Decreased Proliferation Kinetics of Mouse Myoblasts
Steven C. Chen1, Ellie Frett1¤a, Joseph Marx1, Darko Bosnakovski2, Xylena Reed1¤b, Michael Kyba2,
Brian K. Kennedy1,3*
1Department of Biochemistry, University of Washington, Seattle, Washington, United States of America, 2Lillehei Heart Institute and Department of Pediatrics, University
of Minnesota, Minneapolis, Minnesota, United States of America, 3Buck Institute for Age Research, Novato, California, United States of America
Although recent publications have linked the molecular events driving facioscapulohumeral muscular dystrophy (FSHD) to
expression of the double homeobox transcription factor DUX4, overexpression of FRG1 has been proposed as one
alternative causal agent as mice overexpressing FRG1 present with muscular dystrophy. Here, we characterize proliferative
defects in two independent myoblast lines overexpressing FRG1. Myoblasts isolated from thigh muscle of FRG1 transgenic
mice, an affected dystrophic muscle, exhibit delayed proliferation as measured by decreased clone size, whereas myoblasts
isolated from the unaffected diaphragm muscle proliferated normally. To confirm the observation that overexpression of
FRG1 could impair myoblast proliferation, we examined C2C12 myoblasts with inducible overexpression of FRG1, finding
increased doubling time and G1-phase cells in mass culture after induction of FRG1 and decreased levels of pRb
phosphorylation. We propose that depressed myoblast proliferation may contribute to the pathology of mice
overexpressing FRG1 and may play a part in FSHD.
Citation: Chen SC, Frett E, Marx J, Bosnakovski D, Reed X, et al. (2011) Decreased Proliferation Kinetics of Mouse Myoblasts Overexpressing FRG1. PLoS ONE 6(5):
Editor: Mel B. Feany, Brigham and Women’s Hospital, Harvard Medical School, United States of America
Received February 7, 2011; Accepted April 4, 2011; Published May 16, 2011
Copyright: ? 2011 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the Pacific Northwest Friends of FSHD. S.C.C. is supported by the cardiovascular pathology training grant NIH
T32 HL007312. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤a Current address: Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York, United States of America
¤b Current address: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Facioscapulohumeral muscular dystrophy, or FSHD, primarily
affects muscles of the face, shoulders and upper arms. It is the third
most common muscular dystrophy, following Duchenne muscular
dystrophy and myotonic muscular dystrophy, affecting 1 in 20,000
individuals . Onset of muscle weakness in FSHD patients most
commonly occurs between puberty and the second decade of life,
ultimately leading to patients becoming wheelchair-bound [2,3,4].
Compared to the majority of muscular dystrophies, FSHD is unique
in its very low rate of any respiratory or cardiac muscle involvement,
of muscular dystrophy . As such, patients with FSHD typically live
a normal lifespan, but suffer a severely decreased quality of life.
The molecular basis of FSHD is still under debate, although the
genetic event linked with FSHD has been identified to be in the
subtelomeric region on the long arm of chromosome 4 [6,7]. This
region, denoted as 4q35, contains a series of 3.3 kb tandem repeat
elements, which have been termed D4Z4 repeats . Unaffected
individuals have 11 to 150 D4Z4 repeats, but patients with FSHD
have had this region truncated to 10 or less . Efforts to identify
the molecular basis of this disease have been hampered, however,
because the truncation associated with FSHD is not within a well-
characterized gene coding or promoter region.
Multiple models have been proposed to explain how a D4Z4
repeat truncation is linked to FSHD, reviewed in . The primary
model is that the loss of D4Z4 repeats increases expression of a
double homeobox transcription factor DUX4c, a putative gene
centromeric to the D4Z4 repeats and highly homologous to DUX4
[11,12,13]. DUX4c has been shown to be up-regulated in FSHD
biopsies and primary myoblasts, possibly leading to induction of the
MYF5 myogenic regulator, which serves to inhibit differentiation
and activate proliferation [14,15]. In addition, overexpression of
DUX4 in other cell lines has been shown to cause apoptosis and
impair myogenesis in both cell culture models and zebrafish
development [16,17,18]. A recent chromosomal analysis of affected
and unaffected4q35alleles has determined that FSHD is linked to a
single nucleotide polymorphism located distal to the last D4Z4
repeat , which stabilizes the DUX4 transcript through
polyadenylation and may result in elevated protein levels and
cytotoxicity via still unknown mechanisms.
A second model proposes that the loss of D4Z4 repeats may
increase the available pool of a repressive complex comprised of
YY1, HMG2B and nucleolin that is normally bound to D4Z4
repeats. YY1 interacts with Ezh2, a histone lysine methyltransfer-
ase, playing a key role in expression of muscle genes during
embryonic development [20,21] and MeCP2, a methyl CpG
binding protein involved in Rett syndrome . In addition, YY1
may also be able to interact with the chromatin insulator CTCF
. HMGB2 may affect the maintenance of heterochromatic
regions by interacting with SP100B and subsequently HP1,
establishing higher-order chromatin structures [24,25]. In con-
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trast, nucleolin may have an opposite effect on heterochromatin
formation as it serves to decondense chromatin through
displacement of histone H1 . Perturbations in any of these
proteins due to loss of D4Z4 repeats resulting in increased
chromatin accessibility may cause gene deregulation in trans and
play a role in the pathogenesis of FSHD.
A third model suggests that D4Z4 may serve as nucleating sites for
local transcriptional repression involving the previously mentioned
YY1 complex. Loss of D4Z4 may lift repression in cis of the 4q35
region and thus the nearby genes FRG1, FRG2 and ANT1 [27,28].
MAR) and its disassociation from the nuclear matrix in FSHD
patients may change the arrangement ofDNAloop domains, causing
increased transcription of FRG1 and FRG2 [29,30,31]. Presently, it is
unclear as to which or how many of these many non-exclusive
mechanisms play a causal role in the pathogenesis of FSHD.
Previously, it has been observed that there may be increased
transcriptional activation of FRG1, FRG2 and ANT1, the three
genes upstream of the D4Z4 tandem repeat elements, in muscle of
human FSHD patients ; however, these results were not
observed in another patient study . Unfortunately, generating
a relevant mouse model to study FSHD has been exceptionally
difficult because mice do not have D4Z4 repeats in an analogous
chromosomal setting. Acting under the assumption that overex-
pression of FRG1, FRG2 or ANT1 plays a causative role in the
development of FSHD, transgenic mice were generated expressing
each of these individual genes under the human skeletal actin
promoter, specific for expression in muscle, which resulted in the
identification of a potential mouse model for FSHD. In contrast to
transgenic mice overexpressing FRG2 or ANT1, only transgenic
mice overexpressing FRG1 appear to have symptoms characteristic
of muscular dystrophy . It should be noted, however, that the
FRG1 transgenic mouse model that resulted in dystrophic
phenotypes had FRG1 skeletal muscle protein levels considerably
higher than that observed in FSHD patients.
Facioscapulohumeral muscular dystrophy region gene-1 (FRG1)
is an actin-bundling protein associated with muscle-attachment
sites, specifically located to the Z-disc in mature muscle tissue
[34,35]. FRG1 has been shown to play a crucial and specific role in
muscle development of Xenopus laevis, further implicating its
importance in muscle development and maintenance [36,37].
Recently, Bodega et al. showed that the FRG1 gene was
prematurely expressed during FSHD myoblast differentiation,
thereby suggesting that the number of D4Z4 repeats in the array
may affect the correct timing of FRG1 expression .
Based on this work, we hypothesized that the dystrophic
phenotype in FRG1 transgenic mice is caused, at least in part, by
decreased proliferation in the muscle satellite cell population,
which are the cells responsible for maintaining proper muscle
regenerative potential. Satellite cells are often thought of as muscle
stem cells, proliferating when there is a need for either muscle
repair or growth and then differentiating into skeletal muscle. We
have observed that increased levels of FRG1 impair normal
satellite cell proliferation and may contribute to disease progres-
sion by limiting the pool of cells to repair damaged muscle and/or
delaying the kinetics of repair.
Expression of FRG1 in skeletal muscle of mice causes
Mice overexpressing FRG1 have been previously described .
In that study, three lines of transgenic mice overexpressing FRG1
specifically in muscle were generated by using the human skeletal
actin promoter to drive transcription of the human FRG1 cDNA.
These mice develop spinal kyphosis and characteristics of
muscular dystrophy, including increased fibrosis in muscles, lower
body weight, lower muscle weight and decreased cross-sectional
area, reduced tolerance to exercise and mis-splicing of specific
transcripts associated with myotonic dystrophy. Mice used in the
Gabellini et al. study  were kindly provided by Dr. Rossella
Tupler. After establishing an independent colony with continual
back-crossing to C57BL/6 mice, we performed a limited
characterization of the in vivo phenotypes in their highest FRG1
expressing line (H-FRG1TG) as a confirmation of their reported
findings. As expected, these mice begin to show mild spinal
kyphosis and reduced body weight by 4 weeks of age, both of
which become progressively more severe over time.
To verify that FRG1 expression was increased specifically in
skeletal muscle, we performed Western blot analysis on lysates
generated from a variety of tissues from 10-week old mice using a
specific a-FRG1 antibody. Equivalent amounts of total protein
were loaded and increased FRG1 protein was detected in all tested
skeletal muscles of H-FRG1TGmice compared to wild-type
littermate controls (Figure 1A). We observed a specific increase
in the expression of FRG1 in skeletal muscles such as quadriceps,
gastrocnemius and diaphragm muscle, and no increase of FRG1 in
cardiac muscle or other tissues, confirming the skeletal muscle-
specific expression of FRG1. Endogenous expression of FRG1 was
detected in the lung, but there was no significant difference in
expression between H-FRG1TGmice and wild-type littermate
Muscle weights of collected tissues were measured at different
ages and normalized to body weight, to account for runted
phenotype of H-FRG1TGmice. Quadriceps and gastrocnemius wet
muscle weight comprised a smaller percentage of total body weight
in H-FRG1TGmice compared to wild-type mice, but diaphragm
weight showed no significant change (data not shown). This
observation agrees with previous observations demonstrating that
despite FRG1 expression in all skeletal muscle tissue, only specific
muscles may exhibit a dystrophic phenotype [33,38].
To examine the histology of specific muscles, we collected and
cryosectioned muscle tissue from 13-week old H-FRG1TGmice
and wild-type littermate controls. Hemotoxylin and eosin staining
of these sections showed an increased incidence of centrally
located nuclei and increased fiber size variability in affected muscle
tissues, namely the quadriceps, but this was not noted in internal
muscles such as the diaphragm (Figure 1B). From these
experiments we find that overexpression of FRG1 only causes a
dystrophic phenotype in a subset of skeletal muscles. We also
noted that H-FRG1TGmice were able to survive past 1 year of age
(data not shown) despite the worsening of their muscular
dystrophy and other associated phenotypes. The apparent sparing
of the diaphragm muscle of any measurable defect may possibly
replicate the muscle specificity of FSHD in human patients, which
similarly does not appear to affect internal muscles. All of these
observations confirm the muscular dystrophy phenotype in H-
FRG1TGmice as originally characterized.
Effects of FRG1 expression in mouse-derived myoblasts
In principle, dystrophic phenotypes in muscle can arise from
enhanced degeneration, defective regeneration, or both. For
instance, myoblasts from mice lacking A-type lamins that exhibit
signs of muscular dystrophy plate with high viability and
proliferate normally, but ultimately have impaired differentiation
. Thus, muscular dystrophies associated with mutations in the
A-type lamin gene, LMNA, may be associated with decreased
satellite cell differentiation, causing depressed regeneration in
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addition to reduced myofiber stability. As a test for potential
regeneration defects in satellite cells of H-FRG1TGmice, we sought
to determine whether myoblasts from these mice had altered
proliferation or differentiation potential in cell culture. Myoblast
cultures were isolated and cultured by standard techniques ,
which, if treated appropriately, are comprised predominantly of
proliferating myoblasts and can provide an accurate readout of the
regenerative potential of muscle from the H-FRG1TGmice. We
speculated that myoblasts from H-FRG1TGmice may have similar
defects to those from Lmna2/2mice.
We isolated and cultured the satellite cell population from
affected quadriceps and unaffected diaphragm muscle of 18-week
old H-FRG1TGmice and wild-type littermate controls. Since the
quadriceps muscle, but not the diaphragm, exhibited character-
istics of muscular dystrophy, we speculated that there is a specific
defect in satellite cells derived from the thigh. We thus performed a
clonal assay on primary isolated myoblasts in order to measure the
proliferation rates of the isolated satellite cells.
We quantified transcript levels of FRG1 in our isolated satellite
cells by qPCR, and observed increased levels of FRG1 transcript in
both proliferating and differentiated cultures from H-FRG1TG
mice (Figure S1A). This was somewhat surprising as the promoter
driving FRG1 expression, the human skeletal actin promoter, is
reported to be active only after differentiation. We also performed
Western analysis on cell lysates generated from the same satellite
cells to observe protein levels of FRG1. Levels of FRG1 protein
are increased in both proliferating and differentiated cultures of
satellite cells from of H-FRG1TGmice (Figure S1B). In addition,
FRG1 is detected in satellite cell cultures derived from either
diaphragm or thigh muscle (Figure S1C).
For clonal analysis, the tissue-derived satellite cells were plated
at a very low density, 1000 cells per 10 cm dish, and allowed to
grow for a predefined amount of time. At regular intervals, plates
were fixed and nuclei were visualized by staining with methylene
blue. We also stained for myosin heavy chain (MyHC) as a control
to verify that cells did not prematurely differentiate over the course
of the clonal assay, in which bFGF and high serum levels were
maintained. After fixation and staining, the total number of cells
per clone was determined and binned for comparison in a
histogram format. Thigh-derived satellite cells from an 18-week
old H-FRG1TGmouse show a marked decrease in average clone
size compared to those derived from a wild-type littermate control
(Figure 2A). A significant fraction of these cells show arrest in a 2-
cell clone size skewing the distribution compared to the wild-type
thigh-derived satellite cells. This effect may be even more dramatic
considering that single cell clones were not scored in this assay, as
we consider single cell clones may potentially be new clones arising
from detached satellite cells floating away from the original clone
during mitosis. We replicated these observations with an
independent satellite cell culture isolated from 20-week old mouse
limbs obtained directly from Dr. Rossella Tupler which were
comparable to our 18-week old thigh-derived satellite cells at a
similar time point (Figure S2). The proliferative defect was not
replicated in the diaphragm-derived satellite cells, which show a
very similar clone size distribution between the H-FRG1TGand
wild-type C57BL/6 littermates. These findings indicate that FRG1
Figure 1. FRG1 expression and dystrophic phenotype of mice. A) Muscles and tissue from either H-FRG1TGtransgenic mice (FRG1) or wild-
type littermate control (C57BL/6) were collected from 10-week old mice. 300 mg of total protein from tissue lysates isolated from quadriceps muscle
(Q), gastrocnemius muscle (G), diaphragm muscle (D), whole heart (H), lung tissue (Lu), liver tissue (Li) and brain tissue (B) were probed with a-FRG1
antibody after SDS-PAGE. B) Diaphragm and quadriceps muscle from 13-week old H-FRG1TGmouse (FRG1) or wild-type littermate control (C57BL/6)
stained with hemotoxylin/eosin and viewed under 1006magnification. Arrows note location of centrally located nuclei present in FRG1 cross-section.
Scale bar notes 50 mm.
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overexpression leads to a muscle-type specific defect in prolifer-
ation, and correlates with the dystrophic phenotype.
To determine whether there is an age-dependent increase in
severity of the observed proliferation defect, we isolated and
performed a clonal assay on both thigh- and diaphragm-derived
satellite cells isolated from 4-week old mice, which appear
asymptomatic, to compare to the aforementioned data from more
severely symptomatic 18-week old mice. For each of these
populations, we scored multiple time points of a clonal assay, to
more thoroughly assay the proliferative defect. The clone size
distributions of myoblasts from asymptomatic 4-week old mice did
not show any significant proliferative defect when compared to
their 18-week old counterparts at a similar clone size (Figure 2B).
To more easily compare the data from these clone size
distributions, we obtained the average clone size for each of these
populations and calculated the average doubling time of each of
these lines. The data show that the proliferative defects associated
with FRG1 overexpression appear to be age-specific, as myoblasts
obtained from quadriceps muscle of 4-week old H-FRG1TGmice
are indistinguishable from those derived from littermate controls,
whereas satellite cells from 18-week old mice H-FRG1TGmice
have a severe defect (Table 1). These effects are further reflected
in the general health of the mice and severity of muscle dystrophy
at later ages. It is interesting to observe that by 52-weeks of age, we
were unable to successfully culture satellite cells from the thigh
muscle of H-FRG1TGmice, possibly due to either extremely
depressed proliferation rates or exhaustion of the satellite cell
population. It should also be noted that we never observed any
significant differentiation defect in any of our satellite cell cultures
(data not shown). We conclude that the overexpression of FRG1 in
muscle tissue causes muscle-type-specific and age-dependent
impairment in the ability of satellite cells to proliferate when
isolated in cell culture.
Inducible FRG1 expression in C2C12 myoblasts
In addition to experiments performed in mouse-derived satellite
cell culture, we sought to establish an independent cell culture
model system that would be free of any potential artifacts
introduced during generation of the H-FRG1TGmice. Initially,
we performed viral transduction of FRG1 under the CMV
promoter in the pMXIH vector as previously described .
Findings in early passages after selection indicated a proliferative
defect associated with FRG1 overexpression. Unfortunately, we
Figure 2. Clonal analysis of mouse-derived myoblasts. A) Myoblasts isolated from diaphragm (D) or thigh (T) of 18-week old H-FRG1TGmice
(FRG1) or wild-type littermate controls (WT) were cultured and plated at low density. Cells were fixed at regular time intervals and stained for myosin
heavy chain. Total number of nuclei per clone was counted and a representative graph of data from 96-hours post-plating is shown (n=100). B)
Myoblasts isolated from diaphragm (D) or thigh (T) of 4-week old H-FRG1TGmice (FRG1) or wild-type littermate controls (WT) were scored for
proliferation as above at 72-hours post-plating (50,n,80).
Table 1. Doubling times derived from clonal assays.
4 wk. old18 wk. old
Hours post-plating48 h. 72 h. 48 h.96 h. 144 h.
BL/6-D1817.4 19.922.2 25.6
FRG1-D 17.417.725.7 21.924.4
BL/6-T 18.618.8 23 28.126
Average calculated doubling time of myoblasts isolated from 4-week or 18-
week old H-FRG1TGmice (FRG1) or wild-type littermate control (BL/6) at varying
times post-plating. Doubling times were calculated from average clone size
with the following formula: [T *Ln(2)]/Ln(average clone size).
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observed a loss of the proliferative defect over time as measured by
BrdU-positive cells (Figure S3). The loss of defects in proliferation
was accompanied by an increase in cells staining negative for
FRG1, suggesting either that expression was being actively
silenced or, more likely, high FRG1 expressing cells were being
outcompeted over time by lower expressing cells with a faster rate
of proliferation. Given this complication, we adopted another
strategy to drive FRG1 overexpression.
We chose to employ an inducible system to control expression of
FRG1 that would allow us to turn on expression when needed. We
utilized C2C12 myoblasts that have an integrated cassette
expressing FLAG-tagged FRG1 under a tetracycline-responsive
promoter (iC2C12-FRG1) generated as previously described and
kindly provided to us by Dr. Michael Kyba . FRG1 expression
is induced in iC2C12-FRG1 myoblasts by the addition of
doxycycline and levels of induction can be modulated by adjusting
levels of the drug. There is no detectable expression of FRG1 in
uninduced cells as assayed by Western blot of whole cell lysates
using an a-FLAG antibody. In comparison, robust expression of
FRG1 was observed under a variety of induction conditions
ranging from 250 to 1000 ng/mL doxycycline (Figure 3A).
FRG1 is believed to play a role in post-transcriptional mRNA
processing and localizes to nuclear Cajal bodies , therefore we
performed immunofluorescence to investigate whether FRG1 was
properly localized in iC2C12-FRG1 myoblasts after induction.
iC2C12-FRG1 myoblasts were cultured on glass coverslips and
cells were grown for 24 hours in the presence or absence of
doxycycline followed by fixation and staining. Staining with DAPI
and an a-FLAG antibody revealed nuclear localization of FRG1-
FLAG following induction (Figure 3B). There was some variation
in magnitude of expression of FRG1-FLAG on a cell-to-cell basis,
but we observed robust partially punctuate staining in the nucleus,
consistent with possible localization to Cajal bodies.
Interestingly, we observed in our initial characterization of
induced iC2C12-FRG1 myoblasts a similar phenomenon as the
virally transduced C2C12 cells in that after numerous passages in
the presence of doxycycline, induced cells showed much lower
expression levels of FRG1-FLAG (Figure S4) and likely a loss of
any proliferative defect. A similar explanation is likely that lower
FRG1 expressing cells in this non-clonal population ultimately
outcompete higher expressing cells and take over the culture.
Characterizing defects in proliferation following FRG1
expression in iC2C12-FRG1 myoblasts
To investigate the proliferation defect caused by FRG1
overexpression, we conducted a number of assays in iC2C12-
FRG1 myoblasts. We first assayed the proliferation rates of cells in
mass culture in the presence or absence of doxycycline. In order to
determine growth rates, identical numbers of cells were plated in
wells of a 24-well plate and at regular time intervals the total
number of cells per well were counted using a hemocytometer. We
determined that induction of FRG1-FLAG in iC2C12-FRG1
myoblasts has a negative effect on their proliferation rate, as shown
by the increased doubling time when grown in doxycycline
(Figure 4A). Naı ¨ve C2C12 myoblasts were not affected by
exposure to doxycycline (data not shown), demonstrating that the
phenotype is likely specific to FRG1 overexpression.
To further investigate the proliferative defect of iC2C12-FRG1
myoblasts expressing FRG1, we utilized flow cytometry to
determine the fraction of cycling cells in specific phases of the
cell cycle. Actively proliferating iC2C12-FRG1 myoblasts grown
in either the presence or absence of doxycycline were fixed and
stained with DAPI. Subsequent flow cytometry analysis of the cells
for DNA content indicated an increased fraction of FRG1
overexpressing iC2C12-FRG1 myoblasts in G1-phase, coupled
with a corresponding decrease in the fraction of cells in S-phase
(Figure 4A). These findings suggest that cells overexpressing
FRG1 are delayed in transit through G1. We believe that this shift
in cell cycle profile may at least in part explain the gross
proliferation defect that we have observed.
One potential weakness of just looking at a cell cycle phase
distribution is that absolute cell cycle length is indeterminate in a
mass culture cell cycle profile. To address this issue, we performed
a mitotic shakeoff assay to synchronize proliferating iC2C12-
FRG1 myoblasts so that transit through specific cell cycle phases
could be measured. Since cells progressing through mitosis are less
adherent to culture dishes, this technique provides a way to
synchronize cells in the absence of any drugs to mediate cell cycle
arrest. After being plated in the presence or absence of doxycycline
for a period of 24 hours, mitotic cells were isolated by mild shaking
and rocking for a period of 20 minutes to generate synchronized
cultures. Fixation at 2-hour intervals followed by subsequent
analysis by flow cytometry for DNA content was performed to
Figure 3. Expression of FRG1 in iC2C12-FRG1 myoblasts. A) Western blot of lysates from iC2C12-FRG1 myoblasts before induction with
doxycycline or after induction for 24 hours with concentrations ranging from 250 ng/mL to 1000 ng/mL using a-FLAG antibody. b-actin loading
control shown below. B) Localization of FRG1 by immunofluorescence in iC2C12-FRG1either uninduced or induced with 500 ng/mL doxycycline for
24 hours. DAPI stain is represented in the blue channel and a-FRG1 antibody staining is represented in the green channel.
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Figure 4. Characterization of proliferation defect in iC2C12-FRG1 myoblasts. A) Mass culture doubling times calculated from
hemocytometer counts of iC2C12-FRG1 myoblasts with or without induction of expression by doxycycline over 120 hours. Below are the cell cycle
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track cells as they progressed through the cell cycle. We observed
that FRG1 overexpressing cells exhibited a consistent 1–2 hour
delay in exit from G1 and entry into S-phase (Figure 4B &
Table 2). This finding is consistent with the FACS analysis of
asynchronous cells, which indicated that a higher percentage of
FRG1 overexpressing cells were in G1 phase. It should be noted
that it is difficult to monitor progression beyond one cell cycle in this
assay since the iC2C12-FRG1 myoblasts rapidly lose synchronicity.
Cell cycle specific factors are affected by FRG1 expression
of the mammalian cell cycle, inhibiting E2F-dependent transcrip-
tion and maintaining cells in G1-phase when hypophosphorylated.
Phosphorylation by cyclin-dependent kinases interferes with the
capacity of pRb to repress E2F-dependent transcription, permitting
S-phase entry [43,44]. Since FRG1 overexpression led to increased
G1 phase occupancy, we examined the phosphorylation state of
pRb as an indicator of these altered cell cycle kinetics. We observe
via Western blot that compared to naı ¨ve C1C12 or uninduced
iC2C12-FRG1 myoblasts, levels of 807/811 phosphorylated pRb
are lower in induced iC2C12-FRG1 myoblasts (Figure 5A). Total
levels of pRb appear lower, however this is likely an artifact of the
antibody used, since it is known to have somewhat greater affinity
for hyperphosphorylated pRb. Decreased pRb phosphorylation is
consistent with a decreased proliferation rate and increased G1
In this study, we demonstrate decreased proliferation rates of
myoblasts expressing FRG1, an attribute that could contribute to
the long term reduction in muscle regenerative potential and
muscular dystrophy observed in transgenic mice overexpressing
FRG1. We have verified expression and muscular dystrophy in the
H-FRG1TGmouse and seen that thigh-derived myoblasts, but not
diaphragm-derived myoblasts, from these animals demonstrate a
proliferative defect by clonal analysis. We also find that induction
of FRG1 in myoblasts by a tetracycline-responsive system
negatively affects proliferation as determined in cell cycle profiles
measured by flow cytometry and hypophosphorylation of pRb.
Reduced myoblast proliferation is not commonly linked to
muscular dystrophy, which is more classically attributed to death
of muscle fibers, such as in Duchenne muscular dystrophy , or
a combination of enhanced fiber degeneration and defective
differentiation kinetics such as in Lmna2/2mouse models [39,46].
However, there have been reports of depressed proliferation
kinetics in Duchenne muscular dystrophy myoblasts, compound-
ing the existing mechanisms of muscular dystrophy . Since the
proliferation defect gets more severe in myoblasts isolated from H-
FRG1TGmice of increasing age, it is difficult to differentiate
between two models: (1) that the proliferation defect precedes
onset of the dystrophic phenotype or (2) that the defect derives
from reduced satellite potential with age resulting from increasing
strain on satellite cells to repair damage.
Our findings in C2C12 cells may indicate that a cell cycle defect
can occur as a primary result of FRG1 overexpression. However, it
ultimately remains unclear precisely how FRG1 impacts cell cycle
progression. FRG1 has been shown to localize to Cajal bodies in
the nucleus, where it is reported to regulate RNA processing
[42,48]. Misprocessing of RNA transcripts has also been linked to
myotonic dystrophy [49,50]. One possible hypothesis for induction
of G1 arrest by FRG1 involves altered splicing of transcripts
encoding cell cycle components. For instance, altered processing of
the cyclin E RNA produced different isoforms of the protein with
different affinities for Cdk2 [51,52,53].
One contentious observation regarding overexpression of FRG1
in patients with FSHD is that other research groups have been
unable to replicate the results published showing increased FRG1
transcript in affected muscle . Some studies of myoblasts
subjected to microarray analysis or measuring RNA transcription
of FRG1 have not yielded any results showing an increase of FRG1
transcript [54,55], while similar experiments with qRT-PCR done
in other groups have shown an increased trend . Recently,
however, Bodega et al. reported that overexpression of FRG1 in
profiles of uninduced iC2C12-FRG1 myoblasts as well as after 24 hours and 72 hours in the presence of doxycycline. Cell cycle profiles were
calculated from DNA content analysis by flow cytometry on proliferating myoblasts that were fixed and DAPI stained. *denotes statistical significance
p,0.05, **p,0.005. B) Cell cycle profiles of synchronized iC2C12-FRG1 myoblasts over 24 hours in the absence or presence of doxycycline showing
fraction of cells in G1, S or G2-phase. Comparison of data is plotted in a line graph fashion showing G1 in green, S in blue and G2 in red to
demonstrate the generalized lag exhibited by iC2C12-FRG1 myoblasts expressing FRG1. Uninduced iC2C12-FRG1 myoblasts are graphed with a
dashed line, while iC2C12-FRG1 myoblasts with doxycycline-induced FRG1 expression are graphed with a solid line. n.20,000 cells analyzed for each
Table 2. Raw cell cycle profile data.
2468 10 121416 18 20 2224
ICE FRG1 2 2dox
%G186.4182.4 74.5848.95 21.034.31 20.55 40.3553.3556.66 43.65 29.16
%S3.11412.52 34.5374.9492.66 32.41 14.4320.47 27.68 48.95 51.91
%G210.4813.612.9 16.52 4.033.0347.04 45.22 26.1815.667.4 18.93
ICE FRG1 + +dox
%G187.8387.6780.81 66.74 38.2512.04 16.5 34.7556.4164.9650.97 39.37
%S 2.311.82 7.8321.0449.4587.96 52.06 18.2411.67 19.1135.2 46.6
%G29.8610.51 11.3612.22 12.3031.44 47.0131.9215.93 13.83 14.23
Values from WinCycle analysis of synchronized ICE-FRG1 myoblasts in the presence or absence of doxycycline-induced FRG1 expression showing fraction of cells in G1, S
or G2-phase of the cell cycle plotted in table form to emphasize differences. N.20,000 analyzed for each timepoint.
FRG1 Overxpression Impairs Myoblast Proliferation
PLoS ONE | www.plosone.org7May 2011 | Volume 6 | Issue 5 | e19780
muscle biopsies is not a uniform finding and may depend on the
composition and age of the muscle biopsy, as FRG1 is only
upregulated during an early state of differentiation into myotubes.
Regardless of whether the levels of FRG1 are increased or not in
FSHD patients, the H-FRG1TGmouse is a valuable tool for
studying the mechanics of muscular dystrophy, though it is
important to note that FRG1 expression in these mice is many fold
higher than observed in any human patients. Ultimately, FRG1
remains relatively poorly characterized and our findings may help
to further elucidate its function.
It has been reported that myoblasts isolated from human muscle
biopsies exhibit a morphological difference upon differentiation
. The authors observed that, compared to differentiated wild-
type myotubes, FSHD myotubes were thinner, less branched, more
disorganized and were comprised of fewer myoblasts as measured
by total number of nuclei per myotube. Converse to our findings,
theydidnot note any proliferation defects intheirmyoblastcultures,
which may be attributed to differences in their isolation technique,
sample sources and/or assay techniques. Regardless of the caveats,
this raises the possibility of a compound defect in both proliferation
and differentiation as a mechanism for the development of FSHD.
One disparity in our data may be the difference observed
between the proliferative defect described in mass culture
(Figure 4A) and that seen by mitotic shakeoff analyzed by flow
cytometry (Figure 4B). Although the difference seen by mitotic
shakeoff is less severe than that observed by mass culture doubling
time, it is worth noting that the cells are kept under different
conditions in each of these assays. In mass culture, myoblasts find
themselves in much denser conditions with self-conditioned media.
In the mitotic shakeoff, myoblasts are in a much less dense
environment and exposed to fresh media and growth factors. It is
possible that, given enough time, the cell cycle profiles of myoblasts
isolated by mitotic shakeoff would exhibit a more severe defect with
increased cell density, but it is impossible to determine this within
the time frame whereby cells remain synchronized.
In summary, our experiments have demonstrated the expression
of FRG1 in mouse muscle causes a tissue-specific and age-
dependent proliferative defect in the satellite cell population,
possibly playing a part in the development of muscular dystrophies
and FSHD in humans.
Materials and Methods
Animal care and genotyping
High-expressing FRG1 transgenic mice (H-FRG1TG) were kindly
provided by Dr. R. Tupler . DNA was extracted from tail
samples by digestion with TENS solution (50 mM Tris pH 7.5,
100 mM EDTA pH 8.0, 400 mM NaCl, 0.4% SDS, proteinase K
0.5 mg/mL) followed by ethanol precipitation of genomic DNA.
PCR genotyping of FRG1 mice was performed with the following
primers: HSA-FRG1-59 59-GAT CTA GCG GCC GCC ATG
GCC GAG TAC TCC TAT GTG AAG TCT-39 and HSA-
FRG1-39 59-GCG CGC TTA ATT AAT CAC TTG CAG TAT
CTG TCG GCT TTC A-39. Mice were bred and maintained
under specific pathogen-free conditions. All experiments were
performed in compliance with the University of Washington
Institutional Animal Care and Use Committee protocol #2362-04.
Analysis of FRG1 expression
Tissue lysates were generated from quadriceps, gastrocnemius,
diaphragm, heart, liver, lung and brain tissue isolated from H-
FRG1TGmice and wild-type littermate controls. Homogenization
was done in 5 mL of ice-cold RIPA buffer supplemented with
protease inhibitors (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1%
NP-40, 0.25% deoxycholate, 1 mM Na3VO4, 1 mM NaF, 1 mM
PMSF, 1 mg/mL aprotinin, 1 mg/mL leupeptin) per gram of tissue
for 30 seconds using the Omni-Tip system (OMNI International).
Samples were incubated on ice for 30 minutes, spun down at
16,500 rcf for 15 minutes at 4uC, followed by transferring of
supernatant and recentrifugation for an additional 10 minutes to
generate tissue lysates.
RNA was isolated from both proliferating and differentiated
mouse muscle satellite cell cultures using the RNAqueous kit
(Ambion Inc). qPCR was performed using primers as previously
described . Cell lysates were also generated from the
aforementioned muscle satellite cell cultures by homogenization
in RIPA buffer as described above.
Histological analysis of muscle
Quadriceps, gastrocnemius, soleus and diaphragm were isolated
from H-FRG1TGmice and wild-type littermate controls, blotted
briefly on filter paper and weighed on an analytical balance.
Tissues were mounted in Tissue-Tek OCT compound (Sakura
Finetek), frozen in a liquid nitrogen-cooled bath of isopentane and
stored at 280uC. Cryosections were collected using a Leica
CM1850 for hemotoxylin and eosin staining. Stained sections
were viewed and photographed under 1006 magnification on a
Zeiss Axiovert 200 M. Analysis of images was performed using
Isolation of mouse muscle satellite cells and cell culture
To isolate satellite cells, 4-week and 18-week old FRG1 or wild-
type mouse thigh or diaphragm muscle tissue was dissected away
and placed in chilled Growth Media (GM) consisting of Ham’s
F10C+15% horse serum (Atlanta Biologicals Lot D0195)+50 mg/
ml gentamicin with the addition of 0.25 mg/ml Fungizone.
Connective tissue and fat were dissected away from muscle tissue
under a dissecting microscope and muscle was weighed on an
analytical balance. Isolation of the myoblastic population was
performed as previously described , with the following
modifications and details. Diced muscle was minced for 2 minutes
using curved-tip scissors and treated with 50 mL of 0.05% trypsin-
EDTA (Gibco) diluted in Hank’s saline per mg of tissue. The
tissue/trypsin mixture was incubated at 37uC and pipetted
thoroughly every 5 minutes for 20 minutes total. The trypsinized
tissue mixture was then passed through a 70 mm cell strainer (BD
Falcon) to reduce fibroblast contamination and the strainer was
rinsed with an additional 100 mL of F10C per mg of tissue. The
flowthrough was then centrifuged at 800 rpm (Sorvall Legend T)
for 5 minutes and plated on gelatin-coated dishes in GM+6 ng/ml
Figure 5. Phosphorylation of pRb is perturbed by FRG1
expression. Western blot analysis for pRb, pRb Ser807/811 and
FRG1 in iC2C12-FRG1 cells grown in the presence or absence of
doxycycline. b-actin loading control shown below.
FRG1 Overxpression Impairs Myoblast Proliferation
PLoS ONE | www.plosone.org8 May 2011 | Volume 6 | Issue 5 | e19780
basic fibroblast growth factor (bFGF)+0.25 mg/ml Fungizone .
The satellite cell cultures were observed carefully and differentially
passaged to eliminate any fibroblast contamination over 5
passages. To differential passage, satellite cells were treated to a
mild trypsinization and observed under a microscope until the
round satellite cells were seen detaching from the plate. Because of
their morphological differences, satellite cells will detach from the
dish before the flattened and larger fibroblasts. Once the satellite
cells were observed detaching, the trypsin solution was immedi-
ately removed and plated to isolate the satellite cell population.
The established mouse-derived satellite cell cultures were
cultured on 10 cm collagen-coated tissue culture plates in GM
with 4 ng/ml bFGF. Cells were supplemented with fresh bFGF
every 12 hours to inhibit differentiation of satellite cells into
myocytes. Passaging occurred when cells reached 56105cells in a
10 cm dish by rinsing with Saline A (10 mM dextrose, 30 mM
HEPES, 3 mM KCl, 130 mM NaCl, 1 mM Na2HPO4-7H2O),
trypsinization and replating at 56104in a new 10 cm dish.
iC2C12-FRG1 myoblasts were generated as described and
generously provided by Dr. Michael Kyba at the University of
Texas Southwestern . iC2C12-FRG1 myoblasts were main-
tained in DMEM (Hyclone)+10% FBS (Atlanta Biologicals Lot
A0087) taking care to passage before confluency to avoid loss of
the myoblastic population. Induction of FRG1-FLAG in iC2C12-
FRG1 myoblasts was achieved by incubating cells with 500 ng/
mL doxycycline for 24 hours.
Clonal analysis of proliferation
To perform clonal assays, proliferating satellite cells were
trypsinized and plated at 1000 cells per 10 cm dish in GM with
6 ng/mL bFGF to prevent premature differentiation. Cells were
left undisturbed to minimize the formation of satellite clones and
fixed with AFA (50% ethanol, 5% formalin, 5% acetic acid) at
regular intervals. Myoblast clones were then immunostained for
myosin heavy-chain (MyHC) using mouse monoclonal MF-20,
biotinylated rabbit anti-mouse IgG, streptavidin and biotinylated
horseradish peroxidase (Vector Labs, Inc.), followed by counter-
staining with 1% methylene blue as previously described .
Clones were then scored by visualization at 256/506magnifica-
tion on a dissecting microscope for both number and percent
differentiation. Doubling times were calculated with the formula
‘‘Doubling Time=[Time post-plating *Ln(2)]/Ln(average clone
Viral transduction of C2C12 myoblasts
Expression vectors for FRG1 were generated in the pMXIH
vector as described previously . 293T cells were transfected
with either pMXIH or pMXIH-FRG1 in combination with the Q-
ampho packaging plasmid using calcium phosphate. Virus-
containing media were filtered through a 0.45 mm filter and
applied to C2C12 myoblasts to generate C2C12-pMXIH vector
control myoblasts and C2C12-FRG1 myoblasts which should
stably express FRG1 protein.
Flow cytometry and other proliferation assays
To assay doubling time of iC2C12-FRG1 myoblasts, prolifer-
ating cells were grown in the presence or absence of doxycycline to
induce expression of FRG1. 2000 cells were plated per well of a 24-
well dish. One well of cells plated in this dish per condition was
trypsinized every 24 hours over 120 hours and total cell number
was counted in triplicate on an improved Neubauer hemocytom-
eter. Cell number was graphed and exponential curve fit was
performed using Microsoft Excel to determine the growth constant
and calculate the doubling time.
For BrdU staining of virally-transduced C2C12 myoblasts, after
viral transduction, cells were plated on glass coverslips and pulsed
with BrdU cell proliferation reagent (Amersham) for 60 minutes.
Cells were then fixed in 4% formalin, permeabilized with 0.5%
Triton X-100 in PBS and blocked in 5% goat serum, 5% horse
serum, 0.2% Tween-20, 0.2% fish skin gelatin in PBS. a-BrdU
mouse antibody (BD Biosciences) was diluted 1:350 in PBS and
incubated for 1 hour at 37uC followed by goat a-mouse antibody
conjugated to AlexaFluor488 diluted 1:400. Coverslips were
mounted using Vectashield (Vector Labs).
For flow cytometry assays, proliferating iC2C12-FRG1 myo-
blasts were also plated in 10 cm dishes for cell cycle profile
analysis. After trypsinization, cell pellets were resuspended in
DAPI solution (2 mM CaCl2, 22 mM MgCl20.1 mg/mL BSA,
0.1% Nonidet P-40, 10 mg/mL DAPI, 10% DMSO) and run on
an InFlux flow cytometer (Cytopeia). 20,000 cells were run
through the flow cytometer measuring DNA content. Analysis was
done using WinCycle (Phoenix Flow Systems) to determine cell
cycle phase distributions as previously described .
For determining initial effects of FRG1 expression in synchro-
nized cells, proliferating iC2C12-FRG1 myoblasts either exposed
to doxycycline for 24 hours previously or grown in the absence of
doxycycline were synchronized by mitotic shakeoff as described
. Three 10-cm dishes were tapped vigorously against a hard
surface to shake off mitotic cells, incubated with gentle rocking for
20 minutes to prevent reattachment, then tapped vigorously again
to further dislodge mitotic cells, and finally the medium containing
mitotic cells was centrifuged and plated in a single 60-mm dish.
Twelve 60-mm dishes of synchronized cells were collected and
placed either in the presence or absence of doxycycline. Every
2 hours after the initial plating, cells were trypsinized and pellets
were fixed and resuspended in the same DAPI solution used for
flow cytometry to cover a total period of 24 hours. DNA content
was measure as previously mentioned using flow cytometry, with
20,000 cells being analyzed.
Immunofluorescence and immunoblotting
iC2C12-FRG1 myoblasts with or without the induction of
FRG1-FLAG were grown on round glass coverslips, fixed in a 4%
formalin solution and stained with mouse monoclonal a-FLAG
antibody (Sigma) at 1:10,000 dilution. For cell lysates, proliferating
satellite cells or iC2C12-FRG1 myoblasts were lysed in RIPA
buffer at 4uC, incubated on ice for 30 minutes, and centrifuged at
16,500 rcf for 10 minutes to remove cell debris. Cell lysates were
run on a 10% SDS-PAGE gel, transferred to nitrocellulose
overnight at 4uC in a Tris-glycine buffer and incubated overnight
at 4uC with one of the following primary antibodies. Monoclonal
a-FRG1 at 1:1000 (Abnova clone 4A5), mouse monoclonal pRb at
1:1000 (BD Pharmingen clone 14001A), rabbit anti-mouse a-pRb
Ser795 at 1:1000 (Cell Signaling), rabbit anti-mouse a-pRb
Ser807/811 at 1:1000 (Cell Signaling), or mouse monoclonal a-
b-actin at 1:10000 (Abcam). Western blotting was completed the
next day using the corresponding secondary antibody of donkey
anti-rabbit HRP at 1:10000 (Amersham Biosciences) or rabbit
anti-mouse HRP at 1:10000 (Amersham Biosciences). Blots were
visualized using ECL substrate (PerkinElmer) on Kodak Biomax
Light (Sigma) film.
derived myoblasts. A) Levels of FRG1 in 20-week old
diaphragm-derived myoblasts from either H-FRG1TG(FRG1) or
wild-type littermate control (C57BL/6) as assayed by qPCR
Examination of FRG1 expression in muscle-
FRG1 Overxpression Impairs Myoblast Proliferation
PLoS ONE | www.plosone.org9 May 2011 | Volume 6 | Issue 5 | e19780
normalized to GAPDH. Proliferating cultures were judged to have
less than 5% differentiated cells while differentiated cultures
exhibited greater than 70% differentiation. *indicates no expres-
sion detected. B) Western analysis on proliferating and differen-
tiated diaphragm-derived myoblast cultures from 20-week old
mice as described above probing for total FRG1 levels. b-actin
loading control shown below. C) Western analysis on proliferating
satellite cell cultures from diaphragm or thigh of 4-week old mice
probing for total FRG1 protein levels. b-actin loading control
myoblasts. Myoblasts isolated from hindlimb of 18-week old H-
FRG1TG(FRG1) or wild-type littermate controls (WT) were
cultured and plated at low-density. Total number of nuclei per
clone were counted at 120-hours post-plating (n=100). Similarly
myoblasts isolated from dissected hindlimbs of 20-week old H-
FRG1TGmouse (FRG1) or wild-type littermate controls (WT) were
subjected to this procedure in the lower figure showing total
number of nuclei per clone at 120-hours post-plating (n=13 for
FRG1 line, n=73 for WT line).
Additional clonal analysis of mouse-derived
duced C2C12 myoblasts. C2C12 mouse myoblasts transduced
with either vector control (pMXIH) or a FRG1-expressing
construct (FRG1) were scored for incorporation of BrdU after
Loss of proliferative defect in virus-trans-
60-minute pulse to determine % of S-phase cells. Transduced
myoblasts show proliferative defect at early passages (passage 8)
but lose the phenotype over time.
myoblasts over time. iC2C12-FRG1 myoblasts were cultured
and maintained with or without the presence of 500 ng/mL
doxycycline to induce FRG1 expression. Immunofluorescence with
an a-FLAG antibody (green) and DAPI staining (blue) reveals loss
of FRG1 expression after ,20 passages under induction condi-
tions, but robust expression with an acute induction of 24 hours.
Loss of FRG1 expression in iC2C12-FRG1
The authors would like to thank Rubysue Mangalindan, Ashot Safarli and
other members of the Ladiges lab in the Department of Comparative
Medicine for handling and maintenance of mouse colonies. Flow
cytometry and assistance with analysis was graciously provided by Donna
Prunkard in the Rabinovitch lab, Department of Pathology, UW.
Conceived and designed the experiments: SCC BKK. Performed the
experiments: SCC EF JM XR. Analyzed the data: SCC EF JM XR BKK.
Contributed reagents/materials/analysis tools: DB MK. Wrote the paper:
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FRG1 Overxpression Impairs Myoblast Proliferation
PLoS ONE | www.plosone.org11 May 2011 | Volume 6 | Issue 5 | e19780