Ca2+ transients activate calcineurin/NFATc1 and initiate fast-to-slow transformation in a primary skeletal muscle culture

Hans-Peter Kubis, Nina Hanke, Renate J. Scheibe, Joachim D. Meissner, and Gerolf Gros

Zentrum Physiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany

Submitted 20 August 2002 ; accepted in final form 11 February 2003


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The calcineurin-mediated signal transduction via nuclear factor of activated T cells (NFATc1) is involved in upregulating slow myosin heavy chain (MHC) gene expression during fast-to-slow transformation of skeletal muscle cells. This study aims to investigate the Ca2+ signal necessary to activate the calcineurin-NFATc1 cascade in skeletal muscle. Electrostimulation of primary myocytes from rabbit for 24 h induced a distinct fast-to-slow transformation at the MHC mRNA level and a full activation of the calcineurin-NFATc1 pathway, although resting Ca2+ concentration ([Ca2+]i) remained unaltered at 70 nM. During activation, the calcium transients of these myocytes reach a peak concentration of ~500 nM. Although 70 nM [Ca2+]i does not activate calcineurin-NFAT, we show by the use of Ca2+ ionophore that the system is fully activated when [Ca2+]i is >=150 nM in a sustained manner. We conclude that the calcineurin signal transduction pathway and the slow MHC gene in cultured skeletal muscle cells are activated by repetition of the rapid high-amplitude calcium transients that are associated with excitation-contraction coupling rather than by a sustained elevation of resting Ca2+ concentration.

muscle plasticity; NFATc1; resting calcium concentration


FAST-TO-SLOW TRANSFORMATION is the adaptational response of adult skeletal muscle cells to permanent work load. During this process, "fast" muscle cells change their contractile and metabolic properties to those characteristic for "slow" muscle cells. Important aspects of this process are a switch from fast to slow myosin isoforms and from enzymes of anaerobic metabolism to those of oxidative metabolism (17). The adaptational process is accomplished by the induction of slow muscle gene expression and the repression of fast muscle gene expression. Extending preceding studies of Chin et al. (4), we have shown (14) that the signal transduction for the induction of the slow myosin heavy chain (MHC) I gene during fast-to-slow transformation is mediated by the calcineurin-nuclear factor of activated T cells (NFATc1) pathway. An unanswered question in the regulation of this signal transduction pathway during fast-to-slow transformation of skeletal muscle is the following: what type of calcium signal is responsible for the activation of the calcineurin-NFATc1 pathway? For T cells, it has been shown that the intracellular Ca2+ concentration ([Ca2+]i) must increase in a sustained fashion to activate the calcineurin-NFAT pathway (6). Sreter et al. (19) have shown that chronic electrostimulation of rabbit skeletal muscle in vivo produces a lasting increase in [Ca2+]i along with fast-to-slow transformation, and it appeared possible that a long-term rise in resting [Ca2+]i constitutes the signal that activates the calcineurin-NFATc1 pathway in muscle. Such a mechanism has indeed recently been postulated by Olson and Williams (17). In the present study, we use a primary skeletal muscle culture that we have described previously (10) 1) to develop the properties of an adult fast muscle in culture, 2) to be transformed into myotubes with slow properties when [Ca2+]i is raised by Ca2+ ionophore for an extended period (10), and 3) to be transformed in a similar fashion by in vitro electrostimulation (11, 14). With this culture system, we demonstrate here that electrostimulation for 24 h leads to a sustained nuclear localization of NFATc1 in skeletal muscle cells and to a fast-to-slow transformation at the myosin mRNA level, whereas the resting [Ca2+]i is not changed. Elevation of resting [Ca2+]i during electrostimulation of the myocytes occurs after several days but does not appear to contribute to calcineurinNFATc1 activation. It is concluded that suitable repetition of the rapid, high-amplitude Ca2+ transients associated with muscle stimulation, activation, and contraction is able to activate the calcineurin-NFATc1 system and, subsequently, induce upregulation of MHC I along with a fast-to-slow transformation of the skeletal muscle cells in primary culture.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. Myoblasts from the hindlimbs of newborn New Zealand White rabbits were isolated and cultured on microcarriers (CultiSpher-GL; Percell Biolytica, Astorp, Sweden) in suspension as described previously (10, 11). After between 14 and 28 days, myotubes were collected from microcarriers as reported (10). Some experiments were carried out with Xestospongin, which was supplied by Calbiochem (San Diego, CA), or with calcium ionophore A-23187 from Sigma-Aldrich. All other chemicals were purchased either from Invitrogen (Karlsruhe, Germany), Sigma-Aldrich, or Merck (Darmstadt, Germany).

Animal experiments were carried out according to the guidelines of the local Animal Care Committee (Bezirksregierung Hannover).

Electrostimulation. After the myotubes had been growing on microcarriers for 14 days under resting conditions, some of them were electrostimulated in the culture flasks with a pattern consisting of cycles of 1 Hz for 15 min followed by a 30-min pause. This cycle was continuously repeated for several days. Voltage applied at the platinum electrodes was chosen to achieve maximal contraction amplitudes of the myotubes [details as reported before (11)]. The polarity of the platinum electrodes was changed every second to avoid electrophoretic separation of the serum proteins in the cell culture medium. The medium of stimulated cultures contained 1 mM N-acetylcysteine as a radical scavenger.

Northern blot analysis. Total RNA isolation, RNA separation on 1.2% agarose-formaldehyde gels, hybridization with MHC cDNA probes, and autoradiography were done as described previously (15). For detection of slow MHC I mRNA, the 3'-terminal 450-bp HinfI fragment from rabbit MHC I cDNA (1) was used. Fast MHC IId mRNA was estimated with the 3'-terminal PstI fragment of the rabbit MHC IId cDNA (13) that is specific for fast MHC isoform IId (20). For normalization of Northern blots, 18S rRNA was detected with the 5.8-kb HindIII fragment of 18S rDNA (9) and {beta}-actin mRNA was hybridized with the 1.8-kb BamHI fragment of human {beta}-actin cDNA (16). Autoradiography exposure times were 5 days, 3 days, and 8 h, respectively, for MHC mRNA, {beta}-actin mRNA, and 18S rRNA.

NFATc1 immunofluorescence. Myotubes grown on microcarriers were detached from the carriers by incubation with Accutase (PAA, Austria) for 60 min. After sedimentation of the microcarriers, the myotubes in the supernatant were centrifuged at 330 g for 5 min and resuspended in Dulbecco's modified Eagle's medium (DMEM)/10% neonatal calf serum. Cells were then seeded on glass coverslips and cultured for two days. It was verified (data not shown) by immunofluorescence using monoclonal antibodies (Accurate Chem. and Sci.) that during these two days, the myotubes retained the expression pattern of fast and slow MHC that they had developed during the preceding culture on microcarriers. Electrostimulation of myotubes on coverslips with 1 to 3 stimulation cycles [= (1, 2, 3) x 45 min] was performed in 24-well plates, whereas stimulation with cycles repeated for 24 h and 40 h was performed in petri dishes (diameter 6 cm). Again, platinum electrodes were used for electrostimulation. Settings of the stimulator were chosen by microscopical control of myotube contractions. After the stimulation protocol was finished, cells were washed and fixed with 3% paraformaldehyde (PFA) or 100% methanol and permeabilized in 0.1% Triton X-100 as described previously (14). Incubation with goat anti-NFATc1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA; this antibody reacts with both the phosphorylated and the dephosphorylated form of NFATc1) for 30 min was followed by incubation with fluorescein isothiocyanate (FITC)-labeled anti-goat IgG secondary antibody (Santa Cruz Biotechnology). Immunostained myotubes were photographed in an inverted fluorescence microscope (Leica, Wetzlar, Germany) at a magnification of x400 (objective x40, NA 1.25). For quantification of the subcellular localization of NFATc1, a total of at least 300 cells were inspected to generate one of the columns in Figs. 2, 3, and 4. Each cell was classified as exhibiting nuclear staining only, cytoplasmic staining only, or both nuclear and cytoplasmic staining.



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Fig. 2. Single cell Ca2+ transients of primary skeletal muscle cells. A: averaged transient of 30 transients of single myocytes during electrostimulation. B: series of single cell transients electrostimulated with 1 Hz.

 


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Fig. 3. Nuclear import of NFATc1 (nuclear factor of activated T cells) in myocytes under treatment with various concentrations of Ca2+ ionophore A-23187. A: fluorescence image of purely cytoplasmic (left), simultaneously cytoplasmic and nuclear (center), and exclusively nuclear NFATc1 staining (right). Bar = 10 µm. B: quantification of NFATc1 nuclear import with ionophore concentrations between 0.5 and 10 x 108 M.

 


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Fig. 4. NFATc1 subcellular localization after electrostimulation for various times. Percentage of NFATc1-stained cells under control conditions (bar A), after three 45-min cycles, each consisting of 15-min stimulation at 1 Hz followed by 30 min pause (bar B), after 24-h continuous repetition of the same cycle (bar C), and after 40-h continuous repetition of this cycle (bar D). Black columns indicate the percentage of cells exhibiting exclusively nuclear NFATc1 staining, gray columns give the percentage of cells that have both cytoplasmic and nuclear staining, and white columns indicate the percentage of cells that exhibit cytoplasmic staining only. The cells were fixated after the 30-min pause that followed the last 15-min stimulation interval. At least 300 cells were counted for each condition (bars AD). Staining was achieved with a primary NFATc1 antibody and a secondary FITC-labeled anti-goat IgG antibody.

 

Determinations of [Ca2+]i. Resting [Ca2+]i and Ca2+ transients were measured with the fluorescent Ca2+-sensitive indicator fura 2 (Molecular Probes, Leiden, Netherlands). Myotubes cultured as described in NFATc1 immunofluorescence were washed three times with Ringer solution (145 mM NaCl, 2.5 mM KCl, 1 mM MgSO4, 10 mM HEPES, 10 mM glucose, and 1.8 mM CaCl2, pH 7.4) and incubated with Ringer solution containing 2 µM fura 2-acetoxymethyl ester (AM) for 30 min at 37°C. After fura 2 loading, the solution was exchanged for Ringer solution and cells were washed again twice and then incubated at 37°C for 40 min to allow further deesterification of fura 2-AM. The experimental setup consisted of an inverted microscope (Axiovert 135 TV, Zeiss, Jena, Germany) with the objective LD Achroplan 40 x 0.6 (Zeiss, Jena, Germany) connected to a photomultiplier (PMT 01–710, PTI) measuring fluorescence emission at 510 nm. Excitation wavelengths were 340 and 380 nm produced by a Xenon lamp (UXL-75XE, Ushio). To estimate resting [Ca2+]i, fluorescent light was recorded from an area on a glass coverslip that contained an average number of 30 myocytes. Measurement of Ca2+ transients was performed on single cells. Background fluorescence was determined before fura 2 loading at each excitation wavelength over an identical number of myocytes and was substracted from subsequent fluorescence measurements. The value of Rmax, i.e., the fura 2 fluorescence ratio F340/F380 at full calcium binding of the dye, was determined by incubating the cells in Ringer solution with saponin [0.002% (wt/vol), 2–5 min according to Carroll et al. (3)], and a CaCl2 concentration of 2.5 mM. Incubation time was optimized by continuous ratio measurement after addition of saponin. Rmax was found to be 11.4 ± 1.4 (n = 5). Rmin, the fluorescence ratio F340/F380 at zero calcium binding in vivo, was estimated by incubating the cells in Ca2+-free Ringer solution with 0.5 mM EGTA for 1 h at 37°C, followed by an incubation for 30 min in the same buffer with 10 µM ionomycin. Rmin was found to be 0.67 ± 0.1 (n = 37). The fura 2/Ca2+ dissociation constant Kd was determined in vitro as described (8) and was found to be Kd = 166 ± 6 nM and Sf2/Sb2 = 4.44 (Sf2: n = 37; Sb2: n = 5). The measured fluorescence ratios R were used with Rmax, Rmin, Kd, and Sf2/Sb2 to calculate the intracellular Ca2+ concentrations as published by Grynkiewicz et al. (8): [Ca2+] = Kd (Sf2/Sb2) (R –Rmin)/(Rmax – R). Fura 2 calibration was performed daily.


    RESULTS
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[Ca2+]i during electrostimulation and under treatment with Ca2+ ionophore. Table 1 shows the effect of 24 h of electrostimulation on resting [Ca2+]i levels. The stimulation pattern consisted of cycles (15-min stimulation at 1 Hz followed by a pause of 30 min of rest) that were continuously repeated. It is apparent that this stimulation pattern after having been applied for 24 h has no effect on resting [Ca2+]i. Figure 1 illustrates, however, that after prolonged electrostimulation with the same pattern for 1 or 2 wk, myotubes do develop resting [Ca2+]i levels that are elevated above control levels by about 50%. Figure 1 also shows that under control conditions, a fall occurs in [Ca2+]i with increasing age of the myotubes, whereas in stimulated myocytes no change over the original level is observed. We conclude that continued electrostimulation results in a lasting elevation of resting [Ca2+] over control levels, but it does so only after having been applied for more than 24 h.


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Table 1. Resting [Ca2+]i after 24 h of stimulation

 


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Fig. 1. Resting intracellular Ca2+ concentration ([Ca2+]i) in myocytes as a function of time of culture. {bullet}, Stimulated myocytes; {square}, unstimulated myocytes of identical age. Stimulation pattern: continuous repetition of a cycle comprising 15 min of electrostimulation at 1 Hz and a subsequent pause of 30 min. The arrow depicts the start of electrostimulation. *Significant: ANOVA (P < 0.05); symbols represent mean values ± SD for n = 6–10 experiments.

 

Figure 2A shows a Ca2+ transient obtained by superimposing 30 transients recorded from single cells. This averaged transient possesses a peak Ca2+ concentration of ~500 nM and a 75% decay time of 250 ms. An example of a measurement of a series of single cell transients during electrostimulation of the myocytes with 1 Hz is given in Fig. 2B. It is apparent in Fig. 2B that Ca2+ levels return to base level between stimuli and that no significant change in peak Ca2+ occurs during the stimulation train for up to 30 min. Between stimulation intervals, the resting Ca2+ remained at control levels for at least 24 h, as evident from the measurements reported above.

Table 2 addresses the question whether the myoinositol 1,4,5-trisphosphate (IP3) receptor Ca2+ channel of the sarcoplasmic reticulum affects resting Ca2+ levels in myotubes with and without electrostimulation. Blocking the channel with Xestospongin reduces resting Ca2+ levels under control conditions from about 60 to 40 nM, suggesting that a continuous flux of Ca2+ from the sarcoplasmic reticulum via this channel contributes to the Ca2+ level in the sarcoplasm under resting conditions. However, Xestospongin does not alter the lack of an effect of 20 min of continuous electrostimulation (1 Hz) on the resting Ca2+ level.


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Table 2. Influence of Xestospongin on resting [Ca2+]i in myocytes

 

Table 3 gives the results of a titration of the intracellular resting Ca2+ level of the myotubes with Ca2+ ionophore A-23187 that had been added to the medium 4 h before Ca2+ measurement. Age of the culture was 16 days, and [Ca2+]i rose from a control level of about 60 to about 120 nM with 5 · 108 M ionophore and to ~150 nM with 10 · 108 M ionophore. It may be noted that after prolonged exposure of the myotubes to the ionophore, e.g., for 24 h, Ca2+ levels began to decline somewhat compared with the levels of Table 3, probably due to the onset of compensatory mechanisms (data not shown). The titration with ionophore was used to study the dependence of NFATc1 activation on the intracellular Ca2+ level (see Fig. 3).


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Table 3. [Ca2+]i after incubation with calcium ionophore A-23187 for 4 h

 

Nuclear translocation of NFATc1 by ionophore treatment and electrostimulation. Figure 3 demonstrates that NFATc1 translocation in the present system can be induced by a sustained increase in resting [Ca2+] as it is generated by the continuous presence of Ca2+ ionophore. Figure 3A shows the intracellular distribution of NFATc1 fluorescence with staining in the cytoplasm only and lack of staining in the nuclei (left) as it is seen in control conditions. Addition of Ca2+ ionophore results in an intermediate activation of NFATc1 with simultaneously nuclear and cytoplasmic staining (Fig. 3A, center) or a pronounced activation with exclusively nuclear staining (Fig. 3A, right). Graded increases in [Ca2+]i were induced by addition of various concentrations of Ca2+ ionophore A-23187 to the culture 4 h before fixation of the cells for immunofluorescence. It is apparent that A-23187 induces NFATc1 activation in a dose-dependent fashion, with 5 x 108 M ionophore producing an about 50% activation of the NFATc1 system. This ionophore concentration is seen in Table 3 to raise [Ca2+]i from 60 to 120 nM. Full activation of NFATc1 is achieved with 10 x 108 M ionophore and a resting intracellular Ca2+ level of 150 nM.

Figure 4 shows the effect of electrostimulation for various times on the activation and translocation of NFATc1 into the nucleus. The age of the myotubes studied was 23 days, and all cells were transferred onto coverslips after the third week. Cells of Fig, 4, bars B–D, were then exposed to electrostimulation either with three cycles (each cycle consisting of 15-min stimulation at 1 Hz and 30-min pause) (bar B) or with this same cycle repeated continuously for 24 h (bar C)orfor 40 h (bar D). The fixation of cells for NFATc1 localization was done 30 min after the end of the last stimulation interval in all cases (i.e., at the end of the last cycle). Whereas under resting conditions NFATc1 was almost exclusively cytoplasmic and thus inactive (Fig. 4, bar A), Fig. 4, bar B, shows that 30 min after only 3 stimulation intervals, 20% of the myotubes exhibited exclusively nuclear NFATc1 staining (this number amounts to about 60% when determined immediately after the third stimulation interval; see Ref. 11). This represents a significant activation of the calcineurin-NFAT pathway. After 24 h of continuous repetition of the cycles, the degree of activation is markedly enhanced (60% of the cells show exclusively nuclear staining; again, this number is considerably higher, almost 100%, immediately after the last stimulation interval). The increase in activation after prolonged repetition of the stimulation cycle is seen because 30 min after each stimulation interval, some NFATc1 remains in the nuclei (e.g., see Fig. 4, bar B) and is added to the NFATc1 that is translocated during the next stimulation interval. Thus, from cycle to cycle, the activation of NFATc1 increases until a plateau of near-maximal activation (close to 100% at the end of the stimulation interval, 60% after the 30-min pause) is reached. This plateau does not increase further between 24 and 40 h of continued repetition of the stimulation cycles (Fig. 4, bars C and D). Thus, although three subsequent cycles achieve a moderate activation only, long-term repetition of the same cycle produces a very high degree of activation. The stimulation-induced import of NFATc1 can be totally blocked by cyclosporin A as reported earlier (11, 14). This shows that calcineurin activation is an essential step for generating nuclear import of the transcription factor.

Figure 5 illustrates the effect of Xestospongin on nuclear translocation of NFATc1. All cultures were grown on microcarriers for 3 wk and were then transferred onto coverslips. Activation of NFATc1 in controls was low with and without Xestospongin, but immediately after 20 min of continuous electrostimulation NFATc1 it was almost fully activated, no matter whether Xestospongin was present or not. This suggests that the decrease in resting [Ca2+] that is affected by Xestospongin (Table 2) does not influence the calcineurin-dependent nuclear import of NFATc1.



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Fig. 5. Effect of Xestospongin on NFATc1 nuclear translocation. Percentage of NFATc1-stained cells without electrostimulation (0) and immediately after 20-min continuous electrostimulation with 1 Hz (20). Experiments were done either in the absence (–) or in the presence (+) of 1 µM Xestospongin that had been added 2 h before the stimulation was started. Note that fixation of cells was done immediately after the end of the stimulation interval. Hence, nuclear staining is more pronounced than in Fig. 3, where a 30-min pause was interposed between the end of the stimulation and fixation.

 

MHC pattern under electrostimulation. Figure 6 shows that 24 h of electrostimulation of the myotube culture with 15 min at 1 Hz/30-min pause cycles results in an incipient transformation of MHC mRNA from a "fast" toward a "slow" pattern with a clear increase in MHC I mRNA and a decrease in MHC IId mRNA. Thus 24 h stimulation 1) leads to the strong activation of NFATc1 as seen in Fig. 4, bar C; and 2) is sufficient to initiate the process of fast-to-slow transformation at the level of MHC mRNA expression. Continuation of electrostimulation with the same pattern for 14 days results in a complete switch to MHC I mRNA expression with loss of MHC IId mRNA (Fig. 7).



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Fig. 6. Effect of 24 h of electrostimulation on myosin mRNA pattern of myotubes in culture. Northern blots for slow myosin heavy chain (MHC) I mRNA. Lane 1, 14 day-old control culture; lane 2, same culture after electrostimulation for 24 h. Northern blots for fast MHC IId mRNA; lane 3, 14-day-old control culture; lane 4, same culture after electrostimulation for 24 h. Stimulation protocol as in Fig. 1. Normalization by probes for 18S rRNA and {beta}-actin mRNA.

 


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Fig. 7. Effect of 14 days of electrostimulation on myosin mRNA pattern. Northern blots for MHC I mRNA: lane 1, 28-day-old control culture; lane 2, culture with electrostimulation during the last 14 days. Northern blots for MHC IId mRNA: lane 3, 28-day-old control culture; lane 4, culture with electrostimulation during the last 14 days. Stimulation protocol as in Fig. 1. Normalization by probes for 18S rRNA and {beta}-actin mRNA.

 


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Ca2+ signaling under short-term electrostimulation (<=24 h). Electrostimulation of the present primary skeletal muscle culture for 24 h has not only caused a sustained translocation of NFATc1 into the nuclei (Fig. 4) but also resulted in a clearly elevated expression of the slow myosin chain MHC I mRNA and a decreased expression of the fast myosin chain MHC IId mRNA (Fig. 6). We have previously shown that the upregulation of MHC I under electrostimulation (as well as that under calcium ionophore treatment) can be suppressed by the calcineurin inhibitor cyclosporin A (11, 14). The observed change in myosin expression is a crucial part of fast-to-slow fiber-type transformation as it occurs in vivo under chronic electrostimulation (17) and has also been demonstrated to occur during a 14-day exposure of the present primary muscle culture to Ca2+ ionophore A-23187, which results in a sustained elevation of [Ca2+]i (10, 15).

The first step in the chain of events leading to calcineurin activation, NFATc1 nuclear import and switching on the MHC I gene, is a Ca2+ signal. This is confirmed by the observation of Kubis et al. (10) and Meissner et al. (15) that an ionophore-mediated permanent increase in resting [Ca2+]i indeed induces muscle transformation. It has been shown in T cells that an elevation of [Ca2+]i above 400 nM lasting for 2 h is required to commit T cells to activation (21). Dolmetsch et al. (6) have also shown that in T cells, a sustained [Ca2+]i elevation is required for activation of the calcineurin-NFATc1 pathway. In addition, Dolmetsch et al. (5) have observed that at identical time-averaged [Ca2+]i, oscillating Ca2+ levels increase NFAT-dependent gene expression in Jurkat T cells more efficiently than a sustained increase in [Ca2+]i.

Which kind of Ca2+ signal initiates myotube transformation under electrostimulation? Sreter et al. (19) have shown very early that chronic electrostimulation in vivo is associated with a long-term rise in [Ca2+]i. Olson and Williams (17) later postulated that such a sustained rise in [Ca2+]i may initiate the process of muscle transformation. In contrast with this view, the present data show that the level of resting [Ca2+]i of myotubes is not increased after 24 h of electrostimulation (Fig. 1, Table 1). Nevertheless, NFATc1 is almost fully translocated into the nuclei already after 20 min of stimulation (Fig. 5) and remains so after 24 and 40 h (Fig. 4), and an increase in MHC I mRNA and a decrease in MHC IId mRNA expression are detectable after 24 h. We conclude that the myotubes' calcineurin/NFATc1 system is able to sense the short intracellular Ca2+ transients that are associated with each electrical stimulation and myotube contraction. In our cell culture, these Ca2+ transients have a 75% decay time of about 250 ms and reach peak Ca2+ concentrations of ~500 nM. These short transients occur continuously at a frequency of 1 Hz during each 15-min stimulation interval. In myotubes, this pattern appears to almost fully activate NFATc1 and upregulate the transcription of slow myosin mRNA. This implies that increased contractile activity, when it occurs for sufficiently long periods in the present culture (see Ref. 11), can initiate fast-to-slow muscle transformation at the myosin level by virtue of the Ca2+ transients responsible for excitation contraction coupling. This result is noteworthy because it has not been shown before that brief Ca2+ transients can activate calcineurin/NFATc1. The Ca2+ oscillations that Dolmetsch et al. (6) found quite effective in Jurkat T cells had rather large amplitudes of between 200 and 1000 nM, occurred at low frequencies between 0.01 and 0.001 Hz, and lasted for ~0.5 to 1 min per spike.

The Ca2+ transients measured here have longer decay times than adult muscle fibers harvested by proteolytic digestion (3). The shorter duration of the Ca2+ transient in adult fibers may contribute to the failure of continuous stimulation at 1 Hz to induce nuclear import of green fluorescent protein (GFP)-NFAT construct, as reported by Liu et al. (12). In contrast, series of Ca2+ transients of longer duration, as seen in our culture system, are apparently able to activate calcineurin-induced NFATc1 import at 1 Hz (Ref. 11 and this study). This occurs although the Ca2+ transients in the present culture are not superimposed at 1 Hz, as shown in Fig. 2B. It may be noted that the brief transients in the present model can presumably have this effect because the peak Ca2+ concentration during each muscle activation reaches ~500 nM, which by far exceeds the level of 120 nM that is necessary to induce 50% activation of the calcineurin-NFATc1 system when a sustained elevation of [Ca2+]i is produced by ionophore.

It has been proposed that the IP3 receptor in skeletal muscle may serve to modulate [Ca2+]i (7). By the association of calcineurin with the IP3 receptor-FK506 binding protein (FKBP 12) complex (2), the IP3 Ca2+ channel may conceivably be involved in the activation of the calcineurin/NFAT cascade. The data of Table 2 show that indeed the IP3 inhibitor Xestospongin affects the resting [Ca2+]i, but Xestospongin does not affect the efficiency of electrostimulation to induce NFATc1 nuclear import. We conclude that the IP3 receptor is not a major factor in the activation of calcineurin/NFATc1-mediated slow MHC expression.

Ca2+ signaling under chronic electrostimulation (>24 h). Figure 1 shows that chronic electrostimulation lasting for >24 h does lead to a rise in intracellular resting Ca2+ levels above control levels. This elevated Ca2+ level persists for the observed time of 14 days of chronic electrostimulation (Fig. 1). During this time, NFATc1 remains mainly nuclear and the process of muscle transformation approaches completion as seen from the myosin mRNA pattern of Fig. 7. It may be noted that this time is also sufficient to approach completion of fast-to-slow transformation at the level of MHC protein expression (10).

What is the nature of the Ca2+ signal that maintains the transformation process over this extended period of time? The present results suggest that the resting [Ca2+] of ~75 nM, although increased by 50% over control values of ~50 nM, is not the responsible signal. The evidence for this comes from Fig. 3B in conjunction with Table 3. Figure 3B shows that exposure of the myotubes to 5 x 108 M A-23187 causes an approximately half-maximal activation of NFATc1. At this ionophore concentration, Table 3 shows the resting [Ca2+]i of the myotubes to be 120 nM, i.e., significantly higher than [Ca2+]i = 75 nM as established under chronic electrostimulation. To achieve nearly full activation of NFATc1, a resting [Ca2+]i of 151 nM at an ionophore concentration of 10 x 108 M is required (Fig. 3B, Table 3). Thus neither full nor half-maximal nuclear import of NFATc1 is accomplished by the resting Ca2+ levels seen in Fig. 1 during chronic electrostimulation. Again, the conclusion is that during chronic as well as short-term electrostimulation, the signal for activation of calcineurin/NFATc1 is constituted by the sequences of rapid Ca2+ transients associated with muscle activation and contraction. It is possible that the moderate but sustained increase in the level of resting [Ca2+]i mediates other events associated with fast-to-slow transformation that are not linked to calcineurin/NFATc1, such as metabolic adaptation of the muscle fiber (14).


    ACKNOWLEDGMENTS
 
We thank Drs. C. Brownson, R. Guntaka, P. K. Umeda, and A. Wittinghofer for generous gifts of plasmids. We thank E.-A. Haller and A. Jacobs for excellent technical assistance.

This work was supported by the Deutsche Forschungsgemeinschaft (Gr 489/13).


    FOOTNOTES
 

Address for reprint requests and other correspondence: H.-P. Kubis, Zentrum Physiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany, (E-mail: Kubis.HansP{at}mh-hannover.de).

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.


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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