1 Généthon, CNRS URA 1922-1923, 91002 Evry; 2 Laboratoire de Physiologie, Atelier de Régénération Neuromusculaire, Faculté de Médecine Saint-Antoine, 75012 Paris, France; and 3 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
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ABSTRACT |
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Lack of functional calpain 3 in humans is a cause
of limb girdle muscular dystrophy, but the function(s) of calpain 3 remain(s) unknown. Special muscle conditions in which calpain 3 is
downregulated could yield valuable clues to the understanding of its
function(s). We monitored calpain 3 mRNA amounts by quantitative RT-PCR
and compared them with those of -skeletal actin mRNA in mouse leg muscles for different types of denervation and muscle injury. Intact
muscle denervation reduced calpain 3 mRNA expression by a factor of 5 to 10, while
-skeletal actin mRNA was reduced in a slower and less
extensive manner. Muscle injury (denervation-devascularization), which
leads to muscle degeneration and regeneration, induced a 20-fold
decrease in the mRNA level of both calpain 3 and
-skeletal actin.
Furthermore, whereas in normal muscle and intact denervated muscle, the
full-length transcript is the major calpain 3 mRNA, in injured muscle,
isoforms lacking exon 6 are predominant during the early regeneration
process. These data suggest that muscle condition determines the
specific calpain 3 isoform pattern of expression and that calpain 3 expression is downregulated by denervation.
calpain; -skeletal actin; regeneration; reverse
transcriptase-polymerase chain reaction
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INTRODUCTION |
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LIMB GIRDLE MUSCULAR DYSTROPHY type 2A (LGMD2A) belongs to a group of autosomally inherited muscular dystrophies characterized by progressive symmetrical atrophy and weakness of the proximal limb muscles. The LGMD2A gene encodes calpain 3 (26), which is the only member of the calpain family whose deficiency is associated with a defined phenotype in humans. Calpains are nonlysosomal Ca2+-dependent cysteine proteases (25), which may be ubiquitous proteins such as µ- and m-calpain (17), or tissue-specific calpains, like calpain 3, also called nCL1, p94, or CAPN3, which were initially reported to be skeletal muscle specific (29). The relationship between the deficiency in calpain 3 and the occurrence of LGMD2A is a unique opportunity to address the function(s) of this particular protease, and of the calpains in general. Calpains have been implicated in a wide variety of processes such as apoptosis (32), cell division (35), and myogenic differentiation (18). Recent data suggest that a lack of calpain 3 can lead to myonuclear apoptosis in vivo (3), although the pathophysiological role of this enzyme remains to be demonstrated.
Because its natural substrates are unknown, insight into calpain 3's functions can be gained by studying its expression profiles during embryogenesis. The appearance of calpain 3 mRNA during skeletal muscle development is a relatively late event. It occurs later than that of other muscle-specific proteins and is subsequent to muscle innervation in both human and mouse myogenesis. In humans, transcripts of calpain 3 in the trunk and limb muscles have been detected at the eighth week of embryonic development (11). This developmental stage is broadly equivalent to embryonic day 13.5 (E13.5) in the mouse, when calpain 3 transcripts can be visualized in paraxial muscles (10). Thus it seems likely in humans and mouse that innervation could be a prerequisite for calpain 3 mRNA expression in the muscle.
The response of the gene repertoire to muscle injury can also be
informative for the comprehension of protein functions. In fact, the
pattern of expression for a number of mRNAs of muscle proteins has been
shown to be similar during embryogenesis and during muscle regeneration
after injury (2, 9, 16, 28). Therefore, skeletal muscle
regeneration after injury and the innervation of regenerated muscle
fibers provide a valuable model for evaluating the role of these events
in inducing the transcription of specific mRNAs. For instance, it was
previously shown that the pattern of -skeletal actin mRNA expression
during muscle regeneration in the muscle autografting model is similar
to that seen during embryonic development (8),
particularly at the time of muscle innervation (27).
In an attempt to study the muscle status in which calpain 3 is
required, we first investigated the effect of transient and chronic
denervation of intact healthy adult mouse muscles to determine whether
muscle innervation modulates the expression of calpain 3 mRNA in a
similar way to that of -skeletal actin. Second, to provide
additional information on the role of calpain 3 expression during
myogenesis, we followed its expression after muscle injury both with
and without reinnervation. The expression study was limited to the mRNA
level because no specific mouse calpain 3 antibodies are available for
quantitative studies. We also examined exon splicing events because it
has been shown that alterations involving the calpain 3-specific
regions (NS, IS1, and IS2) and/or the Ca2+-binding site
occur during normal development (15, 23).
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MATERIALS AND METHODS |
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Animals
Male Swiss mice weighing 18-22 g (CEAL, Ardenay, France) were kept in a room at constant temperature with natural night-day cycles and fed pellets and water ad libitum. All protocols were conducted according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). Surgical procedures were performed under chloral hydrate anesthesia (3.5%, 0.3 ml ip). Anesthetized animals were killed by cervical dislocation.Muscle Denervation
The dorsal skin of the thigh was cut and the posterior muscles divided to show the sciatic nerve. A chronic denervation was obtained by cutting a 5-mm section of the sciatic nerve (SNS). To avoid regeneration, the proximal end of the nerve was ligatured. In contrast, to allow nerve regeneration, the sciatic nerve was crushed (SNC) for 10 s at midthigh with no. 5 Dumont forceps.Muscle Injury
The injury delivered to the right extensor digitorum longus (EDL) was a denervation devascularization (DD) (1). The anterior compartment of the hindlimb, which contains the tibialis anterior (TA) and EDL, was exposed. The distal tendon of the EDL was dissected with the aid of a microscope, and a surgical thread (5/0) was slid under and shimmied up to the proximal tendon to disrupt all the vascular and nerve supplies. In addition, the proximal and distal tendons were crushed successively with a forceps for 40 s to complete muscle anoxia. The EDL was then gently laid into its natural bed and the skin was sutured. DD injury is difficult to perform on the TA because of the absence of a proximal tendon in this muscle, and the lesion area never extends to the entire TA.Experimental Protocol
The animals were randomly assigned to one of four experimental groups. Treatments applied were: a single SNC, a single SNS, a DD of the right EDL, and an SNS performed in addition to DD (DDS). In each group, additional animals were sham operated, and their muscles were used as control. In these animals, the skin was cut and muscles or sciatic nerves were visualized, but no treatment was applied. At selected days, up to 21 days after denervation or muscle injury, the muscles of three mice from each experimental group were excised: the TA muscle in treatments SNC and SNS and the EDL muscle in treatments DD and DDS. Chronic denervation was insured at the time of muscle excision by visualization of abnormal gait of the limb and by verifying the discontinuity of the sciatic nerve at the thigh. After dry blotting, muscles were immediately frozen in liquid nitrogen and stored atRNA Preparation
Each muscle (25-100 mg) was homogenized into a FastRNA (Bio101) tube that contained silica/ceramic matrix. One milliliter of RNA PLUS (Bioprobe) extraction solution was added, and the tube was placed in a FastPrep (Bio101) instrument for 20 s at a speed rating of 6. Total RNA was then extracted according to the Bioprobe protocol. These samples were dissolved in ultrafiltered water, and their concentrations were determined by measuring the optical density at 260 nm with a Beckman spectrophotometer. RNA integrity was checked on a 1% agarose gel.Reverse Transcription Reaction
One microgram of each total RNA sample was used in reverse transcription reactions performed with the SuperScript II RT (GIBCO BRL) using random hexamer primers. The reaction was carried out at 42°C for 50 min. The RT was inactivated by incubation for 15 min at 70°C.Oligonucleotide Primers and TaqMan Probes
Oligonucleotide primers and TaqMan probes were designed using Primer Express (Perkin Elmer Applied Biosystem) and Oligo 4.0 (Primer Analysis software). The sequences used were from the mouse TFIID (GenBank accession no. D01034), the mouse capn3 gene (GenBank accession no. X92523) and the mouse skeletal actin mRNA sequence (GenBank accession no. M12866). The TaqMan probe consisted of an oligonucleotide with a 5' reporter dye and a downstream, 3' quencher dye. The fluorescent reporter dye, 6-carboxyfluorescein, was covalently linked to the 5' end of the oligonucleotide. This reporter dye was quenched by 6-carboxytetramethyl rhodamine, which was located at the 3' end. Primer pairs and TaqMan probes used are as follows. TFIID Probe (M654TFIID.p): 5'-TGTGCACAGGAGCCAAGAGTGAAGA-3' Forward primer (M616TFIID.a): 5'-ACGGACAACTGCGTTGATTTT-3' Reverse primer (M724TFIID.m): 5'-ACTTAGCTGGGAAGCCCAAC-3' Calpain 3 Probe (M884CAPN3.p): 5'-TGCCAAGCTCCATGGCTCCTATGAAG-3' Forward primer (M811CANP3.a): 5'-ACAACAATCAGCTGGTTTTCACC-3' Reverse primer (M954CANP3.m): 5'-CAAAAAACTCTGTCACCCCTCC-3'Quantitative PCR
The technique used to estimate relative values of mRNA levels of the three tested genes is based on real-time detection of PCR products by measuring the increase of fluorescence due to TaqMan probe degradation (14). This probe anneals at a specific position between the forward and reverse primer sites. The degradation of the hybridized probe is due to the nucleolytic activity of the ampliTaq Gold DNA polymerase during each polymerization step and does not interfere with the exponential accumulation of PCR products (22). The probe degradation induces an increase in fluorescence of the reporter dye due to the reduction of the fluorescent resonance energy transfer. Fluorescence emission is monitored in real time during the PCR. The increase of fluorescence is related to the initial number of copies through a particular parameter, the threshold cycle (Ct). It is defined as the PCR cycle at which the fluorescence signal rises above a predetermined baseline (threshold) value. The threshold value must be low enough to correspond with the exponential phase. It has been shown that under these conditions, Ct is related to the initial number of template copies (14).The PCR amplifications were performed using 1 µl of each reverse transcription reaction product diluted in a reaction buffer containing 1× TaqMan buffer, 4-6 mM MgCl2, 2.5 units of ampliTaq Gold DNA polymerase, 200 nM primers (forward and reverse), and 100 nM TaqMan probe in a final volume of 50 µl. Cycling conditions consisted of an ampliTaq Gold activation step at 95°C for 10 min followed by 40 cycles of 2 steps: 15 s of denaturation at 95°C and 60 s of annealing at 60°C. The PCR was performed on an ABI PRISM 7700 sequence detector (Perkin Elmer Applied Biosystem), allowing automatic data collection of the fluorescence emission. The mRNA level of each sample was determined as an average from data obtained from two independent PCRs, each including duplicates. The target mRNA content of 30 samples was measured simultaneously in one assay (96-well plate) with linear standard samples included.
Normalization of Quantitative PCR
Two series of control samples were included in each assay. The first consisted of a series of five successive fivefold dilutions of an individual reverse transcription product from total RNA extracted from an untreated muscle. This series of controls allowed the estimation of the PCR efficiency by interpolating the slope of the curve relating the Ct parameter obtained for each point with the relative concentration of cDNA (dilution of reverse transcription product). Theoretically, the PCR efficiency is 100% when the number of copies doubles at each PCR cycle. After testing different couples of primer pairs in various magnesium concentrations, we kept the pair that gave the highest PCR efficiency (>90%).The second series of controls consisted of five different RT products from various amounts of mouse total skeletal muscle RNA (respectively, 1 µg, 100 ng, 10 ng, 1 ng, and 0.1 ng) mixed with an appropriate amount of total RNA from the worm Eisenia foetida andrei to keep the final amount of RNA in the RT mix to 1 µg. RNA from this worm was chosen because it is easy to obtain and the gene sequences are divergent enough from the mouse gene sequences not to compete at the PCR level. With these controls, a standard curve was produced by associating the Ct with its corresponding mRNA relative concentration.
To account for variations due to RNA extraction and the RT reaction,
the measured levels of calpain 3 and -skeletal actin mRNAs were
correlated with those of TFIID mRNAs. TFIID is a transcription factor
that has been used as an endogenous control (4, 20). Because the TFIID gene is ubiquitously expressed, we consider the mRNA
level of TFIID to be proportional to the quantity of total RNA of all
types of cells included in the sample. Results were expressed as the
ratio of the mRNA level of each gene of interest (calpain 3 and
-skeletal actin) to the mRNA level of TFIID.
Detection of Calpain 3 Isoform
Each cDNA was amplified by PCR with calpain 3-specific primer pairs covering specific IS1 sequences, which include exon 6, and IS2 sequences, which include exons 15 and 16 as described in Ref. 15.Detection of Apoptosis
The TdT-mediated dUTP nick end labeling (TUNEL) method (an in situ cell death detection kit) was used according to the manufacturer's recommendations (Boehringer). ![]() |
RESULTS |
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Changes in levels of calpain 3 and -skeletal actin mRNAs were
first investigated in intact muscles that were denervated either transiently or chronically. The transient denervation is obtained with
an SNC, and the chronic denervation is obtained with an SNS. Second,
changes in levels of calpain 3 and
-skeletal actin mRNAs were
monitored in two treatments of a second type involving a direct muscle
injury called DD. The muscle suffers ischemia, which induces
necrosis followed by regeneration. One treatment (DD) allows
reinnervation, while the other (DDS) prevents it because it includes,
in addition to DD, an SNS. For each treatment, the mRNA levels in three
muscles of three different mice were measured at eight different dates
following the treatments (days 1, 3, 5, 7, 9, 11, 14,
and 21).
Histology
Ten-micrometer-thick cross sections stained with hematoxylin and eosin were microscopically examined. Normal intact muscle exhibits fibers with peripheral nuclei and a polygonal shape. The nucleus is nonperipheral in ~1% of the fibers. Interstitial nuclei (mainly fibroblasts) are rare and scattered between the fibers. The fiber size is variable but never exceeds 50 µm (Fig. 1A). After a DD treatment, the muscle first exhibits a stage of degeneration that is followed by regeneration. By days 1-3 after injury, inflammatory cells appear between the fibers at the periphery of the muscle. In the central part of the muscle, most of the fibers are pale without nuclei, and inflammatory cells are undetectable (Fig. 1B). By days 3 and 4, the fibers are invaded centripetally by inflammatory cells, indicating a phenomenon of phagocytosis. Some fibers (1 to 4 layers) remain intact at the periphery of the muscle. By days 5 and 6, inflammatory cells become rare and small, round regenerating fibers (myotubes) with central nuclei, and basophilic cytoplasm can be observed at the junction between the ring of intact fibers and the central zone. Their number increases and their size enlarges because they invade the central zone centripetally. Reinnervation of all the fibers (intact and regenerated ones) occurs by days 11-14 (21). By day 21, regenerated fibers show a polygonal shape, a central nucleus, a near normal size, and a fascicular organization (Fig. 1C). The lack of reinnervation of the regenerated fibers results in an atrophy of the fibers, which exhibit mainly peripheral nuclei (Fig. 1D), contrasting with the centrally nucleated regenerated fibers that are allowed to be reinnervated (Fig. 1C).
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Three days after a nerve injury of an intact muscle (SNS and SNC treatment), the appearance of the muscle is normal (Fig. 1, E and F). Twenty-one days after the SNC treatment corresponding with the crush of the sciatic nerve, the appearance is also unchanged because the denervation is only transient (Fig. 1G). In the case of chronically denervated muscle corresponding with the SNC treatment, fiber atrophy is detectable 21 days after the nerve lesion (Fig. 1H).
Calpain 3 and -Skeletal Actin mRNA Levels
in Denervated Intact Muscles: SNC and
SNS Treatments
Calpain 3.
After SNC treatment, the relative levels of calpain 3 mRNA reached a
minimal level of 10% at day 3, compared with control levels
(Fig. 2). Later, from days 5 to 11, the levels stabilized at ~40%. At day
14, the levels increased and eventually reached 80% of the
control levels at day 21.
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-Skeletal actin.
In SNC treatment,
-skeletal actin mRNA levels stay at ~100% at
day 1. On the following days, between days 3 and
11, these levels stabilize at 60-80% of the control
values. At day 14, the levels are higher than the 100%
control levels and reach 160% at day 21.
Calpain 3 compared with -skeletal actin.
For both SNC and SNS treatment, 3 days after nerve injury, the levels
of calpain 3 mRNA clearly decrease in a quicker and stronger manner
than the levels of
-skeletal actin mRNA. In the following days, this
discrepancy results in a distinct plateau for each mRNA level. Between
days 11 and 14, the levels of both calpain 3 and
-skeletal actin mRNA simultaneously increase in SNC, but relatively
low levels are maintained in chronically denervated muscles (SNS).
Calpain 3 and -Skeletal Actin mRNA Levels in
Regenerating Muscles: DD and DDS
Treatments
Calpain 3.
In DD treatment, the levels of calpain 3 mRNA drop to ~10%
of the control values 1 day after injury and stay there until the fifth
day (Fig. 3). The levels then
progressively increase to reach 30% between days 7 and
14. At day 21, the levels reach ~60%.
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-Skeletal actin.
In DD treatment, the levels of
-skeletal actin mRNA drop to ~30%
of control level on the first day and 10% on the 5th day after muscle
injury. Subsequently, one sees an increase in
-skeletal actin mRNA
levels. The levels approach 30% at day 7 and further increase to reach 70 and 80%, respectively, between days 9 and 14. At day 21, the average levels return to
almost control levels. If one compares individual animals, the amounts
of
-skeletal actin mRNA are even higher than in two out of three
control animals (data not shown).
Calpain 3 compared with -skeletal actin.
Muscle injury results in a similar steep decrease for both calpain 3 and
-skeletal actin mRNA expression on the first day following
injury. From days 3 to 7, both mRNA levels slowly
increase from 10 to 30%. At day 9, only the
-skeletal
actin mRNA level dramatically climbs from 30 to 70%, with the DD
treatment in which innervation occurred but not with the DDS treatment.
Calpain 3 mRNA levels remained low (~50%) until day 21 in
both DD and DDS, just as
-skeletal actin did in DDS. The
-skeletal actin pattern is thus very reminiscent of the profile
exhibited under these conditions by calpain 3 mRNAs.
Detection of Specific Isoforms
We observed differences in the gel profiles of the PCR products covering the IS1 region (Fig. 4). A strong expression of isoforms lacking exon 6 is observed from days 1-7, compared with untreated muscles in both DD and DDS treatments with a peak at day 5. After day 7, this fragment was detected at trace levels and was only visible in PAGE (data not shown). The SNC and SNS treatments, which affect the innervation of the muscle without direct muscle injury, do not lead to an increase in the expression of isoforms lacking exon 6. No difference was observed in the gel profiles of the PCR products covering the IS2 region after any of the treatments (data not shown).
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Detection of Apoptosis
Deficiency of calpain 3 in humans has been correlated with the presence of myonuclear apoptosis (3). Given that denervation of muscles could also induce apoptosis (33), we investigated the presence of apoptotic myonuclei in our model of intact mouse muscles treated by an SNS or an SNC. No significant increase of apoptotic myonuclei after SNC treatment and after SNS treatment was detected on TUNEL staining on TA biopsies (data not shown). ![]() |
DISCUSSION |
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Only a few studies have used molecular tools to investigate the expression profiles of the genes specific for different muscular dystrophies in detail. Such "molecular physiology" could be of great value to better comprehend the role and function of these genes in developing, resting, and active muscles and to provide new insights into the development of treatments for muscle disorders involving these genes. This study summarizes our attempts to address the molecular physiology of calpain 3 in a murine experimental model. Although LGMD2A is not a neurogenic disease, the use of denervated muscles allowed us to test the influence of the motor nerve on the expression of the calpain gene. In the same manner, our experimental degeneration of the muscle provides some insight into the regulation of calpain 3 expression during muscle fiber regeneration but does not explain why the lack of calpain 3 results in muscle degeneration in LGMD2A.
Three main observations emerged from this study. 1) Muscle
denervation decreases calpain 3 mRNA expression more strongly and faster than that of the skeletal muscle marker -skeletal actin. 2) Neither muscle regeneration nor innervation of
regenerated fibers activated the expression of calpain 3 mRNA, whereas
the
-skeletal actin mRNA level was increased at this time.
3) The isoforms lacking exon 6 become predominant during the
regeneration process after muscle injury, whereas no spliced isoforms
are detected in denervated muscles.
Method To Quantify Specific mRNAs
In this study, we used a "real-time" quantitative RT-PCR method to measure the relative levels of two types of mRNAs in mouse skeletal muscle: calpain 3 andIt is also worth noting that the experimental design does not allow discrimination between de novo transcription and maintenance of mRNA populations. Meanwhile, it provides a valuable estimate of steady-state levels of the particular mRNA examined at a given time.
Calpain 3 Expression in Denervated Intact Muscle
Chronic muscle denervation results first in disuse of the muscle and later in the occurrence of muscle atrophy. The dramatic decrease of calpain 3 mRNA expression (down to 10% of control value) after denervation suggests that calpain 3 plays a minor role in unused muscle fibers or that its maintenance at normal levels could be deleterious to this unused muscle. The presence of calpain 3 could also be necessary in functioning muscle to inhibit one or several active processes known to occur in denervated muscles. In this case, its decrease might launch some active process such as proteolysis that causes fiber atrophy (12) or an increase in the level of polyubiquitin and proteasomes (24). Calpain 3 expression might thus be an antagonist to the expression of other proteases.The discrepancy of mRNA expression between calpain 3 and -skeletal
actin observed at the time of denervation is also observable at the
time of reinnervation. A few days after the muscles have been
reinnervated (i.e., the 14th day after nerve crush injury),
-skeletal actin mRNA expression increases over the control value, and it is still 160% overexpressed at day 21, whereas
calpain 3 mRNA reaches only 80% at days 14 and
21 after injury. A boosted synthesis of contractile
filaments is probably needed after reinnervation to compensate for the
muscle atrophy, which may not be the case for calpain 3. Another
explanation could be that the reinnervation increases the stability of
-skeletal actin mRNAs.
Calpain 3 Expression in Degenerating-Regenerating Muscle
The denervation-devascularization treatment induces a degeneration-regeneration of muscle fibers after ischemia and is derived from the initial model of the EDL muscle-free grafts in the cat and rat (8). Because mouse muscles are too small to permit sutures of their tendons, Anderson (1) proposed devascularizing the mouse EDL by pinching the tendons with forceps after cutting the vascular pedicle. The results of calpain 3 mRNA expression in this model show that an impressive feature following muscle injury is the strongly reduced levels (5-10% of control value) of both calpain 3 andCalpain 3 Isoforms
Because the quantitative estimates reflect the overall calpain 3 mRNA population, irrespective of the relative contribution of each of its splice isoforms, additional tests using conventional RT-PCR were performed to assess their modulation. Only the splicing of exon 6 is observed, but it is striking how it seems to be correlated with the proliferation and differentiation of the MPC. The splicing of exon 6 does not seem to be affected by innervation, since DD and DDS treatments yield similar patterns, and no splicing events are shown in intact muscle following denervation. In a previous study (10), it was shown that the exon 6 splicing event is detectable in mouse embryos from E12.5 to E17.5, i.e., at the time of myoblast proliferation. Moreover, in C2C12 cell culture, the isoform lacking exon 6 is predominant at the myoblast stage, whereas the isoform containing exon 6 is predominant at the myotube stage (15). So, correlation between the predominance of the isoform lacking exon 6 with muscle regeneration might indicate a specific role for this isoform in the MPC. Such a role could have a link with the fact that this particular exon 6 splicing event affects the autolytic activity of calpain 3 (15). On the other hand, we showed that the mRNA level of all the isoforms combined is low during the regeneration process. Therefore, the predominance of the isoforms lacking exon 6 might just reflect the fact that only mature fiber cells (innervated or not) express full isoforms, whereas mononuclear cell types express isoforms lacking exon 6. Such cells, including macrophages and MPC, are indeed predominant during the early stages of the regeneration. This is in agreement with the hypothesis that MPC cells expressing MyoD and Myf-5 need leukemia inhibitory factor release from infiltrating/resident macrophages to expand their compartment before the expression of myogenic regulatory factor (MRF) 4, to differentiate and to form in myotubes (28). The splicing of calpain 3 exon 6 could be dependent on the expression on MyoD and/or Myf-5 and downregulated by MRF4.Apoptosis in Denervated Muscles
A specific process has been described in denervated muscles, namely, the increased presence of apoptotic muscle fibers (33, 36). Numerous apoptotic nuclei were also recently observed in muscle biopsies from LGMD2A patients (3). In both cases, calpain 3 levels may be so reduced that they may no longer have a normal activity that would prevent apoptosis. It has been suggested that the absence of calpain 3 would lead to accumulation of inhibitoryConclusions
Together, the results of the experiments on denervated and/or regenerating muscle indicate that normal expression of calpain 3 mRNA requires the presence of the motor nerve, which innervates the muscle. This expression is regulated either by muscle activity or by a diffusible factor provided by motor nerve terminals at the end plate. Although calpain 3 is a protease, these results show that it may play a role in the regulation of the atrophy without being directly involved in the protein degradation per se, but rather by blocking its start-up. Calpain 3 seems to have a paradoxal role because it does not seem to act as a primary protease, although it is more abundant than the ubiquitous calpains at the mRNA level by a factor of 10 (30). This is consistent with the fact that absence of functional calpain 3 in humans is associated with atrophy of the limb girdle muscles. Our data confirm the hypothesis that calpain 3 has a regulatory role instead of a degradative one. It has recently been shown that calpain 3 mRNA is decreased in a rat model of cachexia, whereas proteases like m-calpain and proteasome subunits mRNA are increased (6). A transgenic mouse overexpressing interleukin-6 displays muscle atrophy with a decrease of calpain 3 mRNA. Calpain 3 seems to play a counterregulatory role (34).Expression of the complete calpain 3 isoform is not upregulated during the myogenic processes involved in muscle regeneration. But, the isoform lacking the autolytic site region (exon 6) seems to be predominant during the early regeneration process. These data are in agreement with the myogenic development expression pattern in utero. Finally, muscle reinnervation, intact or regenerated, induces a slow return to control values. Therefore, the mature complete isoform of calpain 3 seems rather to play a role in the housekeeping of intact innervated fibers or a regulatory role, while the isoform in which exon 6 is skipped could play a role during MPC multiplication and differentiation. Further investigations are needed to clarify this point.
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ACKNOWLEDGEMENTS |
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We thank Prof. Michel Vidaud for initiating us in the quantitative RT-PCR technique. We thank Régis Gluzman for assistance. We also thank Susan Cure for helpful assistance in writing the manuscript.
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FOOTNOTES |
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This work was supported by grants from the Association Française contre les Myopathies (AFM). D. Stockholm is the recipient of an AFM fellowship.
Address for reprint requests and other correspondence: J. S. Beckmann, Généthon, CNRS URA 1922, 1 bis rue de l'Internationale, BP 60, 91002 Evry, France (E-mail: beckmann{at}weizmann.ac.il).
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.
Received 21 September 2000; accepted in final form 19 December 2000.
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