hindlimb suspension induces the expression of multiple myosin heavy chain isoforms in single fibres...

Hindlimb suspension induces the expression of multiple

myosin heavy chain isoforms in single ®bres of the rat

soleus muscle

Y . O I S H I , 1 , 3 A . I S H I H A R A , 2 H . Y A M A M O T O 3 and E . M I Y A M O T O 3

1 Laboratory of Muscle Physiology, Faculty of Education, Kumamoto University, Kurokami, Kumamoto

2 Laboratory of Neurochemistry, Faculty of Integrated Human Studies, Kyoto University, Kyoto,

3 Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto, Japan

ABSTRACT

To examine the expression patterns of myosin heavy chain (MHC) isoforms in single fibres of the

soleus muscle following weightlessness, 10-week-old male Wistar rats were subjected to hindlimb

suspension for 4 weeks. Hindlimb suspension resulted in reduced body weight and absolute and

relative mass of the soleus muscle compared with controls (P < 0.01). A total of 975, 892 and 1098

single ®bres from pre-suspended controls, age-matched controls and suspension groups,

respectively, were subjected to MHC analyses using SDS-PAGE. Single ®bres containing only MHC I

decreased (87.9 vs. 67.9%, P < 0.05) and single ®bres containing only MHC IIa disappeared after

hindlimb suspension. On the contrary, single ®bres containing multiple type II MHC isoforms were

observed as follows: 10.1% single ®bres contained MHCs IIa and IId; 14.1% contained MHCs I, IIa

and IId; and some (1.4%) expressed the MHC IIb isoform with MHCs IIa and IId. The relative content

(%) of each MHC isoform in MHC hybrid single ®bres was calculated using densitometer scanning.

The MHCs IIa and IId hybrid single ®bres contained the same amount of MHC IIa (51.3 � 6.3%) and

MHC IId (48.7 � 6.3%). In the MHCs I, IIa and IId hybrid single ®bres, the percentage of MHC IIa was

distributed in a wide range (�80%), whereas the percentage of MHC IId was a relatively low range

(�40%), and the relative content of MHC I was inversely correlated with that of MHC IIa and MHC

IId, respectively. The ®bre type composition of suspended soleus muscle, analysed by histochemical

myosin ATPase staining, was changed, with a decrease in the percentage of type I ®bres and an

increase in that of type IIA ®bres. Our results indicate that hindlimb suspension induces multiple type

II MHC expression in the soleus single ®bres and suggest that the single ®bres containing multiple

type II MHC isoforms should be classi®ed into type IIA.

Keywords hybrid single ®bre, SDS-PAGE.

Received 26 May 1997, accepted 16 September 1997

The myosin adenosine triphosphatase (ATPase) activity

of a muscle ®bre is largely determined by the myosin

heavy chain (MHC) isoform expressed in the ®bre

(Bottinelli et al. 1994) because the MHC molecule

contains the catalytic site for ATPase activity

(Sivaramakrishnan & Burke 1982). The MHC isoforms

in adult rat skeletal muscles at normal conditions are

divided into four types: IIa, IId (IIx), IIb and I, in order

of increasing mobility as separated by electrophoresis,

while the order of myo®brillar ATPase activity is MHC

IIb > IId (IIx) = IIa > I (Bottinelli et al. 1994). The

histochemically identi®ed ®bre types I, IIA, IIB and

IID (IIX) are represented by the MHC isoforms I, IIa,

IIb and IId (IIx), respectively (Staron & Pette 1987a,b,

Termin et al. 1989b). The maximal shortening velocity

(Vmax) of each ®bre correlates with the relative content

of fast-type MHC isoforms (Reiser et al. 1985a,b,

Schiaf®no & Reggiani 1996).

Hindlimb suspension is an experimental model that

can simulate weightlessness or unloading of the skel-

etal muscle and result in muscle atrophy, especially in

the slow-twitch soleus muscle (Thomason & Booth

1990, Roy et al. 1991). Some research has suggested

that the proportion of MHC IIa isoform in the soleus

muscle increased after hindlimb suspension (McDon-

ald & Fitts 1993, Leterme et al. 1994), whereas other

Correspondence: Yasuharu Oishi, Laboratory of Muscle Physiology, Faculty of Education, Kumamoto University, Kumamoto 860, Japan.

Acta Physiol Scand 1998, 162, 127±134

Ó 1998 Scandinavian Physiological Society 127

research has suggested that it is the proportion of

MHC IId that increased (Takahashi et al. 1991,

Campione et al. 1993, Oishi 1993). Type IId (IIx)

MHC is coded with a distinct mRNA and the ®bre

which contains MHC IId (IIx) is classi®ed as type IID

(IIX) by histochemistry (DeNardi et al. 1993). The

MHC IId (IIx) isoform is normally observed in the

respiratory diaphragm muscle (BaÈr & Pette 1988,

LaFramboise et al. 1990) and in fast-twitch muscles

such as the tibialis anterior (Termin et al. 1989a,

LaFramboise et al. 1990). Galler et al. (1994) reported

that several types of MHC hybrid single ®bres which

contain MHC IId and either IIa or IIb isoforms were

observed in the fast-twitch vastus lateralis and psoas

muscles. However, MHC IId (IIx) was not detected in

the slow-twitch soleus muscle during the normal

stages of rat growth and ageing (Sugiura et al. 1992).

We have previously reported on the expression of the

MHC IId isoform in the soleus after 4 weeks of

hindlimb suspension (Oishi 1993) and the disappear-

ance of MHC IId during 4 weeks of recovery (Oishi

et al. 1994). These results have indicated that the MHC

IId isoform was synthesized in the soleus muscles

during soleus unloading. However, it is unknown

whether or not only the MHC IId (IIx) is expressed,

or if the MHC IId (IIx) is co-expressed with other

isoforms in a single ®bre of the soleus muscle after

hindlimb suspension. There were no previous studies

that reported an increase in type IID (IIX) ®bres

which contained only the type IId (IIx) MHC isoform

in the soleus muscle after hindlimb suspension. Only a

few studies have reported an increase in hybrid single

®bres which contained several types of MHC isoforms

after the unloading of the soleus muscle. For example,

Talmadge et al. (I996) demonstrated, using an

immunohistochemical technique, that single ®bres

which contained two or three types of MHC isoforms

increased after 14 days of hindlimb suspension or a 14

day space ¯ight.

In the present study, we examined the single ®bres

for MHC expression patterns using the electrophoretic

technique in control and hindlimb suspended soleus

muscles. This technique is useful to examine the MHC

expression patterns at a single ®bre level and it is

possible to determine the quantitative contents of each

MHC isoform expressed. As a result, the present study

showed that many single ®bres expressed multiple type

II MHC isoforms after 4 weeks of hindlimb suspen-

sion, and those single ®bres had a characteristic ex-

pression pattern of each MHC isoform examined by

densitometer scanning. The ®bre type composition was

also calculated by histochemical myosin ATPase ana-

lyses.

MATERIALS AND METHODS

Animal care and treatment

Thirteen male 10-week-old Wistar rats, weighing be-

tween 317.5 and 333.7 g, were randomly separated into

3 groups: pre-suspended 10-week-old controls (n = 4);

age-matched 14-week-old controls (n = 4), and 4 week

suspension (n = 5). All animals were housed in a con-

trolled environment with 12 h of light and 12 h of

darkness at a room temperature of 22 � 2 °C. Animals

were given food and water ad libitum. The rats in the

suspension group were subjected to hindlimb suspen-

sion at 10 weeks of age for 4 weeks according to the

model by Morey (1979) with a slight modi®cation

(Oishi 1993). After completing the experiment, the

animals were anaesthetized with sodium pentobarbital

(60 mg kg)1, i.p.), and the soleus muscles on both sides

were removed, weighed and quickly frozen in isopen-

tane cooled with liquid nitrogen. The experimental

protocol, animal care and treatment were approved by

the committee for animal studies at Kumamoto Uni-

versity.

Single ®bre dissection

The right soleus muscle was soaked for 30 min in a

relaxation solution containing 5.56 mM ATP, 5.04 mM

MgCl2, 4 mM EGTA, 10 mM PIPES and 137 mM KCl,

at pH 6.8. The single ®bres were dissected under a

microscope and then incubated for 5 min at 50 °C in

40 lL of lysis buffer (BaÈr & Pette 1988) containing

10% (w/v) glycerol, 5% (v/v) b-mercaptoethanol, 2.3%

(w/v) SDS, and 60 mm Tris±HCl, at pH 6.8. An ali-

quot of the sample (20 lL) was subjected to electro-

phoresis.

Electrophoresis

The MHC isoforms of single ®bres were analysed by

sodium dodecyl sulfate-polyacrylamide gel electropho-

resis (SDS-PAGE) according to the method by Sugiura

& Murakami (1990) with a slight modi®cation (Oishi

1993). SDS-PAGE was continued for 24 h at a constant

current of 11 mA at 4 °C using a 1 mm thick separating

gel with a linear gradient of 5±8% acrylamide and 30±

40% glycerol, and a 1 mm thick stacking gel with 3.5%

acrylamide and 35% glycerol. After the SDS-PAGE, the

gel was silver-stained. The proportion of single ®bres

was expressed as a percentage of total single ®bres an-

alysed in each group on the basis of the MHC expres-

sion patterns. Furthermore, the relative content of each

MHC isoform in the MHC hybrid single ®bre was

scanned with a Pharmacia LKB Ultrascan laser scanning

densitometer and expressed as a percentage of total

MHC contents within the single ®bre.

Hindlimb suspension and myosin heavy chain isoforms á Y Oishi et al. Acta Physiol Scand 1998, 162, 127±134

128 Ó 1998 Scandinavian Physiological Society

Histochemistry

Serial cross-sections, 10 lm thick, at the widest portion

of the left muscle belly were cut in a cryostat main-

tained at )20 °C and mounted onto glass microscope

slides. The sections were air-dried for 1 h and then

stained for ATPase activity following acid (pHs 4.3 and

4.5) and alkaline (pH 10.3) preincubations (Brooke &

Kaiser 1970). Then the sections were dehydrated in a

graded series of ethanol, followed by two changes in

xylene and then coverslipped. The soleus muscle ®bres

were classi®ed into several types: type I (positive for

pHs 4.3 and 4.5 preincubations and negative for pH

10.3 preincubation), type IIA (positive for pH 10.3

preincubation and negative for pHs 4.3 and 4.5 prein-

cubations), and intermediate type IC or IIC (positive

for pHs 4.3, 4.5 and 10.3 preincubations with some

differences in staining intensity). The ®bre type com-

position was calculated by analysing all ®bres in the

cross-section.

Statistics

Means and standard deviations (SD) were calculated

from individual values using standard procedures. The

statistical differences between the control and experi-

mental groups were analysed using the Student's t-test.

Differences were considered signi®cant at P < 0.05.

RESULTS

Body and soleus muscle weight

Table 1 shows the body and soleus muscle weights of

the control and suspended groups. The body wt in the

control group increased from 320.7 � 8.4 g in the 10-

week-old rats to 445.5 � 29.3 g in the 14-week-old rats,

whereas the body wt in the suspended group was lower

than that of the 14-week-old control group and similar

to that of the 10-week-old control group. The soleus

muscle weight increased from 129 � 4 mg in the 10-

week-old group to 161 � 34 mg in the 14-week-old

control group. The soleus muscle weight of the sus-

pended group was 45.7 and 56.5% lower than that of

the 10-week-old and 14-week-old control groups, res-

pectively. The relative muscle weight per body wt in the

suspended rats was signi®cantly lower than both con-

trol groups.

MHC expression patterns in the single ®bres

using SDS-PAGE analyses

A total of 975, 892 and 1098 single ®bres were analysed

by SDS-PAGE from pre-suspended controls (10-week-

old), age-matched controls (14-week-old), and 4 week

suspended soleus muscles, respectively. The control

soleus muscles contained two types of MHC isoforms ±

fast-type MHC IIa and slow-type MHC I ± and their

distribution patterns were as follows: pure MHC I, pure

MHC IIa and a mix of both isoforms (Fig. 1a). In

addition, MHCs I and IIa hybrid single ®bres in the

control muscles contained various amounts of each

isoform as follows: I/IIa (MHC I > MHC IIa) or IIa/I

(MHC IIa > MHC I) (Fig. 1a). In contrast to controls,

the single ®bres of the suspended soleus muscle

showed different patterns (Fig. 1b). The synthesis of

MHC IId isoform was observed in single ®bres with

MHC IIa (shown as IIa/IId), or MHCs I and IIa

(shown as I/IIa/IId).

The proportions of single ®bres with a given MHC

composition for each group are shown in Table 2. In

the control soleus muscle, the percentage of single ®-

bres containing only the MHC I isoform was

76.8 � 8.1% in the 10-week-old control group and

87.9 � 5.5% in the 14-week-old control group, and the

single ®bres containing only the MHC IIa isoform was

15.7 � 9.6% in the 10-week-old controls and

4.7 � 2.9% in the 14-week-old controls. The percent-

ages of MHCs I and IIa hybrid single ®bres were the

same in both control groups (7.5 � 4.1% in the 10-

week-old control, and 7.4 � 3.9% in the 14-week-old

control). After hindlimb suspension, single ®bres con-

taining only the MHC I isoform decreased to

67.9 � 6.5% and ®bres containing only the MHC IIa

isoform disappeared (Table 2). On the other hand,

hindlimb suspension induced unique types of MHC

hybrid single ®bres: 10.1 � 1.4% of single ®bres con-

tained MHCs IIa + IId and 14.1 � 2.9% contained

MHCs I, IIa and IId (Fig. 1b and Table 2). Further-

more, some single ®bres expressed the MHC IIb iso-

form with type IIa and IId MHC isoforms (shown as

IIa/IId/IIb in Fig. 1c and Table 2).

Table 1 Body and soleus weightsGroup n Body wt (g) Soleus wt (mg) Soleus/body wt (mg g)1)

10-week-old cont. 4 320.7 (8.4) 129 (4) 0.40 (0.015)

14-week-old cont. 4 445.5 (29.3) 161 (34) 0.36 (0.065)

4 week suspension 5 338.4 (32.0)## 70 (14)**,## 0.21 (0.045) **,##

Values are means (SD). cont., control. **, P < 0.01 compared with 10-week-old cont.; ##, P < 0.01

compared with 14-week-old cont.


Acta Physiol Scand 1998, 162, 127±134 Y Oishi et al. á Hindlimb suspension and myosin heavy chain isoforms

Relative content (%) of each MHC isoform in MHC hybrid

single ®bres analysed by densitometer scanning

The MHC hybrid single ®bres were densitometorically

scanned to determine the relative content (%) of each

MHC isoform. Figure 2 shows the relative content of

each MHC isoform in MHCs I and IIa hybrid single

®bres of control and suspension groups. In the 10-

week-old control (Fig. 2a, n = 72), the percentage of

MHC IIa was widely distributed, from 17.3 to 90.2%,

and 79.2% of single ®bres (n = 57 of 72) contained

>55% of MHC IIa. In contrast, the 14-week-old con-

trol (Fig. 2b, n = 30) and the 4 week suspension

(Fig. 2c, n = 47) groups contained relatively low per-

centages of MHC IIa and high percentages of the MHC

I isoform compared with the 10-week-old controls. All

MHC I and IIa hybrid single ®bres in the 14-week-old

controls and 91.5% (n = 43 of 47) in suspension

groups contained <60% of MHC IIa (Fig. 2b & c).

Only in the MHC IIa and IId hybrid single ®bres

observed in the soleus muscle after hindlimb suspen-

sion were the relative contents of MHC IIa and MHC

IId similar, being 51.3 � 6.3 and 48.7 � 6.3%, res-

pectively (Fig. 3, n = 55).

Furthermore, 14.1% of soleus single ®bres con-

tained MHC I, IIa and IId isoforms after hindlimb

suspension, as shown in Table 2 and Fig. 1(b). Figure 4

shows the relationship between the relative contents of

each MHC isoform in those hybrid single ®bres

(n = 108). The content of MHC IIa was widely dis-

tributed (�80%), whereas the content of MHC IId was

relatively low (�40%, Fig. 4a). The relative content of

MHC I was inversely correlated to both MHC IIa and

MHC IId (Fig. 4b & c).

Table 2 Proportion of single ®bres (%) containing various expression patterns of MHC isoforms in the soleus muscle in control and hindlimb

suspended rats

MHC expression patterns in single ®bres

Group I I + IIa IIa I + IIa + IId IIa + IId IIa + IId + IIb

10-week-old cont. (n = 975) 76.8 (8.1) 7.5 (4.1) 15.7 (9.6) 0 0 0

14-week-old cont. (n = 892) 87.9 (5.5) 7.4 (3.9) 4.7 (2.9) 0 0 0

4 week suspension (n = 1098) 67.9* (6.5) 6.5 (2.9) 0 14.1 (2.9) 10.1 (1.4) 1.4 (2.5)

Values are mean (SD). cont., control; n, total number of single ®bres analysed. *, P < 0.05 compared with 14-week-old cont.

Figure 1 MHC expression patterns of

soleus single ®bres of control (a) and

suspended (b) rats. Marker protein contains

four types of MHCs derived from the

gastrocnemius and plantaris muscles of the

control rats. Note that differences were

observed in the MHC expression patterns

between control (a) and suspension (b)

groups. Some of the single ®bres of the

suspended soleus muscle also contained

pure MHC I or MHCs I and IIa as shown

in Table 1, although those patterns did not

appear here. The single ®bres which

expressed the MHC IIb isoform with

MHCs IIa and IId observed in the

suspended soleus are shown in panel (c) as

IIa/IId/IIb. The super®cial portion of the

gastrocnemius muscle (Sup. Gast.) of

control rat was used as a marker for the

MHCs IId and IIb.



Fibre type composition based on myosin ATPase staining

In order to examine the effects of hindlimb suspension

on the ®bre type composition of the soleus muscle,

histochemical myosin ATPase staining was performed

in the control and suspension groups. The soleus

muscles of the 10-week-old controls (Fig. 5a) and

suspended animals (Fig. 5c) contained types I, IIA and

some intermediate type IIC or IC ®bres, while only two

types of ®bres, types I and IIA, were observed in the

14-week-old control animals (Fig. 5b). The ®bre type

composition decreased in the percentage of type I ®-

bres (Fig. 6, bottom panel) and increased in that of type

IIA ®bres (Fig. 6, middle panel) after hindlimb sus-

pension. The percentage of intermediate type IC or IIC

®bres did not change (Fig. 6, upper panel).

DISCUSSION

The primary objectives in the present study were to

observe multiple type II MHC isoforms, including

types IId and IIb, expressed in the soleus single ®bres

after 4 weeks of hindlimb suspension, and to calculate

the relative content of each MHC isoform in MHC

hybrid single ®bres. We observed four types of MHC

hybrid single ®bres: I + IIa, IIa + IId, I + IIa + IId,

and IIa + IId + IIb, as shown in Table 2. The MHCs

IId and IIb, neither of which was observed in the

controls, were newly synthesized during hindlimb sus-

pension. On the other hand, the single ®bres containing

only the MHC IIa isoform, which were observed in

15.7% of the pre-suspension control group, disap-

peared. These results and the fact that the MHC IId or

MHC IIb was always co-expressed with the MHC IIa

isoform may suggest that the synthesis of the MHC IId

or MHC IIb occurred in the pure MHC IIa ®bres

during hindlimb suspension. In addition, based on the

study by Talmadge et al. (1996), who suggested the

MHC conversion in rat soleus muscle during the un-

loading state as I ® IIa ® IIx ® IIb, the following

scheme for MHC conversion may be considered from

our results: IIa ® IIa + IId ® IIa + IId + IIb.

Figure 2 Percentage distribution of each MHC isoform in the MHC

I and IIa hybrid single ®bres of the 10-week-old (a) and 14-week-old

(b) control groups, and the 4 week suspension group (c). Note that

many hybrid single ®bres in the 10-week-old controls were MHC IIa

dominant, whereas most hybrid single ®bres in the 14-week-old

control and suspension groups were MHC I dominant.

Figure 3 Percentage distribution of each MHC isoform in the MHC

IIa and IId hybrid single ®bres observed in the 4 week suspended

soleus muscle.


Acta Physiol Scand 1998, 162, 127±134 Y Oishi et al. á Hindlimb suspension and myosin heavy chain isoforms

The MHC pro®les of the control soleus muscle

shifted from types IIa to I between 10-week-old and

14-week-old rats (Table 2). This MHC conversion

during normal growth in rat soleus muscles can be

explained by the ®ndings of Kugelberg (1976), who

observed the shift of motor units from type IIA to I. In

addition, 10-week-old and 14-week-old control muscles

contained similar proportions of MHCs I and IIa hy-

brid single ®bres, shown in Table 2, but the former was

MHC IIa dominant and the latter was MHC I dominant

(Fig. 2a & b). These results indicate that within the

MHC I and IIa hybrid single ®bres, the MHC pheno-

type shifted from type IIa to I. In the 4 week suspen-

sion group, the MHC I was dominant in the MHC I

and IIa hybrid single ®bres (Fig. 2c). Furthermore, the

proportion of single ®bres containing pure type I MHC

decreased and those containing types I, IIa and IId

MHC increased by 14.1% (Table 2). From these results,

the following scheme for MHC conversion during

hindlimb suspension may be possible: I ® I + IIa ®I + IIa + IId.

Our ®nding that some soleus single ®bres expressed

the MHC IIb isoform after 4 weeks of hindlimb sus-

pension was consistent with previous studies. For ex-

ample, Fauteck & Kandarian (1995) and Talmadge et al.

(1996) observed the appearance of the MHC IIb iso-

form in the rat soleus muscle after 28 and 14 days of

hindlimb unweighting, respectively. Also, Diffee et al.

(1991b) reported an increase in type IIb MHC mRNA in

the soleus muscle after 2 weeks of hindlimb suspension.

The increase in the fast-type MHC IId and/or IIb iso-

form in the soleus following hindlimb unweighting in-

dicates that the MHC pro®les shift toward a `fast'

phenotype. This phenomenon is con®rmed by the fact

that hindlimb suspension induces an increase in the

maximal shortening velocity and myosin ATPase activ-

ity in the soleus muscle (Gardetto et al. 1989, Diffee et al.

1991a, McDonald & Fitts 1993, Fauteck & Kandarian

1995, Widrick et al. 1996). Co-expressions of MHCs

IIa+IId, I + IIa + IId and IIa + IId + IIb in the

soleus single ®bres observed in the present study were

also reported by Talmadge et al. (1996) using an

immunohistochemical technique after 14 days of hind-

limb suspension with additional MHC combinations, i.e.

I + IIx, I + IIa + IIx + IIb. Their results and those of

the present study suggest that prolonged inactivity or

unloading in the soleus muscle induces multiple type II

MHC expression in the soleus single ®bres.

Concerning the increase in the ratio of type IIA

®bres by histochemical analyses after hindlimb sus-

pension, Templeton et al. (1988) suggested this was

caused by a shift of ®bres from type I to IIA. Similarly,

McDonald et al. (1994) suggested a ®bre type conver-

sion from type I to IIA on the basis of MHC pro®les.

In the control soleus muscles of the present study, the

percentage of type I or type IIA ®bres was similar to

that of the MHC I or MHC IIa isoform (Table 2,

Fig. 6), respectively, indicating that the ®bres contain-

ing pure MHC I or pure MHC IIa were classi®ed as

type I or type IIA ®bres, respectively. However, these

Figure 4 Relationship between the percentage distribution of each

MHC isoform in the MHC I, IIa and IId hybrid single ®bres of the

4 week suspended soleus muscle.



similarities were not maintained after hindlimb sus-

pension, because type IIA ®bres increased as shown by

histochemical myosin ATPase staining (Fig. 6), and the

single ®bres containing pure MHC IIa disappeared, as

shown by SDS-PAGE analyses (Table 2). On the

contrary, 25.6% of type II MHC hybrid single ®bres

(IIa + IId, I + IIa + IId and IIa + IId + IIb) were

observed. These results suggest that the single ®bres

containing multiple type II MHC isoforms were clas-

si®ed into type IIA. Therefore, we conclude that the

histochemically identi®ed type IIA ®bres that increased

after hindlimb suspension were not caused by the

complete shift of MHC pro®les from type I to IIa, but

by the production of multiple type II MHC isoforms.

In summary, 4 weeks of hindlimb suspension in-

duces the expression of multiple type II MHC isoforms

in single ®bres of the rat soleus muscle. The MHC

hybrid single ®bres contained two or three type II

MHC isoforms and the proportion of each MHC iso-

form had a characteristic pattern. Combining the results

from histochemical myosin ATPase and SDS-PAGE

analyses in the single ®bres, the ®bres containing

multiple type II MHC isoforms were classi®ed into type

IIA. These results indicate that the weightlessness or

unloading of the anti-gravity soleus muscle induces the

expression of fast-type II MHC isoforms in the single

®bres accompanied by muscle atrophy.

The authors are grateful to Dr Robert J. Talmadge for his critical

review of the manuscript. This study was partially supported by a

grant from the Ministry of Education, Science, Sports and Culture of

Japan to YO (grant no. 06780088).

Figure 6 Percentage distributions of type I (bottom panel), type IIA

(middle panel) and intermediate type IIC or IC (top panel) ®bres in

the soleus muscles of the control and suspended rats. Cont., control

group; Sus., suspension group. ##, P < 0.01 compared with the

values of both the 10- and 14-week-old controls.

b

Figure 5 Transverse sections of rat

soleus muscles in the 10-week-old

control (a), 14-week-old control (b) and

hindlimb suspended (c) rats. ATPase

staining following preincubation at pHs

10.3 (a1, b1 and c1), 4.5 (a2, b2 and c2),

and 4.3 (a3, b3 and c3). 1, type I ®bres; 2,

type IIA ®bres; 3, type IIC ®bres; 4,

type IC ®bres. The intermediate type

IIC or IC ®bres reacted positively to all

preincubation media, but had some

differences in staining intensity. The

scale bar on panel (c1) indicates 100 lm

for all panels.


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hindlimb suspension induces the expression of multiple myosin heavy chain isoforms in single fibres...

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