Neuromuscular Disorders 19 (2009) 241249

Contents lists available at ScienceDirect

Neuromuscular Disorders
journal homepage: www.elsevier.com/locate/nmd

Review

Mammalian animal models for Duchenne muscular dystrophy

Raffaella Willmann a, Stefanie Possekel b, Judith Dubach-Powell b, Thomas Meier b, Markus A. Ruegg a,*
a
b

Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

Santhera Pharmaceuticals, Hammerstrasse 47, CH-4410 Liestal, Switzerland

a r t i c l e

i n f o

Article history:
Received 9 October 2008
Received in revised form 24 November 2008
Accepted 27 November 2008

Keywords:
Duchenne muscular dystrophy
Animal models
Mouse
Dog
Cat
GRMD
mdx

a b s t r a c t
Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease that affects boys and leads to early
death. In the quest for new treatments that improve the quality of life and in the search for a possible
denitive cure, the use of animal models plays undoubtedly an important role. Therefore, a number of
different mammalian models for DMD have been described. Much knowledge on the molecular
mechanisms underlying the disease has arisen from studies in these animals. However, the use of different models does not often allow a direct comparison of results obtained in preclinical trials and therefore
hinders a straightforward translational research. In the frame of TREAT-NMD, a European Network of
Excellence addressing the fragmentation in the assessment and treatment of neuromuscular diseases,
we compare here the currently used mammalian animal models for DMD with the aim of selecting
and recommending the most appropriate ones for preclinical efcacy testing of new therapeutic
strategies.
2008 Elsevier B.V. All rights reserved.

1. Introduction
Neuromuscular disorders cover a broad range of diseases associated with progressive muscle weakness and/or neurodegeneration, and are frequently affecting patients at young age. Almost
all of these hereditary diseases have an incidence of less than 5/
10,000 and are often referred to as orphan diseases. In Duchenne
muscular dystrophy (DMD) the underlying genetic defects are
now well understood. However, current treatment options remain
limited. Challenges encountered in the attempt to develop efcacious therapeutic interventions are often based on the fact that
the gene defect per se cannot be corrected because of considerable
technical hurdles. Thus, alternative approaches including pharmacological treatment options or exon-skipping are currently under
evaluation as potential therapeutic strategies.
In contrast to most neuromuscular pathologies, several distinct
mammalian models are available for DMD. One of the goals of
TREAT-NMD, a European Network of Excellence, is to evaluate
the multitude of animal models, readouts and measurement protocols used in preclinical research with the aim of reaching a consensus within the international community as to which models,
readouts and protocols are most appropriate for efcacy testing
of potential new treatments. This will open the possibility to
design and conduct experiments that can be compared between

* Corresponding author. Tel.: +41 61 267 22 23; fax: +41 61 267 22 08.
E-mail address: markus-a.ruegg@unibas.ch (M.A. Ruegg).
0960-8966/$ - see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.nmd.2008.11.015

different laboratories thus allowing proof-of-concept studies. We

believe that these measures will increase the value of preclinical
data collected for regulatory lings with health authorities, and
will accelerate the preclinical stage of drug development.
In a rst step, animal models need to be thoroughly analyzed
and compared with a special emphasis on their suitability in
proof-of-concept studies. An appropriate animal model should allow a reliable prediction of the response to a given therapeutic
intervention in humans. In the case of genetic diseases, the underlying genetic defect in the animal model should be comparable to
the defect in patients. Moreover, a profound knowledge of all pathological characteristics of the animal model is crucial for its reliable
use. The present review addresses these points by providing a comprehensive overview of the mammalian models currently in use
and proposing a choice of animal models most appropriate for testing treatment options at the preclinical stage. For this reason, the
review will not discuss animal models created to study basic
mechanisms of the disease. Suggestions and guidelines to standardize readouts and assessment protocols in the selected animal
models (see also [1]) represent the next goals for TREAT-NMD
and will therefore be subject of future consensus papers.
2. Human pathology
DMD is an X-linked recessive disease that occurs with an incidence of 1 in 3500 boys. Human dystrophin, which is mutated in
DMD, was identied more than 20 years ago. Dystrophin is a
protein of 427 kDa encoded by a giant gene of more than 2.5 Mb.

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R. Willmann et al. / Neuromuscular Disorders 19 (2009) 241249

Fig. 1. The dystrophin gene and its protein products (adapted form [87]). There are seven promoters of the dystrophin gene. The corresponding protein product, Dp427, is
found in brain (B), muscle (M) and Purkinje neurons (P) and results in full-length dystrophin. The other promoters lead to shortened dystrophin. The C-terminal region,
expressed in all isoforms, contains protein domains that form the binding sites for some of the components of the dystrophin-associated protein complex: the WW domain
(WW), the cysteine-rich ZZ domain (ZZ) and the coiled coil domain (CC).

Its transcription is regulated in a tissue-specic manner by several

promoters (Fig. 1). In healthy muscle, dystrophin is located at the
subsarcolemma and interacts with a number of membrane
proteins forming the dystrophin glycoprotein complex (DGC) (for
review see [2]), whose function is to stabilize muscle cell membrane during cycles of contraction and relaxation. Moreover, several lines of evidence indicate a role of the DGC in the regulation
of mechanosensitive calcium channels, especially in the subsarcolemmal space, that inuence calcium homeostasis and signaling
(for review see [3]). About two thirds of all DMD patients have
large deletions in the dystrophin gene, mainly involving two hot
spots located around exons 4553 and exons 230. As a consequence, the essential C-terminal region that binds to the DGC is often truncated. In cases where portions of the central part of the
protein are missing but the C-terminal region is maintained, the
protein is partially functional. The resulting disease is milder and
dened as Becker muscular dystrophy [4]. In one third of DMD
cases, the disease is caused by small deletions or point mutations
that introduce a stop codon, resulting in very little or no dystrophin production [5], either because of transcript or protein
instability.
Absence of dystrophin, as encountered in DMD, leads to disintegration of the DGC, instability of the muscle cell membrane and
uncontrolled inux of calcium. This initiates a set of downstream
pathological processes that ultimately lead to disintegration of
muscle cell proteins, cell damage and progressive muscle wasting.
The subsequent regeneration causes the appearance of bers with
different sizes and with centralized nuclei. Later, muscles become
replaced by broblasts and fat cells, which results in collagen
deposition (brosis).
DMD patients typically appear normal at birth but display
elevated levels of creatine kinase in the blood. The disease
usually manifests at the age of 25 years when the delay in
reaching motor milestones (e.g. climbing stairs) becomes apparent. Later, pseudohypertrophy of the calf muscles and weakness
of proximal limb muscles develop, inevitably leading to wheelchair dependence. Respiratory insufciency and cardiomyopathy
are predominant factors contributing to early morbidity and
mortality.

3. Mouse models for DMD

3.1. mdx mouse
A naturally occurring dystrophin-decient mutant was rst described in 1984 in a colony of C57BL/10 mice (C57BL/10ScSnJ) [6]
and has since been referred to as the mdx-mouse. This mouse,
now called C57BL/10ScSn-Dmdmdx/J, is readily available from commercial breeders and thus widely used in basic and translational
research. It carries a point mutation in exon 23 of the mouse dystrophin gene introducing a premature stop codon, which leads to
the absence of full-length dystrophin. This type of mutation accounts for approximately one third of the mutations found in
DMD patients (see above).
Mdx mice have a slightly shorter life span as compared to wildtype controls [7] (Table 1). The muscle degeneration in mdx mice
differs from DMD patients in that it appears in waves and is not a
continuum. At rst, a marked degeneration and regeneration of
muscle bers is observed at a young age (24 weeks [8]), which results in an increase in the number of newly differentiating myobers characterized by centralized nuclei and an increased
heterogeneity in myober size. In parallel, necrosis is observed in
muscles at this early stage [9,10] while it decreases again after 60
days [11]. Subsequently, loss of muscle tissue is slow and general
muscle weakness in cage-reared animals is not evident until later
in life [12]. Fibrosis in most mdx mouse muscles is less pronounced
than in DMD patients with the exception of the diaphragm muscle
[1315]. Although absolute muscle force of limb muscles remains
comparable to unaffected mice, the relative force normalized to
body weight decreases by approximately 20% and 50% at the age
of 810 weeks and 4 months, respectively [10,14,16]. However, others could not conrm this nding [17,18], which is probably based
on variations in the mice strains (e.g. body weight, cooperation of
the animals in force measurement assays) and on different experimental procedures. Similarly, a reduced force of contraction has
also been reported in isolated muscle preparations although the
lack of standardized conditions makes it again difcult to compare
the results obtained by different groups [1922]. These discrepancies clearly show that the availability of standardized experimental

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R. Willmann et al. / Neuromuscular Disorders 19 (2009) 241249

Table 1
Qualitative and quantitative measurements in mdx mouse model
Parameter
General
Life span

Clinical signs at onset

First signs of pathology
First signs of cardiomyopathy
Breeding
Kyphosis
Body weight
Muscle Physiology
Fore-limb strength (g)

Fore-limb strength per body weight (g

strength/g BW)

Twitch force (mN)

Normalized twitch force (mN/mm2 CSA)

Tetanic force (mN)

Normalized tetanic force (mN/mm2 CSA)

Muscle Physiology
Force drop after eccentric contractions

Force drop after fatigue protocol

Normalized peak force (mN/mm2 CSA)
Biochemistry
CK

Hydroxyproline (brosis marker)

Intracellular [Ca2+]

Respiratory Physiology
Respiratory rate
Response to hypercapnia/hypoxia

Parameter

mdx

mdx
reduced by 17% in females and 19% in
males [7]
23m [65]
CK levels increase, necrosis,
regeneration
2-3w
6m
normal
9m [22]
normal, increases after 8w [14]
drops by 25% after 6m [65]
12w: 35g [10,14,68]
4w: 95g;
8-12w: 156g [17]
10w: 30g [10]
4w: 103g;
8-12w: 138g [17] r
1.5 at 8w [14]
1.6 at 3w, 10w [69]
1.1 at 10w [10]
75 at 4m (whole body) [16]
5.34 at 4w, 6.5 at 8w [70]
4.59 at 4w, 5.07 at 8w [70]r
Edl, 16w: 130 [72]
Edl, 8w: 12 [20]
Sol, 8w: 12 [20]
Stm, 8w: 50 [46]
Edl, 10w: 50 [19]
Sol, 10w: 40 [19]
Dia, 10w: 20 [19]
Dia, 7w: 5.8 [22]
Stm, 8w: 6.6 [46]
Edl, 8w: 82 [20]
Edl, 90d: 50.2 [16]
Edl, 16w: 370 [72]
Edl, 8w: 52 [20]
Sol, 8w: 57 [20]
Edl, 10w: 170 [20]
Edl, 6w: 200 [21]
Sol, 10w: 200 [19]
Dia, 10w: 130 [19]
Dia, 7w: 32 [22]
Edl, 3-32w: decreased [74]
Edl, 90d: 147 [16]
Edl, 16w: 72% [21]
Edl, 90d: 69% [16]
Edl, 10w: -41% [19]
Sol, 10w: 75% [19]
Edl: 272 [19]
1200 U [42]
5000 U [16]r8000 U/l [15]r10000/
12000 U/l [17,40]
Dia, 24w: 19 lg/mg [75]
Dia, 7w: 2.5 lg/mg [22]
Int, 7w: 0.9 lg/mg [22]
normal in Fdb, Sol at 4-9w (in [76])
normal in isolated Fdb bers [31]
60 lM (higher) in Edl at 8-12w [18]
80 nM (2x higher) in Edl at 8-12 w [18]
r

Ca2+ sparks after hypotonia

Parvalbumin

Table 1 (continued)

Fdb: irreversible at 4w [77]

fast muscles: mRNA increases [32]
TA, 24w: 9 lg/mg [33]
Gas, 24w: 7 lg/mg [33]
Sol, 24w: 50 ng/mg [33]
Edl, 32w normal levels [74]
altered at 16m [23]
reduced at 7m [78]

Histology
% of centronucleated bers

Histology
Fiber size

Fibrosis

Necrosis

Fiber type composition

Cardiac Physiology
Dilated cardiomyopathy (LVEDD/LVESD in mm by
echocardiography)

Heart rate

Force output (mN/mm2)

Heart brosis (% of area)

Hydroxyproline content
ECG
Left ventricular developed pressure (mm Hg)
Rate of pressure development (mm Hg/s)
Rate of relaxation (mmHg/s)

TA, 7w: 50% [79]

Dia, 7w: 25% [79]
Sol, 7w: 56% [79]
hind-limb, 4-14w:
average 35% [8]
Edl, 6w: 58%;
TA, 90d : 75% [16]
Edl, 10-12w: 64% [80]
TA, 10-12w: 74% [80]
Dia, 10-12w: 60% [80]
Fdb, 12w: 25% [81]
Gas, 8-10w: 50% [40] r
TA, 3w: 302 [79] t
Dia, 3w: 254 [79] t
TA, 7w: 403 [79] t
Dia, 7w: 433 [79] t
Sol, 7w: 267 [79] t
Dia at 16m [13]
Glut at 24w [14]
TA, 8-13w: 6% [15]
TA, 8-13w: 9% [15] r
TA, 3w: 20% [9]
Edl, Bic, 10w: 5% [10]
TA, Sol, Edl: peaks at 3060d [84],
decreases after 60d [11]
Gas, 8-10w: 20% [17] r
Edl: 50% I, 50% IIA [20]
Edl: 80% IIB, 20% IIX [19]
Sol: 65% IIA, 35% I [19]
Dia: 90% IIX, 5% IIB [19]
TA 90d: 57% IIB [16]
3.6/2 at 42w (increased)
[24]
3.7/2.6 at 24m (no
change) [29]
3.9/2.9 at 9-10m [27]
3.83 at 10m [30]
decreased, 612 bpm at
42w [24]
460 bpm at 9-10m [27]
454 bpm at 10m [30]
25 at 8w [85]
7% at 7m [24]
8% at 12m [29]
12% at 24m [29]
1.68% at 11w [41]
6.23% at 11w [41] r
0.7-3.4% at 10m [30]
4.5 lg/mg at 15m [28]
4 lg/mg at 12-14m [26]
abnormal at 6 and 12m
[25]
80 at 15m [28]
2500 at 15m [28]
2000 at 15m [28]

Abbreviations used are: Bic: biceps, Dia: diaphragm, Edl: extensor digitorum longus,
Fdb: exor digitorum brevis, Gas: gastrocnemius, Glut: gluteus, Int: intercostals, Pl:
plantaris, Sol: soleus, Stm: sternomastoid, TA: tibialis anterior. d: days; w: weeks; m:
months; y: years; BW: body weight; CSA: cross sectional area; CK: creatine kinase;
LVEDD; Left ventricular end diastolic diameter; LVESD: left venticular end systolic
diameter; bpm: beats per minute.
r exercised mdx.
s after 5th elongation [19].
t measured as the minimal Ferets diameter, units in variance coefcient (vc) [79].

protocols would be of advantage both to elucidate basic aspects of

the pathology and to test emerging therapeutic strategies. Respiratory pathology is evident in 16-month-old mice but not in 4month-old mice, showing a worsening of respiratory capacity with
age, in line with the increased histological damage of respiratory

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muscles [23]. Abnormal cardiac function is also seen in the mdx

mouse sharing several characteristics of DMD-associated cardiomyopathy [24]. For example, changes in the electrocardiogram
(ECG) and echocardiogram have been reported in mice that are older than 6 months [2527]. This change in cardiac function is paralleled by an increase in myocardial brosis and the occurrence of
foci of myocardial necrosis and inammation [24,2830]. Mdx mice
have served as a model for several studies on calcium homeostasis
and alterations thereof. Basal cytosolic calcium levels were reported to be identical in mdx and wild-type mice [31] showing a
very robust calcium homeostasis, possibly due to elevated expression of the calcium-buffer parvalbumin [32,33] (see Table 1). Nevertheless, [Ca2+] in the subsarcolemmal compartment [34] and in
the sarcoplasmic reticulum [35] was increased and levels of inositol
1,4,5-triphosphate (IP3) and of the IP3 receptor were higher in
mouse and human dystrophic cell lines [36].
Muscle pathology in the mdx mouse can be aggravated by
forced exercise, a method introduced to allow a better evaluation of the efcacy of experimental therapies. Several exercise
paradigms have been described [3739]. Muscle damage of exercised mdx animals is increased as reected by an increased number of muscle bers with centralized nuclei and increased
brosis [17,40] (Table 1). Plasma creatine kinase levels are further elevated, calcium homeostasis is more severely impaired
and fore-limb strength is generally reduced [15,17,18,40] while
body weight remains unchanged. Cardiomyopathy has been described for the exercised mdx mouse [41], however, a systematic
analysis of the impact of exercise on cardiac pathology is still
lacking.

3.4. mdx double mutants

3.2. mdx2Cvmdx5Cv

3.4.2. mdx; MyoD/

MyoD is a myogenic regulatory factor that plays an important
role during muscle differentiation. Therefore, MyoD/ mice were
crossed with mdx mice in an attempt to obtain mice with a reduced
capacity to regenerate [47]. Mutant mdx; MyoD/- mice uniformly
display marked muscle dystrophy. Specically, by 35 months of
age, mdx; MyoD/ mice develop a kyphosis/scoliosis of the spine
similar to DMD patients and an abnormal waddling gait (Table
2). The animals become progressively less active with concomitant
weight loss and they die prematurely at 12 months of age [47].
Moreover, they develop severe cardiomyopathy at the age of 10
months with foci of necrosis that are not observed in MyoD/ mice
[48] and therefore this mutant has been proposed as a model for
DMD-associated cardiomyopathy.

In 1989, a series of mdx variants (termed mdx2Cv, mdx3Cv, mdx4Cv,

and mdx5Cv) were recovered from male mice (C3H.X25  C57BL/
6Ros) treated with the chemical mutagen N-ethyl-nitrosourea that
were crossed with C57BL/10Sn.mdx female animals [42]. The mice
display muscle pathology and elevated serum creatine kinase levels that do not differ from the original mdx mutant (Table 2). Muscle cross-sections showed degeneration/regeneration, centrally
nucleated bers, necrosis, cellular inltration and brosis in the
diaphragm [42]. The variation in ber size was found to be larger
than in mdx mice [43]. Pseudomyotonia, detected by electromyography, and some brosis were detected in the hearts of mdx2Cv and
mdx3Cv at the age of 4 weeks [42]. The mdx3Cv strain also displays an
abnormal breeding phenotype with reduced neonatal survival [43].
The pathology in this series of mice (mdx2Cvmdx5Cv) at older age
(>6 months) is not reported.
3.3. mdx52
To create a mouse model with large deletions in the dystrophin gene like those found in two thirds of human patients, exon
52 was disrupted on a C57BL/6J background [44]. The mice do
not show skeletal muscle weakness or behavioral changes until
18 months of age. At 4 months of age, tibialis anterior and extensor digitorum longus muscles of the mutant mice are paler and
about 1.5 times larger than corresponding wild-type muscles
(Table 2). Fibrosis is evident in diaphragm, soleus and extensor
digitorum longus. A marked variation of ber size with some
necrotic spots is also observed at 4 months and centronucleated
bers make up 90% of the whole bers at this age. Although
heart and brain are partially decient for dystrophin, no pathological changes are observed in these organs. Overall, the dystrophic phenotype of 4-month-old mdx52 mice is similar to
age-matched mdx mice; no data are, however, available on
disease progression with age [44].

3.4.1. mdx; utr/

In an attempt to aggravate the pathology of the mdx model and
to study utrophin-based gene therapy strategies, utrophin mutant
mice (on C57B5/129J background) were crossed with C57BL/10
mdx mice to obtain double knock-out mice [45,46]. Mice lacking
both dystrophin and utrophin share many phenotypical hallmarks
with DMD. The mice have a markedly reduced life span of 420
weeks, severe muscle weakness with joint contractures, kyphosis
and a pronounced growth retardation that starts around weaning
(Table 2). Necrotic bers and connective tissue are visible in the
diaphragm already 6 days after birth and centrally nucleated bers
are evident at 2 weeks. Interestingly, the diaphragm is not more affected than that of the mdx mice, suggesting that utrophin does not
compensate for the lack of dystrophin in this muscle [45,46]. The
skeletal muscles appear normal at birth but show rst signs of
degeneration/regeneration already after 2 weeks. Although the
dystrophic pathology is similar to mdx mice at 45 weeks of age,
it then remains severe in contrast to the improvements seen in
mdx mice. By 10 weeks of age, brosis is evident in tibialis anterior
muscle. Occasionally, larger necrotic areas are observed in the
heart [46]. The neuromuscular and the myotendinous junctions
in mdx; utr/ mice show a marked reduction in membrane folds,
presumably as a result of the lack of utrophin [45,46]. Therefore,
both the disease progression and the phenotype of these double
mutant mice resemble the progression of muscle dystrophy in
DMD patients and the interstitial brosis of skeletal muscle lends
itself to assess the efcacy of anti-brotic treatments.

3.4.3. mdx; adbn/

Mdx mice that lack the DGC protein a-dystrobrevin were created to investigate the role of a-dystrobrevin in signaling and in
conferring muscle stability [49]. The double mutant mice have a
reduced life expectancy of about 810 months and show moderate
dystrophic changes including necrotic bers, mononuclear inltrates, ber size variability and brosis (Table 2). The percentage
of centronucleated bers in the tibialis anterior muscle is higher
than 90% and some cardiomyopathy was also observed. In general,
these mice show a more severe pathology than mdx mice but not as
severe as that of mdx; utr/ mice.
3.4.4. mdx; a7 integrin/
Several lines of evidence indicate that a7b1 integrin, the main
integrin expressed in skeletal muscle, acts in synergy with the
DGC to connect the extracellular matrix to the cytoskeleton [50].
To test this, a7 integrin and dystrophin double mutant mice were
generated [51]. The double knockout mice show no phenotypic difference from wild-type and mdx at birth, but their pathology worsens rapidly and they die prematurely at 2428 days of age (Table
2). Mice start to lose weight at 15 days of age and show a marked

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R. Willmann et al. / Neuromuscular Disorders 19 (2009) 241249

Table 2
Qualitative and quantitative measurements in mouse models other than mdx
Parameter

mdx2Cv-5Cv

mdx52

mdx; utr-/-

mdx; myoD-/-

mdx; adbn-/-

mdx; a7 integrin-/-

mdx; PV-/-

General
Life span
Clinical signs at onset

nd
nd

nd
nd

24-28d [51]
nd

1y [16]
nd

18m [44]
none [44]
nd

12m [47]
waddling gait,
less active [47]
nd
nd
nd

8-10m [49]
nd

nd
nd
abnormal in
mdx3Cv [43]
nd
nd

6-20w [45,46]
small size, joint
contractures [14]
at birth
8 w [46]
none

nd
nd
nd

nd
nd
nd

nd
nd
nd

nd
nd

present [66]
14 g at 4w, 18 g at
8w [46,66]

3-5m [47]
nd

nd
nd

18d [51]
reduced after 15d
compared to mdx
[51]

nd
nd

nd
nd

nd
nd

250 g at 8w [14]
1.8 at 8w [14]

nd
nd

nd
nd

nd
nd

nd
nd

nd
nd

nd
nd

nd
nd

nd
nd

nd

nd

nd

nd

nd

Edl, 90d: 113.6 [16]

nd
Force drop after eccentric
contraction s
Force drop after fatigue protocol nd
nd
Normalized peak force (mN/
mm2 CSA)

nd

nd

nd

nd

Edl, 90d: 54.3% [16]

nd
nd

Stm, 8w: 30 [46]

Edl, 9w: 5 [73]
Edl, 10w: 40 [73]
Sol, 10w: 25 [73]
Dia, 10w:15 [19]
Stm, 8w: 4.9 [46]
Edl, 10w: 130 [19]
Sol, 10w: 100 [19]
Dia, 1w: 90 [19]
Edl, 9w: 65% [19]
Edl, 10w: -76% [19]
Sol, 10w: 50% [19]
Edl, 217 [19]

nd
5.1 at 4m (whole
body) [16]
nd
Edl, 90d: 39.8 [16]

nd
nd

nd
nd

nd
nd

nd
nd

First signs of pathology

First signs of cardiomyopathy
Breeding
Kyphosis
Body weight

Muscle physiology
Fore-limb strength (g)
Fore-limb strength per body
weight (g strength/g BW)
Twitch force (mN)
Normalized twitch force (mN/
mm2 CSA)

Normalized tetanic force (mN/

mm2 CSA)

Biochemistry
CK

nd

elevated

nd

nd

nd

8000U [16]

Intracellular [Ca2+]

1200-6000 U
[42]
nd

nd

nd

nd

nd

nd

Fdb bers at 90d: 69.8

nM [16]
Gas at 90d: 9 lmol/g
dry weight [16]

Respiratory physiology
Respiratory rate

nd

nd

nd

nd

nd

higher [51]

nd

Histology
% of centronucleated bers

yes [42]

present [47]

TA, 1m: 90%

[49]

Pl 10 and 20d: 2.5%

and 10% [51]

TA, 90d: 87% [16]

Fiber size

nd

Fibrosis

some [42]

hind-limb, 15d :
variations [51]
hind-limb from day
15 [51]

Edl, 90 and 180d :

hypertrophy [16]
nd

Necrosis

nd

Necrotic/regenerative areas

yes [42]

Fiber type composition

nd

Edl, TA, Dia, 4.5m: Dia, 8w : 40% [45]

90% [44]
TA, 35d: 75% [82]
TA, 10w: 90% [46]
Edl, 4w: 30% [66]
Edl, 8w: 70% [66]
Edl, 9w: 90% [73]
Sol, 4w: 60% [66]
Sol, 8w: 70% [66]
Dia, *: 65% [83]
Edl, *: 85% [83]
TA, *: 80% [83]
*
: age unknown
TA & Edl, 4m:
nd
Hypertrophy [44]
Dia & TA 4.5m:
Sol, 8w: extensive
minimal [44]
[66]
Sol, Bic, Glu, 24w:
extensive [14]
TA, 10w: present
[46]
Dia, TA present at begins at 2w [46]
4.5m [44]
present in Dia at 6d
[45]
nd
Edl, 4w: 12% [66]
Edl, 8w: 8% [66]
Sol, 4w: 25% [66]
Sol, 8w: 20% [66]
nd
Edl: 65% IIB, 35% IIX
[19]
Sol: 55% IIA; 45% I
[19]
Dia: 80% IIX, 5% IIB
[19]

attenuated
nd
hypertrophy [47]
nd
nd

nd

TA, 1m: small nd

areas [49]

nd

nd

nd

nd

nd

nd

nd

nd

TA, 90d: 46% IIB [16]

(continued on next page)

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R. Willmann et al. / Neuromuscular Disorders 19 (2009) 241249

Table 2 (continued)
mdx2Cv-5Cv

mdx52

mdx; utr-/-

mdx; myoD-/-

mdx; adbn-/-

mdx; a7 integrin-/-

mdx; PV-/-

Pseudo-myotonia [42]

nd

nd

5m [48]

nd

nd

nd

nd

nd

nd

Force output (mN/mm )

nd

nd

nd

nd

nd

nd

Heart brosis (% of area)

nd

nd

prominent
8w [46]
15 at 8w
[85]
nd

none at 20-24d
[51]
evident at 20-24d [51]

evident at 10m
in 50% [48]

nd

nd

nd

Parameter
Cardiac physiology
Dilated cardiomyopathy (LVEDD/
LVESD in mm by echocardiography)
Necrosis (histological)
2

nd

Abbreviations used are: Bic: biceps, Dia: diaphragm, Edl: extensor digitorum longus, Fdb: exor digitorum brevis, Gas: gastrocnemius, Pl: plantaris, Sol: soleus, Stm: sternomastoid, TA: tibialis anterior. d: days; w: weeks; m: months; y: years; BW: body weight; CSA: cross sectional area; CK: creatine kinase; LVEDD: Left ventricular end diastolic
diameter; LVESD: left ventricular end systolic diameter; nd: not determined.
s after 5th elongation [19].

kyphosis at 18 days of age. Fiber size variation and brosis is observed in hind-limb muscles from day 15 onwards and a pervasive
dystrophy develops in the following days. Histological analysis
shows that exor muscles are generally more affected than extensor muscles. Interestingly, in these muscles a loss of ber number
and a general size reduction is observed together with a low number of centronucleated bers, suggesting that impaired regeneration may be responsible for the loss of muscle mass. Mice
eventually die of respiratory failure and cardiomyopathy. The
aggravated pathology of mdx; a7 integrin/ mice suggests that
the ne tuning of a7 integrin levels may be responsible for the different clinical severity of dystrophin deciency in human and
mouse [51]. In conjunction with the nding that overexpression
of a7 integrin in mdx mice ameliorates the disease [52], these data
indicate a compensatory function of a7 integrin in dystrophindecient muscle.

were crossed with PV knockout (PV/) mice [16]. Such mdx; PV/
mice are very similar to mdx mice (Table 2). The extensor digitorum
longus muscle shows a marked hypertrophy at 90 and 180 days
that exceeds that observed in mdx. Moreover, the decrease in the
number of type IIb bers and the increase in the number of centronucleated bers are more pronounced in mdx; PV/ mice than in
mdx mice. In addition, muscles from 90-day-old double mutants
are weaker than those from mdx when tested in vivo and in vitro.
In summary, although the high levels of PV in mdx mice may contribute to the rather mild pathology, it is not sufcient to explain
the difference to DMD patients.

3.4.5. mdx; PV/

Compared to DMD patients and dog models thereof, mdx mice
show a relatively mild pathology. To test whether this difference
might be due to the high concentrations of the potent calcium-buffer parvalbumin (PV) in the skeletal muscles of rodents, mdx mice

In the golden retriever, the best characterized dog model, the

disease results from a single base pair change in the 30 consensus
splice site of intron 6, leading to skipping of exon 7 and alteration
of the reading frame in exon 8, which creates a premature stop
[53]. Unlike the dystrophin-decient mdx mouse, GRMD dogs suf-

4. Canine models for DMD

4.1. Golden retriever (GRMD)

Table 3
Qualitative and quantitative measurements in the Golden Retriever Muscular Dystrophy (GRMD) dog model
Parameter

Severe/less severe

General
Life span
Clinical signs at onset
First signs of pathology
First signs of cardiomyopathy
Breeding
Kyphosis
Body weight

10d [55]/1-3y, in rare cases up to 6y [55]

weak, dehydrated, hypothermic [55]/inability to open claw, less active, abnormal gait [55]
at birth/6-9w [55]
nd/1 y or more [55]
none/normal [55]
nd/6m [55]
reduced at all ages [55]/ reduced at all ages [55,67]

Muscle physiology
Fore-limb strength per body weight (g strength/kg BW)
Twitch force (N)
Normalized twitch force (N/kg BW)

nd/hind-limb, 3m: 5.8 [71]

nd/hind-limb, 6m: 0.8 (exion), 1.57 (extension) [67]
nd/hind-limb, 6m: 0.064 (exion), 0.112 (extension) [67]

Muscle physiology
Tetanic force (N)
Normalized tetanic force (N/kg BW)

nd/hind-limb, 6m: 5.89 (exion), 12.82 (extension) [67]

nd/hind-limb, 6m: 0.47 (exion), 0.965 (extension) [67]

Biochemistry
CK

100-500 x normal level at 1-2d [55]/increased from 3w; 6-8w: 100 x normal level [55]

Histology
Fiber size
Fibrosis
Necrosis

nd/trunk, face muscles, 9-12w: atrophy [55]

nd/in prox. muscles at 14 m [55] in cranial sartorius at 5 m [56]
severe, many muscles [55]/nd

Cardiac physiology
Dilated cardiomyopathy (LVEDD/LVESD in mm by echocardiography)
Heart brosis (% of area)

nd/34/20 at 25w [58]

nd/6.3% collagen at 25w [58] prominent at 1-1.5y [86]

Abbreviations used are: d: days; w: weeks; m: months; y: years; BW: body weight; CK: creatine kinase; LVEDD: Left ventricular end diastolic diameter; LVESD: left
ventricular end systolic diameter; nd: not determined.

R. Willmann et al. / Neuromuscular Disorders 19 (2009) 241249

fer from a rapidly progressing, fatal disease similar to DMD. There

is, however, a large variation in disease severity as some pups survive only for a few days, while others are ambulant for months or
even years [54]. To account for this variation, we distinguish
between the severe and the less severe pathology in Table 3.
Incomplete muscle repair in GRMD results in progressive weakness
and gait abnormalities at the age of 69 weeks leading to muscle
atrophy, brosis and contractures by 6 months (Table 3). The
growth of affected pups is delayed and their walking ability is impaired. The dogs become less active than non-affected littermates
at about 9 months of age. At this time, their gait is still uncoordinated and their limbs are abducted [55]. Severely affected dogs
have difculties to rise and can walk only a few steps. At the age
of 12 months, pharyngeal and esophageal dysfunctions start to develop and respiratory capacity is decreased. Kyphosis develops by
the 6th month of age. Moreover, as seen in DMD patients, GRMD
dogs display selective muscle involvement although the most affected muscles in dogs (i.e. tongue, masticatory and trunk muscles)
are different to those of humans [56]. Myocardial involvement is
much more evident in the golden retriever than in other animal
models, matching very closely the cardiac complications encountered in DMD patients. Atrophy already appears during the neonatal period [55], although histological changes are generally not
present up to 3 months of age [57]. Systolic dysfunction and left
ventricular dilation becomes apparent after several months or
years [57], although Tissue Doppler Imaging has allowed the detection of severely impaired systolic and diastolic indices in young
dogs (25 11 weeks) [58]. Recently, NMR techniques have been
applied to assess differences between dystrophic and healthy muscles in the dog, a method that may be useful to monitor a potential
therapeutic response [59].
4.2. Beagle (CXMDJ)
Beagle dogs were mated with an affected golden retriever to obtain a breed of a smaller size, which is an advantage for some therapeutical approaches. One of these strains has been established in
Japan (CXMDJ [60]). GRMD and CXMDJ present very similar phenotypes. The survival rate of beagle dogs carrying the dystrophin
mutation is increased compared to golden retriever and pup mortality is low, although it tends to increase after a few generations.
In general, large breed dogs develop more severe clinical signs than
small breed dogs [55]. The diaphragm muscle is affected shortly
after birth while in limb muscles abnormalities become evident
only after 2 months of age [61]. Cardiac involvement in the CXMDJ
dogs is milder and has a slower progression than that described for
GRMD dogs. Although the CXMDJ model has not been widely used
and thus is not yet characterized in detail, it would seem to be an
attractive alternative to the golden retriever, particularly because
of its improved survival rate and because the beagle dog is the species of choice for non-rodent toxicology studies in drug
development.
4.3. Feline models for DMD
The only other natural dystrophin deletion described in a mammalian model occurs in the dystrophic cat, which lacks approximately 200 kb of the dystrophin gene [62]. The hypertrophic
feline muscular dystrophy (hfmd) animal model is characterized
by increased levels of creatine kinase in the blood at the age of
45 weeks although muscle involvement becomes apparent only
at the age of 1014 weeks. The name of the disorder reects the
development of extensive muscle hypertrophy. The cats eventually
die due to compression of the esophagus by the hypertrophied diaphragm or because of impaired water intake caused by glossal
hypertrophy [63], which then leads to renal failure. Histological

247

analysis has revealed the presence of local foci of necrosis and

regeneration but no signs of brosis were seen [63]. Although cardiac failure is rare, the hearts are hypertrophic in 9-month-old cats
[64]. Thus, the pathology of hfmd cats does not resemble DMD as
closely as the GRMD dog because it lacks the hallmarks of generalized muscle wasting.

5. Conclusions
Several aspects must be considered when evaluating the animal
models for use in the preclinical testing of potential new treatment
options. The following criteria play a signicant role:
 The genetic basis of the disease should be the same both for
humans and the animal model.
 The model should reiterate key hallmarks of the human disease.
For DMD, these include: muscle weakness, respiratory insufciency, cardiomyopathy, histological changes in skeletal muscle
(e.g. centralized nuclei, ber size variations).
 The animals should be readily (i.e. commercially) available, easy
to handle and maintainable in standard laboratory housing
conditions.
 Disease progression should be well characterized and reference
values should be available in the scientic literature to allow a
precise outcome analysis.
 The phenotype should be robust and reproducible over generations to allow the use of standard readout parameters and
ensure comparability of outcomes.
Taking all these aspects into consideration, double mutant mice
like mdx mice decient for MyoD, utrophin, parvalbumin or a7
integrin do not resemble the genetic background of DMD patients
and are therefore less appropriate to predict therapeutic effects.
Mdx2Cv-mdx5Cv, mdx52, the beagle and the cat models, while bearing the genetic defect found in patients, are not yet characterized
in detail and the available literature is limited; moreover, the
pathology observed in mdx52 and in cats is milder and differs considerably from the human disease. Therefore, the mdx and GRMD
mammalian model should be considered currently as the best suited animal models for preclinical therapy testing.
The mdx mouse model has considerable advantages, particularly because of the large number of papers published on this animal model resulting in a good understanding of muscle, cardiac
and respiratory pathology. Moreover, the reproducibility of the
phenotype, the commercial availability and the relatively low costs
of maintenance are further important advantages. Finally, this animal model has been used to demonstrate efcacy of several potential treatment strategies including small molecule pharmaceuticals
as well as gene correction and cell replacement approaches.
One important drawback of GRMD is the high degree of variability in disease severity between breeds and among littermates
although they all carry the same genetic mutation. Therefore, special care must be taken in choosing the control animals and control
experiments. Moreover, a rather high number of animals is required to substantiate any conclusions about the treatment efcacy. Additional disadvantages are the high expense of breeding,
the necessity for a veterinary facility and the large amount of compound required to treat large-sized animals. However, dog models
closely resemble the human disease, are beginning to be appropriately characterized and have already been used in a few tests on
treatment efcacy.
Therefore, we recommend the mdx mouse as the model of
choice for preclinical tests and proof-of-concept studies, whereas
we recommend the use of GRMD in well controlled experimental
settings, when using complementary tests, such as those used in

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R. Willmann et al. / Neuromuscular Disorders 19 (2009) 241249

supportive therapy for respiratory and cardiac functions, or as an

alternative to mdx for specic experimental therapeutic approaches such as the use of stem cells or the testing of viral
vectors.
Acknowledgements
This manuscript was compiled within the frame of Activity 7
Accelerate preclinical phase of new therapeutic treatment development of the European Network of Excellence TREAT-NMD
(Contract No. 036825). We thank our network partners as well as
the collaborators of the Club of Interest. In particular, we are grateful for input and comments on the manuscripts to: Annemieke
Aartsma-Rus, Reginald Bittner, Stephan Blot, Serge Braun, Kay Davies, Annamaria De Luca, Patrice Denee, Arnaud Ferry, Jean-Marie
Gillis, Herve Laouenan, Hanns Lochmller, Marie Montus, JeanMarc Raymackers, Volker Straub, Christian Thirion, Jon Tinsley
and Maaike van Putten.
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