Depolarization-induced slow calcium transients activate early genes in skeletal muscle cells

Maria Angélica Carrasco, Nora Riveros, Juan Ríos, Marioly Müller, Francisco Torres, Jorge Pineda, Soledad Lantadilla, and Enrique Jaimovich

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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


    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 epsilon -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.


    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). [alpha 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 DH5alpha cells. Purified plasmids were digested with EcoRI, and the products were labeled with [alpha -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).

egr-1 message was detected by using a 578-bp fragment prepared by RT-PCR from total RNA extracted from rat skeletal muscle cells in culture. The primers used were 5'-AGCTTCCGCCGCCGCAAGAT-3' and 5'-TAACGAGAAGGCGCTGGTGGAG-3'. Product was labeled as described for other probes.

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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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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Fig. 1.   K+ depolarization increases messenger RNAs of early genes c-fos, c-jun, and egr-1. Total RNAs were isolated from rat myotubes in primary culture depolarized by K+ at the times indicated, and c-fos, c-jun, and egr-1 mRNA levels were analyzed by Northern blot. The results were normalized to GAPDH expression and presented as percentages of untreated control cells (means ± SE); 5-10 experiments were analyzed for c-fos and c-jun, and 3 experiments were analyzed for egr-1. *P < 0.05, **P < 0.001, compared with untreated controls (ANOVA followed by Dunnett's multiple comparison post test).

An alternative technique used in this work to analyze early gene expression, semiquantitative RT-PCR, gave results similar to Northern blotting. An example for c-fos and c-jun is shown in Fig. 2A; the kinetics of mRNA increase were clearly demonstrated in this case. Values for eight experiments using RT-PCR and nine experiments using Northern blotting did not differ significantly. The values (means ± SE) for each gene, expressed as percentages with reference to a normalized 100% control, were, for c-fos, 230.5 ± 17.15% (n = 8) using RT-PCR and 196.7 ± 26.3% (n = 9) using Northern blotting; for c-jun, 215.5 ± 14.55% (n = 8) using RT-PCR and 183.9 ± 12.8% (n = 9) using Northern blotting; and for egr-1, 216.8 ± 9.89% (n = 8) using RT-PCR and 237.3 ± 49.76% (n = 3) using Northern blotting.


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Fig. 2.   A: RT-PCR analysis of c-fos and c-jun mRNA levels. Results from a single experiment are shown to illustrate the mRNA kinetics following K+-induced depolarization. This technique was used for results shown and quantified in B and in Figs. 4 and 8. B: the increase in c-fos, c-jun, and egr-1 mRNA levels obtained after high K+-induced depolarization is equivalent with 2 different protocols. Myotubes were either incubated in high K+ for different times and collected for analysis, or exposed to high K+ for only 1 min, changed to resting condition medium, and collected for analysis at variable times. RT-PCR analysis of mRNA was performed. With both protocols, the maximal stimulation was obtained at 15 min (only time shown).

In rat myotubes in primary culture, changing the K+ concentration from 4.7 to either 47 or 84 mM resulted in depolarization, with a change in membrane potential from -42 mV to about -12 or +1 mV, respectively, inducing a similar calcium increase (13). Under these conditions, the depolarization-induced increase in intracellular calcium lasts for tens of seconds and then calcium levels return to basal (13, 14). According to these observations, we have compared the results obtained with two protocols that differ in the exposure time to elevated K+. In one protocol, myotubes were incubated in high-K+ medium for just 1 min, re-fed with resting condition medium, and collected for analysis at various times. The other protocol consisted of continuous exposure of the rat myotubes to the depolarizing K+ concentration; that is, after the initial depolarization-induced calcium release, the plasma membrane was kept depolarized for the duration of the experiment. The results obtained with both protocols at the 15 min phosphorylation peak were equivalent and are shown in Fig. 2B.

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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Fig. 3.   BAPTA-AM inhibits the depolarization-induced slow calcium transient and ERK and cAMP-response element-binding protein (CREB) phosphorylation. Relative fluorescence intensity from fluo 3-loaded myotubes is shown. Fluorescence images of a rat myotube loaded with fluo 3 were obtained as described in MATERIALS AND METHODS. A region of the cell was selected, and fluorescence intensity was quantified using previously described software (7). High K+-containing solution (47 mM) was perfused when indicated (arrow). A: myotube in control conditions; images were acquired every 232 ms. At least 2 major components are evident in the fluorescence signal, a fast signal [associated with excitation-contraction (E-C) coupling] and a slow one linked to cytoplasmic and nuclear calcium increases. B: myotubes incubated and treated in the same conditions described for A but with Oregon green-BAPTA used as a calcium indicator. Upon K+ depolarization, only a fast calcium transient is present in the records for 2 different cells, shown as open and closed circles. C: fluo 3-loaded myotubes preincubated for 30 min in the presence of 50 µM BAPTA-AM (records for 2 different cells shown as open and closed circles) and depolarized in the presence of the chelator. Images were acquired every 500 ms. Note that in both cases the slow component of the calcium signal was almost completely inhibited. D: rat myotubes were preincubated with 100 µM BAPTA-AM under resting conditions and depolarized by exposure to high K+ concentration for the times indicated. Western blots for both phosphorylated ERKs and CREB, as well as their respective loading controls, are shown. Similar results were obtained in 2 independent experiments.

To directly relate the slow calcium transient to regulation of early gene expression, we conducted experiments in cells loaded with BAPTA-AM in the same conditions as those depicted in Fig. 3. A significant inhibition of the enhancement in mRNA expression after K+ depolarization was observed in BAPTA-AM-incubated cells compared with untreated controls for all three early genes tested (Fig. 4A). The same type of inhibition was obtained when cells were treated with the IP3 inhibitor 2-APB (Fig. 4B), a drug already shown to be capable of inhibiting the slow calcium transient and both ERK and CREB phosphorylation (21). Xestospongin C, a toxin reported to block IP3 receptors (7), also reduced enhancement of c-fos mRNA by 60% and c-jun mRNA by 67% (not shown).


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Fig. 4.   Inhibition of the depolarization-induced slow calcium transient diminishes the increase in early gene mRNA levels. Myotubes were pretreated either with vehicle or 100 µM BAPTA-AM (A) or 50 µM 2-aminoethoxydiphenyl borate (2-APB; B) and depolarized with high K+. The mRNA levels were determined by semiquantitative RT-PCR. Results obtained at 15 min of exposure to high K+ in the control or experimental series were expressed as percentages of the corresponding control (no depolarization). Values are means ± SE. *P < 0.05, **P < 0.001, compared with the increase obtained in control conditions (Student's t-test for paired data).

In muscle cells treated with 10 µM ryanodine, a condition that inhibits the fast calcium transient, sparing the slow calcium signal, there was no significant change on the increase in ERK or CREB phosphorylation induced by depolarization. P-CREB mean values from two experiments performed with myotubes depolarized in the absence or presence of ryanodine, expressed as percentages with reference to a normalized 100% control, were 301.0 and 289.0% after 5 min of depolarization and 297.0 and 243.0% after 10 min of depolarization, respectively. P-ERK percentages (means ± SE, n = 3) in the absence or presence of ryanodine for myotubes exposed to 5 min of depolarization were 196.3 ± 21.7 and 197.0 ± 24.9% for P-ERK1, and 168.0 ± 11.1 and 166.7 ± 8.0% for P-ERK2.

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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Fig. 5.   Absence of extracellular calcium inhibits neither ERK or CREB phosphorylation nor the increase in c-jun or c-fos mRNA levels. Depolarization was performed in either the presence of 3 mM calcium or the absence of calcium plus the addition of 0.5 mM EGTA. A: Western blot of phosphorylated ERK1/2 and total ERK2. These results are representative of 3 independent experiments. B: CREB phosphorylation and total CREB as control for loading. Similar results were obtained in 3 experiments. C: myotubes were depolarized in either the presence (+Ca) or absence (-Ca) of extracellular calcium. Values (means ± SE, n = 3) represent the maximal induction of c-fos and c-jun mRNA levels obtained by Northern blot.

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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Fig. 6.   Caffeine stimulates ERK and CREB phosphorylation and induces an increase in c-fos and c-jun mRNAs. A: Western blot of phosphorylated ERKs from myotubes incubated with 10 mM caffeine. Total ERK2 is shown as control for loading. Results are representative of 3 experiments. B: Western blot of phosphorylated and total CREB. A representative experiment is shown. C: myotubes were either depolarized with K+ or treated with 10 mM caffeine. Northern blots were performed with samples obtained from 3 independent experiments. The results were obtained with either depolarization or caffeine at 15 min of stimulation and are presented as percentages (means ± SE) of the untreated control.

In differentiated myotubes, caffeine generates long-lasting, massive calcium transients that give rise to propagated, slow calcium waves (9). To interpret the effects of caffeine, two questions must be answered. First, considering that caffeine normally acts through activation of ryanodine receptors, is the calcium pool involved in slow calcium signals also being depleted by caffeine? When myotubes were incubated with 1 µM thapsigargin, a slow calcium transient, product of calcium pump inhibition, could be seen in cells incubated in the absence of extracellular calcium (Fig. 7A). Under these conditions, caffeine did not elicit further calcium increase (Fig. 7A). Thapsigargin alone did induce an increase in ERK and CREB phosphorylation, but no further increase was evident upon treatment with both thapsigargin and caffeine (Fig. 7, B and C). In thapsigargin-treated cells, high K+-induced depolarization did not elicit a calcium transient, and neither induced any increase in P-ERK or P-CREB levels (not shown).


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Fig. 7.   Intracellular calcium store depletion by thapsigargin (TPG) prevents stimulation of ERK and CREB phosphorylation by caffeine. A: relative fluorescence intensity from fluo 3-loaded myotubes was determined as described in MATERIALS AND METHODS. The response to the sarco(endo)plasmic reticulum Ca2+-ATPase pump inhibitor TPG was determined in a medium containing no calcium plus the addition of 0.5 mM EGTA. Note the calcium transient elicited by TPG and the absence of calcium increase upon addition of caffeine. When external (10 mM) Ca2+ was added, a large calcium influx was apparent. B and C: rat myotubes were preincubated for 5 min in the absence of external calcium (medium containing 0.5 mM EGTA). TPG (1 µM) was added for 4 min, followed by caffeine or vehicle for 1 min. The effect of caffeine was assessed by incubating the myotubes for 1 min, also in resting medium containing no calcium. ERK phosphorylation (B) or P-CREB levels (C) were analyzed as described in MATERIALS AND METHODS. Data are reported as ERK phosphorylation or P-CREB immunoreactivity represented as average fold increases (means ± SE, n = 3) over basal level.

Second, does caffeine or calcium released by caffeine also activate IP3 receptors? We stimulated calcium release with caffeine in the presence of 2-APB, considering that in our system this compound inhibited the slow calcium component that is mediated by IP3 receptor activation. P-CREB levels were examined in myotubes exposed to 10 mM caffeine for 1 min in the presence of 50 µM 2-APB (myotubes were preincubated for 30 min with either 2-APB or vehicle). The values (means ± SE), expressed as percentages with reference to a normalized 100% control, were 258.3 ± 27.4% in myotubes exposed to caffeine, decreasing to 135.3 ± 8.9% in myotubes exposed to caffeine and 2-APB (n = 3, P < 0.05).

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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Fig. 8.   Inhibition of the MEK/ERK pathway impairs the increase in CREB phosphorylation and the increases in c-fos, c-jun, and egr-1 mRNA levels induced by depolarization. Rat myotubes were pretreated with vehicle (DMSO) or with 10 µM U-0126 for 30 min and depolarized in the absence or presence of U-0126. A: Western blot of phosphorylated ERK1/2 and of total ERK2. B: Western blot of phosphorylated and total CREB. Similar results were obtained in 4 experiments. C: the level of c-fos, c-jun, and egr-1 mRNAs was assessed by RT-PCR in 3 independent experiments. The maximal stimulation with depolarization, obtained at 15 min of exposure to high-K+ medium, is compared with that obtained in the presence of the MEK inhibitor. *P < 0.05, **P < 0.001.

SB-203580, a p38 MAPK inhibitor, was also tested. SB-203580 (10 µM) decreased P-CREB levels and c-fos and c-jun mRNA levels. P-CREB values (means ± SE) from three experiments performed with myotubes depolarized for 5 min in the absence or presence of the inhibitor, expressed as percentages of a normalized 100% control, were 260.5 ± 25.3 and 166.9 ± 21.41%, respectively (P < 0.05). The value (mean ± SE), expressed as a percentage of a normalized 100% control, was 234.3 ± 15.3% (n = 3) for c-fos and was reduced to 159.0 ± 15.0% (P < 0.001) in the presence of the inhibitor. c-jun mRNA levels changed from 213.0 ± 10.1% under control conditions (n = 3) to 162.0 ± 6.9% (P < 0.01) in the presence of the p38 MAPK inhibitor.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 9.   Scheme depicting pathways that link calcium increase induced by depolarization to activation of the early genes c-fos and c-jun. The scheme is based on results from the present study (except dotted line). Activation of ERK1/2, CREB, c-fos, and c-jun by depolarization requires calcium (slow component). Pharmacological inhibition of ERKs and p38 results in decreased activation of CREB and early genes. In the case of pharmacological inhibition of CaMKs, only a partial effect on c-fos could be detected. Results on egr-1 were not included because the effects of CaMKs and p38 inhibition were not assessed.

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.


    ACKNOWLEDGEMENTS

We thank Manuel Estrada for help with image acquisition and analysis.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abu-Shakra, S, Cole AJ, and Drachman DB. Nerve stimulation and denervation induce differential patterns of immediate early gene mRNA expression in skeletal muscle. Mol Brain Res 18: 216-220, 1993[ISI][Medline].

2.   Abu-Shakra, S, Cole AJ, and Drachman DB. Cholinergic stimulation of skeletal muscle cells induces rapid immediate early gene expression: role of intracellular calcium. Brain Res Mol Brain Res 26: 55-60, 1994[ISI][Medline].

3.   Adams, L, and Goldman D. Role for calcium from the sarcoplasmic reticulum in coupling muscle activity to nicotinic acetylcholine receptor gene expression in rat. J Neurobiol 35: 245-257, 1998[ISI][Medline].

4.   Araya, R, Liberona JL, Riveros N, Powell J, Carrasco MA, and Jaimovich E. Dihydropyridine receptors as voltage sensors for the depolarization-evoked, IP3R-mediated, slow calcium signal in skeletal muscle cells. J Gen Physiol 121: 3-16, 2003[Abstract/Free Full Text].

5.   Aronson, D, Dufresne SD, and Goodyear LJ. Contractile activity stimulates the c-jun NH2-terminal kinase pathway in rat skeletal muscle. J Biol Chem 272: 25636-25640, 1997[Abstract/Free Full Text].

6.   Chomczynski, P, and Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

7.   Estrada, M, Cardenas C, Liberona JL, Carrasco MA, Mignery G, Allen PD, and Jaimovich E. Calcium transients in 1B5 myotubes lacking ryanodine receptors are related to inositol trisphosphate receptors. J Biol Chem 276: 22868-22874, 2001[Abstract/Free Full Text].

8.   Favata, M, Hiruichi KY, Manos EJ, Daulerio AJ, Stradley DA, Fesser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, and Trzaskos JM. Identification of a novel inhibitor of mitogen-activated protein kinase. J Biol Chem 273: 18623-18632, 1998[Abstract/Free Full Text].

9.   Flucher, BE, and Andrews SB. Characterization of spontaneous and action potential-induced calcium transients in developing myotubes in vitro. Cell Motil Cytoskeleton 25: 143-157, 1993[ISI][Medline].

10.   Freyssenet, D, Di Carlo M, and Hood DA. Calcium-dependent regulation of cytochrome c gene expression in skeletal muscle cells. J Biol Chem 274: 9305-9311, 1999[Abstract/Free Full Text].

11.   Hamaya, Y, Takeda T, Dohi S, Nakashima S, and Nozawa Y. The effects of pentobarbital, isoflurane, and propofol on immediate-early gene expression in the vital organs of the rat. Anesth Analg 90: 1177-1183, 2000[Abstract/Free Full Text].

12.   Hunter, T. Signaling---2000 and beyond. Cell 100: 113-127, 2000[ISI][Medline].

13.   Jaimovich, E, Reyes R, Liberona JL, and Powell JA. IP3 receptors, IP3 transients, and nucleus-associated Ca2+ signals in cultured skeletal muscle. Am J Physiol Cell Physiol 278: C998-C1010, 2000[Abstract/Free Full Text].

14.   Jaimovich, E, and Rojas E. Intracellular Ca2+ transients induced by high external K+ and tetracaine in cultured rat myotubes. Cell Calcium 15: 356-368, 1994[ISI][Medline].

15.   Kubis, HP, Haller EA, Wetzel P, and Gros G. Adult fast myosin pattern and Ca2+-induced slow myosin pattern in primary skeletal muscle culture. Proc Natl Acad Sci USA 94: 4205-4210, 1997[Abstract/Free Full Text].

16.   Michel, JB, Ordway GA, Richardson JA, and Williams RS. Biphasic induction of immediate early gene expression accompanies activity-dependent angiogenesis and myofiber remodeling of rabbit skeletal muscle. J Clin Invest 94: 277-285, 1994[ISI][Medline].

17.   Osbaldeston, NJ, Lee DM, Cox VM, Hesketh JE, Morrison JFJ, Blair GE, and Goldspink DF. The temporal and cellular expression of c-fos and c-jun in mechanically stimulated rabbit latissimus dorsi muscle. Biochem J 308: 465-471, 1995[ISI][Medline].

18.   Osman, AA, Pendergrass JK, Maezono K, Cusi K, Pratipanawatr T, and Mandarino L. Regulation of MAP kinase pathway activity in vivo in human skeletal muscle. Am J Physiol Endocrinol Metab 278: E992-E999, 2000[Abstract/Free Full Text].

19.   Pette, D. Training effects on the contractile apparatus. Acta Physiol Scand 162: 367-376, 1998[ISI][Medline].

20.   Pilegaard, H, Ordway GA, Saltin B, and Neufer PD. Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab 279: E806-E814, 2000[Abstract/Free Full Text].

21.   Powell, JA, Carrasco MA, Adams DS, Drouet B, Rios J, Muller M, Estrada M, and Jaimovich E. IP3 receptor function and localization in myotubes: an unexplored Ca2+ signaling pathway in skeletal muscle. J Cell Sci 114: 3673-3683, 2001[ISI][Medline].

22.   Puntschart, A, Wey E, Jostardndt K, Vogt M, Wittwer M, Widmer HR, Hoppeler H, and Billeter R. Expression of fos and jun genes in human skeletal muscle after exercise. Am J Physiol Cell Physiol 274: C129-C137, 1998[Abstract/Free Full Text].

23.   Rolli, M, Kottlyarov A, Sakamoto KM, Gaestel M, and Neininger A. Stress-induced stimulation of early growth response gene-1 by p38/stress-activated protein kinase 2 is mediated by a cAMP-responsive promoter element in a MAPKAP kinase 2-independent manner. J Biol Chem 274: 19559-19564, 1999[Abstract/Free Full Text].

24.   Ryder, JW, Fahlman R, Wallberg-Henriksson H, Alessi DR, Krook A, and Zierath JR. Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. J Biol Chem 275: 1457-1462, 2000[Abstract/Free Full Text].

25.   Sakamoto, K, and Goodyear LJ. Invited review: intracellular signaling in contracting skeletal muscle. J Appl Physiol 93: 369-383, 2002[Abstract/Free Full Text].

26.   Sheng, M, Mc Fadden G, and Greenberg ME. Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB. Neuron 4: 571-582, 1990[ISI][Medline].

27.   Spina, RJ, Chi MM, Hopkins MG, Nemeth PM, Lowry OH, and Holloszy JO. Mitochondrial enzymes increase in muscle in response to 7-10 days of cycle exercise. J Appl Physiol 80: 2250-2254, 1996[Abstract/Free Full Text].

28.   Wu, GY, Deisseroth K, and Tsien RW. Activity-dependent CREB phosphorylation: convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 98: 2808-2813, 2001[Abstract/Free Full Text].


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