CUGBP1 overexpression in mouse skeletal muscle
reproduces features of myotonic dystrophy type 1
Amanda J. Ward1,2, Mendell Rimer4, James M. Killian3, James J. Dowling5
and Thomas A. Cooper1,2,∗
1Department of Pathology and Immunology,2Department of Molecular and Cellular Biology and3Department of
Neurology, Baylor College of Medicine, Houston, TX 77030, USA,4Department of Neuroscience and Experimental
Therapeutics, Texas A&M Health Science Center College of Medicine, College Station, TX 77843, USA and
5Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA
Received April 25, 2010; Revised June 8, 2010; Accepted June 29, 2010
The neuromuscular disease myotonic dystrophy type I (DM1) affects multiple organ systems with the major
symptoms being severe muscle weakness, progressive muscle wasting and myotonia. The causative
mutation in DM1 is a CTG repeat expansion in the 3′-untranslated region of the DM protein kinase (DMPK)
gene. RNA transcribed from the expanded allele contains the expanded CUG repeats and leads to the nuclear
depletion of Muscleblind-like 1 (MBNL1) and to the increased steady-state levels of CUG-binding protein 1
(CUGBP1). The pathogenic effects of MBNL1 depletion have previously been tested by the generation of
MBNL1 knockout mice, but the consequence of CUGBP1 overexpression in adult muscle is not known. In
a DM1 mouse model expressing RNA containing 960 CUG repeats in skeletal muscle, CUGBP1 up-regulation
is temporally correlated with severe muscle wasting. In this study, we generated transgenic mice with
doxycycline-inducible and skeletal muscle-specific expression of CUGBP1. Adult mouse skeletal muscle
overexpressing CUGBP1 reproduces molecular and physiological defects of DM1 tissue. The results from
this study strongly suggest that CUGBP1 has a major role in DM1 skeletal muscle pathogenesis.
The most common cause of adult-onset muscular dystrophy is
myotonic dystrophy type I (DM1) with an estimated incidence
of 1 in 8000 individuals worldwide. DM1 is highly variable
both in the severity and in the type of symptoms affecting
skeletal, cardiac and smooth muscle as well as the endocrine
and central nervous systems. Involvement of the skeletal
muscle is complex and includes myotonia, tissue insulin resist-
ance, muscle weakness and atrophy/wasting that primarily
affects distal limb muscles early in the disease with proximal
progression (1,2). Respiratory failure resulting from progress-
ive muscle weakness is the major cause of death in individuals
affected by DM1 (3,4). The mechanisms underlying the
process of muscle wasting are largely undefined.
DM1 is caused by a CTG trinucleotide repeat expansion in
the 3′-untranslated region of the DM protein kinase (DMPK)
gene (5). The mutation does not primarily cause disease
through loss of DMPK function, but rather through the gain
of RNA function. The CTG repeat expansion is transcribed
and the CUG repeat RNA is retained in the nucleus as
nuclear foci (6). Two pathways are affected by the accumu-
lation of this toxic RNA. First, the RNA-binding protein
Muscleblind-like 1 (MBNL1) binds to and is sequestered by
the CUG repeat RNA, causing its depletion from the nucleus
up-regulated in DM1 myoblasts, heart and skeletal muscle
(8,9) through a PKC-mediated phosphorylation event which
stabilizes the protein (10). MBNL1 and CUGBP1 antagonisti-
cally regulate alternative splicing transitions during normal
heart and skeletal muscle development (11). CUGBP1 also
plays roles in translational control and mRNA stability (12).
The depletion of MBNL1 and increased expression of
CUGBP1 results in widespread transcriptional and post-
transcriptional changes, including alterations in mRNA stab-
ility and alternative splicing (11–14).
The best-characterized post-transcriptional change in DM1
is the misregulation of alternative splicing. Global mRNA
∗To whom correspondence should be addressed. Tel: +1 7137983141; Fax: +1 7137985838; Email: firstname.lastname@example.org
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Human Molecular Genetics, 2010, Vol. 19, No. 18
Advance Access published on July 5, 2010
profiling studies in DM1 mouse models using splicing-
sensitive microarrays found alterations in more than 200 spli-
cing events (13). In the majority of cases examined so far, the
misregulation is due to the disruption of a program of develop-
mentally regulated splicing such that embryonic splice var-
iants are aberrantly expressed in adult tissues. The abnormal
expression of specific genes is directly responsible for the
onset ofsome diseasesymptoms.
example is the muscle-specific chloride channel 1 (CLCN1)
which is mis-spliced in DM1 tissue resulting in mRNA degra-
dation. The lack of chloride channel function in adult muscle
produces myotonia (15–18). Recent evidence suggests that
aberrant expression of ryanodine receptor (RyR1) with exon
70 exclusion in adult DM1 skeletal muscle may alter exci-
tation–contraction coupling and promote muscle degeneration
(19,20). However, solid links between many symptoms in the
skeletal muscle system and individual alternative splicing or
transcriptional events remain to be established.
We previously generated a DM1 mouse model (EpA960/
muscle-specific expression of RNA containing 960 interrupted
CUG repeats within the natural position of DMPK exon 15
(DMPK-CUG960 RNA) (21). Induction of DMPK-CUG960
RNA in skeletal muscle reproduced many disease features
including characteristic histological abnormalities, myotonia,
decreased muscle function, severe muscle wasting, MBNL1
co-localization with nuclear foci, CUGBP1 up-regulation
and misregulated alternative splicing (21). The muscle
wasting phenotype was temporally correlated with increased
CUGBP1 expression, suggesting that CUGBP1 may contribute
to, or be primarily responsible for, muscle wasting in DM1.
Other data also support this correlation, as follows: (i) splicing
events responsive to CUGBP1 but not MBNL1 reverted to
embryonic patterns, demonstrating a downstream consequence
of elevated CUGBP1; (ii) a DM1 mouse model expressing
CUG250 RNA in the context of the human skeletal actin
gene (HSALR) has neither increased CUGBP1 nor a strong
muscle degeneration phenotype (22); (iii) MBNL1 knockout
mice (MBNL1DE3/DE3) do not exhibit severe muscle wasting
suggesting that MBNL1 depletion alone is not able to repro-
duce this disease feature (23); (iv) constitutive transgenic
overexpression of CUGBP1 in developing mouse skeletal
muscle results in impaired skeletal muscle differentiation
and dystrophic muscle histology (24,25); (v) CUGBP1 overex-
pression in Drosophila somatic muscles generated loss of
muscle integrity (26), and (vi) inducible expression of exogen-
ous CUGBP1 in adult mouse cardiomyocytes reproduces DM1
cardiac symptoms (27). Cumulatively, these data provide a
strong rationale for testing the hypothesis that CUGBP1
plays a pathogenic role in skeletal muscle wasting observed
To determine whether CUGBP1 overexpression in adult
mouse skeletal muscle is sufficient to reproduce features of
DM1 including skeletal muscle wasting, we generated trans-
genic mice with doxycycline-inducible and skeletal muscle-
specific expression of CUGBP1. Mice with an 8-fold induction
of CUGBP1 in skeletal muscle exhibited decreased muscle
weight, impaired muscle performance and dystrophic muscle
histology characteristic of DM1, including a large fraction of
myofibers containing central nuclei. Additionally, we show
that many alternative splicing events misregulated in human
DM1 skeletal muscle are also misregulated in mouse muscle
overexpressing CUGBP1. These data show that CUGBP1
up-regulation in skeletal muscle is pathogenic and is sufficient
to reproduce functional and molecular features of DM1.
CUGBP1 overexpression is sufficient to produce a muscle
phenotype reminiscent of DM1
We previously generated a doxycycline-responsive mouse
line, TRECUGBP1, which encodes an N-terminal Flag-tagged
human CUGBP1 LYLQ isoform located downstream of a
tetracycline-responsive element and a minimal CMV promoter
(27). TRECUGBP1 mice were crossed to a mouse line with
skeletal muscle-specific expression of a modified reverse tetra-
cycline transactivator (rtTA), MDAFrtTA. These mice express
rtTA2S-M2, a modified rtTA with an increased doxycycline
sensitivity and lower basal activity, from the rat myosin
light chain 1/3 promoter/enhancer (28). At 2–3 months of
induced by feeding doxycycline-containing food (+dox).
Two groups of uninduced controls were used: (i) bitransgenic
MDAFrtTA/TRECUGBP1 mice fed a normal diet (–dox) and
(ii) monotransgenic TRECUGBP1 or MDAFrtTA (+dox)
mice. Both control groups produced similar results in all
experiments performed and were therefore grouped for statisti-
MDAFrtTA/TRECUGBP1 mice given food containing 2 g
dox/kg food achieved short-term robust expression of
CUGBP1. Serial dilution analysis conducted on gastrocnemius
muscle protein lysate following 2 weeks of induction showed
an 8-fold increase in CUGBP1 (Fig. 1A). Western blotting
expressed from the exogenous transgene specifically in the
skeletal muscle and no bands of the correct molecular
weight were detected in any other organ tissue tested
(Fig. 1B). The bands in the liver and kidney protein lysates
were deemed non-specific because they were not recognized
by CUGBP1 antibody (data not shown). Additionally,
CUGBP1 was up-regulated in the predominately fast twitch
quadriceps and triceps, as well as the slow twitch soleus
muscle (Fig. 1C). There was some induction of CUGBP2
and MBNL1, potentially due to the expression within regener-
ating myofibers, supported by the up-regulation of embryonic
myosin heavy chain (eMHC), a marker of regeneration
(Fig. 1C). CUGBP1 protein induction was not maintained
over time, and by 8 weeks on dox diet, CUGBP1 was only
up-regulated 2–3-fold (Fig. 1D).
MDAFrtTA/TRECUGBP1 mice had a strong, observable
phenotype by 2 weeks on dox diet exhibiting impaired move-
ment, abnormal gait and an 18% reduction in total body
weight compared with uninduced control mice (Supplemen-
tary Material Movie S1 and Fig. 2A). Furthermore, there
was a 30% reduction in gastrocnemius muscle weight by 4
weeks on dox diet (Fig. 2B). Muscle function was assessed
in MDAFrtTA/TRECUGBP1 mice by an acute graded tread-
mill assay over the 8-week time course on dox diet. During
the time points of high CUGBP1 induction, namely 1–4
Human Molecular Genetics, 2010, Vol. 19, No. 183615
weeks on dox diet, MDAFrtTA/TRECUGBP1 mice exhibited
a significant reduction in muscle function (Fig. 2C). However
by 8 weeks of induction, when CUGBP1 expression levels are
(Fig. 2C). These data demonstrate a very tight correlation
between the level of CUGBP1 expression and the severity of
muscle functional properties.
Histological examination of uninduced MDAFrtTA/TRE-
CUGBP1 gastrocnemius muscle by hematoxylin and eosin
staining showed normal muscle histology with peripherally
located nuclei and no evidence of myofiber atrophy or regen-
eration (Fig. 3A). By 2 weeks on dox diet, the muscle his-
tology was strikingly abnormal with an abundance of
myofibers with centrally located nuclei (.10% of the
sample) (Fig. 3B). A high fraction of myofibers containing
central nuclei is one of the most characteristic changes
observed in DM1 muscle histology (1). MDAFrtTA/TRE-
CUGBP1 (+dox, 2 weeks) histology also showed myofiber
atrophy, increased variation in myofiber size, evidence of
degeneration marked by the presence of small myofibers
with inflammatory infiltrate and pyknotic nuclear clumps
(Fig. 3B and Supplementary Material, Fig. S1). Similar
changes were seen after 4 weeks on dox diet, but by 8
weeks the histological abnormalities were milder, only
occasionally showing central nucleated myofibers (Fig. 3C
and data not shown).
Type I fiber atrophy and type II predominance have been
previously reported in patient biopsies in DM1 (1). Using
immunolabelling on MDAFrtTA/TRECUGBP1 (+dox) gas-
trocnemius muscle to define fiber types, we did not detect
any notable differences between type I and type II fibers in
terms of myofiber size or abundance compared with uninduced
control muscle (Supplementary Material, Fig. S2).
To make a direct histological comparison between adult
muscle overexpressing CUGBP1 or DMPK-CUG960 RNA,
(+dox) mice was compared side by side with gastrocnemius
mice 4 weeks after the induction of DMPK-CUG960RNA.
Similarto CUGBP1 overexpressing
HSA-Cre-ERT2(+tam) mice showed myofiber atrophy, myo-
fiber size variation, degenerative changes and an even greater
Figure 1. MDAFrtTA/TRECUGBP1 mice display CUGBP1 up-regulation in skeletal muscle upon dox administration. (A) Western blot for CUGBP1 following
serial dilution of gastrocnemius muscle protein lysates from induced MDAFrtTA/TRECUGBP1 (+dox, 2 weeks) mice show an 8-fold elevation in CUGBP1
expression compared with uninduced control mice. (B) Western blot using anti-Flag antibody to detect exogenous CUGBP1 shows that the expression is specific
to the skeletal muscle of MDAFrtTA/TRECUGBP1 (+dox, 2 weeks) mice. Ponceau staining of the blot was used as the loading control. (C) CUGBP1 is highly
expressed in all MDAFrtTA/TRECUGBP1 (+dox, 2 weeks) skeletal muscles tested including the gastrocnemius, quadriceps, triceps and soleus muscles.
Western blots for CUGBP2 and MBNL1 and the regeneration marker eMHC are also shown. Both GAPDH and Ponceau stain were used as loading controls.
(D) CUGBP1 western blot over the 8-week time course on dox diet shows short-term induction in MDAFrtTA/TRECUGBP1 mice. CUGBP2 and MBNL1
protein expression are also shown.
3616Human Molecular Genetics, 2010, Vol. 19, No. 18
presence of centrally nucleated myofibers [Fig. 3D and as
described previously (21)]. The histopathological abnormal-
ities present in both MDAFrtTA/TRECUGBP1 (+dox)
muscle and EpA960/HSA-Cre-ERT2(+tam) muscle are con-
sistent with a dystrophic process and are very similar to
those seen in patients with classical DM1. Some of these fea-
tures, in particular the abundance of central nuclei, are
observed much more frequently in DM1 than other muscular
dystrophies and therefore serve as a valuable diagnostic attri-
MDAFrtTA/TRECUGBP1 mice given food containing 2 g
dox/kg food induce CUGBP1 expression 8-fold above
endogenous levels but this level of expression was not sus-
tained beyond 2 weeks. In DM1 skeletal muscle tissue,
CUGBP1 levels are elevated 2–4-fold (8,9). We therefore
tested whether a lower dosage of doxycycline would provide
sustained elevation of CUGBP1 levels comparable to that
observed in DM1 skeletal muscle. MDAFrtTA/TRECUGBP1
mice induced by a low dosage dox diet (0.05 g dox/kg food)
had approximately a 2-fold increase in CUGBP1 levels in
the gastrocnemius muscle with stable protein expression for
up to 6 months (Fig. 4A). The muscle had no detectable
expression of the regeneration marker, eMHC, consistent
with an impaired regeneration response in DM1 patients
(Fig. 4A) (29,30). This low induction of CUGBP1 was not
enough to induce changes in alternative splicing (Supplemen-
tary Material, Fig. S3). However, MDAFrtTA/TRECUGBP1
mice with a 2-fold increase in CUGBP1 did have mild
muscle histology with scattered myofibers containing centrally
located nuclei, particularly by 4–6 months on dox diet, com-
pared with uninduced control mice (Fig. 4B). There was little
variability in myofiber size and few small regenerating myofi-
bers were present (Fig. 4B).
Overexpressing CUGBP1 in mouse skeletal muscle
reproduces misregulation of alternative splicing that is
characteristic of DM1
A distinctive molecular feature of DM1 skeletal muscle is the
misregulation of alternative splicing, such that there is
increased expression of embryonic splice variants in adult
tissues. Detailed analysis of developmentally regulated
alternative splicing events was previously conducted in
mouse heart tissue overexpressing
MBNL1 knockout mice (11). Whereas some events were
jointly regulated by both CUGBP1 and MBNL1, others
responded only to an increase in CUGBP1 or loss of
MBNL1, and are referred to as CUGBP1-responsive or
MBNL1-responsive events, respectively.
To determine whether MDAFrtTA/TRECUGBP1 (+dox, 2
weeks) mouse muscle recapitulated the misregulation of
alternative splicing observed in DM1, we analyzed 19 alterna-
tive splicing events previously demonstrated to be misregu-
lated in DM1 patient skeletal muscle (20,21,24,31,32). Nine
of the splicing events were significantly misregulated in skel-
etal muscle overexpressing CUGBP1, showing increased
expression of the embryonic splice variant in adult tissues
for all nine events (Fig. 5A). Splicing of H2afy exon 6 was
regulated by both CUGBP1 and MBNL1 in mouse heart
tissue, whereas three of the events (Ank2 exon 21, Capzb
exon 8 and Fxr1h exon 15) were previously demonstrated to
be CUGBP1-responsive events in mouse heart (11). Addition-
ally, we showed that these three CUGBP1-responsive events
are misregulated in induced EpA960/HSA-Cre-ERT2mouse
muscle which has increased expression of CUGBP1, but not
in HSALRor MBNL1DE3/DE3muscle without CUGBP1
up-regulation (21). Since these three alternative splicing
Figure 2. Induced MDAFrtTA/TRECUGBP1 mice exhibit reduced muscle
weight and muscle function. (A) Total mouse body weight at 1, 2, 4 and 8
weeks following CUGBP1 induction relative to the pre-induction weight.
Induced mice (open circles, n ≥ 5) have a significant reduction in body
weight when compared with uninduced control mice (closed circles, n ≥ 5),
∗∗P , 0.001. (B) Induced mice (open bars, n ¼ 8 muscles) have a significant
reduction in gastrocnemius muscle weight when compared with uninduced
control mice (closed bars, n ¼ 8 muscles),∗∗P , 0.001. Only male mice
were used for this assay and muscle weights were normalized to the length
of the tibia. (C) Mice were subject to an acute treadmill exercise regime at
each time point and the time to drop-off is represented in a box plot. The
top and bottom of each box denote the 75th and 25th percentiles, respectively,
and the median is shown by the bold middle line. Induced mice (red boxes,
n ≥ 4) have a significant reduction in muscle function at 1–4 weeks on dox
diet compared with uninduced control mice (black boxes, n ≥ 4), P , 0.001.
Human Molecular Genetics, 2010, Vol. 19, No. 183617
events change in response to CUGBP1 overexpression but not
MBNL1 depletion, up-regulation of CUGBP1 is essential for
at least some molecular features of DM1. Furthermore, these
data show that overexpression of CUGBP1 is sufficient to
reproduce some of the embryonic splicing patterns that are
observed in DM1 skeletal muscle.
The remaining 10 splicing events tested showed little or no
misregulation in MDAFrtTA/TRECUGBP1 (+dox) mouse
muscle (Fig. 5B). For four events (Titin exon Zr4, Capn3
exon 16, Nrap exon 12 and Dystrophin exon 78), there was
only a weak developmental regulation between newborn
limb and adult control in the mouse. One event (Sorbs1
exon 23) was previously characterized as a MBNL1-
responsive event in mouse heart (11) and was therefore not
expected to change in muscle overexpressing CUGBP1. Spli-
cing of Clcn1 exon 7a is well characterized in other DM1
mouse models and its splicing misregulation is responsible
for myotonia (15,16,18). Clcn1 exon 7a is not misregulated
in MDAFrtTA/TRECUGBP1 (+dox) muscle, and in agree-
ment with this result, six out of six mice tested by electromyo-
graphy did not show evidence of myotonia (data not shown).
Alternative splicing events that are misregulated in DM1
patients, but not in MDAFrtTA/TRECUGBP1 (+dox) mice
are likely MBNL1-responsive events or are effects secondary
to other disease processes.
The expression of CUG repeat RNA in individuals with DM1
induces alterations in two RNA binding proteins; namely, the
depletion of MBNL1 and up-regulation of CUGBP1. The role
of MBNL1 in disease pathogenesis has been well established
through the generation and characterization of MBNL1 knock-
out mice (23). Although MBNL1 knockout is sufficient for the
development of myotonia and dystrophic muscle histology,
MBNL1 knockout mice do not possess a strong muscle
wasting phenotype (23). More recently, transcriptome profil-
ing in MBNL1 knockout mice has revealed widespread
changes in transcription, RNA processing and mRNA decay
which largely overlap with changes in transgenic mice gener-
ated by the expression of CUG250RNA in the context of the
Figure 3. Induced MDAFrtTA/TRECUGBP1 mice exhibit DM1-like histological features. Light microscope analysis of gastrocnemius muscle stained with
hematoxylin and eosin. (A) Uninduced control mice have normal myofiber size and peripherally located nuclei. Scale bar: 20 mm. (B and C) Induced
MDAFrtTA/TRECUGBP1 mice have an abundance of central nuclei (arrows) and evidence of degeneration (small fibers with inflammatory infiltrate, double
asterisks) by 2 and 4 weeks on dox diet. (D) Skeletal muscles from EpA960/HSA-Cre-ERT2mice 4 weeks after the induction of DMPK-CUG960RNA show
similar changes as CUGBP1-overexpressing muscle, including hypotrophic myofibers with central nuclei (arrows) and degenerative changes (double asterisks).
All panels are 40× magnification.
3618Human Molecular Genetics, 2010, Vol. 19, No. 18
human skeletal actin gene (HSA-CUG250 RNA) (13,14).
Neither MBNL1 knockout mice nor transgenic mice expres-
sing HSA-CUG250 RNA have increased CUGBP1 levels.
These studies indicate that MBNL1 can reproduce some fea-
tures of DM1 independent of alterations in CUGBP1
So what is the role of CUGBP1, if any, in DM1 pathogen-
esis? CUGBP1 protein levels are increased in DM1 heart and
skeletal muscle (8,9,33), but whether this increase plays a
primary role in muscle degeneration or is simply an effect sec-
ondary to muscle damage is not known. Several studies using
transgenic mice support a pathogenic function of CUGBP1.
First, mice expressing DMPK-CUG5RNA in skeletal muscle
had elevated levels of CUGBP1 with no change in MBNL
expression or sequestration and showed characteristic DM1
histopathology, myotonia and splicing abnormalities (34).
Second, induction of DMPK-CUG960RNA in mouse skeletal
muscle resulted in a severe muscle wasting phenotype that cor-
related with an increase in CUGBP1, which has not been
mimicked by MBNL1 depletion alone (21). Third, within
6 h following induction of DMPK-CUG960RNA in mouse
cardiac muscle, CUGBP1 levels were increased and pharma-
cological inhibition of CUGBP1 up-regulation prevented the
DM1-like heart phenotype (35,36). Fourth, we recently
showed that 4-fold overexpression of exogenous CUGBP1 in
mouse cardiac tissue reproduced molecular, histopathological
and functional changes observed in DM1 mouse models and
individuals with DM1 (27).
In this paper, we tested the consequence of transgenic
expression of exogenous CUGBP1 in adult mouse skeletal
muscle. We showed that an 8-fold increase in CUGBP1
resulted in decreased muscle weight and impaired muscle
function. By histology, the muscle was reminiscent of DM1
muscle biopsies with a large fraction of myofibers containing
centrally located nuclei, pyknotic nuclear clumps and evidence
of muscle degeneration. We estimate that less than 10% of the
gastrocnemius muscle exhibits regeneration consistent with
the low expression of eMHC. In contrast, some of the misre-
gulated splicing changes assayed from gastrocnemius tissue
show nearly full reversion to the embryonic patterns. There-
fore, we conclude that the large extent of the molecular and
phenotypic changes cannot be explained solely based on non-
specific responses to muscle regeneration rather than direct
effects of CUGBP1 expression.
CUGBP2 and MBNL1 were also increased in MDAFrtTA/
TRECUGBP1 mice with an 8-fold increase in CUGBP1.
Although it is possible that elevated MBNL1 may have
some consequences on the skeletal muscle phenotype in
these mice, MDAFrtTA/TRECUGBP1 mice fed a low dose
dox diet with only a 2-fold increase in CUGBP1 did not
exhibit elevated MBNL1 and yet show histological abnormal-
A number of alternative splicing events misregulated in
DM1 skeletal muscle were shown to be regulated by
CUGBP1 and not MBNL1 in transgenic and knockout mice
(11). It is likelythat
CUGBP1-responsive events in DM1 tissues are a molecular
signature of elevated CUGBP1, independent of MBNL1
depletion. In this study, we showed that nine misregulated
alternative splicing events identified in DM1 skeletal muscle
are also misregulated in the MDAFrtTA/TRECUGBP1
(+dox, 2 weeks) mice with the expression of the embryonic
splice variants in adult muscle for all events. Many of the
affected transcripts have important functions in skeletal
muscle, and their aberrant splicing may have adverse conse-
quences on muscle integrity and function. It has already
been reported that alternative splicing of RyR1 exon 70
alters excitation–contraction coupling (19) and this exon is
misregulated in CUGBP1 overexpressing muscle. These data
suggest that down-regulation of CUGBP1 in DM1 mouse
models expressing DMPK-CUG repeat RNA even in the pres-
ence of MBNL1 depletion may ameliorate the disease pheno-
A progressive loss of inducibility occurred in the
MDAFrtTA/TRECUGBP1 mice fed 2 g dox/kg food for 8
weeks, possibly due to the induction of post-transcriptional
down-regulation by high CUGBP1 expression or increased
Figure 4. Long-term expression of CUGBP1 in MDAFrtTA/TRECUGBP1
mice induced with a low dosage dox diet (0.05 g dox/kg food). (A) Western
blot of protein extracted from gastrocnemius muscle shows a 2-fold
up-regulation of CUGBP1 in MDAFrtTA/TRECUGBP1 mice (two mice per
time point) fed a low dosage dox diet compared with uninduced controls
(one mouse per time point). The induction is stable over the 6-month time
course. There is no detectable expression of the regeneration marker,
eMHC. (B) Hematoxylin and eosin staining of gastrocnemius cross-sections
from low dosage dox-induced MDAFrtTA/TRECUGBP1 mice shows an
increase in myofibers containing centrally located nuclei (arrows) (20× mag-
Human Molecular Genetics, 2010, Vol. 19, No. 183619
doxycycline metabolism by the liver. The inability to main-
tain long-term induction of CUGBP1 was overcome using a
lower doxycycline dosage (0.05 g dox/kg food), but we
only achieved a 2-fold up-regulation of CUGBP1 by this
method. Although there were some mild histological features
in the MDAFrtTA/TRECUGBP1 mice induced with the
lower doxycycline dosage diet, there were not other features
of DM1 including misregulated alternative splicing. In DM1
tissue and cell cultures, CUGBP1 is stabilized by hyper-
phosphorylation (10). It may be possible that in addition to
increasing CUGBP1 steady-state levels, hyperphosphoryla-
tion alters the protein activity or function and this may be
necessary for mediating the effect of CUGBP1 on muscle
wasting at lower levels of the protein. Although this very
low, stable induction of CUGBP1 over a long time course
did not produce a severe muscle wasting phenotype, it may
more accurately represents the progressive nature of muscle
atrophy in DM1.
The results presented here demonstrate that aberrant
expression of CUGBP1 in adult skeletal muscle is pathogenic
and produces a phenotype reminiscent of DM1 skeletal
muscle. In the future, it will be important to determine the
alterations that occur downstream of CUGBP1 up-regulation
and which of those events are responsible for the specific
phenotypes observed in CUGBP1 overexpressing muscle
and individuals with DM1. Although some alternative spli-
cing and translational targets of CUGBP1 have already
been identified, global transcriptome profiling will undoubt-
edly identify new CUGBP1 targets with vital roles in skeletal
MATERIALS AND METHODS
TRECUGBP1 (line 3413) transgenic mice were generated as
previously described and maintained on an FVB background
(27). MDAFrtTA transgenic mice express a modified rtTA
from the myosin light chain 1/3 promoter/enhancer and were
maintained on a mixed C57BL/6× DBA background (28).
All mice used in these studies were F1 progeny from TRE-
CUGBP1 × MDAFrtTA matings. At 2–3 months of age,
bitransgenic MDAFrtTA/TRECUGBP1 mice were given free
access to food containing 0.05 g dox/kg food (Teklad Harlan
Inc., Madison, WI, USA) or 2 g dox/kg food (Bio-Serv,
Frenchtown, NJ, USA). Whenever possible, littermate TRE-
CUGBP1 or MDAFrtTA (+dox) or
CUGBP1 (–dox) controls were used.
Mice were placed in an AccuPacer treadmill with an electric
shock stimulus grid (AccuScan Instruments, Inc.) and muscle
function was assessed by a graded treadmill protocol. The
starting treadmill speed was 10 m/min and the pace was
increased by 2 m/min every 2 min until a maximum speed
of 30 m/min was reached. Exhaustion was declared the
third time a mouse spent 3–5 s on the shock grid without
returning to the treadmill. The experiment was stopped
immediately at the time of exhaustion or 30 min, whichever
Figure 5. Induced MDAFrtTA/TRECUGBP1 mice reproduce the characteristic alternative splicing misregulation observed in DM1 patients. (A) MDAFrtTA/
TRECUGBP1 (+dox, 2 weeks) mice have increased expression of the embryonic splice variants in adult gastrocnemius muscle for the indicated alternative
splicing events,∗P , 0.01 and∗∗P , 0.001. t-Test for statistical significance was performed between adult uninduced and induced mice. (B) Alternative splicing
events that are not significantly misregulated in induced MDAFrtTA/TRECUGBP1 gastrocnemius muscle. Newborn limb (n ¼ 1, pooled limbs from 12 mice),
adult uninduced (n ¼ 4, individual) and adult induced (n ¼ 4, individual).
3620 Human Molecular Genetics, 2010, Vol. 19, No. 18
Tissues were fixed overnight in 10% formalin and then stored
in 70% ethanoluntil sectioning.
paraffin-embedded and cut in cross-section at 10 mm. Hema-
toxylin and eosin staining was done according to the standard
procedures. Immunohistochemistry for slow twitch and fast
twitch myosin heavy chain isoforms was performed with
monoclonal anti-myosin slow clone NOQ7.5.4D (Sigma) and
monoclonal anti-myosin fast clone MY-32 (Sigma) antibodies.
RNA isolation and RT–PCR
Total RNA was isolated from skeletal muscle using TRIzol
reagent (Invitrogen) with the Omni TH homogenizer (Omni
International). RNA integrity was assessed on a formaldehyde
gel. cDNA was generated from 4 mg of RNA using oligo(dT)
and AMV reverse transcriptase (Life Sciences, Inc.). The PCR
program for all transcripts was 958C for 1 min 15 s (958C for
45 s, 578C for 45 s, 728C for 1 min), 25 cycles, 728C for
10 min. For the Serca1 and Cypher transcripts, the program
was modified to include only 20 cycles, and for the Clcn1 tran-
script, 26 cycles was used. All primer sequences were
designed to flank the alternative exon and are provided in Sup-
plementary Material, Table S1. After separation on a 5% non-
denaturaing acrylamide gel, band products were quantified
using the Kodak Gel Logic 2200 (Rochester, NY, USA) and
Molecular Imaging software. The percentage of exon inclusion
was calculated as [exon inclusion band/ (exon inclusion
band + exon exclusion band)] × 100 with a correction factor
for the amount of ethidium bromide bound per base pair of
Protein isolation and western blot
Skeletal muscle was subject to dounce homogenization in lysis
buffer [10 mM HEPES pH 7.5, 0.32 M sucrose, 5 mM MG132,
5 mM EDTA, protease inhibitor cocktail tablet (Roche) and
1% SDS]. Protein concentration from the supernatant was
determined with the BCA protein assay kit (Thermo Scienti-
fic). Protein samples (50 mg each) were separated on a 10%
SDS–PAGE gel and transferred to Immobilon-P membrane
(Millipore), after which the membranes were blocked and
probed with one of the following antibodies: CUGBP1 clone
3B1 (M. Swanson, 1:1000) and CUGBP2 clone IH2 (M.
Swanson, 1:500) followed by goat anti-mouse light chain
(Jackson ImmunoResearch, 1:10 000); MBNL1 (C. Thornton,
1:1000) followed by goat anti-rabbit (Calbiochem, 1:10 000);
eMHC F1.652 (Developmental Studies Hybridoma Bank,
1:200) and GAPDH (Abcam, 1:5000) followed by sheep anti-
mouse (Jackson ImmunoResearch 1:10 000).
All statistical analysis was performed with GraphPad InStat
software. When two groups were compared, a two-tailed Stu-
dent’s t-test with equal or unequal variance was performed as
appropriate, and the significance level was set at P-values less
than 0.05 for all statistical analyses. All data are expressed as
mean+standard error of the mean.
Supplementary Material is available at HMG online.
We thank Donnie Bundman for technical assistance in gener-
ating the TRECUGBP1 transgenic line and Xander Wehrens
(Baylor College of Medicine, Houston, TX, USA) for gener-
ous access to the treadmill apparatus. All histology was per-
formed by the Center for Comparative Medicine pathology
core facility at Baylor College of Medicine, with a special
thanks to Bilqees Bhatti for her expertise.
Conflict of Interest statement. None declared.
This work was supported by the National Institutes of Health
predoctoral NRSA fellowship (F31NS067740 to A.J.W.),
National Institutes of Health (R01AR45653, R01GM076493
to T.A.C.), the Muscular Dystrophy Association (T.A.C.),
and set-up funds from Texas A&M Health Science Center
College of Medicine (M.R.).
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