Institut National de la Santé et la Recherche Médicale Unité 99, Hôpital Henri Mondor, F-94010 Créteil, France
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ABSTRACT |
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Changes in the activity and in the expression of adenylyl cyclase (AC) were examined in mouse skeletal muscle after denervation and during development. Four isoforms of AC (AC2, AC6, AC7, and AC9) were detected by Northern blot analysis in gastrocnemius muscle, AC9 being the most abundant. After denervation, the levels of AC2 and AC9 mRNA decreased, whereas those of AC6 and AC7 increased. AC activity in response to several neurotransmitters was increased after denervation. During development, AC activity was high in fetus and neonate and declined in the adult; the sensitivity of AC activity to various neurotransmitters was the highest on the third postnatal day. The levels of AC6 and AC7 mRNAs were high on the third postnatal day and then decreased in adult, paralleling the decline in AC activity. All the characteristics of AC expression and activity in fetus and neonate resembled those observed in denervated adult muscle. These results indicate that changes in AC activity and AC mRNAs play an important role in the various physiopathological states of skeletal muscle, especially during muscle atrophy.
fast-twitch muscle; neurotransmitters; G proteins; Northern blot; in situ hybridization
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INTRODUCTION |
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ADENYLYL CYCLASES (ACs) catalyze the conversion of ATP to cAMP and play an important role in neurotransmission in the central nervous system as well as in the regulation of muscle contraction. Many neurotransmitters, including ACh and norepinephrine, transduce their signal into a cell by activating G protein-coupled receptors that modulate AC activity; changes in the intracellular cAMP, in turn, affect cAMP-dependent protein kinases (PKAs).
Genes for at least nine distinct mammalian ACs have been reported
(reviewed in Refs. 29, 34, 62). The nine isoforms have been cloned,
sequenced, and characterized. All of them are activated by forskolin,
with the possible exception of AC9, and by the GTP-bound -subunit of
the stimulatory G protein (Gs
). However, other regulatory properties vary among the different isoforms:
for example, their susceptibility to protein kinase C (PKC) and PKA and
to the GTP-bound
-subunit of the inhibitory G protein
(Gi
) differ; PKC has been
implicated in the stimulation of AC2 and AC5 but markedly inhibits the
activity of AC6 (38, 41, 43, 75); PKA inhibits the activity of both AC5
and AC6 (33). AC1 and AC8 are specific for neural tissues and are
activated by calmodulin in the presence of calcium (11, 40, 72). AC5, essentially expressed in heart and striatum, and AC6 can be inhibited by physiologically relevant concentrations of calcium (32, 37), whereas
the calcium-mediated inhibition of AC3 activity in vivo is due to a
direct phosphorylation of AC3 by a calmodulin kinase II (69, 70). AC2,
AC4, and AC7 are insensitive to calcium but are activated by the
-subunits of the heterotrimeric G proteins (43, 61);
moreover, they seem to be preferred substrates for PKC isoenzymes (35,
68). Finally, AC9 is not regulated by either calcium or
-subunits
(55). It is likely that the diversity of these regulatory mechanisms
accounts for the tissue-specific pattern of cAMP synthesis and, in
particular, for the occurrence of subtle regulation by cross talk or
convergence between independent signaling pathways.
Mammalian adult skeletal muscles are composed of heterogeneous populations of fibers that differ in their biochemical and structural characteristics due to the expression of an alternate subset of genes (reviewed in Refs. 15, 21). These fibers have been divided into four major classes on the basis of their expression of distinct isoforms of contractile proteins. Slow-twitch fibers (type I) express myosin heavy chain (MHC) type I and use mainly oxidative metabolism, whereas fast-twitch fibers (type II) express MHC IIa, IIb, or IIx and are mainly oxidative (type IIa) or glycolytic (types IIb and IIx) (reviewed in Ref. 52). The basis for such a muscle diversity is the existence of a heterogeneous population of myoblasts, differing in their gene expression, but evidence also exists that suggests gene expression in skeletal muscle is primarily determined by the stimulation pattern imposed on the fiber by its motoneuron.
Muscle denervation has been used as a model to study the molecular mechanisms by which muscle activity regulates synaptic protein expression. Dramatic changes occur in the morphological and biochemical properties of a skeletal muscle after section of its motor nerve (14, 24, 46). Some of these changes are thought to result from the immobility of the muscle after denervation, but deficiency of trophic factors resulting from the denervation may also be involved. This process is an important component of many human neurological diseases and has been extensively studied (26, 27). For example, after denervation there is a marked increase in extrajunctional ACh receptor (AChR) density, as well as an increase in the levels of mRNAs coding for the receptor subunits (4, 25, 49, 57), and denervation of skeletal muscle activates the expression of AChR genes (66). One candidate for a second messenger linking the action of nerve factors to AChR biosynthesis is cAMP. Analogs of this nucleotide, as well as activators of AC, stimulate AChR accumulation in cultured myotubes (6, 7), and cAMP is one of the second messengers that mediate the increase in AChR number elicited by calcitonin gene-related peptide (CGRP), a neuronal messenger coexisting with the classical transmitter ACh (42). As an early consequence of denervation, the expression of immediate early genes and the cAMP metabolism are affected, though differently in the different types of muscles. mRNA levels of the fos and jun protooncogenes are increased rapidly after denervation of the lower leg muscles of adult mice (1, 5). Moreover, the mRNAs for four muscle-specific regulatory factors, MyoD, myogenin, Myf-5, and muscle regulatory factor 4 (MRF4), which play an important role in controlling skeletal muscle development by regulating muscle-specific gene expression, rapidly increase following denervation of adult skeletal muscle. This increase can be suppressed by subsequent direct electrical stimulation (2, 9, 17, 67). In rat gastrocnemius muscle, AC activity was found to be increased by two- to fourfold from 14 to 28 days after sciatic nerve section (30, 51, 53). Moreover, chronic motor nerve pacing results in an increase in the AC activity; this increase is time dependent and appears to be consistent with the fiber type-specific differences in cAMP signaling (39, 65).
To date, little is known about the AC isoform expression and distribution in skeletal muscle. To explore the potential role of the ACs in gene regulation and signaling in muscle, we investigated 1) the expression of the different AC transcripts in normal adult gastrocnemius muscle on denervation of the sciatic nerve and during development, 2) the changes in AC activity in response to several neurotransmitters in these different conditions, and 3) the distribution of AC isoforms by in situ hybridization in mouse skeletal muscle before and after denervation.
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MATERIALS AND METHODS |
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Animals and Tissue Preparation
C57BL/6 mice were purchased from Janvier. Male adult mice (aged 2 mo) weighing 20-25 g were used for the denervation study. Under pentobarbital anesthesia (60 mg/kg body wt, intraperitoneal injection), denervation was carried out by excision of a 5-mm piece of the left sciatic nerve at the midthigh region. Ten or thirty days after denervation, mice were decapitated and the gastrocnemius muscles were excised (D10 and D30, respectively). The contralateral muscle was used as a control (C10 or C30). No changes were found between C10 and C30 in AC activity assay and Northern blot analysis. Therefore, we denote the control muscle as simply C. For the fetal studies, time-mated females were decapitated after 19 days of gestation (E19). Mice at postnatal day 3 (PN3), PN21, and PN60 (adult) were decapitated, and their gastrocnemius muscles were rapidly removed. The muscles dissected on E19 or PN3 from the hindlimbs of fetal mice from a given litter were pooled and considered as a single preparation, respectively. These muscles may have contained minor amounts of bone residue that were difficult to remove during dissection.AC Activity Assay
Isolated muscles were immediately homogenized in 10 volumes of ice-cold buffer containing (in mM) 50 Tris · HCl (pH 7.6), 2 EDTA (pH 7.6), 1 dithiothreitol, and 0.1 phenylmethylsulfonyl fluoride, and then they were centrifuged at 150 g for 10 min at 4°C. The pellet was discarded, and the supernatant was centrifuged at 17,000 g for 30 min at 4°C. The pellet that was obtained was resuspended in the same buffer and resubjected to the same centrifugation step. This procedure was repeated three times. The final pellet was resuspended in an appropriate volume of the same buffer and stored atBefore the AC activity assay was started, the frozen membranes were
thawed and centrifuged at 17,000 g for
30 min at 4°C. The pellet was resuspended in an ice-cold membrane
buffer containing (in mM) 50 Tris · HCl (pH 7.6), 2 dithiothreitol, and 2 EGTA. The washing process was repeated twice.
Finally, the pellet was resuspended with the membrane buffer at a
concentration of ~1 mg protein/ml. AC activity was measured as
described previously (29, 44), with some modifications. The standard
reaction mixture contained (in mM) 50 Tris · HCl (pH
7.6), 1 EGTA, 5 MgCl2, 1 ATP containing
[-32P]ATP [1 × 106 counts/min
(cpm)], 1 cAMP containing
[8-3H]cAMP (10,000 cpm), and 5 phosphocreatine, with 250 µg/ml creatine phosphokinase,
10 µM GTP, and 30 µg of membrane protein in a final volume of 60 µl with or without test agents. Test agents were diluted with a 50 mM
Tris · HCl buffer (pH 7.6) and added to the reaction
mixture at the concentrations indicated in the text. Reactions were
initiated by addition of membranes and run for 10 min at 37°C. The
reactions were terminated by addition of 200 µl of 0.5 M HCl,
followed by boiling for 7 min and neutralization by 200 µl of 1.5 N
imidazole. cAMP formed during the incubation was separated by alumina
column and corrected for recovery of added
[3H]cAMP. Protein was
measured by the method of Bradford (8), with BSA as the standard. CGRP
(rat), salmon calcitonin (sCT), and amylin fragment 8-37 (human)
were purchased from Sigma.
Probes
Probes specific for each of the AC subtypes were chosen from regions where the sequences are the most divergent. Probes specific for rat AC2, AC5, and AC6 were obtained by PCR, using mRNA from rat brain and the following specific oligonucleotides: the primers 5'-GGGAAGATTAGTACCACGGAT-3' (sense) and 5'-AGGAGAAGCCA AGGATGGACG-3' (antisense) for the 334-bp rat AC2 probe (20), 5'-CAGAGAACCAACTCCATTGGACACAATCCG-3' (sense) and 5'-CACACAGGCGTAGATCACAGATATAAACAC-3' (antisense) for the 459-bp rat AC5 probe (54), and 5'-TGCTGCTGGTCACCGTGCTCAT-3' (sense) and 5'-GGACGCTAAGCAGTAGATCATAGTGTCAA-3' (antisense) for the 493-bp rat AC6 probe (55). The amplified fragments were subcloned into the Sma I site of pGEM3 (Promega) for the rat AC2 or into the cloning site of pCRII (Invitrogen) for the rat AC5 and AC6 fragments. A 722-bp fragment cloned in pCRII and a 739-bp fragment cloned in pGEM3 were derived from human AC7 (nt 2453-3175; membrane-spanning domains 7-12) (50) and mouse AC9 (nt 1-739) (54), respectively. Various restriction fragments (1738, 1900, 1748, and 280 bp) of rat cDNAs coding for GscDNA probes were used for Northern blot analysis. The cDNA fragments
were labeled with
[-32P]dCTP, using a
standard random primer reaction (Megaprime, Amersham).
cRNA probes were used for in situ hybridization. Before in vitro
transcription was started, the pGEM3 plasmids containing the 334-bp
fragment of the rat AC2 and the 739-bp fragment of the mouse AC9 were
linearized with EcoR I, the pCRII
plasmids containing the 493-bp fragment of the rat AC6 and the 772-bp
fragment of the human AC7 were linearized with
Xho I and
EcoR V, respectively, and the pSP65
plasmid containing the 295-bp fragment of PKA subunit RI was
linearized with EcoR I, to prepare
antisense cRNA probe. The in vitro transcription was carried out at
42°C for 90 min in a 20-µl reaction mixture containing 1 µg of
linearized plasmid templates and SP6 RNA polymerase in the presence of
[
-33P]UTP (>3,000
Ci/mmol, 1 mCi = 37 MBq; ICN). The
33P-labeled probes were separated
from free nucleotides by spin column (Sephadex G-50, fine) and then
concentrated by ethanol precipitation.
Northern Blot Analysis
Total RNAs were prepared by the guanidinium thiocyanate-phenol extraction method (13). Poly(A)+ RNAs were isolated by the batch method with the use of the oligo(dT) cellulose. Ten micrograms of poly(A)+ RNA were electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde as previously described (44) and transferred overnight onto Hybond N+ membrane (Amersham) by capillary blotting. The 0.24- to 9.5-kb RNA ladder from GIBCO BRL was used as the molecular mass standard. The membranes were hybridized for 2 h at 65°C (rapid hybridization buffer, Amersham) with 2 × 106 cpm/ml of probe and washed in 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% SDS at room temperature for 20 min, followed by 1× SSC-0.1% SDS, 0.5× SSC-0.1% SDS and 0.1× SSC-0.1% SDS at 65°C for 30 min, respectively, and were then exposed to Kodak XAR film with intensifying screens atIn Situ Hybridization
In situ hybridization was performed essentially as described previously (44), with some modifications. Briefly, the excised gastrocnemius muscles were embedded immediately in Tissue-Tek OCT (Miles, Elkhart, IN) and quick frozen atData Analysis
Northern blots were performed at least three times for each AC type, G protein, and myogenin, and quantification was done by densitometric scanning and computer analysis with a laser densitometer (Ultroscan XL, LKB Instruments, Bromma, Sweden). For each AC type, G protein, and myogenin, the results were normalized using a GAPDH probe, and the values for the control and adult muscle were set arbitrarily as 100% for comparison after denervation and during development, respectively (see Figs. 4-7). To determine the relative levels of AC isoforms mRNAs, in each experiment, the length and the specific activity of the probes as well as the exposure time were taken into account (see Table 1). The values were expressed as arbitrary units per 100 bp of the probe per 24 h of exposure and normalized to GAPDH values, also corrected in the same way. Statistical comparisons were made by ANOVA followed by Student's two-tailed, nonpaired t-test. A value of P < 0.05 was considered to be significant. Data are presented as means ± SE. ![]() |
RESULTS |
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AC Activity Assay in Control and Denervated Muscle
After denervation, the weight of the gastrocnemius muscle decreased gradually, reaching 70 and 40% of that of the control, contralateral muscle 10 and 30 days after denervation (D10 and D30), respectively.We first measured the basal and forskolin-, isoproterenol-,
epinephrine-, and ACh-stimulated AC activities in the control and
denervated muscles (Fig. 1).
Basal AC activity in denervated muscle (D10 and D30) was higher than
that in control muscle. The AC activity was 8.4 ± 0.34 pmol · min1 · mg
1
in membranes from control animals and 11.97 ± 1.67 and 15.68 ± 1.56 pmol · min
1 · mg
1
in membranes from D10 and D30, respectively. Forskolin, isoproterenol, and epinephrine stimulated AC activity in a concentration-dependent manner. Compared with the basal AC activity, AC activity stimulated by
forskolin (100 µM) was increased to 3.1-fold in the control and to
4.6- and 7.0-fold in D10 and D30, respectively. AC activity stimulated
by isoproterenol (10 µM) was increased to 2.9-fold in the control and
to 3.9- and 4.6-fold in D10 and D30, respectively, compared with the
basal AC activity. AC activity stimulated by epinephrine (100 µM) was
increased to 2.5-fold in the control and to 3.8- and 4.1-fold in D10
and D30, respectively, compared with the basal AC activity. On the
other hand, ACh inhibited AC activity in a concentration-dependent
manner. Compared with the basal AC activity, AC activity was decreased
to 50% by ACh (100 µM) in the control and to 16 and 8% in D10 and
D30, respectively.
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Furthermore, because CGRP, sCT, and amylin have been reported to specifically increase AC activity in skeletal muscle, we assessed their effects in control and denervated muscle (Fig. 2). These three peptides stimulated AC activity in a concentration-dependent manner. Compared with the basal AC activity, AC activity stimulated by CGRP (10 µM) was increased to 1.9-fold in the control and to 2.5- and 3.8-fold in D10 and D30, respectively. AC activity stimulated by sCT (10 µM) was increased to 1.6-fold in the control and to 2.2- and 3.5-fold in D10 and D30, respectively, compared with the basal AC activity. AC activity stimulated by amylin (10 µM) was increased to 1.7-fold in the control and to 2.0- and 2.9-fold in D10 and D30, respectively, compared with the basal AC activity.
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AC Activity Assay in Developing Muscle
We measured AC activity in the absence of stimuli (basal) or in the presence of 100 µM forskolin, 10 µM isoproterenol, 100 µM epinephrine, 100 µM ACh, 10 µM CGRP, 10 µM sCT, or 10 µM amylin at different ages (E19, PN3, PN21, and adult). The basal AC activities at these points were 12.4, 14.8, 10.6, and 8.4 pmol · min
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Northern Blot Analysis in Control and Denervated Muscle
Expression of AC2, AC6, AC7, and AC9 mRNAs. AC isoform mRNAs in mouse gastrocnemius muscle were resolved as single bands of 4.2, 6.5, 7.5, and 8.5 kb for AC2, AC6, AC7, and AC9, respectively, as described in other tissues (20, 54, 55, 68). We did not detect any AC5 mRNA. After 6 days of exposure, AC4 mRNA was barely detectable both in control and in denervated gastrocnemius muscle (not shown). Moreover, none of the calcium/calmodulin-stimulated AC mRNAs were observed either in rat or in mouse skeletal muscle. GAPDH mRNA, used as an internal control in all these experiments, migrated as a unique 1.5-kb band in mouse gastrocnemius muscle; its expression was constant after denervation, as observed in rat skeletal muscle (56). The levels of expression of AC2 and AC9 were decreased after denervation. Compared with control muscle, the levels of AC2 and AC9 mRNA were lower by 43.3% (D10) and 39.1% (D30) and by 35.9% (D10) and 30.6% (D30), respectively. In contrast, compared with control muscle, the levels of AC6 and AC7 mRNA were higher by 1.3-fold (D10) and 5.1-fold (D30) and by 4.4-fold (D10) and 10.8-fold (D30), respectively (Fig. 4). The relative levels of these four types of AC mRNA in control and D30 muscles were in the following orders: AC9 > AC2 > AC7 > AC6 in control muscles and AC7 > AC6 > AC9 > AC2 in D30 muscles (Table 1). The relative loss of AC9, compared with the other isoforms, may explain the higher sensitivity to forskolin of the total AC activity assayed in the denervated muscles.
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Expression of Gs,
Gi-1
,
Gi-2
,
Gi-3
, and myogenin mRNAs.
G protein and myogenin isoform mRNAs in mouse gastrocnemius muscle were
resolved as single bands of 1.8, 2.4, 3.5, and 1.6 kb for
Gs
,
Gi-2
,
Gi-3
, and myogenin,
respectively, as described in previous reports (17, 36). We did not
detect Gi-1
mRNA. The levels of
expression of the four detectable mRNAs were increased after
denervation. Compared with control muscle, the levels of Gs
,
Gi-2
,
Gi-3
, and myogenin mRNA were
higher by 5.4-fold (D10) and 9.6-fold (D30), by 1.5-fold (D10) and
4.5-fold (D30), by 1.4-fold (D10) and 3.7-fold (D30), and by 30.9-fold
(D10) and 61.1-fold (D30), respectively (Fig.
5). The increased level of expression of
myogenin mRNA corresponded with a previous report (17).
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Northern Blot Analysis in Developing Muscle
Expression of AC2, AC5, AC6, AC7, and AC9 mRNAs. Northern blot analysis of the relative levels of AC2, AC5, AC6, AC7, and AC9 mRNAs was performed to determine whether the levels of these messengers changed during development. The results are shown in Fig. 6, which depict the overall changes in the relative level of each RNA after correction for the GAPDH signal. The pattern of change in the RNA expression was different for the AC isoforms examined. The levels of expression of AC2 and AC9 were increased gradually during development. The relative ratios of AC2 and AC9 RNA to GAPDH messenger were calculated as 2.0-, 2.3-, and 2.6-fold increases and 2.2-, 3.3-, and 4.2-fold increases over the values for E19, PN21, and adult muscles, respectively. On the other hand, the levels of expression of AC6 and AC7 mRNAs were increased 1.1- and 1.4-fold to peak levels at PN3, respectively, followed by a decrease to adult levels. We did not detect AC5 mRNA at any stage during development.
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Expression of Gs,
Gi-1
,
Gi-2
, and
Gi-3
mRNAs.
The expression of G protein mRNAs was examined by Northern blot
analysis during development. We did not detect
Gi-1
mRNA during development.
The levels of expression of the other three G protein mRNAs decreased
gradually during development. The relative ratios of these mRNAs to
GAPDH messenger in adult muscle were decreased by 22, 10, and 27%
compared with the values at E19 for Gs
,
Gi-2
, and
Gi-3
, respectively (Fig.
7).
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In Situ Hybridization in Control and Denervated Muscle
Figure 8 shows X-ray film images of a series of control or denervated gastrocnemius muscle sections 10 days after operation, hybridized with the AC2, AC6, AC7, AC9, and PKA regulatory subunit RI
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DISCUSSION |
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The experiments presented in this study demonstrate that four AC isoforms (AC2, AC6, AC7, and AC9) are expressed in mouse gastrocnemius muscle, AC9 and AC2 being the most abundant. It is striking that skeletal muscle does not express a detectable amount of AC5, which is the predominant form in mature cardiac myocytes (19, 63). After denervation, the pattern of changes in the mRNA expression for these four ACs was markedly altered: the levels of AC2 and AC9 mRNA decreased, whereas those of AC6 and AC7 increased. Using in situ hybridization, we showed that the distribution of the four AC isoforms detected was uniform and diffuse both in normal and denervated muscle. In parallel with the changes in the AC expression program, we observed an increase in AC activity and a hypersensitivity to various effectors such as ACh, calcitonin, CGRP, amylin, isoproterenol and epinephrine, probably related to changes in the expression of G proteins. It is interesting to note that the characteristics of the AC isoform expression and activity in the fetus and the neonate resemble those observed in denervated adult skeletal muscle. Namely, compared with adult muscle, the levels of AC6 and AC7 mRNAs are high, those of AC2 and AC9 mRNAs are low, and the AC activities in response to various agents are high.
The patterns of AC isoform expression are different in skeletal, cardiac, and smooth muscles. We have shown that in the rat uterus five AC isoforms (AC2, AC4, AC6, AC7, and AC9) are expressed, with AC6 being the most abundant. The level of expression of these ACs increases during the course of pregnancy and diminishes near term and after delivery. The highest level of expression for each type of AC is consistently seen on the 17th day of pregnancy (59). In cardiac muscle, we (19), as well as Tobise et al. (63), reported an age-related change in the myocardial transduction pathway determined by a shift from a predominantly AC6 to a predominantly AC5 pattern. It is therefore obvious that the various AC genes are controlled by a unique combination of transcription factors in the different muscle cell types. The overall consequences of the changes in the AC pattern for muscle function are difficult to assess at present: AC2 and AC7 are closely related, and the only major difference between AC9 and AC6 is a potential weak inhibition by calcium of the latter.
Many studies have established that motoneuron activity influences gene
expression in developing and adult skeletal muscle fibers. The nerve
provides a trophic influence on the muscle by one or more specific
transmitter substances and/or electrical activity. The neural
dependence is further substantiated by the fact that neonatal
denervation retards the development of fast-contracting muscle fibers
(18). Gastrocnemius muscle contains 75-100% fast-twitch muscle
fibers, depending on the species (28, 67). Denervation of fast-twitch
muscles, such as extensor digitorum longus and tibialis anterior,
results in a relative increase in the proportion of myofibers
expressing slow myosin and slow troponin I (67, 68). A number of other
phenotypic changes also follow denervation of fast-twitch muscles,
including decreased myosin ATPase activity, increased levels of enzymes
involved in oxidative pathways, prolonged contraction time, and
increased proportion of slow-twitch fibers (27, 60, 64). Denervation
results in muscle atrophy, and the denervated muscle becomes
hypersensitive to the neurotransmitters present in the nerves that are
destroyed, as the consequence of increases in neurotransmitter
receptors. Although most skeletal muscle genes are expressed at similar
levels in electrically active innervated muscle and in electrically
inactive denervated muscle, a small number of genes, including those
encoding AChR, the neural cell adhesion molecule (NCAM),
and myogenin, are expressed at significantly higher levels in
denervated than in innervated muscles (cf. references cited in Ref.
74). Denervation of adult muscle causes a dramatic increase in the
sensitivity of the fibers to ACh, which is directly attributable to
increases in the levels of -,
-,
-, and
-subunit RNAs
within 24-48 h and the expression of the embryonic type of
nicotinic AChR (nAChR) (2). As an early event following denervation of
adult rat hindlimb muscles, a rapid increase in the level of muscle
regulatory factors (MyoD, myogenin, Myf-5, and MRF4) has been described
(2, 67). The increase in myogenin transcripts in the denervated muscle
is similar to the rapid increase in myogenin expression observed when
myoblasts are committed to differentiate in culture. The increase in
the myogenin mRNA expression is maintained even 30 days after
denervation, when we observed an increase of AC6 and AC7 and of
-subunits of the heterotrimeric G proteins
(G
s,
G
i-2, and
G
i-3). Genes similar to those
that are active during muscle cell development may be switched on
following a nerve injury, including AChRs, creatine kinase, and
phosphoglyceromutase (3, 45). The return of these parameters to normal
after reinnervation is variable and may be incomplete (23). It thus
appears that, after denervation of skeletal muscle, a genetic program
that includes myogenic differentiation and protooncogene expression is
activated (1, 5, 71).
Before innervation, AChR are detected over the entire surface of the
myotube. Once the motor nerve ending has contacted the muscle cells,
AChR density increases rapidly at the NMJ while its biosynthesis
declines in the extrajunctional regions (12). Outside the junction, the
electrical activity evoked by the motor nerve suppresses the AChR gene
expression primarily by a reduction of the transcription rates of AChR
genes. At the NMJ, the degradation rate of AChRs is accelerated on
denervation but can be stabilized by reinnervation or by cAMP. This
effect is mediated through the PKA-dependent pathway (58, 73). The NMJ
plays an important role in the course of development and maturation of
skeletal muscle. Using in situ hybridization, we have shown that the
distribution of the four AC isoforms detected was uniform and diffuse
in the muscle and could not be detected preferentially in the NMJ,
where the PKA RI subunit and AChR mRNAs are distributed (12, 31). After denervation, RI
subunit mRNA increases specifically at the
level of the NMJ and the program of AC expression is modified: AC2 and
AC9 mRNAs decrease, whereas AC6 and AC7 mRNAs increase, without any
apparent modification in their distribution. Imaizumi-Scherrer et al.
(31) showed that PKA subunit RI
mRNA is localized at the NMJ and, in
addition, demonstrated that RI
recruits a C
subunit to the
junction, providing the potential for local responsiveness to cAMP at
this site. Thus PKA is implicated in the establishment and/or
maintenance of the unique pattern of gene expression occurring at the
junction or in the modulation of synaptic activity via protein
phosphorylation. Although the mRNA localization of the various ACs is
diffuse, it is possible that a specific distribution of the different
AC isoforms exists, playing an as yet unidentified but important role
at the NMJ. However, this is still impossible to determine due to the
absence of good antibodies specific for each isoform.
Because the mouse gastrocnemius muscle is composed of almost 100% type II fast-twitch fibers and because denervation results in an increased amount of slow-twitch fibers, we can assume that AC2 and AC9 are restricted to fast-twitch fibers and that AC7 and AC6 might be associated with the reprogramming after destruction of the motoneuron contacts. Torgan and Kraus (65) also showed that expression of AC2 in the muscle fiber significantly correlates with the percentage of fast-twitch muscle fibers: electrical motor nerve pacing, which induces fiber type transformation of fast-twitch to slow-twitch fibers, induces a decrease in AC2 mRNA preceding the fiber type conversion of metabolic and contracting proteins. This transformation is accompanied by an alteration in gene expression for proteins of energy metabolism and sarcoplasmic reticulum function and by switches in contractile protein isoform expression (39). Therefore, our results indicate that innervation is necessary to maintain the high levels of AC2 and AC9 transcripts and is also required to repress the AC6 and AC7 genes to the low levels found in the control muscle.
At the receptor level, denervation induces the appearance of new AChRs in the muscle membrane outside the endplate (47). cAMP plays a major role in this regulation, since it is one of the second messengers responsible for the increase in AChR number elicited by CGRP. More recently, it has been suggested that nerve regulation of the AChR degradation occurs also via a PKA-dependent pathway (73). In the present study, the basal and stimulated AC activity to various agents at D30 was higher than that at D10, a parallel change being observed in the expression levels of ACs (AC6 and AC7) as well as of G protein mRNAs. Taking these findings together, the changes in AC activity and sensitivity to various agents induced by denervation were due not only to an increase in the numbers of AC molecules as a consequence of increased levels of mRNAs coding for ACs (especially, AC6 and AC7) but also to changes in the receptor-G protein complex occurring together with muscle atrophy.
In conclusion, our studies illustrate the differential expression of AC2, AC6, AC7, and AC9 in skeletal muscle after denervation. Among these isoforms, AC9 and AC7 are the predominant subtypes in normal and denervated muscle, respectively. The expression of these AC isoforms is altered during development, but the pattern of their changes is different. A parallel decrease in mRNAs encoding for AC6 and AC7 and in AC activity during development might indicate that these two isoforms are the major factors contributing to the decline in the activity in the adult animals. Therefore, we suggest that changes in AC activity, determined by the content of specific isoforms, play an important role during muscle atrophy and possibly in other various physiopathological states of the skeletal muscle.
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ACKNOWLEDGEMENTS |
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We thank Dr. I. Espinasse [URA 1159, Centre National de la
Recherche Scientifique (CNRS)] for the gift of all the G protein plasmids, Dr. M. C. Weiss (Unité de Génétique de la
Différenciation, URA 1149, CNRS, Département de Biologie
Moléculaire, Institut Pasteur) for the gift of PKA subunit RI
plasmid, and Dr. D. Daegelen (Génétique et Pathologie
Moléculaires de la Différenciation Musculaire, INSERM
Unité 129) for the gift of myogenin plasmid. We are also thankful
to Drs. G. Guellaen, Y. Laperche, R. Barouki, J. F. Laycock and S. Lotersztajn for critical reading of the manuscript and to E. Grandvilliers for expert secretarial assistance.
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FOOTNOTES |
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This work was supported by INSERM and the Université Paris-Val de Marne.
Y. Suzuki is a recipient of a fellowship from the Fondation pour la Recherche Médicale. T. Shen is a recipient of a fellowship from the People's Republic of China.
Present addresses: Y. Suzuki, Department of Obstetrics and Gynecology, Fukushima Medical College, Fukushima, 960-12 Japan; T. Shen, c/o Dr. Rebecca Morris, Lankenau Medical Research Center, 100 Lancaster Ave. West, Wynnewood, PA 19096.
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. §1734 solely to indicate this fact.
Address reprint requests to J. Hanoune.
Received 15 January 1998; accepted in final form 5 March 1998.
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