Skeletal muscle HSP72 response to mechanical unloading: influence of endurance training

Skeletal muscle HSP72 response to mechanical unloading:

influence of endurance training

D. Desplanches,1 L. Ecochard,1 B. Sempore,1 M.-H. Mayet-Sornay1 and R. Favier1,2

1 Unit�e Mixte de Recherche 5123 Centre National de la Recherche Scientifique, Laboratoire de Physiologie, Universit�e Claude

Bernard, Lyon Cedex 08, France

2 Equipe Mixte INSERM 0221, Laboratoire de Bio�energ�etique Fondamentale et Appliqu�ee, Universit�e Joseph Fourier, Grenoble Cedex

09, France

Received 28 July 2003,

accepted 4 December 2003

Correspondence: Dr R. Favier,

EMI 0221, Laboratoire de

Bio�energ�etique Fondamentale et

Appliqu�ee, Universit�e Joseph

Fourier, BP 53X, 38041 Grenoble

Cedex 09, France.

Abstract

Aims:1 It has been shown that increased contractile activity results in heat

shock protein 72 (HSP72) accumulation in various skeletal muscles. By

contrast, there is no consensus for muscle HSP72 response to muscle disuse

for short duration (5–8 days). On the basis of a greater constitutive HSP72

expression in slow-twitch muscles we tested the hypothesis that mechanical

unloading for a longer period (2 weeks) would affect this phenotype to a

greater extent. Secondly, we evaluated the effects of a physiological muscle

heat shock protein (HSP) enhancer (endurance training) on HSP response to

unloading and muscle remodelling.

Methods: Adult male Wistar rats were assigned randomly to four groups:

(1) sedentary weight-bearing; (2) hindlimb-unloaded (HU) via tail suspension

for 2 week; (3) trained on a treadmill (6 week) and (4) trained 6 week and

then HU for 2 week.

Results: Unloading resulted in a preferential atrophy of slow muscles [soleus

(SOL), adductor longus (AL)] and a slow-to-fast fibre transition with no

change in HSP72 level. HSP72 levels were significantly lower in fast muscles

[extensor digitorum longus (EDL) and plantaris (PLA)], and did not change

with mechanical unloading. Endurance training was accompanied by a small

(SOL) or a large (EDL, PLA) increase in HSP72 level with no change in AL.

Training-induced accumulation of HSP72 disappeared with subsequent

unloading in the SOL and PLA whereas HSP72 content remained elevated in

EDL.

Conclusion: The results of this study indicate that (1) after 2 weeks of

unloading no change occurred in HSP72 protein levels of slow-twitch muscles

despite a slow-to-fast fibre transition; and (2) the training-induced increase of

HSP72 content in skeletal muscles did not attenuate fibre transition.

Keywords heat shock protein, hindlimb suspension, muscle fibre, treadmill

exercise.

Cells exposed to various environmental and/or meta-

bolic stresses respond by the rapid transcription and

subsequent translation of a set of highly conserved

polypeptides referred to as heat shock proteins (HSP).

The major HSPs have been classified according to their

molecular sizes, some of them being constitutively

expressed (e.g. HSC73) whereas other isoforms (e.g.

HSP72) are not generally observed in normal unstressed

cells and were initially considered to be strictly indu-

cible. In skeletal muscles, the HSP72 isoform seems to

be proportional to the percentage of type I fibres with

the slow oxidative muscle [soleus (SOL)] having the

Acta Physiol Scand 2004, 180, 387–394

� 2004 Scandinavian Physiological Society 387

highest HSP72 levels (Locke et al. 1991, 1994). The

physiological significance of HSP72 accumulation in

slow-twitch muscles remains largely unknown but

this protein exerts fundamental housekeeping and

homeostatic functions, acting as molecular chaperone,

and playing a major role in the control of protein

maturation and adequate protein folding, transport,

translocation and degradation (Georgopoulos & Welch

1993, Locke 1997).

In contrast to a rather extensive literature on the effects

of sustained increased contractile activity on HSP72

levels (Neufer et al. 1996, Hernando & Manso 1997,

Ecochard et al. 2000, Gonzalez et al. 2000, Mattson

et al. 2001, Ornastsky et al. 19952 , Samelman 2000), the

influence of muscle disuse on HSP72 expression has been

only scarcely examined and the conclusions are rather

contradictory. Initially Kilgore et al. (1994) reported

that pectoralis muscle of the blue-winged teal expresses

high levels of HSP72 during periods of both decreased

and increased muscle use. On the contrary, Naito et al.

(2001) reported a substantial decrease in muscle HSP72

content following 8 days of hindlimb unloading (HU).

By using a combination of procedures to reduce muscle

loading (hindlimb suspension, tenotomy) and/or neuro-

muscular activation (denervation), Oishi et al. (2001)

have shown that HSP72 levels were not affected by

5 days of unloading alone but decreased substantially

when HU was superimposed to tenotomy and denerva-

tion. These data suggest that the levels of HSP72 in

skeletal muscles are clearly dependent upon the intensity

of mechanical unloading and/or neuromuscular activa-

tion. In this respect, muscle unloading up to 4–5 weeks

was found to result in drastic reduction in electromyo-

graphic (EMG) activity (Blewett & Elder 1993) and

enhanced muscle atrophy with a large slow-to-fast fibre

shifting in slow, but not in fast muscles (Desplanches

et al. 1987, 1990, Thomason et al. 1987, Thomason &

Booth 1990). Alterations in muscle fibre typology could

be determinant for HSP72 expression during muscle

atrophy since HSP72 is expressed in proportion to the

type I/b-myosin heavy chain (MHC) muscle fibres (Locke

et al. 1991, 1994, Locke 19973 ).

In this study, we questioned whether the mitigated

HSP72 response to 5–8 days of HU (Naito et al. 2001,

Oishi et al. 2001) was linked to the short unloading

duration. Indeed, extending HU up to 4–5 weeks resul-

ted in a further histological, biochemical and electro-

physiological alterations in slow muscles (Desplanches

et al. 1987, 1990, Thomason et al. 1987, Thomason &

Booth 1990, Blewett & Elder 1993, Schulte et al. 1993).

Based on muscle atrophy (Desplanches et al. 1987,

1990), rate of protein breakdown (Thomason & Booth

1990), slow myosin content (Thomason et al. 1987),

fibre typology (Desplanches et al. 1987) and EMG

activity (Blewett & Elder 1993), 2 weeks seems to be

the optimum duration for studying the effects of HU on

skeletal muscle properties. We reasoned that HSP72

response to HU could be clearly demonstrated if the

drastic decrease in slow fibre/myosin content (Desp-

lanches et al. 1987, Thomason & Booth 1990) was the

primary stimulus for HSP72 response to mechanical

unloading. We examined two slow antigravity muscles

(SOL, adductor longus (AL)], which are known to

display both muscle atrophy and slow-to-fast fibre

transition after unloading (Desplanches et al. 1987,

Thomason & Booth 1990). The results were compared

with those obtained on two fast muscles [plantaris

(PLA), extensor digitorum longus (EDL)], which display

only minimal atrophy and fibre shifting with unloading

(Desplanches et al. 1987, Thomason & Booth 1990).

The second aim of the study was to evaluate the effects

of a natural physiological muscle HSP enhancer (en-

durance training) on HSP response and muscle atrophy

to subsequent HU. Indeed, endurance training resulted

in HSP72 accumulation in various skeletal muscles

(Ecochard et al. 20004 , Gonzalez et al. 2000, Samelman

2000, Mattson et al. 2001) and Naito et al. (2001)

suggested that HSP72 accumulation by whole body

hyperthermia was responsible for the attenuation of HU

muscle atrophy. On the basis of a higher protein content

and fractional protein synthesis in trained- as compared

with sedentary-HU rats (Munoz et al. 1994), we hypo-

thesized that HSP72 could play a role in the control of

muscle remodelling during reduced contractile activity.

Methods

Animals

The present investigation was performed following the

recommendations provided by the European Conven-

tion for the protection of Vertebrate Animals used for

Experimental and Scientific purposes (Strasbourg,

19855 ). Male Wistar rats from IFFA-CREDO (Saint

Germain sur l’Arbresle, France) were housed one per

cage in a temperature controlled room at 22 � 2 �C,

with a dark–light cycle of 12–12 h. They were provided

with a standard rat chow and tap water ad libitum, and

acclimatized to their new environment for 1 week. They

were randomly assigned to four groups: a control

sedentary weight-bearing group (S-WB, n ¼ 15), a

sedentary hindlimb-unloaded group (S-HU, n ¼ 15),

an endurance-trained weight-bearing group (T-WB,

n ¼ 15), and a group initially endurance-trained and

then hindlimb-unloaded (T-HU, n ¼ 15).

Hindlimb suspension

Half of the rats (S-HU and T-HU groups) were

subjected to 2 weeks of hindlimb unloading using the

388 � 2004 Scandinavian Physiological Society

Muscle HSP72 expression with training and suspension Æ D Desplanches et al. Acta Physiol Scand 2004, 180, 387–394

tail suspension model (Desplanches et al. 1987, 1990).

Briefly, an orthopedic tape surrounding the cleaned tail

was connected to the top of the cage where a swivel

allowed 360� rotation. The unloaded rats were elevated

in a head-down position so that the hindlimbs could not

reach the cage floor or walls whereas the forelimbs

allowed movements and access to food and water.

Training

Half of the rats (T-WB and T-HU groups) were

submitted to endurance training as described pre-

viously (Abdelmalki et al. 1996). Exercise duration

and intensity were increased progressively and involved

running 5 days per week on a motor-driven treadmill

(TecMachine, Andr�ezieux-Bouth�eon, France). Initially

the rats ran for 10 min at 30 m min)1, 10% slope.

After 1 week at this intensity of exercise, the duration

of bouts was progressively increased, so that the

animals were running 35, 60 and 85 min after 2, 3

and 4 weeks of training respectively. During the last

2 weeks of training, rats ran 90 min day)1 at

30 m min)1, 10% slope.

Tissue sampling

To limit acute effects of exercise on muscle adaptations,

trained rats were sacrificed 24 h after the last bout of

running. They were anaesthesized with pentobarbital

sodium (50 mg kg)1 i.p.). On the basis of fibre type

distribution, selected muscles of the hindlimb were

removed and weighed. Slow oxidative (SOL, AL) and

fast oxidative-glycolytic (PLA, EDL) muscles were

frozen in isopentane, cooled in liquid nitrogen and

stored at )80 �C.

Biochemical analysis

Fifty milligram frozen samples of muscle were homo-

genized in a ground-glass homogenizer with 1 mL of

Tris (hydroxymethyl)aminomethane 25 mm (pH 7.4) at

0 �C to determine spectrophotometrically citrate syn-

thase (CS) activity as previously described (Abdelmalki

et al. 1996, Ecochard et al. 2000). For relative quan-

tification of HSP72, we performed polyacrylamide gel

electrophoresis and immunoblotting. Protein concen-

tration was measured using a standard method (Lowry

et al. 1951) and 200 lL of muscle homogenate con-

taining either 20 lg (slow muscles) or 50 lg (fast

muscles) of proteins were mixed with 200 lL of buffer

containing 40 mm Tris(hydroxymethyl)aminomethane

pH 6.8, 1% sodium dodecyl sulphate (SDS), 6%

glycerol, and 1% b-mercaptoethanol. This mixture

was then heated at 100 �C for 15 min, and subjected to

one-dimensional SDS–polyacrylamide gel electrophor-

esis (SDS–PAGE) with a 5% stacking and 12.5%

resolving gels for 12 h. After electrophoretic separ-

ation, proteins were transferred at a constant voltage to

nitrocellulose membranes. After protein transfer, the

membranes were blocked for 2 h, then incubated 2 h

with a monoclonal antibody specific for HSP72 (SPA

810, StressGen6 Biotechnologies, Victoria, BC, Canada)

and then reacted with the secondary antibody (goat

anti-mouse immunoglobulin G conjugated to horse-

radish peroxidase, Bio-Rad). HSP72 were visualized by

the enhanced chemiluminescence (ECL) detection

method (RPN 2106, Amersham Pharmacia Biotech

Europe GmBH, Saclay, France7 ). To confirm relatively

even loading among samples, the superior gel portion

not used for immunoblotting was stained with 0.5%

Coomassie Brilliant Blue. Transfer efficiency was veri-

fied by staining polyacrylamide gels with 0.5% Coo-

massie Brilliant Blue G and nitrocellulose membrane

with Ponceau S. Bands from blots were quantified

using a Kodak EDAS 120 system including digital

camera and image analysis software (Eastman Kodak,

Rochester, NY,8 USA). For analysis of the ECL films,

the mean density of each band was taken to

calculate the amount in each sample and the data are

expressed numerically as integrated optical density

(arbitrary units).

Histochemical analysis

The midbelly of the muscle was cut perpendicular to its

longitudinal axis into serial 10-lm thick cross-sections

with a cryotome ()30 �C) and stained for the myosin

adenosine triphosphatase (ATPase). After preincubation

at pH 4.4 in acid buffer (50 mm acetic acid) with

25 mm CaCl2 for 4 min at 25 �C, the ATPase reaction

was carried out in buffer (pH 9.4) with 18 mm CaCl2and 2.7 mm ATP at 37 �C for 20 min. Muscles fibres

were classified into three major types (I, IIa, IIb) and

intermediate fibre types (IIc, IIab). With the myosin

reaction, type IIc displays an intermediate behaviour in

pH sensitivity between types I and IIa, and type IIab

between IIa and IIb fibres. Fibre type distribution is

expressed as the number of fibres of each type relative to

the total number of fibres. Measurements were made on

1000 fibres per section for each animal.

Statistical analysis

Data are expressed as mean � SE. Statistical compar-

isons between groups were calculated using two-way

analysis of variance (Statview 4.02, Abacus Concepts,

Berkeley, CA, USA). Fisher’s protected least significant

difference for multiple comparisons was used post hoc

when significant F-ratios were obtained and significance

was accepted at the P < 0.05 level.


Acta Physiol Scand 2004, 180, 387–394 D Desplanches et al. Æ Muscle HSP72 expression with training and suspension

Results

To take into account growth stunting induced by both

HU and endurance training, muscle weights are reported

on a relative basis (i.e. per 100 g body weight, Table 1).

After unloading, the SOL and AL weights were �50

and 70% of controls. The PLA weight of unloaded

group was �10% less than control whereas the EDL

was similar in both S-WB and S-HU groups. Endurance

training prior to unloading failed to prevent atrophy in

the SOL, AL and EDL and even increased PLA muscle

loss with HU (Table 1).

Total protein content (calculated from protein con-

centration and muscle weight) was significantly reduced

by HU in the SOL (54–59%), the AL (32–56%) and the

PLA (8–23%) but remained unchanged in the EDL

whether the rats were kept sedentary or endurance-

trained (Table 1). Total CS activity was also signifi-

cantly decreased in the SOL ()60%) and the PLA

()30%) muscles in response to unloading.

HSP72 levels

The HSP72 level was clearly influenced by muscle

function (Fig. 1). In S-WB rats, high levels of HSP72

were found in slow muscles (SOL and AL) as compared

with fast muscles like the EDL and PLA. HSP72 level

remained unaffected by 2 weeks of HU in both slow

(SOL, AL) and fast (EDL, PLA) muscles from sedentary

rats.

Following 6 weeks of endurance training, HSP72

increased moderately but significantly in the SOL

(�50%) whereas HSP72 levels were drastically

enhanced in the EDL (�500%) and in the PLA

(�300%). HSP72 remained unchanged in the slow AL

by 6 weeks of training.

When HU follows endurance training, HSP72 content

returned towards sedentary levels in the SOL and

PLA whereas it remained substantially elevated in the

EDL.

Histological and biochemical properties of the muscles

Although HSP72 levels were significantly higher in

slow as compared with fast muscles (Fig. 2a), changes

in HSP72 expression with hindlimb unloading and/or

training in the SOL were not directly related to

changes in muscle typology. Indeed, following 2 weeks

of hindlimb unloading, the SOL and AL displayed a

reduction of type I fibres and a simultaneous increase

in intermediate type IIc fibres whether the rats

remained sedentary or were previously trained

(Table 2). In fast muscles, hindlimb unloading induced

an increase in type IIab fibres accompanied by a

decline either in type IIb fibres (EDL) or in type

IIa (PLA).

In trained animals, the percentage of type IIa fibres

was significantly lower in SOL whereas in fast twitch

muscles, the reduced percentage in type IIb fibres was

counterbalanced by a significant increase in type IIa

(EDL, PLA) and in type IIab (PLA) fibres. Training prior

to unloading did not modify the fibre transition

observed in HU slow muscles. Fibre alterations induced

by training were still observed after 2 weeks of HU in

PLA but not in EDL muscle.

The efficiency of the training programme was eval-

uated by determination of CS activitiy (Table 1).

Training alone resulted in a significant increase in CS

activity in AL and PLA but remained unchanged in SOL

and EDL muscles. In AL, the training-induced increase

in oxidative capacity was significantly reduced by

subsequent HU. In PLA, CS activity was higher in

T-HU than in S-HU rats and did not differ from S-WB

animals (Table 1). There was, however, no significant

correlation between HSP72 levels and CS activity

(Fig. 2b).

Table 1 Influence of hindlimb unloading and training on

muscle weight, total protein content (mg muscle)1) and citrate

synthase activity. Values are mean values � SE

Muscle weight

(mg g)1 BW)

Total protein

content (mg)

Citrate synthase

activity

(lmol min)1 g)1

protein)

SOL

S-WB 40.7 � 1.3 36.9 � 2.3 136.0 � 6.4

T-WB 44.7 � 1.3� 35.5 � 2.2 151.1 � 7.7

S-HU 21.5 � 0.9* 15.2 � 1.4* 109.7 � 4.3*

T-HU 22.4 � 0.9* 16.5 � 0.9* 132.1 � 13.0

AL

S-WB 24.6 � 0.9 27.7 � 0.8 116.9 � 2.7

T-WB 24.9 � 0.7 21.4 � 1.4 151.1 � 9.7�

S-HU 16.4 � 0.6* 12.2 � 1.0* 120.1 � 7.8

T-HU 17.0 � 0.5* 14.7 � 1.6* 134.7 � 8.6

EDL

S-WB 43.8 � 1.0 36.8 � 2.1 133.6 � 6.5

T-WB 44.7 � 0.7 37.5 � 1.6 134.3 � 4.3

S-HU 42.8 � 0.9 36.6 � 1.2 126.5 � 6.5

T-HU 41.5 � 0.5* 34.1 � 1.7 123.5 � 5.8

PLA

S-WB 84.3 � 1.3 80.8 � 4.9 124.5 � 8.1

T-WB 86.0 � 2.3 68.5 � 4.5 185.9 � 15.9�

S-HU 74.4 � 1.8* 62.3 � 1.8* 88.3 � 3.3*

T-HU 70.3 � 1.0*,� 62.8 � 2.4 118.0 � 4.2*,�

S-WB, sedentary weight-bearing; S-HU, sedentary hindlimb-

unloaded; T-WB, trained weight-bearing; T-HU, trained

hindlimb-unloaded rats. SOL, soleus; AL, adductor longus;

EDL, extensor digitorum longus; PLA, plantaris.

*Significantly different from WB group within the same

training status; �significantly different from S group within the

same loading status.



Discussion

The main purposes of this study were to determine the

influence of 2 weeks of HU on skeletal muscle HSP72

content and to evaluate the effects of endurance training

on HSP72 and muscle remodelling during subsequent

unloading.

Unloading and skeletal muscle HSP72 levels

Recently, it was reported that 8 days of hindlimb

suspension in adult rats resulted in SOL atrophy and

decreased level expression of HSP72 (Naito et al.

2001). Although a mechanistic explanation was not

provided in that study, it seems possible that reduced

HSP72 content with unloading was linked to the fibre

type transition classically reported with HU (Desp-

lanches et al. 1987, 1990, Thomason et al. 1987,

Thomason & Booth 1990). Indeed, in rat skeletal

muscles, HSP72 expression is somewhat related to the

proportion of slow type I/b-MHC muscle fibres

(Fig. 2a, Locke et al. 1991, 1994). On the basis of a

progressive decrease in type I fibres in SOL from the

first to the fifth week of HU ( Desplanches et al. 1987),

we expected to see a lower HSP72 level after 2 week

than after 1 week of suspension in SOL muscle (Naito

et al. 2001). Contrary to that expectation, we found a

similar HSP72 content in the SOL from S-WB and

S-HU rats. The inability of HU to affect HSP72

content in slow muscles was confirmed by examining

another slow-twitch muscle, the AL (adducting and

extending the hip during posture maintenance and

Adductor longus

0

20

4060

80

100

120

S-WB S-HU T-WB T-HU

HSP

72 (

% o

f S-

WB

leve

l)

Soleus

0

50

100

150

200

S-WB S-HU T-WB T-HU

HSP

72 (

% o

f S-

WB

leve

l) † *

Plantaris

0

100

200

300

400

500

S-WB S-HU T-WB T-HU

HSP

72 (

% o

f S-

WB

leve

l) †

*

EDL

0

200

400

600

800

S-WB S-HU T-WB T-HU

HSP

72 (%

of

S-W

B le

vel)

†

† *

Figure 112 Influence of hindlimb suspension and training on HSP72 level in rat skeletal muscles. Note that HSP72 contents are

expressed as a percentage of HSP72 levels in control sedentary, weight-bearing group. Values are mean values � SE. S-WB,

sedentary weight-bearing; S-HU, sedentary hindlimb-unloaded; T-WB, trained weight-bearing; T-HU, trained hindlimb-unloaded

rats. *Significantly different from WB group within the same training status; �significantly different from S group within the




locomotion) which is known to display muscle fibre

transition after unloading as the SOL (Vijayan et al.

1998). In AL, HSP72 level remained unaffected in

spite of a reduced percentage of slow fibres with HU

(Table 2). Collectively, these data suggest that the

marked fibre transition induced by mechanical unload-

ing in slow muscles is not accompanied by alteration

in HSP72 content.

It has been postulated that the decrease in muscle

HSP72 after 1 week of HU could explain both the

reduced polypeptide elongation (Ku et al. 1995) and

decline in the rate of protein synthesis observed within

the first days of muscle disuse imposed by HU (see

review in Thomason & Booth 1990). Nevertheless,

Oishi et al. (2001) reported recently that 5 days of HU

alone did not affect HSP72 content in the SOL, but a

drastic reduction in neuromuscular activity induced by

superimposition of HU, tenotomy and denervation

decreased HSP72 levels. These data suggest that some

factors linked to the level of contractile activity are

determinants for HSP72 accumulation.

Many of the mechanisms that have been considered

as possibly controlling muscle atrophy during HU are

consistent with a rapid decrease in protein synthesis

followed by a progressive increase in protein degrada-

tion to reach a peak after 14 days of mechanical

unloading (Thomason & Booth 1990). Although we

did not measure protein turnover in the present

experiment, we found that total protein content

(Table 1) was drastically decreased in slow muscles in

accordance with our previous data (Desplanches et al.

1990). In that study, using quantitative electron micr-

oscopy, we found that both absolute myofibrillar and

mitochondrial volume were drastically decreased after

HU, in agreement with the reduced CS activity reported

in this study (Table 1). We suggest that the low HSP72

content reported after 1 week (Naito et al. 2001)

returned to control level on the second week of tail

0

5000

10000

15000

20000

25000

0 25 50 75 100HSP

72 c

onte

nt (

arbi

trar

y un

its)

Type I fiber (%)

y= 93.4x + 3069; R2=0.51

0

5000

10000

15000

20000

25000

30 60 90 120 150 180 210HSP

72 c

onte

nt (

arbi

trar

y un

its) y=15.6x + 5392; R2=0.0059

CS activity (µmol min–1 mg–1 protein)

Figure 2 Relationship between HSP72 content and muscle

fibre typology (a) or oxidative capacity (b). Note that slow

muscles (soleus and adductor longus) possess a higher content

of HSP72 than fast muscles (PLA and EDL). HSP72 values

are expressed in arbitrary units. There was no significant

correlation between HSP72 content and oxidative capacity

(as assessed from citrate synthase activity) of skeletal muscles.

Similar relationships can be obtained on each muscle (SOL,

AL, PLA, EDL) whether the rats were kept sedentary, trained,

and/or suspended.

Table 2 Influence of hindlimb unloading and training on

muscle typology. Data are expressed as the percentage of each

fibre type

Type I Type IIc Type IIa

SOL

S-WB 91.1 � 3.2 2.3 � 09 6.6 � 3.0

T-WB 96.0 � 1.9 3.4 � 1.6 0.6 � 0.4�

S-HU 80.5 � 3.2* 15.6 � 2.6* 3.8 � 1.1

T-HU 77.9 � 4.5* 21.2 � 4.9* 0.9 � 0.5�

AL

S-WB 81.1 � 1.8 2.6 � 1.3 16.3 � 1.5

T-WB 79.6 � 2.5 3.2 � 1.4 17.0 � 1.4

S-HU 75.3 � 2.4* 9.8 � 0.9* 14.7 � 5.5

T-HU 69.8 � 1.9* 11.0 � 2.4* 19.2 � 3.7

Type I Type IIa Type IIab Type IIb

EDL

S-WB 5.8 � 0.4 19.7 � 1.3 6.9 � 0.7 67.6 � 1.6

T-WB 6.8 � 0.4 24.3 � 1.5� 6.5 � 0.9 62.4 � 1.2�

S-HU 6.9 � 0.5 21.7 � 1.2 10.8 � 0.6* 60.6 � 0.9*

T-HU 5.8 � 0.4 24.1 � 1.6 10.8 � 1.6* 59.3 � 1.9

PLA

S-WB 11.0 � 0.7 23.1 � 1.1 7.9 � 1.5 58.0 � 0.7

T-WB 11.5 � 1.4 29.0 � 2.4� 12.0 � 0.5� 47.5 � 2.7�

S-HU 12.1 � 1.0 20.1 � 0.6* 10.0 � 0.8 57.8 � 1.4

T-HU 12.1 � 0.8 21.0 � 1.0* 21.9 � 1.4*� 45.1 � 2.7�

S-WB, sedentary weight-bearing; S-HU, sedentary hindlimb-

unloaded; T-WB, trained weight-bearing; T-HU, trained

hindlimb-unloaded rats; SOL, soleus; AL, adductor longus;

EDL, extensor digitorum longus; PLA, plantaris.

*Significantly different from WB group within the same

training status; �significantly different from S group within the




suspension because of a progressive increase in the rate

of protein breakdown (see Thomason & Booth 1990).

A maintained HSP72 level in the SOL after 2 weeks of

suspension would be in keeping with the role of

chaperone of HSP72 for preventing aggregation and

assisting refolding of denatured proteins (Georgopoulos

& Welch 1993). HSP72 content in fast-twitch muscles

(EDL, PLA) was similar before and after unloading

whereas these muscles displayed a slight muscle fibre

shifting characterized by a decrease in types IIa and IIb

in PLA and EDL muscle, respectively (Table 2).

Influence of prior training on HSP72 level during

unloading

Six weeks of endurance training resulted in HSP72

accumulation in all but one (AL) muscles examined

(Fig. 1). HSP72 content was slightly increased in the

SOL (50%, P < 0.01) whereas HSP72 was drastically

accumulated in the PLA (�300%, P < 0.001) and in the

EDL (�500%, P < 0.001). These data are in agreement

with previous data obtained after several weeks of

endurance training (Ecochard et al. 2000, Samelman

20009 , Mattson et al. 2001). Interestingly, we found that

the increase in HSP72 content with training was

significantly higher in fast (PLA, EDL) than in slow

(SOL, AL) muscles in agreement with the recent data of

Gonzalez et al. (2000). Nevertheless, there was no

significant correlation between HSP72 level and oxida-

tive capacity (Fig. 2b). Muscle-specific differences in

HSP expression have been recently reported after heat

shock (Locke 200010 , Locke & Tanguay 1996) but the

reasons for this differential induction of HSP72 remain

to be determined.

HSP72 accumulation in the SOL and PLA muscles

from trained animals was totally abolished by subse-

quent unloading whereas the slow AL was basically

insensitive to increased and/or decreased contractile

activity. The training-induced increase of HSP72 con-

tent in the EDL was significantly reduced by 2 weeks of

suspension, but HSP72 levels remained significantly

elevated (threefold increase) above sedentary weight-

bearing rats. The mechanisms implicated in the main-

tenance of a high HSP72 level in the EDL from

endurance trained rats after unloading are not readily

apparent but could be a consequence of the limb

positioning taken by suspended rats. Indeed, the posi-

tion of the hindlimb during suspension was such that

the ankle usually is extended and outstretched caudally

(Alford et al. 1987). It is thus conceivable that stretch-

ing the EDL during the unloading period after training

prevented the return of HSP72 level toward sedentary

control. This hypothesis is somewhat supported by the

determinant role of muscle length on the muscle

responses to mechanical unloading (Leterme et al.

1994, Ohira et al. 1997) and by an increased EMG

response to HU in anterior muscles of the leg (Alford

et al. 1987). Alternatively, the balance between HSP72

synthesis and degradation in the EDL might be different

from those of the PLA and SOL muscles.

In this study, we found that accumulation of HSP72

in SOL muscle by prior training (Fig. 1) failed to

attenuate muscle atrophy (Table 1). These data con-

trast with those reported in a previous study where

HSP72 accumulation with whole body hyperthermia

(41 �C for 60 min) reduced muscle HU atrophy (Naito

et al. 2001). Some factors may explain these apparent

discrepancies. It is likely that the rather long heat

shock exposure used by Naito et al. (2001) resulted in

extensive muscle damage and large HSP72 accumula-

tion. Indeed, short-term exposure to 41 �C (10 min) is

not accompanied by a significant increase of HSP72 in

SOL muscle (Locke 200011 ). Secondly, our rats were

suspended for 2 weeks and thus displayed a signifi-

cantly greater atrophy than after 7 days of muscle

disuse (Desplanches et al. 1987, Thomason & Booth

1990).

In conclusion, the present results demonstrate that (1)

hindlimb suspension for 2 weeks was not accompanied

by significant changes in basal HSP72 level in both slow

(SOL and AL) and fast (EDL, PLA) muscles; (2)

endurance training resulted in a significant increase in

HSP72 level in various muscles whether slow (SOL) or

fast (EDL, PLA); (3) the training–induced increase of

HSP72 content in slow muscle did not affect fibre

transition. Surprisingly, endurance training elevated

HSP72 levels in fast muscles (EDL, PLA) and the

HSP72 response to subsequent unloading was clearly

different in these muscles. It is hypothesized that limb

positioning of the EDL muscle during HU prevented the

return of HSP72 to levels similar to those obtained in

sedentary WB animals.

This study was supported by CNES grant no. 793/CNES/99/

7678 (DD, RF).

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Skeletal muscle HSP72 response to mechanical unloading: influence of endurance training