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Katsumasa Yamashita-Goto, Ryoko Okuyama, Masanori Honda, Kensuke

Kawasaki, Kazuhiko Fujita, Takahiro Yamada, Ikuya Nonaka, Yoshinobu Ohira

and Toshitada Yoshioka
J Appl Physiol 91:417-424, 2001.

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J Appl Physiol
91: 417–424, 2001.

Maximal and submaximal forces of slow fibers in human

soleus after bed rest


Department of Physiology, St. Marianna University School of Medicine,
Kawasaki City, Kanagawa 216-8511; 2Department of Orthopaedic Surgery, University of
Tokai, Isehara City, Kanagawa 259-1193; 3National Center for Neurology and Psychiatry,
Kodaira City, Tokyo 187-8551; 4Department of Physiology and Biomechanics, National
Institute of Fitness and Sports, Kanoya City, Kagoshima 891-2393; and 5Aomori
University of Health and Welfare, Aomori City, Aomori 030-8505, Japan
Received 3 May 2000; accepted in final form 5 March 2001

Yamashita-Goto, Katsumasa, Ryoko Okuyama, skeletal muscles (16, 26, 36, 42). Increased loading of
Masanori Honda, Kensuke Kawasaki, Kazuhiko Fujita, skeletal muscles, such as occurs with exercise training

Downloaded from jap.physiology.org on July 25, 2007

Takahiro Yamada, Ikuya Nonaka, Yoshinobu Ohira, or mechanical stretch, stimulates protein synthesis
and Toshitada Yoshioka. Maximal and submaximal forces and often results in hypertrophy. In contrast, de-
of slow fibers in human soleus after bed rest. J Appl Physiol
creased loading, such as occurs during exposure to
91: 417–424, 2001.—The effects of 2 and 4 mo of bed rest, with
or without exercise countermeasures, on the contractile proper- actual microgravity or a simulation model such as bed
ties of slow fibers in the human soleus muscle were examined. rest, results in a loss of muscular protein and/or atro-
Mean fiber diameters were 8 and 36% smaller after 2 and 4 mo phy, especially in primary antigravity muscles such as
of bed rest, respectively, than the pre-bed rest level. Maximum the soleus (27, 28). Animal studies have shown that
tetanic force (Po), maximum activated force (Fmax) per cross- chronic periods of unloading result in a shift from
sectional area (CSA), and the common-logarithm value of free oxidative to glycolytic metabolic profile (12, 17, 49),
Ca2⫹ concentration required for half-maximal activation transition of fiber types from slow to fast (3, 4, 11, 38),
(pCa50) also decreased after 2 and 4 mo of bed rest. In contrast, increase in shortening velocity (9, 15, 47), decrease in
maximum unloaded shortening velocity (Vo) was increased af- force-generation capacity (9, 47, 48), and alteration in
ter 2 and 4 mo of bed rest. After 1 mo of recovery, fiber excitation-contraction coupling (7, 20, 25, 31, 37, 50),
diameters, Po, Fmax per CSA (P ⬎ 0.05), and pCa50 were in-
such as increased rate of Ca2⫹ uptake and leakage to
creased and Vo decreased toward pre-bed rest levels. Effects of
knee extension/flexion exercise by wearing an anti-G Penguin and from sarcoplasmic reticulum (50), in hindlimb
suit for 10 h daily, and the effects of loading or unloading of the muscles, e.g., the soleus, tibialis anterior, and vastus
plantar flexors with (Penguin-1) or without (Penguin-2) placing lateralis.
the elastic loading elements of the suit, respectively, were in- There are a few reports regarding the effects of
vestigated during ⬃2 mo of bed rest. In the Penguin-1 group, decreased activity on the contractile properties of hu-
mean fiber diameter, Po, Fmax per CSA, Vo, and pCa50 were man soleus muscle fibers (3, 28, 33, 44–46). For exam-
similar before and after bed rest. However, the responses of ple, an increase in unloaded shortening velocity (Vo)
fiber size and contractile properties to bed rest were not pre- and/or decrease in force production associated with
vented in the Penguin-2 group, although the degree of the fiber atrophy were observed after 17 days of bed rest
changes was less than those induced by bed rest without any (44, 45). However, the contractile responses to long-
countermeasure. These results indicate that long-term bed rest
results in reductions of fiber size, force-generation capacity, and
term unloading are still unclear. Furthermore, the
Ca2⫹ sensitivity, and enhancement of shortening velocity in development of countermeasures to prevent muscle
slow fibers of the soleus. The data indicate that continuous atrophy during long-term unloading is important for
mechanical loading on muscle, such as stretching of muscle, is clinical medicine, as well as space medicine. Muscle
an effective countermeasure for the prevention of muscular deconditioning, such as an atrophy and decrease in
adaptations to gravitational unloading. maximum force, of knee extensors and/or plantar flex-
human soleus fibers; contractile properties; countermeasure ors during 20 or 14 days of bed rest was prevented by
daily isotonic leg-press exercise (3 s ⫻ 30 repetitions
with 30-s interval; Ref. 1) or by concentric/eccentric
MECHANICAL LOADING IS ONEof the primary factors regu- plantar flexion (5 sets of 6–10 repetitions every other
lating the synthesis and degradation of proteins in
The costs of publication of this article were defrayed in part by the
Address for reprint requests and other correspondence: Y. Ohira, payment of page charges. The article must therefore be hereby
School of Health and Sport Sciences, Osaka Univ., Toyonaka City, marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
Osaka 560-0043, Japan (E-mail: ohira@space.hss.osaka-u.ac.jp). solely to indicate this fact.

http://www.jap.org 8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society 417

day; Ref. 5), respectively. However, the duration of the The Penguin suit group was divided into two subgroups:
bed rest period was relatively short in these studies. Penguin-1 (n ⫽ 4) and Penguin-2 (n ⫽ 3). Subjects in the
Therefore, the present study was performed to exam- Penguin-1 group wore the full assembly of the Penguin suit,
ine the effects of long-term bed rest on the contractile which included all of the elastic loading elements, during the
10-h period. Subjects in the Penguin-2 group also wore the
properties of fibers from the human soleus muscle and
full assembly of the Penguin suit, except that the elastic
to determine whether a specific loading paradigm could loading elements at the ball of the foot were disconnected.
prevent any bed rest-associated adaptations. Therefore, in Penguin-1 subjects, ⬃60–70 N of force were
applied to the foot, i.e., the distal tarsal bones at the ball of
the foot, when the subject was plantar flexing the ankle or
Subjects and experimental design. The Institute of Biomed- when the plantar flexor muscles were relaxed. No such resis-
ical Problems (IBMP) in Moscow, Russia, performed this tive forces on the plantar flexors were imposed by the suit in
study for the National Space Development Agency of Japan the Penguin-2 group. For the Penguin suit groups, the soleus
(NASDA) (Contract no. 8H0-046, The Performance of muscle was biopsied twice, i.e., ⬃2 wk before bed rest and
NASDA’s Bed Rest Study at IBMP Facility). Thirteen immediately after ⬃2 mo (Penguin-1) or ⬃1.5 mo (Penguin-2)
healthy Russian male volunteers participated in this study. of bed rest.
All subjects were evaluated clinically and were considered to Muscle biopsy. During bed rest, the subjects were carried
be in good physical condition. All subjects were informed to the medical treatment room for performance of the muscle
about the possible risks in taking muscle biopsies, and a biopsies. Biopsies were taken from the left soleus with the
signed, informed consent was obtained from each subject. subjects in a prone position by using the procedures de-
Physical characteristics of subjects in each group are shown scribed by Bergstrom (8). Local anesthesia was induced via
in Table 1. The Human Use Committees at IBMP, NASDA, subcutaneous injection of 4 ml of a 2% lidocaine hydrochlo-
and St. Marianna University School of Medicine approved ride solution. A skin incision (5 mm) was made, and a Berg-

Downloaded from jap.physiology.org on July 25, 2007

this study. strom sterile needle was inserted toward the center of mus-
Detailed experimental protocols have been described in cle. The biopsy sample was divided into several portions. One
our group’s previous papers (27, 28). The study consisted of of the sample portions was longitudinally tied to a glass bar
two separate experiments. The first experiment was per- by use of surgery thread and was placed in a cold 50%
formed to study the effects of 4 mo of bed rest without any (vol/vol) glycerin solution (4°C) for 24 h. The glycerin solution
exercise countermeasure and 1 mo of recovery on the con- was exchanged with freshly prepared glycerin solution, and
tractile properties of slow-type fibers in soleus muscle. Six the muscle tissues in the solution were kept at ⫺20°C for
volunteers (“control” group) were subjected to 4 mo of bed 8–10 wk before the contractile analyses were performed.
rest at a 6° head-down-tilt position. Muscle biopsies were Because the samples were not treated for skinning imme-
taken from soleus muscles four times, i.e., ⬃3 wk before bed diately after biopsy by using Triton X-100, for example, the
rest, after ⬃2 and ⬃4 mo of bed rest, and after ⬃1 mo of muscle fibers were kept in glycerin solution to skin for 8 wk
normal ambulatory recovery. The subjects were fed three at ⫺20°C before the analyses of contractile properties, and all
times per day, and daily total energy intake was ⬃3,000 kcal groups were treated in the same way. The analyses were
for each subject. The temperature and humidity in the room completed within 2 wk, and the order of analysis was random
were maintained within a normal range, and the day-night across the groups.
cycle was regulated at 8 AM and 6 PM. Analytical procedures. On the day of the experiment, first
The second experiment was designed to determine the a bundle was placed in relaxing solution for 15 min and then
effects of exercise countermeasures performed during the bed individual fibers were carefully dissected from the bundle. A
rest period on the same muscle properties as described above. single muscle fiber (⬃5 mm long) was transferred to an
Seven subjects were assigned to one of two Penguin suit experimental chamber (0.3 ml volume) filled with the relax-
groups. They were subjected to a 2-mo bed rest at 6° head- ing solution. The relaxing solution (47, 48) consisted of 10
down-tilt position. All subjects in the second experiment wore mM EGTA, 3.5 mM MgATP, 1.5 mM Mg2⫹, and 20 mM
an anti-G Penguin suit for 10 h/day (11 AM to 9 PM) and PIPES at pH 7.0 (20°C). The ionic strength was adjusted to
additionally performed knee extension and flexion exercise in 0.2 M with potassium methane sulfonate. One end of the
a supine position in bed against a resistance of 100 N for the fiber was secured with T-shaped aluminum clips to a semi-
last 15 min of each hour. The subject’s shoes were equipped conductor transducer element (AE801, Aksjeselskapet
with rollers on the heels, which slid on a smooth surface of a Mikro-Elektronikk), and the other end was attached to a
platform with the ankle maintained at a relatively constant hook connected to a servomotor (G120D, General Scanning,
position. Thus this exercise emphasized knee, not ankle, Watertown, MA).
movement. The legs were relaxed, keeping the knee at an All experiments were performed at 20 ⫾ 1°C. The Ca2⫹
extended position during the remaining 45 min. concentration of the activating solution was adjusted be-
tween pCa 7.0 and pCa 4.0 (47, 48). Before the mechanical
experiments, the fibers were secured in the analytical appa-
ratus and treated with a skinning solution containing 1%
Table 1. Physical characteristics of subjects (vol/vol) Triton X-100 for 3 min. Isometric tension was re-
corded digitally at 12 kHz by an analog-to-digital converter
Age, yr Weight, kg Height, cm (AD216, Nittobo Acoustic Engineering) and stored on a hard
disk connected to a personal computer (PC-9821Ap, NEC) for
Control (n ⫽ 6) 30.8 ⫾ 3.1 80.0 ⫾ 7.6 181.0 ⫾ 2.0 later analysis.
Penguin-1 (n ⫽ 4) 30.5 ⫾ 1.9 72.0 ⫾ 3.3 180.0 ⫾ 3.0 Sarcomere length was adjusted to 2.5 ⫾ 0.2 ␮m by use of a
Penguin-2 (n ⫽ 3) 33.3 ⫾ 2.9 70.0 ⫾ 6.8 176.0 ⫾ 2.0 laser diffraction pattern and was monitored using a micro-
Values are means ⫾ SE. Control, bed rest for 4 mo without any scope and charge-coupled device camera connected to the
exercise countermeasure. See MATERIALS AND METHODS for description microscope. If any irregularity in the sarcomere was ob-
of the Penguin-1 and Penguin-2 groups. served, the fiber was discarded. The fiber diameter, absolute

maximally activated force (Po), relative maximally activated expression (Table 2). However, the data from these fibers
force (Fmax) per fiber cross-sectional area (CSA), Vo, and Ca2⫹ were not used because the distribution of fibers having these
sensitivity of the myofilaments were determined for each MHC profiles was low and not always detected in some
fiber. The CSA of a fiber was calculated from the two diam- samples. The number of fibers analyzed in each muscle was
eters in two perpendicular projections. Assuming that the seven to eight because of the small sample size.
shape of muscle fiber is a cylinder, we measured the width Statistical analysis. All values are expressed as means ⫾
and then thickness of fiber by shifting the stage of the SE. For the first experiment, the values were analyzed by
microscope. Fiber CSA then was calculated by using width using ANOVA followed by Scheffé’s post hoc test. For the
and thickness as reported by Frontera and Larsson (13). second experiment, one-tailed paired t-tests were used for the
The pCa-tension relationships were determined by activat- comparison between pre- and post-bed rest. ANOVA followed
ing the fibers in a series of solutions containing free Ca2⫹ by Scheffé’s post hoc test was used for comparisons of the
concentrations (pCa; 7.0, 6.5, 6.0, 5.5, 5.0, and 4.0). After percent change after 2 mo of bed rest, relative to the pre-bed
washing the muscle fiber in preactivating solution containing rest levels, between the control and Penguin suit groups.
2 mM EGTA, pCa solution was applied. Then, the fiber was Statistical significance was set at P ⬍ 0.05.
relaxed by washing in the relaxation solution after the ten-
sion reached a plateau level. The Fmax was determined in RESULTS
pCa 4.0 solution first. Then the fiber was relaxed and Ca2⫹
solution was applied from a lower concentration (pCa 7.0) to Effects of bed rest without countermeasure. Figure 1
a higher concentration. After application of a pCa solution, shows the responses of the fiber diameter (A), Po (B),
the sample was washed in relaxing solution containing 10 Fmax per CSA (C), and Vo (D) to 4 mo of bed rest
mM EGTA and in preactivating solution containing 2 mM without any countermeasure. The patterns of response
EGTA, and then the next pCa solution was applied. The
experiment was terminated when the Fmax decreased more
for fiber diameter, Po, and Fmax per CSA were similar.
Mean fiber diameters were ⬃8 and 36% smaller after 2

Downloaded from jap.physiology.org on July 25, 2007

than 10% in pCa 4.0 solution, the resting tension did not
return to zero, or irregularity in the sarcomeres was ob- and 4 mo of bed rest than before bed rest, respectively.
served. Fiber size was smaller after 4 mo of bed rest than after
Hill plot analysis was used to determine the Ca2⫹ sensi- 2 mo of bed rest (27%) and was similar to pre-bed rest
tivity of individual fibers. The Ca2⫹ sensitivity of fibers was levels after 1 mo of recovery.
evaluated by the concentration of Ca2⫹ for half-maximal The mean Po (mN) values were 38 and 76% lower
activation (pCa50). The Vo was determined by the slack test after 2 and 4 mo of bed rest than the pre-bed rest level,
procedure. The times required for the redevelopment of force respectively. In addition, the mean Po was 38% less
after five to six imposed slack steps [each step ⬍20% of fiber after 4 than 2 mo of bed rest. These values recovered to
length (FL)] were plotted against the corresponding slack
length, and the points were fitted with a linear regression
pre-bed rest levels within 1 mo after bed rest. The Fmax
line. The slope of this line, which is Vo, was normalized to the per CSA was 27 and 42% lower after 2 and 4 mo,
FL and expressed as FL per second (FL/s). respectively, of bed rest than before bed rest. There
After the mechanical measurements, the fiber segments was no significant difference between the mean Fmax
were removed from the apparatus; solubilized in 0.03 ml of per CSA after 2 or 4 mo of bed rest (15% lower at 4 mo,
an SDS buffer solution consisting of 0.1 M Tris 䡠 HCl (pH 8.8), P ⬎ 0.05). After 1 mo of recovery after 4 mo of bed rest,
5 mM EDTA, 10% SDS, and 50 mM DTT; boiled for 3 min; there was a tendency for Fmax per CSA to increase, but
and stored at ⫺80°C until subsequent gel electrophoresis the value was still lower than the pre-bed rest level
analyses. The fiber sample solutions were run on 12% acryl- (P ⬎ 0.05).
amide gels and stained by Bio-Rad Silver Stain Plus (Bio- Vo was significantly higher after 2 and 4 mo of bed
Rad, Hercules, CA). The expression of myosin heavy chain
(MHC) isoforms in the same fibers was determined (27). NIH
rest, i.e., by 82 and 97%, respectively, than that before
Image was used to quantify the relative content of each bed rest. There were trends (P ⬎ 0.05) for the Vo to be
isoform. In the present study, all data were determined from higher after 4 than 2 mo of bed rest and to approach
fibers expressing only type I MHC with the total number of pre-bed rest levels after 1 mo of recovery.
fibers used equal to 25 per group. Some of the isolated fibers Figure 2 illustrates the pCa-tension relationships
contained either I⫹IIa, I⫹IIa⫹IIx, IIa, or IIa⫹IIx MHC after 2 and 4 mo of bed rest. Before bed rest, the free

Table 2. Percent fiber-type distribution based on myosin heavy chain expression

I I ⫹ IIa I ⫹ IIa ⫹ IIx IIa IIa ⫹ IIx

Control group
Pre-bed rest 91.6 ⫾ 3.3 2.5 ⫾ 1.2 0 5.4 ⫾ 2.0 0
2 Mo bed rest 83.3 ⫾ 5.0 2.5 ⫾ 1.4 1.3 ⫾ 0.5 8.3 ⫾ 2.2 4.6 ⫾ 1.5
4 Mo bed rest 75.8 ⫾ 4.4 4.2 ⫾ 2.2 4.6 ⫾ 2.4 12.5 ⫾ 3.0 5.0 ⫾ 2.0
1 Mo after bed rest 86.6 ⫾ 5.2 4.6 ⫾ 2.3 3.3 ⫾ 1.6 2.9 ⫾ 1.2 4.2 ⫾ 1.8
Penguin-1 group
Pre-bed rest 91.2 ⫾ 4.1 2.2 ⫾ 0.5 0 6.0 ⫾ 2.0 0
2 Mo bed rest 82.3 ⫾ 5.2 1.9 ⫾ 0.6 6.5 ⫾ 2.2 9.0 ⫾ 1.9 4.4 ⫾ 1.7
Penguin-2 group
Pre-bed rest 92.1 ⫾ 3.2 2.2 ⫾ 0.5 0 5.8 ⫾ 1.6 0
1.5 Mo bed rest 83.1 ⫾ 5.0 2.7 ⫾ 0.8 1.5 ⫾ 0.4 8.0 ⫾ 2.2 5.0 ⫾ 2.0
Values are means ⫾ SE. Number of fibers analyzed: 7–8 per muscle.

Fig. 1. Changes in contractile profiles of slow soleus

muscle fibers after 2 and 4 mo of bed rest and 1 mo of
recovery. A: fiber diameter. B: absolute Ca2⫹-activated
maximal force (Po). C: relative Ca2⫹-activated maximal
force (Fmax) per cross-sectional area. D: unloaded short-
ening velocity. Values are means ⫾ SE. FL, fiber
length. Significantly different from *pre-bed rest level,

2 mo of bed rest, and §4 mo of bed rest (P ⬍ 0.05).

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Number of fibers: 25 per group.

Ca2⫹ concentration required for pCa50 in the control in the control group were ⫺8, ⫺38, ⫺27, and ⫹83%,
group was 5.92 (1.2 ⫾ 0.1 ␮mol). The values of pCa50 respectively. These parameters were similar before
were lower after 2 and 4 mo of bed rest, i.e., 5.79 (1.6 ⫾ and after bed rest in the Penguin-1 group.
0.2 ␮mol) and 5.68 (1.7 ⫾ 0.2 ␮mol), respectively, than The pCa50 level was lower in the Penguin-1 (5.93,
the pre-bed rest level. There was no significant differ- 1.2 ⫾ 0.1 ␮mol) than the Penguin-2 group (5.77, 1.7 ⫾
ence in pCa50 between 2 and 4 mo of bed rest. After 1 0.2 ␮mol) after 2 mo of bed rest (Fig. 2). There also was
mo of recovery, the pCa50 level was increased (5.89, a significant difference in the pre- and post-bed rest
1.3 ⫾ 0.1 ␮mol) relative to the level obtained at the end pCa50 values for the Penguin-2 group but not for the
of the 4 mo of bed rest period and was similar to the Penguin-1 group.
pre-bed rest level.
Effects of loading and/or knee exercise during bed DISCUSSION
rest. The countermeasures performed during bed rest
in the Penguin-1 and -2 groups had different effects on Effects of bed rest without countermeasures. To our
the contractile properties (Figs. 2 and 3). Before bed knowledge, this is the first report of the effects of 2 and
rest, there were no significant differences in fiber di- 4 mo of bed rest on the contractile properties of single
ameter (Fig. 3A), Po (Fig. 3B), Fmax per CSA (Fig. 3C), muscle fibers in the human soleus muscle. In the
and Vo (Fig. 3D) between Penguin-1 and -2 groups (Fig. present study, the Fmax per CSA and Po, as well as fiber
3). The countermeasures performed by the Penguin-2 diameter, were significantly decreased and the Vo sig-
group ameliorated but did not prevent the adaptations nificantly increased after 2 and 4 mo of bed rest. These
to bed rest observed in the control group (Fig. 1). For contractile responses to long-term bed rest are similar
example, the percent changes from pre-bed rest levels to the results obtained after 17 days of spaceflight and
in fiber diameter, Po, Fmax per CSA, and Vo after 2 mo bed rest in human subjects (44–46).
of bed rest in the Penguin-2 group were ⫺8 (P ⬎ 0.05), The Fmax is thought to be dependent on fiber size,
⫺25, ⫺15, and ⫹36%, respectively. These adaptations because larger fibers have relatively more cross bridges

number of cross bridges per CSA in atrophied muscle

fibers. Because the cross bridge is formed between
thick and thin filaments and is the origin of force, Fmax
is dependent on the number of cross bridges per CSA of
a maximally activated muscle. A decrement in thin
filament density after 17 days of spaceflight or bed rest
has been reported (33, 34, 46). Therefore, the decrease
in Fmax per CSA after bed rest may reflect a decrement
in thin filament density.
Other factors, such as the transition from weakly to
strongly bound cross bridges, may be related to the
decrement in Fmax after bed rest. Active force in muscle
fibers may be generated by the transition of cross
bridges from a weakly to a strongly bound state (21). In
addition, the chemomechanical efficiency of cross
bridges may affect the Fmax. Chemomechanical effi-
ciency is the energy-transferring efficiency when chem-
ical energy released in association with one molecule
ATP breakdown is converted into physical energy by
one cross bridge. A decrease in this efficiency may
cause a large reduction of force development per CSA.

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However, whether changes in cross-bridge transition
patterns or in chemomechanical efficiency of cross
bridges are induced after bed rest is unknown.
The mean Vo of type I fibers in the soleus muscle has
been reported to increase by 30% over control values
after 17 days of bed rest (44). In the present study, the
Vo of type I fibers was more than 70% faster after 2 and
4 mo of bed rest than before bed rest. Vo is thought to
be dependent on the MHC and myosin light chain
isoform profiles and the myosin ATPase activity of the
fiber (22, 32, 39). In hindlimb-unloaded rats, the rela-
tive composition of the fast MHC isoforms increases
significantly in the predominantly slow soleus muscle
(9–11), and this appears to be closely related to an
increase in Vo (11). Previously, our laboratory reported
that 2 and 4 mo of bed rest resulted in a small percent-
age (⬃10–20%) of soleus muscle fibers transforming
from a slow to a fast phenotype in the same subjects
(27, 28). An ⬃40% decrease of slow-twitch fibers was
also observed after hindlimb suspension of rats (10). In
Fig. 2. Tension-pCa relationship of slow soleus muscle fibers after 2 the present study, however, all fibers used expressed
and 4 mo of bed rest and 1 mo of recovery. Number of fibers: 25 per only the slow MHC isoform. Therefore, fiber type trans-
group. formation cannot explain the observed increase in Vo
after bed rest. One possibility for the increase in Vo
may be related to the kinetic transitions of cross-bridge
and can generate higher forces than smaller fibers. The states. For example, Widrick et al. (46) proposed the
relative Fmax per square meter of fiber CSA was 27 and possibility that changes in lattice spacing induced by
42% lower after 2 and 4 mo, respectively, of bed rest decreased activity results in a reduction in the internal
than before bed rest. Although an ⬃8 and 36% decre- drag, which, in turn, increases the velocity of shorten-
ment in fiber diameter was observed after 2 and 4 mo ing. However, the precise mechanism(s) for the eleva-
of bed rest (Fig. 1A), respectively, the Po was 38 and tion of Vo in slow-twitch fibers after bed rest remain
76% lower for the same time periods (Fig. 1B). Thus undefined.
factors other than fiber atrophy influenced the de- It is generally accepted that Ca2⫹ sensitivity is re-
crease in Po. This observation is consistent with the lated to Ca2⫹ affinity of troponin C (18, 30, 41) and/or
report of Widrick et al. (44) of an ⬃10% decrease in the cooperativity between thin and thick filaments (20,
Fmax per CSA after 17 days of bed rest. The larger 24). Slow-type troponin C isoform is expressed in slow
decrease in Fmax per CSA in the present study most fibers and has two high- and low-affinity binding sites
likely reflects the longer experimental period. for Ca2⫹. One of the low-affinity sites is usually inac-
The mechanism(s) for the reduction in Fmax per CSA tive (40). Modulations of Ca2⫹ sensitivity during peri-
is (are) unknown. One possibility is a reduction in the ods of decreased activity have been reported (23, 25,

Fig. 3. Effects of knee extension/flexion exercise with or

without loading on ankle plantar flexors by wearing a
Penguin suit on the contractile properties of slow soleus
muscle fibers during 2 mo of bed rest. A: fiber diameter.
B: Po. C: Fmax per cross-sectional area. D: unloaded
shortening velocity. Values are means ⫾ SE. *Signifi-
cantly different from pre-bed rest level (P ⬍ 0.05).
Number of fibers: 25 per group.

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50). Recently, a depression in Ca2⫹ sensitivity after Effects of loading and/or exercise during bed rest.
bed rest (45) and spaceflight (46) has been reported. The countermeasure effects of two types of resistive
The right shift in pCa-tension relationship could be knee extension/flexion exercise induced by wearing
related to an increased filament spacing due to a selec- Penguin suits were determined. In the Penguin-1
tive loss of actin filaments (45). It has been reported group, continuous resistance was provided at the
that the cooperativity between filaments is affected by knee and ankle. In the Penguin-2 group, the loading
troponin T isoforms (14, 19, 29). An elevation in the on the ankle was removed. Therefore, although there
intracellular free Ca2⫹ concentration in atrophied so- were no differences in the duration and type of knee
leus muscles in a resting state has been reported (20). extension/flexion exercise between the two groups,
Intracellular Ca2⫹ concentration may affect the affin- loading at the ankle induced passive stretch of soleus
ity of troponin C for Ca2⫹. Experiments using glycer- muscle in the Penguin-1 but not the Penguin-2
inated fibers cannot evaluate Ca2⫹ movement via the group.
sarcoplasmic reticulum, which is related to Ca2⫹ sen- The resistive exercise with loading in the Penguin-1
sitivity. It has been reported, however, that sarcoplas- group prevented the adaptations in the contractile
mic reticulum function is altered by periods of de- properties observed in the control group after 2 mo of
creased activity (7, 20, 25, 31, 37, 50). The protein and bed rest. However, the same type of exercise performed
mRNA levels of the “fast” isoform of the sarco(endo)- by the Penguin-2 group, in which no resistive forces on
plasmic reticulum Ca2⫹-ATPase are markedly in- the plantar flexors were imposed, was less effective. It
creased in the rat soleus muscle during 28 days of is clear that the passive stretch of soleus for 10 h/day
hindlimb unloading (37). The sarco(endo)plasmic retic- during bed rest in Penguin-1 group had a beneficial
ulum Ca2⫹-ATPase pre-mRNA and mRNA transcript effect. The stretching of the muscles, not necessarily
levels in soleus have been shown to increase by two-, the knee extension/flexion exercise, during bed rest
three-, and sevenfold after 2, 4, and 10 days of hind- was effective to prevent the alteration of contractile
limb unloading, respectively (31). properties.

In the Penguin-2 group, the changes in fiber diame- 5. Bamman MM, Hunter GR, Stevens BR, Guilliams ME, and
ter, Fmax per CSA, Ca2⫹ sensitivity, and Vo after 2 mo Greenisen MC. Resistance exercise prevents plantar flexor de-
conditioning during bed rest. Med Sci Sports Exerc 29: 1462–
of bed rest were smaller than those observed after bed 1468, 1997.
rest alone (control group). It has been reported that 6. Bangart JJ, Widrick JJ, and Fitts RH. Effect of intermittent
intermittent exercise could partially prevent the alter- weight bearing on soleus fiber force-velocity-power and force-pCa
ation of the contractile properties during periods of relationship. J Appl Physiol 82: 1905–1910, 1997.
7. Bastide B, Conti A, Sorrentino V, and Mounier Y. Proper-
decreased activity (2, 6, 35, 43). Therefore, the type of ties of ryanodine receptors in rat muscles submitted to unloaded
exercise, for example, tonic or phasic, is an important conditions. Biochem Biophys Res Commun 270: 442–447, 2000.
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ing-related changes in muscle properties. Invest 14, Suppl 68: 1–110, 1962.
The present data suggest that mechanical loading by 9. Caiozzo VJ, Haddad F, Baker MJ, Herrick RE, Prietto N,
and Baldwin KM. Microgravity-induced transformations of
stretching of muscle, not necessarily the contraction of myosin isoforms and contractile properties of skeletal muscle.
muscle reported by Akima et al. (1) and Bamman et al. J Appl Physiol 81: 123–132, 1996.
(5), is a useful countermeasure for prevention of decon- 10. Caiozzo VJ, Baker MJ, McCue SA, and Baldwin KM. Sin-
ditioning in slow muscle fibers with antigravity func- gle-fiber and whole muscle analyses of MHC isoform plasticity:
interaction between T3 and unloading. Am J Physiol Cell Physiol
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tonic activity due to stretching. It is also indicated that 11. Campione M, Ausoni S, Guezennec CY, and Schiaffino S.
stretching has to be considered as a possible counter- Myosin and troponin changes in rat soleus muscle after hindlimb
measure for prevention of deconditioning in antigrav- suspension. J Appl Physiol 74: 1156–1160, 1993.
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E, Manchester JK, and Lowry OH. Effects of microgravity
load, duration, and/or exercise frequency required as and tail suspension on enzymes of individual soleus and tibialis

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the countermeasure is still unclear. anterior fibers. J Appl Physiol 73: 66S–73S, 1992.
In conclusion, effects of 2 and 4 mo of bed rest, with 13. Frontera WR and Larsson L. Contractile studies of single
or without exercise countermeasures, on the contrac- human skeletal muscle fibers: a comparison of different muscles,
permeabilization procedures, and storage techniques. Muscle
tile properties of slow fibers in the human soleus mus-
Nerve 20: 948–952, 1997.
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CSA, and pCa50 were reduced after 2 and 4 mo of bed tion and isoforms of myosin heavy chain and troponin-T of rat
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and Vo remained unchanged. Thus it is suggested that Substrate profile in rat soleus muscle fibers after hindlimb
continuous mechanical loading on muscle, such as unloading and fatigue. J Appl Physiol 88: 473–478, 2000.
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nese Space Utilization Promotion Center, Tokyo), C. Sekiguchi Ca2⫹ transients in mouse soleus muscle after hindlimb unload-
(NASDA, Tsukuba City, Japan), B. S. Shenkman, and I. B. Koz- ing and reloading. J Appl Physiol 87: 386–390, 1999.
lovskaya (Dept. of Neurophysiology, IBMP). Acknowledgement is 21. Jurian FJ and Morgan DL. Variation of muscle stiffness with
extended to Dr. R. R. Roy (Brain Research Institute, University of tension during tension transients and constant velocity shorten-
California at Los Angeles) for careful reading and advice for the ing in the frog. J Physiol (Lond) 319: 193–203, 1981.
completion of this paper. 22. McDonald KS and Fitts RH. Effect of hindlimb unweighting
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