From the INSERM U129, Institut Cochin de Génétique
Moléculaire, Université René Descartes Paris V, 24 rue du Faubourg Saint Jacques, 75014 Paris, France, the
§ Laboratoire de Neurobiologie, URA-CNRS 1448, Université René Descartes Paris V, 45 rue des
Saints-Pères, 75006 Paris, France, and the ¶ URA CNRS 1283, Centre Hospitalo-Universitaire Saint Antoine, 27 rue de Chaligny,
75012 Paris, France
Muscle activity is known to modulate the muscle
fiber phenotype. Changes in muscle activity (normal or experimentally
induced) lead to modifications of the expression status of several
muscle-specific genes. However, the transcription regulatory elements
involved in the adaptative response are mainly unknown. The aldolase A muscle-specific promoter, pM, is expressed in adult fast twitch muscle
with a preferential expression in fast glycolytic-2B fibers. Its
activity is induced during postnatal muscle maturation, suggesting a
role of nerve and/or muscle activity. Indeed, denervation of gastrocnemius in newborn mice prevented the activation of the promoter in this muscle, despite the nerve-independent
formation of 2B fibers. Although the nerve was necessary for pM onset
during development, denervating the gastrocnemius in adults had only mild effects on pM activity. By contrast, a transgene including the pM
proximal regulatory sequences that are sufficient to reproduce the 2B
fiber-specific expression of the endogenous promoter was shown to be
highly sensitive to both neonatal and adult denervation. Transgenes
containing muscle-specific pM proximal promoter elements were used to
delineate the regulatory elements involved in this response to
innervation and changes in the contractile activity pattern. Nerve- and
activity-dependent elements could be localized in the
130-base pair-long proximal promoter region of the human aldolase A
gene.
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INTRODUCTION |
Adult skeletal muscle is a heterogenous tissue composed of
different types of myofibers, endowed with different metabolic and
contractile properties (1, 2). This diversity is characterized at the
molecular level by the selective expression of different sarcomeric
protein isoforms and sets of metabolic enzymes, which results in the
physiological specialization of muscle. Usually, myofibers can be
classified in four major types according to the MyHC1
isoform expressed: type 1 (slow twitch,
oxidative), type 2A (fast twitch,
oxidative), type 2B (fast twitch, glycolytic), and type 2X (fast
twitch, mixed oxidative-glycolytic). Muscle differentiation first
proceeds independently of the nerve, and fiber diversity is thought to
arise from distinct myoblast populations (3). However, accumulating
evidence shows that later during development, nerve instructions
modulate the specialization of muscle fibers. Moreover, adult muscle
fibers are plastic entities whose phenotype can change and adapt in
response to external influences including relative muscle usage, motor
neuron activity, energy source availability, and hormonal status (4).
The neuronal influence seems to be transmitted to skeletal muscle
fibers mainly via contractile activity, and chronic low frequency
electrical stimulation of muscle was shown to cause a transformation of
fast fibers into slow fibers (5-7). These data suggest that nerve and
muscle contractile activity play a major role in the final
specialization of adult muscle fibers. Adaptative changes to extrinsic
factors are accompanied by modifications of the expression patterns of
multiple sarcomeric proteins and metabolic enzymes, which are
controlled in part at the transcriptional level (8). The precise
transduction pathways and regulatory mechanisms coupling external
stimuli and muscle phenotypic plasticity are still poorly understood.
As a first step to investigate such mechanisms, it is necessary to
characterize the regulatory sequences and corresponding
transcription factors involved in the response of genes whose
transcription is affected by these stimuli.
Aldolase A is a glycolytic enzyme that is highly expressed in skeletal
muscle and whose expression was shown to be modulated in response to
alterations in contractile activity in the chicken (9), in the rat
(10), and in the rabbit (11). Aldolase A expression is achieved through
the use of alternative promoters that have distinct tissue and
developmental specificities (12-17). The muscle-specific promoter, pM,
is highly activated during postnatal muscle maturation, and in adults,
pM-derived transcripts accumulate in fast twitch glycolytic muscles and
to a much lower level in slow twitch muscles (14, 18, 19). We have
previously shown that the proximal regulatory sequences (bp
310 to
+45 relative to the transcription start site) of the human aldolase A
muscle-specific promoter are sufficient to target the expression of a
chloramphenicol acetyltransferase reporter gene (pM310CAT transgene) to
fast twitch muscles of transgenic mice, in a pattern mimicking its
preferential expression in 2B fibers (20).
In this paper we have investigated the role of innervation and muscle
activity on both the murine endogenous muscle pM promoter and the
pM310CAT transgene and define the regulatory sequences responsive to
these stimuli. To examine the role of the nerve, sections of the
sciatic nerve were performed either on newborn mice (before the
induction of the promoter) or on adult mice (when the promoter is fully
active); whereas neonatal denervation drastically affects the
endogenous murine pM activity, only a mild effect is observed after
adult denervation, suggesting that the nerve is required during a
critical period of neonatal development. However, in both cases, the
expression of the transgene is highly sensitive to denervation, showing
that the proximal sequences of the human pM promoter contain a very
efficient nerve-response element (NRE). To further investigate the
influence of muscle activity on aldolase A expression, we crossed our
transgenic mice with ADR mice that were affected by a recessive
myotonia (21). ADR mice constitute a model of muscle hyperactivity (22,
23) showing biochemical changes reminiscent of those observed in
chronically stimulated muscle (24). Expression of the pM310CAT
transgene is severely reduced in ADR both in formerly fast and slow
muscles, suggesting that some elements of the pM promoter respond to
muscle activity regardless of the fiber type. Studies of transgenes
harboring shorter portions of the pM regulatory sequences enabled us to further delineate the sequences responsible for the nerve-response and
the contractile activity-dependent expression elements to a
small DNA region of 130 bp.
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EXPERIMENTAL PROCEDURES |
Transgenic and ADR Mice--
The transgenic mice pM310CAT,
M-tkCAT, and
AT-tkCAT have been described elsewhere (20, 25). The
mice were propagated in the B6/CBA background, and positive carriers of
the transgene were identified by Southern blot analysis as described
previously (20). CAT activity was assayed as described previously (20), using various amount of protein extracts and different times of incubation to keep the enzyme activity in a linear range and to ensure
detection of activities ranging from 0.1 to 10,000 cpm/µg/min of
reaction.
The ADR mouse colony originated from the kind gift of a couple of ADR
heterozygote mice by Dr. Harald Jockusch. The mutation was originally
in the A2G background, but this background was lost as the mice were
bred with the aldolase A transgenic mice. Comparison of ADR and
wild-type mice was always carried on littermate animals. Detection of
the adr allele was performed by polymerase chain reaction on
about 500 ng of tail genomic DNA using a specifically designed couple
of oligonucleotides (one in the muscle chloride channel CPC1 gene, and
the other in the transposon whose insertion has disrupted the gene
(21)). The sequences of the primers used were: adr sense,
5'-CTGTCCAACCTAAACTCTCAAGC-3'; and adr antisense, 5'-TCCTACCGCATCCTCAGCAA-3'. Denaturation was performed at 95 °C for
2 min followed by 30 cycles of polymerase chain reaction (1 min at
94 °C, 45 s at 59 °C, 2 min at 72 °C). The reaction was performed using 50 pmol of each primer, 250 µM of each
dNTPs in Taq polymerase buffer (Eurogentec) supplemented
with 1.5 mM MgCl2. The specific amplified DNA
fragment (591 bp) was then detected by migration of the polymerase
chain reaction product on an 1% agarose gel. Heterozygous and
homozygous mice were easily distinguished by the evident myotonic
phenotype.
Denervation of Mice--
Newborn mice were anaesthetized by
brief exposure in ice, whereas intraperitoneal injection of avertin was
used for older mice. The sciatic (or femoral) nerve was then exposed
and surgically cut at two locations, and a section of the nerve was
removed to prevent reinnervation during the time of the experiment. In
all cases, for surgical and controls procedures, mice were handled in
accordance with French and European laws governing animal use for
experimental purposes.
Northern Blots--
Total RNAs were prepared by the guanidium
thiocyanate single-step procedure (26). Northern blot analysis was done
as described previously (14). The murine aldolase A pM-derived
mRNAs (M-mRNA)-specific probe corresponds to the M-specific
exon of the mouse aldolase A gene (18). The myoglobin probe was
synthesized by reverse transcription-polymerase chain reaction and
corresponds to the third exon of the gene (27). MyHC-2B transcripts
were detected using a 76-bp fragment of the 3'-untranslated region of
the rat gene (28); in this case, hybridization was performed at
55 °C (in these conditions, there is no cross-hybridization with
other MyHC as shown by the absence of signal in soleus (see Fig. 5) and
diaphragm (not shown), which are devoid of 2B fibers). Normalization was performed by hybridization with the ribosomal (18 S) probe R45 (14)
or by staining the membrane with methylene blue (29).
Histochemical Determination of Muscle Fiber Types--
Skeletal
muscles were dissected from adult mice obtained by crossing the
pM310CAT mice (B6/CBA background) with ADR mice (A2G background).
Wild-type and ADR homozygous mice were issued from the same litter.
Muscles were frozen in isopentan at liquid nitrogen temperature and
cross-sectioned in a microtome cryostat. The 5-10-µm-thick unfixed
serial sections were reacted with either BA-D5 (MyHC-1), SC-71
(MyHC-2A), BF-F3 (MyHC-2B), or BF-35 (all MyHC types except 2X)
monoclonal antibodies (30) (provided by Regeneron, Pharmaceuticals, Tarrytown, NY) and then treated as described previously (31).
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RESULTS |
Innervation Is Required for pM Activation during
Development--
In mouse, pM is activated late during postnatal
development (after day 10 after birth) (14), during a period that
coincides with the end of polyinnervation and establishment of adult
contractile activity (32). We first tested whether innervation is
required for this postnatal pM activation. For this purpose, distal
hindlimb muscles of newborn mice including gastrocnemius (a fast muscle mainly composed of 2B fibers in the adults) were denervated by section
of the sciatic nerve. Six weeks later, we compared the amount of
M-mRNAs in the denervated gastrocnemius and in the nonoperated contralateral muscle. As shown in Fig. 1,
although M-mRNAs were easily detected in the innervated
gastrocnemius, they were only barely detectable in the denervated
gastrocnemius. This down-regulation of pM activity could result from
muscle atrophy and/or improper fiber specialization caused by
denervation. Because M-mRNAs are mostly expressed in 2B fibers (18)
and thus coexpressed in adults with transcripts of the MyHC-2B gene, we
checked by Northern blots (Fig. 1) and immunohistochemistry (not shown)
whether the formation of 2B fibers and concomitant accumulation of
MyHC-2B transcripts is altered following denervation. In agreement with
previous works on rats (33, 34) we found that denervation leads only to
a small decrease of the amount of MyHC-2B transcripts. Thus muscle atrophy induced by denervation did not prevent appearance of the 2B
fibers in which pM is normally active. Therefore, contrary to postnatal
accumulation of MyHC-2B transcripts, which is mostly innervation-independent, the presence of the nerve is required for the
strong activation of pM.

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Fig. 1.
Innervation is required for the developmental
accumulation of aldolase A M-mRNAs but not MyHC-2B mRNAs.
Transcripts derived from the mouse endogenous pM promoter or from the
MyHC-2B gene were detected by Northern blot in gastrocnemius of mice
that have suffered from a section of the sciatic nerve on day 1 postpartum. Both denervated gastrocnemius (lane
D) and contralateral nonoperated muscles (lane C) were
analyzed 6 weeks after section of the nerve. 10 µg of total RNA from
pooled muscles of five independent mice have been used. The membrane
was stained with methylene blue to control RNA amount and quality and
sequentially hybridized with probes for M-mRNAs, MyHC-2B mRNAs,
and ribosomal 18 S RNA (R45).
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The Activation of the pM310CAT Transgene Is Greatly Impaired by
Denervation--
The regulatory elements of the proximal region of the
human and the mouse promoters are highly conserved (17). We have
previously shown that the pM310CAT transgene containing 355 bp of the
human aldolase A pM promoter (bp
310 to +45 relative to the
transcription start site) reproduced the fast muscle specificity of the
endogenous gene in all transgenic lines analyzed (20). To see whether
this transgene can also reproduce the nerve-dependent
activation of the murine pM promoter, the sciatic nerve of newborn
transgenic pM310CAT mice (line 98) was sectioned, and CAT reporter
activity was measured 6 weeks later in both denervated gastrocnemius
and soleus (a slow muscle devoid of 2B fibers) and in control
contralateral muscles (Fig.
2A). Denervation led to an
about 100-fold decrease of CAT activity in the denervated
gastrocnemius, showing that the pM310CAT transgene activation in this
fast muscle requires the presence of the nerve as was observed for the
endogenous murine pM promoter. Interestingly, although transgene
expression was much less affected by denervation in the soleus (a
less than 10-fold decrease is observed), a preferential expression
still persisted after denervation in the (fast) gastrocnemius muscle
compared with soleus.

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Fig. 2.
Denervation altered the developmental
activation of the pM310CAT transgene. Denervation was performed by
section of the sciatic nerve on newborn (day 1 postpartum)
transgenic mice. A, transgene activity in contralateral and
denervated gastrocnemius (fast twitch, G) and soleus (slow
twitch, SOL) muscles analyzed 6 weeks after section of the
nerve. The histograms show on a logarithmic scale the means and
standard deviations of CAT activity (four animals for each bar).
B, at various times in postnatal development, CAT enzymatic
activity was measured in denervated gastrocnemius (white
symbols) and in the contralateral innervated muscle (black
symbols). For a given time, symbols of the same shape correspond
to muscles from the same animal (at least three different animals were
assayed for a given time). The mean CAT activities in denervated and
control muscles were represented by curves. A log scale was
used on the y axis.
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Taking advantage of the great sensitivity of the CAT enzymatic assay,
we could follow transgene activation in denervated gastrocnemius and
control contralateral at various times of development after neonatal
denervation (Fig. 2B). Denervation-related down-regulation of transgene expression was already effective at 15 days, suggesting that innervation is required for pM expression even before its full
activation. Surprisingly, a first step of this activation (between 15 and 25 days) was observed in denervated gastrocnemius with a fold of
increase similar to what is observed in the control innervated muscles.
However, denervation prevented further activation of the transgene
after day 25, leading to the 100-fold difference between denervated and
contralateral muscle at day 40-50 after birth.
Taken together, these observations show that innervation is crucial for
the high level postnatal expression of the aldolase A muscle-specific
promoter in hindlimb fast twitch muscles and that the 355 bp of the
human pM promoter are sufficient to reproduce this
nerve-dependent activation. In addition, it suggests that distinct pathways are involved in the activation of different genes in
the same type of myofibers, with some pathways depending on innervation
(early pM developmental onset) while others seems to be
innervation-independent (activation of the MyHC-2B gene).
Denervation of Adult Transgenic Mice Induces an Early Shut-down of
the pM310CAT Transgene Expression--
Then we examined whether nerve
activity is also required for the maintenance of pM expression in an
adult muscle. Hindlimb muscles of adult transgenic mice from two
independent pM310CAT lines (lines 20 and 98) were denervated by section
of the sciatic nerve. Because the great stability of the CAT protein
could mask a rapid effect of denervation, we performed Northern blots
experiments to detect CAT mRNAs in denervated and innervated
muscles at various times after denervation (Fig.
3). In control nondenervated muscles, two
CAT mRNAs could be observed that correspond to the use of different
polyadenylation sites in the SV40 3' end of the pM310CAT construct. As
illustrated in Fig. 3, no more CAT transcripts could be detected in
muscles 3 or 7 days after denervation. The diminution of CAT
transcripts was faintly detectable as early as 12 h after the
nerve section (not shown) and was already marked 24 h after denervation of the gastrocnemius for one of the two CAT mRNAs, which is probably the less stable one (Fig. 3A). The
induction of the myogenin gene, which is strongly induced after muscle
denervation (35, 36), was used here as a control of effective
denervation. The induction of myogenin after denervation could be
reproduced with the CAT gene under the control of myogenin regulatory
sequences (37), strenghtening the idea that the loss of CAT mRNAs
that we observed in our denervation experiments is due to a
transcriptional control of pM rather than changes in CAT mRNA
stability. Thus denervation of gastrocnemius in adult mice leads to a
dramatic and nearly immediate shut-down of the pM310CAT transgene
expression.

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Fig. 3.
Denervation in adult mice induced a fast
diminution of transgene expression. Northern blots were used to
follow the time course effect of denervation on pM310CAT transgene
activity at the mRNA level. For each time following denervation,
three adult mice (6-8 weeks old) from the two independent transgenic
lines 20 and 98 were analyzed, and 10 µg of total RNAs from
denervated (denerv.) or contralateral gastrocnemius
(control.) were used. A, 3 days after
denervation. Hybridization was performed successively with a CAT probe
(giving two bands corresponding probably to alternative polyadenylation
sites) and with a myogenin-specific probe to control denervation
efficiency. The diminution of the CAT mRNAs was already detectable
as early as 24 h after denervation on one of the two CAT
transcripts (probably the less stable, marked by an
arrowhead). B, 7 days after denervation: the blot
was successively hybridized with a CAT probe, a murine aldolase A
M-mRNA-specific probe, and a MyHC-2B probe. Normalization of the
RNAs was performed by staining the membrane with methylene blue.
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The activity of the endogenous murine aldolase A muscle promoter was
affected by neonatal denervation. Surprisingly, in adult mice, the
section of the sciatic nerve had only a minor effect on the amount of
M-mRNAs (2-4-fold decrease after 7 days of denervation) as for
MyHC-2B mRNAs (Fig. 3B). This could result from a very high stability of M-mRNAs. Alternatively, and perhaps more
probably, this could result from the influence of additional regulatory sequences that could maintain the expression of the endogenous pM
promoter in absence of innervation and that are not present in the
pM310CAT transgene.
Use of the adr Mutation to Test the Role of Muscle Hyperactivity on
pM Expression--
Electrical stimulation of muscle was shown to lead
to a lowered expression of aldolase A in rat and rabbit (10, 11). In rabbit, a qualitative modification in the pattern of aldolase A
transcripts was also observed (11). Through the use of alternative promoters, aldolase A gene gives rise to multiple transcripts of
different sizes (alternative first exons have different lengths) (12,
16, 19, 38). The changes observed by Williams et al. (11),
before the identification of the different promoters, probably
reflected a decline in M-mRNAs, the remaining transcripts corresponding to longer mRNAs controlled by the other ubiquitous promoter.
Muscle stimulation is rather difficult to perform on mice and usually
results in milder changes in these animals than in other species (rats
or rabbits). To overcome this difficulty, we have taken advantage of
the ADR mutant strain. The ADR mice carry a mutation in the muscle
chloride channel ClC1 gene (21), which causes a reduction of the
chloride conductance of their muscles. As a consequence, voluntary
movements are followed by electrical after-activity leading to a series
of "after-contractions" independent of neural transmission (24).
The chronic electrical activity of this myotonia is similar to a
chronic stimulation at low frequency (as normally observed in slow
twitch fibers). Indeed, the expression pattern of several genes is
modified in the ADR mice in a similar fashion to those in chronically
stimulated muscles (22-24). In particular, the expression pattern of
MyHC isoforms is changed in ADR mice, with a disappearance of MyHC-2B
replaced by the 2X, 2A, and 1 isoforms and a shift to an oxidative
phenotype (22, 24, 39).
By successive breeding, we transferred the pM310CAT transgene from line
20 and line 98 onto the adr background. Then we compared the
transgene activity in ADR homozygous mice to their wild-type (or
heterozygous) littermates and investigated how the expression pattern
of fiber-specific genes is changed by the adr mutation (Fig.
4).

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Fig. 4.
Fiber type transitions induced by the
adr mutation. A comparative immunohistochemical
analysis of the fiber types composition was performed on the
gastrocnemius (Gastroc.) and soleus from adult wild-type
(wt) or ADR mice in the adr/transgenic
background. Serial sections from comparable areas allowing
visualization of both muscles were stained immunohistochemically with a
panel of MyHC-specific monoclonal antibodies that stain fibers
expressing respectively MyHC-1 (A and E), MyHC-2A
(B and F), and MyHC-2B (D and
H), or all fibers except those expressing MyHC-2X
(C and G). In the ADR background, the
gastrocnemius became almost homogeneously composed of 2X fibers, with
some rare 2B fibers.
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A comparative immunohistochemical analysis of the gastrocnemius of
wild-type and ADR/transgenic mice (Fig. 4) illustrated the almost
complete disappearance of 2B fibers in ADR gastrocnemius (rare 2B
fibers were observed (data not shown), but they represented less than
1% of their normal proportion), which were replaced mostly by 2X
fibers. By contrast, the fiber composition of the ADR/transgenic soleus
was not significantly modified (Fig. 4). Northern blot analysis of the
normally glycolytic muscles (vastus lateralis and gastrocnemius) in
ADR/transgenic mice confirmed the disappearance of MyHC-2B transcripts
with a concomitant increase of myoglobin expression (marker of
oxidative fibers) (Fig. 5). The
endogenous murine pM promoter was only weakly (2-3-fold) affected by
the ADR phenotype (Fig. 5). Previous studies of the pattern of
expression of the endogenous pM have suggested that it is expressed both in 2B and 2X fibers rather than in 2B only as is the case for the
pM310CAT transgene. The observation that pM expression level is only
slightly modified in the ADR mice is consistent with this point. In
contrast, the proximal regulatory sequences of pM were highly sensitive
to ADR-linked changes in muscle activity, because the activity of the
pM310CAT transgene is reduced by more than 100-fold in ADR mice
(observation made on two independent transgenic lines) (Fig.
6). This drastic decrease of transgene activity could parallel the transition of the 2B fibers to 2X fibers.
However, interestingly, pM310CAT activity is also strongly diminished
in the ADR soleus, a slow twitch muscle that is devoid of 2B fibers and
whose fiber composition was found unchanged in our conditions. This
suggests that hyperactivity-induced changes are not only related to
fiber type transition and that the contractile hyperactivity also acts
directly on pM310CAT transcription level.

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Fig. 5.
Endogenous pM activity is weakly affected by
the ADR phenotype. To monitor the phenotypic changes induced by
the adr hyperactivity, a Northern blot analysis was
performed, using 10 µg of total RNA prepared from vastus lateralis
(VL), gastrocnemius (G), or soleus
(SOL) from four pooled ADR (adr) or wild-type
(wt) mice. The blot was successively hybridized with
MyHC-2B, endogenous aldolase A (M), myoglobin, and 18 S
specific (R45) probes.
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Fig. 6.
pM310CAT transgenes activity is altered in
ADR mice. Transgene activity was measured in normally fast twitch
(gray) or slow twitch (white) muscles of pM310CAT
transgenic mice homozygotes for the adr allele
(adr) and compared with activity in wild-type
(wt) littermates. Muscles used were the gastrocnemius
(G), vastus lateralis (VL), tibialis anterior
(TA), and soleus (SOL). The histograms show for
two pM310CAT lines, on a logarithmic scale, means and standard
deviations of CAT activity (three to four animals for each bar).
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The Regulatory Sequences Necessary for Contractile Activity-induced
Activation of pM Were Delineated to the
164 to
35 bp Promoter
Fragment--
The data presented here showed that the
310/+45
sequences of pM contained a NRE and an activity-dependent
response element (ADRE). To further shorten the region containing the
NRE and the ADRE (which may be the same sequence), we studied the
sensitivity to denervation of transgenic mice harboring smaller
fragments of the pM regulatory sequences. Fragments encompassing the
235/
35 and
160/
35 promoter fragments cloned upstream of the
herpesvirus simplex thymidine kinase
105 promoter and the CAT gene
gave the M-tk and
AT-tk transgenes, respectively (depicted in Fig.
7), which display the same pattern of
expression than pM310CAT (25). As for the pM310CAT transgene, adult
denervation of gastrocnemius caused a rapid and strong decrease of the
CAT mRNAs in both M-tk and
AT-tk transgenic lines compared with
mRNAs from the contralateral muscle (Fig. 7). These results
demonstrate that the NRE is included in the DNA fragment extending from
bp
164 to bp
35 relative to the pM transcription start site. In
addition, the
AT-20 transgene activity was also strongly reduced by
at least 100-fold in the ADR background compared with wild-type mice
(not shown), suggesting that the ADRE is also included in this short
DNA fragment.

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Fig. 7.
Denervation response of M-tk and AT-tk
transgenes. Activity of two transgenes containing only
subfragments of the pM regulatory region cloned upstream the thymidine
kinase minimum promoter (tk-105) was measured in control
contralateral (lanes C) and denervated (lanes D)
gastrocnemius 3 days after surgical section of the sciatic nerve on
adult transgenic mice. Northern blot was performed with 10 µg of
total RNA from three different mice for each line and hybridized with a
CAT-specific probe. Normalization of the RNAs was obtained by staining
the membrane with methylene blue. The absolute activity is different
for the pM310CAT, M-tk, and AT-tk lines, but in each case, no signal
was found in denervated muscle. A schematic representation of the
transgenes used and of the regulatory motifs found within pM promoter
is given.
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DISCUSSION |
Nerve- and Activity-dependent Specialization of Muscle
Fibers--
Nerve and muscle activity not only exert a trophic effect
on muscle fibers but are also involved in their final specialization by
modulating the expression of sarcomeric proteins and metabolic enzymes
(1, 2). To obtain new insights in this process, we have investigated
the effects of modifications in the contractile activity on the fast
muscle-specific promoter (pM) of the aldolase A gene. For this purpose,
we used transgenic mice harboring a transgene (pM310CAT) that
reproduces this preferential expression in 2B fibers
(20).2 The consequences of
modifications in the contractile activity resulting either from the
absence of the nerve or from a genetically induced hyperexcitability
could thus be studied on both the murine endogenous pM promoter and
transgene expression.
Although MyHC-2B mRNAs accumulated in gastrocnemius even in the
absence of innervation (Fig. 1 and Refs. 33 and 34), denervation prevented the developmental increase of murine aldolase A M-mRNAs. Thus, postnatal activation of the murine aldolase A muscle-specific promoter in fast muscles requires a nerve-derived signal, whereas MyHC-2B expression is mostly nerve-independent. However, although activation of murine pM was prevented by denervation of newborn hindlimb muscles, its expression was only moderately affected by
denervation of adult muscles. Therefore, innervation seems to be
crucial for only a short period of time during the activation of
promoter activity. Similar observations had already been reported for
the MyHC-2A gene (34).
The proximal regulatory sequences of human pM (transgene pM310CAT) were
sufficient to reproduce the nerve-dependent onset of the
endogenous gene but do not maintain expression when innervation is
suppressed in adult muscle. This suggests that different regulatory regions are involved in the nerve-dependent stimulation
during development and in the nerve-independent maintenance of the
promoter activity and that the latter sequences are missing in the
transgene. In the vicinity of the pM promoter lies a strong ubiquitous
enhancer that is associated to the other alternative aldolase A pH
promoter. This enhancer was previously shown to spread pM activity in a broader range of fast fibers than what is observed when the transgene includes only the 2B-specific proximal pM regulatory sequences (14,
20). This ubiquitous enhancer could be able, as well, to stimulate an
already active pM promoter in fast twitch muscles even in the absence
of innervation (denervation of adult mice) but would be unable to
participate to its developmental onset.
Interestingly, we observed that denervation does not prevent an
induction of pM310CAT during development but rather acts by strongly
diminishing its activity. Thus, the increased expression of pM that
takes place during postnatal muscle maturation is only partially
controlled by the nerve. This developmental increase coincides with a
peak in thyroid hormone that is known to promote muscle maturation (40)
and favor the fast twitch glycolytic phenotype maturation (41). Action
of thyroid hormone is nerve-independent (33), and thus activation of pM
in fast twitch muscle could result from the combined effects of
innervation and thyroid hormone.
Muscle hyperactivity (as observed in ADR mutant mice) has been
described to cause a transition toward an oxidative phenotype and a
disappearance of the fast 2B fibers (42-44). Indeed we observed that
in ADR/transgenic mice, the gastrocnemius is now composed almost
exclusively of MyHC-2X fibers. In contrast to the endogenous pM
promoter activity, which is moderately affected by the adr mutation, pM310CAT expression is highly diminished in all ADR muscles
that we analyzed, including the slow twitch soleus. This discrepancy
could result, here too, from the presence in the vicinity of pM
proximal sequences of regulatory elements that are missing in the
transgene (and as previously suggested for denervation sensitivity, the
ubiquitous H enhancer is a likely candidate). These sequences that were
shown to spread pM expression to 2X fibers (20) could thus attenuate
the effects observed when only the proximal 2B-specific promoter
sequences are present, as in the pM310CAT transgene. However, the
observation that the transgene expression is also greatly diminished in
the soleus supports a fiber type-independent effect of the contractile
hyperactivity.
Taken together, these results highlight the major but complex influence
of nerve and muscle contractile activity on the maturation of skeletal
muscle. Genes expressed within the same fibers may respond
differentially to those extrinsic factors. Our observations support the
idea that multiple transcription programs are involved in fiber
specificity, each responding probably to different clues that could be
extrinsic (nerve and contractile activity) but also intrinsic to the
fiber (contribution of myoblasts potentially committed to form a
particular type of myofiber). This has been previously suggested by the
studies of the expression patterns of muscle sarcomeric genes during
regeneration in the presence or the absence of nerve (45) or after
muscle stimulation (43, 44). Recently, it has been shown that in
chicken myotubes, the expression of the myosin heavy chain 2 slow gene
depends on both intrinsic (type of myoblasts) and extrinsic
(nerve-induced contraction) factors (46). The present study shows that
the precise regulation of pM results from the combination of different
transcriptional regulatory elements associated with these different
programs whose relative contribution may evolve during muscle
maturation.
Although pM310CAT transgene could reproduce most aspects of the
endogenous pM control (fiber 2B preferential expression and nerve-dependent activation during development), it appeared
much more sensitive to modifications in nerve and contractile activity in adults. These discrepancies illustrate the composite nature of the
elements participating to the control of a muscle gene, each of them
directing a portion of the overall expression pattern of the gene (for
a review see Ref. 47). This study in transgenic mice identifies in the
proximal regulatory sequences of the pM promoter a regulatory module
that was both sufficient to direct 2B fiber-specific expression and to
integrate signals originating from the nerve and from the pattern of
contractile activity that modulates its level of activity.
Transcriptional Response to Nerve and Contractile
Activity--
The finding that the pM310CAT transgene expression is
regulated by contractile activity is supported by the observation that denervation and hyperactivity in the ADR mice caused a dramatic decrease of its expression. Therefore, in the short
310/+45 fragment lie a NRE and an ADRE, and our results suggest that both elements are
included in the
164/
35 region. Although denervation and hyperactivity lead to different alterations in the expression of a
battery of muscle proteins, some similar changes were also observed, as
increased amounts of myogenin mRNAs (22), and we cannot exclude
that their similar effects on pM310CAT transgene transcription results
from the triggering of a common transduction pathway; the NRE and the
ADRE could therefore correspond to one single DNA element. Among the
other genes whose activity is controlled by nerve-derived electrical
activity, attention has been mostly focused on those that were induced
by denervation, like the acetylcholine receptor subunits genes or the
myogenin gene (37, 48-52). The NRE of the genes encoding acetylcholine
receptor subunits has been delineated, and it was found that some
E-boxes were necessary for denervation-induced expression of those
genes (48, 51, 52). This importance is supported by the fact that the
myogenic basic helix-loop-helix factors that bind E-boxes are induced
by denervation (35, 36). However, E-boxes are not sufficient to give
denervation responsiveness because many genes that are regulated by
E-boxes are not controlled by nerve activity (48). The bp
164 to
35
pM region contains an E-box that is bound mostly by USF/MLTF but not
MyoD proteins in gel mobility shift assays using skeletal muscle
nuclear extracts (53). Mutation of this E-box has been shown to reduce
pM310CAT activity in transgenic mice but does not alter its
fiber-specific expression (25, 54); one can imagine that the myogenic
basic helix-loop-helix factors that are overexpressed after denervation
could compete with USF/MLTF for their common binding site and cause a
reduction of promoter activity. Nevertheless, this region contains
other binding sites that have been shown to be important for promoter
activity (including a MEF3 motif, overlapping MEF2 and NFI sites, and a
M1 sequence bound by nuclear receptors (25, 31, 54) and that could
serve as targets of the nerve- and activity-dependent
pathways. The demonstration that the small
164/
35 regulatory region
is sufficient to confer both fiber specificity and nerve- and
contractile activity-dependent expression in skeletal
muscles should facilitate the identification of the key elements
involved in nerve- and activity-dependent regulation of
muscle-specific genes.
We thank Dr. H. Jockusch and Dr. J. Bartsch
for the gift of the ADR mice and advice, Dr M. Buckingham for the gift
of MyHC-2B probe, and Dr J.-P. Concordet for critical reading of the
manuscript. We are grateful to Isabelle Lagoutte, Arlette Dell'Amico,
and Hervé Gendrot for skillful care of the mice used in this
study.