Effect of 17 days of bed rest on peak isometric force and
unloaded shortening velocity of human soleus fibers
J. J.
Widrick1,
J. G.
Romatowski1,
J. L. W.
Bain2,
S. W.
Trappe3,
T. A.
Trappe3,
J. L.
Thompson2,
D. L.
Costill3,
D. A.
Riley2, and
R. H.
Fitts1
1 Department of Biology,
Marquette University, Milwaukee 53201;
2 Department of Cellular
Biology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin
53226; and 3 Human Performance
Laboratory, Ball State University, Muncie, Indiana 47306
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ABSTRACT |
The purpose of this study was to examine the effect of prolonged
bed rest (BR) on the peak isometric force
(Po) and unloaded shortening
velocity (Vo)
of single Ca2+-activated muscle
fibers. Soleus muscle biopsies were obtained from eight adult males
before and after 17 days of 6° head-down BR. Chemically
permeabilized single fiber segments were mounted between a force
transducer and position motor, activated with saturating levels of
Ca2+, and subjected to slack
length steps. Vo
was determined by plotting the time for force redevelopment vs. the
slack step distance. Gel electrophoresis revealed that 96% of the pre-
and 87% of the post-BR fibers studied expressed only the slow type I
myosin heavy chain isoform. Fibers with diameter >100 µm made up
only 14% of this post-BR type I population compared with 33% of the
pre-BR type I population. Consequently, the post-BR type I fibers
(n = 147) were, on average, 5%
smaller in diameter than the pre-BR type I fibers
(n = 218) and produced 13% less
absolute Po. BR had no overall
effect on Po per fiber
cross-sectional area
(Po/CSA), even though half
of the subjects displayed a decline of 9-12% in
Po/CSA after BR. Type I
fiber Vo
increased by an average of 34% with BR. Although the ratio of myosin
light chain 3 to myosin light chain 2 also rose with BR, there was no
correlation between this ratio and
Vo for either the
pre- or post-BR fibers. In separate fibers obtained from the original
biopsies, quantitative electron microscopy revealed a 20-24%
decrease in thin filament density, with no change in thick filament
density. These results raise the possibility that alterations in the
geometric relationships between thin and thick filaments may be at
least partially responsible for the elevated
Vo of the post-BR
type I fibers.
contractile properties; non-weight bearing; skeletal muscle
atrophy; muscle disuse; spaceflight
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INTRODUCTION |
IN THE RAT, THE absence of normal weight-bearing
activity results in a rapid decline in the mass of the hindlimb
extensor muscles. As little as 6 days of spaceflight or 7 days of
ground-based hindlimb suspension is sufficient to reduce the soleus
mass of this species by 25-35% (2, 8). These atrophied soleus
muscles display altered contractile characteristics, including a
reduction in peak force per unit muscle mass, an increase in maximal
shortening velocity, and a reduction in peak power output (2, 8). At least a portion of this change in soleus function is the result of
alterations in processes of muscle contraction that lie distal to
sarcoplasmic reticulum Ca2+
release (9, 21, 22, 34).
Humans exposed to chronic non-weight bearing may also experience a
reduction in lower limb muscular performance. Declines in voluntary
peak isometric ankle extensor torque ranging from ~15-40% have
been reported following long- and short-term spaceflight and prolonged
bed rest (14, 19). However, due to the complexity of the intact
neuromuscular system, it is often difficult to ascribe these changes in
human muscle performance to specific physiological mechanisms. The
development of effective interventions to combat reductions in muscle
function will require a more complete understanding of how the
physiological processes of muscle contraction are altered by chronic
non-weight bearing.
In the present study, we used single chemically permeabilized muscle
fiber segments, activated with saturating levels of
Ca2+, to investigate whether
prolonged bed rest altered cross-bridge mechanisms of muscle
contraction. We were specifically interested in whether the peak
isometric force (Po) and
unloaded shortening velocity
(Vo) of single
soleus fibers were affected by bed rest, since
Po falls and
Vo rises for rat
soleus fibers following non-weight bearing (9, 22, 34). The soleus was
selected for study because, in the rat, single fibers from this
slow-twitch muscle show the greatest functional responses to the
absence of weight bearing activity (9). The duration of bed rest
examined in this study, 17 days, corresponded with the duration of the
National Aeronautics and Space Administration (NASA) Life and
Microgravity Sciences (LMS) Space Shuttle mission (STS-78, June 20 to
July 7, 1996). This was done to allow future evaluation of bed rest as
a ground-based model of spaceflight, since similar single fiber experiments are being conducted on muscle fibers obtained before and
after the LMS mission.
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METHODS |
Subjects.
This study was approved by the Institutional Review Boards at Marquette
University, the Medical College of Wisconsin, Ball State University,
and NASA Ames Research Center. Eight adult males were selected from a
pool of volunteers to serve as subjects. Each subject underwent a
medical examination and provided written informed consent before
participation. The mean (±SE) age, height, and pre-bed rest body
mass of these individuals were 43 ± 3 years, 182 ± 2 cm, and 82.2 ± 4.3 kg, respectively. For simplicity, we have
identified these individuals as subjects
1-8. To facilitate comparison to other studies,
the present identification numbers 1-8 correspond to NASA
identification numbers 428, 439, 435, 249, 405, 457, 374, and 420, respectively.
Bed rest and tissue sampling procedures.
Subjects were continuous residents at the Human Research Facility, NASA
Ames Research Center (Moffett Field, CA) for 39 days. Their residence
was divided into a 14-day ambulatory control period, a 17-day bed rest
period, and a 8-day ambulatory recovery period. Subjects remained at a
continuous 6° head-down tilt throughout the bed rest period.
Compliance was monitored 24 h per day by the staff of the Human
Research Facility. All activities, including eating, bathing, excretory
functions, and physiological testing, were performed in this position
using specially designed facilities and equipment.
One to three days before the start of the control period, a muscle
sample was obtained from the left soleus of each subject using the
needle biopsy procedure. During the control and bed rest periods,
subjects underwent physiological testing using similar equipment and
procedures that had been developed for the NASA LMS mission. This
testing, which was performed by each subject on two separate days
during the control period (days 2 or
3 and days
7 or 8) and on three
separate occasions during bed rest (days 2 or 3,
days 8 or
9, and days
13 or 14), included
the determination of right ankle extensor isometric torque using a
specially designed isokinetic dynamometer and measurement of maximal
oxygen consumption during supine cycle ergometry. During each
isokinetic testing session, subjects performed 175 voluntary
contractions at angular velocities ranging from 0 to 300°/s, broken
down as follows: 12 contractions at peak torque (isometric), ~26
contractions at >50% but
80% of peak torque, ~77 contractions
at >25% but
50% of peak torque, and ~60 contractions at
25%
of peak torque. A complete description of these testing procedures has
been presented by Trappe et al. (32, 33). A post-bed rest biopsy sample
was obtained from the right soleus on the final day of bed rest before reambulation.
Pre- and post-bed rest muscle biopsies were placed on saline-soaked
gauze and divided into several portions. One portion, used for the
single-fiber functional experiments described in this study, was
immediately submerged in cold skinning solution [composition in
mM: 125 potassium propionate, 20.0 imidazole (pH 7.0), 2.0 ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 4.0 ATP, 1.0 MgCl2,
and 50% glycerol (vol/vol)] and shipped overnight at 4°C to
Marquette University, where on arrival it was stored at
20°C. A second portion was pinned at a mild stretch and
immersion fixed in a 0.1 M cacodylate buffer (pH 7.2) consisting of 4%
glutaraldehyde and 2% paraformaldehyde with 5 mM calcium chloride.
This sample was shipped overnight at 4°C to the Medical College of
Wisconsin for osmium postfixation and embedding for electron microscopy
as previously described (27).
Single fiber functional experiments.
The compositions of the relaxing and activating solutions were
determined with the computer program of Fabiato and Fabiato (4) and the
stability constants (adjusted for temperature, pH, and ionic strength)
compiled by Godt and Lindley (11). Each solution contained (in mM) 7 EGTA, 20 imidazole, 14.5 creatine phosphate, 1 free
Mg2+, 4 free MgATP, and sufficient
KCl and KOH to produce a total ionic strength of 180 mM and a pH of
7.0. The free Ca2+ concentrations
of the relaxing and activating solutions had pCa values (where pCa =
log free Ca2+
concentration) of 9 and 4.5, respectively.
All contractile measurements were conducted within the initial 28 days
after the biopsy procedure. The methods used to mount the single fiber
segments were the same as those used previously in this laboratory (9,
22, 34, 35). Briefly, a single fiber segment was isolated from a muscle
bundle, transferred to an experimental chamber, and mounted between an
isometric force transducer (Cambridge model 400; Cambridge Technology,
Watertown, MA) and a direct current position motor (Cambridge model
300B; Cambridge Technology). The experimental apparatus was attached to
the stage of an inverted microscope during the experiments. Sarcomere
length was adjusted to 2.5 µm, and the fiber segment length (FL) was
recorded. A Polaroid photograph was taken of the fiber while it was
briefly suspended in air, and fiber width was measured at three points
along the photograph. The mean of these measurements was defined as
fiber diameter by assuming the fiber takes on a circular cross section
when suspended in air (23). Prior to study, the fiber was briefly
bathed in relaxing solution containing 0.5% Brij-58 (polyoxyethylene
20 cetyl ether; Sigma Chemical, St. Louis, MO).
The fiber was activated by transfer from its original chamber,
containing relaxing solution, into an adjacent chamber containing activating solution. These solutions were maintained at 15°C
throughout data collection. Output from the force transducer and
position motor was directed to a digital storage oscilloscope before
being amplified and interfaced to a personal computer. Custom software performed on-line analysis and stored data to disks.
Absolute peak force (in mN) was calculated as the difference between
resting force, measured while the fiber was in relaxing solution, and
the maximal force obtained during activation at pCa 4.5. Normalized
peak force (in kN/m2) was
defined as the absolute peak force divided by the fiber cross-sectional
area (CSA). Peak stiffness was determined by oscillating the fiber (1.5 kHz) at an amplitude that produced a 0.05% peak-to-peak change in FL
(
length) as previously described (34). Peak elastic modulus
(stiffness/fiber CSA) was calculated as [(
force in pCa 4.5
force in pCa 9.0)/(
length)] × (FL/fiber CSA).
Vo was determined
by the slack test procedure as previously performed in this laboratory
(9, 22, 34, 35). The times required for the redevelopment of force
after five to six imposed slack steps (each
20% of FL) were plotted
against the corresponding slack length, and the points were fitted with
a linear least squares regression line. The slope of this line was
Vo that was
normalized to the length of the fiber and expressed as FL per second.
Single fiber gel electrophoresis.
After the contractile measurements were made, the fiber segment was
solubilized in 10 µl of 1% sodium dodecyl sulfate sample buffer and
stored at
80°C. To determine fiber myosin heavy chain (MHC)
composition, ~0.5 nl of fiber volume was run on a Hoefer SE 600 gel
system consisting of a 3% (wt/vol) acrylamide stacking gel and
a 5% (wt/vol) separating gel (21). To determine myosin light
chain (MLC) expression, ~1 nl of fiber volume was loaded on a gel
consisting of a 3.5% acrylamide stacking gel and a 12% acrylamide
separating gel (21). All gels were silver stained as described by
Giulian et al. (10). A flatbed scanner with a transparency adapter was
used to store an image of each gel on computer disk. Image analysis
software (SigmaGel, Jandel Scientific Software) was used to quantify
the relative levels of MLC1,
MLC2, and
MLC3 in each fiber.
Electron microscopy.
Longitudinal and thin cross sections (~60 nm) were cut from bundles
of fibers and stained with uranyl acetate and lead citrate before
examination and photographing in a JEOL 100 CXII electron microscope.
Myofilament densities and sarcomere length were determined from cross
and longitudinal sections, respectively. Final myofilament density
values were adjusted for variations in sarcomere lengths on an
individual subject basis (1). Interpretations were made on 40 pre- and
40 post-bed rest well-fixed fibers not damaged during dissection (5 pre- and 5 post-bed rest fibers per subject). Processing artifacts in
pre-bed rest controls did not generate the ultrastructural changes
observed for the post-bed rest tissues in this report.
Data analysis.
Results are presented as mean ± SE. Pre- and post-bed rest means
were compared with analysis of variance. Statistical significance was
accepted at P < 0.05.
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RESULTS |
Single fiber MHC expression.
Contractile properties and MHC composition were determined for a total
of 395 single soleus fibers (227 pre-bed rest, 168 post-bed rest). As
illustrated in lane 1 of Fig.
1, the 5% polyacrylamide gel
system resolved the slow type I MHC isoform and the two fast MHC
isoforms present in adult human skeletal muscle (29). Overall, 218 of
the pre-bed rest fibers, or 96% of the group total, and 147 of the
post-bed rest fibers, or 87% of the group total, expressed type I MHC
exclusively. The remaining fibers expressed either a fast MHC isoform
(7 pre- and 16 post-bed rest fibers with subject 5 accounting for 11 of the fast post-bed rest fibers)
or coexpressed slow and fast isoforms (2 pre- and 5 post-bed rest
fibers). Typically, the fast isoform present in these fibers was type
IIa, although a small number of post-bed rest fibers expressed the type
IIx isoform. Because the vast majority of the pre- and post-bed rest fiber populations expressed the type I MHC exclusively, the present analysis focuses entirely on the contractile properties of these fibers.

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Fig. 1.
Representative 5% polyacrylamide gel illustrating myosin heavy chain
(MHC) identification in human single muscle fibers.
Lane 1 was loaded with a human
skeletal muscle myosin standard. Each of the remaining lanes was loaded
with a single human post-bed rest soleus fiber. Values for unloaded
shortening velocity
(Vo) of fibers
in lanes 2-4 were 1.03, 0.72, and
0.65 fiber segment lengths (FL)/s, respectively.
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Type I fiber diameter and absolute force.
A positive correlation was observed between peak
Ca2+ activated force and fiber
diameter for both the pre-bed rest (r = 0.80, P < 0.05) and post-bed rest
(r = 0.61, P < 0.05) populations (Fig. 2). However, on average, the post-bed rest
fibers were 5% smaller in diameter (P < 0.05) and produced 13% less peak absolute force (P < 0.05) than the pre-bed rest
fibers (Table 1).

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Fig. 2.
Relationship between fiber diameter and peak
Ca2+-activated force.
A: pre-bed rest type I fibers.
B: post-bed rest type I fibers. Peak
force (mN) was significantly correlated
(P < 0.05) with fiber diameter for
both groups (pre-bed rest, r = 0.80;
post-bed rest, r = 0.61). Number of
fibers per group is same as in Table 1.
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Table 1.
Diameter, peak absolute force, peak normalized force, and unloaded
shortening velocity of pre- and post-bed rest type I fibers
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Figure 2 suggests that the reduction in the mean diameter of the
post-bed rest population was due mainly to a decline in the number of
large-diameter fibers making up this group. This is confirmed by the
histograms in Fig. 3, in which fibers with
diameters >100 µm made up only 14% of the post-bed rest population
in comparison to 33% of the fiber population before bed rest. There
was no indication that bed rest further reduced the diameter of the
smallest fibers, as only three post-bed rest fibers, or 2% of the
post-bed rest population, were observed with diameters that were less
than the diameter of the smallest of the pre-bed rest fibers. Only five of the eight subjects showed fiber atrophy with bed rest (Fig. 4A).
However, the slope of the regression line in Fig.
4A was considerably <1.00,
indicating that those subjects with the largest-diameter pre-bed rest
fibers showed the greatest decline in fiber diameter with bed rest.

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Fig. 3.
Frequency distributions of fiber diameter.
A: pre-bed rest type I fibers.
B: post-bed rest type I fibers. Data
were obtained by collapsing the individual data points in Fig. 2 into
diameter bins of 5 µm. Note that fibers with diameters >100 µm
made up 33 and 14% of the pre-bed rest and post-bed rest populations, respectively.
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Fig. 4.
Relationship between pre-bed rest
(x-axes) and post-bed rest
(y-axes) type I fiber diameter
(A, in µm), absolute peak isometric force (Po,
B, in mN), normalized
Po
(C, in
kN/m2), and
Vo
(D, in FL/s) for the individual
subjects. Symbols represent means ± SE values for a particular
subject (with the no. adjacent to symbols indicating subject's
identification number). Line of identity denoted by solid diagonal
line. Significant correlations (P < 0.05) were observed between pre- and post-bed rest diameter (r = 0.79), absolute
Po
(r = 0.78), and normalized
Po
(r = 0.82). There was no significant
relationship between pre- and post-bed rest
Vo. Slopes of the
least squares regression lines (dotted lines) fit to the pre- and
post-bed rest data were 0.59, 0.50, and 1.02 for
A-C,
respectively. Subject sample sizes were as follows (pre-bed rest,
post-bed rest, respectively): subject
1 = 33, 13; subject 2 = 31, 19; subject 3 = 27, 21;
subject 4 = 20, 22;
subject 5 = 27, 7;
subject 6 = 25, 19;
subject 7 = 32, 24; and
subject 8 = 23, 22.
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Changes in peak absolute force followed a pattern similar to that
observed for fiber diameter. Half of the fibers in the pre-bed rest
population, but only 27% of the post-bed rest population, produced
peak forces >1.0 mN (Fig. 5). Fibers from
all but one subject produced less average force after bed rest vs.
before (Fig. 4B).

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Fig. 5.
Frequency distributions of peak
Ca2+-activated force.
A: pre-bed rest type I fibers.
B: post-bed rest type I fibers. Data
were obtained by collapsing the individual data points in Fig. 2 into force bins of 0.2 mN. Fibers with peak force >1 mN made up 51 and
27% of the pre-bed rest and post-bed rest populations, respectively.
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Peak normalized fiber force.
On average, peak absolute force declined in proportion to fiber atrophy
and there was no change in peak normalized force following bed rest
(Table 1). The linear regression line describing the relationship
between pre- and post-bed rest mean values for individual subjects has
a slope of 1.02 and almost overlaps the line of identity (Fig.
4C). However, four individuals
displayed a 9-12% reduction in normalized force and one a drop of
4%.
Type I fiber peak stiffness.
Stiffness was determined on 104 pre-bed rest and 89 post-bed rest
fibers obtained from six of the eight subjects
(subjects 1-4,
7, and
8). Peak elastic modulus decreased
by 22% with bed rest (for pre-bed rest, 2.51 ± 0.07 kN/m2 × 104; for post-bed rest, 1.95 ± 0.06 kN/m2 × 104; P < 0.05). Because no
difference in peak normalized force was observed between these 104 pre-
and 89 post-bed rest fibers, the ratio of peak isometric tension to
elastic modulus increased by 20% with bed rest (51 ± 1 for pre-bed
rest and 61 ± 1 for post-bed rest;
P < 0.05).
Type I fiber Vo.
The average Vo of
the post-bed rest type I soleus fibers was 34% greater than the
pre-bed rest type I mean (Table 1). This overall increase in
Vo was the result
of a group of high-velocity type I fibers in the post-bed rest
population that was absent in the pre-bed rest population (Fig.
6). A closer examination of Fig. 6 reveals
that only one pre-bed rest type I fiber had a
Vo that exceeded
1.80 FL/s. In contrast, 25 of the post-bed rest type I fibers,
or 17%, had a Vo
>1.80 FL/s. Every subject showed an overall increase in
Vo with bed rest
(Fig. 4D). There was no significant
relationship between pre-bed rest and post-bed rest
Vo for the eight
subjects.

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Fig. 6.
Frequency distributions of fiber
Vo.
A: pre-bed rest type I fibers.
B: post-bed rest type I fibers. Number
of fibers for each distribution is same as in Table 1. Only 1% of the
pre-bed rest fibers had a
Vo >1.8 FL/s
compared with 17% of the post-bed rest fibers.
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Fiber MLC composition.
Figure 7 is a representative 12% gel
illustrating the MLC isoform expression of three soleus fibers. In this
example, the two soleus fibers expressing type I MHC expressed only
slow isoforms of MLC1 and
MLC2. Table
2 is a summary of the MLC composition of
182 pre- and 90 post-bed rest type I fibers, all of which expressed only the slow MLC1 and
MLC2 isoforms. Note that the
average Vo values
of the pre- and post-bed rest fibers making up this subset were
representative of the entire pre- and post-bed rest fiber population.

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Fig. 7.
Representative 12% gel illustrating myosin light chain (MLC)
expression in human single muscle fibers. Single fiber in
right lane expressed type II MHC and
fast isoforms of MLC1 and
MLC2. Two remaining fibers
expressed type I MHC and slow isoforms of MLC1 and
MLC2. Note lower level of
MLC3 expression in type I
fibers.
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There was no difference in the relative expression of
MLC1s,
MLC2s, or
MLC3 between pre- and post-bed
rest fibers. Because each myosin S-1 expresses one regulatory MLC
(MLC2) and one essential MLC
(either MLC1 or
MLC3), we examined possible
changes in the essential MLC composition of these single fibers by
calculating the ratio of MLC3 to
MLC2s (17). This ratio was
elevated 25% in the post-bed rest population. However, there was no
relationship between fiber
Vo and the
MLC3-to-MLC2s
ratio either before or after bed rest (Fig.
8).

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Fig. 8.
Relationship between
Vo and the
MLC3 content of type I fibers.
A: pre-bed rest fibers.
B: post-bed rest fibers.
MLC3 expression is expressed
relative to the regulatory light chain content
(MLC2s). No significant
relationship (P > 0.05) was observed
between Vo and
MLC3 expression for both the
pre-bed rest and post-bed rest populations. Mean values for these data
are compiled in Table 2.
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Myofibrillar ultrastructure.
Electron micrographs of longitudinally sectioned pre- and post-bed rest
soleus fibers are presented in Fig. 9.
After bed rest, the A bands of soleus myofibrils exhibited normal
density, whereas the I bands appeared "moth-eaten." This
morphological alteration indicates that actin filaments were eliminated
to a greater extent than myosin filaments with bed rest. An extensive
ultrastructural quantitation of the thick and thin filament
concentrations demonstrated no average change in thick filament density
with bed rest but a 20% reduction in thin filament density in the I
band and a 24% reduction in thin filament density in the A band (Table
3).

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Fig. 9.
Electron micrographs of longitudinal sections of pre-bed rest
(A) and post-bed rest
(B) soleus fibers. There is a
decreased myofibril diameter in the post-bed rest fibers as indicated
by the shorter Z lines. Sarcomeres in the post-bed rest fiber
(B) are missing myofilaments in the
I band regions (arrows), giving the fiber a moth-eaten appearance.
Magnification: ×17,750.
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DISCUSSION |
Effect of bed rest on fiber diameter and peak isometric force.
After 17 days of bed rest, the average diameter of the type I fibers
obtained from the soleus (measured at a consistent sarcomere length of
2.5 µm) was reduced by 5%. This change, which is equivalent to a
10% decline in fiber CSA, is almost identical to the 9% reduction in
average fiber CSA obtained from the morphological analysis of sectioned
muscle biopsy samples from these same eight subjects (32).
Interestingly, we observed a bed rest-induced reduction in the
percentage of large-diameter soleus fibers, i.e., those with diameters
>100 µm. Because membrane permeabilization is associated with a
20% increase in fiber diameter (12), these large fibers would have in
vivo diameters of ~83 µm or greater. Related to this, we found that
subjects with the largest-diameter pre-bed rest fibers displayed the
greatest degree of post-bed rest fiber atrophy. Similarly, Edgerton et
al. (3) noted that the greatest fiber atrophy after an 11-day
spaceflight was experienced by those astronauts with the
largest-diameter preflight fibers.
Our results suggest that the largest pre-bed rest type I fibers either
were more susceptible to non-weight bearing and underwent the greatest
atrophy or were more likely to express fast MHC isoforms after bed rest
and, therefore, eliminated from our post-bed rest analysis. The fact
that the percentage of type I fibers fell from 96 to 87% with bed rest
would lend support to this second mechanism. However, 67% of the
post-bed rest type II or I/II fibers were obtained from a single
individual (subject 5). If these 12 post-bed rest fibers are eliminated from analysis, type I fibers then
account for 95% of the post-bed rest population, a value virtually
identical to the pre-bed rest result. The possibility that our post-bed rest type I fiber distributions were biased by this one individual is
supported by a morphological analysis of >3,000 sectioned and histochemically stained fibers from these same subjects in which no
pre- to post-bed rest change in fiber type distribution was observed
(32). Furthermore, the three individuals who had the largest pre-bed
rest fibers and displayed the most fiber atrophy (subjects 2,
7, and
8) accounted for a combined total of
one pre-bed rest and two post-bed rest type II or type I/II fibers.
These observations do not support the idea of a selective conversion of
the largest type I pre-bed rest fibers to type II or I/II post-bed rest
fibers but rather that the largest type I fibers were indeed more
susceptible to atrophy.
Because peak Ca2+-activated
absolute force was directly correlated with fiber diameter before and
after bed rest, the more pronounced atrophy of the largest fibers
reduced the percentage of fibers producing relatively high force
(>1.0 mN), with essentially no change in the proportion of fibers
producing relatively low force. Overall, the average decline in peak
force production was proportional to the loss of fiber CSA, although
one-half of the subjects displayed reductions in normalized force of
~10%.
Effect of bed rest on fiber
Vo.
On the basis of 5% polyacrylamide gel electrophoresis, all of the pre-
and post-bed rest fibers making up this study expressed the adult type
I MHC exclusively. The average
Vo of these
fibers rose by 34% with bed rest. This increase is quantitatively
similar to the change in
Vo noted for rat
type I soleus fibers following 14-21 days of hindlimb suspension
(9, 22, 34).
The average Vo of
the pre-bed rest fibers, 0.86 FL/s, was greater than the value
of 0.52 FL/s we previously reported for 61 human type I soleus
fibers (35). However, an examination of individual mean values
indicates that several subjects were in quite good agreement with our
previous work (35) as well as our preliminary results from the
astronauts of the LMS mission (Widrick and Fitts, unpublished
observations). In contrast, some subjects (e.g.,
subjects 2 and
4) displayed values that were
considerably greater than expected. Although we have no clear
explanation for this wide range in pre-bed rest
Vo, it should be
noted that the eight subjects were extremely heterogeneous in terms of
their occupational and recreational activity levels and their pre-bed rest fitness levels. These factors could have contributed to the wide
variability in Vo
observed in this study. Despite greater than expected variation in
pre-bed rest type I fiber
Vo, all subjects displayed an increase in this variable after bed rest. Furthermore, there was no significant relationship between a subject's pre-bed rest
Vo and the extent
to which this variable rose with bed rest (Fig.
4D).
Vo is thought to
be limited by the rate of actomyosin cross-bridge detachment (16), a
process that has been shown to vary with MHC (17, 26) and MLC (15, 30)
isoform expression and the physical properties of the myofilament
lattice (23). However, it is not clear which of these processes (or
combination of processes) is responsible for the elevated post-bed rest
Vo observed in
this study.
In the laboratory rat, non-weight bearing is associated with an
increase in the number of soleus fibers coexpressing type I and II MHCs
(21). Therefore, one possibility is that our gel conditions failed to
identify small amounts of fast MHC coexpressed by the post-bed rest
fibers. However, Reiser et al. (25, 26) have concluded that
Vo is a nonlinear
function of the relative proportions of slow and fast MHC isoforms
present in a fiber, since slower-cycling cross bridges act as an
internal drag on the rate of detachment of faster cycling cross bridges
and thereby negate some of the effects of faster-cycling cross bridges.
Recent studies conducted on human type IIa, IIa/IIb, and IIb fibers
(17, 18) and on rhesus monkey type I, I/IIa, and IIa fibers (R. H. Fitts, S. C. Bodine, J. G. Romatowski, and J. J. Widrick, unpublished observations) suggest that
Vo remains
relatively unchanged until the expression of a faster MHC isoform
reaches 20-30%, or more, of total MHC. Although it is possible
that our electrophoretic conditions failed to detect
2.5% of the
total MHC present in a particular fiber (22), this appears to be an
order of magnitude below that required to increase
Vo by 34%.
A second possibility is that bed rest modified MLC composition, since
alterations in regulatory and essential MLCs are known to have
modulatory effects on fiber
Vo (15, 30). This
mechanism is not supported by the results of the present study. First,
all of the type I pre- and post-bed rest fibers expressed similar levels of the slow MLC2 isoform.
Second, although MLC3 content increased following bed rest, levels of this essential light chain were
unrelated to Vo
for both the pre- or the post-bed rest type I fibers. In this regard,
our pre-bed rest results are in agreement with the work of Larsson and
Moss (17), who also reported no relationship between
MLC3 levels and
Vo in human
slow-twitch muscle fibers.
Another possibility is that bed rest induced de novo or reexpression of
an alternative slow MHC isoform. Such an alternative MHC isoform could
be responsible for the elevated
Vo of the
post-bed rest fibers if this isoform had a greater intrinsic
Vo than the adult
-MHC and comigrated with the adult
-MHC on our gels. However, even when we employed gel conditions previously shown to resolve two
slow MHC isoforms in young adult rat whole soleus muscles (5), we
observed only one slow MHC isoform. Thus it seems unlikely that the
increased post-bed rest
Vo was induced by
the expression of a second slow MHC previously observed by Fauteck and
Kandarian (5).
A final possibility pertains to the relationship between myofilament
geometry and fiber shortening velocity. Riley et al. (27) have reported
that, after hindlimb suspension, myofibrils of rat soleus fibers
exhibit a moth-eaten appearance, suggesting a reduction in myofilament
packing. Because it is known that fiber Vo is extremely
sensitive to changes in myofilament lattice spacing (23), we quantified
the density of thick and thin filaments in a separate group of
histologically fixed pre- and post-bed rest fibers obtained from the
original muscle biopsies. Whereas thick filament density
remained constant, thin filament density declined by
20-24% in the post-bed rest fibers. In an idealized myofilament,
this would be approximated by the loss of one thin filament for every
six thin filaments, i.e., a single thick filament would be surrounded
by five thin filaments instead of the normal six. Because the area
occupied by these five filaments remains constant (no change in myosin
density), plane geometry dictates that the average distance from thin
to thick filaments increases.
One limitation of this approach is that we do not have myofilament
density measurements and physiological data on the same fibers.
However, the observed increase in
Vo, the decline
in elastic modulus, and the rise in the ratio of
Po to elastic modulus are all
consistent with an increase in the distance between thin and thick
filaments (13, 23). Furthermore, a relatively wide change in
Vo coupled with a
much smaller change in Po is
similar to results obtained from single fiber preparations in which
filament lattice spacing is experimentally altered (23).
Comparison to previous human studies.
To our knowledge, this is the first study to investigate the effects of
bed rest on the functional properties of human soleus fibers.
Previously, Larsson et al. (18) studied human vastus lateralis fibers
obtained after 42 days of bed rest and found a reduction in peak
normalized force with no change in type I fiber
Vo. Comparisons
between these two studies must take into account differences in
experimental design, particularly the muscles studied and the duration
of bed rest. For instance, the soleus and the vastus lateralis are
under different anatomic constraints during the bed rest model of
non-weight bearing. The subjects in the present study were observed to
frequently extend and maintain the foot at a 40° plantar flexed
position during bed rest (24). Consequently, the soleus was maintained
at a shorter than normal length for much of the bed rest period. In
contrast, the vastus lateralis is either at its normal weight-bearing
length or stretched (if subjects flex the knee) during bed rest. It is
well established that muscles maintained in neutral or lengthened
positions do not display the same changes in protein metabolism (20),
ultrastructure (27), or contractile function (28) observed for muscles
maintained at short lengths. Single fibers obtained from muscles that
are chronically shortened may therefore display different contractile characteristics than fibers from muscles maintained at neutral or
lengthened positions.
In the rat hindlimb suspension model, rates of protein degradation and
synthesis follow distinct time courses (31). It seems likely that human
skeletal muscle protein metabolism may also vary with the duration of
bed rest. The fact that we observed no reduction in normalized peak
force whereas Larsson et al. (18) observed a 40% reduction in this
variable could be the result of an increase in net protein degradation
as bed rest is extended beyond 17 days. Interestingly, one-half of our
subjects showed a bed rest-induced reduction in normalized force of
9-12%, suggesting that, in many individuals, the loss in
contractile protein had begun to exceed fiber atrophy by the 17th day
of non-weight bearing.
Finally, to mimic the LMS space shuttle mission, the present subjects
performed loaded contractile activity during the isokinetic and aerobic
capacity testing sessions. On the basis of the observations of Edgerton
et al. (3), it seems unlikely that the aerobic capacity test affected
our results. The five sessions of isokinetic testing conducted between
the pre- and post-bed rest biopsies averaged out to only 28 contractions per day. Furthermore, only 4 of these 28 contractions were
performed above 50% of peak (voluntary) isometric force and of these 4 only 2 were performed as maximal isometric contractions. Clearly, this
was insufficient activity to prevent the atrophy and functional changes
noted in the single fibers of this study. However, we have no way of
knowing whether the observed responses would have been exacerbated if
this isokinetic testing activity had been absent.
Comparison to previous animal studies.
The most widely studied model of non-weight bearing is ground-based rat
hindlimb suspension, with the soleus being the most frequently studied
muscle. The 34% increase in post-bed rest type I soleus fiber
Vo in the present
study is very similar to the 30-50% increase reported for rat
type I soleus fiber after 14 days of hindlimb suspension (9, 22, 34).
However, the decline in the absolute force of
Ca2+-activated rat type I fibers
(22, 34) is four times greater than the absolute force deficits noted
for the human fibers in the present study. In addition, rat type I
soleus fibers show a significant reduction in peak normalized force
after non-weight bearing (9, 22, 34). The greater force decline in rat
fibers may be the result of interspecies differences in the net rates of protein degradation during non-weight bearing (6, 31).
Individual responses to bed rest.
Subject 1 appeared to react to bed
rest in a manner that was inconsistent with the responses of the other
seven subjects. Subject 1 displayed no
atrophy and the largest increases in both normalized
Po and
Vo with bed rest.
This individual was also the only subject to show an increase in
absolute Po with non-weight bearing. It is unclear why this individual responded in this way. Regardless of the mechanism involved, elimination of
subject 1 had no effect on a
reanalysis of the fiber diameter,
Po, and
Vo data of the
remaining seven individuals [for 185 pre- and 134 post-bed rest
type I fibers, diameters were 96 ± 1 and 90 ± 1 µm
(P < 0.05), absolute
Po values were 1.02 ± 0.02 and
0.85 ± 0.02 mN (P < 0.05),
normalized Po values were 140 ± 2 and 137 ± 3 kN/m2
(P > 0.05), and
Vo values were
0.85 ± 0.03 and 1.10 ± 0.05 (P < 0.05), respectively].
Summary and conclusions.
Seventeen days of bed rest produced an average decline in type I fiber
diameter of 5% and a reduction in peak
Ca2+-activated force of 13%, with
relatively large-diameter, and therefore high-force-producing, fibers
being most susceptible to atrophy. On average, fiber atrophy was
proportional to reductions in
Ca2+-activated force so that peak
force per fiber CSA was unchanged. After bed rest,
Vo and
MLC3 content were elevated 34 and
25%, respectively, in single fibers expressing type I MHC. However, MLC3 content was unrelated to
Vo in these
slow-twitch fibers. The mechanism underlying the elevated post-bed rest
Vo is unclear. Possibilities include the expression of an alternative type I MHC or
changes in the geometric properties of the myofilament lattice. Support
for the second of these mechanisms comes from our finding of a
20-24% reduction in thin filament density following bed rest.
 |
ACKNOWLEDGEMENTS |
This study was supported by NASA Grant NAS9-18768 (to R. H. Fitts). D. A. Riley received partial salary support from NASA Grant
NAG2-956.
 |
FOOTNOTES |
Address for reprint requests: R. H. Fitts, Marquette Univ., Dept. of
Biology, Wehr Life Sciences Bldg., PO Box 1881, Milwaukee, WI
53201-1881.
Received 14 August 1996; accepted in final form 17 July 1997.
 |
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