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 J Appl Physiol
91: 417–424, 2001.

Maximal and submaximal forces of slow fibers in human

soleus after bed rest

KATSUMASA YAMASHITA-GOTO,1 RYOKO OKUYAMA,1 MASANORI HONDA,1

KENSUKE KAWASAKI,1 KAZUHIKO FUJITA,1 TAKAHIRO YAMADA,2
IKUYA NONAKA,3 YOSHINOBU OHIRA,4 AND TOSHITADA YOSHIOKA1,5
1
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,
and Toshitada Yoshioka. Maximal and submaximal forces
of slow fibers in human soleus after bed rest. J Appl Physiol
91: 417–424, 2001.—The effects of 2 and 4 mo of bed rest, with
or without exercise countermeasures, on the contractile proper-
ties of slow fibers in the human soleus muscle were examined.
Mean fiber diameters were 8 and 36% smaller after 2 and 4 mo
of bed rest, respectively, than the pre-bed rest level. Maximum
tetanic force (Po), maximum activated force (Fmax) per cross-
sectional area (CSA), and the common-logarithm value of free
Ca2⫹ concentration required for half-maximal activation
(pCa50) also decreased after 2 and 4 mo of bed rest. In contrast,
maximum unloaded shortening velocity (Vo) was increased af-
ter 2 and 4 mo of bed rest. After 1 mo of recovery, fiber
diameters, Po, Fmax per CSA (P ⬎ 0.05), and pCa50 were in-
creased and Vo decreased toward pre-bed rest levels. Effects of
knee extension/flexion exercise by wearing an anti-G Penguin
suit for 10 h daily, and the effects of loading or unloading of the
plantar flexors with (Penguin-1) or without (Penguin-2) placing
the elastic loading elements of the suit, respectively, were in-
vestigated during ⬃2 mo of bed rest. In the Penguin-1 group,
mean fiber diameter, Po, Fmax per CSA, Vo, and pCa50 were
similar before and after bed rest. However, the responses of
fiber size and contractile properties to bed rest were not pre-
vented in the Penguin-2 group, although the degree of the
changes was less than those induced by bed rest without any
countermeasure. These results indicate that long-term bed rest
results in reductions of fiber size, force-generation capacity, and
Ca2⫹ sensitivity, and enhancement of shortening velocity in
slow fibers of the soleus. The data indicate that continuous
mechanical loading on muscle, such as stretching of muscle, is
an effective countermeasure for the prevention of muscular
adaptations to gravitational unloading.
human soleus fibers; contractile properties; countermeasure
MECHANICAL LOADING IS ONEof the primary factors regu-
lating the synthesis and degradation of proteins in
Address for reprint requests and other correspondence: Y. Ohira,
School of Health and Sport Sciences, Osaka Univ., Toyonaka City,
Osaka 560-0043, Japan (E-mail: ohira@space.hss.osaka-u.ac.jp).
or mechanical stretch, stimulates protein synthesis
and often results in hypertrophy. In contrast, de-
creased loading, such as occurs during exposure to
actual microgravity or a simulation model such as bed
rest, results in a loss of muscular protein and/or atro-
phy, especially in primary antigravity muscles such as
the soleus (27, 28). Animal studies have shown that
chronic periods of unloading result in a shift from
oxidative to glycolytic metabolic profile (12, 17, 49),
transition of fiber types from slow to fast (3, 4, 11, 38),
increase in shortening velocity (9, 15, 47), decrease in
force-generation capacity (9, 47, 48), and alteration in
excitation-contraction coupling (7, 20, 25, 31, 37, 50),
such as increased rate of Ca2⫹ uptake and leakage to
and from sarcoplasmic reticulum (50), in hindlimb
muscles, e.g., the soleus, tibialis anterior, and vastus
lateralis.
There are a few reports regarding the effects of
decreased activity on the contractile properties of hu-
man soleus muscle fibers (3, 28, 33, 44–46). For exam-
ple, an increase in unloaded shortening velocity (Vo)
and/or decrease in force production associated with
fiber atrophy were observed after 17 days of bed rest
(44, 45). However, the contractile responses to long-
term unloading are still unclear. Furthermore, the
development of countermeasures to prevent muscle
atrophy during long-term unloading is important for
clinical medicine, as well as space medicine. Muscle
deconditioning, such as an atrophy and decrease in
maximum force, of knee extensors and/or plantar flex-
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
plantar flexion (5 sets of 6–10 repetitions every other
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.

http://www.jap.org 8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society 417
418 HUMAN SOLEUS FIBER ADAPTATIONS TO BED REST
day; Ref. 5), respectively. However, the duration of the
bed rest period was relatively short in these studies.
Therefore, the present study was performed to exam-
ine the effects of long-term bed rest on the contractile
properties of fibers from the human soleus muscle and
to determine whether a specific loading paradigm could
prevent any bed rest-associated adaptations.
MATERIALS AND METHODS
Subjects and experimental design. The Institute of Biomed-
ical Problems (IBMP) in Moscow, Russia, performed this
study for the National Space Development Agency of Japan
(NASDA) (Contract no. 8H0-046, The Performance of
NASDA’s Bed Rest Study at IBMP Facility). Thirteen
healthy Russian male volunteers participated in this study.
All subjects were evaluated clinically and were considered to
be in good physical condition. All subjects were informed
about the possible risks in taking muscle biopsies, and a
signed, informed consent was obtained from each subject.
Physical characteristics of subjects in each group are shown
in Table 1. The Human Use Committees at IBMP, NASDA,
and St. Marianna University School of Medicine approved
this study.
Detailed experimental protocols have been described in
our group’s previous papers (27, 28). The study consisted of
two separate experiments. The first experiment was per-
formed to study the effects of 4 mo of bed rest without any
exercise countermeasure and 1 mo of recovery on the con-
tractile properties of slow-type fibers in soleus muscle. Six
volunteers (“control” group) were subjected to 4 mo of bed
rest at a 6° head-down-tilt position. Muscle biopsies were
taken from soleus muscles four times, i.e., ⬃3 wk before bed
rest, after ⬃2 and ⬃4 mo of bed rest, and after ⬃1 mo of
normal ambulatory recovery. The subjects were fed three
times per day, and daily total energy intake was ⬃3,000 kcal
for each subject. The temperature and humidity in the room
were maintained within a normal range, and the day-night
cycle was regulated at 8 AM and 6 PM.
The second experiment was designed to determine the
effects of exercise countermeasures performed during the bed
rest period on the same muscle properties as described above.
Seven subjects were assigned to one of two Penguin suit
groups. They were subjected to a 2-mo bed rest at 6° head-
down-tilt position. All subjects in the second experiment wore
an anti-G Penguin suit for 10 h/day (11 AM to 9 PM) and
additionally performed knee extension and flexion exercise in
a supine position in bed against a resistance of 100 N for the
last 15 min of each hour. The subject’s shoes were equipped
with rollers on the heels, which slid on a smooth surface of a
platform with the ankle maintained at a relatively constant
position. Thus this exercise emphasized knee, not ankle,
movement. The legs were relaxed, keeping the knee at an
extended position during the remaining 45 min.
Table 1. Physical characteristics of subjects
Body
Age, yr Weight, kg Height, cm
Control (n ⫽ 6) 30.8 ⫾ 3.1 80.0 ⫾ 7.6 181.0 ⫾ 2.0
Penguin-1 (n ⫽ 4) 30.5 ⫾ 1.9 72.0 ⫾ 3.3 180.0 ⫾ 3.0
Penguin-2 (n ⫽ 3) 33.3 ⫾ 2.9 70.0 ⫾ 6.8 176.0 ⫾ 2.0
Values are means ⫾ SE. Control, bed rest for 4 mo without any
exercise countermeasure. See MATERIALS AND METHODS for description
of the Penguin-1 and Penguin-2 groups.
The Penguin suit group was divided into two subgroups:
Penguin-1 (n ⫽ 4) and Penguin-2 (n ⫽ 3). Subjects in the
Penguin-1 group wore the full assembly of the Penguin suit,
which included all of the elastic loading elements, during the
10-h period. Subjects in the Penguin-2 group also wore the
full assembly of the Penguin suit, except that the elastic
loading elements at the ball of the foot were disconnected.
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
when the plantar flexor muscles were relaxed. No such resis-
tive forces on the plantar flexors were imposed by the suit in
the Penguin-2 group. For the Penguin suit groups, the soleus
muscle was biopsied twice, i.e., ⬃2 wk before bed rest and
immediately after ⬃2 mo (Penguin-1) or ⬃1.5 mo (Penguin-2)
of bed rest.
Muscle biopsy. During bed rest, the subjects were carried
to the medical treatment room for performance of the muscle
biopsies. Biopsies were taken from the left soleus with the
subjects in a prone position by using the procedures de-
scribed by Bergstrom (8). Local anesthesia was induced via
subcutaneous injection of 4 ml of a 2% lidocaine hydrochlo-
ride solution. A skin incision (5 mm) was made, and a Berg-
Downloaded from jap.physiology.org on July 25, 2007
strom sterile needle was inserted toward the center of mus-
cle. The biopsy sample was divided into several portions. One
of the sample portions was longitudinally tied to a glass bar
by use of surgery thread and was placed in a cold 50%
(vol/vol) glycerin solution (4°C) for 24 h. The glycerin solution
was exchanged with freshly prepared glycerin solution, and
the muscle tissues in the solution were kept at ⫺20°C for
8–10 wk before the contractile analyses were performed.
Because the samples were not treated for skinning imme-
diately after biopsy by using Triton X-100, for example, the
muscle fibers were kept in glycerin solution to skin for 8 wk
at ⫺20°C before the analyses of contractile properties, and all
groups were treated in the same way. The analyses were
completed within 2 wk, and the order of analysis was random
across the groups.
Analytical procedures. On the day of the experiment, first
a bundle was placed in relaxing solution for 15 min and then
individual fibers were carefully dissected from the bundle. A
single muscle fiber (⬃5 mm long) was transferred to an
experimental chamber (0.3 ml volume) filled with the relax-
ing solution. The relaxing solution (47, 48) consisted of 10
mM EGTA, 3.5 mM MgATP, 1.5 mM Mg2⫹, and 20 mM
PIPES at pH 7.0 (20°C). The ionic strength was adjusted to
0.2 M with potassium methane sulfonate. One end of the
fiber was secured with T-shaped aluminum clips to a semi-
conductor transducer element (AE801, Aksjeselskapet
Mikro-Elektronikk), and the other end was attached to a
hook connected to a servomotor (G120D, General Scanning,
Watertown, MA).
All experiments were performed at 20 ⫾ 1°C. The Ca2⫹
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%
(vol/vol) Triton X-100 for 3 min. Isometric tension was re-
corded digitally at 12 kHz by an analog-to-digital converter
(AD216, Nittobo Acoustic Engineering) and stored on a hard
disk connected to a personal computer (PC-9821Ap, NEC) for
later analysis.
Sarcomere length was adjusted to 2.5 ⫾ 0.2 ␮m by use of a
laser diffraction pattern and was monitored using a micro-
scope and charge-coupled device camera connected to the
microscope. If any irregularity in the sarcomere was ob-
served, the fiber was discarded. The fiber diameter, absolute
 HUMAN SOLEUS FIBER ADAPTATIONS TO BED REST
maximally activated force (Po), relative maximally activated
force (Fmax) per fiber cross-sectional area (CSA), Vo, and Ca2⫹
sensitivity of the myofilaments were determined for each
fiber. The CSA of a fiber was calculated from the two diam-
eters in two perpendicular projections. Assuming that the
shape of muscle fiber is a cylinder, we measured the width
and then thickness of fiber by shifting the stage of the
microscope. Fiber CSA then was calculated by using width
and thickness as reported by Frontera and Larsson (13).
The pCa-tension relationships were determined by activat-
ing the fibers in a series of solutions containing free Ca2⫹
concentrations (pCa; 7.0, 6.5, 6.0, 5.5, 5.0, and 4.0). After
washing the muscle fiber in preactivating solution containing
2 mM EGTA, pCa solution was applied. Then, the fiber was
relaxed by washing in the relaxation solution after the ten-
sion reached a plateau level. The Fmax was determined in
pCa 4.0 solution first. Then the fiber was relaxed and Ca2⫹
solution was applied from a lower concentration (pCa 7.0) to
a higher concentration. After application of a pCa solution,
the sample was washed in relaxing solution containing 10
mM EGTA and in preactivating solution containing 2 mM
EGTA, and then the next pCa solution was applied. The
experiment was terminated when the Fmax decreased more
than 10% in pCa 4.0 solution, the resting tension did not
return to zero, or irregularity in the sarcomeres was ob-
served.
Hill plot analysis was used to determine the Ca2⫹ sensi-
tivity of individual fibers. The Ca2⫹ sensitivity of fibers was
evaluated by the concentration of Ca2⫹ for half-maximal
activation (pCa50). The Vo was determined by the slack test
procedure. The times required for the redevelopment of force
after five to six imposed slack steps [each step ⬍20% of fiber
length (FL)] were plotted against the corresponding slack
length, and the points were fitted with a linear regression
line. The slope of this line, which is Vo, was normalized to the
FL and expressed as FL per second (FL/s).
After the mechanical measurements, the fiber segments
were removed from the apparatus; solubilized in 0.03 ml of
an SDS buffer solution consisting of 0.1 M Tris 䡠 HCl (pH 8.8),
5 mM EDTA, 10% SDS, and 50 mM DTT; boiled for 3 min;
and stored at ⫺80°C until subsequent gel electrophoresis
analyses. The fiber sample solutions were run on 12% acryl-
amide gels and stained by Bio-Rad Silver Stain Plus (Bio-
Rad, Hercules, CA). The expression of myosin heavy chain
(MHC) isoforms in the same fibers was determined (27). NIH
Image was used to quantify the relative content of each
isoform. In the present study, all data were determined from
fibers expressing only type I MHC with the total number of
fibers used equal to 25 per group. Some of the isolated fibers
contained either I⫹IIa, I⫹IIa⫹IIx, IIa, or IIa⫹IIx MHC
Table 2. Percent fiber-type distribution based on myosin heavy chain expression
I I ⫹ IIa
Control group
Pre-bed rest 91.6 ⫾ 3.3 2.5 ⫾ 1.2
2 Mo bed rest 83.3 ⫾ 5.0 2.5 ⫾ 1.4
4 Mo bed rest 75.8 ⫾ 4.4 4.2 ⫾ 2.2
1 Mo after bed rest 86.6 ⫾ 5.2 4.6 ⫾ 2.3
Penguin-1 group
Pre-bed rest 91.2 ⫾ 4.1 2.2 ⫾ 0.5
2 Mo bed rest 82.3 ⫾ 5.2 1.9 ⫾ 0.6
Penguin-2 group
Pre-bed rest 92.1 ⫾ 3.2 2.2 ⫾ 0.5
1.5 Mo bed rest 83.1 ⫾ 5.0 2.7 ⫾ 0.8
Values are means ⫾ SE. Number of fibers analyzed: 7–8 per muscle.
419
expression (Table 2). However, the data from these fibers
were not used because the distribution of fibers having these
MHC profiles was low and not always detected in some
samples. The number of fibers analyzed in each muscle was
seven to eight because of the small sample size.
Statistical analysis. All values are expressed as means ⫾
SE. For the first experiment, the values were analyzed by
using ANOVA followed by Scheffé’s post hoc test. For the
second experiment, one-tailed paired t-tests were used for the
comparison between pre- and post-bed rest. ANOVA followed
by Scheffé’s post hoc test was used for comparisons of the
percent change after 2 mo of bed rest, relative to the pre-bed
rest levels, between the control and Penguin suit groups.
Statistical significance was set at P ⬍ 0.05.
RESULTS
Effects of bed rest without countermeasure. Figure 1
shows the responses of the fiber diameter (A), Po (B),
Fmax per CSA (C), and Vo (D) to 4 mo of bed rest
without any countermeasure. The patterns of response
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
and 4 mo of bed rest than before bed rest, respectively.
Fiber size was smaller after 4 mo of bed rest than after
2 mo of bed rest (27%) and was similar to pre-bed rest
levels after 1 mo of recovery.
The mean Po (mN) values were 38 and 76% lower
after 2 and 4 mo of bed rest than the pre-bed rest level,
respectively. In addition, the mean Po was 38% less
after 4 than 2 mo of bed rest. These values recovered to
pre-bed rest levels within 1 mo after bed rest. The Fmax
per CSA was 27 and 42% lower after 2 and 4 mo,
respectively, of bed rest than before bed rest. There
was no significant difference between the mean Fmax
per CSA after 2 or 4 mo of bed rest (15% lower at 4 mo,
P ⬎ 0.05). After 1 mo of recovery after 4 mo of bed rest,
there was a tendency for Fmax per CSA to increase, but
the value was still lower than the pre-bed rest level
(P ⬎ 0.05).
Vo was significantly higher after 2 and 4 mo of bed
rest, i.e., by 82 and 97%, respectively, than that before
bed rest. There were trends (P ⬎ 0.05) for the Vo to be
higher after 4 than 2 mo of bed rest and to approach
pre-bed rest levels after 1 mo of recovery.
Figure 2 illustrates the pCa-tension relationships
after 2 and 4 mo of bed rest. Before bed rest, the free
I ⫹ IIa ⫹ IIx IIa IIa ⫹ IIx
0 5.4 ⫾ 2.0 0
1.3 ⫾ 0.5 8.3 ⫾ 2.2 4.6 ⫾ 1.5
4.6 ⫾ 2.4 12.5 ⫾ 3.0 5.0 ⫾ 2.0
3.3 ⫾ 1.6 2.9 ⫾ 1.2 4.2 ⫾ 1.8
0 6.0 ⫾ 2.0 0
6.5 ⫾ 2.2 9.0 ⫾ 1.9 4.4 ⫾ 1.7
0 5.8 ⫾ 1.6 0
1.5 ⫾ 0.4 8.0 ⫾ 2.2 5.0 ⫾ 2.0
420 HUMAN SOLEUS FIBER ADAPTATIONS TO BED REST
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).
Number of fibers: 25 per group.
Ca2⫹ concentration required for pCa50 in the control
group was 5.92 (1.2 ⫾ 0.1 ␮mol). The values of pCa50
were lower after 2 and 4 mo of bed rest, i.e., 5.79 (1.6 ⫾
0.2 ␮mol) and 5.68 (1.7 ⫾ 0.2 ␮mol), respectively, than
the pre-bed rest level. There was no significant differ-
ence in pCa50 between 2 and 4 mo of bed rest. After 1
mo of recovery, the pCa50 level was increased (5.89,
1.3 ⫾ 0.1 ␮mol) relative to the level obtained at the end
of the 4 mo of bed rest period and was similar to the
pre-bed rest level.
Effects of loading and/or knee exercise during bed
rest. The countermeasures performed during bed rest
in the Penguin-1 and -2 groups had different effects on
the contractile properties (Figs. 2 and 3). Before bed
rest, there were no significant differences in fiber di-
ameter (Fig. 3A), Po (Fig. 3B), Fmax per CSA (Fig. 3C),
and Vo (Fig. 3D) between Penguin-1 and -2 groups (Fig.
3). The countermeasures performed by the Penguin-2
group ameliorated but did not prevent the adaptations
to bed rest observed in the control group (Fig. 1). For
example, the percent changes from pre-bed rest levels
in fiber diameter, Po, Fmax per CSA, and Vo after 2 mo
of bed rest in the Penguin-2 group were ⫺8 (P ⬎ 0.05),
⫺25, ⫺15, and ⫹36%, respectively. These adaptations
Downloaded from jap.physiology.org on July 25, 2007
in the control group were ⫺8, ⫺38, ⫺27, and ⫹83%,
respectively. These parameters were similar before
and after bed rest in the Penguin-1 group.
The pCa50 level was lower in the Penguin-1 (5.93,
1.2 ⫾ 0.1 ␮mol) than the Penguin-2 group (5.77, 1.7 ⫾
0.2 ␮mol) after 2 mo of bed rest (Fig. 2). There also was
a significant difference in the pre- and post-bed rest
pCa50 values for the Penguin-2 group but not for the
Penguin-1 group.
DISCUSSION
Effects of bed rest without countermeasures. To our
knowledge, this is the first report of the effects of 2 and
4 mo of bed rest on the contractile properties of single
muscle fibers in the human soleus muscle. In the
present study, the Fmax per CSA and Po, as well as fiber
diameter, were significantly decreased and the Vo sig-
nificantly increased after 2 and 4 mo of bed rest. These
contractile responses to long-term bed rest are similar
to the results obtained after 17 days of spaceflight and
bed rest in human subjects (44–46).
The Fmax is thought to be dependent on fiber size,
because larger fibers have relatively more cross bridges
 HUMAN SOLEUS FIBER ADAPTATIONS TO BED REST
Fig. 2. Tension-pCa relationship of slow soleus muscle fibers after 2
and 4 mo of bed rest and 1 mo of recovery. Number of fibers: 25 per
group.
and can generate higher forces than smaller fibers. The
relative Fmax per square meter of fiber CSA was 27 and
42% lower after 2 and 4 mo, respectively, of bed rest
than before bed rest. Although an ⬃8 and 36% decre-
ment in fiber diameter was observed after 2 and 4 mo
of bed rest (Fig. 1A), respectively, the Po was 38 and
76% lower for the same time periods (Fig. 1B). Thus
factors other than fiber atrophy influenced the de-
crease in Po. This observation is consistent with the
report of Widrick et al. (44) of an ⬃10% decrease in
Fmax per CSA after 17 days of bed rest. The larger
decrease in Fmax per CSA in the present study most
likely reflects the longer experimental period.
The mechanism(s) for the reduction in Fmax per CSA
is (are) unknown. One possibility is a reduction in the
421
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.
Downloaded from jap.physiology.org on July 25, 2007
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
the present study, however, all fibers used expressed
only the slow MHC isoform. Therefore, fiber type trans-
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
states. For example, Widrick et al. (46) proposed the
possibility that changes in lattice spacing induced by
decreased activity results in a reduction in the internal
drag, which, in turn, increases the velocity of shorten-
ing. However, the precise mechanism(s) for the eleva-
tion of Vo in slow-twitch fibers after bed rest remain
undefined.
It is generally accepted that Ca2⫹ sensitivity is re-
lated to Ca2⫹ affinity of troponin C (18, 30, 41) and/or
the cooperativity between thin and thick filaments (20,
24). Slow-type troponin C isoform is expressed in slow
fibers and has two high- and low-affinity binding sites
for Ca2⫹. One of the low-affinity sites is usually inac-
tive (40). Modulations of Ca2⫹ sensitivity during peri-
ods of decreased activity have been reported (23, 25,
422 HUMAN SOLEUS FIBER ADAPTATIONS TO BED REST
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.
50). Recently, a depression in Ca2⫹ sensitivity after
bed rest (45) and spaceflight (46) has been reported.
The right shift in pCa-tension relationship could be
related to an increased filament spacing due to a selec-
tive loss of actin filaments (45). It has been reported
that the cooperativity between filaments is affected by
troponin T isoforms (14, 19, 29). An elevation in the
intracellular free Ca2⫹ concentration in atrophied so-
leus muscles in a resting state has been reported (20).
Intracellular Ca2⫹ concentration may affect the affin-
ity of troponin C for Ca2⫹. Experiments using glycer-
inated fibers cannot evaluate Ca2⫹ movement via the
sarcoplasmic reticulum, which is related to Ca2⫹ sen-
sitivity. It has been reported, however, that sarcoplas-
mic reticulum function is altered by periods of de-
creased activity (7, 20, 25, 31, 37, 50). The protein and
mRNA levels of the “fast” isoform of the sarco(endo)-
plasmic reticulum Ca2⫹-ATPase are markedly in-
creased in the rat soleus muscle during 28 days of
hindlimb unloading (37). The sarco(endo)plasmic retic-
ulum Ca2⫹-ATPase pre-mRNA and mRNA transcript
levels in soleus have been shown to increase by two-,
three-, and sevenfold after 2, 4, and 10 days of hind-
limb unloading, respectively (31).
Downloaded from jap.physiology.org on July 25, 2007
Effects of loading and/or exercise during bed rest.
The countermeasure effects of two types of resistive
knee extension/flexion exercise induced by wearing
Penguin suits were determined. In the Penguin-1
group, continuous resistance was provided at the
knee and ankle. In the Penguin-2 group, the loading
on the ankle was removed. Therefore, although there
were no differences in the duration and type of knee
extension/flexion exercise between the two groups,
loading at the ankle induced passive stretch of soleus
muscle in the Penguin-1 but not the Penguin-2
group.
The resistive exercise with loading in the Penguin-1
group prevented the adaptations in the contractile
properties observed in the control group after 2 mo of
bed rest. However, the same type of exercise performed
by the Penguin-2 group, in which no resistive forces on
the plantar flexors were imposed, was less effective. It
is clear that the passive stretch of soleus for 10 h/day
during bed rest in Penguin-1 group had a beneficial
effect. The stretching of the muscles, not necessarily
the knee extension/flexion exercise, during bed rest
was effective to prevent the alteration of contractile
properties.
 HUMAN SOLEUS FIBER ADAPTATIONS TO BED REST
In the Penguin-2 group, the changes in fiber diame-
ter, Fmax per CSA, Ca2⫹ sensitivity, and Vo after 2 mo
of bed rest were smaller than those observed after bed
rest alone (control group). It has been reported that
intermittent exercise could partially prevent the alter-
ation of the contractile properties during periods of
decreased activity (2, 6, 35, 43). Therefore, the type of
exercise, for example, tonic or phasic, is an important
factor as the countermeasure for prevention of unload-
ing-related changes in muscle properties.
The present data suggest that mechanical loading by
stretching of muscle, not necessarily the contraction of
muscle reported by Akima et al. (1) and Bamman et al.
(5), is a useful countermeasure for prevention of decon-
ditioning in slow muscle fibers with antigravity func-
tion. This effect may be induced by a stimulation of
tonic activity due to stretching. It is also indicated that
stretching has to be considered as a possible counter-
measure for prevention of deconditioning in antigrav-
ity muscles. However, the effective or minimum work-
load, duration, and/or exercise frequency required as
the countermeasure is still unclear.
In conclusion, effects of 2 and 4 mo of bed rest, with
or without exercise countermeasures, on the contrac-
tile properties of slow fibers in the human soleus mus-
cle were examined. The fiber diameter, Po, Fmax per
CSA, and pCa50 were reduced after 2 and 4 mo of bed
rest. The Vo was increased in response to 2 and 4 mo of
bed rest. After 1 mo of reambulation period, however,
these changes were generally recovered. These results
indicate that long-term inactivity causes the reduction
of fiber size, force generation, and Ca2⫹ sensitivity, and
the elevation of Vo in single muscle fibers of human
soleus. As a result of daily muscle stretching during 2
mo of bed rest, fiber diameter, Po, Fmax per CSA, pCa50,
and Vo remained unchanged. Thus it is suggested that
continuous mechanical loading on muscle, such as
stretching of muscles, is effective as the countermea-
sure for prevention of muscular adaptation to gravita-
tional unloading.
We express special thanks to Drs. R. Izumi, K. Yasukawa (Japa-
nese Space Utilization Promotion Center, Tokyo), C. Sekiguchi
(NASDA, Tsukuba City, Japan), B. S. Shenkman, and I. B. Koz-
lovskaya (Dept. of Neurophysiology, IBMP). Acknowledgement is
extended to Dr. R. R. Roy (Brain Research Institute, University of
California at Los Angeles) for careful reading and advice for the
completion of this paper.
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