Báo cáo khoa học: Calpain 3: a key regulator of the sarcomere? pot

MINIREVIEW
Calpain 3: a key regulator of the sarcomere?
Ste
´
phanie Duguez, Marc Bartoli and Isabelle Richard
Ge
´
ne
´
thon, CNRS UMR8115, Evry, France
Introduction
Calpains (EC 3.4.22.17) are nonlysosomal cysteine pro-
teases with activity that is calcium dependent (for a
detailed review of calpains, see [1]). The most well-
known ones are the ubiquitous heterodimeric calpains
(l and m), which have been known since the eighties,
and calpain 3, loss-of-function mutations of which lead
to limb-girdle muscular dystrophy type 2A (LGMD2A,
MIM No. 253600 [2]).
LGMD2A is one of the most common LGMDs,
accounting for about 35% of cases, with a prevalence
estimated to be 1 : 15 000–1 : 150 000 depending on
the area (for a complete list of publications reporting
mutations, see Table 1 and supplementary Doc S1;
[3,4]). To date, 286 distinct pathogenic calpain 3 muta-
tions (13 nonsense, 74 deletion⁄ insertion, 38 splice site,
and 161 missense) have been characterized in the lit-
erature and the Leiden database ( />capn3_home.html). They are distributed along the
entire length of the gene (Fig. 1). Patients with
LGMD2A, like other LGMD2 patients, classically pre-
sent with progressive muscle weakness and atrophy of

the shoulder and pelvic girdle musculature, an elevated
serum creatine kinase activity and a degener-
ation ⁄ regeneration pattern in muscular biopsy samples
[5]. Interestingly, patients homozygous for null muta-
tions usually have no protein and a more severe phe-
notype, suggesting that a correlation between clinical
phenotype and genotype may exist.
Calpain 3 is the only calpain known to cause a
monogenic disease, and its implication in LGMD2A
underscores its crucial role in muscle homeostasis.
Recently, significant progress has been made in the
comprehension of its mode of regulation and its poss-
ible function in muscle. This review summarizes the
current knowledge about the calpain 3 gene and pro-
tein, as well as the disease pathogenesis.
The calpain 3 gene and its expression
The human calpain 3 gene is located on chromosome
15q15.1-q21.1 and covers a genomic region of 138 kb
(Ensembl gene ID ENSG00000092529; Fig. 1). The
Keywords
calpain 3; limb girdle muscular dystrophy
type 2A; skeletal muscle
Correspondence
I. Richard, Ge
´
ne
´
thon, CNRS UMR8115,
1 rue de l’Internationale, 91000 Evry, France
Fax: +33 1 60 77 86 98

Tel: +33 1 69 47 29 38
E-mail:
(Received 23 March 2006, accepted 18 May
2006)
doi:10.1111/j.1742-4658.2006.05351.x
Calpain 3 is a 94-kDa calcium-dependent cysteine protease mainly
expressed in skeletal muscle. In this tissue, it localizes at several regions of
the sarcomere through binding to the giant protein, titin. Loss-of-function
mutations in the calpain 3 gene have been associated with limb-girdle mus-
cular dystrophy type 2A (LGMD2A), a common form of muscular dystro-
phy found world wide. Recently, significant progress has been made in
understanding the mode of regulation and the possible function of cal-
pain 3 in muscle. It is now well accepted that it has an unusual zymogenic
activation and that cytoskeletal proteins are one class of its substrates.
Through the absence of cleavage of these substrates, calpain 3 deficiency
leads to abnormal sarcomeres, impairment of muscle contractile capacity,
and death of the muscle fibers. These data indicate a role for calpain 3 as a
chef d’orchestre in sarcomere remodeling and suggest a new category of
LGMD2 pathological mechanisms.
Abbreviation
LGMD2A, limb-girdle muscular dystrophy type 2A.
FEBS Journal 273 (2006) 3427–3436 ª 2006 The Authors Journal compilation ª 2006 FEBS 3427
predominant product of this gene is encoded by 24
exons corresponding to a 3316-bp mRNA and is
principally expressed in adult skeletal muscle in
fast-twitch and slow-twitch fibers [2,6]. Accordingly,
the phenotype of LGMD2A affects both types of
fiber [7].
In addition to the main product, multiple alternative
transcripts have been detected in human, mouse, rat

and rabbit tissues, but usually with an expression level
100- to 1000-fold lower (for listing of isoforms, see
[8,9]). Some of these transcripts are expressed from an
additional alternative ubiquitous promoter known to
be present in human and mouse genomes or from a
lens-specific promoter detected in mouse, rat and rab-
bit genomes but absent from the human genome. As
the phenotype observed in patients with LGMD2A is
muscle-restricted, we will not discuss the role of cal-
pain 3 outside skeletal muscle.
Structure of the calpain 3 protein
Translation of the main calpain 3 gene product leads to
a 94-kDa protein of 821 amino acids consisting of
a short N-terminal region (domain I), a papain-type
proteolytic domain (domains IIa and IIb), a C2-like
domain (domain III) and a calcium-binding domain
composed of five EF-hands (domain IV) [6,10] (Fig. 1).
In addition, calpain 3 possesses three unique sequences
not found in any other calpains, NS (N-terminal
sequence), IS1 and IS2 (inserted sequences 1 and 2).
NS is a 20–30 amino-acid N-terminal domain rich in
proline encoded by exon 1. This region is in domain I
which corresponds to a regulatory propeptide found in
various cysteine proteinases [11]. IS1 is a polypeptide of
about 50 amino acids encoded by exon 6 and embed-
ded in the proteolytic domain. It contains three auto-
lytic sites: Y274, N292 and Y322. As a consequence,
Table 1. Publications reporting calpain 3 mutations in chronological order (for full reference details see supplementary Doc S1). The geo-
graphic origin of patients reported in each publication is indicated in the second column. A website reporting calpain 3 mutations is indicated
at the end of the table.

Publication Country
Richard et al. 1995 PMID: 7720071 Brazil, France, Reunion Island
Dincer et al. 1997 PMID: 9266733 Turkey
Richard et al. 1997 PMID: 9150160 France, Israel, Italy, Turkey, USA
Haffner et al. 1998 PMID: 9452114 Germany
Penisson-Besnier et al. 1998 PMID: 9655129 Brazil, France, Reunion Island
Kawai et al. 1998 PMID: 9771675 Japan
Urtasun et al. 1998 PMID: 9762961 Spain
Chou et al. 1999 PMID: 10102422 Italy, Mexico, Poland, USA
Passos-Bueno et al. 1999 PMID: 10069710 Brazil
Minami et al. 1999 PMID: 10567047 Japan
Richard et al. 1999 PMID: 10330340 Bulgaria, Canada, France, Germany, Greece, USA, Italy, Japan,
Lebanon, the Netherlands, Poland, Russia, Spain, Switzerland,
Turkey, UK, USA, Vietnam
Pogoda et al. 2000 PMID: 10679950 Russia
Chae et al. 2001PMID: 11525884 Japan
Pollitt et al. 2001PMID: 11297944 UK
de Paula et al. 2002 PMID: 12461690 Brazil
Vainzof et al. 2003 PMID: 12890817 Brazil
Chrobakova et al. 2004 PMID: 15351423 Czech Republic
Canki-Klain et al. 2004 PMID 14981715 Croatia
Cobo et al. 2004 PMID: 15757244 Spain
Fanin et al. 2003 PMID: 14578192 Italy
Fanin et al. 2004 PMID: 15221789 Italy, UK
Fanin et al. 2005 PMID: 15725583 Italy
Georgieva et al. 2005 PMID: 16001438 Bulgaria
Milic et al. 2005 PMID: 16100770 Croatia
Piluso et al. 2005 PMID: 16141003 Italy
Saenz et al. 2005 PMID: 15689361 Brazil, France, Reunion Island, Spain
Todorova et al. 2005 PMID: 15733273 Germany

Leiden Muscular Dystrophy pagesª (Calpain-3) (last
modified 26 September 2004)
Skeletal muscle calpain 3 S. Duguez et al.
3428 FEBS Journal 273 (2006) 3427–3436 ª 2006 The Authors Journal compilation ª 2006 FEBS
calpain 3 in which IS1 is deleted no longer autolyzes,
although it is still proteolytically competent [8]. Cal-
pain 3 autolysis occurs rapidly in heterologous cells or
inadequately extracted muscle samples and presumably
after physiological activation in living muscle [12,13].
It generates a small fragment of 30 kDa and a large
C-terminal fragment, the size of which ranges from 60
to 55 kDa depending on the extent of autolysis. IS2 is
a peptide of about 80 amino acids encoded by exons
15–16 and located between domain II and domain III.
A basic PVKKKKNKP sequence encoded by exon 15
seems to act as a nuclear translocation signal at least in
human and COS-7 cells [12,14]. IS2 has been demon-
strated to be important in the control of the activity of
calpain 3, as exon 15 deletion leads to a Ca
2+
inde-
pendence of autolytic activity, and exon 16 deletion
leads to loss of substrate proteolysis [8,15].
Because of the rapid autolysis of calpain 3, it has so
far been impossible to obtain crystals of the full mole-
cule. However, Jia and colleagues established a 3D
model of calpain 3 based on the known structure of
m-calpain [10]. The model shows that the proteolytic
domain can be subdivided into two globular subdo-
mains (domain IIa and IIb), forming a catalytic cleft

at their interface. As in ubiquitous calpains, domain
III of calpain 3 fits a C2 motif. In this model, IS1 and
Fig. 1. Calpain 3 gene, mRNA and protein. Upper panel: the human calpain 3 gene structure (GenBank accession number AF209502.1).
Arrows labeled ‘Pub’, ‘Pm’ and ‘Pl’ represent alternative promoters expressing calpain 3 variants in all tissues, skeletal muscle and lens,
respectively. Sole exons encoding for the muscle-specific variant are represented. Middle panel: Localization and distribution of the 289
LGMD2A mutations along the calpain 3 transcript. The24 exons are numbered and represented by a green box. (s) indicates missense muta-
tions; q nonsense mutations; ⁄ splice site mutation; fi large deletion; r in-frame deletion; m frameshift deletion; . insertion and
complex
mutation. Lower panel: schematic representation of the calpain 3 protein with its four domains and specific insertions (NS, IS1 and IS2).
S. Duguez et al. Skeletal muscle calpain 3
FEBS Journal 273 (2006) 3427–3436 ª 2006 The Authors Journal compilation ª 2006 FEBS 3429
IS2 have been structured as loops protruding out of
the globular core structure. However, Diaz and col-
leagues have shown that, instead of protruding, IS1 is
composed of an a-helix flanked by loops that close the
catalytic cleft, blocking its access to substrates and
inhibitors [16].
Recently, other structural analyses have revealed
that calpain 3 could homodimerize through its penta
EF-hand domain [17]. The dimer would be in a tail to
tail orientation, placing the catalytic domains at both
ends. This homodimerization is reminiscent of the het-
erodimeric structure of the ubiquitous calpains, the
large subunits of which associate with a small subunit
of 30 kDa [18]. It has been suggested that the small
subunit may act as a chaperone or that dissociation
from the catalytic subunit is part of the activation pro-
cess of the ubiquitous calpains [19,20]. Therefore, the
interesting observation of calpain 3 dimerization raises
the question of whether and how it can intervene in

the regulation of calpain 3 activation or binding to
partners.
Subcellular localization of calpain 3
Insights into the subcellular localization of calpain 3
have come from a yeast two-hybrid screening in which
calpain 3 was found to bind to the I-band and M-line
regions of titin [21,22]. This extremely large molecule
spans half the sarcomere from the Z-disc to the M-line
and participates in the construction and overall elasti-
city of the myofibrils [23]. The binding of calpain 3 to
the I-band was restricted to the Ig83 immunoglobulin-
like domain of titin which is located in the N2A
region, close to the extensible PEVK domain (Fig. 2).
In the M-line, it was mapped to a unique region
flanked by two immunoglobulin C2 motifs encoded by
Mex5, the next to last exon of titin (Fig. 2). The min-
imal region for the binding to N2A of calpain 3
involves the IS2 region, comprising residues 570–639
[22]. Concerning the binding to the M-line, no minimal
domain has been found [21]. However, the replacement
of exon 1 by the lens-specific first exon abolished the
binding to both sites, whereas the absence of IS1 and
IS2 seems to increase the binding [8]. In conclusion,
the mechanism of calpain 3 binding to the two titin
regions is apparently different, suggesting distinct phy-
siological functions.
Subsequent immunolocalization studies carried out
in humans and mice confirmed that calpain 3 is
localized in several regions of the sarcomere. In
addition to the N2A and M-line location, calpain 3

also seems to be localized in the Z-disc [13,22,24].
Beside these localizations, calpain 3 has also been
found at the costameres and myotendinous junctions
in mouse muscle and in the nucleus in human mus-
cle [13,14].
Regulation of the proteolytic activity
of calpain 3
As a cytoplasmic protease, the activity of calpain 3
must be tightly regulated temporally and spatially to
be effective and to avoid unwanted damage. Besides
control at the transcriptional level and regulation via
Fig. 2. Schematic representation of titin. Diagram of titin with its different domains and the calpain 3 binding and cleavage sites. White
circles represent calpain 3 and black arrowheads the sites where calpain 3 cleaves titin. The question mark represents putative calpain 3
binding in the Z-disc region.
Skeletal muscle calpain 3 S. Duguez et al.
3430 FEBS Journal 273 (2006) 3427–3436 ª 2006 The Authors Journal compilation ª 2006 FEBS
its compartmentalization, another interesting regula-
tory mechanism has been identified at the protein level
[13,16]. When extracted from fresh muscle, calpain 3 is
seen mainly in an unprocessed form that has been
shown to correspond to an inactive protein [13,16,25].
Once activated, it first undergoes intramolecular pro-
teolysis at one autolytic site in IS1. Then, calpain 3
can intermolecularly proteolyze the other sites, remov-
ing the IS1 loop and leaving the two other parts of the
molecule associated through noncovalent bounds.
Thus, IS1 acts as an inhibitory peptide of calpain 3
activity.
In contrast with the situation in muscle, calpain 3
is fully active when expressed in nonmuscle cells [13].

Therefore, it can be postulated that the muscle inhibi-
tion arises through interaction with a muscle-
specific protein, a good candidate for which is its
partner, titin. Interestingly, when calpain 3 was over-
expressed in muscle, either in transgenic mice or by
gene transfer, no obvious phenotype was seen, indica-
ting a high buffering capacity of the muscle [26,27].
On the other hand, overexpression of calpain 3 in
muscular dystrophy with myositis (mdm) mice, a
strain carrying a deletion of several amino acids in
the Ig83 domain of titin, aggravates the phenotype,
suggesting the necessity for N2A binding to control
calpain 3 activity [28,29]. However, coexpression with
the N2A region in COS-7 cells does not by itself
impair the proteolytic activity of calpain 3 [30]. How-
ever, downstream of N2A, titin has a domain pre-
senting some homology with calpastatin, an inhibitor
of the ubiquitous calpains [31]. In addition, in tibialis
muscular dystrophy, a muscle disease caused by speci-
fic mutations in the M-line titin, calpain 3 was
reduced or absent as an unprocessed protein [32,33].
Taken together, these observations pinpoint titin as a
reservoir of inactive calpain 3 molecules and suggest
that dissociation from titin corresponds to calpain 3
activation.
The question remains what is the signal leading to
calpain 3 activation? The main activation signal of the
ubiquitous calpains is Ca
2+
. After much debate, the

Ca
2+
dependency of calpain 3 activity is now well
established thanks to research with mutants and
in vitro analyses of the catalytic subdomains [15,34–
36]. However, Ca
2+
cannot be considered a signal for
calpain 3 because a trace amount is sufficient for acti-
vation [12]. Another signal that has been excluded is
exercise, as calpain 3 is down-regulated after eccentric
exercise and does not autolyze with exhaustive or
endurance exercises in humans [37,38]. Therefore, fur-
ther studies are still needed to obtain a clear picture of
the calpain 3 activation signal.
Calpain 3 substrates
Coexpresssion experiments and in vitro studies have led
to the identification of numerous proteins that can be
cleaved by calpain 3, including titin, filamin C, vinexin,
ezrin and talin [13,39,40]. Although in vivo confirmation
is awaited, the fact that these proteins are located in the
vicinity of calpain 3 renders them likely physiological
substrates. The absence of a consensus sequence at the
cleavage sites indicates that there is no specificity and
suggests that calpain 3 cleaves destructured regions as
the ubiquitous calpains do. The pattern of cleaved prod-
ucts suggests limited proteolysis as a means to irrevers-
ibly modulate the function of substrates (Fig. 3). For
example, the cleavage of filamin C at the extreme
C-terminus abolishes the interaction with c-sarcoglycans

and d-sarcoglycans and dissociates the dimerization
domain from the rest of the molecule [39]. Another
example comes from talin. The ubiquitous calpains
cleave talin at almost the same position as calpain 3
[41]. This cleavage induces a 16-fold increased affinity
for integrin b-3, showing that calpains may induce an
increase in the function of their substrates [42]. These
data pinpoint a putative function for calpain 3 in the
adjustment of cytoskeleton ⁄ membrane links.
Lack of proteolysis of substrates as the
origin of LGMD2A pathogenesis
Fine correlation analyses of LGMD2A mutations with
perturbations on calpain 3 features may be the first
Fig. 3. Calpain 3 substrates. Four calpain 3 substrates with the pro-
posed cleavage sites (black arrows). The main domains and binding
sites are shown. FERM, band F, ezrin ⁄ radixin ⁄ moesin; SoHo, sor-
bin homology; SH3, Src homology 3. To obtain a description of
these domains, see the InterPro website at the EMBL-EBI: http://
www.ebi.ac.uk/interpro/.
S. Duguez et al. Skeletal muscle calpain 3
FEBS Journal 273 (2006) 3427–3436 ª 2006 The Authors Journal compilation ª 2006 FEBS 3431
step to understanding the pathogenesis of the disease.
The numerous patients who present two null mutations
leading to the absence of the protein together with the
recessive pattern of inheritance clearly indicates that
LGMD2A is due to a deficiency in the function of cal-
pain 3. This piece of evidence has further been valid-
ated by the reproduction of the phenotype when the
gene has been knocked-out in mice. However, analysis
of patient biopsy specimens showed that a proportion

of them have normal calpain 3 expression on western
blot (in particular for the patients homozygous for the
mutations T184M, G222R, G496R, S606L, R490W,
R490Q, R489Q and R461C). However, further analysis
showed that some of these mutations could be associ-
ated with impairment of autolytic activity, and there-
fore indicative of perturbation of calpain 3 function
[34].
To add more complexity, it has been shown that
S606L, a mutation located in IS2, leads to a normal
calpain 3 level, with autolytic activity as well as correct
subcellular localization [7]. Interestingly, in vitro analy-
sis of other LGMD2A missense mutations (S744G and
R769Q) indicated that they retain autolytic activity as
well [15]. Even though they have the ability to cleave
calpain 3 intermolecularly, they are no longer able to
cleave the endogenous fodrin in transfected COS-7
cells. These data indicate that, in these mutants, the
intramolecular and intermolecular proteolysis is not
affected and suggests a problem in substrate recogni-
tion. Others mutations were shown to impair titin
binding, including the fully active R448H, D705G
mutants [10,40]. In those cases, it is possible that the
resulting abnormal compartmentalization may have
the consequence of preventing the cleavage of sub-
strates. Taken together, these observations are consis-
tent with the hypothesis that LGMD2A pathogenesis
is related to the loss of proteolytic activity against sub-
strates. A preliminary requirement to confirm that this
is true in vivo would require the identification of a con-

dition in which a physiological cleavage of substrates
could be observed.
Calpain 3 activity is needed in fully
mature myofibers
Calpain 3 is not essential for building functional mus-
cles, as indicated by the fact that muscles of patients
develop normally and that the mean age of onset of
the disease is in the second decade. Along with this
fact, expression of the full-length calpain 3 during both
human and mouse skeletal muscle development is a
relatively late event, subsequent to muscle innervation
and therefore to myoblast proliferation and fusion
[43]. It was also consistently observed that its expres-
sion is concomitant with the appearance of neoformed
myotubes and reinnervation in the regeneration pro-
cess occurring after experimental degeneration [44,45]
and with myoblast differentiation during in vitro myo-
genesis in C2C12 cells [8,45,46]. It can be concluded
that calpain 3 is not required for myoblast prolifer-
ation and fusion, in contrast with the ubiquitous cal-
pains, nor for the regulation of muscle regeneration
and reinnervation. The function of calpain 3, which
manifests itself as proteolysis of substrates, seems to be
important during the life of fully differentiated fibers;
its absence leads to degeneration and death of the
fibers.
Calpain 3 in the life and death
of myofibers
The ubiquitous calpains have been shown to partici-
pate in the initial proteolytic events that accompany

muscle wasting, whereas calpain 3 can be excluded
from this process for several reasons. First, calpain 3
deficiency results in muscle atrophy in LGMD2A. Sec-
ondly, in two models of cachexia (transgenic mice
overexpressing interleukin 6 and Yoshida AH-130 rat
ascites hepatoma), calpain 3 mRNA has been shown
to be down-regulated [47,48]. Thirdly, calpain 3 is also
down-regulated during the atrophic phase seen after
nerve section [44]. In all cases, calpain 3 activity corre-
lates negatively with muscle degradation, again in
contrast with the ubiquitous calpains which show a
positive correlation [49].
We can also state that the myofiber degeneration
observed in patients with LGMD2A is not related to
membrane disruption, in contrast with other muscular
dystrophies caused by mutations in proteins of the
dystrophin–glycoprotein complex (for a relatively
recent review, see [50]). In fact, there is a normal
amount and correct localization of sarcolemmal pro-
teins such as dystrophin, sarcoglycans and merosin in
LGMD2A [51–54]. Even if some Evans blue-positive
cells can occasionally be seen in calpain-3-deficient
muscles, they probably reflect dying fibers rather than
membrane permeability. Furthermore, no increased
numbers of Evans blue-positive cells after exercise and
no deficiency in membrane resistance of stretched iso-
lated muscles have been observed [55].
Beside membrane fragility, a second pathogenic
mechanism leading to LGMD2 has been identified in
the form of deficiency in membrane repair [56]. This

defect was observed in LGMD2B due to mutations
in dysferlin, a member of the newly described ferlin
family [57]. It is interesting to note that there is a
Skeletal muscle calpain 3 S. Duguez et al.
3432 FEBS Journal 273 (2006) 3427–3436 ª 2006 The Authors Journal compilation ª 2006 FEBS
secondary reduction in dysferlin in calpain 3-deficient
muscle [52] and that an interaction has been identified
between calpain 3 and dysferlin [58]. However, the lack
of Evans blue-positive cells argues against the partici-
pation of calpain 3 in the repair process.
Another mechanism is needed to explain LGMD2A
pathology. Indeed, interesting observations can be put
together to link calpain 3 deficiency with abnormal
sarcomere organization. First, biopsy samples from
LGMD2A patients and calpain 3-deficient mice
present aspecific ultrastructural changes such as the
presence of lobulated fibers, fragmentation and disor-
ganization of myofibers [34,40,59]. Secondly, calpain 3-
deficient primary myotubes from knock-out mice
lacked well-organized sarcomeres and presented a mis-
incorporation of adult myosin heavy chain [40].
Finally, antisense oligonucleotides against calpain 3 led
to immature Z discs and diffuse distribution of
a-actinin in myotubes [60]. Altogether, these data sug-
gest a role for calpain 3 in sarcomere maintenance in
mature muscle cells.
During adult life, skeletal muscles must constantly
adapt to respond to metabolic, mechanical or hormo-
nal conditions. These adaptations involve altered
patterns of both protein synthesis and protein degrada-

tion and promote changes in contractile and metabolic
proteins to optimize muscle function [61]. Considering
the highly organized structure of the muscles, the
exchange of myofibrillar proteins during these proces-
ses, known as sarcomere remodeling, necessitates the
intervention of proteolytic systems. Indeed, numerous
studies have shown that the ubiquitous calpains inter-
vene in the initial phase of myofibril disassembly, and
the ubiquitin ⁄ proteasome system is in charge of the
degradation of proteins that are no longer needed
[49,62,63]. Interestingly, the recovery phase subsequent
to unloading is associated with an increase in calpain 3
expression, whereas calpain 3-deficient muscles failed
to regain their full weight under this condition [64]. In
addition, there is an increase in ubiquitination of pro-
teins in reloading that is not seen in the absence of
calpain 3. It is noteworthy that a reduction in the
expression of several ubiquitin ⁄ proteasome system
components was observed in calpain 3-deficient mice
[65]. In conclusion, it is possible that calpain 3 defici-
ency impairs the remodeling response consequent to
perturbation of the ubiquitin ⁄ proteasome system.
These abnormal sarcomeres seem to have a twofold
effect: (a) a decrease in the force-generating capacity
of the fibers related to impaired contractility of the
muscle fibers [55]; (b) an increase in cellular stress as
indicated by the up-regulation of heat-shock proteins
in the muscles of knock-out mice and the presence of
apoptotic myonuclei in patients and mice [14,54,64].
However, it is not possible to know whether the per-

turbation of the apoptosis-controlling pathway of NF-
jB observed in patients with LGMD2A is subsequent
to the stress response, to the adjustment of the nuclei
number to the volume of the atrophying fibers, or is a
direct consequence of the lack of calpain 3 activity on
the NF-jB ⁄ IjBa pathway.
Conclusion
Ten years ago, the gene responsible for LGMD2A was
identified as coding for the enigmatic protease, cal-
pain 3. This finding was the starting point for molecu-
lar diagnosis for patients and had the consequence of
designating LGMD2A as a common form of muscular
dystrophy. The recognition of LGMD2A is still a chal-
lenge at the protein level as some mutations maintain
the protein but in an inactive form and secondary
reductions are observed in a number of muscular dys-
trophies. From a therapeutic point of view, treatment
of this recessive disease by gene transfer can be pro-
posed and tested based on information about the gene
involved. Indeed, we recently demonstrated the safety
and efficacy of adeno-associated virus (AAV)-mediated
calpain 3 cDNA transfer in a mouse model of
LGMD2A [26]. However, gene therapy still has some
obstacles that remain to be worked out before it can
become a therapeutic solution in human beings. Hope-
fully, identification of the role of calpain 3 will eventu-
ally lead to an understanding of the pathogenesis of
the disease and proposals of original pharmacological
treatment for this disorder. In fact, we are on the verge
of understanding the full extent of calpain 3 regulation

and physiological function. In addition to its regula-
tion of transcription, alternative splicing and subcellu-
lar compartmentalization, calpain 3 has an interesting
and unusual internal zymogenic mechanism of activa-
tion that is unique in the protease world. In mature
innervated fibers, calpain 3 seems to play a role in sar-
comere remodeling by cleaving cytoskeletal proteins
during muscular adaptation. This role is in agreement
with the cytoskeletal nature of the known in vitro sub-
strates of calpain 3. Identifying the signal that triggers
its activity is the next step for the validation of its phy-
siological substrates and determination of the conse-
quences on muscle regulation. Placing calpain 3 in the
context of the biological pathway in which it acts will
then make it possible to envisage how and when to
intervene therapeutically to bypass this pathway or
compensate for its perturbation.
Overall, calpain 3 can be envisaged as a ‘chef-d’
orchestre’ in the homeostasis of the muscle sarcomere.
S. Duguez et al. Skeletal muscle calpain 3
FEBS Journal 273 (2006) 3427–3436 ª 2006 The Authors Journal compilation ª 2006 FEBS 3433
From this proposed role, it can be postulated that
deregulation of sarcomere remodeling would constitute
the origin of LGMD2A pathogenesis. It suggests the
existence of a new pathogenic mechanism besides
membrane fragility and membrane repair which may
also be applied to other muscular dystrophies caused
by mutations in sarcomeric proteins.
Acknowledgements
We would like to acknowledge Dr Nathalie Daniele,

Dr Susan Cure and Dr Oliver Danos for critical read-
ing of the manuscript. This work was supported by the
Association Franc¸ aise contre les Myopathies.
References
1 Goll DE, Thompson VF, Li H, Wei W & Cong J (2003)
The calpain system. Physiol Rev 83, 731–801.
2 Richard I, Broux O, Allamand V, Fougerousse F,
Chiannilkulchai N, Bourg N, Brenguier L, Devaud C,
Pasturaud P, Roudaut C, et al. (1995) Mutations in the
proteolytic enzyme calpain 3 cause limb-girdle muscular
dystrophy type 2A. Cell 81, 27–40.
3 Urtasun M, Saenz A, Roudaut C, Poza JJ, Urtizberea
JA, Cobo AM, Richard I, Garcia Bragado F, Leturcq
F, Kaplan JC, et al. (1998) Limb-girdle muscular dys-
trophy in Guipuzcoa (Basque Country, Spain). Brain
121, 1735–1747.
4 Fanin M, Nascimbeni AC, Fulizio L & Angelini C
(2005) The frequency of limb girdle muscular dystrophy
2A in northeastern Italy. Neuromuscul Disord 15, 218–
224.
5 Fardeau M, Eymard B, Mignard C, Tome FM, Richard
I & Beckmann JS (1996) Chromosome 15-linked limb-
girdle muscular dystrophy: clinical phenotypes in
Reunion Island and French metropolitan communities.
Neuromuscul Disord 6, 447–453.
6 Sorimachi H, Imajoh-Ohmi S, Emori Y, Kawasaki H,
Ohno S, Minami Y & Suzuki K (1989) Molecular clon-
ing of a novel mammalian calcium-dependent protease
distinct from both m- and mu-types. Specific expression
of the mRNA in skeletal muscle. J Biol Chem 264,

20106–20111.
7 Jenne DE, Kley RA, Vorgerd M, Schroder JM, Weis J,
Reimann H, Albrecht B, Nurnberg P, Thiele H, Muller
CR, et al. (2005) Limb girdle muscular dystrophy in a
sibling pair with a homozygous Ser606Leu mutation
in the alternatively spliced IS2 region of calpain 3.
Biol Chem 386 , 61–67.
8 Herasse M, Ono Y, Fougerousse F, Kimura E,
Stockholm D, Beley C, Montarras D, Pinset C,
Sorimachi H, Suzuki K, et al. (1999) Expression and
functional characteristics of calpain 3 isoforms
generated through tissue-specific transcriptional and
posttranscriptional events. Mol Cell Biol 19, 4047–4055.
9 Kawabata Y, Hata S, Ono Y, Ito Y, Suzuki K, Abe K
& Sorimachi H (2003) Newly identified exons encoding
novel variants of p94 ⁄ calpain 3 are expressed ubiqui-
tously and overlap the alpha-glucosidase C gene.
FEBS Lett 555, 623–630.
10 Jia Z, Petrounevitch V, Wong A, Moldoveanu T, Da-
vies PL, Elce JS & Beckmann JS (2001) Mutations in
calpain 3 associated with limb girdle muscular dystro-
phy: analysis by molecular modeling and by mutation in
m-calpain. Biophys J 80, 2590–2596.
11 Suzuki K & Sorimachi H (1998) A novel aspect of cal-
pain activation. FEBS Lett 433, 1–4.
12 Sorimachi H, Toyama-Sorimachi N, Saido TC,
Kawasaki H, Sugita H, Miyasaka M, Arahata K,
Ishiura S & Suzuki K (1993) Muscle-specific calpain,
p94, is degraded by autolysis immediately after transla-
tion, resulting in disappearance from muscle. J Biol

Chem 268, 10593–10605.
13 Taveau M, Bourg N, Sillon G, Roudaut C, Bartoli M
& Richard I (2003) Calpain 3 is activated through auto-
lysis within the active site and lyses sarcomeric and
sarcolemmal components. Mol Cell Biol 23, 9127–9135.
14 Baghdiguian S, Martin M, Richard I, Pons F, Astier C,
Bourg N, Hay RT, Chemaly R, Halaby G, Loiselet J,
et al. (1999) Calpain 3 deficiency is associated with myo-
nuclear apoptosis and profound perturbation of the
IkappaB alpha ⁄ NF-kappaB pathway in limb-girdle
muscular dystrophy type 2A. Nat Med 5, 503–511.
15 Ono Y, Shimada H, Sorimachi H, Richard I, Saido TC,
Beckmann JS, Ishiura S & Suzuki K (1998) Functional
defects of a muscle-specific calpain, p94, caused by
mutations associated with limb-girdle muscular dystro-
phy type 2A. J Biol Chem 273, 17073–17078.
16 Diaz BG, Moldoveanu T, Kuiper MJ, Campbell RL &
Davies PL (2004) Insertion sequence 1 of muscle-specific
calpain, p94, acts as an internal propeptide. J Biol Chem
279, 27656–27666.
17 Ravulapalli R, Diaz BG, Campbell RL & Davies PL
(2005) Homodimerization of calpain 3 penta-EF-hand
domain. Biochem J 388, 585–591.
18 Graham-Siegenthaler K, Gauthier S, Davies PL & Elce
JS (1994) Active recombinant rat calpain II. Bacterially
produced large and small subunits associate both in vivo
and in vitro. J Biol Chem 269, 30457–30460.activity and
a novel mode of enzyme activation. EMBO J 18, 6880–
6889.
19 Hosfield CM, Elce JS, Davies PL & Jia Z (1999) Crystal

structure of calpain reveals the structural basis for
Ca(
2+
)-dependent protease
20 Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia Z &
Davies PL (2002) A Ca(
2+
) switch aligns the active site
of calpain. Cell 108, 649–660.
Skeletal muscle calpain 3 S. Duguez et al.
3434 FEBS Journal 273 (2006) 3427–3436 ª 2006 The Authors Journal compilation ª 2006 FEBS
21 Kinbara K, Sorimachi H, Ishiura S & Suzuki K (1997)
Muscle-specific calpain, p94, interacts with the extreme
C-terminal region of connectin, a unique region flanked
by two immunoglobulin C2 motifs. Arch Biochem
Biophys 342, 99–107.
22 Sorimachi H, Kinbara K, Kimura S, Takahashi M, Ishi-
ura S, Sasagawa N, Sorimachi N, Shimada H, Tagawa
K, Maruyama K, et al. (1995) Muscle-specific calpain,
p94, responsible for limb girdle muscular dystrophy type
2A, associates with connectin through IS2, a p94-specific
sequence. J Biol Chem 270, 31158–31162.
23 Horowits R (1999) The physiological role of titin in
striated muscle. Rev Physiol Biochem Pharmacol 138,
57–96.
24 Keira Y, Noguchi S, Minami N, Hayashi YK &
Nishino I (2003) Localization of calpain 3 in human
skeletal muscle and its alteration in limb-girdle muscular
dystrophy 2A muscle. J Biochem (Tokyo) 133, 659–664.
25 Anderson LV, Davison K, Moss JA, Richard I,

Fardeau M, Tome FM, Hubner C, Lasa A, Colomer J
& Beckmann JS (1998) Characterization of monoclonal
antibodies to calpain 3 and protein expression in muscle
from patients with limb-girdle muscular dystrophy type
2A. Am J Pathol 153, 1169–1179.
26 Bartoli M, Roudaut C, Martin S, Fougerousse F, Suel
L, Poupiot J, Gicquel E, Noulet F, Danos O & Richard
I (2006) Safety and efficacy of AAV-mediated calpain 3
gene transfer in a mouse model of limb-girdle muscular
dystrophy type 2A. Mol Ther 13, 250–259.
27 Spencer MJ, Guyon JR, Sorimachi H, Potts A,
Richard I, Herasse M, Chamberlain J, Dalkilic I,
Kunkel LM & Beckmann JS (2002) Stable expression
of calpain 3 from a muscle transgene in vivo: imma-
ture muscle in transgenic mice suggests a role for
calpain 3 in muscle maturation. Proc Natl Acad Sci
USA 99, 8874–8879.
28 Garvey SM, Rajan C, Lerner AP, Frankel WN & Cox
GA (2002) The muscular dystrophy with myositis
(mdm) mouse mutation disrupts a skeletal muscle-
specific domain of titin. Genomics 79, 146–149.
29 Huebsch KA, Kudryashova E, Wooley CM, Sher RB,
Seburn KL, Spencer MJ & Cox GA (2005) mdm mus-
cular dystrophy: interactions with calpain 3 and a novel
functional role for titin’s N2A domain. Hum Mol Genet
14, 2801–2811.
30 Kinbara K, Ishiura S, Tomioka S, Sorimachi H, Jeong
SY, Amano S, Kawasaki H, Kolmerer B, Kimura S,
Labeit S, et al. (1998) Purification of native p94, a mus-
cle-specific calpain, and characterization of its autolysis.

Biochem J 335, 589–596.
31 Maruyama K, Endo T, Kume H, Kawamura Y,
Kanzawa N, Nakauchi Y, Kimura S, Kawashima S &
Maruyama K (1993) A novel domain sequence of
connectin localized at the I band of skeletal muscle
sarcomeres: homology to neurofilament subunits.
Biochem Biophys Res Commun 194, 1288.
32 Haravuori H, Vihola A, Straub V, Auranen M, Richard
I, Marchand S, Voit T, Labeit S, Somer H, Peltonen L,
et al. (2001) Secondary calpain3 deficiency in 2q-linked
muscular dystrophy: titin is the candidate gene.
Neurology 56, 869–877.
33 Hackman P, Vihola A, Haravuori H, Marchand S,
Sarparanta J, De Seze J, Labeit S, Witt C, Peltonen L,
Richard I & Udd B (2002) Tibial muscular dystrophy is
a titinopathy caused by mutations in TTN, the gene
encoding the giant skeletal-muscle protein titin. Am J
Hum Genet 71, 492–500.
34 Fanin M, Nascimbeni AC, Fulizio L, Trevisan CP,
Meznaric-Petrusa M & Angelini C (2003) Loss of cal-
pain-3 autocatalytic activity in LGMD2A patients with
normal protein expression. Am J Pathol 163, 1929–1936.
35 Rey MA & Davies PL (2002) The protease core of the
muscle-specific calpain, p94, undergoes Ca
2+
-dependent
intramolecular autolysis. FEBS Lett 532, 401–406.
36 Garcia Diaz BE, Gauthier S & Davies PL (2006) Ca(
2+
)

dependency of calpain 3 (p94) activation. Biochemistry
45, 3714–3722.
37 Feasson L, Stockholm D, Freyssenet D, Richard I,
Duguez S, Beckmann JS & Denis C (2002) Molecular
adaptations of neuromuscular disease-associated pro-
teins in response to eccentric exercise in human skeletal
muscle. J Physiol 543, 297–306.
38 Murphy RM, Snow RJ & Lamb GD (2006) mu-calpain
and calpain-3 are not autolyzed with exhaustive exercise
in humans. Am J Physiol Cell Physiol 290, C116–C122.
39 Guyon JR, Kudryashova E, Potts A, Dalkilic I, Brosius
MA, Thompson TG, Beckmann JS, Kunkel LM &
Spencer MJ (2003) Calpain 3 cleaves filamin C and
regulates its ability to interact with gamma- and delta-
sarcoglycans. Muscle Nerve 28, 472–483.
40 Kramerova I, Kudryashova E, Tidball JG & Spencer
MJ (2004) Null mutation of calpain 3 (p94) in mice
causes abnormal sarcomere formation in vivo and
in vitro. Hum Mol Genet 13, 1373–1388.
41 Hayashi M, Suzuki H, Kawashima S, Saido TC &
Inomata M (1999) The behavior of calpain-generated
N- and C-terminal fragments of talin in integrin-
mediated signaling pathways. Arch Biochem Biophys
371, 133–141.
42 Yan B, Calderwood DA, Yaspan B & Ginsberg MH
(2001) Calpain cleavage promotes talin binding to the
beta 3 integrin cytoplasmic domain. J Biol Chem 276,
28164–28170.
43 Fougerousse F, Durand M, Suel L, Pourquie O, Delezo-
ide AL, Romero NB, Abitbol M & Beckmann JS (1998)

Expression of genes (CAPN3, SGCA, SGCB, and TTN)
involved in progressive muscular dystrophies during
early human development. Genomics 48, 145–156.
S. Duguez et al. Skeletal muscle calpain 3
FEBS Journal 273 (2006) 3427–3436 ª 2006 The Authors Journal compilation ª 2006 FEBS 3435
44 Stockholm D, Herasse M, Marchand S, Praud C,
Roudaut C, Richard I, Sebille A & Beckmann JS (2001)
Calpain 3 mRNA expression in mice after denervation
and during muscle regeneration. Am J Physiol Cell
Physiol 280, C1561–C1569.
45 Miyabara EH, Aoki MS, Soares AG & Moriscot AS
(2005) Expression of tropism-related genes in regenerat-
ing skeletal muscle of rats treated with cyclosporin-A.
Cell Tissue Res 319, 479–489.
46 Nakashima K, Yamazaki M & Abe H (2005) Effects of
serum deprivation on expression of proteolytic-related
genes in chick myotube cultures. Biosci Biotechnol
Biochem 69, 623–627.
47 Busquets S, Garcia-Martinez C, Alvarez B, Carbo N,
Lopez-Soriano FJ & Argiles JM (2000) Calpain-3 gene
expression is decreased during experimental cancer
cachexia. Biochim Biophys Acta 1475, 5–9.
48 Tsujinaka T, Fujita J, Ebisui C, Yano M, Kominami E,
Suzuki K, Tanaka K, Katsume A, Ohsugi Y, Shiozaki
H, et al. (1996) Interleukin 6 receptor antibody inhibits
muscle atrophy and modulates proteolytic systems in
interleukin 6 transgenic mice. J Clin Invest 97, 244–249.
49 Bartoli M & Richard I (2005) Calpains in muscle wast-
ing. Int J Biochem Cell Biol 37, 2115–2133.
50 Lapidos KA, Kakkar R & McNally EM (2004) The

dystrophin glycoprotein complex: signaling strength
and integrity for the sarcolemma. Circ Res 94, 1023–
1031.
51 Vainzof M, de Paula F, Tsanaclis AM & Zatz M (2003)
The effect of calpain 3 deficiency on the pattern of
muscle degeneration in the earliest stages of LGMD2A.
J Clin Pathol 56, 624–626.
52 Chrobakova T, Hermanova M, Kroupova I, Vondracek
P, Marikova T, Mazanec R, Zamecnik J, Stanek J,
Havlova M & Fajkusova L (2004) Mutations in Czech
LGMD2A patients revealed by analysis of calpain3
mRNA and their phenotypic outcome. Neuromuscul
Disord 14, 659–665.
53 Pollitt C, Anderson LV, Pogue R, Davison K, Pyle A &
Bushby KM (2001) The phenotype of calpainopathy:
diagnosis based on a multidisciplinary approach. Neuro-
muscul Disord 11, 287–296.
54 Richard I, Roudaut C, Marchand S, Baghdiguian S,
Herasse M, Stockholm D, Ono Y, Suel L, Bourg N,
Sorimachi H, et al. (2000) Loss of calpain 3 proteo-
lytic activity leads to muscular dystrophy and to
apoptosis-associated IkappaBalpha ⁄ nuclear factor
kappaB pathway perturbation in mice. J Cell Biol 151,
1583–1590.
55 Fougerousse F, Gonin P, Durand M, Richard I &
Raymackers JM (2003) Force impairment in calpain
3-deficient mice is not correlated with mechanical dis-
ruption. Muscle Nerve 27, 616–623.
56 Bansal D, Miyake K, Vogel SS, Groh S, Chen CC,
Williamson R, McNeil PL & Campbell KP (2003)

Defective membrane repair in dysferlin-deficient muscu-
lar dystrophy. Nature 423, 168–172.
57 Bashir R, Britton S, Strachan T, Keers S, Vafiadaki E,
Lako M, Richard I, Marchand S, Bourg N, Argov Z,
et al. (1998) A gene related to Caenorhabditis elegans
spermatogenesis factor fer-1 is mutated in limb-girdle
muscular dystrophy type 2B. Nat Genet 20, 37–42.
58 Huang Y, Verheesen P, Roussis A, Frankhuizen W,
Ginjaar I, Haldane F, Laval S, Anderson LV, Verrips
T, Frants RR, et al. (2005) Protein studies in dysferlin-
opathy patients using llama-derived antibody fragments
selected by phage display. Eur J Hum Genet 13,
721–730.
59 Chae J, Minami N, Jin Y, Nakagawa M, Murayama K,
Igarashi F & Nonaka I (2001) Calpain 3 gene muta-
tions: genetic and clinico-pathologic findings in limb-
girdle muscular dystrophy. Neuromuscul Disord 11,
547–555.
60 Poussard S, Duvert M, Balcerzak D, Ramassamy S,
Brustis JJ, Cottin P & Ducastaing A (1996) Evidence
for implication of muscle-specific calpain (p94) in
myofibrillar integrity. Cell Growth Differ 7, 1461–1469.
61 Attaix D, Mosoni L, Dardevet D, Combaret L, Mirand
PP & Grizard J (2005) Altered responses in skeletal
muscle protein turnover during aging in anabolic and
catabolic periods. Int J Biochem Cell Biol 37, 1962–
1973.
62 Huang J & Forsberg NE (1998) Role of calpain in ske-
letal-muscle protein degradation. Proc Natl Acad Sci
USA 95, 12100–12105.

63 Cao PR, Kim HJ & Lecker SH (2005) Ubiquitin-protein
ligases in muscle wasting. Int J Biochem Cell Biol 37,
2088–2097.
64 Kramerova I, Kudryashova E, Venkatraman G &
Spencer MJ (2005) Calpain 3 participates in sarcomere
remodeling by acting upstream of the ubiquitin-protea-
some pathway. Hum Mol Genet 14, 2125–2134.
65 Combaret L, Bechet D, Claustre A, Taillandier D,
Richard I & Attaix D (2003) Down-regulation of genes
in the lysosomal and ubiquitin-proteasome proteolytic
pathways in calpain-3-deficient muscle. Int J Biochem
Cell Biol 35, 676–684.
Supplementary material
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part of the online article:
Doc S1. Supplementary references.
This material is available as part of the online article
from
Skeletal muscle calpain 3 S. Duguez et al.
3436 FEBS Journal 273 (2006) 3427–3436 ª 2006 The Authors Journal compilation ª 2006 FEBS