Fast-to-slow transformation and nuclear import/export kinetics of the transcription factor NFATc1 during electrostimulation of rabbit muscle cells in culture

doi:10.1113/jphysiol.2002.017574

Fast-to-slow transformation and nuclear import/export kinetics of the transcription factor NFATc1 during electrostimulation of rabbit muscle cells in culture

Hans-Peter Kubis, Renate J. Scheibe, Joachim D. Meißner, Gunther Hornung and Gerolf Gros

Zentrum Physiologie, Medizinische Hochschule Hannover, D-30623 Hannover, Germany

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

Contractile activity imposed by chronic electrical stimulation of a primary skeletal muscle cell culture grown on microcarriers over several days led to an increase of slow myosin heavy chain I (MHCI) and a decrease of fast MHCII expression at mRNA and protein levels, indicating an ongoing fast-to-slow transformation. Only patterns with periods of continuous stimulation of 5 min in a 45 min cycle were capable of inducing a fibre type transformation, and this was independent of the applied stimulation frequency over the range 1-10 Hz. We have shown before that the calcineurin-NFATc1 signalling pathway is indispensable in mediating MHCI upregulation during fibre type transformation. Therefore, subcellular localization of NFATc1 was studied immunocytochemically. This revealed that only one stimulation train lasting for 5 min was sufficient to induce nuclear import of this factor, which was about complete after 20 min of continuous stimulation. For both induction of NFATc1 import and MHCI mRNA upregulation, the minimum stimulation interval of 5 min was sufficient and stimulation frequency was not crucial between 1 and 10 Hz. Repetition of stimulation cycles, with pauses ( 40 min) shorter than the time required for complete export of NFATc1, led to an accumulation of NFATc1 in the nuclei with each cycle and thus to an amplification of the transformation signal during extended periods of electrostimulation. The temporal behaviour of NFATc import/export appears to determine the effectiveness of various electrostimulation protocols in inducing fast-to-slow fibre transformation.

(Received 24 January 2002; accepted after revision 30 March 2002)
Corresponding author H.-P. Kubis: Zentrum Physiologie, Medizinische Hochschule Hannover, D-30623 Hannover, Germany. Email: kubis.hansp{at}mh-hannover.de

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Adult skeletal muscle is composed of different fibres, which are classified as slow-twitch oxidative (type I), fast-twitch oxidative/glycolytic (type IIa) and fast-twitch glycolytic (types IId/x and IIb). One of the most striking features of the highly differentiated fibre is its plasticity. Its physiological and biochemical properties can be influenced by the pattern of innervation (Buller et al. 1960; Salmons & Sreter, 1976), by an imposed electrical stimulation (Salmons & Vrbová, 1969), by the level of physical activity (Salmons & Henriksson, 1981), by passive stretch (Goldspink et al. 1992; Russell & Dix, 1992), and by the hormonal state (reviewed in Pette & Staron, 2001). The adaptation to functional demands is a well-documented process, known as fast-to-slow or slow-to-fast transformation. The most widely used experimental model for studying muscle plasticity is low frequency chronic electrical stimulation that leads to a fast-to-slow transformation in vivo (Pette & Vrbová, 1999). Changes in the expression of a large number of genes, such as those coding for proteins of the contractile apparatus, Ca²⁺-handling proteins and metabolic enzymes have been demonstrated during fibre transformation (Pette & Vrbová, 1992; Williams & Neufer, 1996). In addition, a few studies conducted in vitro with cultured skeletal muscle cells from the rat have shown an influence of electrostimulation on the expression of myosin heavy chain (MHC) isoforms that depended on the pattern of stimulation, but myotubes in these studies had not yet reached the fully adult state (Düsterhöft & Pette, 1990; Naumann & Pette, 1994). Sreter et al. (1987) have demonstrated a long lasting elevation of intracellular Ca²⁺ levels during electrically induced fast-to-slow transformation of rabbit fast muscles in vivo, giving rise to speculation about Ca²⁺ as a mediator of fast-to-slow transformation. Nevertheless, little is known about the cellular signal transduction pathways leading to the observed changes in gene expression during electrically induced transformations.

To investigate the signalling pathways underlying fibre type-specific gene expression, we have previously established a primary rabbit skeletal muscle cell culture (Kubis et al. 1997). Growing on gelatin bead microcarriers in suspension, myotubes develop a fast adult expression pattern with fast MHC and fast myosin light chain (MLC) isoforms after 2 weeks of culture. We have shown that a fast-to-slow transformation of myotubes in this culture system can be induced by the addition of Ca²⁺ ionophore (Kubis et al. 1997; Meissner et al. 2000), which indicates the importance of increased intracellular Ca²⁺ levels for fibre transformation. Indeed, Chin et al. (1998) have demonstrated the involvement of the Ca²⁺-calmodulin-regulated protein phosphatase calcineurin in controlling fibre type-specific gene expression of troponin I slow (TnIs) and myoglobin. This transcriptional activation appears to be mediated by NFAT, the nuclear factor of activated thymocytes, which constitutes a family of transcription factors (Crabtree, 1999). The NFAT factors are located in an inactive/ phosphorylated form in the cytoplasm and, upon dephosphorylation by activated calcineurin, translocate into the nucleus where they activate gene expression (Rao et al. 1997). In adult myotubes, only the isoform NFATc1 can undergo nuclear translocation (Abbott et al. 1998). Calcineurin is associated with NFAT in the nucleus to prevent rapid rephosphorylation by kinases and subsequent nuclear export (Beals et al. 1997; Chow et al. 1997; Masuda et al. 1998; Zhu & McKeon, 2000). It has also been shown that calcineurin-dependent regulation of the TnIs promoter is in addition mediated by the transcription factor myocyte enhancer factor 2 (MEF-2) (Wu et al. 2000). We have recently demonstrated that calcineurin is involved in controlling the expression of MHCI, but not of MHCIId, during Ca²⁺ ionophore- and electrostimulation-induced fast-to-slow transformations, indicating that different regulatory mechanisms underlie the expression of slow and fast MHC isoforms during the process of transformation (Meissner et al. 2001). We have further demonstrated that upon addition of Ca²⁺ ionophore, NFATc1 translocates into the nucleus in a calcineurin-dependent manner. Recently, Liu et al. (2001) have demonstrated in fibres from mouse skeletal muscle that the nuclear translocation of NFATc1-GFP fusion protein can also be induced by electrostimulation.

In the present study, we have investigated which patterns of electrostimulation (cycle length 45 min, stimulation interval per cycle variable between 1.5 and 15 min) are capable of inducing fast-to-slow transformation in muscle cells grown on microcarriers. Simultaneously, we have examined the subcellular localization of endogenous NFATc1 in the myotubes during electrostimulation. We show that some stimulation patterns are in fact able to induce fast-to-slow transformation, but specific combinations of duration of both the stimulation periods and the resting periods are required. Furthermore, we demonstrate that continuous stimulation trains of a defined minimal duration (> 1.5 min and 5 min) are sufficient for some translocation of NFATc1 as well as for the induction of slow MHCI isoform expression. Interestingly, the stimulation frequency does not seem to be a crucial parameter between 1 and 10 Hz. The increase in activated nuclear NFATc1 appears to mediate fast-to-slow transformation at the level of MHCI. Its kinetic behaviour can explain why certain stimulation patterns are effective in fast-to-slow transformation in vitro and possibly also in vivo, while others are not.

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture

Newborn New Zealand White rabbits were killed by decapitation. Skeletal muscles from the hindlimbs were isolated and the muscle fibres destroyed by mincing the muscles with scissors. The remaining myoblasts were isolated by tryptic digestion and separated from fibroblasts as described previously (Kubis et al. 1997). Myoblasts were diluted to a final density of 8 times 10⁵ cells ml^-1 in Dulbecco's modified Eagle's medium (DMEM) with 10 % neonatal calf serum (NCS). A volume of 15 ml of the cell suspension was poured into a 260 ml culture flask together with 0.04 g microcarriers (CultiSpher-GL; Percell Biolytica, Astorp, Sweden). Cells were grown at 37 °C with 8 % CO₂ in air and 95 % humidity while gently shaken to improve O₂ supply to the cells. After 24 h the cell suspension was diluted to a cell concentration of 4 times 10⁵ cells ml^-1. After 3 days the culture medium was completely changed to Skeletal Muscle Growth Medium, 5 % fetal calf serum (FCS) with supplements (cat. no. C-23160, PromoCell, Heidelberg, Germany) for 5 days while the medium was replaced every second day. On day 9, the medium was changed back to DMEM with 10 % rabbit serum (PAA, Linz, Austria). Half of the medium was replaced every second day. Myotubes were collected from microcarriers after different times of culture as described previously (Kubis et al. 1997). For isolation of total RNA, the washing steps were omitted. Myotube samples designated for estimation of metabolic enzymes and MHC isoforms were frozen in liquid nitrogen and kept at -80 °C.

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

Electrostimulation

After 14 days myotubes growing on microcarriers were electrostimulated with different stimulation patterns (I-V, Fig. 1) for 1 or 2 weeks. An electrostimulation device was designed in collaboration with the electronic shop, Hannover Medical School (Hannover, Germany). Stimulator settings were adjusted under visual control of the contractions of the myotubes using a stimulus duration of 2.5 ms. Voltage on the power supply was chosen to achieve maximal contraction amplitudes. 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.

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Figure 1. Electrostimulation patterns
Schematic representation of different stimulation patterns (I-V), applied to the primary skeletal muscle cells from day 14 of culture. Each stimulation pattern was repeated for the number of days as indicated in the figure.

Northern blot analysis

Total RNA isolation was performed according the method of Chomczynski & Sacchi (1987), using the RNAwiz RNA isolation system (Ambion, Inc., Austin, TX, USA). RNA was separated on 1.2 % agarose-formaldehyde gels, transferred to nitrocellulose and hybridized with MHC cDNA probes as described previously (Meissner et al. 2001). Briefly, cDNAs were labelled with [³²P]dCTP using random hexamers as primers (Feinberg & Vogelstein, 1983) and hybridization was performed for 18 h at 42 °C in the same solution containing 1 times 10⁶-5 times 10⁶ c.p.m. ml^-1 of labelled probes. Cross-reactivity was minimized by washing the blots twice for 30 min under high stringency conditions (65 °C, 0.2 times SSC, 0.5 % SDS; 1 times SSC (stock sodium citrate solution = 0.15 M NaCl, 15 mM sodium citrate, pH 7.0). For detection of slow MHCI mRNA, the 3' terminal 450 bp HinfI fragment from rabbit MHCI cDNA (Brownson et al. 1992) was used. Fast MHCIId and IIb mRNA were estimated with the 3' terminal PstI fragments of the rabbit MHCIId and IIb cDNAs, (Maeda et al. 1987), specific for fast MHC isoforms IId and IIb, respectively (Uber & Pette, 1993); 18S rRNA was detected with the 5.8 kb HindIII fragment of 18S rDNA (Katz et al. 1983). Autoradiography was performed with intensifying screens at -80 °C with exposure times from 1 to 5 days.

We found no differences in the total RNA content in our culture system between cultures exposed to the different electrostimulation patterns, which is in contrast to the increase in total RNA that has been reported for electrostimulated muscles in vivo (Brownson et al. 1988).

Myosin heavy chain electrophoresis

Cells were homogenized by sonication (30 times 1 s with 2 W at 0 °C), pelleted and extracted as described previously (Kubis et al. 1997). After centrifugation of extracts at 20 000 g for 20 min, supernatants were diluted 1:10 with ice cold water. The solutions were kept overnight at 2 °C to precipitate the actomyosin. The suspension was then centrifuged at 20 000 g for 30 min and actomyosin pellets were solubilized again in extraction buffer. SDS-PAGE was performed according to the method of Kubis & Gros (1997) with minor modifications using a slab gel (15 cm times 22 cm) with 1 mm gel thickness with two stacking gels (3.5 and 6.5 %) and two separating gels (6.5 and 8.5 %) containing increasing amounts of glycerol from 3 to 35 %. Myosin extracts were diluted (1:7) with sample buffer (Cannon-Carlson & Tang, 1997) and heated for 8 min at 95 °C, and samples were loaded and run under the following conditions at 4 °C: the first step was a constant current of 12 mA for 24 h, and the second step was a constant current of 3 mA for 26 h. Gels were silver-stained according to Heukeshoven & Dernick (1985). Dried gels were scanned and relative MHC contents were quantified using an Image Master System from Amersham Pharmacia Biotech (Freiburg, Germany).

Immunofluorescence studies

Myotubes grown on microcarriers were detached from the carriers by digestion with 0.175 % trypsin in bicarbonated salt solution (BSS), pH 7.9, 1.8 mM CaCl₂, and 0.8 mM MgSO₄, centrifuged at 800 g for 5 min and resuspended in DMEM-10 % NCS. Cells were then seeded on glass coverslips and cultured for 2 days. We have verified (data not shown) by immunofluorescence using monoclonal antibodies (Accurate Chem. & Sci. Corp., Westbury, USA) that during these 2 days the myotubes retained the expression pattern of fast and slow myosin heavy chains that they had developed during the culture on microcarriers. Electrostimulation of myotubes with 1-3 stimulation cycles was performed in 24-well plates, while stimulation with cycles repeated for 24 h was performed in Petri dishes (diameter 9 cm). 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) and 100 % methanol and permeabilized in 0.1 % Triton X-100 as described previously (Meissner et al. 2001). Incubation with goat anti-NFATc1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 30 min was followed by incubation with fluorescein isothiocyanate (FITC)-labelled anti-goat IgG secondary antibody (Santa Cruz Biotechnology, Inc.). Immunostained myotubes were photographed under an inverted fluorescence microscope (Leica) at a magnification of times 400 (objective times 40, NA 1.25). For the quantification of the subcellular localization of NFATc1 given in Figs 6-9; a total of at least 300 cells were counted for each column. Subnuclear localization of MEF-2, together with that of NFATc1, was visualized using a confocal laser scanning microscope (Leica DMIRBE) and Image Space software (Leica TCS-NT). After incubation with rabbit anti-MEF-2 antibody (Santa Cruz Biotechnology, Inc.) for 30 min, MEF-2 was visualized with a tetramethyl-rhodamine (TRITC)-labelled anti-rabbit IgG antibody. The MEF-2 antibody used reacts with MEF-2A and, to a lesser extent, with MEF-2C and 2D.

Statistics

Results are expressed as means ± S.D. The statistical significance of differences was estimated using Dunn's post test following a Kruskal-Wallis test.

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Effectiveness of different patterns of chronic electrostimulation in initiating fast-to-slow transformation in primary skeletal muscle culture

Electrostimulation of primary skeletal muscle cultures growing on microcarriers was started on day 14 of the culture, when the myotubes had reached an almost exclusively adult fast MHC pattern (Kubis et al. 1997). As shown in Table 1, after 3 and 4 weeks of culture, control myotubes expressed predominantly MHCIId, the main MHC isoform in fast adult skeletal muscle in rabbits (Aigner et al. 1993). Different stimulation patterns (Fig. 1) were tested for their ability to initiate fast-to-slow transformation. All patterns consisted of cycles of 45 min duration, which were continuously repeated for 1 or 2 weeks. Each 45 min cycle was composed of ON periods (stimulation train) and OFF periods (pause) of different duration. During ON periods myotubes were electrostimulated with impulses of 2.5 ms duration and an impulse frequency of 1, 5 or 10 Hz. During OFF periods myotubes were permitted to rest.

In experiments with pattern I (Fig. 1, I), myotubes were stimulated with 10 Hz for 1.5 min followed by a rest period of 43.5 min. SDS-PAGE revealed a MHC isoform pattern similar to that of controls with mainly MHCIId and some MHCIIb expressed and no MHCI protein detectable (Table 1). Consistent with this, Northern blot analysis demonstrated the expression of MHCIId mRNA in control and stimulated cells (Fig. 2A, lanes 3 and 5), but only traces of MHCI mRNA (Fig. 2B, lanes 1 and 3). Thus, stimulation pattern I did not induce a transformation even though it was performed chronically for 2 weeks.

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Figure 2. Northern blot analysis of MHC mRNA from control and electrostimulated primary skeletal muscle cells
Myotubes were grown for 28 days without stimulation (A, lanes 1 and 3, and B, lane 1) or were electrostimulated from day 14 on for a further 14 days with 1 Hz for 15 min, stimulus duration 2.5 ms, followed by a pause of 30 min in a 45 min cycle (A, lanes 2 and 4, and B, lane 2), or with 10 Hz for 1.5 min, stimulus duration 2.5 ms, followed by a pause of 43.5 min in a 45 min cycle (A, lane 5 and B, lane 3). A, total RNA was probed on Northern blots using the ³²P-labelled 3' terminal PstI fragment of MHCIIb cDNA (lanes 1 and 2) or the 3' terminal Pst I fragment of MHCIId cDNA (lanes 3-5) or18S rDNA. B, total RNA was probed on Northern blots using the ³²P-labelled 3' terminal HinfI fragment of MHCI cDNA or 18S rDNA. The positions of 18S rRNA (1.9 kb) and 28S rRNA (4.8 kb) on the ethidium bromide-stained gels are indicated.

The second stimulation pattern (Fig. 1, II) applied the same total number of stimuli per cycle upon the myotubes as the first pattern; however, cells were stimulated with 1 Hz for 15 min followed by an OFF period of 30 min. After 1 or 2 weeks of stimulation, these myotubes expressed remarkable amounts of MHCI protein and reduced levels of MHCIId protein (Table 1). The high levels of MHCIIa protein seen in Table 1 are in accordance with the transition sequence MHCIId right MHCIIa right MHCI in rabbits in vivo (Pette & Vrbová, 1992). Similarly, Northern blot analysis revealed induction of MHCI gene transcription (Fig. 2B, compare lane 2 with lane 1) and diminished MHCIId and IIb mRNA levels (Fig. 2A, lanes 2 and 4, compare with lanes 1 and 3, respectively) in stimulated versus control cells after 2 weeks. Thus, the same total number of stimuli per cycle as in pattern I led to a fibre type transformation when applied by a pattern with lower frequency and longer ON period.

To investigate the possible impact of the length of the ON period on the transformation, we again applied the same total number of stimuli to the muscle cells as in patterns I and II, but reduced the length of ON and OFF periods (Fig. 1, III). Within one 45 min cycle cells were stimulated three times for 5 min with 1 Hz, each stimulation interval being followed by an OFF period of 10 min. After 2 weeks, a clear increase in slow MHCI and MHCIIa protein was detected. At the mRNA level, a strong expression of slow MHCI (Fig. 3A, lane 3) and a largely reduced transcription of fast MHCIId (Fig. 3B, lane 3) were observed, whereas control cells expressed only traces of MHCI (Fig. 3A, lane 1) and strong MHCIId transcription (Fig. 3B, lane 1). Thus, reduction of the ON period to a duration of 5 min followed by a 10 min OFF period, while keeping the total number of stimuli per 45 min cycle constant, also resulted in a fast-to-slow transformation.

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Figure 3. Effect of electrostimulation with different patterns on MHC mRNA expression
Myotubes were grown for 28 days without stimulation (lanes 1) or were electrostimulated from day 14 on for a further 14 days with 1 Hz for 5 min, stimulus duration 2.5 ms, followed by a pause of 40 min in a 45 min cycle (lanes 2), with 1 Hz for 5 min, followed by a pause of 10 min in a 15 min cycle (lanes 3), with 1 Hz for 15 min, followed by a pause of 30 min in a 45 min cycle (lanes 4), with 10 Hz for 5 min, followed by a pause of 40 min in a 45 min cycle (lanes 5), or with 1 Hz continuously (lanes 6). A, total RNA was probed on Northern blots using the ³²P-labelled 3' terminal HinfI fragment of MHCI cDNA or 18S rDNA. B, total RNA was probed on Northern blots using the ³²P-labelled 3' terminal PstI fragment of MHCIId cDNA or 18S rDNA. The positions of 18S rRNA (1.9 kb) and 28S rRNA (4.8 kb) on the ethidium bromide-stained gels are indicated.

To study possible effects of different frequencies on muscle transformation, we then used one short ON period of 5 min in a 45 min cycle. The cultures were stimulated with either 1 Hz or 10 Hz causing a 10-fold difference of the total number of stimuli applied. As shown in Fig. 3, Northern blot analysis revealed that both stimulation frequencies led to an increase of slow MHCI mRNA (Fig. 3A, lanes 2 and 5) and a reduction of fast MHCIId mRNA (Fig. 3B, lanes 2 and 5) compared to the controls (Fig. 3A and B, lanes 1), despite the short ON periods and long duration of the OFF periods. In addition, stimulation with 5 Hz for 5 min (Fig. 1, IV) also resulted in a clear increase in MHCI and MHCIIa protein together with a decrease in MHCIId protein (Table 1) compared to unstimulated cells. This demonstrates that pattern IV is also capable of inducing a fibre type transformation. Thus, a 5 min ON period within a 45 min cycle is sufficient to cause fibre type transformation, independent of the frequency and number of stimuli applied.

Continuous electrostimulation of myotubes with 1 Hz (Fig. 1, V) for 8 or 14 days was also effective in increasing the expression of slow MHCI protein (Table 1). The strong level of MHCI mRNA (Fig. 3A, lane 6) and weak level of fast MHCIId mRNA expression (Fig. 3B, lane 6) after 14 days of continuous stimulation demonstrates an advanced state of fast-to-slow transformation.

Changes in MHC mRNA levels have been detected early (within a few days) after the start of electrostimulation of rabbit and rat fast-twitch muscle in vivo and in vitro (Brownson et al. 1988; Kirschbaum et al. 1990; Liu & Schneider, 1998). As shown in Fig. 4, stimulation of the myotubes in the present primary cell system with pattern II for 24 h resulted an increase of the MHCI mRNA (Fig. 4B, compare lane 2 with lane 1) and a concomitant decrease of MHCIId mRNA (Fig. 4A, compare lane 2 with lane 1), showing clearly that 24 h of stimulation are sufficient to switch on the fast-to-slow transformation process.

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Figure 4. Northern blot analysis of MHC mRNA after electrostimulation for 24 h
Myotubes were grown for 16 days without stimulation (lanes 1) or electrostimulated (1 Hz for 15 min, stimulus duration 2.5 ms, followed by a pause of 30 min in a 45 min cycle) for 1 day from day 15 (lanes 2). A, total RNA was probed on Northern blots using the ³²P-labelled 3' terminal PstI fragment of MHCIId cDNA or 18S rDNA. B, total RNA was probed on Northern blots using the ³²P-labelled 3' terminal HinfI fragment of MHCI cDNA or 18S rDNA. The positions of 18S rRNA (1.9 kb) and 28S rRNA (4.8 kb) on the ethidium bromide-stained gels are indicated.

Effects of the different patterns of electrostimulation on the subcellular localization of NFATc1

We have previously shown that calcineurin is involved in the regulation of slow MHCI, but not of fast MHCIId, during the electrostimulation-induced fast-to-slow transformation in primary muscle cell culture (Meissner et al. 2001). In addition, we have demonstrated a calcineurin-dependent translocation of NFATc1 during a Ca²⁺ ionophore-induced fast-to-slow transformation. In an attempt to understand why certain stimulation patterns are effective in inducing a transformation while others are not, we analysed the subcellular localization of NFATc1 during electrostimulation with different stimulation patterns. The localization of endogenous NFATc1 was determined by immunofluorescence studies using an anti-NFATc1 antibody. The myotubes were grown for 14 days on microcarriers and then transferred onto glass coverslips where they were grown for 2-3 more days. They were stimulated continuously at 1 or 10 Hz for various times. Figure 5A shows (left hand side) that in unstimulated cultures NFATc1 staining is found only in the cytoplasm of the myotubes, indicating the presence of the inactive form. In contrast, after cultures had been continuously electrostimulated at 1 Hz for 30 min, NFATc1 staining was localized exclusively in the nuclei of the myotubes (shown on the right), indicating that electrostimulation-induced translocation to the nucleus has occurred. Figure 5B shows the nuclear export of NFATc1; 40 min after the end of stimulation (on the right), NFATc1 staining is nearly exclusively detectable in the cytoplasm. In the middle of the figure intermediate stages of distribution of NFATc1 staining illustrate the ongoing nuclear import and export.

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Figure 5. Immunofluorescence analysis of the subcellular localization of NFATc1 in electrostimulated primary skeletal muscle cells
Myotubes were grown for 14 days on microcarriers and for 2 additional days on glass coverslips. The cultures were then electrostimulated with a single stimulation train with 1 or 10 Hz for 30 min. A, NFATc1 staining was analysed at the beginning (time 0 min, on the left) and the end (time 30 min, on the right) of the stimulation train. In addition, intermediate stages of NFATc1 translocation are shown. B, NFATc1 staining was analysed at the end of the stimulation train (time 0 min, on the left) and after 40 min of resting time (time 40 min, on the right). In addition, intermediate stages of NFATc1 translocation are shown. Immunofluorescence analysis was performed with an anti-NFATc1 polyclonal antibody and fluorescence was detected with an inverted fluorescence photomicroscope (Leitz). Scale bar, 10 µm.

To quantify the import and export kinetics of NFATc1, we counted the cells that had cytoplasmic staining only, nuclear staining only or both cytoplasmic and nuclear staining. In this way we investigated the kinetics of the nuclear import of NFATc1 after continuous stimulation at 1 or 10 Hz lasting for different periods (Fig. 6). Figure 6 shows that nuclear import of NFATc1 was nearly complete after 20 min of continuous stimulation. Figure 6 illustrates further that, after stimulation for 1.5 min with 1 or 10 Hz, NFATc staining was almost exclusively found in the cytoplasm, indicating that no translocation had occurred. However, after stimulation for 5 min with 1 or 10 Hz, significant nuclear staining was found, indicating translocation of NFATc1. This is consistent with the data on the expression of MHCI (Table 1 and Fig. 2 and Fig. 3), which show that MHCI expression is not induced by patterns using stimulation periods of 1.5 min but is induced by patterns using stimulation periods of 5 min. Thus, these data indicate a close correlation between MHCI expression and NFATc1 translocation. Addition of the calcineurin inhibitor cyclosporin A (CsA) prior to stimulation completely abolished nuclear import of NFATc1 (Fig. 6), indicating that nuclear translocation of NFATc1 in electrostimulated primary muscle cells is indeed dependent on the activation of calcineurin. In conclusion, the calcineurin-regulated translocation of NFATc1 is independent of the stimulation frequency but requires a minimal duration of the stimulation period of between 1.5 and 5 min.

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Figure 6. Immunofluorescence analysis of the nuclear import of NFATc1 in myotubes electrostimulated for different time periods
Myotubes were grown for 14 days on microcarriers and for 2 additional days on glass coverslips. Cells were then electrostimulated with a single stimulation train with 1 Hz (left hand side) or 10 Hz (right hand side) for different time periods as indicated in the figure. Electrostimulation was performed in the presence or absence of CsA (500 ng ml^-1) as indicated. Immunostaining was performed with anti-NFATc1 antibody at the time points indicated. White bars represent the percentage of cells with cytoplasmic staining only, black bars show the percentage of cells with nuclear staining only, and grey bars represent the percentage of cells with cytoplasmic plus nuclear staining. All bars represent the mean of three independent experiments.

The nuclear export of NFATc1 occurring after the end of a continuous stimulation with 1 Hz for 20 min is seen in Fig. 7 to be somewhat slower than the import and is almost complete after 40 min. In addition, it is seen that the kinetics of export of NFATc1 were not altered in the presence of CsA. The slow export kinetics of NFATc1 led us to investigate the subcellular localization of NFATc1 after repeated stimulation cycles (Fig. 8). Myotubes were stimulated with one or more 40 min cycles comprising a 10 min at 1 Hz ON period and a 30 min OFF period. Immediately after the first 10 min stimulation period (left hand panel in Fig. 8) nuclear NFATc1 had risen markedly but not maximally, and fell to a very low level during the subsequent 30 min pause. When myotubes had gone through two of the stimulation cycles, nuclear NFATc1 was somewhat higher immediately after the second 10 min stimulation period, and fell again during the subsequent pause, but the level at the end of the pause was significantly higher than that seen after the first cycle. The third panel in Fig. 8 shows the NFATc1 levels after the third 10 min stimulation period. Again, the nuclear NFATc1 was higher at the end of the 30 min pause than it was in the two previous cycles. The likely explanation of this gradual accumulation of NFATc1 with each cycle is that, because a 30 min pause is too short to achieve complete export of nuclear NFATc1, some NFATc1 remains in the nucleus after each cycle and adds up to a markedly increased NFATc1 level after several cycles have been undergone. The significance of this behaviour is borne out in the right hand panel of Fig. 8. After the same stimulation cycles have been repeated for 24 h, this protocol is able to generate a nearly complete translocation of NFATc1 into the nucleus during the stimulation phase, and even to maintain a more than half-maximal translocation by the end of the 30 min pause. It is likely that this phenomenon contributes to entertaining a strongly continuing fibre type transformation process under conditions of discontinuous exercise.

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Figure 7. Immunofluorescence analysis of nuclear export of NFATc1 after cessation of electrostimulation
After 14 days on microcarriers and 2 more days on glass coverslips myotubes were electrostimulated with a single stimulation train with 1 Hz for 10 min. After cessation of the stimulation cells were stained for NFATc1 at the time points indicated. Bars represent the percentage of cells with cytoplasmic, nuclear, or cytoplasmic plus nuclear staining. All bars represent the means of three independent experiments. After cessation of stimulation, CsA (500 ng ml^-1) was added and NFATc1 staining was determined after 15 and 40 min as indicated.

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Figure 8. Immunofluorescence analysis of NFATc1 nuclear export after repeated stimulations
Myotubes grown for 14 days on microcarriers and for 2 days on glass coverslips were electrostimulated with 1 Hz for 10 min followed by an OFF period of 30 min in a 40 min cycle. The stimulation cycle was repeated for 24 h. Cells were stained with an anti-NFATc1 antibody during pause periods after the first three stimulations and the last stimulation after 24 h at the time points indicated. Bars represent the percentage of cells with cytoplasmic, nuclear, or cytoplasmic plus nuclear staining. All bars represent the mean of three independent experiments.

NFATc1 and MEF-2 are thought to interact in the calcineurin-dependent activation of the expression of some slow fibre type genes (Olson & Williams, 2000b). We therefore investigated the subnuclear localization of NFATc1 and MEF-2 in electrostimulated myotubes by confocal microscopy. In contrast to NFATc1, MEF-2 is always localized in the nucleus (Black & Olson, 1998). Figure 9 shows a confocal fluorescence image of a representative nucleus after 24 h of repeated stimulation (1 Hz for 10 min ON/30 min OFF). The picture was taken at the end of the last cycle, after the last 30 min resting period. At that time about 50 % of myotubes showed completely nuclear and about 40 % exhibited nuclear plus cytoplasmic staining of NFATc1 (see Fig. 8, right hand panel). The intranuclear distribution of NFATc1 (Fig. 9A) was visualized with a FITC-labelled secondary antibody. The intranuclear distribution of MEF-2 (Fig. 9B) was visualized with a TRITC-labelled secondary antibody. The images were taken from a myotube with nuclear plus cytoplasmic staining of NFATc1 and demonstrate that neither transcription factor was homogeneously distributed but both showed a punctate pattern of fluorescent foci. The overlay of both images (Fig. 9C) shows an overlap of NFATc1 and MEF-2 in several foci as indicated by the yellow spots. A similar overlap was found in cells with exclusively nuclear staining of NFATc1 after stimulation for 24 h (data not shown).

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Figure 9. Immunofluorescent confocal microscopy after dual immunostaining for NFATc1 and MEF-2 in electrostimulated myotubes
After 14 days on microcarriers and 2 days on glass coverslips cells were stimulated at 1 Hz (10 min ON/30 min OFF in 40 min cycles) over 24 h. After the end of the last ON period cells were allowed to rest for an additional 30 min before they were fixed and stained with anti-NFATc1 (A) and anti-MEF-2 polyclonal antibody (B). NFATc1 and MEF-2 were visualized by FITC- and TRITC-labelled secondary antibody, respectively. Images were collected in separate channels to prevent overlap of fluorescence. C, overlay of both channels; the yellow colour indicates areas of co-localization of NFATc1 and MEF-2. Scale bar, 10 µm.

DISCUSSION

Top
Abstract
Introduction
Methods
Results
Discussion
References

This report describes studies on the fast-to-slow transformation of adult primary skeletal muscle cultures grown on microcarriers induced by chronic electrical stimulation. Electrostimulation of myotubes with different patterns revealed that an ON period of 5 min in a 45 min stimulation cycle was sufficient to induce MHCI expression and to reduce MHCIId expression at the mRNA and protein levels, demonstrating an ongoing fast-to-slow transformation. In contrast, shorter ON periods of 1.5 min in a 45 min cycle failed to induce a fast-to-slow transformation. In addition, an insufficient duration of the ON period could not be overcome by raising stimulation frequency from 1 Hz to 10 Hz. The data indicate that (i) initiation of a fast-to-slow transformation process requires a minimum duration of the ON period used during each stimulation cycle and (ii) the total number of stimuli applied is not the decisive parameter for the fibre type switch.

In a study performed previously by Naumann & Pette (1994), primary skeletal muscle cultures from the rat were electrostimulated with different impulse patterns and these were reported to exert different effects on the expression of MHC isoforms. Bursts (ON periods) of 250 ms duration, applied to the culture every 1 s for a total of 20 days, induced the formation of small amounts of the adult slow MHCI. The same bursts given every 4 s for 20 days promoted expression of MHCI more significantly. In contrast, 250 ms bursts given every 100 s for 20 days induced the expression of MHCIId and IIb and of the neonatal MHC to a greater extent than the two other stimulation protocols, but did not promote the expression of MHCI at all. It may be noted that myotubes in this study had not reached the fully adult state under both unstimulated and stimulated conditions, and embryonic MHC remained a major isoform. The authors concluded that expression of MHCI in their system depends on the burst frequency rather than impulse frequency applied during the bursts. These results may be in agreement with the present observations. When the 250 ms bursts were repeated every 100 s (corresponding to a burst frequency of 0.01 Hz), no signs of fast-to-slow transformation were observed by Naumann & Pette. This may simply be a consequence of the fact that this burst is too short to initiate a transformation, as the present data would predict. When the same burst was repeated every 1 or 4 s, i.e. was applied at frequencies of 1 or 0.25 Hz, some degree of fast-to-slow transformation became apparent. This may reflect the fact that for such short bursts, as presumably for single stimuli, a minimum frequency of >> 0.01 Hz is required under conditions of continuous stimulation in order to effect a fibre type switch; 0.25 Hz may be this minimum frequency and 1 Hz is shown here to be sufficient. In the present study no attempt has been made to define the required minimum frequency.

Jarvis et al. (1996) reported that under continuous electrostimulation in vivo an increase in stimulation frequency from 2.5 to 10 Hz leads to an increase in the extent of fibre transformation. This may suggest that, at sufficiently great burst periods, the extent of transformation may additionally be modulated by the stimulation frequency or the total workload, respectively. This aspect has not been pursued in the present study.

Hennig & Lømo (1985) have reported a detailed analysis of the firing patterns of individual motor units in the rat in vivo. Motor units of the slow soleus muscle exhibited impulse bursts of up to 8 min duration followed by relatively short pauses between bursts of 1 s on average (frequency around 20 Hz), while bursts in the fastest motor units of the extensor digitorum longus lasted no longer than 1-4 s with pauses of 1 min on average (frequency around 80 Hz). The present results suggest indeed that the 'slow' firing pattern can be expected to maintain MHCI expression on the basis of the long burst duration, while the 'fast' pattern would not be expected to induce MHCI expression because burst duration is far too short. It thus appears conceivable that in vivo the dependence of muscle transformation on the firing pattern is similar to that observed here in vitro.

The calcineurin-NFAT signalling pathway has been implicated in activating some fibre type-specific genes like TnIs and myoglobin (Chin et al. 1998). It has been suggested that calcineurin-dependent regulation of the TnIs promoter depends on both NFAT and MEF-2 binding sites (Wu et al. 2000). It has been proposed that calcineurin acts as a Ca²⁺ sensor that couples prolonged changes in Ca²⁺ levels to reprogramme muscle gene expression (Olson & Williams, 2000a). In accordance with this hypothesis, we have recently demonstrated that the calcineurin inhibitor CsA prevents the Ca²⁺ ionophore-induced upregulation of MHCI and nuclear translocation of NFATc1 in myotubes (Meissner et al. 2001). In contrast, downregulation of MHCIId during the Ca²⁺ ionophore-induced fast-to-slow-transformation was not reversed by CsA. Similarly, CsA abolished the upregulation of MHCI in electrostimulated myotubes but again did not influence the downregulation of MHCIId. Together, these earlier data indicate that the calcineurin-NFATc1 pathway is involved in the regulation of MHCI, but not of MHCIId, during fast-to-slow-transformation and calcineurin alone is not sufficient to mediate the complete transformation.

The involvement of calcineurin in the upregulation of MHCI led us to investigate the subcellular localization of the endogenous NFATc1 in electrostimulated primary myotubes. Electrostimulation caused translocation of NFATc1 from the cytoplasm into the nuclei and addition of CsA prevented the nuclear import, indicating that calcineurin is involved in the electrostimulation-induced translocation of NFATc1. Nuclear translocation of NFATc1 was fast, a single stimulation train of 1 Hz or 10 Hz for 5 min was already sufficient to cause a significant import, whereas 1.5 min stimulation was not enough to induce import. After continuous stimulation at 1 Hz for 20 min, nuclear import was nearly complete. It is not clear what the mechanistic basis for the observed minimum stimulation period might be. Our findings also demonstrate that the export kinetics of NFATc1, determined subsequently to a 20 min stimulation period, are somewhat slower than the import kinetics. Similarly, Shibasaki et al. (1996) reported a fast nuclear import (t_1/2 about 7.5 min) and a slower nuclear export (t_1/2 about 12 min) of transfected NFAT4 in BHK fibroblasts. In our myotube cultures, the export of NFATc1 was not fully complete 40 min after cessation of the stimulation. More importantly, we could demonstrate that from cycle to cycle an accumulation of activated NFATc1 occurred in the nuclei when the 40 min stimulation cycles were repeated. The degree of accumulation is expected to depend on the length of the resting phases between the stimulation intervals. After 24 h of continuously repeated stimulation cycles (1 Hz for 10 min ON/30 min OFF), almost all myotubes exhibited exclusively nuclear NFATc1 staining at the end of the ON period. Even 30 min later, after the 30 min OFF period, about half of the myotubes still had exclusively nuclear NFATc1 staining and about another 40 % had combined nuclear plus cytoplasmic staining. By contrast, at the end of the OFF period of a single cycle without repetition less than 10 % of the nuclei had any NFATc1 staining at all. It is obvious that the cycle-to-cycle accumulation of NFATc1 can act as a powerful amplification mechanism that guarantees a high level of transcription even when contractile activity is moderate and discontinuous. By confocal microscopy, we also demonstrated that after 24 h of repeated electrostimulation cycles, nuclear NFATc1 localization overlaps with MEF-2 localization. These studies were performed with the endogenous transcription factors and a similar overlap was not observed after a single stimulation train of 1 Hz for 30 min (data not shown). NFATc1 and MEF-2 have been considered to interact in the calcineurin-dependent activation of the expression of some slow fibre type genes (Olson & Williams, 2000b). It appears possible that the observed spots of overlap of the localizations of the two transcription factors represent such an interaction slow fibre genes.

The import and export kinetics of NFATc1 observed in this study are in excellent agreement with the requirements for an electrostimulation pattern that is successful in inducing of fast-to-slow transformation at the level of MHCI. This is apparent in several aspects of the results given above: (i) an ON period of 1.5 min in a 45 min cycle was not sufficient to initiate transformation independently of the frequency. This corresponds with the finding that NFATc1 import was not initiated by stimulation periods of 1.5 min, either at 1 or at 10 Hz; (ii) ON periods of 5 min or more were effective in inducing both transformation and NFATc1 nuclear import; (iii) increased ON periods resulted in an increased expression of MHCI as well as increased nuclear import of NFATc1; (iv) all effective electrostimulation patterns applied resting periods of 40 min, which appears to be consistent with the observation that nuclear export of NFATc1 was not yet quite complete after 40 min. Longer pauses would be expected to impair the transformation efficiency drastically, although this remains to be shown.

Recently, Liu et al. (2001) have reported that electrostimulation of fibres from skeletal muscles of the adult mouse with either continuous 10 Hz stimulation or stimulation with cycles of 10 Hz for 5 s, followed by 45 s pauses, and repetition of these cycles for 2 h, induced an import of NFATc-GFP fusion protein. In contrast, continuous stimulation with 1 Hz as well as stimulation cycles of 50 Hz for 0.1 s, followed by pauses of 50 s, and repetition of the cycles for 2 h, failed to induce NFATc import. In contrast, from the present results we would expect both discontinuous patterns to be ineffective in activating NFAT because the stimulation periods are << 1.5 min, while both continuous patterns should be effective. In fact, we have shown here that continuous 1 Hz stimulation not only activates NFATc1 but also increases expression of MHCI at protein and mRNA levels. The NFAT import kinetics observed by Liu et al. (2001) are in good agreement with those observed in this study and with those reported by Shibasaki et al. (1996) in BHK fibroblasts. However, we find NFAT export to be almost complete after 40 min, which agrees with the half time given by Shibasaki et al. (1996), whereas Liu et al. (2001) report that the intranuclear foci of NFATc-GFP fusion protein persist for more than 2 h. The question arises whether the NFATc-GFP fusion protein behaves differently in terms of export kinetics from the native NFATc1 observed in the present study. Liu et al. (2001) report lack of co-localization of NFAT and MEF-2 after 30 min stimulation. This agrees with our finding after the same stimulation period, while after 24 h stimulation we do find co-localization. As we also find the first signs of MHCI upregulation (and MHCII downregulation) after 24 h stimulation, this might support the idea that interaction of MEF-2 and NFATc1 is required to turn on the MHCI gene.

We conclude that several important features of the behaviour of MHCI expression under electrostimulation can be perfectly explained by the kinetics of nuclear import and export of NFATc1. The present data clearly suggest that the electrostimulation-induced import of NFATc1 into the nuclei is a crucial step in the signal transduction mediating fast-to-slow transformation at the MHCI level.

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

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Acknowledgements

We are grateful to Drs C. Brownson, R. Guntaka, P. K. Umeda, and A. Wittinghofer for their generous gifts of plasmids. We thank E.-A. Haller and A. Jacobs for excellent technical assistance. R.J.S. and J.D.M. thank Dr W. H. Müller for helpful discussions. We thank the Deutsche Forschungsgemeinschaft for financial support of this project (Gr 489/13).

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ABBOTT, K. L., FRIDAY, B. B., THALOOR, D., MURPHY, T. J. & PAVLATH, G. K. (1998). Activation and cellular localization of the cyclosporin A-sensitive transcription factor NF-AT in skeletal muscle cells. Molecular Biology of the Cell 9, 2905-2915	[Abstract/Full Text]
AIGNER, S., GOHLSCH, B., HÄMÄLÄINEN, N., STARON, R. S., UBER, A., WEHRLE, U. & PETTE, D. (1993). Fast myosin heavy chain diversity in skeletal muscles of the rabbit: heavy chain IId, not IIb predominates. European Journal of Biochemistry 211, 367-372	[Abstract]
BEALS, C. R., SHERIDAN, C. M., TURCK, C. W., GARDNER, P. & CRABTREE, G. R. (1997). Nuclear export of NFATc enhanced by glycogen synthase kinase-3. Science 275, 1930-1933
BLACK, B. L. & OLSON, E. N. (1998). Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF-2). proteins. Annual Reviews of Cell and Developmental Biology 14, 167-196	[Abstract/Full Text]
BROWNSON, C., ISENBERG, H., BROWN, W., SALMONS, S. & EDWARDS, Y. (1988). Changes in skeletal muscle gene transcription induced by chronic stimulation. Muscle and Nerve 11, 1183-1189	[Medline]
BROWNSON, C., LITTLE, P., JARVIS, J. C. & SALMONS, S. (1992). Reciprocal changes in myosin isoform mRNAs of rabbit skeletal muscle in response to the initiation and cessation of chronic electrical stimulation. Muscle and Nerve 15, 694-700	[Medline]
BULLER, A. J., ECCLES, J. C. & ECCLES, R. M. (1960). Interactions between motoneurons and muscles in respect of the characteristic speeds of their responses. Journal of Physiology 150, 417-439
CANNON-CARLSON, S. & TANG, J. (1997). Modification of the Laemmli sodium dodecylsulfate-polyacrylamide gel electrophoresis procedure to eliminate artifacts on reducing and nonreducing gels. Analytical Biochemistry 246, 146-148	[Medline]
CHIN, E. R., OLSON, E. N., RICHARDSON, J. A., YANG, Q., HUMPHRIES, C., SHELTON, J. M., WU, H., ZHU, W., BASSEL-DUBY, R. & WILLIAMS, R. S. (1998). A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type. Genes and Development 12, 2499-2509	[Abstract/Full Text]
CHOMCZYNSKI, P. & SACCHI, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162, 156-159	[Medline]
CHOW, C.-W., RINCÓN, M., CAVANAGH, J., DICKENS, M. & DAVIS, R. J. (1997). Nuclear accumulation of NFAT4 opposed by the JNK signal transduction pathway. Science 278, 1638-1641
CRABTREE, G. R. (1999). Generic signals and specific outcomes: signaling through Ca²⁺, calcineurin, and NF-AT. Cell 96, 611-614	[Medline]
DÜSTERHÓFT, S. & PETTE, D. (1990). Effects of electrically induced contractile activity on cultured embryonic chick breast muscle cells. Differentiation 44, 178-184	[Medline]
FEINBERG, A. P. & VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132, 6-13	[Medline]
GOLDSPINK, G., SCUTT, A., LOUGHNA, P. T., WELLS, D. J., JAENICKE, T. & GERLACH, G. F. (1992). Gene expression in skeletal muscle in response to stretch and force generation. American Journal of Physiology 262, R356-363	[Medline]
HENNIG, R. & LØMO, T. (1985). Firing patterns of motor units in normal rats. Nature 314, 164-166	[Medline]
HEUKESHOVEN, J. & DERNICK, R. (1985). Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6, 103-112
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