Satellite cell loss and impaired muscle regeneration
in selenoprotein N deficiency
Perrine Castets1,2,3,4,5, Anne T. Bertrand1,2,3,4, Maud Beuvin1,2,3,4, Arnaud Ferry1,2,3,4,
Fabien Le Grand6,7, Marie Castets8, Guillaume Chazot8, Mathieu Rederstorff9, Alain Krol9,
Alain Lescure9, Norma B. Romero1,2,3,4, Pascale Guicheney1,5and Vale ´rie Allamand1,2,3,4,∗
1UPMC Univ Paris 06, IFR14, Paris F-75013, France,2CNRS, UMR7215, Paris F-75013, France,3Inserm, U974,
Paris F-75013, France,4Institut de Myologie, Paris F-75013, France,5Inserm, U956, Paris F-75013, France,6Institut
Cochin, Universite ´ Paris Descartes, CNRS, UMR8104, Paris F-75014, France,7Inserm, U1016, Paris F-75014,
France,8CNRS, UMR5238, Universite ´ de Lyon, Centre Le ´on Be ´rard, Lyon F-69008, France and9Architecture et
Re ´activite ´ de l’ARN, Universite ´ de Strasbourg, CNRS, IBMC, Strasbourg F-67084, France
Received September 21, 2010; Revised and Accepted November 24, 2010
Selenoprotein N (SelN) deficiency causes a group of inherited neuromuscular disorders termed SEPN1-
related myopathies (SEPN1-RM). Although the function of SelN remains unknown, recent data demonstrated
that it is dispensable for mouse embryogenesis and suggested its involvement in the regulation of ryanodine
receptors and/or cellular redox homeostasis. Here, we investigate the role of SelN in satellite cell (SC) func-
tion and muscle regeneration, using the Sepn12/2mouse model. Following cardiotoxin-induced injury, SelN
expression was strongly up-regulated in wild-type muscles and, for the first time, we detected its endogenous
expression in a subset of mononucleated cells by immunohistochemistry. We show that SelN deficiency
results in a reduced basal SC pool in adult skeletal muscles and in an imperfect muscle restoration following
a single injury. A dramatic depletion of the SC pool was detected after the first round of degeneration and
regeneration that totally prevented subsequent regeneration of Sepn12/2muscles. We demonstrate that
SelN deficiency affects SC dynamics on isolated single fibres and increases the proliferation of Sepn12/2
muscle precursors in vivo and in vitro. Most importantly, exhaustion of the SC population was specifically
identified in muscle biopsies from patients with mutations in the SEPN1 gene. In conclusion, we describe
for the first time a major physiological function of SelN in skeletal muscles, as a key regulator of SC function,
which likely plays a central role in the pathophysiological mechanism leading to SEPN1-RM.
Satellite cells (SCs) are essential for adult muscle homeostasis,
growth and repair. They are maintained in a quiescent state
under basal conditions, but upon injury, they are activated,
enter the cell cycle and give rise to a population of muscle
precursors that proliferate, differentiate and then fuse to
form new fibres, allowing muscle architecture restoration (1).
In parallel, a subset of SCs is able to return to its quiescent
state, thereby replenishing the initial pool and allowing
repeated repair of the tissue (2). Most SCs are identifiable by
the expression of the paired box transcription factor Pax7,
whereas expression of the myogenic regulatory factors Myf5,
MyoD and Myogenin defines muscle precursors upon acti-
vation and as differentiation begins (1,3). Self-renewing cells
are described as activated Pax7+/MyoD+cells that maintain
Pax7 expression, lose that of MyoD and exit from the cell
cycle (4–6). Recently, the Pax7+/Myf52satellite ‘stem-like’
cells were proposed to ensure the expansion of the SC popu-
lation, whereas the Pax7+/Myf5+SCs commit to the myogen-
esis differentiation programme (7). Several transcription
factors and signalling pathways have been described to regu-
late SC activation and the balance between proliferation
and differentiation (1), whereas the mechanisms controlling
cell-cycle exit towards a quiescent state remain poorly
∗To whom correspondence should be addressed at: Inserm, U974, Institut de Myologie, Groupe Hospitalier Pitie ´-Salpe ˆtrie `re, 47, bd de L’Ho ˆpital, 75651
Paris Cedex 13, France. Tel: +33 142165707; Fax: +33 142165700; Email: firstname.lastname@example.org
# The Author 2010. Published by Oxford University Press. All rights reserved.
For Permissions, please email: email@example.com
Human Molecular Genetics, 2011, Vol. 20, No. 4
Advance Access published on December 2, 2010
at BIUS Jussieu on June 8, 2011
Selenoproteins are defined by a specific selenocysteine (Sec)
residue inserted in their peptidic sequence via a recoded UGA
codon (8). Most of them are involved in cell redox homeosta-
sis, owing to the great reactivity of the Sec amino acid (9).
Among them, selenoprotein N (SelN) is the only selenoprotein
known to be responsible for a human genetic disorder. Indeed,
SelN deficiency, due to mutations in the SEPN1 gene, causes
SEPN1-related myopathies (SEPN1-RM), characterized by a
generalized early-onset muscle atrophy, myotendinous con-
tractures and muscle weakness, mostly affecting axial
muscles and leading to severe scoliosis, spine rigidity and res-
piratory insufficiency (10–13). Although some data have been
recently obtained regarding the function of SelN, the mechan-
isms underlying the pathology remain largely unknown. A
higher oxidant activity and susceptibility to oxidative stress
have been detected in vitro in SelN-deficient human cells
(14). Inaddition, physical
between SelN and ryanodine receptors (RyR) suggest that
SelN may regulate the latter by modulating the redox state
of their reactive cysteines (15). However, SelN most likely
has additional functions as its expression pattern is ubiquitous
and predominant in developing tissues, contrasting with that of
RyR in mammals (16). Although SelN appeared essential for
zebrafish development (15,17), SelN-deficient mice displayed
normal embryogenesis (16) and postnatal growth (Rederstorff
et al., in preparation).
Here, we established that SelN plays a major role in SC
function, including the generation and/or the maintenance of
the SC pool in skeletal muscles. In the mouse, SelN deficiency
leads to a reduced SC pool in adult muscles, impaired regen-
eration and SC exhaustion during this first round of regener-
ation, hence preventing further restoration of the tissue. Most
relevant to the human pathology, we show that muscle biopsies
from SEPN1-RM patients specifically display a drastic
reduction in the number of SCs. Taken together, these
results point towards a novel SelN-dependent mechanism in
muscles, most likely central in the pathomechanism of
Adult SelN-deficient muscles display a reduced SC pool
To gain insights into the role of SelN in SC function, we first
characterized the size of the SC pool in SelN-deficient
muscles, in 15–21-day, 4- and 10-month-old mice. At these
ages, Sepn12/2mice were indistinguishable from wild-type
(WT) littermates (Rederstorff et al., in preparation). Pax7
immunolabelling on Sepn12/2tibialis anterior (TA) cryosec-
tions revealed that the total number of SCs was unaltered in
the younger mice, whereas it was significantly reduced com-
pared with WT at 4 and 10 months (Fig. 1A). Accordingly,
at 4 months of age, Pax7 transcript expression was decreased
by 41% in Sepn12/2TA compared with WT (Fig. 1B). Like-
wise, on freshly isolated extensor digitorum longus (EDL) and
TA fibres, the number of SCs per fibre was reduced by 40% in
4-month-old Sepn12/2mice (Fig. 1C). Altogether, these find-
ings demonstrate that skeletal muscles from adult Sepn12/2
mice exhibit a reduced SC pool.
SelN is highly expressed during cardiotoxin-induced
Following cardiotoxin (CTX) injury in hindlimb compartments
of adult WT mice, Sepn1 expression was increased by 3- and
6-fold at 24 h and 3 days, respectively, the latter corresponding
to the maximal expression detected, with a progressive
decrease observed thereafter (Fig. 2A). These results were
reinforced by western blotting, which demonstrated an impor-
tant increase in SelN expression between days 3 and 10 after
injury (Fig. 2B). As increased oxidative constraints are
known to occur during muscle regeneration (18), the
expression of other selenoproteins was also quantified after
injury. A major up-regulation was detected only for SelP tran-
scripts, which peaked at 5 days after injection (Supplementary
Material, Fig. S1). We also observed a decrease in the
expression of SelW transcripts from 3 to 7 days after injury,
probably related to the loss of mature myofibres, and an
increase in Sep15 and SelS transcript levels at 3 days that
may be linked with their role in inflammatory cells (19) (Sup-
plementary Material, Fig. S1).
eration, immunohistochemical analyses were performed on
regenerating muscle sections with a previously characterized
SelN antibody (20). No signal was detected in adult uninjured
WT muscles, notably in mature fibres and quiescent Pax7+
cells (Supplementary Material, Fig. S2A). At 3 and 5 days after
injury, SelN expression was observed in numerous mononu-
cleated cells surrounding the regenerating fibres, with no signal
in newly formed eMHC+fibres (Fig. 2C). Similar results were
obtained on late embryonic (E18) muscle sections: abundant
mononucleated cells were detected around muscle groups and
SelN in regenerating muscles, immunolabelling with different
specific cell markers was performed. Co-immunostaining
revealedthatSelN wasexpressedin somePax7+cells,withvari-
able signalintensity(Fig. 2C).A faint signalwas alsodetectedin
in muscle precursors (Supplementary Material, Fig. S2A). Con-
sistently, by quantitative reverse transcription polymerase chain
reaction (qRT-PCR),Sepn1 transcriptswerealmostundetectable
in quiescent murine SCs, using cells freshly purified by Fluor-
escence Activated Cell Sorting (FACS) (a7integrin+/CD312/
Sca12/CD452) previously described in (21), whereas a strong
expression was detected upon activation (SC-derived myoblasts)
Among the SelN+cells detected in regenerating muscles,
some of them were CD45+at 3 days, with only a few remaining
CD45+at 5 days (Supplementary Material, Fig. S2A). Using the
in some macrophages at 3 days after injury, whereas no
cell type and regeneration timing (22). Lastly, only some SelN+
cells were shown to be BrdU+, thereby indicating no correlation
between SelN expression and cell proliferation (Supplementary
Material, Fig. S2A).
At 7 days after injury, SelN expression was still detected in
numerous mononucleated cells, most of which were located
Human Molecular Genetics, 2011, Vol. 20, No. 4695
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outside the basal lamina (Supplementary Material, Fig. S2A).
Nevertheless, a faint signal was also observed in a subset
of Pax7+cells (Supplementary Material, Fig. S2A). At this
stage, SelN+cells were desmin2, CD452and F4/802(data
not shown). At later stages (15 and 30 days after injury),
almost no SelN+cells could be detected in the muscle sections
(data not shown).
These results indicate that, during regeneration, SelN
up-regulation is related to its expression in mononucleated
cells, such as inflammatory cells, activated SCs and most
likely other cell types, including mesenchymal cells and/or
SelN deficiency leads to massive muscle atrophy
by impairing its ability to sustain successive
The regenerative capacity of adult Sepn12/2skeletal muscles
was evaluated following single (SI) and double (DI) injuries in
the hindlimb muscle compartments. Uninjured TA muscles
from 4-month-old WT and mutant mice displayed identical
mass, contractile properties and morphology (Table 1 and Sup-
plementary Material, Fig. S3A–C). Similarly, in mutant and
WT muscles injured only once (SI7, SI15 and SI30), no differ-
ence was observed in the injured-to-contralateral TA mass
ratio, tetanic forces and muscle section area (Table 1). The
number of regenerated fibres per mm2, their size distribution
and the number of myonuclei per fibre were comparable in
WT and Sepn12/2mice from 7 to 30 days after injury
(Fig. 3A and B and Supplementary Material, Fig. S3A–F).
However, 15 days after injury, necrotic and calcified fibres
were present only in Sepn12/2muscles (Fig. 3A and B). Fur-
thermore, fat deposition was observed in mutant mice as early
as 5 days, with significant 3- and 6-fold increases in its area at
7 and 30 days, respectively, compared with WT (Fig. 3C).
These data indicate that SelN deficiency leads to imperfect
muscle regeneration: although restoration of the fibres is
observed, calcification and adipogenesis indicate a defect in
In contrast, 7 and 30 days following the second injury
(DI7 and DI30), a significant reduction of the injured-to-
contralateral muscle mass ratio was observed in Sepn12/2
mice (Table 1). In addition, a 38% reduction in absolute force
P0 was detected in Sepn12/2muscles at 30 days, whereas
specific forces were similar to that of WT (Table 1). This indi-
cates that the reduction in the absolute force of Sepn12/2
muscles was due to its atrophy rather than to a decrease in
the contractile properties of the regenerated fibres. The section
area of Sepn12/2TA was 1.6-fold smaller than WT, at 7 and
30 days after the second injury (Table 1 and Fig. 3A). At the
cellular level, rare regenerating fibres were detected at 7 days
after injury in mutant muscles, the major part of the tissue
being occupied by fibrosis, adipocytes and necrotic fibres
(Fig. 3B and C). At 30 days, necrosis was no longer observed
in Sepn12/2mice, but fat deposition and fibrosis were still
strikingly increased compared with WT muscles that exhibited
an almost complete regeneration (Fig. 3).
Collectively, these results indicate that SelN deficiency
affects the efficiency of muscle restoration following one
injury and totally prevents further regeneration of the tissue.
SCs are lost in SelN-deficient muscles during regeneration
As SC renewal during muscle regeneration is essential for effi-
cient repeated repair of the tissue (2), we investigated the be-
haviour of the SC population during the first round of
regeneration. The number of Pax7+nuclei was first quantified
on injured TA sections by immunostaining and expressed per
muscle unit area (mm2) or as a percentage of total nuclei
located under the basal lamina. At 5 days after injury, no sig-
nificant difference was detected in the SC number between
WT (306+38 cells/mm2) and mutant (258+38 cells/mm2).
In contrast, at 7 days, we observed a pronounced reduction
in the SC population of Sepn12/2TA (94+2 cells/mm2or
15.0+0.3%) compared with WT (198+13 cells/mm2or
24.7+1.2%) that worsened until 30 days after injury
(Fig. 4A). At this latter time point, in WT, the number of
SCs has returned to basal levels, in keeping with self-renewal
Figure 1. AdultSepn12/2musclesexhibita reductionintheirSCpopulation.(A) TAmusclecryosectionsfrom15–21-day,4-and10-month-old micewereimmu-
(arrows), revealing correct localization of the SCs. The number of Pax7+nuclei was expressed as a percentage of total nuclei located under the basal lamina (n ¼ 3,
∗P , 0.03).Scalebar,50 mm.(B)qRT-PCRanalysisofPax7transcriptsin4-month-oldTAmuscles(n ¼ 3,∗P , 0.04).(C)QuiescentSCswerequantifiedonsingle
fibres isolated from 4-month-old WT and mutant EDL and TA muscles (n ≥ 5 mice, .330 cells,∗P , 0.03). All data are represented as mean+SEM.
696Human Molecular Genetics, 2011, Vol. 20, No. 4
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of the resident pool. In contrast, in Sepn12/2mice, the number
of SCs was strikingly reduced compared with uninjured
mutant muscles and injured WT muscles (Fig. 4A). It is
worth noting that the few remaining SCs exhibited a correct
localization, under the basal lamina. These findings were
also observed in regenerating soleus muscles and were
further confirmed using an antibody against M-cadherin,
another marker of SCs (data not shown). These data indicate
that a massive loss of SCs occurs between days 5 and 7
after injury in Sepn12/2mice.
To further investigate this precocious depletion of SCs, we
quantified Pax7 transcript expression from 3 to 30 days after
Figure 2. SelN is highly expressed during CTX-induced muscle regeneration. (A) Sepn1 transcript expression was quantified by qRT-PCR in uninjured (Ctr) and
injured TA muscles at different time points after injury. All values are normalized to the 18s rRNA and expressed as fold increase over uninjured muscles (n ¼ 3,
∗P , 0.03). (B) SelN protein expression was quantified by western blot in uninjured (Ctr) and injured WT muscles (n ≥ 3,∗P , 0.02 from uninjured). Protein
loading was monitored using a pan-actin antibody. Due to slight variations in the control loading, protein levels were quantified based on the protein concentration
and expressed as fold increase over uninjured muscles. All data are mean+SEM. (C) Immunohistochemistry was performed against SelN on WT and mutant
muscle sections at 3 (panel iv) and 5 days (panels i–iii, v). Co-staining was performed with eMHC (i, ii), Pax7 (iii) and F4/80 (iv, v). Double positive cells are
indicated with arrows; SelN+-only cells with arrowheads and cells with barely undetectable or no signal for SelN with open arrows. Sepn12/2muscle sections (ii)
were used as negative control. Scale bar, 50 mm. (D) qRT-PCR analysis of Sepn1 mRNA in freshly FACS-sorted SCs (quiescent—SC) and activated cells (myo-
Table 1. SelN deficiency leads to muscle atrophy following two injuries.
Section area (mm2)
The injured-to-contralateral uninjured mass ratios, the absolute (P0) and specific (P0/m) maximal tetanic forces measured in situ, and muscle section area are
informed. Measurements were performed in uninjured muscles (basal), 7, 15 or 30 days following the first injury (SI7, 15, 30) and 7 or 30 days following the
second injury (DI7, 30). Basal TA mass (mg) in WT versus Sepn12/2mice: 40.1+2.2 versus 38.5+1.2 for females; 53.8+2.5 versus 57.4+1.8 for males.
∗P , 0.02;∗∗P , 0.002.
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injury. Although Pax7 expression was similarly increased at 3
days in control and mutant muscles, the extent of Pax7
up-regulation was lower in Sepn12/2TA from 5 days after
injury. At 15 and 30 days, the expression levels detected in
mutant muscles were reduced by 5-fold compared with WT and
by 2-fold in comparison to uninjured Sepn12/2muscles
(Fig. 4B). Similarly, we showed that Myf5 expression was
significantly reduced in mutant muscles as early as 5 days and
cious loss of Pax7/Myf5 transcript expression in Sepn12/2
muscles, preceding the depletion of SCs detected by immunos-
taining. In contrast, MyoD and Myogenin expression, which
regeneration studied (Supplementary Material, Fig. S3G and H).
Altogether, these data indicate that the basal defect in the
SCs in uninjured Sepn12/2muscles worsens drastically
during muscle regeneration, leading to exhaustion of the SC
pool in regenerated Sepn12/2muscles.
SelN deficiency affects SC function in vitro and increases
the proliferation of muscle precursors
The cause of the drastic SC depletion observed in regenerating
SelN-deficient muscles was then investigated. Cell death by
apoptosis was ruled out by Terminal deoxynucleotidyl Trans-
ferase Biotin-dUTP Nick End Labeling staining and quantitat-
ive measurement of the caspase-3 activity in muscles
following the first injury (Supplementary Material, Fig. S4).
We then analysed the cell fate decision of SCs in the
absence of SelN, by isolating EDL fibres that were cultured
for 24, 48 and 72 h. According to Zammit et al. (5), immunos-
taining was performed to distinguish between non-committed
(Pax7+-only), activated (Pax7+/MyoD+) and committed
(MyoD+-only) cells (Fig. 5A). Almost all SCs were activated
at 24 and 48 h on both mutant and WT fibres (data not shown).
After 72 h in culture, a significant 63% reduction in the Pax7+/
MyoD2cell population was observed in mutant compared
with WT (Fig. 5A and B). These findings suggest that SelN
deficiency affects the cell fate choice of SCs in the context
of isolated fibres.
In light of the comparable pool of progenitors detected at
5 days after injury in mutant and WT, which contrasts with
the basal defect in the SC population of Sepn12/2muscles,
we hypothesized that SelN deficiency may lead to an enhanced
proliferation of muscle precursors. By performing BrdU/
Desmin immunostaining in vitro, we demonstrated that the
proliferation rate of Sepn12/2primary myoblasts cultured in
growth medium was increased by 60% (Fig. 5C). In contrast,
when cultured in low serum-containing medium, mutant
cells exhibited a reduction in their proliferation rate and a
fusion index similar to that of WT, indicating an efficient
differentiation (Fig. 5C and D).
Lastly, immunostaining for MyoD and Ki67 performed on
TA sections at 3 days after injury revealed a 55% increase
in the proportion of cycling muscle precursors (Ki67+/
MyoD+) in injured Sepn12/2muscles compared with WT
(Fig. 5E). Moreover, 5 days after injury, the proportion of
Pax7+cells that were cycling (Ki67+/Pax7+) or proliferating
(BrdU+/Pax7+) was significantly
muscles (Fig. 5F and G).
Taken together, these data demonstrate that SelN deficiency
leads to an enhanced proliferation of muscle precursors,
whereas their commitment towards differentiation appears
Muscle biopsies from SEPN1-RM exhibit a striking SC loss
To investigate whether the loss of SCs also occurred in human
SelN-deficient muscles, Pax7 immunostaining was performed
Figure 3. SelN deficiency abolishes the repeated regenerative capacity of skeletal muscles. (A, B) TA muscles were stained with HE following an SI (SI7/15: 7
and 15 days after injury) or a DI (DI7/30: 7 or 30 days after second injection). White arrowhead indicates calcified fibres, also shown with Alizarin Red coloration
(enlarged inset); black arrowheads, necrotic fibres; arrows, adipocytes. (C) Fat deposition was detected by Oil Red O staining and quantified relative to the total
muscle area, in injured TA muscles at SI5, SI7, SI15, SI30, DI7 and DI30 (n ≥ 3,∗P , 0.05,∗∗P , 0.008). Scale bar: 500 mm (A, C); 100 mm (B).
698Human Molecular Genetics, 2011, Vol. 20, No. 4
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on 8 genetically confirmed SEPN1-RM, 14 pathological
control and 7 age-matched control muscle biopsies (Sup-
plementary Material, Table S1).
In the control cohort, the number of SCs decreased with age,
ranging from 18 to 8 SCs per 100 myonuclei in muscle biopsies
from 3–10- and 30–40-year-old individuals, respectively
(Fig. 6A and B). By comparison, Pax7 staining on all
SEPN1-RM muscle biopsies revealed a drastically reduced SC
number at all ages. Moreover, we observed that this defect wor-
sened with age, with 1.5-, 2- and 10-fold reductions detected in
muscle biopsies from 3-, 11–23-, and 35-year-old patients,
respectively, and a complete exhaustion of the SC pool in the
biopsy from the oldest patient (Fig. 6A and B). Conversely,
patients with confirmed mutations in the RYR1 gene, as well
as other pathological controls, exhibited a proportion of SCs
very similar to that of control individuals (Fig. 6B).
These striking observations strongly suggest that the early
and progressive depletion of the SC population is a specific
feature of SEPN1-RM.
Skeletal muscles possess a high regenerative potential depen-
dent on the resident quiescent muscle progenitors (SCs) that
are able both to reconstruct fibres and to self-renew, allowing
the maintenance of the initial pool of SCs (2). In this study, we
investigated whether SelN, deficient in SEPN1-RM, may play
a critical role in muscle regeneration and SC function, taking
advantage of the Sepn12/2murine model ((16); Rederstorff
et al., in preparation). The results we have obtained point to
a decisive role of SelN in SC maintenance and consequently
in the regenerative capacity of skeletal muscles.
A reduced basal number of SCs was detected in Sepn12/2
adult muscles, suggesting that SelN is involved in SC homeo-
stasis independently from the regeneration context. This may
reflect the fact that SelN deficiency impairs the generation of
the uncommitted cell population entering into quiescence
during the perinatal period or leads to a progressive loss of
the quiescent SCs from 1 to 4 months of age (4,23).
Figure 4. SelN deficiency leads to premature SC depletion. (A) Injured muscle cryosections immunostained for Pax7 (red) and laminin (green), 7 and 15 days
following an SI (SI7/15). Nuclei were stained with DAPI (blue). Scale bar, 50 mm. The number of Pax7+nuclei was expressed as a percentage of total nuclei
located under the basal lamina (n ¼ 3,∗P , 0.03 compared with WT,#P , 0.002 compared with uninjured KO muscles). qRT-PCR analysis of Pax7 (B) and
Myf5 (C) transcripts in uninjured TA (Ctr) and injured muscles from 3 to 30 days after injury (n ¼ 3,∗P , 0.05 compared with WT,#P , 0.04 compared with
uninjured KO muscles).
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Following an SI, we demonstrated that in the absence of
SelN, muscle regeneration was imperfect, although the
number and size of the regenerated fibres seemed unperturbed.
Alteration of the local tissue environment in which the
regeneration process occurs could be responsible for the calci-
fication, residual necrotic fibres and fat deposition observed
in knock-out (KO) muscles (24). These defects could also
reflect a perturbation in the behaviour of the committing SC
population that would be compatible with the observed
muscle regeneration (25–27). It is possible that fat deposition
may also be due to an initial imbalance between muscle and
adipocyte progenitors (28,29).
Following the second injury, muscle regeneration was com-
pletely abolished in mutant muscles, leading to drastic muscle
atrophy, with only rare newly formed fibres and massive fat
deposition. We established that this inability of the mutant
muscles to undergo repeated cycles of regeneration was
associated with the drastic depletion of the SC pool occurring
during the first regeneration event. Failure to reconstitute the
quiescent SC population during regeneration could be due to
(i) cell death, (ii) the inability to generate a sufficient pool
of muscle progenitors during the regenerative process, (iii) a
perturbed fate determination of these cells from a self-
renewing potential to myogenic differentiation or trans-
differentiation andlastly(iv)a defectin theniche
repopulation. We first ruled out, in vivo, increased cell death
as a mechanism for SC depletion in Sepn12/2regenerating
muscles, as well as in 15–21-day-old mice (data not shown).
Second, despite an initial defect in the SC number in the
muscles of mutant mice, a comparable pool of Pax7+cells
was observed at 5 days after injury in WT and mutant
muscles, which was related to an enhanced proliferation of
Sepn12/2muscle precursors. This indicates that following
injury, SCs were able to fully expand the pool of muscle pro-
genitors in the absence of SelN. Accordingly, one could
hypothesize that SelN may limit the cycling of cells and that
the early hyper-proliferation of SCs in SelN mutants is associ-
ated with their inability to enter into quiescence (Supplemen-
tary Material, Fig. S5). Indeed, we established that Sepn12/2
muscle precursors exhibit enhanced cycling and proliferation
rates that could be interpreted as a compensatory mechanism
with regard to the initial reduced SC number, but also as a cau-
sative process because this was also detected in cultured
primary myoblasts. Interestingly, cell differentiation appeared
unaltered both in vivo and in vitro, suggesting that SelN plays
a key role in the reversible cell-cycle exit towards quiescence
but not in the permanent cell-cycle exit towards differen-
tiation. Overall, SelN deficiency may alter the cell fate
decision of the pool of activated SCs, as defined by Zammit
et al. (5) and as suggested by the results obtained on isolated
Figure 5. SelN deficiency impairs SC dynamics and enhances proliferation of muscle precursors. (A) EDL-isolated single fibres were cultured for 72 h and immu-
nostained for MyoD and Pax7. Nuclei are detected by DAPI staining (blue). Arrows, black and white arrowheads indicate Pax7+-only, Pax7+/MyoD+and
MyoD+-only cells, respectively. Scale bar, 10 mm. (B) The number of Pax7 and/or MyoD-positive cells was quantified relative to the total number of myogenic
cells on single fibres at 72 h (n ≥ 3 mice, .1400 cells,∗P , 0.02 from WT). (C, D) Primary myoblast cultures were obtained from isolated EDL fibres and
cultured in growth (GM) or differentiation (DM) medium. (C) Proportion of BrdU+cells in the total number of Desmin+cells in GM and DM (n ≥ 3,∗P ,
0.03). (D) Fusion index quantified in control and mutant cultures at 24, 48 or 72 h in DM (n ≥ 3). (E) The proportion of cycling muscle precursors (MyoD+/
Ki67+) in injured muscles 3 days after injury was quantified after immunostaining on cryosections, with regard to the total number of MyoD+cells (n ¼ 3,
∗P , 0.02). The proportion of cycling (Pax7+/Ki67+, F) or proliferating (Pax7+/BrdU+, G) Pax7+cells was quantified after immunostaining performed on
TA sections, 5 days after injury (n ¼ 3,∗P , 0.05).
700Human Molecular Genetics, 2011, Vol. 20, No. 4
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fibres. Hence, almost all Sepn12/2muscle progenitors may
commit towards differentiation, with only rare cells maintain-
ing Pax7 expression and exiting from cell cycle to return into
quiescence and renew the SC pool (Fig. S5). Based on the
model of Kuang et al. (7) and as suggested by the quantifi-
cation of Pax7 and Myf5 transcript expression, SelN deficiency
could alter the balance between the ‘stem-like’ cell population
and the committed pool, and/or their dynamics during regener-
ation, notably by affecting the symmetric versus asymmetric
division processes. Lastly, as SC dysfunction also occurs in
regeneration-independent contexts, we hypothesize that a
defect in the niche repopulation is unlikely to be the major
process leading to SC loss in the regenerating mutant
muscles, but this remains to be demonstrated.
When comparing the defects observed in the absence of
SelN during the first cycle of regeneration, both the SC loss
and the cellular hypotheses described above could be related
to an intrinsic alteration of the SC homeostasis (suggesting a
cell-autonomous mechanism) and/or to perturbations of the
response). Both the basal defect in the SC pool and the modi-
fied in vitro SC dynamics suggest a mechanism independent of
the injury context. However, due to the fact that complete SC
exhaustion occurs following one injury, the involvement of the
necrosis/regeneration conditions in the aggravation of this SC
loss should not be excluded. This could reflect either an
increased sensitivity of SelN-deficient SCs to the ‘hostile’
environment of the regenerating muscles or abnormal acute
conditions in the absence of SelN. This latter point is sup-
ported by the up-regulation of SelN expression during regener-
ation, which was associated with myogenic, but also
non-myogenic (including macrophages) cells, in accordance
with a cell non-autonomous mechanism. Indeed, the role of
pro- and anti- inflammatory macrophages in regulating both
myogenesis and regeneration has been described and supports
the hypothesis that SC depletion could be due to extrinsic SelN
deficiency (22). Similarly, it has recently been established that
fibro/adipogenic progenitors and endothelial cells are also
involved in muscle progenitor dynamics and, although we
could not demonstrate that SelN is expressed in these cells,
they could be associated with the SC defect observed in the
absence of SelN (28,30,31).
Recent data have suggested that SelN participates in the
regulation of RyR in mature fibres (15). RyR and the IP3
receptor, another calcium channel, have been described to be
expressed in SCs (32–34), but no data have yet been obtained
regarding their physiological role in SCs. Nevertheless, invol-
vement of calcium-associated pathways in SC dynamics has
been suggested (35–37), thus providing a possible link
between the proposed role of SelN in Ca2+homeostasis and
SC maintenance. As SelN localizes to the endoplasmic reticu-
lum (ER) (20), its suggested reductase activity (38) may
modify other proteins, either secreted or located at the
plasma membrane or in the ER, notably by modulating their
oxidative state. Effectors of pathways involved in SC self-
renewal or in the regulation of the inflammatory response
are therefore interesting candidates to be considered.
Most importantly, we established that muscles from
SEPN1-RM patients display a reduced SC population,
already detectable in a 3-year-old patient. This defect was
specifically identified in SEPN1-RM muscles and notably
was not observed in Central Core Disease biopsies. In light
of the results obtained in the mouse, we hypothesize that
this defect is related to an abnormal generation and/or main-
tenance of the quiescent SC population. Thereby, one could
suppose that early SC exhaustion in humans is most likely a
central part of the pathomechanism leading to SEPN1-RM.
In particular, we suggest that the premature depletion of the
pool of muscle progenitors that participate in muscle growth
could limit both the size and number of developing fibres, a
key feature of muscle atrophy. We also propose that this
early SC loss could play a role in the midlife decline of the
muscular function observed in patients, due to the limited turn-
over capacity of SelN-deficient muscle tissues. Based on the
clinical variability observed within SEPN1-RM, we believe
that severalother factors maybeinvolved inthe
Figure 6. The SC population is reduced in SEPN1-RM muscle biopsies. (A) Muscle biopsies from two SEPN1-RM patients (P2, P5) and two age-matched control
individuals (CT2 and CT4) immunostained for Pax7 (red) and laminin (green). Nuclei were stained with DAPI (blue). Scale bar, 100 mm. (B) The number of SCs
in muscle biopsies from SEPN1-RM patients and pathological controls was expressed as a percentage of total nuclei located under the basal lamina and compared
with that of age-matched control individuals. Unless specified, the deltoid muscle was biopsied. F, foot muscle; IC, InterCostal muscles; PV, ParaVertebral
Human Molecular Genetics, 2011, Vol. 20, No. 4 701
at BIUS Jussieu on June 8, 2011
pathomechanism and modulate the consequences of the SC
dysfunction described herein.
MATERIALS AND METHODS
To induce complete necrosis of both TA and soleus muscles,
6.7 and 13 mg of CTX (Latoxan, Valence, France) were
injected into the hindlimb anterior and posterior compartments
of 4-month-old mice, respectively. Contralateral limbs were
sham-injected with physiological serum. Mice were eutha-
nized 6 h, 24 h, 3, 5, 7, 10, 15 or 30 days following injection
and muscles were harvested. For double regeneration exper-
iments, CTX was injected as described above, 30 days after
the first injection, and muscles were dissected 7 or 30 days
later. In single- and double-injection protocols, BrdU (BD
Pharmingen, Le Pont-De-Claix, France) was injected intraper-
itoneally 1 h before sacrifice. All animal studies were per-
formed in accordance with the European Union Guidelines
for Animal Care.
Single isolated fibres and primary myoblast cultures
Individual fibres were isolated from EDL and TA of
4-month-old mice, as described in (38), and either directly
fixed in 4% paraformaldehyde (PFA) or grown for 1–3 days
in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen,
Cergy Pontoise, France) supplemented with 10% foetal bovine
serum (FBS; Invitrogen, Cergy Pontoise, France) and 0.5%
chicken embryo extract (MP Biomedicals, Illkirch, France),
on horse serum-coated culture dishes. For primary myoblast
cultures, isolated fibres were obtained from EDL of 15–
21-day-old mice and plated on matrigel-coated culture
dishes. Myoblasts were grown in F10-media (Invitrogen,
Cergy Pontoise, France) supplemented with 20% FBS and
5 ng/ml basic fibroblast growth factor, and differentiated in
DMEM 4% horse serum (VWR International, Fontenay-
Quantitative real-time RT-PCR
Total RNA was extracted from muscle cryosections using the
RNeasy Fibrous Tissue Mini Kit (Qiagen, Courtaboeuf,
France). RNA was also isolated from a7integrin+/CD312/
Sca12/CD452FACS-sorted quiescent SCs described in (21)
and from these cells maintained in culture for 3 days. Quanti-
tative PCR was performed on DNAse-treated RNA, reverse
transcribed to cDNA using the SuperScript II First-Strand Syn-
thesis System (Invitrogen), amplified using primers listed in
Supplementary Material, Table S2, with the LighCycler480
SYBR Green I Master mix (Roche, Meylan, France) and ana-
lysed with the LightCycler480 analysis software (Roche,
Meylan, France), as described (16). Data were normalized to
the expression levels of the 18s rRNA.
Western blot analysis
Western blot analysis was performed as previously described
(16). Membranes were stained with Ponceau Red for loading
control and immunoprobed with SelN (ab137; (20)) and
pan-Actin (Sigma, Lyon, France, A2066) primary antibodies.
The in situ isometric contractile properties of TA muscles were
studied as previously described (39). The absolute maximal
tetanic force (P0) was recorded and analysed with the Power-
Lab system (4SP; ADInstruments) and software (Chart 4;
ADInstruments). The specific force (P0/m) was calculated as
follows: P0/m ¼ P0 (N)/muscle mass (g).
The following antibodies were used on 5–8 mm muscle cryo-
sections or single isolated fibres: Pax7 (DSHB, IA, USA),
MyoD (Santa Cruz, Heidelberg, Germany, sc-304), BrdU
(Abcam, Cambridge, UK, ab6326), Ki67 (Dako, Tappes,
France,TEC-3), SelN (ab137
Cambridge, UK, ab6640), embryonic MHC (DSHB, IA,
ab11575). Following microwave oven antigen retrieval (40),
sections were blocked with 3% IgG free bovine serum
albumin and ChromoPure Mouse IgG, Fab Fragments
(Jackson ImmunoResearch, Suffolk, UK). Individual fibres
were fixed with 4% PFA, permeated with PBS, 0.5%
Triton-X100 and blocked in PBS, 0.35% carrageenan, 10%
goat serum and 10% horse serum. Sections and fibres were
incubated with primary antibodies and with appropriate
Pontoise, France) or biotin-conjugated antibodies (Jackson
ImmunoResearch, Suffolk, France) successively. They were
mounted in Vectashield DAPI (Vectorlabs, Peterborough,
UK) and observed with an Axiophot microscope (Zeiss,
Munich, Germany). Images were captured using the MetaView
software (Ropper Scientific, Trenton, NJ).
Eight-micrometre muscle cryosections were fixed with 4%
PFA, stained with haematoxylin/eosin, and dehydrated in
ethanol and xylene before mounting in Eukitt medium. For
lipid coloration, sections were fixed in 4% formaldehyde,
2% CaCl2at 48C during 30 min, stained with 0.5% Oil Red
O during 15 min at 378C, counterstained with haematoxylin
and mounted in gelatine.
Images and statistical analyses
The MetaMorph (Molecular Devices, CA, USA) and NIS
(Nikon) software were used for cell counting and area
measurement. Quantifications were performed at 25× magni-
fication (three section levels for mouse muscles with a
minimum of eight fields and a minimum of six fields for
human biopsies). Results are presented as mean+SEM of
independent samples or animals; n represents the number of
individual experiment (n ≥ 3). Comparisons between groups
were performed using the Student’s t-test with a 0.05 level
of confidence accepted for statistical significance.
702Human Molecular Genetics, 2011, Vol. 20, No. 4
at BIUS Jussieu on June 8, 2011
Supplementary Material is available at HMG online.
We thank Dr Topaloglu (Ankara, Turkey) and Dr Merlini
(Ferrara, Italy) for providing patient biopsies. The help of
C. Gartioux and Drs Gnocchi, Durieux, Vandebrouck and
Brin ˜as with experimental procedures is kindly acknowledged.
We thank Drs Vilquin and Vauchez for gifts of antibodies
and discussions. We also thank Prof. Voit, Drs Bonne,
Butler-Browne, Furling and Mouly for fruitful discussions
and critical reading of the manuscript. We are thankful to Dr
Butler-Browne for her help with English usage. The Pax7 anti-
body developed by Kawakami was obtained from the DSHB
developed under the auspices of the NICHD and maintained
by The University of Iowa, Department of Biology, Iowa
Conflict of Interest statement. None declared.
This work was supported by the Institut National de la Sante ´ et
de la Recherche Me ´dicale (Inserm), the Association Franc ¸aise
contre les Myopathies (AFM) and the University Paris 06
(UPMC). P.C. received PhD fellowships from the Ministe `re
de la Recherche et de l’Enseignement, UPMC and AFM.
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