Zentrum Physiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany
Submitted 20 August 2002 ; accepted in final form 11 February 2003
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ABSTRACT |
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muscle plasticity; NFATc1; resting calcium concentration
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MATERIALS AND METHODS |
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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 -actin mRNA was
hybridized with the 1.8-kb BamHI fragment of human
-actin cDNA
(16). Autoradiography exposure
times were 5 days, 3 days, and 8 h, respectively, for MHC mRNA,
-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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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 01710, 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), 25 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.
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RESULTS |
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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 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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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 BD, 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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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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DISCUSSION |
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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).
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ACKNOWLEDGMENTS |
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This work was supported by the Deutsche Forschungsgemeinschaft (Gr 489/13).
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FOOTNOTES |
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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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