Mechanical stimulation increases expression of
acetylcholinesterase in cultured myotubes
Douglas A.
Hubatsch and
Bernard J.
Jasmin
Department of Physiology, Faculty of Medicine, University of
Ottawa, Ottawa, Ontario, Canada K1H 8M5
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ABSTRACT |
We tested the hypothesis that acetylcholinesterase (AChE)
expression in skeletal muscle cells is increased by passive mechanical stimulation. To this end, primary cultures of myotubes were subjected to repeated cycles of stretch-relaxation for 5 min, 30 min, 3 h, and 24 h, using the Flexercell FX-2000 strain unit. Although mechanical
stimulation did not affect AChE expression at early time points, it led
to a significant increase (42%; P < 0.05) in total AChE activity at 24 h. This increase reflected a general elevation in the activity of all AChE molecular forms as opposed to a
preferential increase in a specific form. Tetrodotoxin (TTX) treatment
did not prevent the increase in AChE expression, whereas nifedipine
partially blocked it. These changes in enzyme expression were
accompanied by increases in the levels of AChE mRNA, suggesting the
involvement of pretranslational regulatory mechanisms. Together, these
results illustrate that, in addition to neural activation and trophic
factors, passive mechanical forces modulate expression of AChE in
skeletal muscle cells. Because TTX did not prevent the increase in AChE
expression, it appears that the effects of mechanical stimulation are
independent of electrical activity, which further indicates the use of
an alternate signaling pathway.
rat; neuromuscular junction; synapse; stretch; mechanical cell
stimulator
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INTRODUCTION |
ACETYLCHOLINESTERASE (AChE) is an essential component
of cholinergic synapses in both central and peripheral nervous systems, since it is responsible for the rapid hydrolysis of acetylcholine released from presynaptic nerve terminals (for review, see Ref. 22). An
interesting feature of this enzyme is that it displays a marked and
complex polymorphism. Indeed, AChE exists as a family of molecular
forms that present distinct structural features and solubility
characteristics. The various molecular forms may be classified as
either homomeric or heteromeric on the basis of their association with
specialized structural subunits. Although a single gene encodes AChE in
vertebrates (see Ref. 22), alternative splicing and distinct processing
of the polypeptide chain account for the multiplicity of AChE molecular
forms expressed in a variety of tissues and subcellular locations. In
skeletal muscle fibers, for example, asymmetric forms of AChE
accumulate preferentially within the synaptic basal lamina (23),
whereas G4 tetramers are highly
concentrated within the perijunctional compartment (10).
AChE is of particular interest with regard to muscle plasticity, since
expression of AChE is markedly influenced by the presence of the motor
nerve. Denervation of muscle fibers, for example, results in a rapid
and pronounced decrease in the content of AChE asymmetric forms (see
Refs. 22, 24). Alternatively, enhanced neuromuscular activation
achieved by exercise training programs and compensatory hypertrophy
leads to significant increases in total AChE activity that, in fast
muscle, are manifested by preferential changes in the levels of the
various molecular forms (7, 13, 14, 29). Several lines of evidence also
suggest that expression of AChE in muscle is modulated by nerve-derived
trophic factors. For instance, chronic application of tetrodotoxin
(TTX) onto the sciatic nerve, which inhibits electrical activity of
muscle fibers without compromising axoplasmic transport, leads to
reductions in AChE activity that are less pronounced than those induced
by denervation (2, 4, 24). Recent studies have further
shown that systemic administrations of ciliary neurotrophic factor (3) or calcitonin gene-related peptide (12) influence expression of AChE in
rat skeletal muscle. Taken together, results from these studies
indicate, therefore, that the regulation of AChE in skeletal muscle
fibers is a multifactorial process that involves, in addition to the
amount and pattern of nerve-evoked electrical activity, the release of
nerve-derived trophic factors.
In recent years, it has become increasingly evident that several cell
types respond to mechanical perturbations of their normal environment
by displaying significant changes in their growth and differentiation
patterns (1, 11, 30). Skeletal muscle is among the tissues known to be
sensitive to mechanical stimulation, since, in response to these
passive forces, muscle cells readily adapt by synthesizing, for
example, large amounts of contractile proteins and by displaying
hypertrophy (11, 31). Given the elevated levels of AChE seen previously
in both exercise-trained and hypertrophied muscles (Refs. 7, 13, 14, 29
and references therein), the possibility that passive mechanical
stimulation also increases AChE expression in muscle may therefore be
envisaged. In the present study, we have thus tested this hypothesis by
determining the impact of passive mechanical forces on AChE expression.
To this end, we subjected cultures of myotubes to repeated cycles of
stretch-relaxation, using a computerized mechanical cell stimulator.
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MATERIALS AND METHODS |
Primary cultures of rat myotubes.
Sprague-Dawley rats weighing ~50 g were anesthetized with
pentobarbital sodium (35 mg/kg ip), and their hindlimb muscles were quickly excised under aseptic conditions. Muscles from four to five
animals were pooled together for each experiment, thus giving an
n of 1. Muscles were kept in ice-cold
phosphate-buffered saline (PBS) until surgery on all animals was
completed. Freshly dissected muscles were then minced in 8 volumes of
minimum essential medium (MEM) containing 15% donor bovine serum
(DBS), 1% penicillin-streptomycin, 0.1% fungizone, and collagenase
(1.79 mg/g of tissue) until fragments of ~2 mm were obtained. The
mixture was then incubated at 37°C for 1.75 h and spun subsequently
at 250 g for 3 min at 4°C. The supernatant was discarded, and the soft pellet was resuspended in MEM
as above, with the exception that dispase (20 mg/g of tissue) was added
instead of collagenase. This mixture was then incubated at 37°C for
45 min. Digested muscle fibers were filtered through a 53-µm nylon
filter, and the filtrate was spun at 4°C for 10 min at 250 g. The final pellet was resuspended in
MEM with DBS and antibiotics. Satellite cells thus obtained were plated
on Matrigel-coated (Collaborative Biomedical Products, Bedford, MA), six-well Flexercell plates (Flexcell International, McKeesport, PA). Cells were kept at 37°C in a water-saturated atmosphere
containing 5% CO2. The medium was
changed every other day until cells reached confluence. At this stage,
DBS was reduced to 2% to promote differentiation of myoblasts into
myotubes. Control cells were also grown on Flexercell plates to avoid
substratum-induced selective expression of proteins. Cells were used
for AChE analysis 8-10 days after initial plating. With this
approach, we observed that at least 50% of the cells are myoblasts and
that >70% of the myoblasts differentiate into myotubes.
Mechanical stimulation.
Installation of the Flexercell FX-2000 strain unit was performed as
described by the manufacturer (Flexcell International). This system
consists of flexible-bottom culture plates, a vacuum base plate, hoses,
and a computer. The computer controls the extent of stretch (measured
as a percentage) imposed on the flexible-bottom culture wells and the
duration of the mechanical stimulation. The actual mechanical
stimulation of the cells occurs by creation of a vacuum below the
flexible-bottom wells. This causes the substratum to deflect downward,
increasing the surface area and stretching the cells growing on it. In
preliminary experiments, rat myotubes were grown on the flexible-bottom
wells stretched by either 12, 20, or 24% using a cyclical pattern of
five stimulations of 2 s on and 2 s off, followed by a 10-s rest
period. Cells were mechanically stimulated at 37°C using this
pattern for 5 min, 30 min, 3 h, and 24 h. In these studies, we noted
that the AChE response was the greatest with a 24% stretch imposed on
the flexible-bottom culture wells (data not shown). This value was
therefore used in all subsequent experiments (see also Refs. 27, 31,
34, 35). Examination of cultured myotubes under a microscope indicated that cell morphology did not change significantly as a result of
mechanical stimulation. For some experiments, cultures of rat myotubes
were subjected to 24% mechanical stimulation for 24 h in the presence
of 10 µM TTX (Sigma, St. Louis, MO) or nifedipine (Miles
Laboratories, Pittsburgh, PA) to block
Na+ or L-type
Ca2+ channels, respectively (see
Refs. 21, 25).
Analysis of AChE.
Myotubes within one set of experiments
(n = 1; control, 5 min, 30 min, 3 h,
24 h) were harvested at the same time. Because they all originated from
the same culture preparation (see Primary cultures of
rat myotubes above), they were of the same
developmental stage. This was achieved by first starting to stretch a
plate of cells for 24 h; 21 h later, another plate of cells from the same culture preparation was stretched, in this case for 3 h. The
protocol was to keep adding plates as time progressed, such that all
cells were stimulated for the appropriate amount of time and ready for
harvesting at the same time.
Cultures of myotubes were washed twice with ice-cold PBS and scraped
into 600 µl of a high-salt buffer containing antiproteolytic agents
[10 mM tris(hydroxymethyl)aminomethane
(Tris) · HCl (pH 7.0), 10 mM EDTA, 1 M NaCl, 1%
Triton X-100, 1 mg/ml bacitracin, and 25 U/ml aprotinin]. Cells
were homogenized on ice for 2 × 15 s with a Polytron (Kinematica,
Littan, Switzerland) set at 1. Homogenates were spun at 20,000 g at 4°C for 15 min, and the resulting supernatants were kept at
80°C.
AChE activity was measured using a modified version of the
spectrophotometric method of Ellman et al. (6), as described previously
(10, 14). Aliquots of 25 or 50 µl from the homogenates were incubated
in 1 ml of a phosphate buffer solution (pH 7.0) containing 7.5 × 10
4 M acetylthiocholine
iodide as the substrate, 5 × 10
4 M dithionitrobenzoic
acid, and 10
5 M
tetraisopropyl pyrophosphoramide (iso-OMPA), a nonspecific cholinesterase inhibitor. Nonspecific hydrolysis was measured at 412 nm
in the presence of both iso-OMPA and the AChE-specific inhibitor
5-bis(4-allyldimethylammonium phenyl)pentan-3-dibromide. Protein concentration was determined using the bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL), as described by the
manufacturer.
Analysis of AChE molecular forms was performed by velocity
sedimentation, as described previously (10, 14). Aliquots (100 or 150 µl) from the homogenates were layered onto 5-20% sucrose gradients. Samples were centrifuged in a Beckman SW41 rotor at 40,000 revolutions/min for 16 h at 4°C. Approximately 45 fractions were
collected from the bottoms of the tubes and assayed for AChE activity.
Activity peaks were assigned different molecular forms according to
their apparent sedimentation coefficients. Analysis of AChE molecular
forms and processing of the raw data were performed as described
elsewhere (14).
RNA extraction and reverse transcription-polymerase chain reaction.
Total RNA from cultured myotubes was extracted using Trizol (GIBCO,
Burlington, ON, Canada), as recommended by the manufacturer. Briefly,
cells were scraped into 500 µl of Trizol and homogenized 2 × 15 s using a Polytron set at 1. After addition of 100 µl of chloroform,
the solution was mixed vigorously and spun at 12,000 g for 15 min at 4°C. The aqueous
layer was then transferred to a fresh tube, and 250 µl of ice-cold
isopropanol were added. For RNA precipitation, the isopropanol mixture
was spun, and the resultant pellets were washed three times with
ice-cold 75% ethanol.
All final pellets were air dried and initially redissolved in 20 µl
of diethylpyrocarbonate (DEPC)-treated water. Total RNA concentration
for each sample was determined at 260 nm. Each sample RNA was
subsequently further diluted to obtain a final concentration of 50 ng/µl. In our standard assays, only 2 µl of the diluted RNA were
reverse transcribed. The reverse transcription (RT) mixture contained 5 mM MgCl2, 1× polymerase
chain reaction (PCR) buffer II (50 mM KCl and 10 mM
Tris · HCl, pH 8.3), 1 mM
deoxynucleotide triphosphates, 20 units ribonuclease inhibitor, 50 units murine leukemia virus reverse transcriptase, and 2.5 mM random
hexamer primers (GeneAmp RNA PCR kit, Perkin-Elmer Cetus Instruments, Branchburg, NJ). RT was performed at 42°C for 45 min, followed by
heating at 99°C for 5 min to terminate the reaction.
To amplify AChE cDNAs, specific synthetic primers based on the rat AChE
cDNA sequence were used (18). These 5' (CTGGGTGCGGATCGGT) and
3' (TCACAGGTCTGAGCAGCGTT) primers amplified a 670-base pair (bp)
target sequence in the T transcript encoding the various AChE molecular
forms expressed in muscle cells (see Ref. 22). Because these primers
are located in different exons, the PCR products that we obtained
originated from the cDNAs and not from contaminating genomic DNA. Egr-1
transcripts were selectively amplified using primers based on the rat
Egr-1 cDNA (16). These 5' (GAGTTGGGACTGGTAGGTGT) and 3'
(GCAACACTTTGTGGCCTGAA) primers amplified a 512-bp target sequence of
the Egr-1 cDNA. PCR was performed by adding 5 µl of the RT mixture to
20 µl of a solution containing 0.625 units AmpliTaq DNA polymerase,
0.25 µg each of the appropriate 5' and 3' primers,
MgCl2 (2 mM), and PCR buffer II
(1×). PCR conditions for AChE consisted of a cycle of
denaturation at 94°C for 1 min and primer annealing and extension
at 70°C for 3 min. A final 10-min elongation step at 72°C was
added after the last cycle. Cycle number for AChE was typically 42. Amplification conditions for Egr-1 transcripts were 94°C for 1 min,
65°C for 1 min, and 72°C for 1 min, followed by a 10-min
elongation step after the last cycle. The cycle number for these
experiments was 38. After amplification, 10 µl of the PCR mixture
were visualized on either a 1 or 1.5% agarose gel containing ethidium
bromide. The molecular mass of the PCR products was estimated by
comparing product size to a 100-bp ladder marker (GIBCO).
Quantitative PCR was performed as previously described elsewhere in
detail (15, 24, 29), under noncompetitive conditions, to determine the
relative abundance of AChE transcripts in control vs. mechanically
stimulated cells. Selected cDNAs were amplified using
32P-end-labeled primers.
Radiolabeled PCR products were visualized on 1.5% agarose gels and
excised using a scalpel. The counts per minute (cpm) present in these
gel bands were determined directly by Cerenkov counting. Because in
these experiments the amount of total RNA subjected to RT-PCR was equal
for all samples (see above), our PCR data for both AChE and Egr-1 are
normalized per unit of extracted total RNA.
We ascertained that, under our PCR conditions, all measurements were
obtained during the linear phase of amplification (see Refs. 15, 24).
This was done by serial dilutions of total RNA and by amplification of
these dilutions for the appropriate number of cycles (see above).
Plotting on a log-log scale the cpm obtained for each dilution vs. the
input RNA yielded a linear curve (data not shown). On the basis of
these results, all RT-PCR experiments aimed at determining the relative
abundance of AChE and Egr-1 transcripts in control and experimental
samples were performed using RNA concentrations that fell within the
linear curve for that particular number of cycles. In each experiment, negative controls consisted of the same RT and PCR mixtures except that
the input RNA was replaced with DEPC-treated water. RT-PCR conditions
(primer concentrations, input RNA, choice of RT primer, cycling
conditions, and so forth) were initially optimized, and they were
identical for all samples. This was highly controlled between samples
for some of these parameters, since we consistently used RT and PCR
master mixes, and control and experimental samples were always run in
parallel. Precautions were taken to avoid contamination from one sample
to another and RNA degradation (use of filtered pipette tips and
gloves, dedicated sets of pipettes for sample preparation and analysis,
specific areas for pre- and post-PCR procedures, and so forth).
Statistical analysis.
Paired Student's t-tests were
performed to evaluate the effects of mechanical stimulation on
expression of AChE. These tests were used to strictly compare the
effects of mechanical stimulation vs. control conditions. Because we
expected to observe an increase in AChE expression following mechanical
stimulation (see introduction), one-tailed
t-tests were used to determine
significant differences among control and experimental groups. The
level of significance was set at P < 0.05. Data are expressed as means ± SE throughout. The
patterns of AChE molecular forms displayed in Figs.
4-6 are representative examples.
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RESULTS |
Mechanical stimulation increases AChE activity in cultured myotubes.
As shown in Fig. 1, total AChE
activity remained initially unaffected by mechanical stimulation. After
24 h of mechanical stimulation, however, there was a significant
increase (42%; P < 0.05) in AChE
activity compared with the levels observed in control myotubes. TTX
treatment of myotubes mechanically stimulated for 24 h did not block
the increase in AChE activity following mechanical stimulation. In
fact, the increase in AChE activity between myotubes subjected to
mechanical stimulation with or without TTX differed by only ~7%
(n = 5). By contrast, nifedipine
blocked ~50% of the AChE increase seen following mechanical
stimulation (Fig. 2).

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Fig. 1.
Total acetylcholinesterase (AChE) activity in myotubes subjected to
mechanical stimulation (MS). Eight- to ten-day-old primary cultures of
rat myotubes were subjected to 24% mechanical stimulation and
harvested 5 min, 30 min, 3 h, and 24 h later. Data are means ± SE. * Significant difference from control (CTL;
P < 0.05, n = 10).
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Fig. 2.
Effects of nifedipine treatment on AChE activity in myotubes subjected
to mechanical stimulation. Primary cultures of rat myotubes were
mechanically stimulated for 24 h in the presence of nifedipine, an
L-type calcium channel blocker. Data (means ± SE) are expressed as
percent of control. * Significant difference between experimental
and control groups (P < 0.05, n = 3). ** Significant
difference between mechanically stimulated and nifedipine + mechanically stimulated groups (P < 0.05, n = 3).
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Mechanical stimulation increases all AChE molecular forms.
To determine whether the increase in AChE activity observed in response
to mechanical stimulation was caused by a general elevation in the
activity of all molecular forms as opposed to an increase in a specific
form, aliquots of muscle homogenates were separated by velocity
sedimentation on sucrose gradients, and AChE activity was determined in
each fraction. Control rat myotube cultures typically displayed a
molecular form profile consisting of forms
A12,
A8,
G4, and
G1 (see, for example, Fig. 4A). After 24 h of mechanical
stimulation, a similar AChE pattern was observed, indicating that this
treatment increased the expression of all molecular forms. Quantitative
analysis revealed that the activity of each molecular form, expressed
as a percent of total activity, did not change following mechanical
stimulation, thereby confirming that this experimental treatment led to
an elevation in the levels of all AChE molecular forms as opposed to a
preferential increase in a specific form (Fig.
3).

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Fig. 3.
Activity of AChE molecular forms in myotubes subjected to mechanical
stimulation. Values represent relative activity of
A12,
A8,
G4, and
G1 forms present in primary
cultures of rat myotubes before (control) and after 24 h of mechanical
stimulation. Data (means ± SE; n = 3) are expressed as percent of total AChE activity.
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As previously observed (see, for example, Refs. 8, 25), TTX treatment
selectively decreased the amount of asymmetric forms present in these
rat myotube cultures (Fig.
4B).
Mechanically stimulating myotubes in the presence of TTX did not induce
the reappearance or upregulation of the asymmetric forms (Fig.
4C). This was in fact confirmed by
analysis of cultures that did not display significant amounts of
spontaneous contractile activity. In these cultures, the levels of
asymmetric forms were already low, and mechanical stimulation did not
induce their expression (Fig. 5).

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Fig. 4.
Effects of tetrodotoxin (TTX) and mechanical stimulation on AChE
molecular forms in rat myotubes. Molecular form profiles of control
myotubes (A), TTX-treated myotubes
(B), and TTX-treated myotubes with
mechanical stimulation for 24 h (C).
Note that asymmetric forms present in control cells disappeared
following TTX treatment (B) and that
these forms did not return following mechanical stimulation
(C).
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Fig. 5.
Effects of TTX and mechanical stimulation on AChE molecular forms in
noncontracting rat myotubes. Molecular form profiles of control
myotubes (A), TTX-treated myotubes
(B), and TTX-treated myotubes with
mechanical stimulation for 24 h (C)
are shown. Note that AChE molecular form profiles did not change in
comparison with controls.
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Mechanical stimulation increases secretion of AChE.
Under control conditions, cultured myotubes secrete
G4 into the culture medium (see
Refs. 22, 25). We thus examined whether mechanical stimulation also
increased secretion of AChE. Analysis of AChE levels in the medium
collected from mechanically stimulated rat myotubes for 24 h revealed a
70% increase (P < 0.05) in total enzyme activity compared with controls (Fig.
6). As observed with AChE associated with
muscle cells (see Figs. 4 and 5), TX treatment of cultured myotubes
failed to prevent the increase in AChE secretion following mechanical
stimulation (68% increase over control levels).

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Fig. 6.
Effects of mechanical stimulation on secretion of AChE. Medium was
collected from rat myotube cultures mechanically stimulated for 24 h.
A: total AChE activity (means ± SE) in media from control and mechanically stimulated myotubes.
B: only
G4 molecular form of AChE was
present in medium from control myotubes.
C: after mechanical stimulation,
pattern of molecular forms secreted remained unchanged.
* Significant difference between control and mechanically
stimulated myotubes (P < 0.05, n = 3).
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To determine whether passive mechanical forces altered the
pattern of AChE molecular form secreted from the myotubes, an aliquot of the medium was separated by velocity sedimentation on sucrose gradients, and each fraction was analyzed for AChE activity.
Figure 6B shows that only the
G4 form of AChE was present in the
medium of these control rat myotubes. Medium collected from myotubes mechanically stimulated for 24 h also contained only the
G4 form of AChE although the
amount had increased significantly (Fig. 6,
B and
C).
Mechanical stimulation increases AChE and Egr-1 mRNA levels.
To determine the effects of mechanical stimulation on levels of AChE
transcripts, quantitative RT-PCR was performed using total RNA
extracted from primary cultures of rat myotubes. Selective amplification of AChE cDNAs produced the expected band of 670 bp.
Analysis of PCR products revealed that AChE transcript levels per
unit of extracted total RNA (see MATERIALS AND
METHODS) increased progressively over the time course
of the experiments (Fig.
7A) and reached a significant 3.5-fold increase at 24 h
(P < 0.05) compared with
control levels (Fig. 7B). The
apparent early increase in AChE mRNA levels thus preceded the increase
seen in total AChE activity (compare Figs. 1 and 7).

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Fig. 7.
Effects of mechanical stimulation on AChE transcript levels in rat
myotubes. A: total RNA obtained from
control myotubes (lane 1) and myotubes mechanically
stimulated for 5 min (lane 2), 30 min
(lane 3), 3 h
(lane 4), and 24 h
(lane 5) was pooled and subjected to
reverse transcription-polymerase chain reaction (RT-PCR) for detection
of AChE mRNAs; , negative control lane;
left, 100-base pair (bp)
ladder. Quantitation of PCR products over the time
course revealed maximal accumulation of AChE transcripts at 24 h
(lane 5).
B: quantitation of individual control
and 24-h samples. * Significant difference between control
myotubes and myotubes mechanically stimulated for 24 h
(P < 0.05, n = 10). CPM, counts/min.
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Because Egr-1 mRNA levels are known to increase in response to
mechanical stress in other cell types (see, for example, Ref. 17), we
also determined whether expression of this immediate-early gene was
increased in rat myotubes following mechanical stimulation. Selective
amplification of Egr-1 cDNAs produced the expected band of 512 bp.
Levels of Egr-1 mRNAs (per unit of extracted total RNA) from
mechanically stimulated cells were found to increase rapidly, peaking
at 30 min and returning toward control levels thereafter (Fig.
8A).
Quantitative analysis revealed a significant 2.3-fold increase
(P < 0.05) over control levels at 30 min (Fig. 8B).

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Fig. 8.
Effects of mechanical stimulation on Egr-1 mRNA levels in rat myotubes.
A: total RNA obtained from control
myotubes (lane 1) and myotubes mechanically
stimulated for 5 min (lane 2), 30 min
(lane 3), 3 h
(lane 4), and 24 h
(lane 5) was pooled and subjected to
RT-PCR for detection of Egr-1 transcripts; , negative control
lane; left, 100-bp ladder.
Quantitation of PCR products over the time course revealed maximal
accumulation of Egr-1 transcripts at 30 min
(lane 5).
B: quantitation of individual control
and 30-min samples. * Significant difference between control
myotubes and myotubes mechanically stimulated for 30 min
(P < 0.05, n = 10).
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DISCUSSION |
Mechanical stimulation of cultured myotubes using different intensities
and frequencies can mimic the mechanical events occurring during
various types of exercise in vivo (30). In the present study, we thus
mechanically stimulated myotubes to determine whether this type of
stimulation could, in addition to electrical activity and trophic
factors (see introduction), increase expression of AChE. Although no
significant changes in the levels of AChE were found up to 3 h after
the onset of mechanical stimulation, we observed a significant increase
in total AChE activity at 24 h, indicating that passive mechanical
stimulation, indeed, increases expression of AChE in cultured rat
myotubes. In addition, our results show that this increase in AChE
expression was caused by a general elevation in the activity of all
AChE molecular forms. Finally, analysis of AChE mRNA levels revealed
that the observed increase in enzyme activity was accompanied by an
increase in the levels of AChE transcripts, indicating the involvement
of a pretranslational regulatory mechanism.
In vivo, skeletal muscles respond to enhanced levels of neuromuscular
activity with a significant increase in AChE enzyme activity. Recent
studies have shown that, in fast muscles, this increase in AChE
expression results primarily from a selective increase in the content
of G4, provided that the
recruitment pattern of the muscle is phasic as opposed to tonic (2, 7,
14, 29). Therefore, in the present study, we also determined whether passive mechanical stimulation influenced preferentially the expression of a specific AChE molecular form. Sedimentation analysis revealed that
the increase in total AChE was due to an elevation in the activity of
all molecular forms. This type of general increase in all AChE
molecular forms resembles that previously seen in response to
compensatory hypertrophy (13, 29). This suggests, therefore, that the
adaptive response of skeletal muscle fibers to mechanical stimulation
is similar to that occurring in hypertrophying muscles. This conclusion
is in fact coherent with other studies that have shown that cultured
myotubes react to mechanical stimulation by displaying cell growth and
hypertrophy (5, 31).
The delayed expression of AChE in response to mechanical stimulation
has been observed previously for other molecules (see Ref. 32). This
delay may be caused by the nature of the mechanogenic signaling pathway
involved in regulating AChE expression. A muscle cell may respond to an
external mechanical signal in a rapid (milliseconds), intermediate
(minutes), or longer (hours, days) fashion (1). The minimum delay of 3 h that we observed for significant AChE induction suggests, in this
case, the involvement of pretranslational regulatory mechanisms (1,
30). Accordingly, we examined the levels of AChE mRNA in response to
mechanical stimulation and found that transcript levels accumulated
progressively over the experimental time course, reaching a significant
3.5-fold increase over controls at 24 h.
The increased expression of AChE mRNAs following mechanical stimulation
may have occurred via two mechanisms. First, mechanical stimulation may
have caused a significant reduction in the degradation rate of AChE
transcripts. This view is in fact strongly supported by the recent
demonstration that, during myogenesis, AChE mRNA levels are upregulated
via an increase in the stability of existing transcripts as opposed to
the transcriptional activation of the AChE gene (9, 21). Alternatively,
mechanical stimulation may have led to an increase in the expression of
specific transcription factors with the subsequent transcriptional
activation of the AChE gene.
Expression of several immediate-early genes such as Egr-1
has already been shown to be exquisitely sensitive to the effects of
mechanical stimulation in various cell types (for example, this study;
for review, see Refs. 1, 17). Furthermore, a recent study has
demonstrated that passive stretch can modulate expression of myogenin
and muscle regulatory factor 4 in skeletal muscle fibers, indicating
that these basic helix-loop-helix transcription factors may regulate
gene expression under these conditions (20). Because the AChE promoter
contains several binding sites for Egr-1 and myogenic factors (19), it
is conceivable that the increased expression of these transcription
factors following mechanical stimulation ultimately transactivates the
AChE gene, thereby leading to an
increased steady-state level of AChE mRNA. This view is particularly
attractive, since Li et al. (19) have previously reported that mutation
of the Egr-1 sites on the mouse AChE promoter results in a marked loss
in the activity of a reporter gene in transfected cells.
Previous studies have highlighted the contribution of electrical
activity in the regulation of AChE expression in vivo as well as in
vitro. Using TTX, a potent Na+
channel blocker, we uncoupled the effects of electrical activity from
those induced by mechanical stimulation on AChE expression. TTX
treatment of cultured myotubes failed to prevent the increase in AChE
expression and secretion following mechanical stimulation. The
inability of TTX to block the increase in AChE expression following
passive mechanical stimulation suggests that the regulatory mechanism
involved in the adaptive response of AChE to mechanogenic forces is
independent of electrical activity. These results are thus consistent
with previous findings that have shown, for example, that TTX treatment
of cultured myotubes subjected to mechanical stimulation also failed to
prevent increases in the expression of cyclooxygenase (34),
phospholipases (33), and other proteins required for cell growth and
hypertrophy (31, 32).
The notion that mechanical stimulation regulates AChE expression via an
electrical activity-independent pathway is corroborated by examination
of the molecular form profiles in cultured myotubes subjected to
passive mechanical forces in the presence of TTX. TTX treatment of
spontaneously contracting myotubes decreased the levels of asymmetric
forms, and mechanical stimulation failed to restore their levels.
Similarly, mechanical stimulation of noncontracting cultures did not
induce the appearance of the asymmetric forms of AChE. Because, in
cultured muscle cells, it is well established that expression of the
asymmetric forms depends largely on electrical activity (see Refs. 8,
25), these results strengthened the notion that mechanical stimulation
increases expression of AChE by a mechanism distinct altogether from
that involved in the activity-linked regulation of AChE.
Cells subjected to mechanical stimulation respond to mechanogenic
forces by exhibiting a variety of changes, including a rise in
intracellular Ca2+ levels. This
rise in intracellular Ca2+ can
occur quickly, increasing by 50% within the first 10 min after the
onset of stimulation (1, 17). Because intracellular Ca2+ entering myotubes via L-type
channels has been shown to be a key modulator of AChE mRNA expression
(21), we determined whether this pathway was involved in the overall
response of AChE to mechanical stimulation. L-type channels were
blocked using nifedipine, and myotubes were mechanically stimulated for
24 h. Nifedipine was able to partially block the increase seen in
response to mechanical stimulation. These results suggest that
Ca2+ indeed plays a role in
modulating AChE expression in response to mechanogenic forces. In
addition, they demonstrate that, although entry of
Ca2+ through L-type channels is
necessary for the full adaptive response to occur, it is not the sole
signal transduction system involved, since the AChE response was only
partially abolished. Additional Ca2+ influx from alternative
sources such as mechanosensitive ion channels and/or distinct
signaling pathways may thus also participate in initiating the overall
adaptive changes in the expression of AChE following mechanical
stimulation.
In conclusion, AChE expression in skeletal muscle fibers is known to be
significantly influenced by the presence of motoneurons. However, the
results of the present study showing that passive mechanical forces
regulate expression of AChE in cultured myotubes offer an
additional mechanism by which AChE levels may be modified. This
mechanism likely plays important functional roles in the control of
AChE expression both in developing muscle fibers and at mature
neuromuscular junctions. During embryogenesis and myogenesis, for
example, mechanical forces have undoubtedly significant physiological ramifications, since, during differentiation of skeletal muscle, developing fibers are likely subjected to the effects of passive mechanical forces as a result of the growth of the bones (28). In
addition, a recent study has demonstrated that neuromuscular junctions
undergo intense mechanical distortions during cycles of muscle
contraction and relaxation (26). Given that AChE expression in this
region of muscle fibers is known to be exquisitely sensitive to neural
influences (15, 24), mechanical stimulation may therefore represent an
additional regulatory factor affecting AChE expression markedly yet
independently of the well-known effects of the motor nerve. In this
context, it would therefore be of interest to determine whether the
expression of other synaptic proteins is also altered in response to
mechanical stimulation.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. David J. Parry for advice about the primary
cultures of rat myotubes.
 |
FOOTNOTES |
This work was supported by grants from the Medical Research Council of
Canada (MRC) and the Ontario Thoracic Society to B. J. Jasmin. B. J. Jasmin is a Scholar of the MRC.
Address for reprint requests: B. J. Jasmin, Dept. of Physiology,
Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, ON,
Canada K1H 8M5.
Received 29 May 1997; accepted in final form 8 August 1997.
 |
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