Instituto de Ciencias Biomédicas and Centro Fondo de Investigación Avanzada en Areas Prioritarias de Estudios Moleculares de la Célula, Facultad de Medicina, Universidad de Chile, Santiago 6530499, Chile
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ABSTRACT |
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The signaling mechanisms by which skeletal muscle electrical activity leads to changes in gene expression remain largely undefined. We have reported that myotube depolarization induces calcium signals in the cytosol and nucleus via inositol 1,4,5-trisphosphate (IP3) and phosphorylation of both ERK1/2 and cAMP-response element-binding protein (CREB). We now describe the calcium dependence of P-CREB and P-ERK induction and of the increases in mRNA of the early genes c-fos, c-jun, and egr-1. Increased phosphorylation and early gene activation were maintained in the absence of extracellular calcium, while the increase in intracellular calcium induced by caffeine could mimic the depolarization stimulus. Depolarization performed either in the presence of the IP3 inhibitors 2-aminoethoxydiphenyl borate or xestospongin C or on cells loaded with BAPTA-AM, in which slow calcium signals were abolished, resulted in decreased activation of the early genes examined. Both early gene activation and CREB phosphorylation were inhibited by ERK phosphorylation blockade. These data suggest a role for calcium in the transcription-related events that follow membrane depolarization in muscle cells.
myotubes; signal transduction; inositol 1,4,5-trisphosphate
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INTRODUCTION |
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SKELETAL
MUSCLE responds to exercise or to electrical stimuli with changes
in gene expression at the level of structural proteins and energetic
metabolism enzymes (19, 20, 27). In recent years, a number
of studies on the early signaling mechanisms that might link skeletal
muscle activity to biochemical and gene regulatory responses have been
reported (24, 25). A major issue concerns the possible
role of calcium in the early events that lead to changes in gene
expression in muscle cells. In rat skeletal muscle cells in primary
culture, decreased transcription of the nicotinic acetylcholine (ACh)
receptor subunit RNAs was reported to occur after treatment with drugs
that release calcium from the sarcoplasmic reticulum, thus arguing in
favor of a role for intracellular calcium in activity-dependent
gene expression in skeletal muscle (3). The effect of
calcium influx through L-type channels induced by the agonist BAY K
8644, meanwhile, was found to reduce expression of the -subunit of
the nicotinic ACh receptor through posttranscriptional mechanisms
(3). The role of calcium has also been approached by
treating cultured skeletal muscle cells with the calcium ionophore A-23187. The increase in intracellular calcium following a prolonged exposure of primary culture to the ionophore induces a change in myosin
from fast to slow isoforms (15). In L69 myotubes, cytochrome c gene expression is activated by intracellular
calcium increase resulting from a 48-h incubation with A-23187
(10).
Although intracellular calcium in skeletal muscle cells has been thoroughly studied in relation to the fast process of muscle contraction, previous work in our laboratory (13, 14) has shown that the calcium increase in skeletal muscle cells induced by high-K+ depolarization is a complex event involving at least two components. After a very fast calcium transient related to excitation-contraction (E-C) coupling, there is a slower transient not related to contraction that lasts several seconds. Whereas the first component is associated with the ryanodine receptor, the second is inhibited by compounds that interfere with the inositol 1,4,5-trisphosphate (IP3) system, suggesting that these signals are mediated by IP3 receptors (7, 21). The dihydropyridine receptor that functions in skeletal muscle as a voltage sensor, and as such has a fundamental role in E-C coupling, is also a voltage sensor for IP3-mediated slow calcium signals in muscle cells (4).
We have determined that high K+-induced depolarization brings about the stimulation of phosphorylation of ERK1/2 and of the transcription factor cAMP-response element-binding (CREB) protein (21). Furthermore, we have found that both responses are inhibited when the slow signal is blocked (21). These results suggested a signaling system mediated by Ca2+ and IP3 that could be involved in regulation of gene expression in skeletal muscle. In the present work, this study has been extended by examining early genes that are upregulated in skeletal muscle by either exercise or electrical stimulation (1, 5, 16, 17, 22). Experiments were also performed to study the contribution of the ERK and other pathways to both CREB phosphorylation and to early gene activation. We have found that the slow calcium transients elicited by K+ depolarization of myotubes are involved in transient increases of mRNA levels of the early genes c-fos, c-jun, and egr-1. We could also determine that the ERK pathway is involved in both CREB phosphorylation and c-fos, c-jun, and egr-1 activation. In addition, the inhibition of another MAPK, p38 MAPK, reduced P-CREB levels and c-fos and c-jun upregulation, whereas the pharmacological inhibition of CaMK only decreased c-fos mRNA levels. Results indicate that slow calcium transients in skeletal muscle cells are related to signaling pathways likely to be part of the early steps in transcriptional activation.
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MATERIALS AND METHODS |
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Materials.
Dulbecco's modified Eagle's medium/F-12 was from Sigma (St. Louis,
MO). Fetal calf serum, calf serum, antibiotics, and antimycotic were
from Life Technologies (Burlington, ON, Canada). Antibodies against
dually phosphorylated forms of ERK-1 and ERK-2 (P-ERK) and CREB
(P-CREB) were from Cell Signaling Technology (Beverly, MA). CREB
antibody and anti-ERK2 were from UBI (Lake Placid, NY). Horseradish
peroxidase (HRP)-conjugated anti-rabbit was purchased from Pierce
(Rockford, IL), and HRP-conjugated anti-mouse was from Sigma. Enhanced
chemiluminescence reagents were from Pierce or Amersham Pharmacia
Biotech (Amersham, UK). The mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor
U-0126, the CaMK inhibitor KN-93, and the IP3 receptor blocker xestospongin C were from Calbiochem (La Jolla, CA). SB-203580, a p38 MAPK inhibitor, was from Biomol (Plymouth Meeting, PA). BAPTA-AM,
Oregon green BAPTA-5N, and fluo 3-acetoxymethyl ester (fluo 3-AM) were
from Molecular Probes (Eugene, OR). 2-Aminoethoxydiphenyl borate
(2-APB) was from Aldrich. Nylon membranes were from Amersham International (Aylesbury, UK). [ 32P]ATP was from NEN.
All other reagents were purchased from either Sigma or Life Technologies.
Primary culture and cell treatment. Cell suspensions were obtained by collagenase treatment of hindlimb muscular tissue from 21 fetal Sprague-Dawley rats. Briefly, the tissue was mechanically dispersed and then treated with 0.2% (wt/vol) collagenase for 15 min at 37°C under mild agitation. The suspension was filtered through Nytex membranes, spun down at low speed, and preplated for 10-15 min for enrichment of myoblasts. Cells were plated on 60-mm culture dishes in a medium composed of DMEM/F-12 (1:1), 10% heat-inactivated calf serum, 2.5% heat-inactivated fetal calf serum, antibiotics, and antimycotic. To eliminate remaining fibroblasts, 10 µM cytosine arabinoside was added when the myoblasts started to align. For differentiation, fetal bovine serum was reduced to 1.5%.
For experiments, myotubes at 6-7 days were cultured for 24-36 h in serum-free medium. Cells were washed with Ca2+- and Mg2+-free PBS and maintained in Krebs-Ringer under resting conditions for 30 min (in mM: 20 HEPES-Tris, pH 7.4, 118 NaCl, 4.7 KCl, 3 CaCl2, 1.2 MgCl2, and 10 glucose). Depolarization was induced by changing to a medium containing 84 mM KCl while the sodium concentration was decreased proportionally to maintain the osmolarity of the solution. When pharmacological inhibitors were used, cells were incubated in their presence for additional 30 min. All experiments were matched with vehicle-treated controls. Cells were never exposed to concentrations higher than 0.1% DMSO, and this concentration had no effect on responses of skeletal muscle cells. Both control and experimental cells were submitted to the same bath changes to discard differences by handling.Western blot analysis. After treatment, cells were solubilized at 4°C in 0.1 ml of lysis buffer containing 50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 5 mM Na3VO4, 20 mM NaF, 0.2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 1 mM benzamidine, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µM pepstatin. After incubation on ice for 20 min, cells were scraped from the dishes, sonicated for 1 min, and left on ice for 30 min. Nuclear and cellular debris were removed by microcentrifuge centrifugation at 17,000 g for 20 min. In determining protein concentration of the supernatants, BSA was used as standard. Aliquots of lysates were suspended in Laemmli buffer, and proteins were resolved in 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Primary antibody incubations using dilutions of 1:1,000 (P-CREB), 1:750 (CREB), and 1:2,000 (P-ERK1/2 or ERK2) were carried out at 4°C overnight. After incubation with HRP-conjugated secondary antibodies for 1.5 h, membranes were developed by enhanced chemiluminescence according to the manufacturer's instructions. To correct for loading, the membranes were stripped and blotted for anti-ERK2 and anti-CREB. After the films were scanned, a densitometric analysis of the bands was performed with the Scion Image program [National Institutes of Health (NIH)].
Northern blot analysis. Total cellular RNA was isolated by the guanidinium isothiocyanate method (6). Samples (15-20 µg) were electrophoresed on 1% agarose-formaldehyde gels, transferred by capillary blotting onto nylon membranes, and immobilized by photocross-linking. Blots were prehybridized for 1 h at 42°C in a buffer containing 50% deionized formamide, 5× SSPE (sodium chloride-sodium phosphate-EDTA)/1% SDS, and 125 µg/ml salmon sperm DNA. Hybridizations with 1 × 108 cpm/ml 32P-labeled cDNA probes were carried out at 42°C overnight in the same solution. Membranes were washed once with 2× SSPE/0.1% SDS solution for 5 min, once with 0.2× SSPE/0.1% SDS for 5 min, and twice with 0.1× SSPE/0.1% SDS at 68°C for 15 min before autoradiographic film exposure. After autoradiography, bands were quantified by densitometry using an NIH program. Ethidium bromide stain of gels before capillarity transfer and reprobing of blots with GAPDH confirmed the integrity of the RNA samples and documented equivalent loading of each lane in gels used for the analysis.
c-DNA probes.
Rat c-fos cDNA, 2.1 kb, subcloned into EcoRI
sites of p-SP65, and rat c-jun cDNA, 1.8 kb, subcloned into
EcoRI of p-Gem-4, were propagated in electrocompetent
Escherichia coli DH5 cells. Purified plasmids were
digested with EcoRI, and the products were labeled with
[
-32P]dATP by using the random primer/Klenow enzyme
method. Plasmids were a kind gift of Dr. Tom Curran (Children's
Research Hospital, Memphis, TN).
Semiquantitative RT-PCR. cDNA was amplified by using c-fos, c-jun, or egr-1 primers, and the DNA concentration was normalized to GAPDH expression. PCR amplification was maintained in the exponential phase for each product. The c-fos primers used were 5'-AGGCCGACTCCTTCTCCAGCAT-3' (sense) and 5'-CAGATAGCTGCTCTACTTTGC-3' (antisense), corresponding to bases 235-533. The c-jun primers used were 5'-GCGCCGCCGGAGAACCTCTGTC-3'(sense) and 5'-CAGCTCCGGCGACGCCAGCTTG-3' (antisense), corresponding to bases 577-1227 (11).
Calcium measurement. Intracellular, ionized calcium images were obtained from rat myotubes with a fluorescence microscope (Olympus) equipped with a cooled charge-coupled device camera and an image acquisition system (Spectra Source MCD 600). Myotubes were washed three times with Krebs buffer (145 mM NaCl, 5 mM KCl, 2.6 mM CaCl2, 1 mM MgCl2, 10 mM HEPES-Na, and 5.6 mM glucose, pH 7.4) to remove serum and then loaded with 5.4 µM fluo 3-AM (from a stock in 20% Pluronic acid-DMSO) or, when indicated, with Oregon green BAPTA-5N, which was then deesterified in the cytoplasm for 30 min at room temperature. Cells were preincubated in resting solution (see below) containing the dye at a 5.4 µM concentration for 30 min at 25°C. Cells attached to coverslips were mounted in a 1-ml capacity perfusion chamber and placed in the microscope for fluorescence measurements after excitation with a filter system.
Fluorescent images were collected every 0.1-2.0 s and analyzed frame by frame with the data-acquisition program (Spectra Source) for the equipment. Cells were incubated in the Krebs buffer (see above) as a resting condition medium. Cells were exposed to high-K+ solutions (47 mM K+, replacing Na+) and depolarized by a fast (~1 s) change of solution using the perfusion system.Statistics. Results are expressed as means ± SE, and the significance of differences was evaluated using Student's t-test for paired data or ANOVA followed by Dunnett's multiple comparison post test.
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RESULTS |
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mRNA levels of the early genes c-fos, c-jun, and egr-1 in rat
myotubes after depolarization.
Time dependence studies of c-fos, c-jun, and
egr-1 mRNA levels performed after the depolarization
procedure revealed a transient twofold increase that peaked about 15 min after treatment for the three mRNAs (Fig.
1). This increase was significant for all three early genes. c-fos and egr-1 mRNA
expression returned to basal levels after 60 min; c-jun mRNA
levels, meanwhile, remained higher than basal at the end of this
period.
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Relationship between the slow calcium transient and ERK or CREB
phosphorylation and early gene expression.
To link early gene activation with the slow calcium transient seen in
myotubes (Fig. 3A), we first
dissociated the fast calcium transient from the slow one, taking
advantage of the difference in cytosolic calcium that each of them
represents. It has been postulated that cytosolic calcium concentration
must be very low during the slow transient, because no contraction was
detected during this period (13) and the use of
ratiometric calcium dyes so indicates (13, 14). To further
stress this point, we used a calcium-sensitive dye (Oregon green
BAPTA-5N) that has much lower affinity for calcium than fluo 3 (20 µM
Kd compared with 0.4 µM in the absence of
Mg2+). Upon depolarization, the fast calcium transient,
capable of reaching micromolar calcium concentrations, can be clearly
seen in Oregon green-5 BAPTA-5N-loaded cells (Fig. 3B), but
the slow calcium transient was not apparent. Taking advantage of this
result, we tried calcium chelation with the cell-permeant chelator
BAPTA-AM. When myotubes were preincubated with both fluo 3 and 100 µM
BAPTA-AM for 30 min, the slow calcium transient was abolished, whereas the fast calcium transient was spared (Fig. 3C).
Preincubation with BAPTA-AM also resulted in decreased levels of both
P-ERK and P-CREB (Fig. 3D).
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ERK and CREB phosphorylation and early gene mRNA levels in the
absence of extracellular calcium.
Calcium transients arising from skeletal muscle cells in primary
culture exposed to high K+ are normally independent of
extracellular calcium (13). However, because calcium entry
through either voltage-gated or store-operated channels is a
possibility in these cells, it is important to assess whether in our
experimental conditions calcium influx participates in the activation
of ERKs, CREB, and early genes. Experiments conducted under resting and
depolarization conditions with medium containing 0.5 mM EGTA and no
added calcium showed that the effects of depolarization remained
essentially the same in calcium-free conditions (Fig.
5). egr-1 mRNA levels were
also increased by depolarization in the absence of extracellular
calcium (not shown).
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Effect of increasing intracellular calcium with caffeine.
Results obtained in BAPTA-AM-loaded cells indicated that cytosolic
calcium has a role in the effects induced by the depolarization treatment. To pharmacologically increase intracellular calcium, we
incubated myotubes with 10 mM caffeine. Exposure to caffeine resulted
in stimulation of ERK1/2 phosphorylation (Fig.
6A), CREB phosphorylation
(Fig. 6B), and c-fos and c-jun mRNA
levels (Fig. 6C). In Fig. 6C, the results from
three independent experiments on myotubes exposed to either caffeine or
high K+ are shown. The mRNA levels were very similar in
both conditions. In one additional experiment (triplicate), the effect
of caffeine on egr-1 mRNA levels also resulted in an
increase similar to that for c-fos and c-jun (not
shown).
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Effect of MAPK inhibition on CREB phosphorylation and early gene activation. Because depolarization of skeletal muscle cells in primary culture activates ERKs, the role of this kinase cascade on early gene expression and CREB phosphorylation was evaluated. To study the role of the ERK signaling cascade, we used U-0126, a specific MEK inhibitor described as a blocker of the phosphorylated and nonphosphorylated forms of MEK1 and MEK2 (8).
U-0126 (10 µM) completely blocked the increase in ERK1/2 phosphorylation (Fig. 8A). Basal P-ERK levels were also decreased by prior exposure to U-0126. As a consequence of this inhibition, P-CREB levels were diminished (Fig. 8B) to values ranging from 8 to 30% of controls as observed in four independent experiments. The c-fos, c-jun, and egr-1 mRNA levels (Fig. 8C) were also largely diminished under these conditions. These results support a role for the MEK-ERK cascade as a link between membrane potential-triggered signals and nuclear events.
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CaMK inhibition decreases c-fos upregulation. KN-93 for CaMK inhibition was also tested. With 10 µM KN-93, there was no significant effect on the increase of P-CREB induced by high K+. The values (means ± SE), expressed as percentages of a normalized 100% control, were 227.0 ± 77.0% in control myotubes and 284.0 ± 81.0% in myotubes exposed to KN-93 (n = 3). Although c-jun mRNA levels were not affected (202.3 ± 3.5% for control and 215.0 ± 14.8% in the presence of KN-93, n = 4), there was a decrease in c-fos mRNA level from the control value of 224.8 ± 7.3 to 169.0 ± 4.6% (n = 4, P < 0.05). egr-1 levels were not assessed.
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DISCUSSION |
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The present study gives further evidence for a link among membrane depolarization, the upregulation of c-fos, c-jun, and egr-1 mRNA levels, and P-ERK and P-CREB levels through a calcium- and IP3-mediated mechanism. We have demonstrated that after stimulation, there is a rapid and transient increase in early gene expression, that ERKs are involved in this upregulation as well as in CREB phosphorylation increase, and that the effects of depolarization are critically dependent on calcium released from IP3-sensitive intracellular stores. We have used cultured myotubes, which constitute a model system that has some of the elements of adult muscle fibers but is also a model for developing muscle cells. Under this scope, the signals we are studying could be interpreted as relevant for muscle cell development and differentiation; confirmation of their presence and role in adult muscle fibers awaits studies in a different system.
In recent years, several studies on the early signaling mechanisms that putatively link skeletal muscle activity to biochemical and gene regulatory responses have focused on early gene expression. Most of immediate early gene products are transcription factors that bind to promoter regulatory elements of a number of downstream genes, so they are likely to be involved in the adaptive responses induced by neural activity and contractile work in skeletal muscle. It has been shown that after exercise, human skeletal muscle upregulates the expression of most members of the fos and jun gene families (22). Upon electrical stimulation of the motor nerve, c-fos, c-jun, and egr-1 mRNAs increase in both rabbit and rat skeletal muscle (1, 5, 16, 17). egr-1 has also been reported to increase in C2C12 cells exposed to either the calcium ionophore A-23187 or the cholinergic agonist carbachol (2). The latter could be blocked by either ryanodine or dantrolene, indicating that calcium released from sarcoplasmic reticulum is involved.
The present data show that depolarization of rat myotubes in primary culture brings about a twofold transient increase in c-fos, c-jun, and egr-1 mRNA levels, with a maximum about 15 min after exposure to a high K+ concentration. It is interesting to note that a depolarization period of 1 min is enough to induce mRNA upregulation, still significant 30 min after stimulation, suggesting that within this minute the voltage sensors (4) involved in the triggering of the cascades probably undergo a single activation process and that this activation is enough to produce the total effect. In fact, 1 min of depolarization was enough to trigger both ERK and CREB phosphorylation detected after 10-20 min (21).
The results obtained after either extracellular (or intracellular) calcium chelation indicate that calcium release from intracellular compartments is involved in the increase of the early genes examined. It is worth noting that calcium increases such as those produced by either thapsigargin or caffeine are able to mimic the effects of depolarization on intracellular signals. As previously indicated, in skeletal myotubes, the calcium increase induced by depolarization involves two components. There is a fast calcium transient, visualized in the whole myotube, and a slow, localized calcium transient that involves both the nuclei and the cytoplasm surrounding the nuclei (13). Whereas the fast component is antagonized by ryanodine, the slow transient is abolished by compounds that interfere with IP3, such as 2-APB, an inhibitor of IP3-induced calcium release, and U-73122, a PLC inhibitor (21). In a dyspedic (1B5) cell line expressing no ryanodine receptors, only the slow calcium signal is induced by depolarization, and it is blocked by either 2-APB or U-73122 and also by the IP3 receptor antagonist xestospongin-C (7). This evidence indicates that the calcium increase visualized at the nuclear level is induced by an IP3-dependent mechanism and that this mechanism operates independently of the fast calcium transient. We have previously found that 2-APB blocked both the slow calcium transient and the increase in ERK and CREB phosphorylation obtained in skeletal muscle cells depolarized by K+ (21). In this study, we have found that both 2-APB and xestospongin C also decreased the depolarization-induced c-fos, c-jun, and egr-1 mRNA increase. The same results have been obtained with the calcium chelator BAPTA-AM. Therefore, the reduction in both the slow calcium transient and the biochemical responses was obtained by two different procedures, both involving intracellular calcium. Results obtained using ryanodine point to the slow calcium signal as the one responsible for this cascade activation. Slow calcium signals result from IP3 receptor activation; the fact that we can elicit an increase of P-CREB, P-ERK, and early genes with caffeine can be interpreted either as a nonspecific effect of the huge calcium rise induced by caffeine or partially to activation of IP3 receptors by calcium, as suggested by the experiments with 2-APB.
Apart from early gene activation, both exercise and electrical stimulation activate MAPKs (25). Increased ERK1/2 phosphorylation occurs in human and rat skeletal muscle submitted to either exercise (18, 25) or sciatic nerve stimulation (5) as well as after contractile activity of isolated rat muscle (24, 25). In agreement with these observations, we have shown increased ERK1/2 phosphorylation in K+-depolarized skeletal muscle cells in primary culture (21). Our results suggest that activated MAPKs, ERK 1/2 and p38, participate in both CREB phosphorylation and in early gene upregulation.
We have previously described CREB phosphorylation after myotube
depolarization (21). P-CREB interacts with consensus
cAMP-response element (CRE) sequences located in the promoter region of
many early genes, including c-fos and egr-1
(23, 26), and of many specific genes as well. More than
one pathway can activate CREB as shown in hippocampal neurons
(28). A fast CaMK activity is involved in early signaling
to CREB, whereas Ras/ERK is involved in the late phase of CREB
phosphorylation (28). In our work, the MEK inhibitor
U-0126 was able to importantly inhibit P-CREB levels after
depolarization, but other pathways could be involved as well. We have
determined that inhibition of the CaMKs did not affect P-CREB levels;
however, p38 inhibition partially decreased CREB phosphorylation.
Interestingly, whereas fos/jun upregulation could
be decreased both by MEK or p38 inhibitors, only c-fos was affected by the CAMK inhibitor KN-93. A scheme illustrating these findings is shown in Fig. 9.
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Signal transduction pathways can be highly complex, and the cross talk between pathways can take place at several levels from the membrane to the nucleus (12). Undoubtedly, our results present a partial view of all the signaling events taking place in the cell; the focus was placed on some pathways that have been shown to respond to either exercise in skeletal muscle or calcium in other excitable cells.
The evidence provided in this study does suggest an important role for activity-induced slow intracellular calcium increase and underlines the need for further studies on calcium-dependent early signaling mechanisms in skeletal muscle cells.
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ACKNOWLEDGEMENTS |
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We thank Manuel Estrada for help with image acquisition and analysis.
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FOOTNOTES |
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This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico 8980010 and Fondo de Investigación Avanzada en Areas Prioritarias 15010006.
Address for reprint requests and other correspondence: M. A. Carrasco, Instituto de Ciencias Biomédicas and Centro FONDAP de Estudios Moleculares de la Célula, Facultad de Medicina, Universidad de Chile, Santiago 6530499, casilla 70005, Santiago, Chile (E-mail: mcarras{at}machi.med.uchile.cl).
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 15, 2003;10.1152/ajpcell.00117.2002
Received 13 March 2002; accepted in final form 13 January 2003.
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