Download Progressive deterioration of muscles in mdx mice

Clinical Science (1993) 84, 145-150 (Printed in Great Britain)
I45
Progressive deterioration of muscles in rndx mice induced
by overload
J. DICK and G. VRBOVA
Department of Anatomy and Developmental Biology, University College London, London, U.K.
(Received 15 ]une/7 September 1992; accepted 22 September 1992)
1. Extensor digitorum longus muscles of C57 BL/10
and mdx mice were overloaded by removing the
synergist tibialis anterior muscle of 9-12-day-old
animals. The effect of this operation on the weight,
contractile properties and force of the extensor digitorum longus muscle was examined at two different
ages, i.e. at 2-3 months (young group) and at 5-8
months (old group). The changes with age in both the
control and overloaded muscles of normal and rndx
mice are also described. The values obtained from the
overloaded muscles were always compared with those
for the control, unoperated extensor digitorum longus.
2. In the normal strain of mice the weight of the
overloaded extensor digitorum longus muscle in the
younger group was increased and it remained higher
in the older animals. In the mdx mice the overloaded
extensor digitorum longus muscles weighed more in
the younger animals but not in the older group of
mice.
3. The twitch and tetanic tensions of the overloaded
muscles were slightly, but not significantly, increased
in the younger group of mdx mice, whereas in the
older animals there was a significant decrease in both
twitch and tetanic tensions.
4. Thus the overloaded muscles from mdx mice
progressively deteriorated with age. In both strains of
mice the overloaded muscles become less fatiguable
with time.
INTRODUCTION
A mouse mutant with an X-linked muscular
dystrophy (mdx) first described by Bulfield et al. [l]
has recently attracted much interest, mainly because
some of the features of this animal model resemble
those of Duchenne muscular dystrophy. The most
important similarity between muscles from patients
with Duchenne muscular dystrophy and those from
the mdx mouse is the absence of the protein dystrophin [2]. The role of this protein in muscle function
is not known.
There is an important difference between the
mouse mutant and the group of human diseases
where dystrophin is absent. This difference concerns
the clinical picture and particularly the progress of
the disease. Whereas in human muscular dystrophies where dystrophin is missing, muscle deterioration is progressive, in the mouse, after an episode
of muscle degeneration and subsequent regeneration
at 2-4 weeks of age, the animals show few clinical
symptoms of the disease [3], although some morphological features of their muscles are abnormal
[4]. The study of the differences between the two
dystrophin-lacking species may help us to pinpoint
those physiological functions of skeletal muscles
that cannot, in the long run, be carried out without
the presence of this protein.
The possibility that dystrophin is essential for a
particular muscle function is indicated by the fact
that there is a preferential involvement of some
muscle groups in patients with Duchenne dystrophy
and relative sparing of other muscle groups. The
muscles affected most severely and early during the
disease are those concerned with weight-bearing,
such as the hip extensors. While there are many
other differences between the locomotor activity of a
mouse and a human, the requirements of weightbearing on mouse muscles might be less severe than
on human muscles, because of the quadrupedal
locomotion and the relatively short step-cycle of the
mouse. It is possible that the deterioration of the
mdx mouse is not progressive because the weightbearing requirements on mouse muscles are less
demanding. Even in the mouse, muscles that are
exposed to continuous use, such as the diaphragm,
do deteriorate with age [S]. In other models of
muscle disease, such as the C57 BL/6 dy2J strain [6]
and the d y / d y ReJ 129 strain, beneficial effects of
reduced load during early development were
reported [7]. Interestingly, increased load had the
opposite effect on muscles from dystrophic mice of
the strain C57 BL/6 dy2J and led to a greater
deterioration of their muscles [8]. Our aim was to
determine whether increased load and functional
demand would affect muscles from mdx mice and
lead to a progressive deterioration.
Key words: contractile properties, mdx mice, overload, progressive deterioration.
Abbreviations: EDL, extensor digitorum longus; SDH,succinate dehydrogenase; TA, tibialis anterior.
Correspondence: D r Gerta Vrbova, Department of Anatomy and Developmental Biology, University College London, Gower Street, London WCIE 6BT, U.K.
146
J. Dick and
METHODS
Animals
Male and female mice of the X-linked mdx
mutant strain and normal mice of the C57 BL/lO
strain were used.
G. Vrbova
Table I. Normal C57 BL/IO mice. Muscle weight, twitch and tetanic
tensions, time to peak twitch tension and time to half relaxation are shown
for control (C) and overloaded (OL) EDL muscles from C57 BLjlO mice
aged 84k4.9 (young) and 186k29.6 (old) days. The mean values k S E M for
control and overloaded (OL) muscles are shown. The number of animals in
each group is 9. Statistical significance: *P<O.O5 compared with animals
from the young group.
Control
Surgical procedures
Mice aged 9-12 days were anaesthetized with
ether, and using sterile precautions the tibialis anterior (TA) muscle was removed from one leg. The
pups were returned to their mothers, weaned and
their extensor digitorum longus (EDL) muscles were
studied at two different ages.
Youna
Muscle wt. (mg)
Twitch tension (9)
Tetanic tension (g)
Time to peak
twitch tension (ms)
Time to half relaxation
(ms)
Old
OL
Younf
Old
10.6k0.60 10.0f0.68
9.2k1.7 7.9k0.77
34k3.3
33k3.6
26+ I.Ol*
l8k 1.3
15.0+ 1.61 15.6k0.86
18.0 k 2.0
20.8
24.6 k 1.8*
9.5k0.86
37 +2.6
21 k l . 6
7.9k1.45
35 k 3.0
26+2.1
+ I .3
24. I k 1.5*
Physiological recordings
The mice were anaesthetized with chloral hydrate
(4.5% solution, 1 m1/100 g body weight, administered
intraperitoneally). The distal tendons of the EDL
muscles of both legs were exposed, dissected free
and prepared for tension recordings. The sciatic
nerve was sectioned and the tibia1 nerve was
crushed distal to the sciatic nerve section in the
popliteal fossa. The legs were secured to a rigid
table and the tendons of EDL muscles were
attached to strain gauges with 0.7 silk threads. The
muscles were adjusted to optimal length (Lo) so as
to obtain maximum twitch tension on electrical
stimulation of the nerve. Contractions were elicited
by stimulating the peripheral stump of the sciatic
nerve with supramaximal stimuli by means of bipolar silver electrodes. Isometric contractions were
displayed on a storage scope and photographed. A
fatigue test was then carried out. The muscles were
stimulated at 40Hz for 250ms every second for
3min. The fatigue index was expressed as a percentage loss of tension after 3min of stimulation. After
completion of these measurements the muscles were
removed and weighed.
Histology
The EDL muscles were frozen in melting isopentane and 10pm thick cross-sections were cut on a
cryostat. The muscles were then stained by a modified haematoxylin-van Gieson method and in some
cases the muscles were stained for succinate dehydrogenase (SDH) activity [S].
RESULTS
Normal C57 BL/IO mice
Changes in control and overloaded muscles with
age. Mice that had their TA removed in one leg
were examined at two ages: the ‘young group’ at
84 f4.9 ( n= 9) days and the ‘old group’ at 186 _+ 29.6
( n= 9) days. Table 1 compares some characteristic
properties of the EDL muscles of the young and old
Fig. I. Weight (a), twitch tension ( b ) and tetanic tension (c) of the
overloaded EDL muscles expressed as percentages of those of the
control, unoperated side, which were taken to be loo”? (broken
line). The normal C57 BLjlO mice
were 8 4 k 4 . 9 (n = ) days old when
examined and the mdx mice (m) were 7 4 k 7 . 6 (n =9) days old. The
vertical bars indicate +SEM.
(a)
group. The weight and tetanic tension of the control
and the overloaded muscles did not change during
this period. The twitch tension of the control muscle
decreased and the time to peak twitch tension and
the time to half relaxation became slower with age.
In the overloaded EDL this change was less pronounced, because the time to peak twitch tension
and the time to half relaxation was already slightly
prolonged in the overloaded EDL of the young
group (see Table 1).
Effect of overload. A direct comparison of muscle
weight and force output between the control and
overloaded muscle in each animal was carried out
and the results are summarized for the young group
in Fig. 1 and for the old group in Fig. 2. The
Figures show that in both the young and old
groups of normal mice, the weights of the overloaded EDL were significantly higher. Table 2 illustrates that the time to peak twitch tension and the
time to half relaxation was significantly slower in
Overloaded muscles of mdx mice
I80
140
CI
e
w
8
100
__
I47
I
as
I
-
2"
60
20
Fig. 2. Weight (a), twitch tension ( b ) and tetanic tension (c) of the
overloaded EDL muscles expressed as in Fig. I, but the animals
were older when examined. The normal C57 BLjlO mice (0)
were
were
186k29.8 (n=8) days old when examined and the mdx mice
217+ 15.8 (n= 19) days old. The vertical bars indicate kSEM.
(a)
Table 2. Ratio x I00 of time to peak twitch tension and time to half
relaxation of control EDL muscles from normal and mdx mice
calculated for each animal. Means k S E M and the numbers of animals
used (n) are given. Statistical significance: *P<0.05 compared with mice
from the old group.
Time to peak
twitch tension
Time to
half relaxation
(ms)
(ms)
Normal
rndx
Normal
mdx
n
117+6.4*
9
101 +63
9
118kl.2*
9
115+8.4
9
Old
n
101 k 6 . 3
9
99.5k5.2
19
look6
9
101 k 3 . 5
Young
19
the overloaded EDL muscle of the young animals
but not in the overloaded EDL muscle of the old
group.
The fatiguability of the overloaded and control
muscles was also compared. Fig. 3 shows an example from such a test where the overloaded muscle
appeared to be less fatiguable. Table 3 summarizes
the results and shows that the decrease in fatiguability is significant only for the group of older
animals.
Mdx mice
Changes in control and overloaded muscles with
age. In the rndx mice the control and overloaded
EDL muscles were studied at two ages: the younger
group was 7 4 k 7 . 6 (n=9) days old and the older
217*15.8 (n=19) days old.
Table 4 compares the various contractile properties of muscles from the young and old group.
Unlike in the normal animals (see Table l), in the
rndx mouse the weight, twitch tension and tetanic
tension of the control EDL muscle increased with
~
I min
Fig. 3. Traces showing examples of a fatigue test from a control (a)
and an overloaded (6) EDL muscle of a hnonth-old normal C57
BL/IO mouse that had the TA muscle removed at 10 days.
Contractions were elicited at 40 Hz for 250ms each second. The traces show
that in this animal the overloaded EDL was more fatigueresistant than the
control EDL.
Table 3. Fatigue index. The fatigue index, i.e. the decrease in force after
3 min of stimulation, was calculated from fatigue tests performed on control
and overloaded (OL) EDL muscle from C57 BLjlO mice aged 84k4.9
(normal young) and l86f29.6 (normal old) days and mdx mice aged
74f7.6 (mdx young) and 217+ 15.8 (mdx old) days. Values are
means +SEM. Statistical significance: *P < 0.05 compared with control
muscle.
Normal young (n=7)
Normal old (n=7)
mdx young (n=6)
mdx old (n = 16)
Control
OL
72+9
67+7
69+9
76+3
53+9
47+4*
48+7
52 7*
+
age, and there was a decrease in the time to peak
twitch tension and a small increase in the time to
half relaxation. In contrast, the overloaded EDL
muscle did not become heavier with age, its tetanic
tension did not increase and both the twitch tension
and the time to peak twitch tension decreased.
The fatiguability of the overloaded EDL muscles
decreased in the old but not in the young group (see
Table 3). This is similar to results obtained in
normal mice.
A comparison of the effect of overload on muscles from
normal and mdx mice
An example of a tension recording from a control
and an overloaded EDL muscle from an older rndx
J. Dick and G. Vrbova
148
Table 4 mdx mice. Muscle weight, twitch and tetanic tensions, time to
peak tension and time to half relaxation are shown for control and
overloaded (OL) EDL muscles from rndx mice aged 74f 15.8 (young) and
217k 15.8 (old) days. The mean values fSEM for control and overloaded
muscles are shown. The number of animals was nine in the young and 19 in
the old group. The values in parentheses are taken from nine rndx mice
aged 214k 17 that had not been previously operated on. Statistical
significance: *P<0.05 compared with animals from the young group.
OL
Control
Young
Muscle wt. (mg)
Twitch tension (9)
Tetanic tension (9)
Time to peak
twitch tension (ms)
Time to half
relaxation (ms)
Old
10.9k1.3 13.5f1.5
(15.0k0.9)
9.2+1.79 12.9+1.3
(I I .4k0.9)
25k4.2 39.8+3.4*
(34.9f2.6)
21.3f 1.7
19k0.7
(21.1 k0.6)
31.5k5.9 39.4k5.9
(20.9k I .O)
Young
Old
13.6f2.4 13.1k1.5
9.1 k 1.7
7.1 k0.6
30.9k5.4 28.9k2.3
22.4f 1.5
19f 1.2
38.8k5.7 39.5f1.5
the muscles from mdx mice were very different from
those of normal mice. Whereas in normal mice the
twitch and tetanic tensions of the werloaded
muscles increased, in muscles from mdx animals the
tensions were markedly reduced in comparison with
the EDL muscles that were not overloaded. Table 4
shows that the time to peak twitch tension and the
time to half relaxation increased in the overloaded
muscle of normal mice, but not in those from mdx
animals.
The mean difference between the young and old
group was 102 days for normal mice and 143 days
for the mdx mice. However, in view of the variability of ages at which the animals of the young and
old group were examined, this difference is likely to
be unimportant.
It could be argued that the control EDL muscles
from mdx mice were also altered as a result of the
unilateral removal of the TA muscles. We therefore
compared the different characteristics of EDL
muscles from nine intact mdx mice aged 214+ 17
days with those of control EDL muscles from the
operated animals. The results from these experiments are included in Table 4. It is clear that there
was no difference between the two groups, and it
was therefore justified to compare the overloaded
EDL with the EDL of the contralateral leg.
Histology
200ms
Fig. 4. Records of isometric twitch and tetanic contractions from a
control ( a ) and an overloaded (6) ED1 muscle of a 7-monthold
mdx mouse. Tetani were elicited at 40, 60, 80 and IOOHz in the control
muscle and 40,60 and 80Hz in the overloaded muscle.
mouse is shown in Fig. 4. It illustrates that the
tension output of the overloaded muscle is less than
that of the control muscle. Figs. 1 and 2 compare
the effect of overload on weight and force output of
EDL muscles from control and mdx mice. Fig. 1
shows that in the young group there was no
appreciable difference between the response of the
EDL muscles from control and mdx mice to overload. However, in the older group (Figs. 2 and 4)
In EDL muscles of the control mice the overload
induced few morphological changes either in the
young or older group of animals. Contrary to
expectation, there was little change in fibre diameter
in muscles from mdx mice. The staining showed the
typical features described by others in this strain of
mice, i.e. internally placed nuclei, 'and signs of
muscle degeneration and regeneration (Fig. 5A). In
the overloaded muscles of the mdx mice these
features were accentuated in the younger group,
whereas in the older animals the EDL muscles had
large areas tilled with mononucleated cells and a
relative increase in connective tissue (see Fig. 5B).
Staining for SDH showed that in the overloaded
muscle the majority of muscle fibres stained more
darkly for this enzyme than in the control EDL
muscle (compare Figs. 5C and 5D). The appearance
of the stain in the muscle fibres from the overloaded
EDL was disorganized.
DISCUSSION
The present results show that the weight and
tension output of EDL muscles of mdx mice rise
with increasing age. This is in contrast with EDL
muscles from control mice of the C57 BL/10 strain,
which did nor show an increase in weight or tension
output over the period studied (84-186 days). Thus
unlike the muscles of patients with Duchenne or
Overloaded muscles of rndx mice
I49
Fig. 5. Frozen transverse sections from control (A and C) and overloaded (B and D) EDL muscles from a 180dayold
mdx mouse. The sections were stained by the haematoxylin-van-Gieson method (A and 8) or for SDH activity (C and D). The bars
represent IOOpm (top panels) and 50pm (bottom panels).
Becker muscular dystrophy, leg muscles from mdx
mice show no progressive deterioration in their
force output. Using completely different criteria,
Carlson and Makiejus [lo] also report that there is
no progressive weakness in the mdx mouse.
However, the present results also show that whereas EDL muscles from normal mice are able to
withstand long-term overload without any loss of
force output, the overloaded muscles from mdx mice
cannot do so and become weaker compared with
their unoperated controls. Thus, these muscles are
unable to adjust in the long term to the increased
demand imposed upon them. This is in contrast to
the results obtained after acute eccentric exercise,
where muscles from mdx mice showed complete
recovery [111.
In spite of the signs of deterioration in tension
output seen in chronically overloaded EDL muscle
from mdx mice, some adaptive changes did take
place in the overloaded muscle. These were similar
to those seen in the overloaded normal EDL
muscle. In both groups of mice the EDL muscle on
the operated side became more fatigue-resistant.
Such adaptive changes to a similar overload were
seen in normal rat EDL muscles and in the rat
occurred within the first 2 weeks [12]. It is interesting that the ability of the muscle to become less
fatiguable as a result of functional overload is so.
well preserved in muscles of mdx mice. Such fatigue
resistance, determined using a similar fatigue test to
that used here, is also apparent in the muscles from
children with Duchenne muscular dystrophy [I131.
This could be related to the fact that at the time of
testing, the muscles from children with Duchenne
muscular dystrophy had been already ‘overloaded’
for some time since they were weaker than those of
children of comparable age [13].
The explanation for the main result of this study,
i.e. how the overload induces muscle deterioration
in the mdx mice, is not clear. It could be related to
the longer Ca2+ transients and increased protein
degradation seen in working muscle fibres from mdx
mice [14]. However, it is interesting that without
the additional load the muscles can toleratate these
prolonged CaZ transients without any apparent
adverse effect. There seems to be therefore a critical
level of load or ‘overwork’ which induces the muscle
to deteriorate. It could be that, in children with
Duchenne muscular dystrophy, due to their weaker
muscles the relative load increases and this may
contribute to the progressive nature of the disease.
Dystrophin may be somehow related to the ability
of muscles to cope with increased functional
demands.
+
ACKNOWLEDGMENTS
We are grateful to the Muscular Dystrophy
Group of Great Britain and Ireland for support,
and to Dr G. Bulfield for supplying some of the
animals.
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