Mechanism of Muscle Atrophy (IV)
Changes of Muscle Protein Contents and Components withMuscle Atrophy Induced by the Tail Suspension of Rats

Yoriko Atomi", Shigeru Yamada',
Hideo Hatta", Yoriko Yamamoto *'*' Department Sports Sciences, College of Arts and Sciences' University of Tokyo" Faculty of Education, Universitv of Tol<yo"'Sagami Women's UniversitY
Abstlact

A state of hypokhesia and hypodynamia has bee[ induced in the hindlimb muscles of the rat(200g) using a suspension model. After 4 weeks, the dow-twitch postuial soleus muscle underwent amuch greatel atrophy thal the fast-twitch phasic plantaris muscle. The protein contant per muscle
weight in atrophied soleus was less 28% compared with that of the control muscle, while that in atrophied plantaris muscle was not significantly different with control muscle (6'574)' Muscle protein compolents were analyzed by SDS-PAGE. A specific and siSnificant lQductions wate seen in bands ofmuscle proteins of molecular weights of 20K-Da and 3OK-Da, both of which are inctuded mote insoleus than in plantaris. Further physiological and biochemical analysis arc now under study'Key word!: muscle protein, attophy, rat, toleus
Introduction
Recent yea$, disuse atrophy of muscle induced by tail suspension model of tats is extetNiYely(s,ll,ls,l6,20'2t), because the method is simple and non-invasive for animals andbeing investigated
rapid effects can be obtained. ln previous reports, we examined the tail suspension model developedby Morey et at.(15) and rcported a marked reductiolr of soleus muscle weight and espccially
myofibrillar protein in soleus muscle using paired rats(2).
protein(6'ro). Slower
The loss of muscle mass in disuse is due at least in part to the toss of muscle
protein synthesis, faster protetl degradation, o! concurrent changes of both ptoc$ses can decrease
the deposition of musclc protein(g). It is known that atrophy of muscle in immobilized mt hindlimbsl0) however, the mechanism to induce muscle,
showed the changes of both ptocesses to be responsible(atrophy can not be elucidated by the analysis of oYemll turnovar studies'A plimary pulposa of this study was to identify the ptotein contert and component of soleusmuscle that chalges specifically in association with muscle atrophy and gowth failure'Methods

A[imsl Fourteen male wister rats (?-8 week, 2009) were divided into 2 groups of tail suspension

group (SG) atrd control group (CG). The suspension was induced by a model of Morcy et al'(15)The method in detail was described in tho previous literature(2). After 4 weeks of tail suspension

26
muscles of hildlimb were excized atrd analyzed.

Anllysis of muscle proteir. Non-collagen protein contcnt of muscles was measuled by dye_binding
a'say (Bio-Rad Chemical Co., Ltd. USA) after the extraction of muscle ptotein withgolution(13).

O.2N NaOH

The measurement of the protein composition in muscle was caried out by sodium dodecyl sulfate(sDs) polyacrylamide gel electrophoresis (PAGE) as describcd by Laemml(.2) *-g

ro% and, rs%(includes 2 M Urea) acrylamide gel preparcd in a slab get electrophoresis apparatus with l.O mm gel
thickness. The muscle of rats was homogenized with a glass homogenizer with lo vol. of o.o3 M T sHCt buffer, pH 7.5, containing 0.25 M sucrose (buffered sucrosc solution). Total muscle protetrs weresolubilized in the sDs sampte buffer(r2) by hcating in boiling water for 2 min. Electrophoresis wasperformed at 25mA constant until the tracking dye had reached the end of gel. proteins in gets werestainad by coomassie blue(3). After electrophoresis and staining for protein were complcted, poryacrylamide gel was placed on glass ptate and scanned for densitometry at 640 nm using a Helena scanner.
Tlble I The body w.ight! of expelimental rats.
Body welBht c)

Inltla1

Group (n)Su"ended(7)190■ 5Control(7)189■ 94 wk
253■ 38拿 拿
363■ 27
拿拿pく o ol

Table 2 ■ o mu

le welghts OF the expe― ental rats

Plantaris(mg)Suspended44■ 3拿 拿拿

Control

163■ 7SolcusPlantans(mゴ 1008 BW)
180■ 14・I蠍 拿308■ 818■ 1● 拿。

4512

71■ 385 ■3

Mean r SE, *a*p<0.001

Results

The mcan body wcight oF SC was stgnrlcantly lower than that oF CC after 4‐

week suspenslon(Table l)The muscle welghts OF soleus and plantais OF SC were smflcantly lowerthan that oF CG(Table 2) No slgnncant difference was found h the muscle welght per body welght■ n plantarlsmuscle bctween SC and Cc,while■ n the soleus muscle of SC was slgnrlcantly lower,cOmpared withthat oF CG The protc■ n content of total soleus ln SG was signiflcantly lower both h absolute andrelative(per bOdy wclght)values(Table 3)But that Of plantarls muscle m SC was lower only habsolute value comparcd with that of CG
The quantitative or qualitat市 e changes in the proteh cOmponents of the muscle were analyzed

by means of SDS‐ PACE(10%)Tlle total and s01ublc protems of the s。

were compared wlth thOse ln control rat(Flg l)The ratiOs(%)。

leus muscle m suspended rat

f30k‐ Da and 20k‐ Da protem hsoluble proteln h suspcnded soleus muscle were slgnlflcantly 10wer than that OF control sOleus(F182)

Table3 The non-coll6gen Protein @ntant of total rnurcl€ of the exPerifientrl mulcles'
Plantaris

suspendedControl

Sole$

Ptantldi(m8/g muscle)(mg/muscle)

50■ 034●258■ 1437 2 ■25攣 拿

114■ 7● ●670± 20158■ 4201■ 4215■ 9

Mean i SE,な 'pく 001

30tDa ProtC■ n20k‐ Da ProtehｇＳ
ぢぢ 出 髯つ二ｏ∽薇ぢ い ﹄SCSC
Fじ l SAS p01yacrylamlde gel(10%)eleCtrophoresisof total muscle protehs(B)andFig.2 The relative decrease of 30k-Da rnd 20k-Da
proteins in soluble fnction. *** p<0.001

Юlublemuscle protch(A)■ n soleus muscle from thesuspcnded (S) and the control (O rats
Arrowsshow 30k Da and 20k― Da protelns

In order to examine the telationship between these two proteins and muscle atlophy, the changesof these two proteins in plantaris muscle were determined (Fig. 3). Both proteins are specificallymuch ilr soleus. No marked change was seen in the components of plantaris muscle'Discussion

proteir
In the present study suspended soleus muscle showed ma'ked rcduction (70%) in muscle
whilesus(pervalues,bodyweighi)
content as well as muscle weight both in absolute and relative
werepended plantads muscle showed only one-third of soleus atrophy, despitc that both musclesin themaintained in shortened position. Passive stretch, that is, the length of muscle mairtainedsp""to,immobolization or inactivity is a vely impo ant facto! influencing muscle atrophy(lo'to'").
(MG)andtibialis
gasttocnemius
et al.(19) examined the architecaual alterations of soleus, mcdial

28Plontoris

SS

樺一ア攀
響一一撃
・Soleus

滋一

CCFig.3 SDs-polyacrylamide gel(15%) electrophoresisof soluble proteins in soleus and ptaltaris
muscles from the suspended (S) and control(C) rats. Arows show 30k-Da atd 2Ok-Da
proteins.

anterioi (TA) muscle immobilized at different lengths. As a result, soleus and MG muscle showed amarked atlophy in the shortened position, and were able to maintain muscle weight, better than whenimmobilized neutmlly, in the lengthened position. The level of the changes of both muscle atrophy
were almost cqual. A same change was not found for TA muscle.

They speculated that the difference
was due to that in the natule of the anatomic characteristics of the muscle-skeletal units
crossing thear*le joint. The histochemical analysis did not show a consistent change due to fiber types.
According to Fetl et al.(?) and Musacchia et ar.(l?), atrophy is more extensive and rapid in a slow-twitch postural muscles than in fast-twitch phasic muscles(6,26). In the suspended model, Tempttol
et al.(21) showed a specific and sigaificant dectine in Type I myosin isozyme content occured withouta chatrge in that of rype II. These data shows a selective damage in slow twitch fiber by suspension
model. Reccntly Loughha e161.(r) examined the effect of passive stretch on the synthesis and degladation rate of soleus and EDL (extensor digitorum longus) muscle and basicaly ageed with previousstudies(l l'17) descdbing a geater atrophy of slow-twitch postual muscle compared with fast-twitch
phasic muscles in response to suspension, although the responses of the different muscle types havepreviously been exaggelated by pranterflexed position and different lengths at which muscles wereheld. They also showed that the extent of the atrcphy in stretched muscle in re$ponse to hypokinesia
and hypodynamia was greatly reduced, although ploteiD breakdown remained elevated in that muscle.

In the present study, specific reductions of 3Ok-Da and 20k-Da soluble proteins were observed in
the suspended atrophied muscle. Since these proteirs contained more in postural tonic soleus'muscle

29
than h Fast phasic plantarls musclc,there mtht be posslblc to havc causc result relationship withspecrlc mleus atrophy The functlon of thcse proteins and the mechanlsm oF the reductlon of these
prote■ ns ln unstretched hypoklncsla and hypodynanlla havc not becn clucldatcd lt sccms that manyfactors lnducc or modify the atrophy of muscle ln the ta■

suspcnsion modcl cxcludmg stretchmg(thelcngth oF muscle)and fiber type, such as thc dccrease of nervous strnulus(1'14)and the hcrcasc ofstrcss on the whole body(the hCreasc of glucocorticoid,1'9) It iS necessary to examine the relatio■
shlp between the 20k‐ Da or 30k‐ Da protcln and these Factors,mentioncd abovc,h order to determhe
the function of these speciflc protelns Further analysls ls now under lnvettlgat10n

ln conclusion, the markcd atrophy was associated wih the decrease of muscle protein ln soleus
muscle after 4‐ week wspenslon Specric reductlons of 30k‐ Da and 20k■ Da proteh were observedsIInllltaneouslyReFerences(1)跡 見順子 ,山 田茂 ,琉 子 友男 ,骨 格 筋 の蛋 白異化 に関す る研究 (第 二 報 )除 神経 お よびデキサ メサ ゾ ン投 与が骨 格 筋 お よび心 筋重量 に及 ぼす 影響 ,東 京大学教養学 部体 育学 紀要 19:57‑62,1985(2)Atomi,Y and S Yamada Mechanism oF muscle proteh deradatlon II: A model oF muscle atrophy with
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