Vol. 279, Issue 5, R1546-R1557, November 2000
Spaceflight effects on single skeletal muscle fiber function
in the rhesus monkey
Robert H.
Fitts1,
Dominique
Desplanches2,
Janell G.
Romatowski1, and
Jeffrey J.
Widrick1
1 Department of Biology, Marquette University, Milwaukee,
Wisconsin 53201; and 2 Laboratoire de Physiologie,
Faculté de Medecine, 69373 Lyon,
France
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ABSTRACT |
The purpose of this investigation was to
understand how 14 days of weightlessness alters the cellular properties
of individual slow- and fast-twitch muscle fibers in the rhesus monkey.
The diameter of the soleus (Sol) type I, medial gastrocnemius (MG) type
I, and MG type II fibers from the vivarium controls averaged 60 ± 1, 46 ± 2, and 59 ± 2 µm, respectively. Both a control
1-G capsule sit (CS) and spaceflight (SF) significantly reduced the Sol
type I fiber diameter (20 and 13%, respectively) and peak force, with
the latter declining from 0.48 ± 0.01 to 0.31 ± 0.02 (CS
group) and 0.32 ± 0.01 mN (SF group). When the peak force was
expressed as kiloNewtons per square meter (kN/m2),
only the SF group showed a significant decline. This group also showed
a significant 15% drop in peak fiber stiffness that suggests that
fewer cross bridges were contracting in parallel. In the MG, SF but not
CS depressed the type I fiber diameter and force. Additionally, SF
significantly depressed absolute (mN) and relative
(kN/m2) force in the fast-twitch MG fibers by 30%
and 28%, respectively. The Ca2+ sensitivity of the type I
fiber (Sol and MG) was significantly reduced by growth but unaltered by
SF. Flight had no significant effect on the mean maximal fiber
shortening velocity in any fiber type or muscle. The post-SF Sol type I
fibers showed a reduced peak power and, at peak power, an elevated
velocity and decreased force. In conclusion, CS and SF caused atrophy
and a reduced force and power in the Sol type I fiber. However, only SF
elicited atrophy and reduced force (mN) in the MG type I fiber and a
decline in relative force (kN/m2) in the Sol type I and MG
type II fibers.
contractile properties; morphology; slow twitch; fast twitch
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INTRODUCTION |
EXPERIMENTS BOTH FROM
COSMOS and space shuttle missions have shown weightlessness to
result in a rapid decline in the mass and force of rat hindlimb
extensor muscles (2, 15). Additionally, despite an
increased maximal fiber-shortening velocity
(Vo), peak power was reduced in rat soleus (Sol)
muscle after a 14-day spaceflight (2). Weightlessness has
also been shown to induce muscle atrophy, increase fatigue, and reduce
the physical work capacity in humans (3, 20). Declines in
voluntary peak isometric ankle extensor torque ranging from 15 to 40%
have been reported after long- and short-term spaceflight and prolonged
bed rest in humans (7, 11). However, it is not clear to
what extent these changes in neuromuscular performance are due to
alterations in neural mechanisms of recruitment, to changes in muscle
architecture and/or fiber-type composition, and/or to alterations in
cellular processes of contraction. To assess the cellular basis of the
functional changes associated with limb unloading in humans, we have
used the single-skinned fiber preparation to study the effects of 17 days of bed rest and weightlessness (19-21). In
addition to a reduced fiber size and force, both bed rest and
spaceflight increased the Vo and reduced the
power in the slow type I fiber of the Sol. The increased velocity was
not caused by an increased expression of fast-type myosin, because the
adapted fibers contained only slow myosin (19, 20).
When comparing the effects of weightlessness on human versus rodent
skeletal muscle, it is clear that species differences exist. For
example, spaceflights as short as 6-9 days have been shown to
cause slow-to-fast-twitch fiber transitions of up to 10% in
rat skeletal muscle, whereas in humans, no significant fiber-type
transition occurred even after a 17-day flight (2, 8, 20).
It has been hypothesized that larger animals such as nonhuman primates
may more closely mimic the human in their musculoskeletal response to
microgravity (1). Consequently, we recently characterized
the contractile properties of single slow and fast fibers prepared from
the medial gastrocnemius (MG) and Sol muscles of adult rhesus monkeys
and compared the results to those obtained in humans and rats (5,
23). In general, the contractile properties of the monkey fibers
were closer in function to the human than the rat. For example,
Vo of the slow type I fiber averaged 0.91, 0.73, and 0.51 fiber lengths/s in the rat, monkey, and human, respectively
(23). Fiber diameter and peak power also showed distinct
species differences with fibers from the monkey and human showing
larger diameters but lower peak power than rat fibers
(23). On the basis of these data, one could hypothesize
that muscle and neuromuscular adaptations to microgravity would be
similar in monkeys and humans.
This investigation was part of a multinational (Russian, French, and
American) integrated project designed to study how the physiological
and behavioral functions of the rhesus monkey respond to
weightlessness. The rhesus biosatellite program was initiated by the
Russian Institute for Biomedical Problems (IBMP) and the Soviet Space
Agency in 1983. The rhesus monkeys studied in this investigation flew
aboard the Bion 11 biosatellite that was the sixth in the series of
biosatellite flights. The specific objectives of our study were to
1) establish the effects of microgravity of the contractile
properties of individual slow- and fast-twitch fibers isolated from the
Sol and MG muscles of the rhesus monkey; 2) integrate our
results with those of others to sort out the relative importance of
changes in motor recruitment versus the reduced loading in leading to
the atrophy and reduced performance of skeletal muscle; and
3) determine the extent to which the microgravity-induced changes in skeletal muscle fiber function mirror those observed in
humans. The latter objective will allow researchers to determine the
extent to which rhesus monkey biosatellite studies relate to
adaptations in humans.
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MATERIALS AND METHODS |
Animal selection, care, and training.
The Bion 11 mission was a 14-day flight (12/24/96 to 1/6/97) that
carried two 4- to 5-kg male rhesus monkeys. The animals were selected
from a flight pool of 12, and the flight selection was based on the
quality of the preflight data, viable implants, and success in
completing motor and behavioral tasks. A description of the animal
selection procedures, the transportation to the launch site, recovery
procedures from the landing site, and the electromyogram (EMG) and
tendon force sensor implants was published elsewhere (14).
The animal care and experiments described here followed the
Principles of Laboratory Animal Care (National Institutes of
Health Publication 85-23, revised 1985) and were approved by the
animal care and use committees at National Aeronautics and Space
Administration(NASA)-Ames Research Center (Moffet Field, CA), the IBMP,
and Marquette University (Milwaukee, WI). Except for 3 days preflight
and during the flight, the animals were housed individually in standard
4.3-ft2 stainless steel cages at the IBMP in Moscow,
Russia. The module within the Bion satellite contained a capsule with
two contoured chairs, the life support systems, and test equipment
including physiological data recording systems. Three days prelaunch,
the two flight monkeys were placed in the flight chairs and lightly secured with a chest harness attached to the back of the chair to limit
leaning forward at the waist (14). The chair also limited movement of the legs at the hip in the sagittal plane, whereas the
lower legs were unsecured (14). The flight capsule and
living conditions in flight were the same as published for the Bion 10, Cosmos 2229 mission (10).
Experimental groups and flight capsule.
In addition to the two flight animals, we studied five vivarium control
and two capsule control animals. All nine monkeys were juvenile male
rhesus monkeys with an average age of 40.3 ± 0.4 mo. The capsule
control group was studied postflight, and the animals were housed in a
capsule identical to the flight capsule for 17 days
(1/24/97-2/9/97) under environmental conditions (temperature, light-dark cycle, food intake) that mimicked those experienced by the
flight animals. The duration of the 17-day capsule sit was identical to
the flight-animal capsule exposure (3-day prelaunch and 14-day flight).
To be consistent with other publications, this group is referred to as
the 14-day capsule-sit (R+17) control group.
Due to the time needed to train the animals and implant biosensors, the
preflight muscle biopsies were obtained ~4.5 mo before the flight.
Any functional changes in the cellular properties of individual slow-
and fast-twitch fibers due to growth during the 4-mo period were
controlled for by the vivarium control group. During flight, the two
flight animals were restrained in contoured chairs within the capsule.
The 1-G, R+17 capsule control animals were studied to evaluate the
effects of the capsule environment independent of spaceflight.
Biopsy procedure.
After general anesthesia (isoflurane), the pre- and postbiopsies were
taken from two independent sites in the right Sol and the MG muscles
using an open-biopsy technique. Both the pre- and postbiopsies were
taken from the right leg muscles as the left leg contained EMG and
tendon force-transducer implants. The procedures used were exactly as
described previously (5). The sites were selected to
ensure that the same muscle fibers were not sampled during the pre- and
postbiopsies and that the fiber-type distribution within each sampled
region was similar (16). The postflight biopsies were
obtained according to the following schedule: the two flight animals
were biopsied at ~24 h after reentry (R+1), which was as soon as
possible after return of the animals from the landing site to Moscow;
two of the vivarium control animals were biopsied on flight days
3 and 4, respectively, and two were biopsied 1 day
after the flight animals. The fifth vivarium control animal was
biopsied the same day as the two R+17 capsule control animals that were
biopsied 24 h after the chair-restraint period.
The biopsied sample was weighed and then divided longitudinally into
three sections. Two of the three sections were used by other
investigators. A small piece (~1 mm thick) was cut from the bottom of
the third section and used for the morphological studies described
below. The remaining tissue was then divided longitudinally, and one
section was used for the skinned-fiber studies described here. The
other section was aligned longitudinally on a small index card and
frozen in liquid nitrogen and used for enzyme and metabolic studies
(R. H. Fitts, V. Grichko, and L. B. De La Cruz, unpublished
observations). The sections to be used for the skinned-fiber
experiments were placed in 4°C skinning solution [composition in mM:
125 K propionate, 20.0 imidazole (pH 7.0), 2.0 EGTA, 4.0 ATP, and 1.0 MgCl2 and 50% glycerol (vol/vol)] and shipped back to
Marquette University (4°C shipper). On arrival at Marquette, the
bundles were placed in fresh skinning solution and stored at 
20°C
for up to 4 wk (21, 22). The single-fiber experiments
described in this manuscript were carried out within this 4-wk period.
Morphometric analysis of myofibrils.
The 1-mm cross-sectional piece was processed for electron microscopy by
fixing in a 6.25% solution of glutaraldehyde. After a 1-h fixation,
samples were cut into small blocks and put into a solution of 1% osmic
acid in 0.06 M veronal-acetate buffer for 2 h. After dehydration
in increasing concentrations of ethanol, the samples were embedded in
Epon (9). The morphometric analysis of the samples was
carried out on ultrathin sections (60-90 nm) from two tissue
blocks randomly chosen from each biopsy. Twenty micrographs per block
and hence 40 micrographs per biopsy were taken with a systematic
sampling procedure in consecutive frames of 200-square mesh grids. A
final magnification of ×24,000 was used to estimate the myofibrillar
density. Contact prints of the 35-mm films were projected on a screen
fitted with a grid C 16 (144 test points). The myofibrillar density was
determined by standard stereological procedures (17). To
determine Z band and sarcomere lengths, a magnification of ×20,000 was
used, and measurements were made on 10 fibers per animal.
Experimental solutions and single fiber preparation.
The composition of the relaxing and activating solutions were exactly
as described previously (21, 22). The relaxing solution had a free Ca2+ concentration of pCa 9.0 (where pCa = log Ca2+ concentration) that contained the following (in
mM): 20 imidazole, 7 EGTA, 14.5 creatine phosphate, 4.74 ATP, 5.40 MgCl2, and 0.016 CaCl2 · 2H2O. The maximal activating
solution, pCa 4.5, contained (in mM) 20.0 imidazole, 7.0 EGTA, 14.5 creatine phosphate, 4.81 ATP, 5.26 MgCl2, and 7.0 CaCl2 · 2H2O. The pH of the relaxing and activating solutions was adjusted to 7.0 with KOH, and total ionic
strength was adjusted to 180 mM with KCl. For the force-pCa experiments, the fibers were activated with a series of solutions ranging from pCa 6.8 to 5.0. These solutions were made by mixing appropriate volumes of the activating (pCa 4.5) and relaxing (pCa 9.0)
solutions (21).
On the day of an experiment, a muscle bundle (Sol or MG) was
transferred to a dissecting chamber containing relaxing solution. A
single fiber was isolated from the bundle and transferred to an
experimental chamber containing relaxing solution. This stainless steel
chamber contained three troughs that allowed the fiber to be moved from
a low-Ca2+ relaxing solution to activating solutions of
various pCa values. A fiber segment (~2 mm long) was mounted between
a force transducer (model 400, Cambridge Technology, Cambridge, MA;
sensitivity 2 mV/mg) and an isotonic direct-current torque motor (model
300H, Cambridge Technology), as described previously in detail
(18). The experimental chamber was mounted on the stage of
an inverted microscope, and temperature was maintained at 15°C.
Sarcomere length was adjusted to 2.5 µm by using an eyepiece
micrometer (×800), and segment length was determined by moving the
microscope stage with a micrometer so that the fiber segment moved
across the visual field of the eyepiece. The segment length was
determined directly from the micrometer displacement. A Polaroid
photograph was taken of the fiber while it was briefly suspended in
air. Fiber diameter was determined at three points along the length of
the photograph, and fiber cross-sectional area was calculated from the mean width, assuming the fiber forms a circular cross section
when suspended in air. Any fiber showing a high degree of striation
nonuniformity or a damaged region was discarded.
Peak fiber force (N and kN/m2), stiffness,
Vo, and the force-velocity, force-power, and
force-pCa relationships were determined exactly as described previously
(5). During the determination of the force-pCa
relationship, fiber stiffness was also determined at all pCa values
tested. Immediately after the determination of
Vo, the rate of tension redevelopment
(ktr) was measured by activating the fiber in
pCa 4.5. Once peak force was obtained, a 400 µm slack for 60 and 20 ms, respectively, for slow and fast fibers was introduced, after which
the fiber was rapidly restretched to mechanically break the
actin-myosin cross bridges (12). The rate constant for the
tension redevelopment was defined as ktr. A
computer program employing a least-squares fit was used to solve for
ktr (12). Figure
1 shows representative slack-unslack
tests for two slow fibers for which ktr = 3.04 and 3.56/s. Due to the need to conserve time such that multiple
fibers could be studied per day, a laser clamp to maintain a constant
sarcomere length during tension redevelopment was not employed.
However, in preliminary experiments, we showed that slow fiber
ktr was the same with and without a laser clamp.
In contrast, fast fibers showed lower values when a clamp was not used.
Apparently, the likelihood of sarcomere nonunifority developing is
higher in fast fibers. Consequently, fast fiber
ktr values are not reported.
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Fig. 1.
Two representative experiments demonstrating the
technique for measuring the peak rate of tension redevelopment
(ktr). The fiber was first maximally activated
in pCa 4.5 and then rapidly slacked and reextended to break all
attached cross bridges. The ktr was then
determined by fitting an exponential to the force record after
reextension. In this example, 2 slow fibers are shown with a
ktr of 3.56 and 3.04/s.
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After the contractile measurements, the myosin heavy (MHC) and light
chain (MLC) composition of each fiber was determined by SDS-PAGE
exactly as described previously (22). On the basis of
their MHC profile, each fiber was identified as slow type I, fast type
II, or a hybrid containing both slow and fast myosin isozymes
(22). Fast type II fibers were considered as a single group and not subdivided into type IIa and IIx, because in monkey fibers, it is not possible to consistently subdivide these isozymes by
SDS-PAGE analysis (5).
Statistical analysis.
Data are presented as means ± SE. Statistical differences between
groups were assessed using a one-way ANOVA with Tukey's post hoc test.
Statistical significance was accepted at P
0.05.
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RESULTS |
Animal weights and fiber diameters.
At the time of the prebiopsies (8/96), the 12 flight candidates had an
average body weight of 4.0 ± 0.1 kg, whereas 4.5-5 mo
latter, at the time of the postbiopsies, these same animals averaged
4.5 ± 0.1 kg. The body weight of the flight animals
(monkeys 484 and 357) and the R+17 controls
(monkeys 501 and 534) declined during the 14-day
flight or capsule sit period. For the two flight monkeys, body weight
declined from 5.1 to 4.5 (monkey 484) and from 4.9 to 4.3 kg
(monkey 357), whereas for the R+17 controls, body weight
dropped from 5.3 to 4.8 (monkey 501) and from 4.5 to 4.3 kg
(monkey 534).
In the preflight analyses, there were no significant differences
between groups in the diameters or contractile properties of either the
slow type I or fast type II fibers isolated from the Sol or MG muscles.
Consequently, all fibers of a given fiber type and muscle were pooled
to form a single preflight mean (Tables 1-5).
The diameters of the type I and II fibers isolated from the
vivarium controls showed no significant difference when the pre- and
postsamples were compared (Table 1). In contrast, a significant decline
in the Sol type I fiber diameter was observed in both the R+17 and
flight groups (Table 1). Both of these groups also showed a reduced MG
type I fiber diameter compared with the precondition, but only flight
produced significant atrophy in this fiber type relative to the
vivarium controls (Table 1). The fast type II fiber diameters were
unaffected by either the capsule sit (R+17) or flight (Table 1). Some
fibers contained both slow type I and fast type II myosin (hybrid
fibers). The hybrid fibers of the Sol and MG responded to spaceflight
and the capsule sit in a manner qualitatively similar to the type I
population of a given muscle. Thus the MG hybrid fibers showed greater
atrophy with spaceflight (36 ± 4 µm) than with the capsule sit
(50 ± 4 µm). Different from the MG type I fibers, both groups
were significantly smaller than the vivarium controls (61 ± 3 µm).
Fiber force and stiffness.
The peak force (N and kN/m2) and stiffness for the
slow type I and fast type II fibers are shown in Table 2. Type I Sol
fibers from both the capsule control (R+17) and the flight animals
showed a significant decline in peak force (N) compared with the
vivarium controls. However, when these data were expressed as
kiloNewtons per square meter, only the flight group showed a
significant decline. Peak force of the types I and II fibers of the MG
were unaffected by the capsule sit, whereas microgravity significantly
reduced the absolute peak force of both fast and slow fibers and the
force per cross-sectional area of the fast type II MG fibers (Table 2).
Hybrid fibers showed a tendency to respond in a manner similar to the
slow type I fiber type. In the Sol, the hybrid fiber peak isometric
force (Po) was 0.39 ± 0.04, 0.30 ± 0.02, and
0.27 ± 0.03 mN in the vivarium, capsule, and flight groups,
respectively, whereas in the MG, the hybrid fiber Po values
were 0.38 ± 0.06 (vivarium), 0.41 ± 0.10 (R+17 capsule),
and 0.21 ± 0.04 (flight) mN. For all groups except the capsule
control, hybrid fibers represented 11-16% of the fiber population
for both the Sol and MG. In the Sol capsule control group, the hybrid
fibers represented 51% of the total population.
Fiber stiffness (Eo) is thought to partially
reflect the number of attached cross bridges. The Sol type I and MG
type II fibers showed a reduced Eo postflight,
but only in the former was the decline significant. The
Po/Eo ratio was not altered by the
capsule sit or spaceflight in either fiber type or muscle (Table 2). This finding suggests that there was no change in the force per cross
bridge, and thus the decline in the peak force per cross-sectional area
(kN/m2) of the Sol type I fiber would seem to be
attributable to a selective loss in the number of myofilaments per
myofibril and/or to a reduced myofibril density per fiber. The electron
micrograph (EM) analysis of myofilament density did not support
the former possibility. The myofilament density of the two preflight
animals was 71.8 and 69.8% compared with 74.0 and 69.7% postflight.
The vivarium control and R+17 capsule-sit groups showed myofilament
densities that were not significantly different from the flight
animals. Although we could not document a flight-induced decline in
myofilament density, the EM analyses supported the fiber diameter
measurements in that the postflight samples from the Sol type I fibers
showed significantly shorter Z-band lengths (Fig.
2). Flight monkey 357 showed
considerably more type I fiber atrophy and loss of force than flight
monkey 484. Flight induced a 39% decline in the peak force
of the Sol slow type I fibers of monkey 357 compared with 22% for monkey 484. This variability in response is perhaps
best observed in Fig. 3, in which peak
force is plotted against fiber diameter for both the pre- and
postflight data. Postflight, the 9 smallest and 11 weakest fibers all
belonged to monkey 357.
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Fig. 2.
Longitudinal sections of soleus (Sol) muscle fibers from the 2 flight animals pre (A)- and postflight (B).
Postflight myofibrillar atrophy is reflected by the reduced Z-band
length. Monkey 357 showed the greatest Z-band length decline
(preflight 0.86 ± 0.05 vs. postflight 0.50 ± 0.04 µm)
compared with monkey 484 (preflight 1.03 ± 0.04 vs.
postflight 0.76 ± 0.06 µm). For monkey 357, the
decrease was underestimated, because the average postflight sarcomere
length was less than the preflight (postflight 2.10 ± 0.01 vs.
preflight 2.28 ± 0.03 µm), whereas for monkey 484,
the reverse was the case (postflight 2.15 ± 0.01 vs. preflight
2.10 ± 0.03 µm).
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Fig. 3.
Relationships between fiber diameter and peak
Ca2+-activated isometric force (Po). Except for
+, which represents the average point for all preflight Sol type I
fibers (growth, capsule, and flight animals), each symbol represents
the results of a single Sol type I fiber.  ,
Monkey 357 preflight type I fibers;  monkey 357 postflight type I fibers;  ,
monkey 484 preflight type I fibers;  ,
monkey 484 postflight type I fibers.
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pCa-force relationship.
The growth period between the pre- and postflight biopsies resulted in
a right shift in the pCa-force relationship for the slow type I fibers
of the Sol and MG. This shift resulted in a significantly higher free
Ca2+ (pCa50) for half-maximal
activation in the postvivarium controls for the slow type I
fibers relative to the preflight group (Table 3). However, neither the
capsule sit nor spaceflight had any effect on the pCa-force
relationship in slow or fast fibers. Thus the activation threshold,
n1 and n2, and the pCa50 values
were not different between these groups and the postvivarium control group (Table 3). Figure 4 compares the
pCa-force relationships (pre- vs. postflight) for Sol type I fibers
from the vivarium control, R+17 capsule sit, and flight monkeys. The
right shift in the relationship was observed in all three groups
documenting that the change was related to growth and not the capsule
sit or spaceflight.
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Fig. 4.
Force-pCa relationships for type I Sol fibers obtained
from pre- (filled symbols, solid lines) and postbiopsies (open symbols,
dashed lines) for growth controls (A), 14-day capsule-sit
group (R+17; B), and spaceflight (C) animals.
Forces observed during submaximal activations (P) were expressed
relative to force obtained at pCa 4.5 (Po). Symbols
represent means. All fibers expressed type I myosin heavy chain (MHC)
on 5% gels.
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Vo and ktr.
In the slow type I fibers of the Sol, there was no significant effect
of growth (post- vs. previvarium), the capsule sit (R+17 vs.
postvivarium), or flight (flight vs. postvivarium) on the Vo or the peak ktr after
a slack-unslack (Table 4). In contrast, both the slow type I and fast
type II fibers of the MG showed a significant decline in fiber
Vo with growth, whereas this variable was not
affected by the capsule sit or spaceflight (Table 4). The
ktr of the MG type I fiber also showed no change
with the capsule sit or flight but declined with growth (Table 4).
The individual fibers studied were all identified as slow type I,
hybrid, or fast type II based on their myosin isozyme pattern on 12%
and 5% SDS gels. Figures 5 and
6 are representative 12% and 5% gels
showing individual fibers and their Vos. Figure
7 shows a histogram of Sol slow type I
fiber Vo for each group (pre vs. post). The
growth controls showed a large number of fibers with
Vos between 0.5 and 1.0 fiber length (FL)/s,
whereas for the postflight group, there was a trend toward more fibers
with Vo values >1.1 FL/s. However, the ANOVA
analysis demonstrated that the number of fibers with
Vo values >1.1 FL/s was not significantly altered by flight. Spaceflight had no effect on the Sol type I fiber
MLC3/MLC2s ratio. On the basis of densitometric
scanning of the 12% SDS gels of the Sol type I fibers shown in Table
3, the postflight MLC3/MLC2s ratio of
0.109 ± 0.013 was not significantly altered from the preflight
value of 0.138 ± 0.029. Additionally, the
Vos of the fast slow fibers
(Vos >1.1 FL/s) could not be explained by an
increased expression of fast troponin T, because there was no
difference between the slow troponin T-to-total troponin T ratio
between postflight type I fibers with relatively high
Vos compared with those with relatively low
Vos (0.96 ± 0.03 vs. 0.97 ± 0.02, respectively). Hybrid fibers from both the Sol and MG (pre-mean value,
1.39 ± 0.11 FL/s) were significantly faster than the slow type I
fiber (P < 0.001) and, similar to the type I fibers from the Sol, were unaffected by growth (1.33 ± 0.22 FL/s), the capsule sit (1.48 ± 0.16 FL/s), or flight (1.23 ± 0.23 FL/s).
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Fig. 5.
Representative 12% SDS-polyacrylamide gels illustrating myosin
light chain (LC) expression in single rhesus Sol (lanes
3-5) and gastrocnemius (lanes 6-14) fibers.
Lanes 1 and 2 are myosin standards from rhesus
Sol and gastrocnemius muscle, respectively. The fiber type and maximal
fiber-shortening velocity (Vo) for each lane
were: (3) type I, 0.64 fiber length (Fl)/s;
(4) hybrid type I and II, 1.73 Fl/s; (5) type
I, 0.88 Fl/s; (6) type II, 2.04 Fl/s; (7)
type II, 2.18 Fl/s; (8) type II, 5.08 Fl/s;
(9) type I, 0.63 Fl/s; (10) type II, 1.86 Fl/s; (11) type I, 0.37 Fl/s; (12) type I,
0.40 Fl/s; (13) type II, 1.70 Fl/s; (14) type
II, 4.79 Fl/s. Nos. in parentheses represent lanes
3-14.
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Fig. 6.
Representative 5% gel demonstrating separation of type
I, IIa, and IIx MHC's in single rhesus Sol (lanes
3-11) and gastrocnemius (lanes 12-14) fibers. Lanes 1 and 2 are myosin standards from rhesus Sol and gastrocnemius muscle,
respectively. The fiber type and Vo for each
lane were: (3) type IIa, 6.74 Fl/s; (4) type
I, 1.35 Fl/s; (5) type I, 1.09 Fl/s; (6) type
I, 0.76 Fl/s; (7) type I, 1.07 Fl/s; (8) type
I, 0.99 Fl/s; (9) type I, 1.37 Fl/s; (10)
type I, 0.59 Fl/s; (11) type I, 0.89 Fl/s;
(12) type IIx, 8.73 Fl/s; (13) type IIx, 6.04 Fl/s; (14) type I, 0.91 Fl/s.
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Isotonic contractile properties.
The isotonic force-velocity and force-power relationships for the
Sol and MG type I fibers for each group are shown in Figs. 8 and
9. From these data,
Vmax, dimensionless parameter
(a/Po), peak power, and the force and velocity obtained at
peak power were determined. The mean values for each group and fiber
type are shown in Table 5. For the Sol type I fiber, these parameters were unaffected by the growth period from the pre- to the postflight biopsy (Fig. 8). Similar to fiber Vo, the Sol
type I fiber Vmax was not significantly
altered by the capsule sit or spaceflight. In contrast, Sol type I
fiber peak power significantly declined after the capsule sit and
flight compared with the vivarium control (Table 5 and Fig. 8). The
decreased peak power was primarily the result of the reduced
Po, but a greater curvature (lower a/Po ratio)
of the force-velocity relationship also contributed to the decline
(Fig. 8). As with fiber diameter and force, monkey 357 showed a greater decline in Sol type I fiber power than monkey 484 (Fig. 10). For monkey
357, Sol type I fiber peak power declined from 7.42 ± 1.13 to 4.92 ± 0.44 µN · FL · s
1, whereas for
monkey 484, in the same fiber type, peak power declined from
6.93 ± 1.1 to 5.73 ± 0.39 µN · FL · s1. Due to the reduced
Po and a/Po ratio, the Sol type I fibers of the
R+17 (capsule sit) and postflight groups shortened at a slower velocity
at any given load. At peak power, the postflight fibers showed a
significant increase in velocity and decrease in force (both in mN and
kN/m2) compared with the vivarium control group (Table 5).
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Fig. 8.
Composite force-velocity and force-power relationships
for type I Sol fibers obtained from pre (solid lines)- and postbiopsies
(dashed lines) for growth controls, R+17 capsule, and space animals.
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Fig. 9.
Composite force-velocity and force-power relationships
for type I medial gastrocnemius fibers obtained from pre (solid lines)-
and postbiopsies (dashed lines) for growth controls, R+17 capsule, and
space animals.
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Fig. 10.
A
comparison of the effects of spaceflight in rhesus monkeys (14-day
flight) and humans (17-day flight) on the diameter and functional
properties of single type I Sol fibers. The symbols represent the mean
relative change [i.e., (postflight mean value/preflight mean
value) × 100%] experienced by each individual monkey
(, monkey 357; ,
monkey 484) or human (). Bars represent the
means (±SE) of the individual relative changes. The spaceflight human
data were obtained from Widrick et al. (20).
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In the MG type I fiber population, growth caused a significant decline
in peak power as well as the force and velocity obtained at peak power
(Fig. 9 and Table 5). However, spaceflight had no significant effect on
the isotonic contractile properties of the MG except for an increase in
the MG type I fiber a/Po ratio.
| |
DISCUSSION |
A major objective of NASA's space program is to conduct a human
exploration of Mars by 2014. For this to become a reality, a number of
important biological problems need to be solved. Primary among these is
the development of effective countermeasures to prevent the loss of
skeletal muscle mass, force, and power (20). To date, the
major limitations have been the high costs of human spaceflight
missions, which necessarily limited their number, and the difficulty of
studying the effects of weightlessness in humans independent of crew
countermeasure programs. One solution to these problems is to fly
animals without countermeasures in considerably less costly unmanned
biosatellites. Results from earlier collaborative experiments between
the United States and Russia, flown as part of the Cosmos biosatellite
program, suggested that the nonhuman primate might adapt to
microgravity in a manner similar to humans (1).
Additionally, we have recently observed that the single-fiber
contractile function of the nonhuman primate is similar to that
observed in humans (23). Thus a primary purpose of this
investigation was to determine the effects of 14 days of microgravity
on the contractile function of individual slow type I and fast type II
fibers in the rhesus monkey and compare the functional alterations with
those observed in humans. If adaptations were the same or similar,
future biosatellite experiments could be designed to explore the
complex interactions of neural recruitment, altered endocrine function,
and cell and molecular change within individual fibers and their
relative importance to the microgravity-induced decline in skeletal
muscle performance.
Fiber diameter and peak force.
Spaceflight induced a 13% and 17% decline in the diameters of the
slow type I fibers of the Sol and MG muscles, respectively. In
contrast, the fast type II fibers of the MG showed no significant change. The extent of Sol type I fiber atrophy observed here for the
rhesus monkey was similar to that found in humans after 11- and 17-day
flights (4, 20). We observed considerable subject variability as the Sol type I fibers from monkey 357 showed
a 16% decline in diameter, whereas the diameter of this fiber type in
monkey 484 was not significantly altered. This subject
variability has also been observed in humans (Fig. 10). In a recent
17-day spaceflight, only three of the four crew members studied
experienced Sol type I fiber atrophy, and considerable intersubject
variability was also observed after an 11-day spaceflight and a 17-day
bed rest (4, 20, 22). In the human studies, a portion of
the variability might have resulted from varying amounts (number and intensity) of exercise countermeasures employed by each subject. Alternatively, there may be intersubject differences in how the neuromuscular system responds to microgravity. One advantage of this
study is that no countermeasures were performed, and thus the observed
changes can be attributed to space travel. The recent publication of
Recktenwald et al. (14) demonstrated that the two flight
monkeys studied here showed a decreased cycle period and EMG burst
duration of the primary extensors (Sol and MG) during locomotion
postflight, whereas the burst amplitude of the tibialis anterior
(flexor muscle) increased. Because this bias toward flexor activity was
not observed in the R+17 monkeys, the authors concluded that the
alterations in the neural regulation of movement could not be
attributed merely to restriction of movement, but were caused by
factors unique to the microgravity environment (14). Recktenwald et al. (14) also reported that the MG tendon
forces were considerably greater post- compared with preflight during locomotion. Collectively, the authors concluded that microgravity induced a reorganization of the recruitment to favor fast versus slow
motor units and flexor versus extensor muscles. This interpretation is
consistent with our findings of selective atrophy of the slow type I
fibers in the same two flight monkeys and with our recent observations
in humans in which we observed greater atrophy of the slow Sol type I
fibers compared with fast MG fibers (Ref. 20 and unpublished
observations). Of particular importance to our results was the finding
that in-flight monkey 357 showed a progressive change from
predominately Sol activity to mainly MG activity over the duration of
the flight, whereas this recruitment ratio remained unchanged in
monkey 484 (Hodgson, personal communication). This suggests that the Sol type I fiber atrophy observed in
monkey 357, but not 484, was a direct result of
the reduced activation of the Sol motor units in monkey 357.
During treadmill walking postflight, both flight and R+17 capsule
monkeys showed a reduced Sol muscle activation compared with preflight,
whereas MG activity increased in the R+17 but not the flight animals
(14). These recruitment pattern differences are consistent
with our observation of significant atrophy of the MG type I fibers in
the postflight but not the R+17 capsule group.
Monkey 357 showed a considerably greater decline in Sol type
I fiber force compared with monkey 484, and in the MG, only
flight reduced peak force (mN). This latter observation plus the
finding that flight, but not the capsule sit, reduced relative force
(kN/m2) in both slow and fast fibers represents an
important difference between simply reducing activity (R+17 capsule
group) and spaceflight. In the latter condition, the reduced activity
was associated with muscle unloading. Our results do not allow us to
make a definitive conclusion about why weightlessness reduced relative
force. The observation that fiber stiffness declined whereas the
Po/Eo ratio remained unaltered
suggests that the decline in peak force (kN/m2) was not
caused by a reduced force per cross bridge. These data are consistent
with the hypothesis that the microgravity-induced decline in relative
force was caused by myofibril atrophy producing a reduced myofibril
density per fiber and/or a decline in the density of myofilaments
within a myofibril. The morphometrical data do not support the latter
possibility, because flight had no significant effect on the density of
myofilaments per myofibril. The possibility exists that a small, but
functionally significant, decline in myofilament density did occur but
that it went undetected by EM analysis. An additional possibility is
that the myofibril and myofilament densities were unaltered but that
the number of functional cross bridges per myofilament decreased.
Whatever the mechanism, it is clear that the decline in relative force
required not only inactivity, but also a zero-load condition.
Fiber shortening velocity.
After a 17-day spaceflight in humans, we observed an ~25% increase
in the Sol type I fiber Vo (20).
This increase was not associated with any change in the myosin isozyme
pattern. We hypothesized that the increased Vo
was caused by a selective loss in the thin filament actin producing an
increased distance between thin and thick filaments. This geometric
change would cause the cross bridge to dissociate sooner, thus reducing
the internal drag in the last phase of the cross-bridge power stroke
(13). In this study, the increase in Sol type I fiber
Vo was less and not significant at the
P0.05 level (postflight vs. vivarium group). Clearly, fiber and myofibril atrophy occurred as a result of microgravity, but
we have no evidence on whether or not there was a selective loss in
actin relative to myosin. The reason for the reduced effect on fiber
Vo in rhesus compared with humans is unknown but
may be related to the shorter duration of this flight. Importantly, spaceflight showed a tendency to increase the number of slow type I
fibers with Vo > 1.1 FL/s (Fig. 7).
Besides a shift to higher velocity type I fibers, whole muscle velocity
would be increased postflight if the percentage of hybrid or fast type
II fibers increased. However, in this study, spaceflight had no
significant effect on the distribution of fiber types isolated for
study in either the Sol or MG. In contrast, a single-fiber analysis
showed a significant decrease in the percentage of fibers expressing type I MHC and a corresponding increase in fibers containing type IIa
MHC after a 17-day spaceflight in humans. However, when fiber type was
determined by histochemical myosin ATPase analysis of the entire biopsy
cross section, no significant change in fiber-type distribution was
observed (20). Collectively these data suggest that if
significant fiber-type shifting from slow type I to fast type II fibers
occurs with weightlessness in nonhuman primates or humans, spaceflight
durations longer than 17 days are required.
Fiber peak power.
The ability of a muscle to perform work is related to the peak power
that can be generated. In all species, due to their considerably higher
shortening velocities, fast type II fibers generate significantly more
peak power than slow type I fibers (6, 24). The results presented here agree with previously published data on rhesus monkeys
and demonstrate that the peak power of slow and fast fibers in the
rhesus monkeys are similar to the corresponding fiber types in humans
and considerably lower than rat fibers (6, 23, 24).
Both spaceflight and the R+17 capsule sit significantly reduced the
peak power of the Sol slow type I fibers. This decline can be entirely
attributed to fiber atrophy and the reduced force-generating capacity
of the fiber. Thus, when peak power was normalized to fiber volume,
power was not affected by the capsule sit or spaceflight. In the
postflight fibers, the velocity obtained at peak power was
significantly higher than the postvivarium control group, and this
served to partially compensate for the reduced fiber force at peak
power in the postflight animals (Table 5).
Conclusions.
The 14-day spaceflight induced muscle fiber atrophy and a decline in
peak force in the slow type I fibers of the Sol and MG. In the former
muscle, the decline in force could not be totally explained by fiber
atrophy because relative force (kN/m2) also
declined. Flight monkey 357 showed the greatest
fiber atrophy and force loss, and these changes were associated with an
in-flight shift in EMG activity from predominately Sol to mainly MG
over the duration of the flight. In comparison, the R+17 capsule-sit group showed type I fiber atrophy and force loss only in the Sol and no
change in relative force in either muscle or fiber type. This
comparison suggests that the loss in relative force requires not only
inactivity, but also a zero-load condition. The decline in type I fiber
peak power after both the capsule sit and flight was entirely explained
by the fiber atrophy. Spaceflight caused a tendency for more
high-velocity slow fibers (Vo > 1.1 FL/s), and, during the development of peak power, the postflight Sol type I
fibers shortened at significantly higher velocities. This adaptation in
velocity compensated in part for the decline in force during the
development of peak power.
These results demonstrate that the contractile properties of single
rhesus monkey fibers and their adaptation to microgravity are similar
to what we have previously observed in humans. As pointed out by
Recktenwald et al. (14), an advantage to studying rhesus
monkeys is that one can maintain chronically implanted intramuscular
electrodes that allow accurate EMG recordings and direct comparisons
between changes in recruitment and cellular alterations within the
individual muscle fibers. With this model, it should be possible to
determine whether microgravity induces an altered recruitment strategy
from predominantly Sol to predominantly gastrocnemius, thus causing Sol
type I fiber atrophy or the Sol type I fiber atrophy occurs first,
which, in turn, initiates an increased gastrocnemius activity.
Perspectives
NASA has planned human exploration of Mars by the year 2014. It is
expected that the trip would require ~6 mo and that the crew would
live on Mars for 18 mo before returning to Earth. Thus the entire
mission would require ~3 yr of exposure to either the microgravity of
space or the gravitational forces of Mars, a planet with approximately
one-third the Earth's gravity. The data presented here demonstrate
that, without countermeasures, significant muscle atrophy and loss in
force and power occur within 14 day of weightlessness with the
antigravity slow type I fiber more susceptible than the fast type II
fiber. Clearly, exercise countermeasures (and perhaps hormone
supplementation) will have to be incorporated to prevent unacceptable
loss of bone and muscle. Even if artificial gravity during flight
proves possible and effective, it is unlikely to alleviate the need for
exercise programs. The current results are in good agreement with
previous work on humans exposed to 17 days of weightlessness (Fig. 10).
In the latter study, the crew participated in an aerobic exercise
countermeasure program consisting of bicycle ergometry and treadmill
running. Despite this, the type I fiber atrophy and the decline in
force were similar to that observed in the current study in which the
monkeys performed no exercise countermeasures. This suggests that the
aerobic exercise countermeasures employed by NASA are ineffective in
preventing muscle wasting and the loss in force and power. Our results
demonstrate that inactivity was enough to elicit fiber atrophy and
reduced absolute force (mN), whereas the decline in relative force
(kN/m2) appeared to require inactivity and unloading.
Undoubtedly, heavy resistance exercise (rather than aerobic or
endurance exercise) of the key antigravity muscles of the legs and back
will be needed to prevent microgravity-induced muscle wasting.
| |
ACKNOWLEDGEMENTS |
The authors thank the NASA support team of Mike Skidmore (Project
Manager), Dr. Richard Grindeland (Project Scientist), and Shawn
Bengston (Science Operation Manager), whose extraordinary efforts made
this work possible. We also thank the members of the Russian Space
Agency and the Institute of Biomedical Problems and, in particular, Dr.
Inessa Kozlovskaya (Russian Project Scientist) for support throughout
this project. Finally, we thank the Centre d'Etudes Spatiales (CNES)
Project Manager Dr. Michel Viso for coordinating the French-United
States collaboration, Dr. Sue Bodine for conducting the muscle
biopsies, and Dr. Hans Hoppeler for assistance in the analysis of the EMs.
| |
FOOTNOTES |
This study was supported by NASA Grant NAG2-636 to R. H. Fitts and CNES Grant 071 N97/CNES 6896 to D. Desplanches.
Address for reprint requests and other correspondence: R. H. Fitts, Marquette Univ., Dept. of Biology, Wehr Life Sciences Bldg.,
P.O. Box 1881, Milwaukee, WI 53201-1881 (E-mail:
robert.fitts{at}marquette.edu).
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.
Received 28 October 1999; accepted in final form 29 June 2000.
| |
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ABSTRACT
INTRODUCTION
