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1
* Department of Pathology, Brown University School of Medicine and The Miriam Hospital, Providence, Rhode Island 02906, USA; and
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI 02912, USA
1Correspondence: Department of Pathology, Brown University School of Medicine, 164 Summit Ave., Providence, RI 02906, USA. E-mail: herman_vandenburgh{at}brown.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: protein turnover • skeletal myofiber • spaceflight • TCA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2)
. The
mechanical forces that a muscle fiber generates to overcome gravity can
be divided into two kinds: active and passive. Active muscle tension
occurs during muscle contractions and results in the shortening of the
myofiber's sarcomeres. Passive tension is tension applied to the
muscle fibers by stretching and causes a decrease in the overlap of the
sarcomeres. Both active and passive tensions are essential for normal
muscle growth (3)
; they stimulate myofiber hypertrophy (4)
and their
loss leads to muscle atrophy (5)
. Reduced generation of active and
passive tensions in space most likely contributes to the muscle wasting
process. Since extensive exercise programs during long-term space
travel slow the rate of muscle atrophy but do not prevent it, other
factors are also likely to influence the wasting process. For example,
muscle atrophy during space travel may result from reduced levels of
circulating hormones such as growth hormone
(GH)2(6)
or increased levels of the catabolic glucocorticoids (7)
. Using a
ground-based hind limb unloading rodent model for space travel-induced
muscle atrophy, Grindeland et al. (8,
9)
have shown a synergistic
effect between GH and tension in attenuating muscle atrophy. The causes
of muscle atrophy during space travel are therefore most likely
multifaceted, with both local and systemic components.
Knowledge at the cellular and molecular level on how
gravity/tension regulates muscle size by altering protein turnover
rates has used tissue culture models to study these processes under
defined in vitro conditions. These in vitro
studies have shown significant interactions between muscle tension and
exogenous growth factors. For example, increased muscle tension in
avian myofibers increases their growth response to insulin-like growth
factor-1 (10)
and protects the muscle cells from the atrophying effects
of the catabolic glucocorticoids (11)
. Tissue-cultured myofibers
therefore provide a unique model for studying the local vs. systemic
interaction of these factors in the space environment and may assist in
the future development of pharmacological agents to attenuate atrophy
(12)
. Studies with isolated cells in tissue culture offer a number of
advantages over animal exercise physiology studies. It is easier in
tissue culture to separate pure mechanical effects from other
regulating factors present in vivo such as innervation,
electrical activity, and levels of circulating hormones (13)
.
Tissue-cultured striated myofibers are uninnervated and can be
maintained electrically quiescent in defined, serum-free medium (14,
15)
. A potential disadvantage of tissue culture studies is that the
skeletal myofibers are neonatal-like rather than adult fibers, based on
morphology (14)
and contractile protein isoform expression (16)
. The
cultured neonatal myofibers may therefore not respond to space travel
in an identical manner to adult myofibers. With present technology,
adult myofibers cannot be maintained in a healthy condition in
vitro for more than several hours. Results obtained from tissue
culture studies will complement those from in vivo studies
(17,
18)
to better understand gravity/tension regulation of skeletal
muscle growth at the molecular level.
In the present study, tissue engineering techniques were used to form
3-dimensional skeletal muscle, organ-like structures containing several
thousand well-aligned postmitotic myofibers capable of generating
directed work (19)
. These bioartificial muscles (BAMs) can be
maintained without atrophy for over 30 days in vitro in
perfusion bioreactor cartridges (20)
and (as shown in this paper)
successfully flown aboard the Space Shuttle in the mid-deck Space
Tissue Loss (STL) Module hardware. BAMs were found to atrophy during
10–12 days of space travel in a similar manner to in vivo
muscle and will thus provide a unique tool for studying the process of
space travel-induced muscle atrophy and for countermeasure development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The cells were plated at a very high density of 5.0 to 7.5 x 106 cells/culture well in 1.0 ml of growth
medium (85/10/5) consisting of Eagle's basal medium (1.0 mg/l glucose)
(Life Technologies, Inc., Gaithersburg, Md.), 10% horse serum
(Sigma Chemical Co., St. Louis, Mo.), 5% chicken embryo extract,
and 50 U/ml penicillin G (Sigma); the medium was changed every 24 h. For BAM formation, special rectangular elastic culture wells (10 mm
W x 20 mm L x 10 mm H) were constructed from
collagen-coated thin silicone rubber membranes (0.01 inches, Silicone
Specialty Fabricators, Inc., Pasco Robles, Calif.), as described
previously (21,
22)
. These wells were mounted on aluminum holding
brackets and stretched to a final length of 31 mm before cell plating.
In one flight experiment (STS77), the cell layer was overlaid with a
type I collagen gel solution (Collaborative Biomedical Products,
Bedford, Mass.) 48 to 96 h postplating in order to enhance BAM
development (14)
. With or without collagen coating, the muscle cell
layers lift up from the culture substratum after 5–10 days in
vitro and the longer outer edges of the tissue roll inward,
forming a cylindrical muscle-like structure (BAM) ~25 mm in length,
2–3 mm in diameter, and attached to small stainless steel screens at
each end of the wells (Fig. 1
A) (22)
. Under these growth conditions, the myoblasts
proliferate and fuse into striated and contractile multinucleated
myofibers, which are aligned parallel to the long axis of the well by
10–14 days in vitro (Fig. 1B
).
|
For the Space Shuttle experiments, the BAMs were fixed in length
in their elastic culture wells with a stainless steel mesh support
(Fig. 1A
), removed from their aluminum holding bracket, and
transferred to 55 ml bioreactor cartridges (6 BAMs/cartridge) ~130 mm
long and 24 mm I.D. The bioreactors cartridges were purchased from Cell
Co. (Gaithersburg, Md.) and modified by removal of the hollow fibers. A
leak-proof screw cap was machined for one end to allow easy
insertion/removal of the BAMs. The cells were therefore in direct
contact with the perfusion medium. The cartridges were filled with
Dulbecco's glucose-enriched (4.5 g/ml) modified Eagle's medium (DMEM)
buffered with 6.0 g/l N-[2-hydroxyethyl]
piperazine-N-[2-ethanesulfonic acid] (HEPES) containing 2.5% (v/v)
horse serum (DMEM –2.5% HS), placed in a Millipore 37°C portable
incubator, and transported from Providence, R.I., to Kennedy Space
Center 2 to 3 days before launch. At Kennedy Space Center, the
cartridges were mounted in two STL Modules (three to four cartridges
per module) and either maintained as ground controls or loaded in a
mid-deck locker of the Space Shuttle. All reagents were of cell culture
grade from Sigma Chemical Co. The BAM cartridges were perfused during
the experiment at 1.5 ml/min with tissue culture medium (DMEM –2.5%
HS). Eighteen BAMs were flown for 9 days on Mission STS66 and 18 BAMs
served as ground controls. The complete experiment was repeated on
Mission STS77 with twenty four BAMs in each group.
The STL Module was developed by the Walter Reed Army Institute of
Research (Washington, D.C.) and designed as a completely automated
system for short duration (7–14 days) Shuttle mid-deck experiments. It
contains four to eight bioreactor cartridges of varying sizes, each
with a separate media reservoir and peristaltic perfusion system. The
STL Module maintains constantly recirculating media (1–20 ml/min)
through a 120 ml/cartridge medium reservoir bag, a
CO2–O2 gas exchange loop,
37°C temperature regulation, a computerized fraction collection
system (1–3 ml/sample), and automated injectable reagent ports (5
ml/cartridge) (23)
. All manipulations (flow rates, medium removal,
reagent injections, temperature/gas monitoring) are preprogrammed into
the STL before flight, thus requiring no on-board assistance from the
astronauts. The CELLMAX QUAD Cell Culture System (Cell Co.) is a
commercially available bioreactor cartridge perfusion system (24)
and
was used in preflight studies to validate all metabolic, biochemical,
and morphological assays performed in flight experiments. Details of
the validation studies are reported in Chromiak et al. (20)
.
Biochemical assays
Medium glucose and lactate analysis
Culture medium samples (1–3 ml) were withdrawn
automatically from the perfusion flow path at varying times during the
flights and stored in the STL Module until the end of the experiments.
Glucose metabolism and lactate production by the BAMs were measured on
aliquots of media with a YSI Glucose/Lactate Model 2000 Analyzer
(Yellow Springs Instruments, Yellow Springs, Ohio). Each sample was
analyzed in duplicate, and the precision of the measurements for both
glucose and lactate was 0.04 g/l.
Total protein, DNA, myosin heavy chain (MHC), and fibronectin
content
For postflight processing, the BAMs were removed from the
bioreactor cartridges and rinsed for 30–40 min with ice-cold Earle's
balanced salt solution on a rotary shaker at 120 rpm. They were
detached from the culture wells, transferred to microcentrifuge tubes,
and stored at -20°C until further analysis. The BAMs were thawed and
sonicated in 1.0 ml ice-cold sucrose buffer (0.25 M sucrose, 0.02 M
KCl, pH 6.8), and aliquots were removed to determine total
noncollagenous protein and DNA content, as described previously (25)
.
For in-flight processing, 50% (w/v) trichloroacetic acid (TCA) was
infused into selected bioreactor cartridges on the last day of flight
to a final concentration of 10% (w/v), and the BAMs were maintained
for 12–24 h in this solution before total protein and DNA analyses
upon return to Earth. This unusual fixation procedure was necessary for
the in-flight assays and the technique was validated in ground-based
studies (20)
.
Fibronectin and MHC content were analyzed by gel electrophoresis on 6%
polyacrylamide gels (PAGE) on postflight processed samples, as
described previously (26)
. Protein bands were visualized with Fast
Stain (Zoion Research, Worcester, Mass.) and scanned by using a
CCD camera interfaced with a video monitor and IBM-compatible computer
that used JAVA Image Analysis software (Jandel Scientific, San Rafael,
Calif.). Protein quantity was derived by comparing band density with a
standard curve of band densities for known standards (range of 0.25
µg to 2.0 µg) included on the same gel. Calculation of protein
content was made with PEAKFIT software (Jandel Scientific).
Protein turnover rates
Protein synthesis rates were determined in-flight and postflight
by [3H]phenylalanine (Phe) incorporation into
cellular proteins. A 5 ml solution containing 1.25 to 2.5 mCi
[3H]Phe (sp. act. 57 Ci/mmol; Amersham) was
injected automatically into the STL Module perfusion system through a
solenoid valve and the BAMs were incubated for 6 h to 10 h at
37°C. For the in-flight procedure, the bioreactors were then injected
with 50% (v/v) TCA to a final concentration of 10% (v/v) and
processed at the end of the experiment as detailed in Chromiak et al.
(20)
. [3H]Phe incorporation was linear over
this time period and a valid measure of protein synthesis rate. Protein
synthesis rates were expressed as [3H]DPM/µg
total noncollagenous protein incorporated into TCA-precipitated
material. The measurement of the synthesis rates of specific proteins
was not possible in these samples due to the TCA technique used to stop
the reaction (20)
. Postflight processed samples could be used to
measure individual protein synthesis rates. Synthesis rates of the
muscle contractile protein MHC and the fibroblast-specific connective
tissue proteins fibronectin and beta-1 collagen were determined in BAMs
by quantitative PAGE as outlined above and in Chromiak et al. (20)
.
For measurement of protein degradation rates, BAMs were incubated
preflight for 48 h in 85/10/5 medium containing 5 µCi/ml
[14C]Phe (sp. act. 479 µCi/µmol, Amersham),
with a change to fresh medium containing 5 µCi/ml
[14C]Phe after the initial 24 h. The BAMs
were rinsed twice over 15 min, transferred to the bioreactor cartridges
in fresh medium, and transported to Kennedy Space Center. Upon arrival,
the medium in the cartridges was replaced with fresh medium, mounted in
the STL Module, and perfusion was begun at 1.5 ml/min. One to three ml
aliquots of medium were collected from the cartridges at varying times
during the experiments for measurement of TCA-soluble
[14C]Phe released from the cells into the
medium. The total DPMs incorporated during the initial 48h labeling
period, along with the DPMs released at each time point, were used to
calculate protein half-lives [T2 = (ln
2)/k, where k is the fraction of protein degraded
per hour]. The values obtained represent a mean protein degradation
rate for all six BAMs in each bioreactor cartridge and are primarily a
measure of the degradation rate of rapidly turning over regulatory
proteins (27)
.
Morphometric measurements
BAMs were fixed in-flight or postflight with HISTOCHOICE, a
water-based formalin substitute (AMRESCO, Solon, Ohio), at 37°C. For
mean myofiber diameter measurements (STS 66 Mission), cells were
stained as whole mounts with either hematoxylin and eosin or by an
enzyme immunocytochemical technique, using an antibody to sarcomeric
tropomyosin (Sigma Chemical, Cat. #T-9283) as described previously
(28)
. The stained cells were viewed as whole mounts with a Zeiss
microscope (total magnification 100x to 1000x) equipped with a
drawing tube attachment focused onto a Numonics 2210 digitizing tablet
interfaced with an IBM-compatible computer. The stained cells and
tablet digitizing mouse could be viewed through the microscope. Three
individuals measured the length and area of each myofiber in randomly
selected whole mount fields and the mean diameters were calculated
using SIGMASCAN software (Jandel Scientific, San Rafael, Calif.). For
mean myofiber cross sectional area measurements (STS 77 Mission), the
BAMs were fixed postflight, embedded in Epon, thin sectioned, and
stained as outlined above. The myofiber cross sectional areas were
measured on randomly selected fields with the morphometric analysis
system described above.
Statistical analyses
Means and standard errors were calculated with SIGMASTAT
software (Jandel Scientific). Time-matched, one-variable data were
compared using t tests, with P<0.05 accepted as
statistically significant. Data from the two flight experiments were
not pooled for statistical analysis, and the figure legends indicate
the flight from which the data were taken.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
In preliminary ground-based studies, the BAMs could be maintained under
tension in bioreactor cartridges in the CELLMAX QUAD Cell Culture
System for up to 30 days with relatively constant metabolic rates and
protein content (20)
. The BAM-STL Module therefore appears to be an
appropriate model system for determining the effects of space travel on
skeletal myofibers.
BAM total noncollagenous protein and DNA content were assayed in two
bioreactor cartridges on day 9 of flight by in-flight TCA fixation and
processing the fixed tissue after recovery on day 10. As shown in
Fig. 2
, total noncollagenous protein and DNA values measured in this manner
were reduced by 16% and 11%, respectively, compared with time-matched
ground controls, but these decreases were not statistically
significant. Protein/DNA ratios were similar in the two groups (Fig. 2C
).
|
The rate of BAM metabolism was determined by collecting medium
fractions at varying times after loading into the STL Module and
measuring medium glucose and lactate levels in the samples at the end
of the experiment. The rate of cellular metabolism was found to be
linear from 72 h prelaunch through 10 days in space, and the
metabolism rate of flight BAMs was not significantly different from
ground control BAMs (Fig. 3A). These results indicate 1) the ability of the
STL Module to maintain the muscle cells in a healthy state for the
length of the experiment and 2) that space travel has little
direct effect on the glucose metabolism of the muscle tissue. Collected
media fractions also were assayed for TCA soluble
[14C]Phe released from prelabeled cellular
proteins to determine total protein degradation rates. As with lactate
production, the rate of total muscle protein degradation was quite
similar for both flight and ground control BAMs (Fig. 3B
).
|
While space travel had little effect on muscle cell metabolism and
protein degradation rates, there was a significant down-regulation of
protein synthesis rates in BAMs on day 9 of flight relative to ground
controls (74% reduction, P<0.01, Fig. 4
A). Return of flight BAMs to Earth resulted in a rapid
elevation of protein synthesis rates compared with ground controls
(Fig. 4A
, 77%, P<0.001). The synthesis rates of
both myofibrillar and connective tissue proteins were increased upon
return to Earth (Fig. 4B
). MHC, fibronectin, and collagen
synthesis rates were significantly increased by 43%, 69%, and 32%,
respectively. The relatively short 6 to 10 h pulse labeling with
[3H]Phe used for these assays is primarily a
measure of the synthesis rate of rapidly turning over regulatory
proteins rather than the synthesis rate of more slowly turning over
structural and contractile proteins, since the large majority of the
[3H]Phe is incorporated into the rapidly
turning over proteins (27)
. Regulatory protein synthesis rates were
therefore quite sensitive to changes in gravitational forces.
|
MHC content was determined by quantitative PAGE on preflight and
postflight BAMs. MHC content increased 42% (P<0.05) in
postflight ground controls relative to preflight BAMs, whereas BAMs in
space for 10 days showed only a 21% nonsignificant increase in MHC
compared with preflight BAMs (Fig. 5
A). In contrast, the accumulation of the extracellular matrix
protein fibronectin was significantly decreased (29% to 33%,
P<0.05) in both ground and flight BAMs compared with
preflight controls (Fig. 5B
). These data indicate that space
specifically attenuated the accumulation of a muscle-specific but not
an extracellular matrix protein.
|
Quantitative morphometric measurement of BAM mean myofiber diameters
(STS66 Mission) and cross sectional areas (STS77 Mission) were
performed to determine whether space travel led to atrophy of the
postmitotic myofibers. In both flight experiments, a significant 10%
to 12% decrease (P<0.002) occurred in flight BAM myofiber
size compared with ground controls (Fig. 6
). Thus, space travel has a direct effect on the accumulation of a
muscle specific protein and on myofiber size.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and a
more modest 15–30% decrease in total protein degradation (30)
. The
muscle cells are spontaneously contractile and under significant
passive and active tensions when grown in vitro, and rapidly
atrophy when these tensions are reduced (31)
. The differentiated
skeletal myofibers in tissue culture also respond to exogenous anabolic
hormones (such as insulin-like growth factors) and catabolic factors
(such as the glucocorticoids) in a manner similar to adult myofibers
in vivo (10)
. Tissue-cultured skeletal muscle cells
therefore represent a good model for studying protein turnover
regulation by mechanical forces and exogenous growth factors, and were
therefore selected for the spaceflight testing.
The responses of the tissue-cultured myofibers to spaceflight were
found to be quite similar to that reported for humans and animals in
space. Thus, there is a 16% to 26% decrease in myofiber
cross-sectional areas after 9 to 15 days in space (32)
, little
alteration in muscle protein degradation rates (33)
or muscle metabolic
rates (34)
, and preferential loss of myofibrillar proteins (35)
. These
alterations mirror the responses of the tissue-cultured myofibers to
space travel. Although it is difficult to directly compare data from
in vivo animal studies with tissue-cultured cells, the
analogous results demonstrate that the BAMs may be useful for testing
potential countermeasure agents. But caution must be used in
extrapolating results obtained with tissue-cultured avian skeletal
muscle cells that are neonatal-like (e.g., express embryonic and
neonatal isoforms of myosin heavy chain) to what happens in adult
mammalian muscle cells.
Muscle unloading on Earth such as occurs in patients confined to a
wheelchair or bed leads to similar pronounced muscle atrophy as seen in
space, primarily by a large, rapid decrease (>50%) in muscle protein
synthesis rates (36,
37)
, similar to that found for muscle during space
travel. Therefore it is not unreasonable to hypothesize that muscle
unloading from reduced gravitational force is a major contributor to
muscle atrophy in space (although this cannot be positively confirmed
without appropriate 1 g controls in space). The avian BAMs are
spontaneously contractile and generate directed force in
vitro through their cytoskeleton and sarcomeres, and this force
generation is important for cell size maintenance (19)
. Recent evidence
in several other space travel tissue culture studies have implicated
alterations in the cytoskeleton and their linkage to second messenger
systems (38,
39)
. The coupling of muscle cell tension to protein
synthesis via the cytoskeleton may therefore have been compromised in
the myofibers flown in space. This linkage is thus a potential, though
unconfirmed, cellular mechanism responsible for the flight-induced
alterations in the protein synthesis rates and muscle atrophy described
in this paper. Other components of space travel may also contribute to
the wasting problem in vivo, such as altered circulating
systemic growth factors or nutrient availability, but these are
unlikely to be contributing factors in the present in vitro
study where constant perfusion with a serum-containing medium was the
same in ground and flight samples. Alterations in
cytoskeletal/sarcomeric network of fibrils in skeletal muscle therefore
present a likely source of the direct effects of space travel on BAM
skeletal myofibers reported in this paper.
It is likely that indirect effects of space travel on skeletal muscle
also contribute to skeletal muscle atrophy in vivo. Reduced
circulating levels of GH occur during spaceflight; using the muscle
hind limb unloading rodent model, a reasonable model for space
travel-induced muscle wasting (40)
, ground-based studies indicate a
synergism between muscle loading and GH in attenuating muscle wasting.
Thus, a combination of injected GH and brief intermittent exercise was
found to be effective together, but not separately, in attenuating
muscle atrophy caused by muscle unloading in hypophysectomized rodents.
Increased muscle cell tension positively modulates the muscle cell's
growth response to insulin-like growth factor 1 (IGF-1), the major
effector molecule for GH's anabolic effects on skeletal muscle (10)
.
Anabolic factors such as GH or IGF-1 are therefore attractive
candidates for attenuating muscle wasting in space, especially since
their primary action is to stimulate muscle protein synthesis rates
(41)
. Effective countermeasures for long-term space travel-induced
muscle wasting may thus entail a modest exercise program coupled to
regular GH/IGF-1 treatment. Chronic delivery of protein growth factors
such as GH are difficult because of their short half-lives and
significant side effects when injected daily in the large
pharmacological doses necessary for effective attenuation of muscle
wasting (42,
43)
. The feasibility of long-term delivery of therapeutic
proteins such as GH from reversibly implanted genetically engineered
cells has been shown to be possible (28)
, and its ability to attenuate
muscle wasting in the hind limb unloading rodent model more effectively
than daily GH injections has been established (44)
. Thus, cell-based
delivery of GH combined with a modest exercise program may be an
effective countermeasure to skeletal muscle wasting in space, and will
be directly tested in future animal flight experiments.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication November 5, 1998.
Revision received February 17, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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