Calpain 3 mRNA expression in mice after denervation and during muscle regeneration

Daniel Stockholm1, Muriel Herasse1, Sylvie Marchand1, Christophe Praud2, Carinne Roudaut1, Isabelle Richard1, Alain Sebille2, and Jacques S. Beckmann1,3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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 alpha -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; alpha -skeletal actin; regeneration; reverse transcriptase-polymerase chain reaction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 at -80°C. These muscles were used for RNA preparation. In addition, some muscles were frozen in isopentane chilled in liquid nitrogen and stored at -80°C. They were cross-sectioned (10 µm thick) with a cryostat and stained with hematoxylin-eosin for microscopic examination.

RNA 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' alpha -Skeletal actin Probe (M230aactine.p): 5'-CCAGAGCAAGCGAGGTATCCTGACCC-3' Forward primer (M171aactine.a): 5'-CGTCACCAGGGTGTCATGG-3' Reverse primer (MH309aactine.m): 5'-TGTAGAAGGTGTGGTGCCAGAT-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 alpha -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 alpha -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in levels of calpain 3 and alpha -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 alpha -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).


View larger version (98K):
[in this window]
[in a new window]
 
Fig. 1.   Histological features of muscle cross sections stained by hematoxylin and eosin (magnification ×400). A: normal extensor digitorum longus (EDL) muscle. B and C: denervated devascularized EDL muscle 3 days (B) and 21 days (C) after injury. D: denervated and devascularized EDL muscle with chronic section of the sciatic nerve at midthigh 21 days after injury. E and F: tibialis anterior muscle 3 days after sciatic nerve crush (SNC; E) or 3 days after section of the sciatic nerve (SNS; F) at midthigh. G and H: same lesion as E and F but 21 days after injury. Note that all nuclei have disappeared in B, and this central part of the muscle is not yet infiltrated by mononuclear cells. After muscle regeneration and reinnervation, most of the fibers are centronucleated with near normal shape and size (C). In the lack of reinnervation, the regenerated fibers are mainly peripherally nucleated with smaller size (D). Concerning the denervation of intact muscles, 3 days after a nerve injury, muscle modifications were not detectable (E and F), but chronic denervation resulted in muscle fiber atrophy 21 days afterward as shown in H.

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 alpha -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.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2.   Calpain 3 and alpha -skeletal actin mRNA levels in intact muscle treated by a crush (SNC) or section (SNS) of the sciatic nerve. Samples were taken from 3 animals on each date (control, 1, 3, 5, 7, 9, 11, 14, and 21 days after nerve injury). Data are expressed as means ± SE.

In SNS treatment, in which the muscle can no longer be innervated, the levels of calpain 3 mRNA dropped down to reach a minimal level of 20% of control levels at day 5. The levels then reached 40% between days 7 and 14 before decreasing to 20% at day 21.

alpha -Skeletal actin. In SNC treatment, alpha -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.

In SNS treatment in which the denervation is irreversible, the levels start to drop at day 3 and stabilize at ~50% of the control values between days 5 and 21.

Calpain 3 compared with alpha -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 alpha -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 alpha -skeletal actin mRNA simultaneously increase in SNC, but relatively low levels are maintained in chronically denervated muscles (SNS).

Calpain 3 and alpha -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%.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   Calpain 3 and alpha -skeletal actin mRNA levels in muscle treated by denervation devascularization without (DD) or with (DDS) sciatic nerve section. Samples were taken from 3 animals on each date (control, 1, 3, 5, 7, 9, 11, 14, and 21 days after muscle injury). Data are expressed as means ± SE.

In DDS treatment, during which the regenerating muscle is not allowed to be reinnervated, the initial drop to 10% at day 1 lasts until day 3 after muscle injury. From day 5 to day 9, calpain 3 mRNA levels slowly increase from ~20% to ~40% and reach a plateau at ~50% between days 11 and 21.

alpha -Skeletal actin. In DD treatment, the levels of alpha -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 alpha -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 alpha -skeletal actin mRNA are even higher than in two out of three control animals (data not shown).

In DDS treatment, the initial drop in alpha -skeletal actin mRNA levels reaches 10% of the control value at day 1. This drop lasts until day 3 after muscle injury. From day 5 to day 9, the levels of alpha -skeletal actin mRNA slowly increase from 10 to 50% and reach a plateau at ~50% between days 11 and 21.

Calpain 3 compared with alpha -skeletal actin. Muscle injury results in a similar steep decrease for both calpain 3 and alpha -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 alpha -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 alpha -skeletal actin did in DDS. The alpha -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).


View larger version (73K):
[in this window]
[in a new window]
 
Fig. 4.   Visualization of PCR fragments specific for the IS1 region, including exon 6+ and exon 6- isoforms on ethidium bromide-stained polyacrylamide gel.

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -skeletal actin. 2) Neither muscle regeneration nor innervation of regenerated fibers activated the expression of calpain 3 mRNA, whereas the alpha -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 and alpha -skeletal actin. This procedure measures PCR product accumulation during the exponential phase of the PCR reaction using a dual-labeled fluorogenic probe. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations. Although reproducible, this technique requires, as for any technique measuring mRNA levels, an endogenous mRNA control to correct experimental variations in individual RT. We chose a gene coding for a protein of the transcription factor TFIID complex, the TATA box binding protein for our endogenous mRNA control. Although it can be considered a housekeeping gene, we selected it from among several sequences such as glyceraldehyde-3-phosphate dehydrogenase or ribosomal 18S sequence because it did not show responses to our treatments and its mRNA "absolute level" was closer to the mRNA level of our gene of interest. The Ct value obtained by real-time quantitative PCR is the cycle for which the signal proportional to the PCR product reaches a fixed threshold. Ct values give a rough idea of the absolute level of the sequence quantified. In our case, the Ct obtained for TFIID, CAPN3, and alpha -skeletal actin were between 25 and 30 cycles, but 18S Cts were closer to 10 cycles.

It 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 alpha -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), alpha -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 alpha -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 and alpha -skeletal actin mRNAs as early as 1 day postlesion. The residual amounts are probably due to the small ring of surviving fibers at the periphery of the muscle. When muscle precursor cells (MPC) were proliferating, differentiating, and fusing into myotubes, calpain 3 mRNA expression remained remarkably low (as did alpha -skeletal actin mRNA), indicating that large amounts of the calpain 3 protease are not required for the myogenic process or for myofiber construction. This observation contrasts with the putative role attributed to the ubiquitous calpains in myolysis (7, 13, 31) as well as in the fusion of MPC to generate multinucleated fibers (19). Moreover, calpain 3 mRNA levels do not seem to be affected by reinnervation of the regenerated fibers. In contrast, reinnervation boosts the expression of alpha -skeletal actin. This discrepancy suggests that regenerating innervated myofibers need alpha -skeletal actin to mature and return to a normal size, whereas calpain 3 is not required or perhaps is even detrimental, at least in these early stages of maturation of the myotubes. The return to control levels of calpain 3 expression is very slow and may require a longer period than 21 days after injury. This suggests that calpain 3 did not regulate muscle regeneration and reinnervation. Its return to normal values, however, is secondary to the occurrence of these events.

Calpain 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 inhibitory kappa Balpha and a sequestration of nuclear factor (NF)-kappa B outside the nucleus, thereby preventing a protection against apoptosis (3). It was, therefore, tempting to check whether the decrease of calpain 3 in our model (denervation of intact muscle) was correlated with an increase of apoptotic nuclei and a change in the NF-kappa B localization. No such changes were detected, however. Perhaps the time scale followed was too short for the establishment of such a response. Indeed, it has been recently shown that DNA fragmentation observed by the TUNEL method occurred in only a very small number of cell nuclei in muscles denervated for 2 and 4 mo (5).

Conclusions

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, JE. Dystrophic changes in mdx muscle regenerating from denervation and devascularization. Muscle Nerve 14: 268-279, 1991[ISI][Medline].

2.   Anderson, JE. Murray L. Barr Award Lecture. Studies of the dynamics of skeletal muscle regeneration: the mouse came back! Biochem Cell Biol 76: 13-26, 1998[ISI][Medline].

3.   Baghdiguian, S, Martin M, Richard I, Pons F, Astier C, Bourg N, Hay RT, Chemaly R, Halaby G, Loiselet J, Anderson LV, Lopez de Munain A, Fardeau M, Mangeat P, Beckmann JS, and Lefranc G. Calpain 3 deficiency is associated with myonuclear apoptosis and profound perturbation of the IkappaB alpha/NF-kappaB pathway in limb-girdle muscular dystrophy type 2A. Nat Med 5: 503-511, 1999[ISI][Medline]. [Corrigenda. Nat Med 5: July 1999, p. 849.]

4.   Bieche, I, Olivi M, Champeme MH, Vidaud D, Lidereau R, and Vidaud M. Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer. Int J Cancer 78: 661-666, 1998[ISI][Medline].

5.   Borisov, AB, and Carlson BM. Cell death in denervated skeletal muscle is distinct from classical apoptosis. Anat Rec 258: 305-318, 2000[ISI][Medline].

6.   Busquets, S, Garcia-Martinez C, Alvarez B, Carbo N, Lopez-Soriano FJ, and Argiles JM. Calpain-3 gene expression is decreased during experimental cancer cachexia. Biochim Biophys Acta 1475: 5-9, 2000[ISI][Medline].

7.   Carafoli, E, and Molinari M. Calpain: a protease in search of a function? Biochem Biophys Res Commun 247: 193-203, 1998[ISI][Medline]. [Corrigenda. Biochem Biophys Res Commun 249: August 1998, p. 572.]

8.   Carlson, BM, and Gutmann E. Contractile and histochemical properties of sliced muscle grafts regenerating in normal and denervated rat limbs. Exp Neurol 50: 319-329, 1976[ISI][Medline].

9.   Carraro, U, Dalla Libera L, and Catani C. Myosin light and heavy chains in muscle regenerating in absence of the nerve: transient appearance of the embryonic light chain. Exp Neurol 79: 106-117, 1983[ISI][Medline].

10.   Fougerousse, F, Bullen P, Herasse M, Lindsay S, Richard I, Wilson D, Suel L, Durand M, Robson S, Abitbol M, Beckmann JS, and Strachan T. Human-mouse differences in the embryonic expression patterns of developmental control genes and disease genes. Hum Mol Genet 9: 165-173, 2000[Abstract/Free Full Text].

11.   Fougerousse, F, Durand M, Suel L, Pourquie O, Delezoide AL, Romero NB, Abitbol M, and Beckmann JS. Expression of genes (CAPN3, SGCA, SGCB, and TTN) involved in progressive muscular dystrophies during early human development. Genomics 48: 145-156, 1998[ISI][Medline].

12.   Furuno, K, Goodman MN, and Goldberg AL. Role of different proteolytic systems in the degradation of muscle proteins during denervation atrophy. J Biol Chem 265: 8550-8557, 1990[Abstract/Free Full Text].

13.   Guttmann, RP, Elce JS, Bell PD, Isbell JC, and Johnson GV. Oxidation inhibits substrate proteolysis by calpain I but not autolysis. J Biol Chem 272: 2005-2012, 1997[Abstract/Free Full Text].

14.   Heid, CA, Stevens J, Livak KJ, and Williams PM. Real time quantitative PCR. Genome Res 6: 986-994, 1996[Abstract].

15.   Herasse, M, Ono Y, Fougerousse F, Kimura E, Stockholm D, Beley C, Montarras D, Pinset C, Sorimachi H, Suzuki K, Beckmann JS, and Richard I. Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events. Mol Cell Biol 19: 4047-4055, 1999[Abstract/Free Full Text].

16.   Kablar, B, Asakura A, Krastel K, Ying C, May LL, Goldhamer DJ, and Rudnicki MA. MyoD and Myf-5 define the specification of musculature of distinct embryonic origin. Biochem Cell Biol 76: 1079-1091, 1998[ISI][Medline].

17.   Kawashima, S, Hayashi M, Saito Y, Kasai Y, and Imahori K. Tissue distribution of calcium-activated neutral proteinases in rat. Biochim Biophys Acta 965: 130-135, 1988[ISI][Medline].

18.   Kwak, KB, Chung SS, Kim OM, Kang MS, Ha DB, and Chung CH. Increase in the level of m-calpain correlates with the elevated cleavage of filamin during myogenic differentiation of embryonic muscle cells. Biochim Biophys Acta 1175: 243-249, 1993[ISI][Medline].

19.   Kwak, KB, Kambayashi J, Kang MS, Ha DB, and Chung CH. Cell-penetrating inhibitors of calpain block both membrane fusion and filamin cleavage in chick embryonic myoblasts. FEBS Lett 323: 151-154, 1993[ISI][Medline].

20.   Laurent, A, Costa JM, Assouline B, Voyer M, and Vidaud M. Myotonic dystrophy protein kinase gene expression in skeletal muscle from congenitally affected infants. Ann Genet 40: 169-174, 1997[ISI][Medline].

21.   Lefaucheur, JP, and Sebille A. The cellular events of injured muscle regeneration depend on the nature of the injury. Neuromuscul Disord 5: 501-509, 1995[ISI][Medline].

22.   Livak, KJ, Flood SJ, Marmaro J, Giusti W, and Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4: 357-362, 1995[ISI][Medline].

23.   Ma, H, Fukiage C, Azuma M, and Shearer TR. Cloning and expression of mRNA for calpain Lp82 from rat lens: splice variant of p94. Invest Ophthalmol Vis Sci 39: 454-461, 1998[Abstract].

24.   Medina, R, Wing SS, and Goldberg AL. Increase in levels of polyubiquitin and proteasome mRNA in skeletal muscle during starvation and denervation atrophy. Biochem J 307: 631-637, 1995[ISI][Medline].

25.   Murachi, T. Intracellular regulatory system involving calpain and calpastatin. Biochem Int 18: 263-294, 1989[ISI][Medline].

26.   Richard, I, Broux O, Allamand V, Fougerousse F, Chiannilkulchai N, Bourg N, Brenguier L, Devaud C, Pasturaud P, Roudaut C, Hillaire D, Passos-Bueno MR, Zatz M, Tischfield JA, Fardeau M, Jackson CE, Cohen D, and Beckmann JS. Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 81: 27-40, 1995[ISI][Medline].

27.   Schwartz, J, Wiesen J, Carlson B, Yamasaki L, Moore M, and Womble M. Increased phenylalanine incorporation in regenerating skeletal muscle grafts. Can J Physiol Pharmacol 64: 199-205, 1986[ISI][Medline].

28.   Seale, P, and Rudnicki MA. A new look at the origin, function, and "stem-cell" status of muscle satellite cells. Dev Biol 218: 115-124, 2000[ISI][Medline].

29.   Sorimachi, H, Toyama-Sorimachi N, Saido TC, Kawasaki H, Sugita H, Miyasaka M, Arahata K, Ishiura S, and Suzuki K. Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. J Biol Chem 268: 10593-10605, 1993[Abstract/Free Full Text].

30.   Sorimachi, H, Tsukahara T, Okada-Ban M, Sugita H, Ishiura S, and Suzuki K. Identification of a third ubiquitous calpain species. Chicken muscle expresses four distinct calpains. Biochim Biophys Acta 1261: 381-393, 1995[ISI][Medline].

31.   Spencer, MJ, Croall DE, and Tidball JG. Calpains are activated in necrotic fibers from mdx dystrophic mice. J Biol Chem 270: 10909-10914, 1995[Abstract/Free Full Text].

32.   Squier, M, and Cohen JJ. Calpain, an upstream regulator of thymocyte apoptosis. J Immunol 158: 3690-3697, 1997[Abstract].

33.   Trachtenberg, JT. Fiber apoptosis in developing rat muscles is regulated by activity, neuregulin. Dev Biol 196: 193-203, 1998[ISI][Medline].

34.   Tsujinaka, T, Fujita J, Ebisui C, Yano M, Kominami E, Suzuki K, Tanaka K, Katsume A, Ohsugi Y, Shiozaki H, and Monden M. Interleukin 6 receptor antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice. J Clin Invest 97: 244-249, 1996[Abstract/Free Full Text].

35.   Yamaguchi, R, Maki M, Hatanaka M, and Sabe H. Unphosphorylated and tyrosine-phosphorylated forms of a focal adhesion protein, paxillin, are substrates for calpain II in vitro: implications for the possible involvement of calpain II in mitosis-specific degradation of paxillin. FEBS Lett 356: 114-116, 1994[ISI][Medline].

36.   Yoshimura, K, and Harii K. A regenerative change during muscle adaptation to denervation in rats. J Surg Res 81: 139-146, 1999[ISI][Medline].


Am J Physiol Cell Physiol 280(6):C1561-C1569
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society