Changes in contractile properties of skeletal muscle during
developmentally programmed atrophy and death
Lawrence M.
Schwartz1 and
Robert L.
Ruff2
1 Department of Biology, University of
Massachusetts, Amherst, Massachusetts 01003; and
2 Department of Neurology and Rehabilitation and Spinal
Cord Injury and Disorders Care Line, Cleveland Veterans Affairs
Medical Center and Case Western Reserve University School of
Medicine, Cleveland, Ohio 44106
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ABSTRACT |
Skeletal muscle atrophy
and death are protracted processes that accompany aging and
pathological insults in mammals. The intersegmental muscles (ISMs) from
the tobacco hawkmoth Manduca sexta are composed of giant
fibers that undergo distinct hormonally-regulated programs of atrophy
and death at the end of metamorphosis. Atrophy occurs during the 3 days
preceding adult emergence and results in a 40% reduction of mass,
whereas death takes place during the subsequent 30 h and results
in the complete loss of the fibers. There are no significant changes in
tetanic force or calcium sensitivity in skinned fiber preparations
during atrophy. However, the size of caffeine-induced contractions fell
by about 50%. With the onset of the death phase, dramatic reductions
occur in ISM: tetanic force, twitch amplitude, resting potential,
caffeine-induced contractions, calcium sensitivity, and Hill
coefficients. Several lines of evidence suggest that ISM atrophy is
caused by an increase in protein turnover without significant
modification of fiber organization. In contrast, ISM death is
accompanied by disorganization of the contractile apparatus and
concomitant loss of contractile function.
Manduca; apoptosis; calcium; degeneration; sarcopenia
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INTRODUCTION |
THE ATROPHY OF SKELETAL
MUSCLE represents a significant clinical problem in a variety of
normal and pathological conditions in humans. Atrophy and subsequent
muscle weakening accompanies the aging process in both humans and
animal models (26). Disuse atrophy is a significant
consequence in a wide range of disorders that result in immobility,
including spinal cord injury, unloading, weightlessness, chronic
glucocorticoid treatment, sepsis, and muscular dystrophies (8,
10, 14, 17, 21, 23, 25, 42, 43, 52). In some cases
subsequent organismal death results from respiratory arrest
(3).
One of the difficulties in studying skeletal muscle atrophy and death
is that they often occur over a protracted time course. For example, it
takes 4-6 wk of unloading before the quadriceps femoris muscle
exhibits a 20% loss of mass in humans (10). Analysis of
atrophy is further complicated because most mammalian muscles are
composed of distinct fiber types that may change over time, especially
during disuse (8, 26).
One animal model system that does not suffer the technical limitations
of multiple fiber types and the prolonged period of atrophy observed
with mammalian skeletal muscle is the intersegmental muscle (ISM) from
the tobacco hawkmoth Manduca sexta (reviewed in Ref.
44). The ISMs arise in the embryo, where they span each of
the abdominal segments. Their contraction serves to telescope abdominal
segments, thus allowing the caterpillar to "inch" along. After
pupation, the ISMs in the first two and last two abdominal segments
die, whereas the muscles in the middle four segments persist throughout
metamorphosis. Three days before adult emergence (day 15 of
pupal/adult development), these remaining ISMs undergo a three day
period of atrophy that results in a loss of 40% of the muscle mass
(49). These same fibers then participate in the emergence
behavior late on day 18 that allows the adult moth to
extricate itself from the pupal cuticle. After emergence, the ISMs
begin to die and disappear during the subsequent 30 h (11, 30).
The precise timings of both ISM atrophy and death are regulated by a
decline in the circulating titer of the insect steroid hormone
20-hydroxyecdysone (20E) (49). Judicious administration of
exogenous 20E on day 15 can block both atrophy and death,
whereas steroid treatment on day 17 can block death.
However, once either of these sequential programs has been initiated,
it cannot be reversed or prevented by exogenous steroid treatment.
Earlier publications described the anatomic and electrophysiological
changes that accompany atrophy and death of the ISMs and related
abdominal muscles (28, 29, 37, 44). In this study, we have
examined changes in the physiological properties of the ISMs at
specific developmental stages: before atrophy (day 15),
after atrophy but before death (day 18), early during death
(4 h postemergence), and late during cell death (15 h
postemergence). The data presented here suggest that muscle
fiber atrophy and death are distinct processes. Although there are
dramatic changes in muscle mass during atrophy, there are few
concomitant changes in the physiological properties of the cells. In
contrast, death results in both a loss of muscle mass and a profound
reduction in physiological function.
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MATERIALS AND METHODS |
Animals.
Manduca sexta were reared and staged as previously described
(49). Animals were dissected under saline to expose the
lateral ISMs (49). The saline had the following
composition (in mM): 9 NaCl, 34 KCl, 4.2 CaC12, 6 MgCl2, 172 dextrose, and 25 sodium phosphate buffer, pH
6.7. ISMs were collected at four times: 1) on day 15 of pupal/adult development, which was 3 days before adult
emergence, 2) before emergence on day 18 of
pupal/adult development, 3) at 4 h after emergence on day
18, and 4) at 15 h after emergence on day 18 of pupal/adult development. All experiments were performed at room
temperature (22 ± 1°C).
Tension measurements.
Single ISM fibers were dissected from the fifth abdominal segment and
attached to a custom-built isometric tension transducer modeled on the
design of Hellam and Podolsky (15). To minimize possible
changes during an experiment, only 1 fiber was used from each animal,
and 5-20 fibers were examined for each time point. Intact fibers
were stimulated with extracellular electrodes.
When tension data was analyzed, the force of contraction was normalized
for fiber cross-sectional area. At the outset of the experiment, the
diameter of each muscle fiber was measured with a microscope positioned
over the recording apparatus. Mechanically skinned fibers (see below)
had an essentially circular cross section, and their area was
calculated accordingly. When examined histologically, intact fibers had
an elliptical cross section, with the minor axis measuring 0.505 times
the major axis. Therefore, contractile force for the intact fibers was
normalized to fiber cross-sectional area by dividing the force by
(diameter)2(0.505)/4.
We studied ISM fibers obtained at each of the four collection
times. We compared data from the different collection times by
using analysis of variance (ANOVA). Alpha was set at 0.05, and
two-tailed tests were employed for all analyses. Subsets of data were
studied with ANOVA to determine whether there were any significant
differences among the groups of fibers studied at the four different
collection times. When significant interactions were present, post hoc
comparisons between different groups were made by using Tukey's
honestly significant difference test for pairwise comparisons, with
Scheffé's S method when more than two means were compared. The
data presented satisfied normality criteria (32).
In some experiments ISM fibers were mechanically skinned by splitting
the fibers longitudinally with dental picks. Skinned fibers were
individually attached to a tension transducer and bathed in solutions
containing differing levels of buffered free calcium, following the
standard notation that pCa =
log free [Ca2+]. The
skinned fiber activating solutions contained (in mM) 1.0 free
Mg2+, 4.0 MgATP, 135 K+, 15 creatine phosphate,
and 12 EGTA. The concentration of the pH buffer MOPS was varied to keep
the ionic strength of the activating solutions at 200 mM. The
concentration of MOPS for each integral pCa used was (in mM) 26 (pCa
4.0), 28 (pCa 5.0), 32 (pCa 6.0), 44 (pCa 7.0), and 50 (pCa 8.0). The
major anion was propionate, and the pH was adjusted to 7.0. Solutions
were mixed at one time, and 20 U/ml creatine kinase was added to the
activating solutions at the time of an experiment. Binding constants of
EGTA and ATP with hydronium, calcium, and magnesium ions were adjusted
for ionic strength and temperature (25, 42). The
concentration of free calcium (pCa 8.0 to pCa 4.0) and other solution
components were calculated as previously described (25,
42).
Each skinned fiber was secured between the flaps of an aluminum foil
clip. The clips were slipped over stainless steel hooks attached to a
micromanipulator and a custom-made force transducer (25).
The micromanipulator was used to adjust each fiber to its rest length.
The transducer had a resonant frequency of 50 Hz, a compliance of 0.4 µm/mg, and a sensitivity of 12 mV/mg and was linear over the range of
0-500 mg. To elicit tension, a fiber was lowered into one of a set
of Plexiglas wells containing 2.0 ml of relaxing (pCa 8.0) or
activating (pCa <7.0) solution. The fiber could be transferred between
wells in <1 s.
Data for the force measurement at each pCa were normalized to the
maximum tension and plotted vs. pCa. A nonlinear least-squares fit of
the Hill equation (16) was calculated from the data from the second tension trial of each fiber. The equation tension/maximum tension = [Ca2+]nH/(KnH + [Ca2+]nH), where K
is the calcium concentration associated with half-maximal tension and
nH is the Hill coefficient, describes the
steepness of the relationship between tension and calcium
concentration. To minimize artifactual reduction of the slope
when pooling data from several different fibers,
nH was calculated from the tension-pCa relationships by shifting individual fiber tension pCa curves along
with the pCa axis so that the pCa for 50% tension for all fibers
coincided. These data were then fit with the Hill equation to determine
nH, the slope parameter. To control for possible variability in the solutions or recording apparatus, chemically skinned
rabbit adductor magnus muscle was tested at the beginning of each
experiment because the calcium sensitivity of this muscle is well known
and does not vary between rabbits (9). Consequently, these
muscle fibers provided a biological calibration for both the transducer
and the activating solutions. Rabbit adductor magnus muscles were
skinned by incubating muscles for 1 wk at
20°C in 50% glycerin at
pCa 8.0. To test the effects of caffeine, intact ISM fibers were
attached to a tension transducer and exposed to 25 mM caffeine in
Manduca saline (49).
Electrophysiological measurements.
Abdomens were removed from animals at various times relative to adult
emergence, rinsed with saline, cut middorsally, and eviscerated. The
abdomens were pinned over a hole in the center of a wax-filled
Plexiglas recording chamber by using a thin bead of high-vacuum silicon
grease (Dow Corning) to create a watertight seal. The spiracles were
aerated through the hole under the preparation. Because the rigidity of
the overlying pupal cuticle prevents spiracle aeration, reliable
recordings could not be obtained with preparations before day 18 of pupal/adult development. The chamber was then flooded with
saline, and the preparation was used for <30 min. Resting potentials
were recorded with glass microelectrodes (7-12 M
) filled with 3 M KCl. Between 20 and 34 fibers were examined at each stage from at
least three different individual animals.
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RESULTS |
Contractile properties of intact fibers.
The contractile properties of intact ISM fibers were tested at various
stages beginning on day 15 of pupal/adult development and
ending 15 h after adult emergence on day 18. Data from
day 15 represented the period before the onset of atrophy,
data from early on day 18 represented the end of the atrophy
phase, and data from 4 and 15 h after emergence represented the
active period of cell death. Individual intact muscle fibers were
dissected free from the animal and attached to an isotonic tension
transducer. When stimulated with 10-ms depolarizing pulses delivered
via extracellular bipolar electrodes, the fibers produced discrete
twitches (data not shown). Between days 15 and
18, there was a 1.6-fold increase in the normalized
individual twitch amplitude displayed by the ISM (Fig.
1A). By 4 h after
emergence, the twitch amplitude had increased 2.1-fold relative to that
on day 15. However, by 15 h after emergence, when the
fibers were well into the degeneration phase, the fibers produced
barely discernible twitches.

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Fig. 1.
Changes in tension generated by individual intact
intersegmental muscle (ISM) fibers. Tension was normalized to the fiber
cross-sectional area. Data were collected on day 15 of
pupal/adult development, on day 18 before emergence, and on
day 18, 4 and 15 h postemergence (PE). A:
twitch tension measured after the fibers were stimulated with bipolar
electrodes with single 10-ms pulses. B: amplitude of tetanic
contraction in individual fibers stimulated with 10-ms pulses at 30 Hz
with bipolar electrodes.
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Stimulation of the fibers with 10-ms pulses at 15 Hz resulted in fused
tetanus (data not shown). To correct for variability between cells, we
stimulated ISM fibers from different developmental stages at 30 Hz.
When tetanic force was normalized for cross-sectional area, the force
generated by fibers on day 15 was the same as that for
muscles on day 18, even though the ISM had lost 40% of their mass during this period (Fig. 1B). With the onset of
degeneration following emergence, ISM force declined rapidly so that by
15 h after emergence it had fallen by 78%. This represented an
actual weakening of the fibers and not just a reflection of reduced
fiber size, because the force was normalized for the cross-sectional area of the fibers (see MATERIALS AND METHODS).
The physiological trigger for skeletal muscle contraction is the
release of calcium from the sarcoplasmic reticulum (34). Calcium release can be initiated by electrical depolarization of the
surface membrane, as was performed above, or by pharmacological means,
such as exposure to caffeine (36). Exposure to 25 mM caffeine generated large tension transients that peaked after ~3 s
(Fig. 2A). Despite the
continued presence of caffeine, the fibers relaxed to baseline levels
within 30 s. The responsiveness of ISM fibers to caffeine changed
during atrophy and degeneration. On day 18, before
emergence, the peak tensions generated by caffeine were only 61% of
those on day 15 (Fig. 2B). By 15 h after
emergence, caffeine-induced contractions were only 10% of the value on
day 15.

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Fig. 2.
Contractions induced by 25 mM caffeine in individual
intact ISM fibers. A: recording from a single fiber exposed
to 25 mM caffeine in Manduca saline starting at the downward
arrow. Time of caffeine withdrawal is noted by the upward arrow. The
small bumps in the record are artifacts due to vibration of the
apparatus when solutions were changed. B: peak amplitude of
caffeine-induced contraction in individual fibers. Data were collected
on day 15 of pupal/adult development, on day 18 before emergence, and on day 18, 4 and 15 h
postemergence. Significance was determined relative to the preemergence
time point on day 18.
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Contractile properties of skinned fibers.
The reduction in ISM responsiveness to caffeine during development
could have resulted from changes in either the physiological behavior
of the sarcoplasmic reticulum or the ability of the contractile apparatus to respond to free calcium. To address this question, individual fibers were mechanically skinned and attached to a tension
transducer. The fibers were initially placed in a chamber containing
10
8 M calcium ions (pCa 8.0) to establish the baseline
tension for unstimulated fibers (Fig. 3).
Fibers were then rapidly transferred to chambers containing different
pCa levels to determine their tension-calcium relationships. Figure 3
shows representative records for ISM fibers from day 15,
day 18 preemergence, and 18 h after emergence. For the
day 15 fiber (Fig. 3A), tension was initiated at
pCa 5.6 (2.5 µM Ca2+). At pCa 5.4 there was a slight
increase in tension, with maximal tension elicited at pCa 5.2. Exposure
to pCa 5.0 did not generate more tension than pCa 5.2. Upon return to
pCa 8.0, the fiber relaxed to baseline tension. For the day 18 fiber (Fig. 3B), tension was again initiated in
response to pCa 5.6. However, this fiber generated a more graded
response than the day 15 fiber to increased levels of free
calcium ions and did not achieve maximal tension until pCa 5.0. Again,
upon return to pCa 8.0, the fiber relaxed to baseline level. By 18 h after emergence, the ISM were quite far along in the degeneration
process. Fibers from this stage did not generate tension until exposed
to pCa 5.2 (6.3 µM). They continued to display slow incremental
increases in tension with each elevation of free calcium ions up to pCa
4.0 (100 µM). Upon return to pCa 8.0 buffer, the fiber relaxed but
did not achieve the baseline level of tension.

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Fig. 3.
Individual tension-pCa records for skinned ISM fibers.
Individual fibers were mechanically skinned and attached to a tension
transducer. Fibers were bathed in solutions with differing levels of
free calcium ions. The pCa values of the activating solutions are
indicated on the abscissa. A: day 15 of
pupal/adult; B: day 18 before adult emergence;
C: 18 h after adult emergence.
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These data suggest that as the ISM undergo atrophy and degeneration,
they become progressively less sensitive to free calcium ions. To
quantify this observation, we generated force-pCa profiles for ISM
fibers from several developmental stages ranging from day 15 to 18 h after emergence (Fig.
4). For each stage, the force-pCa records
were normalized and pooled as described in MATERIALS AND METHODS. On day 15 of development, ISM fibers rapidly
initiated contraction in response to elevations in the concentration of free calcium, with 50% tension achieved at pCa 5.69 (2.04 µM) ± 0.02 (n = 20) (Fig. 4). On day 18, before
emergence, 50% tension was generated at pCa 5.53 (2.95 µM) ± 0.08 (n = 6). At 4 h after emergence, this value
had increased to pCa 5.31 (4.90 µM) ± 0.02 (n = 5). By 18 h after emergence, 50% tension was not achieved until
pCa 4.89 (12.88 µM) ± 0.16 (n = 6). Therefore,
during the 3-day atrophy period (days 15-18
preemergence), ISM fibers required almost 1.5 times more free calcium
to reach the midpoint in their force-pCa curve. During the subsequent
18 h, the fibers became progressively less sensitive to calcium
and required more than six times the levels free calcium to generate
50% tension relative to day 15 fibers.

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Fig. 4.
Changes in force-pCa relationships in skinned ISMs at
various times relative to adult emergence. Curves were normalized for
comparison by defining the maximal tension for each fiber as 100% and
shifting the curves for all fibers at a given stage so that they shared
the same pCa value for 50% tension. ISM fibers were sampled for each
tension-pCa relationship at the following times of pupal/adult
development: day 15 (A), day 18 just
before emergence (B), 3 h postemergence (C),
13 h postemergence (D), and 18 h postemergence
(E).
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Not only did the fibers display an increase in the amount of calcium
required for 50% tension generation, but the nature of their responses
to calcium also changed. Day 15 ISM fibers displayed a steep
relationship between the percent tension generated and the pCa. This
group of fibers had a Hill coefficient of 5.71 (Fig. 4). The Hill
coefficient for tension-pCa relationship just before emergence was
6.11, which was similar to the value for day 15. After
emergence as ISM degeneration progressed, the slope of the force-pCa
curves became progressively more shallow, with Hill coefficients of
4.21 at 4 h after emergence and 2.93 at 15 h after emergence.
Resting potentials.
Before adult emergence, the ISMs maintained a resting potential of
approximately
70 mV (Fig. 5). This
potential was dependent on aeration of the spiracles, because the cells
rapidly depolarized to about
30 mV when air to the spiracles was
withheld (data not shown). With use of the Nernst equation, a resting
potential of
30 mV roughly coincides with the potassium equilibrium
potential (
22 mV), assuming an internal potassium concentration of 84 mM (19).

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Fig. 5.
Changes in resting potential and mass of the ISMs
following adult emergence. Abdomens were isolated from animals at the
stages indicated, and the lateral ISM were exposed. Resting potentials
(solid line) and muscle mass (dashed line) were measured from distinct
groups of animals. All measurements are means ± SE
(n = 5 animals). In the case of resting potential,
10-14 muscle fibers were sampled per animal. Data on muscle mass
are from Ref. 49. Muscle mass index is [mg dry weight of
ISMs/g body wt] × 1,000.
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Late on day 18, coincident with adult emergence, the resting
potential began to decline at a rate of ~2.5 mV/h (Fig. 5). This reduction in membrane potential paralleled the loss of muscle mass
(49). At 15 h after emergence, the muscles still
retained a resting potential even though they were no longer
contractile in response to tactile stimulation (data not shown) and had
lost ~50% of their mass. Therefore, despite extensive degradation of the contractile apparatus, sarcolemmal integrity was maintained until
this time.
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DISCUSSION |
Muscle is a very dynamic tissue that can rapidly undergo anabolism
or catabolism to grow or atrophy, respectively. The ability to shuttle
amino acids in and out of muscle has obvious benefits for the organism,
because muscle can serve as an amino acid reservoir. The underlying
pathways for synthesis and degradation of muscle protein are carefully
regulated (43, 52). Unfortunately, a variety of disorders
in humans can lead to progressive muscle atrophy and, ultimately,
organismal death due to respiratory failure (3). Cachexia
is the hallmark of many wasting disorders, including AIDS, cancer,
muscular dystrophies, and paralysis (52). The time course
for these progressive disorders is protracted and can occur over a
period of months to years. Furthermore, tissue heterogeneity can
complicate the analysis of these disorders. For example, whereas
myofibrillar damage reduces the number of functioning muscle fibers in
Duchenne muscular dystrophy, the mass of the muscle often actually
increases due to the infiltration of fat and connective tissue
(14, 17, 23, 52).
To understand some of the normal physiological processes that can be
employed by animals to regulate skeletal muscle atrophy and death in
response to developmental cues, we have turned to the ISMs of
Manduca. Because the ISMs of Manduca are composed of a pure population of cells that undergo rapid, synchronous atrophy
and death, they constitute an attractive model system for such studies.
In fact, these cells have been used for molecular (18, 31, 46,
47, 51), biochemical (7, 13, 20), structural
(1, 2, 11, 29, 48), and physiological analysis (present
study and Refs. 29, 37).
Day 15 of pupal/adult development to adult emergence on day 18.
From the time of pupation until day 15 of pupal/adult
development, ISM mass remains extremely constant (49). The
fibers vigorously contract in response to tactile stimulation (data not shown), and the ISMs participate in the defensive behaviors of the
pupa. In this study, day 15 ISM fibers generated rapid,
powerful contractions in response to either electrical depolarization
or exposure to caffeine. The calcium-dependent regulatory proteins rapidly responded to increased levels of free calcium and displayed a
strong positive cooperativity in skinned fiber preparations, as
demonstrated by the high value of the Hill coefficient.
The patterns of gene expression in the day 15 ISMs look much
like that of earlier stages. The levels of actin and myosin heavy chain
transcripts are at the same high level observed earlier in development
(45). However, the levels of polyubiquitin mRNA and
ubiquitin-protein conjugates are slightly elevated at this stage
relative to levels on day 14 (13, 47).
Activation of the ubiquitin pathway suggest that whereas rates of
protein synthesis may remain relatively constant, more rapid turnover
of proteins may facilitate the global loss of protein that begins at
this stage of development with the onset of the atrophy program
(44).
Preemergence on day 18 of adult/pupal development.
Adult emergence occurs late on day 18 of pupal/adult
development. The ISMs provide the major motive force that powers the peristaltic waves of abdominal contractions that help propel the adult
moth out of the overlying pupal cuticle. In this study we found that by
early day 18, before the ISMs have initiated the death
program, the muscles have lost ~40% of their mass relative to
day 15 (Fig. 5). During this period the strength of tetanic contraction was reduced due to the gross loss of contractile proteins. However, when tensions were normalized to fiber cross-sectional area,
tetanic force did not change between day 15 and 4 h
after emergence (Fig. 1). This finding suggests that the reduction in muscle mass observed during the atrophy phase reflects a generalized enhancement of protein turnover, rather than the selective targeting of
specific contractile proteins. This hypothesis is supported by the
observation that de novo expression of actin and myosin heavy chains
remains at the same level during atrophy (45).
There are modest changes in the twitch responses of ISM fibers between
day 15 and 4 h after emergence. When normalized to cross-sectional area, the amplitude of twitch tensions increased (Fig.
1), whereas the magnitude of caffeine-induced contraction decreased. The increased strength of individual twitches may
reflect the reduced fiber volume that accompanies atrophy, which in
turn might facilitate faster diffusion rates of calcium between the sarcoplasmic reticulum and the contractile proteins. Putative increases
in this diffusion rate may not impact on tetanic tension. Prior studies
indicated that the sarcoplasmic reticulum and transverse tubular system
undergo only slight swelling just before emergence (1,
29).
At a molecular level, dramatic changes in the patterns of gene
expression precede the initiation of cell death. RNA stability is
significantly reduced in the cytoplasm of day 17 ISMs
relative to other stages of development (5). This may
function to facilitate the rapid accumulation of newly transcribed
death-associated RNAs relative to the high background of housekeeping
transcripts that were present in the cells. Late on day
17/early on day 18, the ISMs become committed to die.
Whereas most transcripts are retained at steady-state levels, some
genes are repressed, such as actin and myosin heavy chain genes
(45). Concurrently, there is an exponential increase in
the levels of several other transcripts, including polyubiquitin
(47), apolipoprotein III (51), proteasome subunits (31, 50), DALP (death-associated LIM-only
protein) (18); SCLP (small cytoplasmic leucine-rich repeat
protein) (22), Acheron (D. Sun, C. Valavanis, and L. M. Schwartz, unpublished observations), and several uncharacterized
transcripts (46). If animals are treated on
day 17 with either 20E (49) or inhibitors of
RNA or protein synthesis (27, 46), these changes in gene expression do not take place and the muscles do not initiate death. Once these changes in gene expression take place however, death becomes
inevitable and cannot be inhibited pharmacologically.
Postemergence.
The death program is initiated essentially coincident with adult
emergence. This can be seen in terms of the structure, function, and
biochemical properties of the muscles. Coincident with emergence, the
muscles begin to lose mass at a rate of ~4%/h. Concomitantly, there
is a slow, progressive depolarization of the fibers at a rate of ~2.5
mV/h. By 24 after emergence, reliable resting potentials can no longer
be recorded (Fig. 5). These data are similar to those of earlier
reports (29), except that the use of aerated preparations
appears to provide a more accurate measure of the resting potential.
At the RNA level, the abundance of specific transcripts is essentially
unchanged from earlier in the day. However, the initiation of the death
program appears to employ a trigger that enhances the rate of
translation for the death-associated transcripts. For example,
polyubiquitin mRNA is rapidly translated, resulting in an exponential
increase in the level of ubiquitin protein (13). This
facilitates ubiquitin-protein conjugation and subsequent proteosome-dependent proteolysis. The ubiquitin/proteasome pathway appears to play the major role in enhancing the degradation of ISM
proteins during death (7, 13, 20).
Although there is little change in the organization of the contractile
apparatus during the atrophy phase, the postemergence period is marked
by profound sarcomere disruption (29). Whole filaments
rapidly disappear with a preferential loss of thick relative to thin
filaments (1). During this same period, mitochondria are
lost and the T-tubule system swells (1, 29).
Not surprisingly, there are physiological consequences that accompany
these dramatic changes in ISM structure (see Fig.
6). The fibers rapidly weaken, even when
force is normalized to cross-sectional area. This is true for twitches,
tetanus, and caffeine-induced contractions. It has been suggested that
calcium ions may be extruded from dying ISMs and accumulate in the
lumen of the swollen T-tubule system (2). If so, this may
account for the reduced ability of the ISMs to generate tension in
response to either depolarization or caffeine exposure. There are also
definitive defects in the ability of the contractile apparatus to
respond to free calcium, independent of its source. In skinned fiber
preparations, higher levels of free calcium are required to initiate
contraction and responses to elevations in pCa are graded. During this
phase, the Hill coefficient is greatly reduced, suggesting reduced
cooperativity in calcium ion binding.

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Fig. 6.
Summary data from the present study and previous work
(13, 46) are schematized to facilitate correlations.
Curves indicate relative changes in each parameter and are not intended
to represent actual levels. The schematized curves for tetanic force,
twitch tension, caffeine-induced contraction, pCa producing
half-maximal tension, and the Hill coefficient are based on data from
the present study. Data for ubiquitin-dependent proteolysis are from
Haas et al. (13). Note: technical limitations do not allow
resting potential measurements to be collected on day 15 animals that are comparable to those obtained with day 18 and older animals, so the dashed line is speculative.
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There are some parallels between the patterns of muscle atrophy and
cell death seen in Manduca sexta and those observed in some
pathological conditions in mammals. Moderate parenteral doses of
glucocorticoids induce reversible muscle atrophy in rats that is
reminiscent of the changes that occur during atrophy (21). For example, steroid-treated rat muscle undergoes a reduction in the
absolute strength of both twitch and tetanic contractions. However,
when strength is normalized to cross-sectional area, there is no net
force reduction (42). The calcium sensitivity of the
contractile proteins in skinned fiber preparations from rats subjected
to glucocorticoid-induced atrophy were altered only slightly compared
with controls (24), results similar to our observations
with atrophying ISMs (Fig. 4). Pharmacologically large doses of
glucocorticoids or systemic sepsis can trigger degeneration of
mammalian skeletal muscle fibers that is associated with marked loss of
contractile proteins (40, 41) and muscle fiber
depolarization (38, 39), changes observed in ISM fibers during the post-adult emergence period (Fig. 5). Several triggers of
skeletal muscle atrophy in mammals involve enhanced ubiquitin-dependent proteolysis (6, 33). In fact, two ubiquitin E3 ligases
have recently been identified that are induced in all models of mouse skeletal muscle atrophy tested (4, 12). The data from
these studies suggest that whereas a variety of manipulations result in
atrophy, enhanced protein turnover mediated by the ubiquitin-proteasome pathway represents a final common event. Nevertheless, these results do
not preclude the possibility that other proteolytic pathways may also
participate in atrophy, such as the calpains (54). Although it is not known whether the calpains are involved in ISM
atrophy, they do participate in muscle atrophy in other arthropods (35).
In summary, the ISMs provide a simple model system for the study of
developmentally regulated skeletal muscle atrophy and death. These two
phases represent distinct developmental programs that are sequentially
induced by physiological triggers. Atrophy appears to be mediated by a
subtle increase in protein turnover relative to synthesis and is not
accompanied by significant changes in either the structure or function
of the muscle, despite a 40% loss of the mass in just 3 days. In
contrast, the ISM death program involves the induction of
stage-specific transcripts and catastrophic alterations in the
contractile apparatus that lead to reduced physiological capabilities.
At present few studies have examined changes that accompany natural,
nonpathological muscle cell death in mammals. Hopefully the insights
gained from analysis of the ISMs may identify key regulatory components
that play subtle but important roles in sarcopenia and other disorders
of muscle.
 |
ACKNOWLEDGEMENTS |
We thank Laura Bolles for technical assistance, Dr. Walter
Stühmer for the computer analysis of the pCa data, and David
Tharp for drawing Fig. 6.
 |
FOOTNOTES |
L. M. Schwartz was supported by National Institutes of Health
grants. R. L. Ruff was supported by the Offices of Medical
Research and Rehabilitation Research and Development of the Research
and Development Service of the Department of Veterans Affairs.
Address for reprint requests and other correspondence:
L. M. Schwartz, Dept. of Biology, Morrill Science Center,
Univ. of Massachusetts, Amherst, MA 01003 (E-mail:
LMS{at}bio.umass.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.
First published January 16, 2002;10.1152/ajpcell.01275.2000
Received 19 October 2000; accepted in final form 8 January 2002.
 |
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