The results showed that: a the components of the ubiquitin–proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystroph
Trang 1muscle pathological remodelling
Lydie Laure1, Laurence Suel1, Carinne Roudaut1, Nathalie Bourg1, Ahmed Ouali2, Marc Bartoli1, Isabelle Richard1and Nathalie Danie`le1
1 Ge´ne´thon-CNRS FRE3087, Evry, France
2 INRA de Theix, Saint Gene`s Champanelle, France
Muscle atrophy can result from disuse of the organ or
be associated with ageing or severe systemic conditions
such as diabetes, AIDS and cancer It is also a feature
common to many hereditary muscle diseases, including
muscular dystrophies (MDs) Duchenne MD (DMD),
caused by mutation in the dystrophin gene, is the most
common form of the disease and is particularly severe: skeletal and cardiac muscles are affected, and the life-span of the patients is seriously impaired [1] Limb girdle MDs (LGMDs) represent another important subgroup of MD, grouped together on the basis of common clinical features: they all primarily and
Keywords
CARP; FoxO1; muscle; p21 WAF1/CIP1 ;
remodelling
Correspondence
I Richard, Ge´ne´thon, CNRS FRE3087, 1 bis
rue de l’Internationale, 91000 Evry, France
Fax: +33 0 1 60 77 86 98
Tel: +33 0 1 69 47 29 38
E-mail: richard@genethon.fr
(Received 31 July 2008, revised 20 October
2008, accepted 24 November 2008)
doi:10.1111/j.1742-4658.2008.06814.x
In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, a-sarcoglycan and dystrophin, respectively) The results showed that: (a) the components of the ubiquitin–proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condi-tion, suggesting that CARP could be a hub protein participating in com-mon pathological molecular pathway(s) Interestingly, the mRNA level of
a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21WAF1/CIP1, is consistently increased whenever CARP is upregulated CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy However, a switch towards fast-twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity The observation that p21WAF1/CIP1 is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle
Abbreviations
AAV2/1, adeno-associated virus 2/1; Ankrd2, ankyrin repeat domain-containing protein 2; CARP, cardiac ankyrin repeat protein; DAPI, 4¢,6-diamidino-2-phenylindole; DMD, Duchenne muscular dystrophy; EDL, extensorum digitorum longus; FoxO, forkhead box protein O;
FP, fluorescent protein; LGMD, limb girdle muscular dystrophy; MAFbx, muscle atrophy F-box protein; MD, muscular dystrophy; MLC-2v, myosin light chain 2v; MLC-f, myosin light chain, fast; MuRF1, muscle RING finger protein 1; NF, neurofilament protein; NF-jB, nuclear factor-jB; qRT-PCR, quantitative RT-PCR; TA, tibialis anterior; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; Ub, ubiquitin; UPS, ubiquitin–proteasome system; YFP, yellow fluorescent protein.
Trang 2predominantly affect proximal muscles around the
scapular and the pelvic girdles [2] About 20 different
forms of LGMD are currently recognized; among the
most frequent are LGMD2A, LGMD2B and the
sarcoglycanopathies (LGMD2C–F), caused by
muta-tion in the calpain 3, dysferlin and sarcoglycan genes,
respectively [2]
Disuse-induced atrophy and MDs might share some
molecular mechanisms that are possibly involved in
skeletal muscle wasting Muscle atrophy results from
the negative balance in the ratio between protein
syn-thesis and protein degradation, hence leading towards
protein wasting One of the key players in the
degrada-tion of myofibrillar proteins is the
ubiquitin–protea-some system (UPS) [3] The elimination process is
initiated by labelling of the targeted proteins with
mul-tiple ubiquitin molecules, and requires the coordinated
action of three classes of enzymes known as E1
(ubiqu-itin-activating enzymes), E2 (ubiquitin-conjugating
enzymes) and E3 (ubiquitin ligases) [4] The ubiquitin–
proteasome cascade is stimulated at many levels in
several conditions leading to muscle wasting: the
expression of proteasome subunits, the hydrolytic
activity, and the general substrates ubiquitination [5]
In particular, the 14 kDa ubiquitin carrier protein E2
(E2-14 kDa) and two recently identified E3s, muscle
atrophy F-box protein (MAFbx; also commonly called
atrogin-1) and muscle RING finger protein 1 (MuRF1;
also named TRIM63), are upregulated in many
skele-tal muscle-wasting conditions [5] During atrophy,
expression of MAFbx and MurF1 is stimulated by the
forkhead box protein O (FoxO) family of transcription
factors, through inhibition of the Akt pathway [6,7]
In addition, it was also shown that transcriptional
stimulation of MuRF1 is under the control of the
nuclear factor-jB (NF-jB) pathway [8]
Even though the literature largely explores the
con-vergent role of the UPS components in atrophy,
mus-cle wasting is a complex mechanism in which specific,
although poorly understood, pathways could play a
role In particular, cardiac ankyrin repeat protein
(CARP) was suggested to be involved in these
pro-cesses CARP, together with ankyrin repeat
domain-containing protein 2 (Ankrd2) and diabetes-related
ankyrin repeat protein, forms a family of transcription
regulators known as muscle ankyrin repeat proteins
These three isoforms share in their C-terminal region a
minimal structure composed of several ankryrin-like
domains possibly involved in protein–protein
inter-action, PEST motifs characteristics of rapidly degraded
protein, and a putative nuclear localization signal
CARP is expressed in both cardiac and skeletal
mus-cles, and was reported to be either upregulated [9] or
downregulated [10,11], depending on the atrophic situ-ation considered, and upregulated in hypertrophic con-ditions in heart [12–17] and in skeletal muscle [18–21] From the functional point of view, in heart cells, CARP overexpression suppresses troponin C and atrial natriuretic factor expression [22], and its interaction with the transcription factor YB1 inhibits the synthesis
of the ventricular-specific myosin light chain 2v (MLC-2v) [23] In vascular smooth muscle cells, increased CARP expression has been demonstrated to be associ-ated with upregulation of the protein p21WAF1/CIP1, an inhibitor of the cell cycle [24] Taken as a whole, these findings suggest that CARP coordinates the expression
of genes involved in cell structure and proliferation, and could play a role during muscle mass variation
In an attempt to identify hub proteins that may be potential diagnostic markers or even therapeutic tar-gets for the correction of muscle wasting, the expres-sion of pivotal proteins involved in all the mechanisms discussed previously was investigated in denervation-induced atrophy, as well as in three animal models of LGMD and in the mdx mouse, a DMD model Our study demonstrates that: (a) the UPS is transiently upregulated after denervation, consistent with its known role in atrophy, but it does not seem to be greatly activated in MD; (b) FoxO1 is a biological marker specific for the LGMD2A murine model; and (c) among all the genes considered, the expression
of CARP, together with its downstream target, p21WAF1/CIP1, appears to be the only one that system-atically increases CARP overexpression in muscle fibres fails to induce an atrophic phenotype, indicating that CARP per se cannot initiate the phenomenon Nonetheless, the switch towards fast-twitch fibres observed in this situation, together with the observa-tion that the p21WAF1/CIP1expression pattern seems to reflect CARP level, suggests that CARP might play a role in muscle plasticity
Results The proteasome pathway components are only transiently upregulated, whereas increased CARP expression is maintained throughout denervation-induced-atrophy
The expression of several factors possibly involved in atrophy was investigated by the evaluation of their mRNA level by quantitative RT-PCR (qRT-PCR) in conditions leading to transitory or definitive atrophy The genes studied were those encoding: (a) two tran-scription factors involved in the control of muscle mass: NF-jB-p65 and FoxO1; (b) several components
Trang 3of the UPS – ubiquitin (Ub), E2-14 kDa, two E3
ubiquitin ligases, MuRF1 and MAFbx, and the C2,
C8 and C9 subunits of the proteasome; and (c) CARP,
a transcriptional regulator associated with perturbation
of muscle mass Transient or chronic denervation of
the posterior limb was induced and the mRNA levels
were measured in tibialis anterior (TA) muscles at four
different times following the initiation of the
treat-ments (days 3, 9, 14 and 21) Atrophy was efficiently triggered by the treatment, as 40% of the TA weight was lost after 21 days of chronic denervation (Fig 1A) When denervation was only transient, the
TA weight also initially decreased, but slowly increased again from day 14 while innervation occurred [25] (sta-tistically higher than chronic denervation from day 14
to day 21, P < 0.05)
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A
B
Fig 1 Effect of transient or definitive
denervation on muscle weight and gene
expression profiles Male mice of the
129SvPasIco strain were treated transiently
(T) by crushing or definitively (D) by section
of the sciatic nerve Samples were taken
from six animals on each date (control, 3, 7,
9, 14 and 21 days after nerve injury) (A)
Weight of TA muscles from control, crushed
and sectioned limbs (n = 6 per time point).
Other muscles of the lower limb, such as
EDL and soleus muscles, present similar
proportional loss of weight P-values are
shown as *P < 0.05 for significance
between control and each time point, and
as h
P < 0.05 for significance between
tran-sient and definitive denervation (B) Each
graph demonstrates the expression level for
a gene of interest (FoxO1, NF-jB-p65, Ub,
E2-14 kDa, C2, C8, C9, MuRF1, MAFbx and
CARP ) as assessed by qRT-PCR in TA
muscles of treated animals (n = 2–6 for
each time point) Results are expressed as
percentage of expression level measured in
the respective sham-operated muscles.
*P < 0.05 and **P < 0.01 for significance
between control and each time point;
h
P < 0.05 for significance between
transient and definitive denervation.
Trang 4The results showed that, with both transient and
definitive denervation, FoxO1, NF-jB-p65 and several
components of the UPS (subunits C2, C8 and C9, and
the two E3s MuRF1 and MAFbx) were immediately
and transiently upregulated, with higher variations in
the case of the two E3s (note the logarithmic scale)
Fig 1B) After this initial increase, their expression
returned rapidly to normal levels, even displaying a
slight reduction for every proteasome subunit
consid-ered (C2, C8 and C9) Ubiquitin mRNA levels
decreased very early during the time course of atrophy,
remaining very low when denervation was definitive,
but progressively increasing again from the start of
reinnervation when the sciatic nerve was only crushed
(Fig 1B) E2-14 kDa expression, which remained
sta-ble when atrophy was only transient, was reduced at
late stages (from day 14) of definitive
denervation-induced atrophy (Fig 1B) CARP expression increased
with atrophy (Fig 1B) Whereas CARP expression
slowly decreased back to control level with the
reduc-tion of atrophy in transient denervareduc-tion, it stayed high
when sciatic nerve regeneration was prevented CARP
upregulation was particularly important, as reflected
by the logarithmic scale
CARP is robustly upregulated in murine MDs,
whereas FoxO1 expression is increased
specifically in C3-null animals
The expression levels of the mRNAs measured in
denervation conditions were also compared by
qRT-PCR in several models of MD: a natural model of
dysferlin deficiency [26], which we backcrossed on a
C57BL/6 background and renamed B6.A/J-dysfprmd
(model for LGMD2B), and three engineered models
deficient in either dystrophin (mdx4Cv [27]), calpain 3
(C3-null; unpublished), or a-sarcoglycan (Sgca-null
[28]), models of DMD, LGMD2A and LGMD2D,
respectively Every strain was used at an age where the
symptoms of the disease are detectable (4 months of
age for all models except C3-null mice, which were
evaluated at 7 months of age) and was compared to its
respective control breed The levels of mRNA
expres-sion were measured in five muscles [quadriceps,
extensorum digitorum longus (EDL), TA, soleus and
psoas], chosen in order to reflect the muscle
impair-ment specificity – which varies between models – and
the type of fibres composing the muscle (see
Experi-mental procedures)
The results of qRT-PCR showed that the level of
NF-jB-p65 was slightly increased in specific muscles of
every murine model, especially in the two most
inflam-matory models, mdx4Cvand Sgca-null (Fig 2) FoxO1
was upregulated to very similar levels in every muscle
of the C3-null strain (about two-fold over control, with
P < 0.05 for quadriceps, EDL and psoas), whereas its expression was slightly decreased in all the other models (Fig 2)
The expression of Ub was not affected in any of the four pathologies considered, whereas that of E2-14 kDa showed a tendency to decrease in several muscles (Fig 2) In the mdx4Cvmodel, the levels of the three proteasome subunits (C2, C8 and C9) were affected, C2 and C8 being downregulated and C9 upregulated Unexpectedly, considering their role in atrophy, neither MuRF1 nor MAFbx expression increased in these animal models, their levels being even significantly reduced in some cases (Fig 2) The most remarkable effect observed herein was robust upregulation of CARP mRNA (note the loga-rithmic scale) in most muscles of all models of MDs (Fig 2) Interestingly, this increase seemed to be higher
in the muscles strongly affected by the pathologies The increase was far more important in the Sgca-null and in the mdx4Cv models, two dystrophies character-ized by a similar pathogenesis and caused by a defect
in one of the components of the dystrophin-associated glycoprotein complex
CARP is expressed at the protein level in myofibres of denervation-induced atrophy models and in mononucleated cells of highly regenerative MD animals
Among all the genes whose expression was investi-gated in the different models of muscle disorder, we demonstrated that the CARP gene was the only one whose expression systematically increased, which is consistent with CARP’s role as a hub protein partici-pating in common pathological molecular pathway(s) CARP protein expression was hence measured by western blot in conditions of denervation-induced atrophy and in murine models of MD (Fig 3) Inter-estingly, we observed that the protein was detected
by western blot provided that the mRNA level reached 60-fold over the basal condition This ele-ment probably accounts for the inability to detect CARP in many conditions in which its mRNA upregulation is indeed important, although not important enough The protein was therefore detected from day 3 in both denervation conditions, remaining high until day 21 when the sciatic nerve was sectioned, but dropping to undetectable levels when reinnervation occurred during transient dener-vation (Fig 3A, upper left panel) As regards the murine models of dystrophies, CARP protein was
Trang 5Fig 2 Gene expression profiles in murine models of MD Each graph demonstrates the expression level for a gene of interest (FoxO1, NF-jB-p65, Ub, E2-14 kDa, C2, C8, C9, MuRF1, MAFbx and CARP ) as assessed by qRT-PCR in quadriceps, EDL, TA, soleus and psoas muscles of C3-null, B6.A/J-dysf prmd , Sgca-null and mdx 4Cv animals (n = 3–4 for each point) Results are expressed as percentage of expres-sion level measured in the respective control muscles (129svPasIco and C57BL/6) P-values for significance between wild-type and deficient animals: *P < 0.05 and **P < 0.01.
Trang 6detected in Sgca-null animals only (Fig 3A, lower
left panel)
In order to clarify CARP cellular distribution within
the muscle, immunodetection of the protein was
performed on sections of muscles from denervated (3 days after denervation), a-sarcoglycan-deficient and dystrophin-deficient animals, and their appropriate control strains Specificity of the CARP antibody was
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Trang 7first confirmed by the very specific staining observed in
cultured HER911 cells transfected with a plasmid
encoding CARP (data not shown) In all sections
(con-trol, denervated and MD animals), intense staining
was seen within scattered clusters of small myofibres
(Fig 3B) No difference in the number of these clusters
was observed between conditions, indicating that the
increase in CARP expression did not originate from
these cells In denervation-induced atrophy, additional
diffuse checked-pattern staining of higher-calibre fibres
was also detected, with a higher intensity in denervated
muscles than in control sections (Fig 3C) Considering
the dystrophic process present in Sgca-null and mdx4Cv
animals, it is difficult to evaluate whether such
upregu-lation also occurred in these models In any case, very
intense foci corresponding to the cytoplasm of small
round cells flanking the muscle fibres were observed in
Sgca-null and mdx4Cv animals (Fig 3D) These cells
expressed Pax7, the first transcription factor activated
during myogenesis (Fig 3E) Immunostaining of the
neurofilament protein (NF) failed to reveal any
colo-calization with CARP (data not shown)
The p21WAF1/CIP1gene expression profile parallels
CARP in both MD and denervation-induced
atrophy models
In an attempt to dissect the molecular mechanisms
activated downstream of the CARP gene, the gene
expression of three relevant target genes chosen on
account of CARP targets in cardiac and vascular
tis-sues was measured by qRT-PCR in both denervation
and MD models: the slow isoform of myosin light
chain MLC-2v [23], its paralogous gene in skeletal
muscle fast fibres, myosin light chain, fast (MLC-f),
and the cell cycle inhibitor p21WAF1/CIP1[24] Although
it was previously reported to be expressed at low levels
in skeletal muscle [29], MLC-2v gene expression
remained undetectable in our conditions (data not
shown) Whereas MLC-f expression was inversely correlated with CARP level in denervated animals, its level was generally unaffected, or even slightly increased, in muscles of MD models (data not shown)
As neither MLC-2v nor MLC-f expression were corre-lated consistently with CARP level, neither of these proteins seems to be involved in the CARP signalling pathway in skeletal muscle In contrast, in both dener-vation and MD models, p21WAF1/CIP1 gene expression paralleled the CARP profile, i.e increased when muscle degeneration occurred, and progressively decreased back to control level during the reinnervation phase of transient denervation (Fig 4) It is worth mentioning that p21WAF1/CIP1 upregulation was of the same order
of magnitude as CARP upregulation, as reflected by the logarithmic scale
CARP overexpression in wild-type mouse TA muscle does not induce atrophy, but alters fibre type composition
Considering that the upregulation of CARP persisted
in definitive denervation and was consistent in MD models, we tried to understand its contribution to these conditions and therefore investigated its func-tion(s) in skeletal muscle A pseudotyped adeno-associ-ated virus 2/1 (AAV2/1) vector in which the CARP coding sequence is fused with the yellow fluorescent protein (YFP) sequence was injected into the TA mus-cle of normal mice One month after injection, direct observation of the skinned injected muscle using con-focal fluorescence microscopy allowed the visualization
of a high level of YFP fluorescence Measurement of the level of CARP mRNA by qRT-PCR confirmed strong expression of the transgene (more than 60 times the level of mRNA in the control experiment,
P < 0.01; Fig 5A,B) This was indeed reflected by the appearance of a band corresponding to CARP expres-sion in western blots (Fig 5C)
Fig 3 Analysis of CARP protein level and cellular localization in denervation-induced atrophy and in murine models of MD (A) In conditions
of both transient (T) and definitive (D) denervation, the level of expression of CARP protein was assessed on equivalent amounts of lysate proteins resolved by western blot The standardization of the loading was verified by Ponceau red staining The expression of CARP protein correlates perfectly with the mRNA profile The expression of CARP protein was estimated by the same method in the psoas muscle (all models but C3-null) or the deltoid muscle (C3-null mice) of the different MD models (comparison made in each case with the adequate wild-type strain) We previously verified that the upregulation of the level of CARP transcripts is similar in both deltoid and psoas in the C3-null strain (five-fold over wild-type control, data not shown) The results show that the upregulation of the expression of CARP protein can be visualized in the Sgca-null model only (B) CARP was detected by specific immunostaining (in green) on transverse sections of control (129svPasIco), denervated (129svPasIco), Sgca-null and mdx 4Cv muscles Staining with dystrophin (in red) was used to delimit the fibres A view of each muscle taken with a 40· objective is presented, showing the very specific staining observed within clusters of small myofibres Scale bars: 30 lm (C) Surface plots representing the density of pixels from whole muscle sections after immunostaining show that CARP expression increases after denervation Original images were processed using IMAGEJ software (8-bit images, Fire look-up table; http://rsb info.nih.gov/ij/) (D) Very intense foci of mononucleated cells are observed in Sgca-null and mdx 4Cv muscles, but not in control muscles Scale bars: 50 lm (E) Costaining for CARP and Pax7 shows that the cells identified in (D) are positive for Pax7.
Trang 8We next investigated whether any phenotype was
apparent following CARP expression In these
condi-tions, the TA muscle weight was not affected
(Fig 5D) The histological appearance of the muscles
was normal (Fig 5E) Morphometric analyses
per-formed on sliced muscles (Fig 5F) revealed no
differ-ences in terms of number or mean diameter of fibres
in comparison with the untreated control, although
the slight switch of the curve detected in the presence
of CARP might reflect a tendency to generate bigger
fibres Muscle sections were negative for terminal
deoxynucleotidyl transferase dUTP nick end labelling
(TUNEL) staining, a marker of apoptosis (data not
shown) As members of the CARP family were
recently suggested to play a role in fibre typing [30],
immunohistochemistry of sections was performed
using an antibody against slow myosin A shift
towards a reduction of slow-twitch fibre type was
observed in the presence of CARP (P < 0.05;
Fig 5G)
Discussion
In this study, in an attempt to identify proteins
involved in the physiopathology of muscle wasting, we
examined the variation in the expression levels of
sev-eral atrophy-associated genes during transient and
definitive denervation and in four models of MD The
main results gained from these studies are: (a) that the
levels of essential components of the UPS are
aug-mented rapidly and transiently during
denervation-induced atrophy, but are not elevated in most MD
muscles; (b) that FoxO1 mRNA expression is
signifi-cantly increased in an LGMD2A model; and (c) that
CARP is robustly upregulated in numerous murine
MD models and in denervation-induced atrophy
First, in line with their documented role in atrophy [31–33], we demonstrated that the expression levels of most investigated components of the UPS increase transitorily during transient and definitive denervation However, the mRNA expression levels of both Ub and E2-14 kDa, previously reported to be upregulated in atrophic conditions [5], do not increase, suggesting that neither protein is rate-limiting in this atrophic situa-tion Consistent with this result, the role of E2-14 kDa has lately been reconsidered, as the inactivation of the corresponding genes does not seem to induce atrophy resistance, at least in the conditions tested [34] In con-trast to the denervation situation, the mRNA expres-sion levels of the UPS elements were almost never increased in the four MDs tested, suggesting that the UPS is not overly activated in these diseases Whether this reflects the slow progression of the diseases with respect to the atrophy phenomenon or weak involve-ment of the UPS in the pathogenesis remains to be determined
Second, FoxO1 was demonstrated to be specifically upregulated in every muscle of the C3-null strain Besides raising the interesting possibility that FoxO1 could be used as a diagnostic marker for LGMD2A, our results indicate that FoxO1 expression increases as
a consequence of the absence of calpain 3, either because of a functional relationship between the two proteins, or by a specific pathophysiological mecha-nism unique to calpain 3 deficiency Regardless of its cause, this upregulation of FoxO1 is very likely to play
an important role in the atrophy observed in this dis-ease, as its in vivo overexpression was previously dem-onstrated to induce reduction of muscle mass [6,35] However, this phenomenon does not seem to proceed through MuRF1 and MAFbx, as their expression levels did not increase in our C3-null strain, but might
p21 WAF1/CIP1
p21 WAF1/CIP1
Fig 4 Gene expression profiles of p21 WAF1/CIP1 after transient or definitive denervation and in murine models of MD The gene expression of p21WAF1/CIP1was measured by qRT-PCR in TA muscles subjected to denervation-induced atrophy (n = 2–6 for each time point), and in quadri-ceps, EDL, TA, soleus and psoas muscles of the four MD models (n = 3–4 for each point) T, transient denervation; D, definitive denervation Results are expressed as percentage of expression level measured in the respective control muscles for MDs (129svPasIco and C57BL/6)
or in the sham-operated muscles for denervation models In every situation, p21WAF1/CIP1gene expression reflects CARP level.
Trang 9instead involve other FoxO1-dependent signalling
cascades, such as the autophagy pathway [36–38] and/
or the control of satellite cell proliferation [39], two
mechanisms important for muscle mass regulation [40]
Provided that upregulation of FoxO1 is found also in LGMD2A patients, it seems highly likely that imped-ing FoxO1 increase and/or inhibitimped-ing its activity might improve the phenotype
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E
Fig 5 Effect of CARP overexpression in muscle (A) One month after intramuscular injection of AAV–CARP–FP into the TA muscle, trans-duction efficiency was visualized by fluorescence microscopy Note that most observable fibres expressed the construct, apart from a few negative fibres, which reflected the fluorescence background level Scale bar: 50 lm (B) The level of CARP transcript overexpression was evaluated by qRT-PCR Results are expressed as percentage of expression level measured in untransduced control muscles n = 5–7;
**P < 0.01 for significance between AAV–CARP–FP-injected TA muscle and contralateral control (C) Expression of CARP protein was evalu-ated by western blot Equivalent amounts of proteins were resolved, and Ponceau red staining was also used to confirm the standardization
of the loading (D) Weights of injected TA muscles (n = 13) were compared to those of control samples No significant difference was observed (E) Histological analyses of muscles Frozen sections of injected TA muscles (right panel) stained with haematoxylin–phloxin–sa-fran show features identical to normal sections (left panel) Scale bars: 20 lm (F) Morphometric analysis of muscles overexpressing CARP The number of fibres and their minimum diameter in injected muscles are not significantly different as compared to the control (n = 4) (G) Slow fibres were detected using slow myosin immunostaining, and their numbers were determined on three slices of the TA muscle mid-section The number of slow fibres is reduced significantly (*P < 0.05) in CARP-expressing muscles as compared to noninjected muscles, indicating that CARP can influence the fibre type (n = 6).
Trang 10Third, the most striking evidence obtained from our
investigation is that CARP expression appears to be
persistently upregulated in denervation-induced
atro-phy and is also elevated in all the MD models
investi-gated This last observation adds to the panel of muscle
pathologies already reported to be associated with an
increase in CARP expression: DMD, spinal muscular
atrophy, facio-scapulo-humeral muscular dystrophy,
amyotrophic lateral sclerosis, and peroxisome
prolifera-tor-activated receptor-induced myopathy [41], as well
as the mdx, Swiss Jim Lambert (SJL) and muscular
dystrophy with myositis (MDM) animal models,
defi-cient respectively in dystrophin, dysferlin and titin [42–
48] Overall, CARP seems to be a general marker of
muscle damage The reason(s) for CARP upregulation
remain(s) obscure, and whether CARP expression
par-ticipates in or represents an attempt to resist the
unre-lenting muscle degeneration is an important issue
It is of interest that CARP is the only protein
show-ing a variation of profile between transient and
defini-tive denervation, with persistence of upregulation in
the latter condition The CARP profile precisely
reflects muscle atrophy, which could be consistent with
the idea that CARP is an important factor in this
mechanism However, several facts support the idea
that CARP probably has no active part to play in
muscle atrophy per se First, there is no consistent
positive correlation between CARP expression and
atrophic situations [9,11], and it can even be upregulated
when skeletal muscle mass increases [18–21] Second,
in our hands, CARP overexpression in a normal
muscle background failed to induce significant changes
in the number and calibre of fibres
Interestingly, although CARP is upregulated to very
similar levels in both denervation and MD models,
two different CARP expression sites are observed, in
Pax7-positive mononucleated cells and within the
cyto-plasm of large myofibres, suggesting that CARP plays
a role in myogenic activation, as well as in mature
fibres It is possible that a common molecular
signal-ling pathway encompassing CARP and p21WAF1/CIP1
occurs at these two locations Indeed, among the three
potential target genes tested herein, the p21WAF1/CIP1
gene is the only one whose expression matches strictly
with CARP level In addition, p21WAF1/CIP1expression
was observed at the same locations (proliferating
myo-blasts [49] and terminally differentiated myotubes [50])
as CARP overexpression First, in the skeletal
myo-genic lineage, p21WAF1/CIP1 upregulation leads to the
irreversible withdrawal of myoblasts from the cell
cycle, stimulates differentiation, and confers protection
against apoptosis [49] However, intense regeneration
is still ongoing in both the Sgca-null and mdx4Cv
mod-els, which suggests that either p21WAF1/CIP1 is not inhibiting the cell cycle or else that the inhibition pro-cess is not entirely efficient Second, p21WAF1/CIP1 has previously been reported to be upregulated within the myonuclei of denervated muscles, a location where it might be required to protect fibres against denerva-tion-induced apoptosis [50] Taken as a whole, the findings in the MD and denervation models studied herein suggest that the systematic upregulation of p21WAF1/CIP1 whenever CARP expression increases might oppose cell proliferation and/or inhibit apop-tosis, thus preventing muscle remodelling
It should also be noted that muscle ankyrin repeat proteins, which include CARP, have recently been sug-gested to be important for sarcomere length stability and muscle stiffness and to have an inhibitory role in the regenerative response of muscle tissue [30] Here,
we showed that CARP overexpression induces a switch towards fast-twitch fibres All of these elements add to the previous observations related to the effects of p21WAF1/CIP1, and support the idea that CARP plays a global role in muscle plasticity Accordingly, the main-tenance of CARP expression during chronic denerva-tion suggests that this protein plays an active part in this static condition and might contribute actively to the prevention of remodelling through blockade of adaptive pathways during deleterious muscle processes Interestingly, besides MDs, CARP has been reported
to be upregulated in many other pathological tissues (hypertrophic hearts [12–17], nephropathic kidneys [51], and wounded epidermis [52]), which suggests that CARP is a widely spread marker of tissue alterations
If the consequences of CARP overexpression prove
to be detrimental for the skeletal muscle, impeding CARP expression would seem to be especially interest-ing, as CARP expression is increased in many different muscle diseases Considering that transforming growth factor-b, tumour necrosis factor-a and interleukin-1a are known stimuli of CARP expression, pharmacologi-cally targeting these pathways might be an option Indeed, as it has already been demonstrated that drug-mediated inhibition of tumour necrosis factor-a [53] or transforming growth factor-b [54] in the mdx mouse model greatly improves the muscle histology, it would
be interesting to investigate the role of CARP in these signalling pathways
Experimental procedures Animals
All mice were handled in accordance with the European guidelines for the humane care and use of experimental