Mechanism of Muscle Protein Degradation

3. A Model of Muscle Atrophy with the Tail Suspension of Rat.
byYoriko Atomi and Shigeru Yamada
Department of Sports Sciences, College of Artsand Sciences, University oI Tokyo.

Abstract

A modification of the Morey tail suspension model was examined to determine atrophic responsesof rat muscle unloading of hindlimbs {or 60 days. The conspicuous decrease in the wet weight of soleus
muscle was observed. Ratios of decrease in muscle weight to that of the control rat after 60 dayssuspension of hindlimbs werc 70o/o for soleus muscle ad 17-28/s for the other muscle groups. Muscle
protein componeDts were analyzed by SDS-PAGE. The conspicuous difierence was seen in bands ofmuscle proteins of molecular weights of 43 k-Da and 200 k-Da, which respectively coirespond to actin
aod myosin heavy'chain in the soleus muscle of the suspended hypokinesia rat. In conclusion, theMorey method of suspension can be used as a suitable model for the evaluatioD of the influence of
hypokinesia on contractile proteins of skeletal muscles.
Key 'Words: Muscle atroPh!, Protein degradadation, Rat
IntroductionDisuse atrophy of muscle is curreotly being investigated with an extensive variety of techniques

including cage restraineor), cast 6xation(2), denervation(6), space fligfi1<tt,ro) and tenotomy(r?). In addition

to these more established techniques, another technique, which was recently developed by Morey(ID forthe purpose of simulating zeto gtavity, has also been found to induce muscle atrophy(r3,Ier. The present
iovestigatioD was undertaken to determine the possibility of the Morey model in order to elucidate themolecular mecbanism of disuse muscle atrophy.

The loss of muscle mass in disuse is due at least in part to the loss of muscle protein(7,r0). Slower
protein syntheses, Iaster protein degradation, or concurrent changes of both processes can decrease thedeposition of muscle protein(e). Although it is known that atrophy of muscle in immobilized rat
hindlimbs showed the changes of both processes to be responsibleo.), the mechanism to induce muscle
atrophy can not be elucidated by the method of overall turnover studies_

A primary purpose of this study was to evaluate this Morey model of tail suspension as a meansof identifying the specific muscle protein that changes specifically in association with muscle atrophy andgrowth failure.

Materials and Methods

Anirnal rnodel
A Rat was subjected to suspension hypokinesia using an apparatus modified from that originallydescribed by Morey(l8). After the rat was lightly anesthetized with ethylether, a 1 mm stainless steel
ring inserted through the second caudal vertebra. The staioless steel ring causes little or no tissue
reaction when it is inserted cleanly through the vertebral body. A stainless suspending wire was attachedto the ring by a swivel that allows the rat 360' mobility. The length of the suspending wire wasadjusted so that the hindlimbs did not touch the cage bottom. This form oI suspension permitted therats to walk on their forelimbs and gain access to food and water (Fig. 1).
Two female Wistar strain rats were individually housed in stainless steel cage, of which the floor
and the front panel were made of wire mesh. Water and food were set on the front panel.
Meagurement of muscle protein compoEition
Measurement of the protein composition in muscle was carried out by sodium dodecyl sulfate (SDS)
polyacrylamide gel electrophyoresis (PAGE) as described by Laemmli(ra) using 10/6 acrylamide gel
prepared in a slab gel electrophoresis apparatus with 1.0mm gel thickness. the muscle of rats was
homogenized with a glass homogenizer with 10 vol. of 0.03 M Tris HCI buffer, pH 7.5, containning 0.25
M sucrose (buffered sucrose solution). Total muscle proteins were solubilized in the SDS sample buffer(1r)by heating in boiling water for 2 min. Electrophoresis was performed at 25 mA constant until thetracking dye had reached the end of the gel. Proteins in the gels were stained Coomassie bltre(s). Afterelectrophoresis and stainning for protein were completed, poryacrylamide gel was placed on a glass plateand scanned for densitometry at 640 nm using a Helena scanner.
Resulta

Body weights of the control and the suspended rat before aod after 60 days of experiment wereFig. I The tail suspension of the rat.

Table 1 Muscle weightg of experimental rats (gram).
ControlMuscles

0 311Plantaris
1Soleus1

Suspended Difference % Difference
0 ル20 3120 229

0 311

0 226

0 157

0 046

0 148

0 044

0 153

0 045

0 138

0 116

10 1420 117

mean0 140

0 116

1 140

1 142

1 456

1 076

1 448

1 1090 575

0 471

0 550

0 471

0 562

0 471

EDL

Castrocnemlus
1TA
10 08627 50 108

706

0 024

16 90 339

23 40 092

163

r: right leg, l: left leg, difierence: diflerence between the control
and the suspended leg.

L5Fig 2 A

SDS-polyacrylamide gel electrophoresis of muscle proteins. The soleus
muscle from the suspendered (S) and control (C) rats were removed at
60 days a{ter operation.
Electrophoretograms o{ muscle proteins traced by a densitometer.
Arrows indicate ihe position of the proteins oI molecular weights
corresponding myosin heavy-chain and actin.

11188 and 289 g, and 197 and 260 g, respectively.

Muscle weights of hindlimbs of rats were shown in Table 1. The wet weight of the soleus muscleof the suspended rat \nas 7oo/o smaller than that of the control rat. The other musclular weights of thesuspended rat were 17 to 28% smaller than those of the control.
The quantitative or qualitative changes in the proteio component of the muscle were analyzed by meansof SDS-polyacrylamide gel electrophoresis. Total proteio of the soleus muscle in suspended rat werecompared with those of the control rat (Fig. 2). Marked difierences in the staining intensity were seenia the protein bands oI molecular weights which correspood to actin and myosin heavy chain. Differences in the amount of these proteins in the muscle preparation weres qualtitated by the densitometric
tracing of the stained gel (Fig. 1-b). Evidently, peak area of tracing of the these proteins was markedly
smaller in the preparation from the suspended muscle than that from the control.
Discussion

The rat adjusted quickly to the suspended position, as indicated by their eating, drinking and grooming
activities, while a little difierence in the growth of the body weight was seen in the suspended rat of theMorey model.

The suspension for 60 days with the Morey method in the present study induced the conspicuous
atrophy of the rat soleus muscle. The duration of 14 to 30 days might be enough for the examinationof the atrophy of the skeletal muscle compared with other data(12'r5 2') obtained by the same Moreymethodo8). This marked decrease observed in toe soleus muscle weight agreed with the soleus atrophy
consistently found in other disuse models of the rat muscle(3'r720) aod with the observation of protein

degradation elicited by the Morey rnodel(r8).

The mechanism oI the disuse muscle atrophy has not yet been elucidated. The analysis of muscle
protein components may give the solution of the mechanism of the disuse muscle atrophy. Our inves-tigation found that the Morey model for hypokinesia might evoke a decrease in the soleus contractile
protein. Templeton et al.(") observed that the Morey model for hypokinesia evoked a decrease in soleus
contractile fuoction and the decrease in type 1 myosin cortent. A reduction of slow-twich fibers in the soleus
has been reported Jrom rats(3) and guinea pigs(6). Small-cage confinement of rats also evoked a decline
ill slow-twich 6bers(2r). Similarly, the vastus intermediate muscles, the slow muscle in the thighanalogous to the soleus showed a decline in the percentage of slow-twich 6bers with immobilization
without a change in fiber oumbers({). According to the analysis of myosin isozyme data by Templeton

et al.(2r), which show no significant change in the fast-twich component alter either 2 or 4 week o{suspension while, showing a cootineous and significant decline in the slow-twich componert, thepreferential slow-twich fiber atrophy was evident.

The marked atrophy oI the rat soleus was evident in our previous study(1), in which determined theefiect oI denervation, compared with other hindlimb muscles Jakubiec-Puka et a1'(r3) have reported
evidence that the denervation atrophy o{ the rat soleus produces a lower propotion of myosin heavy'chain
to actin. Whether a change in a neural activity is related to soleus atrophy with the Morey model will

require further investigation.

In conclusion, the Morey method of suspension can be used as a suitable model {or a simulation of
the influence of hypokinesia on characteristics of the contractile muscle protein in the rat'

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