer bicarbonate buffer (120 mM NaCl, 25 mM NaHCO₃, 4.8 mM KCl, 2.5 mM CaCl₂, 1.25 mM MgSO₄, 1.21 mM KH₂PO₄, pH 7.4) with 1.2 nM chloramphenicol, 0.5 mM cycloheximide, and 5 mM glucose. After a 30-min preincubation period at 37°C in a shaking water bath, muscles were incubated for 2 h in a fresh 3 ml of buffer. Subsequently, muscles were removed and the incubation medium was stored at 20°C. Before analysis, the medium was thawed and centrifuged at 760 g for 10 min at 4°C. Acid-soluble tyrosine was assayed in a 0.2-ml sample of the medium according to the method of Waalkes and Udenfriend (18), as modified previously (16). In vitro protein degradation was calculated from tyrosine accumulation during the 2-h incubation period and expressed per milligram wet weight of soleus.

Lysosomal latency. The aggregate assay of cathepsins B, H, and L was used to evaluate the relative latency of soleus muscle lysosomes according to Odessey (12), as modified previously (17). After weighing, the muscle was minced on ice in 3 ml of 10 mM 3-(N-morpholino)propanesulfonic acid (pH 7.4), 20 mM KCl, 1 mM EDTA, 10 mM sodium pyrophosphate, and 250 mM sucrose and then homogenized in a Duall tube using a motor-driven tissue grinder fitted with a ground glass pestle. The homogenate was diluted with ice-cold buffer to a concentration of 5-6 mg muscle/ml and then centrifuged at 760 g for 5 min at 4°C. For measuring total cathepsin activities, Triton X-100 (0.2% final concentration) was added to a portion of the supernatant solution. Then, a 1.5-ml aliquot of the supernatant solution, with or without Triton treatment, was diluted with 1.5 ml of 50 mM 2-(N-morpholino)ethanesulfonic acid (pH 5.5), and 0.5 mM dithiothreitol. The mixture was warmed to 37°C, and the pH was adjusted to 5.5 using 2 mM HCl (5-15 µl). The reaction was initiated by mixing a 0.6-ml aliquot of the diluted supernatant with 0.1 ml of 2.1 mg/ml polyGAT (a random polymer of glutamate, alanine, and tyrosine; Sigma Chemical). After 40 min of incubation at 37°C, the reaction was terminated by addition of 1.0 ml of 40% TCA. Acid-soluble tyrosine was assayed in 0.5 ml of the reaction mixture and in a blank containing the same amount of tissue extract. Latency was calculated as the difference in activity in the presence and absence of Triton expressed as a percentage of the total activity measured after Triton treatment.

Statistics. Total protein, in vitro protein degradation, and lysosomal latency were plotted as a function of time. An analysis of variance was used to examine the effect of denervation over time. A paired Student's t-test was used to compare the effect of proximal versus distal denervation. All statistical analyses were performed with Statview 4.02 (Abacus Concepts).

	RESULTS

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Time course after denervation. Denervated EDL muscles showed little decline in total protein content (Fig. 1) or in muscle mass (44 ± 1 to 38 ± 2 mg/100 g body wt). Tibial nerve severing (proximal), which does not denervate EDL, produced no decline in total protein content or muscle mass (P > 0.05). On the other hand, sciatic nerve severing (distal), which denervates the EDL, caused a significant (P < 0.05) decline in total protein (Fig. 1) and muscle mass (11%; not shown) in the EDL by 48 h. After 96 h, total protein content of the distally denervated EDL decreased by only 11%, with no further decrease (P > 0.05) in mass. The ability to demonstrate a significant loss of protein in the EDL after severing the sciatic but not the tibial nerve shows our success in this approach to selective denervation.

View larger version (K): [in this window] [in a new window]   Fig. 1.   Total protein content of extensor digitorum longus (EDL) plotted as a function of time after denervation of soleus. Rats were denervated proximal to the soleus in 1 hindlimb and distal to the soleus in the contralateral hindlimb. Number of animals: control, 5; 24 h, 5; 48 h, 11; 72 h, 5; 96 h, 5. x Significantly different from control muscle [analysis of variance (ANOVA), P < 0.05].

Denervation of the soleus resulted in a significant decline in total protein content (Fig. 2) and muscle mass (not shown), except during the first 24 h. Initial soleus mass was 42 ± 1 and 40 ± 1 mg/100 g body wt after 24 h of proximal or distal denervation. By 48 h, total protein content was significantly (P < 0.05) lower in both the distally (11.4%) and proximally (22.6%) denervated soleus than in control muscles, and the proximally denervated muscles had lost significantly (P < 0.05) more protein. Also by 48 h, soleus mass declined to 30 ± 1 mg/100 g body wt in the proximally denervated muscle versus 32 ± 1 mg/100 g body wt with distal denervation. Seventy-two hours after denervation, these differences were still apparent because total protein had declined by 22.8 and 30.8% in the distally and proximally denervated muscles, respectively; respective muscle weights were 26 ± 1 and 30 ± 1 mg/100 g body wt. However, after 96 h, total protein content of the soleus was only 55% of control values in both distally and proximally denervated muscles and muscle mass did not differ (23 ± 1 mg/100 g body wt in both).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Total protein content of soleus plotted as a function of time after denervation. Rats were denervated as in Fig. 1. Number of animals: control, 15; 24 h, 13; 48 h, 11; 72 h, 9; 96 h, 18. x Significantly different from control muscle (ANOVA, P < 0.05). * Significantly different from contralateral limb (paired Student's t-test, P < 0.05).

To determine whether the differences in atrophy between proximal and distal denervation of the soleus could be attributed, in part, to proteolysis, we analyzed in vitro protein degradation. Protein degradation increased in the soleus following denervation (Fig. 3). This increase was first significant (P < 0.05) 48 h after denervation in both proximally and distally denervated soleus muscles. However, at 48 or 72 h protein degradation was faster in the proximally denervated muscle. After 96 h protein degradation in both muscles was 63% faster than in the control muscles. Therefore, the pattern of increased protein degradation following denervation paralleled the decline in total protein content of these muscles.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   In vitro protein degradation measured in soleus plotted as a function of time after denervation. Rats were denervated as in Fig. 1. Number of animals: control, 12; 24 h, 12; 48 h, 12; 72 h, 12; 96 h, 10. x Significantly different from control muscle (ANOVA, P < 0.05). * Significantly different from contralateral limb (paired Student's t-test, P < 0.05).

Because we had shown previously an increased response of lysosomal proteolysis to denervation (17), we monitored the time course of changes in lysosomal latency following proximal or distal denervation of the soleus. Lysosomal latency declined following denervation, indicating an increased response of the lysosome (Fig. 4). This decline in latency was significant by 24 h after either proximal or distal denervation. In innervated muscle, 54% of the lysosomal activity was latent, whereas only 23% was latent at 96 h after denervation. At 48 and 72 h, latency was lower (P < 0.05) in the proximally denervated than in the distally denervated muscles. Therefore, the increased lysosomal response paralleled the decline in total protein (Fig. 2) and the increase of protein degradation (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Lysosomal latency plotted as a function of time after denervation. Rats were denervated as in Fig. 1. Number of animals: control, 17; 24 h, 21; 48 h, 11; 72 h, 9; 96 h, 9. x Significantly different from control muscle (ANOVA, P < 0.05). * Significantly different from contralateral limb (paired Student's t-test, P < 0.05).

	DISCUSSION

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The decline in total protein we found following denervation is consistent with published data. Both the soleus and EDL atrophy following denervation, although the soleus atrophies more rapidly than does the EDL (10). After 4 days, the denervated soleus lost 45% of total protein content, whereas EDL in the distally denervated limb lost only 11% (Figs. 1 and 2). These results are surprising because denervation may affect type IIb fibers more than type I fibers (13) and the EDL contains 38% IIb fibers and only 3% type I fibers whereas the soleus contains 84% type I fibers and no IIb fibers (1).

The increase in in vitro protein degradation we found following denervation also is consistent with previous reports. Protein degradation in rat soleus has been reported to increase by ~80% 4 days after denervation (10). Although our values for protein degradation are slightly higher than those reported previously (10) (0.9 vs. 0.7 nmol Tyr · mg¹ · 2 h¹), we found a similar percent increase in protein degradation 96 h after denervation (Fig. 3).

We have previously demonstrated that denervation decreases lysosomal latency in rat soleus (17). Seventy-two hours after denervation, lysosomal latency declines from 50% in innervated muscles to 35% in denervated muscles. This change in latency is similar to that measured in the present study (Fig. 4). Others have reported an increase in cathepsin B activity in soleus muscle following denervation (7). However, whereas we found a decline of in vitro proteolysis when lysosomal activity was inhibited by chloroquine, methylamine, or leucine methyl ester (17), this other study reported that the addition of the lysosomal inhibitors leupeptin, methylamine, or E-64c did not alter net protein breakdown (7). Dissimilar incubation conditions in the two studies may account for the different results.

Previous studies showed that injection of a crude nerve extract decreases the extent of atrophy in denervated EDL muscles over a period of 1-2 wk (2-4). Denervated rat EDL exhibits a decline in wet weight, total protein content, and cross-sectional area of type IIb fibers after 7 days (3). Injection of a crude sciatic nerve extract partially reverses these consequences of denervation (3), supporting the role of a neurotrophic factor that maintains muscle size. Comparisons of denervated EDL with contralateral muscles that were either immobilized or denervated and injected with nerve extract suggested that loss of the neurotrophic influence is responsible for at least 40% of the atrophy, with the remaining 60% being attributed to disuse atrophy (4). Despite these findings, the mechanism of action of neurotrophic factors remains unclear.

In the present study, distal (long nerve stump) and proximal (short nerve stump) denervation of the rat soleus muscle was used to evaluate the potential role of loss of neurotrophic factors on muscle atrophy. These data showed a parallel pattern for the responses of loss of total protein content (Fig. 2), increase in in vitro protein degradation (Fig. 3), and decrease in lysosomal latency (Fig. 4). For each parameter, at 48 and 72 h after denervation the proximally denervated muscle elicited a greater response. These results are consistent with the hypothesis that a neurotrophic factor released by the nerve may attenuate protein breakdown by lowering lysosomal proteolysis in the innervated soleus. Consequently, with a longer nerve stump, atrophy, accelerated protein degradation, and declining lysosomal latency were somewhat delayed compared with the muscle with a short nerve stump.

In summary, our data support the hypothesis that denervation decreases total protein, in part, via increased protein degradation involving the lysosome. Our data comparing distal and proximal denervation suggest the loss of a neurotrophic factor contributes to this mechanism of protein loss. Current efforts in our laboratory are examining the role of the neurotrophic factor in regulating lysosomal activity.

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