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J. Biol. Chem., Vol. 276, Issue 48, 45243-45254, November 30, 2001
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§¶,¶,
,, and§**
From the Neuromuscular Research Laboratory,
Department of Chemistry and Biochemistry, Laurentian University,
Sudbury, Ontario P3E 2C6, Canada, the Department of Internal
Medicine, University of Texas Southwestern Medical Center, Dallas,
Texas 75390, and the § Faculty of Health Sciences,
University of Western Ontario, London, Ontario N6A 3K7, Canada
Received for publication, June 12, 2001, and in revised form, September 11, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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This study tested the hypothesis that calcineurin
signaling is modulated in skeletal muscle cells by fluctuations
in nerve-mediated activity. We show that dephosphorylation of NFATc1,
MEF2A, and MEF2D transcription factors by calcineurin in all muscle
types is dependent on nerve activity and positively correlated with muscle usage under normal weightbearing conditions. With increased nerve-mediated activity, calcineurin dephosphorylation of these targets
was found to be potentiated in a way that paralleled the higher muscle
activation profiles associated with functional overload or nerve
electrical stimulation conditions. We also establish that muscle
activity must be sustained above native levels for calcineurin-dependent dephosphorylation of MEF2A and MEF2D
to be transduced into an increase in MEF2 transcriptional function, suggesting that calcineurin cooperates with other activity-linked events to signal via these proteins. Finally, examination of individual fiber responses to overload and nerve electrical stimulation revealed that calcineurin-MEF2 signaling occurs in all fiber types but most
readily in fibers that are normally least active (i.e.
those expressing IIx and IIb myosin heavy chain (MHC)), suggesting that signaling via this phosphatase is also dependent upon the
activation history of the muscle cell.
Skeletal muscle fibers display considerable variation in their
size and expression of contractile proteins. Slow/type I fibers display
relatively small diameters and express slow isoforms of myofibrillar
and Ca2+-regulatory proteins, whereas fast/type II fibers
generally display larger fiber girths and express faster isoforms of
these proteins (see Ref. 1 for review). The phenotype of an adult
muscle fiber is malleable and is largely influenced by the amount and
pattern of activity that it receives from its motor nerve (1).
Compelling evidence of this is provided by studies that have induced
transformations in muscle phenotype by cross-reinnervating slow or fast
muscles with their foreign nerve counterparts or by electrically
stimulating these muscles with impulse patterns modeled after foreign
nerve discharges (see Ref. 1 for review). For example, electrical stimulation of a fast twitch muscle with low frequency continuous stimuli, a pattern that mimics the activity of slow motoneurons (2),
causes this muscle to express slower, more energy-efficient contractile
proteins and to become more fatigue-resistant (3, 4). On the other
hand, sporadic, high frequency stimulus trains associated with fast
motoneuron firing (2) induce the expression of faster, more
metabolically costly, contractile proteins in predominantly slow twitch
muscles (5, 6) and cause these muscles to become less resistant to
fatigue (4).
Acetylcholine is released from the nerve terminal with each motoneuron
discharge. The binding of this neurotransmitter to receptor-mediated
ion channels on the target muscle cell induces a depolarization of the
sarcolemma and triggers the release of Ca2+ from the
sarcoplasmic reticulum. It is suggested that as a result of their
distinct discharge profiles, slow and fast motoneurons modulate the
expression of muscle genes via the different patterns of intracellular
Ca2+ that they evoke (7). Frequent muscle depolarizations
elicited in response to the tonic activity of slow motoneurons induce a sustained elevation of muscle Ca2+ (8), whereas the
infrequent, burst firing of fast motoneurons evokes transient spikes in
muscle Ca2+ (9).
Calcineurin, a Ca2+/calmodulin (CaM)-activated1
phosphatase, has been implicated as a
molecular decoder of sustained Ca2+ signals evoked in
muscle cells in response to frequent nerve-mediated depolarizations and
as an integral signaling intermediate in pathways that promote skeletal
muscle fiber hypertrophy and expression of a slower contractile protein
phenotype (for review, see Ref. 10). It is postulated that
nerve-mediated increases in muscle intracellular Ca2+
activate calcineurin and CaM-sensitive kinases which in turn trigger
the action of nuclear factor of
activated T cells c1 (NFATc1) and
myocyte enhancer factor
2 (MEF2) proteins (7, 10, 11). It is thought that these
proteins bind cooperatively with other transcription factors such as
GATA-2 to activate the transcription of slow fiber-specific or
growth-regulatory genes (7, 10, 11). In support of this model,
increasing intracellular Ca2+ levels in cultured myocytes
with ionomycin activates calcineurin (12, 13) and promotes the
acquisition of a slow oxidative phenotype (13, 14). Moreover, NFAT and
MEF2 binding appears essential for the
calcineurin-dependent activation and slow fiber-specific expression of the slow upstream regulatory element enhancer, a transcriptional element that directs the expression of the troponin I
slow gene (11). The clear importance of calcineurin signaling events in
regulating muscle phenotype is further demonstrated by experiments that
prevented fiber growth and fast-to-slow fiber type transitions
(i.e. IIb In the present study, we thus tested the hypothesis that calcineurin
signaling pathways in skeletal muscle cells are sensitive to
nerve-mediated activity. To this end, we investigated whether calcineurin is more extensively activated in more highly recruited muscles under normal weightbearing conditions and whether this activity
is countered by neuronal quiescence. Next, we examined the potentiation
of calcineurin signaling in overloaded plantaris muscles over the time
course when motor unit recruitment levels are rapidly doubled by this
condition (17). Moreover, to provide insight into what specific aspect
of the nerve electrical stimulus (i.e. pulse frequency,
amount, etc.) is key to activating calcineurin in muscle cells, we
contrasted the effectiveness of various exogenous nerve stimulation
paradigms to initiate signaling via this enzyme. The extent of muscle
calcineurin signaling was assessed by measuring the phosphorylation
status of the calcineurin substrates NFATc1, MEF2A, and MEF2D; the
nuclear abundance of NFATc1; and the transcriptional function of
MEF2.
Consistent with our hypothesis, we show calcineurin-mediated
dephosphorylation of NFATc1, MEF2A, and MEF2D to be nerve
activity-dependent and to occur in all muscle types.
Dephosphorylation of these transcription factors by calcineurin
appeared to be positively correlated with muscle usage under normal
weightbearing conditions. Moreover, we provide evidence that muscle
activity must be increased above native levels for
calcineurin-dependent dephosphorylation of MEF2A or MEF2D
to be transduced into an increase in transcriptional function of these
proteins, suggesting that accessory activity-linked signaling events
are required for complete signaling of calcineurin to target genes
(18). Finally, we found that calcineurin-MEF2 signaling was initiated
in all muscle fiber types in response to increased nerve-mediated
activity, but occurred most readily in the least active fibers, those
expressing IIx or IIb MHC, suggesting that modulation of this pathway
is also influenced by the activation history of each muscle cell.
Animals, Surgeries, and Stimulation
Paradigms--
Sprague-Dawley rats (260-300 g) and CD-1 mice (25-35
g) were obtained from Charles River Laboratories (St. Constant, Quebec, Canada). Tg mice (C57BL6) harboring lacZ under the control
of three copies of a high affinity desmin MEF2 consensus element (desMEF2) have been described previously (19). All surgical procedures
were performed under aseptic conditions on animals anesthetized by
intramuscular injection (1.2 µl/g) of 100 mg/ml ketamine
hydrochloride and 10 mg/ml xylazine in a volume ratio of 1.6:1.
Treatment of animals was in accordance with the guidelines established
by the Canadian Council on Animal Care. For denervation and TTX
paralysis experiments, hamstring musculature in the left hip region of
rats was incised to expose the sciatic nerve. In denervated animals, a
5-mm portion of the sciatic nerve was excised. In TTX animals, muscles
in the left hindlimb were paralyzed for 3 or 7 days by chronic
superfusion of the sciatic nerve (0.5 µl/h) with this sodium channel
blocker (350 µg/µl) via a mini-osmotic pump (model 1007D, Alza
Corp., Palo Alto, CA) and attached silicon drug delivery system as
described in detail previously (5). Briefly, a silicon cuff was secured
around the nerve and was connected by silicon tubing to the pump that
was implanted subcutaneously on the animal's back. The efficacy of
paralysis of the triceps surae muscles in TTX animals was verified
twice daily using established ankle reflex criteria (5). Control rats
were implanted with a sham nerve cuff and drug delivery system. For OV
experiments, compensatory hypertrophy of the plantaris was induced in
each hindlimb of desMEF2 Tg and CD-1 mice by surgically ablating the soleus and a major portion of the gastrocnemius muscle (15). In control
mice, the tendons of the soleus and gastrocnemius were separated from
the plantaris tendon but were not severed. For muscle regeneration
experiments, local injury of the plantaris in each hindlimb of CD-1
mice was induced via freezing the muscle. Briefly, the medial hamstring
musculature was incised to expose the distal half of the plantaris.
Metal forceps (2 mm in width) pre-cooled with liquid nitrogen were then
applied to the surface of the muscle for 10 s. For electrical
stimulation experiments, hamstring musculature in the left hip region
of each animal was incised to expose the sciatic nerve. Hindlimb
muscles were activated by electrically stimulating the sciatic nerve at
supramaximal voltage (15 V) with a bipolar electrode using a 10-Hz
continuous or an intermittent 100-Hz stimulus pattern (Grass®
Stimulator, model S48, Grass Instrument, Quincy, MA). For the
latter pattern, 1-s 100-Hz trains were administered once every 30 s, 1 min, 2 min, 5 min, 15 min, or 30 min. Administered stimuli were
0.1-ms square wave pulses. Hip, knee, and ankle joints were fixed
during the stimulation session to ensure that contractions were isometric.
CsA and FK506 Administration and Muscle Removal--
For OV and
electrical stimulation experiments, mice were injected subcutaneously
with either CsA (25 mg/kg), FK506 (3-5 mg/kg), or vehicle
(Cremaphor® EL; Sigma-Aldrich, Oakville, Ontario, Canada)
twice daily, separated by a 12-h interval, for 1-28 days. Injections
were initiated 1 day prior to the beginning of each experiment. Vials
of CsA (50 mg/ml; Novartis, Dorval, Quebec, Canada) were mixed in a
ratio of 1:7 (v/v) with vehicle, whereas FK506 was solubilized in
ethanol (5 µl/mg drug) and then diluted to a final concentration of
0.75 mg/ml with vehicle. To investigate the long term effect of
calcineurin inhibition on the phosphorylation status of NFATc1 and MEF2
in normal weightbearing hindlimb muscles, rats were injected with CsA
(10 mg/kg, n = 3) or vehicle (n = 3)
twice daily, or FK506 (2 mg/kg, n = 2) once daily, for
a period of 28 days. At respective end points, blood was collected for
analyses of CsA and FK506 levels (London Health Science Center, London,
Ontario, Canada). After each experimental condition, muscles were
excised and quick-frozen in melting isopentane pre-cooled with liquid
nitrogen. The 25 mg/kg dose of CsA in mice produced blood levels of
this drug that were comparable with those obtained using a 10 mg/kg
dose in rats (2865 ± 517 ng/ml in mice versus
2137 ± 613 ng/ml in rats) and resulted in a ~60% decrease in
total plantaris calcineurin activity (17.4 ± 0.6 pmol/min/mg
protein in vehicle-treated versus 7.1 ± 0.9 pmol/min/mg protein in CsA-treated mice, n = 3) as
measured by Isotechnika (Edmonton, Alberta, Canada). Administration of FK506 resulted in lower blood levels in rats (13.0 ± 1.7 ng/ml) than in mice (83 ± 14 ng/ml). This lower dosage of FK506 in rats was necessary to ensure the maintenance of the health and body weight
of these animals for the duration of the treatment period.
Western Blot Analysis of MEF2 and NFATc1 Expression--
Whole
cell and nuclear extracts were generated as described previously (18).
For in vitro alkaline phosphatase (AP) reactions, muscle
samples (150 mg) were homogenized in 1 ml of HEPES buffer (4 mM EGTA, 10 mM EDTA, 0.1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride in 50 mM HEPES, pH = 7.4), incubated on ice for 1 h, and then centrifuged for 15 min at
20,000 × g. Each lysate (50 µg) was incubated with 0, 100, or 200 units of AP covalently linked to agarose beads (Sigma-Aldrich) in AP reaction buffer (1 mM
MgCl2, 50 mM Tris-HCl, pH = 8.5) at
30 °C for 15 min. The beads were separated from the reaction mixture
by centrifugation, the supernatant recovered, and then concentrated to
the original volume of added protein using a 5000 nominal molecular
weight limit 4-ml Ultrafree® filter unit (Millipore,
Bedford, MA). The concentration of total protein in whole cell, nuclear
extract, and AP-treated samples was estimated with the Bradford
Microassay (Bio-Rad, Mississauga, Ontario, Canada) using bovine serum
albumin as a standard.
Samples were each diluted in two volumes of 2× Laemmli Buffer
(Bio-Rad), boiled for 3-5 min, and run on a 6-8% SDS-polyacrylamide gel using the MiniProtean III system (Bio-Rad). Proteins were stained
with Coomassie Blue to verify equal loading of samples. Proteins were
transferred onto Hybond-P polyvinylidene difluoride membranes (Amersham
Pharmacia Biotech, Baie d'Urfé, Quebec, Canada) and stained with
Ponceau S Red to verify equal transfer efficiency. Immunoblotting was
performed using standard procedures with working dilutions of the
following primary antibodies: NFATc1 (Affinity Bioreagents, Inc.,
Golden, CO), MEF2A (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and
MEF2D (gift from Dr. R. Prywes). The MEF2D antibody cross-reacts with
MEF2C and was used to estimate the abundance of this protein in whole
cell and nuclear extracts. Membranes were subsequently probed with
horseradish peroxidase secondary antibody conjugates (Sigma), and
labeled proteins were visualized using the ECL plus system and
Hyperfilm ECL (Amersham Pharmacia Biotech). The size of each protein
was estimated using a Kaleidoscope broad range protein standard
(Bio-Rad). MEF2C was used as a gel loading control for normal
weightbearing, denervated, TTX-inactivated, and electrical stimulation
samples, because the expression of this protein did not differ between
muscle types or across these different treatment conditions. For OV
experiments, Coomassie Blue-stained SDS-polyacrylamide gels were used
as loading controls, because MEF2C protein content was found to
increase over the time course of this condition (data not shown).
Quantification of NFATc1, MEF2A, and MEF2D
Phosphorylation--
We used AP-treated samples to determine which
bands were best suited to measure the phosphorylation status of NFATc1,
MEF2A, and MEF2D proteins. Up to 6 NFATc1 bands were detected ranging in molecular mass from 85 to 141 kDa (Fig. 1A, see AP
panels) in untreated samples. Pre-treatment of protein extracts
with AP increased the prevalence of the 85- and 89-kDa species of
NFATc1 (Fig. 1A, closed arrows), suggesting that
detected bands correspond to dephosphorylated (85 and 89 kDa) and
phosphorylated (>89 kDa) forms of this protein. Similarly, AP
treatment reduced both MEF2D and MEF2A bands to single 70-and 67-kDa
species, respectively (Fig. 1A, middle panels,
closed arrows). The density of dephosphorylated (dePO4) and most phosphorylated (most PO4)
band(s) of NFATc1, MEF2D, or MEF2A was determined using
AlphaEaseTM/FluroChemTM software (Alpha Innotech Corp., San Leandro,
CA) and expressed relative to the summed density of all bands for that sample.
Genotyping of desMEF2 Tg Mice and Staining for Immunohistochemistry--
To determine the MHC content of each
fiber, cryosections (10 µm) were cut from the same anatomical
location in each muscle midbelly and recovered onto
Superfrost® Plus microscope slides (Fisher Scientific,
Ottawa, Ontario, Canada) and were processed for immunohistochemistry as
described previously (15). Briefly, sections were first blocked in 5%
goat serum in 25 mM PBS (pH = 7.4) and then probed
with one of the following primary antibodies raised against the
different MHC types: type I (BA-F8), IIa (SC-71), IIb (BF-F3), all MHCs
but IIx (BF-35), and embryonic (F1.652) (15). After three 10-min PBS
rinses, sections were then probed with either horseradish
peroxidase-conjugated anti-mouse IgG or IgM, in the case of BF-F3
(Sigma). Bound antibody complexes were visualized using
diaminobenzidine tetrahydrochloride.
In Vivo DNA Plasmid Injections and Visualization of NFAT-Green
Fluorescent Protein (GFP) Fusion Protein--
Plasmids that encoded
GFP linked to either full-length NFATc1 (NFATc1-GFP) or a truncated
variant of this protein (amino acids 319-716, Calcineurin Dephosphorylation of NFATc1 and MEF2 Is Correlated with
Muscle Usage in Normal Weightbearing Muscles--
To test the
hypothesis that calcineurin signaling is modulated by nerve-mediated
activity, we first investigated whether the calcineurin substrates
NFATc1, MEF2A, or MEF2D are dephosphorylated to a greater extent in the
soleus, a more highly recruited (20) and relatively slower
(i.e. equal proportions of fibers expressing I or IIa MHC)
muscle, versus its synergist, the medial gastrocnemius, a
comparatively less active (20), faster (i.e. proportionately large number of fibers expressing IIx or IIb MHC) plantar flexor under
normal weightbearing conditions. Western blots of whole cell extracts
showed a multiple banding pattern for each of these transcription
factors. We identified dephosphorylated (dePO4, closed arrows) and most phosphorylated (most
PO4, open arrows) forms of these proteins (Fig.
1A). All of these calcineurin
substrates were more extensively dephosphorylated (Fig. 1, A
and B) and expressed at higher levels relative to total
protein (Fig. 1A) in the soleus compared with the medial
gastrocnemius under normal weightbearing conditions. Compared with the
soleus, the medial gastrocnemius displayed at least 2-fold higher
levels of the most PO4 form of all three substrates and
approximately half the levels of the dePO4 form of NFATc1
and MEF2D (Fig. 1B). For MEF2A, the divergence in banding
patterns between these muscles was limited to the higher molecular
weight species of this protein. The soleus also displayed higher
amounts of NFATc1 in nuclear extracts compared with its less active
synergist (Fig. 1C). Interestingly, NFATc1 detected in the
nuclei from both muscles displayed multiple bands, consistent with the
notion that partial dephosphorylation of this protein is sufficient to
induce its nuclear import (18).
To investigate whether the enhanced dephosphorylation and expression of
MEF2A and MEF2D observed in more active muscles (i.e. the
soleus) was associated with an increase in transactivational function
of MEF2, we used Tg mice (desMEF2) that express a lacZ reporter gene driven by three high affinity MEF2 cis-elements from the
desmin promoter (11, 19). These DNA binding sites do not preferentially
bind one specific MEF2 isoform (19) and thus report the combined
transcriptional function of all MEF2 proteins. In contrast to a
previous report of positive MEF2 transcriptional activity in ~15% of
the solei from mice of this line (11), and detection of
To address whether the greater dephosphorylation of NFATc1, MEF2D, and
MEF2A in more active versus less active muscles was calcineurin-dependent, we investigated the influence of
calcineurin inhibitors on the phosphorylation status of these
transcription factors in the rat soleus and medial gastrocnemius.
Consistent with a greater activation of calcineurin in the soleus,
inhibition of this phosphatase by treatment of rats with either CsA or
FK506 enhanced the phosphorylation of NFATc1 in this muscle, but not in
the medial gastrocnemius (Fig.
2A). This was characterized in
the soleus by an accumulation (p < 0.05) of the most
PO4 form of NFATc1 (9 ± 3%, 31 ± 0.5%, and
27 ± 8% in vehicle, CsA, and FK506, respectively) and a
reduction (p < 0.05) of the dePO4 form (22 ± 1%, 4 ± 0.3%, and 2 ± 1% in vehicle, CsA,
and FK506, respectively), approaching the range of values seen in the
normal weightbearing medial gastrocnemius (Fig. 2A). Of
note, CsA treatment also induced a subtle phosphorylation of NFATc1 in
the mouse plantaris (Fig. 5A), a fast twitch muscle that
performs less mechanical work than the soleus, but more work than the
gastrocnemius during locomotion (21). On the other hand, administration
of calcineurin inhibitors enhanced the phosphorylation of MEF2D and
MEF2A in both the soleus and medial gastrocnemius, suggesting that
these calcineurin substrates may be preferentially sensitive to the
activation of this phosphatase. For MEF2D, this was characterized by a
6-fold (p < 0.05) increase in the most PO4
form of this protein in the soleus and a 40% increase in the medial
gastrocnemius (Fig. 2A, open arrows). Although, unlike the mouse, a major difference in MEF2A phosphorylation between
normal weightbearing rat soleus and medial gastrocnemius muscles was
not observed, CsA or FK506 treatment induced the appearance of the most
PO4 form of this protein in both muscle types (Fig. 2A). Taken together, these results suggest that calcineurin
activity is positively correlated with muscle usage and emphasize that signaling via this enzyme is not restricted to muscles displaying a
slow phenotype under normal weightbearing conditions.
Nerve-dependent Regulation of Calcineurin--
To
verify that activation of calcineurin in normal weightbearing muscles
was nerve activity-dependent, we investigated whether neuronal quiescence would mimic the effects of CsA and FK506, and
enhance the phosphorylation of calcineurin substrates. To this end, the
sciatic nerve was either severed or chronically superfused with the
sodium channel blocker TTX. Pharmacological blockade of nerve action
potentials with TTX for either 3 (Fig. 2) or 7 days (data not shown)
mirrored the effect of calcineurin inhibition and induced a subtle
increase in the most PO4 form of NFATc1 in the soleus (five
of six experiments) and a more prominent phosphorylation of MEF2D in
both muscle types (4 of 6 solei and 3 of 5 medial gastrocnemius). These
results thus confirm that calcineurin-mediated dephosphorylation of
these substrates in normal weightbearing muscles is nerve
activity-dependent. In contrast, denervation for 3 (Fig. 2)
or 7 days (data not shown) did not influence the phosphorylation status
of these transcription factors in the soleus or medial gastrocnemius.
The failure of denervation to produce the same response as
TTX-inactivation may be related to the fact that, unlike TTX,
denervation induces marked muscle fibrillations (22) and is associated
with increased leakiness of the sarcoplasmic reticulum and higher
intracellular Ca2+ (23), which would presumably activate
calcineurin independent of nerve activity.
Calcineurin-MEF2 Signaling during Overload--
During functional
compensatory OV, the plantaris sustains a rapid doubling of its
activity levels as measured by EMG (17). This increase occurs gradually
during the first 15 days of OV and then stabilizes at levels that are
2-fold greater than control over 30 days of this condition (17). To
address whether the OV-induced enhancement of the calcineurin
dephosphorylation of MEF2A or MEF2D (18) paralleled the time course of
this increase in muscle recruitment, we assessed the phosphorylation
status of these proteins over 21 days of this condition. Plantaris OV induced a progressive dephosphorylation of both MEF2A and MEF2D compared with sham-operated controls, evidenced by a gradual decrease in the relative amount of the most PO4 form of each of
these proteins that closely matched the time course of increased
plantaris EMG with this condition (17) (Fig.
3B). OV also induced a
progressive increase in MEF2D protein content, whereas the expression
of MEF2A was transiently higher at 5-7 days of OV, returning near
control levels by 14 days of this condition (Fig. 3A). These
adaptations in MEF2A and MEF2D in response to OV appeared mediated by
calcineurin, as they were either fully (i.e. MEF2D) or
largely (i.e. MEF2A) prevented in mice treated with CsA or
FK506 (Fig. 3A, right panels). The matching of
dephosphorylation of MEF2A and MEF2D with OV muscle activation profiles
(17) suggests that signaling of calcineurin to each of these
transcription factors is mediated by the increase in muscle fiber
recruitment associated with this condition. On the other hand, the
difference in timing of MEF2A and MEF2D responses to OV suggests that
signaling of calcineurin to each of these proteins may be subject to
distinct regulatory mechanisms (i.e. activity
versus increases in insulin-like growth factor, etc.).
The absence of MEF2 reporter gene expression in
cross-sections of normal weightbearing muscles, despite a clear
calcineurin-dependent dephosphorylation of MEF2A and MEF2D,
raised the possibility that complete signaling of this phosphatase via
these proteins may only occur when muscle activation is above normal
levels. To test this notion, we investigated whether OV would induce an
increase in MEF2 transcriptional function in the plantaris. Indeed, OV rapidly (within 2 days) induced the expression of the MEF2 reporter in
a large number of plantaris fibers (Fig. 3, C-E). Peak
expression of the MEF2 reporter occurred in plantaris cells at 5-7
days of OV and was largely compromised in mice treated with calcineurin inhibitors (Fig. 3E), thus mirroring the adaptations in
MEF2A protein expression at this time (Fig. 3A). Positive
In accordance with principles related to motor unit recruitment (24), a
majority of the increase in activity in overloaded muscles is sustained
by smaller, lower threshold slow (S) and fast fatigue-resistant (FR)
motor units (respectively associated with fibers expressing I and IIa
MHC). OV also results in a doubling of the normal recruitment levels of
larger, usually silent, higher threshold fast fatigue-intermediate (FI)
and fast fatigable (FF) motor units (respectively associated with
fibers expressing IIx and IIb MHC). To assess whether MEF2
transcriptional function was increased in response to OV and followed
the general recruitment order ((S, type I) = (FR, type IIa) > (FI, IIx) > (FF, IIb)], we examined the MHC phenotype of
In the latter series of experiments, we were unable to assess whether
an increase in activity above normal would induce calcineurin-MEF2 signaling in type I fibers, because these cells are extremely rare in
the plantaris in this line of Tg mice. Indeed, only 1 of 33 desMEF2 Tg
mice examined displayed type I fibers in the plantaris, and none
stained positive for Calcineurin-NFATc1 Signaling in OV Muscles--
To investigate
whether calcineurin signaling to NFATc1 is also driven by increases in
nerve-mediated activity, we assessed the phosphorylation status of
NFATc1 in plantaris whole cell extracts over the time course of OV.
Consistent with MEF2D findings, Western blots showed that OV induced a
rapid, although subtle, dephosphorylation of NFATc1 that was apparent
as early as 1 day after surgery and persisted into the longer term of
this condition (Fig. 5A). This shift
in phosphorylation profile was characterized by an increase (p < 0.05) in the dePO4 band relative to
total NFATc1 protein (from 3 ± 1% in control to 14 ± 6%
in 5-day OV tissues). This dephosphorylation of NFATc1 was also
associated with an enrichment of this protein in nuclear extracts in
the early phase of OV (Fig. 5B). Both whole cell and nuclear
adaptations in NFATc1 were mediated by calcineurin, because they were
prevented in mice administered either CsA (Fig. 5, A
(right panel) and B) or FK506 (data not shown).
To verify that NFATc1 was enriched in the nucleus of OV plantaris
cells, we injected control and OV plantaris muscles with a DNA plasmid
that encodes full-length NFATc1 linked to GFP (NFATc1-GFP) (7). This
approach was used because commercial antibodies generated against
NFATc1 cross-react with other lower molecular weight Rel domain
proteins,3 thus rendering
localization of this protein difficult with conventional immunohistochemistry. By 5 days after injection, a large percentage (>80%) of control plantaris muscle cells, primarily the smaller, more
metabolically active fibers, expressed the GFP fusion protein (data not
shown). Under normal weightbearing conditions, NFATc1-GFP co-localized
with myonuclei in ~10% of all plantaris fibers (Fig. 5C).
The percentage of fibers that displayed nuclear-localized NFATc1-GFP
tended (p > 0.05) to be higher after OV (Fig.
5C), lending modest support to our Western blot findings of
an enrichment of this protein in the nuclear fraction (Fig.
5B) with this condition. Nonetheless, in both control and OV
muscles, the bulk of NFATc1-GFP protein was localized throughout the
cytoplasmic compartment of each fiber with the highest levels found
within the subsarcolemmal region (Fig. 5, compare D and
E). To verify that the GFP tag did not influence the
localization of the NFATc1-GFP fusion protein, normal weightbearing
muscles were injected with a plasmid that encodes a variant of
NFATc1-GFP (
Although for the most part, the dephosphorylation of NFATc1 was subtle
over the time course of OV, a substantial increase in the lowest
molecular weight, dePO4, form of this protein was observed
at 2-5 days of OV (Fig. 5A). Because the timing of this phenomenon coincided with the OV-induced emergence of a small to modest
number of mature (n = 8 ± 2/muscle cross-section)
and nascent (n = 84 ± 33/muscle cross-section)
fibers that displayed central nuclei, we assessed the contribution of
possible regeneration/myogenic-related events to the significant
OV-related dephosphorylation of NFATc1 at 2-5 days. To this end, we
induced fiber regeneration in the distal half of the plantaris via
freeze injury and subsequently examined the phosphorylation and nuclear
localization of NFATc1 in the injured portion of this muscle at 3 days
after injury. We found that regeneration induced a significant
dephosphorylation of NFATc1 that matched the response observed after
2-5 days of OV (Fig. 5, compare A and J).
Regenerating muscle fibers also displayed a prominent nuclear
localization of NFATc1-GFP (Fig. 5, compare H and
I). These data suggest the enhanced dephosphorylation of
NFATc1 that occurred in the short-term of OV may be related in part to
the OV-induced appearance of these nascent fibers. Of note, NFATc1 was
not the only target of calcineurin in regenerating fibers, because an
enhanced dephosphorylation of MEF2A and MEF2D was also observed in
extracts from freeze-injured muscles (Fig. 5J). On the other
hand, involvement of MEF2 in the regeneration process requires further
examination, because expression of the MEF2 reporter was not detected
in regenerating portions of the remnant gastrocnemius in desMEF2 Tg
mice after 5 days of plantaris OV (data not shown). Taken together,
these findings suggest that NFATc1 may have a dual role in OV muscles:
one related to nerve-mediated signaling and the other to the
establishment of nascent fiber phenotype.
Activation of Calcineurin by Nerve Electrical Stimulation--
To
provide insight into what specific aspect of the nerve electrical
stimulus (i.e. pulse frequency, aggregate amount, etc.) is
key to activating this phosphatase in muscle cells, we compared the
ability of various nerve electrical stimulation paradigms to
dephosphorylate NFATc1 and MEF2 proteins in the soleus and medial
gastrocnemius. In contrast to OV, which imposes the largest increases
of activity on smaller, low threshold motor units, electrical stimulation of the sciatic nerve at supramaximal voltage simultaneously activates all motor unit types. We found that activation of the mouse
sciatic nerve for 1 h with 10-Hz continuous stimuli, a frequency typical of S motor units, resulted in a
calcineurin-dependent dephosphorylation of MEF2D in the
fast medial gastrocnemius (Fig. 6A, top).
Interestingly, a similar MEF2D response occurred when this muscle was
activated intermittently for 1 h with bursts of high frequency
(100 Hz) stimuli, a pulse frequency native to FF motor units (Fig.
6A, top), but only when these bursts were
administered in close enough succession (interburst interval
In contrast to findings in the medial gastrocnemius, nerve-mediated
activation of the soleus for 1-2 h with either the 10-Hz continuous or
100-Hz/30 s stimulation patterns did not potentiate the
dephosphorylation of MEF2D (Fig. 6A, bottom),
NFATc1 or MEF2A (data not shown). Likewise, none of the stimulation
paradigms induced detectable expression of the MEF2 reporter in
cross-sections this muscle (Fig. 6E), suggesting the already
high calcineurin activity in these cells may render them refractory to
relatively modest amounts of additional nerve activity. Taken together,
these data further substantiate that the calcineurin pool in the
largest and least active cells is relatively more sensitive to
increases in contractile activity and reinforce the concept that a
significant increase of muscle usage above native levels is required to
fully activate MEF2 in skeletal muscles.
The purpose of the present study was to test the hypothesis that
skeletal muscle calcineurin signaling pathways are sensitive to
nerve-mediated activity. To this end, we investigated whether calcineurin is more active in highly recruited muscles under normal weightbearing conditions and whether signaling via its substrates NFATc1, MEF2A, and MEF2D is potentiated in a way that matched increased
activation profiles of muscles subjected to functional OV. Moreover, to
directly assess the involvement of the electrogenic stimulus in the
activation of this phosphatase in skeletal muscle cells, we examined
whether calcineurin signaling via these substrates would be countered
by neuronal quiescence or enhanced in response to electrical
stimulation of the sciatic nerve. Here, we confirm our hypothesis and
show the extent of calcineurin dephosphorylation of NFATc1 and MEF2 to
be positively correlated with muscle usage in normal weightbearing
muscles. Moreover, this response was nerve activity-dependent, because it was countered by silencing
sciatic nerve action potentials with TTX. We also establish that
complete signaling of this phosphatase via MEF2A or MEF2D proteins
(i.e. leading to detectable increases in MEF2
transcriptional function) largely occurs when nerve activation is
increased above normal levels such as with OV, supporting our
contention that calcineurin likely cooperates with other
activity-linked signaling pathways to effect a response via these
proteins (18). Interestingly, this triggering of MEF2 transcriptional
function by calcineurin occurred in all fiber types in response to
increased activity, but most readily in fibers that are normally the
least active, suggesting that signaling via this phosphatase is also
influenced by the activation history of the muscle cell. Taken
together, these findings provide novel insight toward our
understanding of the nerve activity-dependent modulation of
calcineurin signaling in adult skeletal muscle cells in
vivo.
Calcineurin Dephosphorylation of NFATc1 and MEF2 Is Nerve
Activity-dependent and Correlated with Muscle
Usage--
It is postulated that sustained Ca2+ elevations
such as those that occur in slow muscle fibers in response to tonic
firing of their slow motor nerve are conducive to the activation of
calcineurin (7). It is also thought that this phosphatase is refractory to the brief Ca2+ transients that are evoked in fast muscle
cells in response to phasic firing of their fast motor nerve (7). We
provide evidence that validates this model, in part, by showing that
under normal weightbearing conditions calcineurin is activated to a
greater extent (i.e. more extensive dephosphorylation of
NFATc1, MEF2A, and MEF2D) in the soleus, a muscle that is highly
recruited (20) and displays 2-fold higher resting intracellular
Ca2+ levels compared with less active hindlimb muscle
counterparts (26). Moreover, we provide first time evidence that this
phosphatase is activated in a nerve activity-dependent
fashion, because the dephosphorylation of NFATc1 and MEF2 proteins in
these hindlimb muscles was countered by silencing sciatic nerve action
potentials with TTX. On the other hand, our finding of a subtle
calcineurin-dependent dephosphorylation of these substrates
in the predominantly fast twitch plantaris or medial gastrocnemius
suggests that this enzyme is not completely refractory to the activity
profiles of fast motor units. Indeed, given the dephosphorylation of
NFATc1, MEF2A, and MEF2D was positively correlated with the extent of
recruitment of these plantar flexors (i.e. soleus > plantaris > medial gastrocnemius) under normal weightbearing
conditions (20, 21), we thus refine this model and propose that the
daily aggregate amount of nerve-mediated muscle usage and not the fiber
type per se, is the key factor influencing calcineurin
activity. When these results are considered with findings that
calcineurin-dependent dephosphorylation of NFATc1, MEF2A,
and MEF2D is potentiated in plantaris muscles subjected to OV, they
provide evidence that all three of these transcription factors are
targets of calcineurin downstream of nerve activity.
Muscle Activity above Native Levels Is Prerequisite for Full
Activation of MEF2--
Previous studies of cultured neurons and
skeletal myocytes and whole muscle tissues have shown that
dephosphorylation of MEF2A by calcineurin may be associated with an
increase in the transactivating function of this protein (11,
27).2 Our finding of an absence of MEF2 reporter gene
expression in all normal weightbearing muscles including the soleus,
despite a clear calcineurin-dependent dephosphorylation of
MEF2A and MEF2D, appear to be in sharp contrast to these previous
findings. The fact that in the present study, MEF2 transcriptional
activity was only observed in overloaded plantaris and soleus muscles, and in type IIb fibers that were electrically stimulated in amounts that presumably exceeded their native activity levels, suggest that: 1)
under normal weightbearing conditions, there exist molecular mechanisms
that keep MEF2 from being fully active and, 2) an increase in
nerve-mediated activity initiates signaling events that either relieve
this repression or fully enhance the transcriptional function of MEF2.
Indeed, recent converging lines of evidence suggest that calcineurin
cooperates in a synergistic fashion with
Ca2+/CaM-dependent kinases (CaMKs) to activate
MEF2 proteins in skeletal and cardiac muscle cells (11, 28). Activation
of CaMKs appears to permit MEF2 transcriptional function by promoting
the dissociation of the class II histone deacetylases from the DNA
binding domain of MEF2 proteins (29, 30). In light of these results and
our finding of an absence (this study) or a low amount (~15% of
solei examined; Ref. 11) of MEF2 reporter gene expression in the normal weightbearing soleus of desMEF2 Tg mice, it is tempting to speculate that soleus contractile activity levels, although able to activate calcineurin, may just be below the threshold for CaMK activation. Although the present study did not address whether increased muscle activation above normal is required for complete signaling of calcineurin via NFATc1, previous findings that overexpression of both
calcineurin and CaMK IV are required for full activation of NFAT1 in
T-lymphocytes (31) suggests that this may indeed be the case.
It is proposed that frequent muscle fiber activation and contractile
loading activate calcineurin signaling pathways, which lead to fiber
hypertrophy and the expression of a slower contractile protein
phenotype (15, 18). Our finding of negligible amounts of
calcineurin-MEF2 signaling in the normal weightbearing soleus (this
study and Ref. 11) coincides with previous reports that this
phosphatase has a minor influence on the phenotype of this muscle in
that CsA only induces subtle transitions in MHC expression after long
term administration of this drug (7, 16). Similarly, our observation of
a prominent induction of MEF2 reporter gene activity in both overloaded
plantaris and soleus fibers concurs with our previous findings that
calcineurin-dependent pathways have a profound effect on
the size and MHC phenotype of skeletal muscle cells during compensatory
growth (15, 18). Collectively, this suggests that expression of the
MEF2 reporter is much more effective than the phosphorylation status of
MEF2 or NFATc1 proteins as an indicator of the degree to which
calcineurin signaling pathways are impacting muscle fiber phenotype.
Reporter gene data thus provide additional evidence of an involvement
of MEF2A or MEF2D proteins in the signaling of fiber hypertrophy and
fast-to-slow fiber type transformations in response to OV
(11, 18).
The dephosphorylation and induced transactivational activity of MEF2D
observed in the plantaris in response to OV was likely nerve
activity-dependent, because it followed closely the
reported increase in EMG activity that occurs in this muscle under
similar conditions (17). On the other hand, the MEF2A response to OV was more complex and also involved a transient increase in the expression of this protein at 5-7 days that could not be totally explained by the timing of the increase in muscle recruitment levels
(17). The fact that this latter response was unique to OV muscles, and
was only partially prevented with CsA, suggests that other parallel
signaling pathways, active in the early phase of muscle growth, may
cooperate with calcineurin to amplify signaling via MEF2A. Indeed,
several lines of evidence suggest that insulin-like growth factor
(IGF)-1 may act as a parallel signaling effector of MEF2A and an
inducer of muscle growth. First, muscle IGF-1 levels increase in
vivo in a transient fashion in response to OV (32), the timing of
which just precedes the observed increase in MEF2A transcriptional
function. Moreover, application of IGF-1 to cultured skeletal myocytes
increases intracellular Ca2+ and activates calcineurin (33)
and CaMK (30), both purported to induce hypertrophy of these cells, in
part, via activation of MEF2 (30). The apparent requirement of
accessory signaling events for the induction of MEF2 transcriptional
function in skeletal muscles coincides with our previous
demonstrations, and those of others, that activation of calcineurin is
required, but alone not sufficient, to induce muscle fiber hypertrophy
(18, 34) or fast-to-slow fiber type conversions (18, 35).
Fast Fiber Calcineurin Pool Appears Preferentially Sensitive to
Nerve-mediated Activity--
Muscle intracellular Ca2+
transients evoked by fast nerve activity were originally proposed to be
unsuited for calcineurin activation (7). In contrast to this notion, we
show that calcineurin signaling via MEF2 proteins is actually initiated
most readily in the fastest, least active fibers (those expressing IIx
or IIb MHC) in response to OV or electrical stimulation. Given the
apparent requirement of increased activation for full induction of the
MEF2 reporter, the ready initiation of calcineurin-MEF2 signaling in
IIx and IIb fibers may relate to the fact that these cells are normally recruited for only seconds per day (2) and thus would respond quickly
to what would appear to be a minimal foreign activity stimulus, but for
these cells would be a substantial increase from native activity
profiles. In contrast, slow twitch fibers are recruited for a large
portion of the day (22-33% of daily time; Ref. 2) and would require
more of a deviation from their native activity levels to initiate such
signaling events. This is consistent with the finding that soleus MEF2
reporter expression was refractory to all electrical stimulation
paradigms. Alternatively, the calcineurin pool in IIx and IIb fibers
may be comparatively more sensitive to modulation by motor nerve
activity. Indeed, several lines of evidence support this notion. For
one, the catalytic subunit of calcineurin is expressed at comparatively
higher levels in fast twitch muscles (35), and thus may convey greater
signaling potency to fast fibers. In addition, transcript levels of
myocyte-enhanced calcineurin interacting protein I and II, proteins
that inhibit calcineurin signaling in muscle cells, are preferentially
expressed at lower levels in fast compared with slow twitch skeletal
muscles (36), thereby potentially lowering the threshold for
calcineurin activation in the former cell types. Finally, greater
responsiveness of IIx or IIb fibers to activation via the
calcineurin-MEF2 pathway is also consistent with reports that these
cells are the most sensitive and the first to adapt to increased
nerve-mediated activity (3, 15, 37).
Frequent Nerve-mediated Depolarization of Muscle Cells Is Required
for Calcineurin Activation--
To elucidate which aspect of the
electrogenic stimulus is important in activating calcineurin in
hindlimb muscle cells, we compared the efficacy of various exogenous
nerve electrical stimulation paradigms in the induction of
calcineurin-dependent dephosphorylation of NFAT and MEF2
proteins. The 10-Hz continuous paradigm that we used mimicked the pulse
frequency and tonic nature of slow motor units (2), whereas the
intermittent 100-Hz burst stimulation paradigm mimicked the pulse
frequency native to faster (fast fatigue-resistant and fast fatigable)
motor unit types (2). Although the aggregate activity associated with
1-2 h of 10-Hz continuous electrical stimulation was well below levels
required to initiate calcineurin-MEF2D signaling in the soleus, it was
sufficient to activate this pathway in plantaris and medial
gastrocnemius IIb fibers. The effectiveness of 10-Hz continuous
stimulation in activating calcineurin in these fastest fibers is
consistent with previous findings that this paradigm elicits a
sustained elevation of muscle Ca2+ (0.15-0.3
µM) (8) toward levels known to initiate calcineurin signaling in B-lymphocytes (38). Interestingly, we found that an
intermittent 100-Hz stimulus pattern was just as effective as the 10-Hz
continuous paradigm in inducing a calcineurin-dependent dephosphorylation of MEF2D when the interburst interval was
sufficiently brief ( Calcineurin Substrates Are Differentially Sensitive to Calcineurin
Activation--
Another interesting finding of this study was that
NFATc1 and MEF2A were less readily dephosphorylated compared with MEF2D in response to calcineurin activation. One possible explanation for the
difference in the sensitivity of MEF2 and NFATc1 proteins is that MEF2
resides in the nucleus of muscle cells (39) and thus would be exposed
to a different Ca2+ milieu compared with cytoplasmic
localized NFATc1 (25). Indeed, during excitation-contraction coupling,
the Ca2+ transients evoked in the nucleus of muscle cells
are more sustained (i.e. have slower rise and decay times)
compared with cytoplasmic levels (40), thereby promoting calcineurin
activity. Nonetheless, the fact that dephosphorylation of MEF2D was
more complete than MEF2A in the OV plantaris or in the stimulated
medial gastrocnemius, despite their shared nuclear localization,
suggests that there also exist differences in the sensitivity of these
different MEF2 proteins to activation by calcineurin. The underlying
basis for this difference is not known, but may relate to the fact that the different MEF2 isoforms are divergent in their transcriptional activation domain and thus may not be subject to the same regulation by
putative opposing kinases (39). In this regard, p38 and ERK5 kinases
have been shown to phosphorylate MEF2A, but not MEF2D (41-43).
Nonetheless, the finding that a CsA-sensitive dephosphorylation of
NFATc1 and MEF2A does occur in the normal weightbearing soleus and is
induced in the plantaris by 1 day of OV, suggests that a relatively
more extended period of chronic muscle usage than the exogenous
paradigms that we administered is required to elevate cytoplasmic
Ca2+ levels or to create the cellular conditions necessary
to initiate calcineurin signaling via these substrates in muscle cells.
Nerve-independent Activation of Calcineurin Signaling in Newly
Differentiated Muscle Cells--
Although the
calcineurin-dependent dephosphorylation and nuclear
localization of NFATc1 was clearly sensitive to nerve-mediated activity, the finding of a substantial dephosphorylation of this protein in regenerating muscles at a time when muscle cells would still
be aneural (44), suggests that nerve-mediated increases in
Ca2+ are not the sole requirement for activation of
calcineurin-NFAT signaling in skeletal muscle cells. Thus, our results
support previous demonstrations of an importance of Ca2+
and calcineurin-NFAT signaling in the regulation of myogenic commitment
(45, 46) and muscle regeneration (12, 47). Calcineurin acts via
distinct NFAT isoforms to signal discrete events in myocyte development
such that NFATc3 appears to be involved in the conversion of myoblasts
to myotubes (45), whereas NFATc2 appears important for myotube fusion
(47). Our findings of a prominent nuclear localization of NFATc1 in
nascent muscle fibers in regenerating muscles coincides with reports
that this isoform of NFAT preferentially signals downstream of
calcineurin in fully differentiated muscle cells (12, 45) and suggests
that this protein may play a role in the establishment of the new
myofiber phenotype. Although we did show muscle regeneration to
potentiate the dephosphorylation of MEF2A and MEF2D in the plantaris,
MEF2 reporter gene expression was not detected in nascent fibers in cross-sections of the plantaris or remnant portions of the regenerating gastrocnemius in OV mice, suggesting that calcineurin-MEF2 signaling may play a comparatively minor role at this stage of myogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IIx IIa I) in the overloaded (OV)
plantaris (15), and induced a subtle atrophy and shift toward
expression of faster contractile proteins in the normal weightbearing
soleus (7, 16), by administration of the calcineurin inhibitors
cyclosporin A (CsA) or FK506. Although the importance of
Ca2+ and calcineurin-dependent signaling
pathways in regulating fiber phenotype is established, the role of
nerve-mediated activity as an in vivo upstream modulator of
calcineurin activity in skeletal muscles has yet to be confirmed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Galactosidase
(-gal) Activity--
The desMEF2 Tg mice were identified by
polymerase chain reaction screening of genomic DNA for the presence of
the lacZ transgene using the following primers: sense
(5'-TGCCGTCTGAATTTGACCTG-3') and antisense
(5'-GCATAACCACCACGCTCATC-3'). The lacZ transgene is not
completely penetrant in this line of mice (11). Thus, to ensure that
experimental desMEF2 Tg mice had the potential for lacZ
expression, they were also screened for induction of -gal activity
in the right medial gastrocnemius in response to a paradigm of nerve
electrical stimulation that consistently induces the expression of the
transgene in this muscle: 3 half-hour sessions of intermittent 100-Hz
stimulation (1 train every 30 s), with each session separated by a
4-h interval. The medial gastrocnemius muscle of the electrically
stimulated limb was collected the following day and was quick-frozen in
melting isopentane, pre-cooled in liquid nitrogen. For OV animals, this
muscle was collected during the synergist ablation surgery. To assay
-gal activity, cryosections (14-20 µm) were cut from each muscle
midbelly and were fixed in 1% glutaraldehyde in 0.1 M
phosphate-buffered saline (PBS), pH = 7.4 for 10 min at 4 °C.
Tissue sections were then rinsed three times for 5 min each time in 0.1 M PBS at 4 °C and incubated in the dark at 37 °C for
3-12 h in a solution containing 0.1 M MgCl2, 0.5 M potassium ferricyanide, 0.5 M
ferrocyanide, and 40 mg/ml 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal) in 0.2 M PBS.
After a final two 5-min rinses in PBS, tissues were mounted in 50%
glycerol in PBS. Ninety percent of mice displayed positive staining for
-gal in the right, stimulated medial gastrocnemius, and muscles in
the left, nonstimulated limb of these mice were used for subsequent experiments.
NFATc1-GFP) were
obtained from Dr. E. R. Chin (7). Plasmids were extracted with
chloroform and phenol and ethanol-precipitated overnight (
20 °C).
The following day, DNA was resuspended in a sterile solution of 10%
sucrose in 1× PBS (25 mM, pH = 7.4) at a
concentration of 2 µg/µl. Mice were anesthetized, and the medial
gastrocnemius was incised to expose the distal portion of the
plantaris. Ten µl of this DNA solution was then injected into the
muscle using a 30-gauge insulin syringe. To maximize the cellular
uptake of the plasmid, muscles were pre-injected with 10 µl of 10%
sucrose in PBS, 15 min prior to the injection of DNA. Muscles were
excised from mice at 5 days after injection and then frozen in melting
isopentane. Cryosections (20 °C) of the distal plantaris were cut,
fixed in methanol (20 °C) for 5 min, and then incubated with
bisbenzimide (0.05 µg/ml) in PBS for 30 min in the dark to stain
nuclei. Sections were subsequently rinsed (three times for 10 min each
in PBS) and exposed to UV or fluorescein isothiocyanate illumination to
visualize nuclei (at 465 nm) and NFAT-GFP fusion proteins (at 500 nm), respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Calcineurin-dependent
dephosphorylation of NFATc1, MEF2D, and MEF2A is more prevalent in more
active muscles. A and C, Western blots of
NFATc1, MEF2D, MEF2A, or MEF2C in whole cell (A) or nuclear
extracts (C) prepared from normal weightbearing mouse soleus
(mSOL) or mouse medial gastrocnemius (mMG)
muscles. Samples in the middle and right
panels of A are medial gastrocnemius extracts
that were pretreated with 0 ( ), 100 (+), or 200 (++) units of AP.
Lanes in A represent individual muscles whereas
those in C are pooled muscle samples (n = 3/lane). MEF2C served as a protein loading control. B,
relative abundance of the most phosphorylated (most PO4)
and dephosphorylated (dePO4) form(s) of NFATc1, MEF2D, and
MEF2A, as a percentage of the total amount of detected protein. Values
represent means ± S.E., n = 3-4 muscles/group.
Asterisks denote differences (p < 0.05).
Note that, for mouse medial gastrocnemius MEF2A, double
dePO4 bands were pooled for quantification.
-gal in
Western blots of pooled (n = 5-7 muscles/lane) soleus
samples,2 we did not observe
any positive staining for -gal activity under normal weightbearing
conditions in any soleus cross-sections of the 15 desMEF2 Tg mice
examined, despite the fact that all of these mice displayed potential
for expression of the transgene (data not shown; see "Experimental
Procedures"). Expression of the MEF2 reporter was not detected in
cross-sections of normal weightbearing plantaris and medial
gastrocnemius muscles, nor in Western blots of plantaris, EDL, and
white vastus muscles2 of these Tg mice. These findings thus
suggest that dephosphorylation of MEF2A and MEF2D by calcineurin does
not necessarily lead to an increase in MEF2 transcriptional function in
normal weightbearing muscles.
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Fig. 2.
Effects of calcineurin inhibitors and neural
inactivation on the phosphorylation of calcineurin substrates in target
muscles. A and B, Western blot analysis of
NFATc1, MEF2D, MEF2A, and MEF2C in whole cell extracts prepared from
rat soleus (rSOL) or medial gastrocnemius (rMG)
muscles after 28 days of vehicle (Veh; n = 3), CsA (n = 3), or FK506 (n = 2)
treatments (A) or after 7 days of sham treatment
(n = 3), denervation (DEN, n = 4), or TTX-induced neural inactivation (TTX,
n = 6) (B). Blots in A and
B are representative of three to five independent
experiments. Filled arrows denote the dePO4
form, and open arrows the most PO4 form, of each
protein as determined by treatment of samples with AP (data not shown).
MEF2C served as a loading control for total protein. Note that, in
contrast to the mouse, only one detected dePO4 form of
NFATc1 was detected in rat muscle.
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Fig. 3.
OV induces a
calcineurin-dependent dephosphorylation of MEF2A and MEF2D,
and activation of a MEF2 reporter in the plantaris. A,
Western blot analysis of MEF2A and MEF2D in plantaris whole cell
extracts (n = 3 samples/group) over a time course of OV
(left panel) or after 5 days of OV in the absence
(vehicle-treated) or presence of calcineurin inhibitors, CsA, and FK506
(right panel). Coomassie Blue staining was used as a protein
loading control. Filled arrows denote the dePO4
form, and open arrows the most PO4 form, of each
protein. Western blots are representative of two to five independent
experiments. B, relative abundance of the most
PO4 form (open arrow) of MEF2A and MEF2D
expressed as a percentage of the total amount of detected protein.
Values are means ± S.E., n = 2-3 samples/group.
C and D, cross-sections of plantaris muscles from
desMEF2 Tg mice after normal weightbearing conditions (C) or
after 5 days of OV (D) stained for -gal activity.
E, number of fibers in midbelly cross-sections of desMEF2 Tg
plantaris muscles that stained for -gal activity in control mice and
after a time course of OV, or in pooled muscle sections after 5 days of
OV plus CsA or FK506 treatment. Values are means ± S.E.
(n = 3-5 muscles/group). Note that the time of highest
-gal activity coincides with the peak of MEF2A expression.
-gal staining was detected in a modest number of fibers at 14 or 21 days of OV (Fig. 3E), thus matching the persistent
dephosphorylation of MEF2A and MEF2D in the longer term of this
condition (Fig. 3, A and B).
-gal-positive cells in OV muscle cross-sections of desMEF2 Tg mice.
At the onset of this condition (2 days), the MEF2 reporter was detected
in all three subsets of fast fibers, particularly in those expressing
IIx MHC (Fig. 4A). This
finding suggests that the largest, least active fiber types
(i.e. those expressing IIx or IIb) are initially the most responsive to increases in muscle activation. From day 5 to day 21 of
OV, the majority of cells that were positive for -gal activity were
the small, highly recruited IIa fibers (Fig. 4A; compare B and C), although a significant number of
positive IIx and IIb fibers were still detected. Taken together, these
data suggest that the most important determinant of significant and
sustained MEF2 transactivating function with OV appears to be the
nerve-mediated imposition of fiber recruitment levels substantially
above native levels relative to each cell type. Finally, the finding
that expression of the MEF2 reporter was only detected in rare (~1%)
nascent fibers (n = 84 ± 33/muscle) that are
characteristic of 5-day OV muscles (compare Fig. 4, F and
G) emphasizes that the activity-dependent induction of calcineurin-MEF2 signaling is a phenomenon restricted to
mature muscle fibers.
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Fig. 4.
MEF2 reporter gene expression is rapidly
induced in all fibers with OV. A, percentage of
-gal-positive fibers that expressed IIa, IIx, or IIb MHC after
different durations of OV. Values are means ± S.E.
(n = 2-4 muscles/group). B-G,
cross-sections of plantaris muscles from desMEF2 Tg mice after 5 days
of OV stained for -gal activity (B and F) or
immunolabeled for type IIa MHC (C), all MHCs except IIx MHC
(D), IIb MHC (E), or embryonic MHC
(G). Panels F and G show a region in
one OV muscle that contained regenerating fibers. The
asterisk and triangles in B-E denote
-gal-positive fibers that express IIb or IIx MHC, respectively.
Filled arrows in F and G denote the
same cells in these serial cross-sections. Note that the majority of
-gal-positive cells in panel B express IIa MHC (compare
B and C). Note also the paucity of -gal
activity in regenerating fibers that display positive staining for
embryonic MHC in OV muscles (compare F and G).
H-J, cross-sections of soleus muscles from desMEF2 Tg mice
after 5 days of OV stained for -gal activity (H) or
immunolabeled for type I (I) or IIa MHC (J).
Daggers and the short arrow denote
-gal-positive fibers that express type I and IIa MHC,
respectively.
-gal activity. Thus, in a separate set of
experiments, we investigated whether OV would induce the expression of
the MEF2 reporter in type I fibers in the soleus, a muscle comprising
~50% of this cell type (data not shown). Staining for -gal
activity was detected in both fibers expressing I or IIa MHC in this
muscle after 5 days of OV (compare Fig. 4, H-J), confirming
that all cell types have the potential for induction of the MEF2 reporter.
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Fig. 5.
Calcineurin-NFATc1 signaling is increased in
OV and regenerating muscles. A and B,
Western blot analysis of NFATc1 in plantaris whole cell (A)
and nuclear (B) extracts after different durations of OV
(left panel in A) or after 5 days of OV in the
absence or presence of CsA (right panel in A,
B). Coomassie Blue staining was used as a protein loading
control for blots in A and MEF2C for blots in B.
Results are representative of three independent experiments.
C, percentage of fibers displaying nuclear-localized
NFATc1-GFP after 5 days of control or OV treatment. Values are
means ± S.E. (n = 3 muscles/group).
D-I, cross-sections of control (D-G) or
regenerating (H and I) plantaris muscles injected
with a plasmid encoding GFP linked to either full-length NFATc1
(D, E, H, I) or a truncated
variant of this protein that is constitutively localized to the nucleus
(F and G). Sections of these muscles were first
incubated with bisbenzimide to stain myonuclei and then exposed to
fluorescein isothiocyanate illumination to visualize GFP fusion
proteins (D, F, H), or UV illumination
to visualize myonuclei (E, G, I).
Asterisks in D and E denote examples
of mature fibers in control muscles that display nuclear-localized
NFATc1-GFP. White filled arrows in D-I denote
nuclei that co-localize with GFP fusion proteins. J, Western
blot analysis of NFATc1, MEF2A, and MEF2D in whole cell extracts
prepared from control or regenerating (Regen) plantaris
muscles. Regenerating muscles were collected 3 days after freeze
injury. Lanes represent pooled muscle samples
(n = 3/condition). For Western blots, filled
arrows denote the dephosphorylated, and open arrows,
the most phosphorylated form(s) of these calcineurin substrates.
1-318) (7), lacking a region in the protein that masks
the nuclear localization sequence (25). NFATc1(1-318)-GFP always
co-localized with myonuclei in all muscle fibers examined (compare Fig.
5, F and G), thus supporting the notion that the
GFP tag itself was not responsible for the treatment-induced subcellular localization of these fusion proteins.
1 min) (Fig. 6B). Consistent with MEF2D dephosphorylation
findings, we found that electrical stimulation of desMEF2 Tg mice for
30 min, three times daily (each session separated by 3 h) with
either the 10-Hz continuous or 100-Hz/30 s paradigms consistently
induced an increase in MEF2 transcriptional activity in some fibers
expressing IIb MHC in the medial gastrocnemius (Fig. 6, compare
C and D). These paradigms also induced expression
of the MEF2 reporter in some fibers expressing IIb in the plantaris
(Fig. 6E). Given that the aggregate activity evoked by these
paradigms (1 h and 200 s, respectively) largely exceeds the mean
activity per hour of FF motor units (1.4-8.0 s/h; Ref 2) and
associated IIb fibers, these data thus suggest that calcineurin-MEF2D
signaling in fibers expressing this MHC is initiated when the aggregate
amount of activity exceeds normal levels or when the interval between
activation bursts is sufficiently brief (1 min). In contrast to MEF2D
findings, neither paradigm of stimulation was effective in
dephosphorylating MEF2A or NFATc1 in the medial gastrocnemius muscle
(Fig. 6A, middle panels), even after several
hours (data not shown), suggesting that MEF2D is relatively more
sensitive to nerve-mediated dephosphorylation via calcineurin.
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Fig. 6.
Nerve electrical stimulation induces a
calcineurin-dependent dephosphorylation of MEF2D and
expression of the MEF2 reporter in type IIb fibers. A
and B, Western blots of MEF2D, MEF2A, and NFATc1 in whole
cell extracts of the mouse medial gastrocnemius (MG) or
soleus (SOL) under normal weightbearing conditions
(Control) or after 1 h of 10-Hz continuous or
intermittent 100-Hz burst stimulation of the sciatic nerve, in the
absence or presence of CsA. For the latter pattern, 100-Hz bursts were
administered once every 30 s (in A), 1 min, 2 min, 5 min, 10 min, 15 min, or 30 min (in B). Filled
arrows and open arrows in A and
B, respectively, indicate the dephosphorylated and
phosphorylated form(s) of these proteins. Results in A and
B are representative of at least three independent
experiments. MEF2C served as a loading control. C-E,
cross-sections of mouse medial gastrocnemius (C and
D) or soleus (SOL) and plantaris (PL)
(E) muscles of desMEF2 Tg mice stained for -gal activity
(C and E) or immunolabeled for IIb MHC
(D). Note that -gal-positive cells in C-E
were typed as expressing IIb MHC.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 min), suggesting that the aggregate amount of
activity or the integral of the Ca2+ signal are key to
activating calcineurin, rather than the pulse frequency or associated
amplitude of the Ca2+ transient. Nonetheless, the finding
that MEF2 reporter gene was not induced in slower type I, IIa, or IIx
fiber types by any of these stimulation paradigms supports the
contention that muscle activation above native levels is required to
induce a significant increase in calcineurin signaling via MEF2 proteins.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Prywes for providing the MEF2D and MEF2A, Dr. D. J. Parry for the MHC antibodies, and Dr. E. R. Chin for the NFATc1-GFP plasmid. We also thank the Immunosuppressive Drug Laboratory, London Health Sciences Center (London, Ontario, Canada) for measurement of rodent blood CsA and FK506. Finally, we thank K. Joanisse for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants from the Natural Sciences and Engineering Research Council of Canada (to R. N. M.) and the National Institutes of Health (to R. S. W.).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.
¶ These authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 705-675-1151 (ext. 1010); Fax: 705-673-6508; E-mail: rnmichel@nickel.laurentian.ca.
Published, JBC Papers in Press, September 12, 2001, DOI 10.1074/jbc.M105445200
2 H. Wu, R. Bassel-Duby, and R. S. Williams, unpublished observations.
3 S. Dunn and R. Michel, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
CaM, calmodulin;
OV, overload;
NFAT, nuclear factor of
activated T cells c1;
MEF2, myocyte enhancer factor
2;
TTX, tetrodotoxin;
CaMK, Ca2+/calmodulin-dependent kinase;
EMG, electromyography;
CsA, cyclosporin A;
AP, alkaline phosphatase;
IGF, insulin-like growth
factor;
S, slow;
FR, fast fatigue-resistant;
FI, fast
fatigue-intermediate;
FF, fast fatigable;
PBS, phosphate-buffered
saline;
MHC, myosin heavy chain;
-gal, -galactosidase;
GFP, green
fluorescent protein.
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