Clinical Science (1992) 82,227-236 (Printed in Great Britain)
227
Contractile properties and susceptibility to exerciseinduced damage of normal and rndx mouse tibialis
anterior muscle
P. SACCO; D. A. JONES,J. R. T. DICK* and G. VRBOVA*
Department of Medicine, University College London, The Rayne Institute, London, U.K., and
"Deportment of Anatomy and Developmental Biology, University College London, London, U.K.
(Received 25 Junell2 September 199 I; accepted 20 September 199 I)
1. The functional properties of tibialis anterior muscles
of normal adult (C57BL/10) and age-matched dystrophin-deficient (C57BL/10 mdx) mice have been investigated in situ. Comparisons were made between tibialis
anterior muscle strength, rates of force development and
relaxation, force-frequency responses and fatiguability.
Subjecting rndx and C57 muscles to a regimen of
eccentric exercise allowed the hypothesis to be tested
that dystrophin-deficient muscles are more susceptible to
exercise-induced muscle damage.
2. mdx muscles were, on average, 30% stronger than
C57 muscles and almost 80% heavier, but both had
similar muscle lengths. Thus, although mdx muscles were
stronger in absolute terms, their estimated force per unit
cross-sectional area was significantly less than that of
C57 muscles.
3. The force-frequency relationships of C57 and rndr
muscles differed in that whilst, at 40 Hz, the former
developed 70% of the force developed at 1 0 0 Hz, the
latter developed only 55%of the maximal force. Twitch
force was normal in mdx muscles, but contraction time
was shortened, and the consequent reduction in fusion
frequency probably explains the force-frequency differences observed between the two groups.
4. mdx muscles were less fatiguable than normal
muscles when stimulated repeatedly at a frequency of 40
Hz. It is possible that the lower relative force at 40 Hz in
rndr muscles entailed a lower energy demand and thus a
slower rate of fatigue than seen in normal muscles.
5. Eccentrically exercised C57 muscles showed a large
loss of maximal force for up to 12 days after exercise.
Maximal force loss occurred 3 days after exercise (55%of
non-exercised tibialis anterior muscle), which also corresponded with the period of greatest fibre necrosis. C57
muscles showed a significantly reduced 40 Hz/100 Hz
force-frequency ratio at 1and 3 days after exercise. This
was primarily due to a reduced twitch amplitude rather
than to a change in the time course of the twitch. It is
unlikely, therefore, that the altered contractile charac-
teristics of mdx muscle were a result of the presence of
damaged but otherwise normal fibres.
6. C57 and mdx tibialis anterior muscles displayed
similar degrees of force loss after exercise. Furthermore,
the rate of recovery after the nadir of force loss was very
similar for the two groups. By 12 days after exercise,
force recovered to 76% and 80%of control in C57 and
mdu muscles, respectively. Our findings do not support
the hypothesis that dystrophin-deficient muscle is more
susceptible to exercise-induced muscle damage.
INTRODUCTION
Identification of the protein dystrophin as the missing
or defective gene product in Duchenne and Becker
muscular dystrophies has been a major advance in understanding the pathogenesis of these disorders [ 11.Although
dystrophin is now known to be associated with the sarcolemma1 membrane, its precise role in the muscle fibre and
the reason why its absence leads to dystrophic changes
remain to be elucidated. The discovery that dystrophin is
absent in the mdx strain of mice and that the gene locus
affected is homologous with that responsible for the
human Duchenne and Becker muscular dystrophies [2]
makes the mdx strain a valuable model for investigating
the functional consequences of dystrophin deficiency.
The ma'x mutant was originally described by Bulfield et
af. [3] in a colony of C57BL/10 mice. These animals have
chronically raised serum levels of muscle-specific creatine
kinase and exhibit histological changes characteristic of a
mild form of muscular dystrophy. Dangain & Vrbov5 [4]
reported that muscles from mdx mice undergo an acute
and massive fibre necrosis at approximately 3 weeks of
age with a subsequent regeneration and recovery by 5
weeks, although degeneration and regeneration, on a
more limited scale, continue throughout adult life [4-61. It
is possible that the lack of dystrophin may be reflected in
altered contractile properties [7] and fatiguability of
Key words: contractile properties, dystrophin, eccentric exercise, rndx mice, muscle damage.
Abbreviations: MRR, maximum rate of relaxation: MRTD, maximum rate of tension development; TA, tibialis anterior; TP, time to peak tension.
Correspondence: D r P. Sacco, Department of Medicine, The Rayne Institute, University Street, London WCI E 611, U.K.
228
P. Sacco et al.
dystrophic muscle or in the susceptibility to damaging
exercise and the ability to recover.
We have compared the contractile properties and
fatigue characteristics of mdx tibialis anterior (TA)
muscle with those of normal mice. However, a difficulty in
interpreting these results is the fact that dystrophic
muscles contain populations of both necrotic and regenerating fibres, and it is possible that any abnormalities
may be a reflection of the presence of these fibres. In the
second part of the work presented here we have investigated th'e contractile properties of damaged and regenerating normal mouse muscle and compared these
values with those of mdx muscle.
Exercise in which active muscles are forcibly stretched
(negative work or eccentric contractions) can lead to
considerable loss of force, fibre degeneration and subsequent regeneration [8-101 even in normal muscles, and it
has been suggested that dystrophic muscles may be more
susceptible to the damaging effects of eccentric exercise
[ 111. The recent speculation that dystrophin is a structural
protein, which may serve to stabilize the fibre membrane
against the damaging effects of mechanical stresses
associated with muscle activity [ 121, is consistent with this
hypothesis. In the third part of the work we have tested
the hypothesis that the absence of dystrophin causes mdx
muscle to be more vulnerable to stretch-induced muscle
damage.
MATERIALS AND METHODS
Experimental animals
Myopathic mice ( m d x )were maintained as a breeding
colony from stock donated by Dr G. Bulfield, Department
of Genetics, University of Edinburgh. Normal animals
were obtained from a colony of C57BL/10 mice, this
being the strain in which the mdx mutant arose. Experiments were performed on female animals aged 16-26
weeks (mdx, mean=21.4 weeks) and 16-24 weeks
(normal C57, mean= 21.0 weeks).
Force recordings
All investigations were carried out using the TA
muscle. Animals were anaesthetized by an intraperitoneal
injection of chloral hydrate [4.5% (w/v) solution, 0.01
ml/g body weight]. The distal tendon of the TA was cut
and the proximal end was tied to a Statham force transducer with fine silk suture. The sciatic nerve was isolated
and cut, and the leg was secured by metal pins through the
knee and ankle joints. A bipolar platinum stimulating
electrode was positioned in contact with the peripheral
stump of the sciatic nerve.
Muscles were stimulated via the nerve with square
wave pulses of 100 ,us duration and the voltage was
adjusted so as to give supramaximal stimulation (5-10 V).
An initial tetanic stimulation at 100 Hz for 0.5 s was used
to remove any slack which might be present in the tendon
attachments. The muscle length was then adjusted to give
maximum twitch tension without a significant increase in
resting tension. Data were recorded (a) on a storage
oscilloscope screen, (b) on a Devices chart recorder and
(c) after digitization on a computer database for
subsequent analysis. The latter was used to measure the
time to peak tension (TP), maximum rate of tension
development (MRTD) and maximum rate of relaxation
(MRR). Since the MRTD and MRR are related to the
absolute force of the contraction, they were normalized
for peak force to obtain a value of the percentage change
in peak force/ms.
Stimulation protocol
A force-frequency relationship was determined for
each muscle by stimulating for half a second at 1,40, 60,
80 and 100 Hz. At least 30 s recovery time was allowed
between tetani.
After 5 min recovery, muscles were subjected to a
fatigue protocol consisting of supramaximal stimulation at
40 Hz for 250 ms every second for 3 min. The tetanic
force after 3 min of fatigue was expressed as a percentage
of the maximum tetanic force; this was usually the initial
contraction, but occasionally there was some potentiation
during the first 2-5 s, in which case the greatest value was
used.
Exercise protocol
Eccentric contractions were elicited in the dorsiflexor
muscles of the right foot [13], the contralateral muscles
serving as non-exercised controls. Mice were anaesthetized with an intraperitoneal injection of 4.5% (w/v)
chloral hydrate (0.01 ml/g body weight) and the right knee
was clamped firmly to restrict movement to the lower
limb. The peroneal nerve was exposed just above the knee
and was placed on a small hook electrode. The foot was
taped to a perspex holder, which rotated around a pivot at
the ankle. The foot holder was attached to the rim of a
motor-driven wheel, the rotation of which caused the foot
to travel from full flexion to full extension (approximately
100")and back in 600 ms. The TA was stimulated for 300
ms at 100 Hz using 100 ,us square wave pulses of supramaximal voltage beginning just before and continuing
during the lengthening phase. The shortening phase was
entirely passive. Lengthening contractions were repeated
every 5 s for 240 repetitions, after which the incision was
closed and the animals were allowed to recover. One,
three, six or twelve days after exercise the animals were
again anaesthetized, and the strength, contractile
properties and fatiguability of exercised and contralateral
muscles were determined as described above.
Post-mortem examination
After the fatigue run, the mice were killed by a lethal
dose of chloral hydrate. TA length was measured in situ in
a number of mice. All muscles were carefully excised,
weighed and a portion from the belly of the muscle was
orientated to give transverse sections before being frozen
in isopentane cooled with liquid nitrogen.
Contractile properties and susceptibility to damage of mdx mouse muscle
Histology and histochemistry
Cryostat sections were cut at 8 p m and were stained
with haematoxylin and eosin and for NADH-tetrazolium
reductase.
Morphometry
Fibre areas were measured by using semi-quantitative
image analysis [14] on transverse sections stained with
haematoxylin and eosin.
One-hundred fibres were measured from each TA
muscle in 10 adjacent fields covering the lateral portion of
the muscle. To provide a quantitative estimate of damage
the number of 'damage foci'/field were counted under
x 400 magnification for every second field across the
section (15 fields counted/section). Damage foci were
defined as fibres which were infiltrated by mononuclear
cells.
229
where the TA muscle represented a significantly smaller
portion of body weight (1.5%).
Muscles from C57 mice were normal in appearance,
fibres with internal nuclei being very rare (Fig. la).
Examination of rndx muscles showed a wide variation in
muscle fibre size, foci of fibre necrosis and infiltration
with mononuclear cells (Fig. l b ) . A notable feature of
mdx muscles was that the majority of fibres had one or
more internally placed nuclei1 (Fig. l b ) . Staining for
NADH-tetrazolium reductase showed similar proportions of oxidative and non-oxidative fibres in normal
and rndx muscles. Muscle fibre areas of normal and rndx
TA muscles are shown in Fig. 2 and Table 1. Mean fibre
areas were very similar for normal and mdx muscles, but
the latter showed a greater range of fibre areas, illustrated
by the larger SD (Table 1).Thus 17% of the mdx fibres had
an area less than 2000 pm2 compared with 10% of
normal muscles, whereas 9% of the mdx fibres had an
area greater than 6000 pm2 compared with 1% of the
normal fibres.
Statistics
Values for grouped mean data were compared by using
Student's t-test; where not specifically stated, significance
was set at the 5% level. Results are expressed as
means f SEM.
RESULTS
Muscle size and histology
Details of animal and muscle weights are given in Table
1. rndx mice were, on average, 20% heavier than normal
C57 mice, whereas the individual TA muscles from rndx
mice were 80% heavier than those from normal animals.
Both types of muscles were the same length, so that the
calculated cross-sectional areas of mdx muscles were
greater than those of the normal C57 muscles.
The mdx TA muscle weights were approximately 2.3%
of total body weight, in contrast to the normal group,
Table I. Physical properties of mdx and normal TA muscles.
Values are given as meanstsm except for fibre areas, which are given as
means tso. Abbreviation: CSA, cross-sectional area. Statistical significance: * P <0.05, **P<O.Ol compared with normal muscle.
Animal wt. (9)
TA wt. (mg)
TA wt. as % of body wt.
Muscle length (mm)
Estimated muscle CSA (mm*)
10-l
XFibre area (pm')
mdx muscle
Normal muscle
33.0 i.I.6*
(n=9)
76 k 2**
( n = 17)
2.28 +0.06**
( n = 17)
11.1 t0.4
(n=6)
6.8-10.3**
( n = 17)
3.39 -1 I .37
27.1 i 1.1
(n=8)
( n = 500)
(n=600)
41tl
(n=15)
1.53i0.05
(n=l5)
10.5 f0.3
(n=7)
3.9 i0.7
(n=15)
3.I7 f0.88
Contractile properties
Although maximum isometric force was elicited at a
stimulation frequency of 100 Hz for both normal and
rndx muscles, the relative force generated at submaximal
frequencies differed in the two groups (Fig. 3). The
comparative deficit of force at low frequencies of stimulation in the rndx muscle was particularly marked at frequencies of around 40 Hz, although the force generated
was also significantly smaller at 60 Hz.
Fig. 4 shows the force records for a single twitch of an
rndx and normal TA muscle. Twitches from mdx muscles
reached peak force more rapidly than those from normal
muscle. TP for isometric twitches (Table 2) was, on
average, 4.2 ms shorter in the mdx muscle. This difference was reflected in a 20% higher MRTD normalized
for peak force (Table 2) compared with normal muscles.
MRRs of mdx and C57 muscles showed no significant
differences (Table 2).
mdx muscles developed over 30% more force during
maximum stimulation compared with normal muscle
(1.59kO.08 and 1.17f0.03 N, respectively, mean sf^^^,
P< 0.01). Comparing the maximum force with the
estimated muscle cross-sectional area provides an
estimate of the intrinsic force-generating capacity of a
muscle. Whereas normal muscles generated 0.30 k 0.01
N/mm2, in rndx muscles only 0.24k0.01 N/mm2 was
produced ( P < 0.01). Thus despite the larger absolute
forces generated by mdx muscles, they produced only
80% of normal muscle force per unit cross-sectional area.
Fig. 5 shows the change in isometric force during
repetitive stimulation for an rndx and normal C.57 TA
muscle. Although rndx muscles commonly lost more force
after the first few tetani, the force loss after 3 min of
stimulation was consistently less in rndx muscles. Thus at
the end of the fatiguing protocol, rndx muscles generated,
on average, 48 f 2.2% of fresh force as compared with
42 -t 1.4% for normal muscles ( P <0.05). On the basis of
230
P. Sacco et al.
Fig. I . Histological appearance of C57 (left) and mdx (right) mouse TA muscles before and after eccentric exercise.
((1, 6) Non-exercised; (c, d), I day after exercise; (e, fl, 3 days after exercise; (9,h), 6 days after exercise. Bar=50 pn.
23 I
Contractile properties and susceptibility to damage of mdx mouse muscle
this index, rndx muscles would appear to be significantly
more fatigue-resistant than normal muscles.
aJ
2
P
-
.-E
Exercised and damaged muscles
Both mdx and C57 muscles showed a small decrease in
weight after the damaging exercise, this being greatest at 6
days when the rndx TA muscle weight decreased by 13%
compared with 5% for normal TA muscles.
Histological examination by light microscopy showed
little change in normal muscle 1 day after exercise, except
for the presence of numbers of rounded, eosinophilic
fibres (compare Figs. l a and lc). Three days after
exercise, there was an invasion of the muscle by inflammatory cells accompanied by muscle fibre destruction
(Fig. le). By 6 days (Fig. lg), far fewer inflammatory cells
were present, but there were plentiful small basophilic
cells possessing large central nuclei, which were probably
regenerating myoblasts.
The damage, evident histologically, to the mdx TA
muscles 3 days after exercise (Fig. If) appeared more
extensive than that occurring in the normal C57 muscles
(Fig. le). However, the exercise-induced damage in the
rndx mice was imposed on muscles which were already
showing areas of degeneration and regeneration (Fig. 1b).
Although histological assessment is an imperfect method
of quantifying fibre damage, 3 days after exercise C57
muscles averaged 5.5 damage foci/field, compared with
an average of 6.9 damage foci/field in exercised mdx TA
muscles (Fig. 6), this difference proving significant at the
5% level. However, when the number of damage foci in
the non-exercised mdx muscles was taken into account,
the number of damaged fibres resulting from the exercise
protocol was seen to be equivalent in mdx and C57 TA
muscles.
The loss of force and subsequent recovery was
accompanied by changes in the contractile properties,
which were, in some respects, different for the C57 and
mdx muscles. Fig. 7 shows force-frequency recordings of
exercised and control muscles 3 days after exercise in a
3
E
L
c
0
.”
e
Y
0.20 P
0
I
20
40
60
80
Stimulation frequency (Hz)
I00
I .oo
aJ
2
9
-
0.80
.-E
1
E
L
0.60
0
.-c0
Y
5
0.40
Y
0.20
.I
0
I
20
40
60
Stimulation frequency (Hz)
80
I00
Fig. 3. Mean force-frequency relationships of C57 (a; n=6) and
rndx (b; n=6) mouse T A muscles at 3 days after exercise. (a) 0 ,
Exercised muscle; A , control muscle. (b) 1, Exercised muscle: A,
control muscle. Statistical significance: *f (0.05, **f(0.01 for the
difference between rndx and C57 control muscles; tf (0.05, i t P (0.0 I
for the difference between exercised and control muscles. Values are
meansfSEM.
c57
I
’D
”
c
=
8
”,
E
g
0
1
2
3
4
5
6
7
IO-lxFibre area (pm’)
8
c
(
20 ms
Fig. 2. Fibre areas of rndx and C57 T A muscles (mean fibre areas
are indicated by arrows). 1,mdx, n = 5 muscles, 500 fibres: 0 , C57,
n = 6 muscles. 600 fibres.
Fig. 4. Isometric twitch recordings of rndx ( a ) and C57 (b) mouse
T A muscles
232
P. Sacco et al.
C57 and an mdx mouse. Mean force-frequency
recordings (where force is expressed as a ratio of maximal
force) for all the muscles examined are shown in Fig. 3.
The C57 TA muscles (Fig. 3a) showed a significantly
greater loss of force at sub-maximal stimulation frequencies (1, 40 and 60 Hz), resulting in a shift to the right of
the force-frequency curve. In contrast, no change was
seen in the force-frequency characteristics of mdx
muscles at the same time after exercise (Fig. 7b). A
measure of the shape of the force-frequency curve can be
obtained by taking the ratio of force at 40 Hz compared
with 100 Hz and Fig. 8 shows the time course of changes
in this ratio. In the C57 TA muscles there was a reduction
in the 40 Hz/100 Hz force ratio at 1 and 3 days after
exercise, but the increment in the 40 Hz/100 Hz ratio at 6
days was comparable with that seen for the C57 TA
muscles.
The force-frequency changes in normal muscles 3 days
after exercise were not accompanied by significant
changes in the contraction time of the twitch. There was a
small slowing in the TP of the twitch at 6 days in both the
exercised normal and mdx muscles.
Table 2. Force characteristics of mdx and normal TA muscles.
Values are given as means ~ S E M . Statistical significance: *P c0.05,
**P (0.0 I compared with normal muscle.
Twitch tension (N)
Tetanic tension (N)
Twitch TP (ms)
Normalized MRTD twitch (%/ms)
Normalized MRR twitch (%/ms)
0.5N
I
rndx muscle
Normal muscle
0.50 f O . O l *
( n = 17)
I .59 +0.08**
( n = 17)
17.5 f0.7*
( n = 15)
8.6k 2.5**
( n = 17)
4.5k4.2
(n=17)
0.33k0.01
(n=15)
1.17 k0.03
(n= 15)
21.6 f0.5
( n = 15)
7.1 k 3.5
( n = 15)
3.9f2.7
(n= 15)
Changes in tetanic force after exercise were very
similar for the normal and rndx TA muscles (Fig. 9).Force
loss was greatest at 3 days when forces had declined to
52% (rndx) and 56% (C57) of that of the contralateral
muscles. After 3 days there was a gradual recovery, so
that by 12 days after exercise forces were 79% (mdx)and
76% (C57)of control force.
One and three days after exercise there was a tendency
for normal muscles to show an increase in fatigue resistance. Thus, after 3 days of recovery damaged muscles
developed 45+.3.3% of fresh force after fatigue as
opposed to 4 1k 4.5% for control muscles. However,
these differences were not significant at any time after
exercise. No differences in fatigue resistance were found
between exercised and non-exercised mdx muscles at any
time after exercise.
DISCUSSION
Differences were found in the size, strength and
contractile properties of TA muscles from mdx mice
when compared with those from normal mice of a similar
age.
Muscle size and strength
rndx mice were heavier than the age-matched controls,
as has been reported by others [4, 15, 161, although some
studies have found no differences in either body weight or
muscle mass [17, 181. The larger body mass was not a
consequence of a longer body length. We found the mdx
and C57 TA muscles to be of similar length, and Marshall
et al. [16] have reported that the tibia1 lengths of normal
and mdx mice are the same. When muscle mass was
expressed as a percentage of body weight, the mdx TA
accounted for a greater proportion of body mass than the
TA of normal mice. It would appear, therefore, that the
greater body weight of the rndx mice could be attributed
to larger and heavier muscles.
Histological examination of rndx muscles showed little
evidence of connective tissue proliferation, which is in
c
.
(
30 s
Fig. 5. Changes in isometric force of mdx (a) and C57 (6) mouse TA muscles as a result of stimulation at 40 Hz for
0.25 s every I s for 3 min
Contractile properties and susceptibility to damage of mdx mouse muscle
agreement with the findings of other workers [5, 171.
Marshall et al. [16] used image analysis to measure the
connective tissue content of mdx and normal soleus
muscles, and found that although more connective tissue
could be measured in the endomysial and perimysial
spaces in rndx muscle, the difference only became
substantial in animals which were older than those used in
the present study.
In the absence of any oedematous or connective tissue
infiltration, the greater size of mdx muscles must have
been the result of increased muscle fibre bulk due to fibre
c57
c57
(con.)
(ex.)
mdx
(con.)
mdx
(ex.)
Fig. 6. Number of damage focilmicroscope field in mouse TA
muscle counted at 3 days after exercise. Ninety fields (five muscles)
were counted for each group. Values are meansfsm. Abbreviations:
con., contralateral muscle; ex., exercised muscle.
233
hyperplasia and/or fibre hypertrophy. The distribution of
fibre sizes shown in Fig. 1 suggests that both processes
may be active in the dystrophic muscle as there were
greater numbers of both large and small fibres in the mdx
TA. Anderson et al. [15]examined the extensor digitorum
longus and soleus muscles of young and old m d x and
normal mice and found that the muscles of 4-week-old
rndx mice tend to have fewer fibres than normal, whereas
at 32 weeks rndx mice had a greater number of fibres in
the extensor digitorum longus, but not soleus, muscle.
These observations would suggest that in rndx mice the
larger muscles are a consequence of the dystrophic
process, and not the result of a greater number of fibres at
birth.
In showing genuine muscle hypertrophy, muscle of
rndx mice differs from that of human subjects with
Duchenne muscular dystrophy, where, although there is
enlargement of certain muscles, this is a ‘pseudo-hypertrophy’, with fat and connective tissue accounting for the
increased size [ 191. Fibre splitting and fibre hypertrophy
do occur in Duchenne muscular dystrophy, but it would
appear that these processes are not sufficient to offset the
degenerative process in the human condition. In the rndx
mouse, after the initial phase of massive fibre necrosis, the
fibre hypertrophy and hyperplasia would seem to run
ahead of the degenerative process, resulting in a muscle
which is larger than that of age-matched normal mice.
Further evidence that the larger size of rndx muscle is
due to an increase in contractile material is afforded by
the fact that mdx muscles were stronger, in absolute terms
(30% on average), than the TA muscles from normal
Fig. 7. Force-frequency recordings of mouse C57 (top) and mdx (bottom) mouse TA muscles at 3 days after
exercise (right) compared with the control contralateral muscle (left)
P. Sacco et al.
Contractile characteristics and fatiguability
0.80 -
..................................c 5 7
0.50
--
0.40 7
0
1
I
2
4
6
8
Time after exercise (days)
10
12
The contractile characteristics of nidx TA muscles
were found to differ from those of normal muscles. As a
proportion of the maximum tetanic force, less force was
generated at intermediate frequencies of stimulation. The
ratio of twitch force to maximum tetanic force was similar
in mdx and normal muscles, but the time course of the
mdx twitch was of shorter overall duration. The consequence of the faster twitch was that the fusion frequency
was higher for rndx TA muscles. The reason for this
behaviour is not clear, but does not appear to be related
to differences in fibre-type composition between mdx and
normal muscles. although the stain for NADH-tetrazolium reductase that we have used is not an infallible
guide to fibre-type composition. In a separate study
(J. R. T. Dick & G. Vrbovi, unpublished work) it was
found that the TP of the twitch of extensor digitorum
longus muscles increased significantly with age in normal
mice, but no such slowing of the twitch occurred in rndx
extensor digitorum longus muscles. Thus older mdx
muscles appear to maintain the contractile properties of
muscles from younger mice.
Dystrophic muscle fibres differ from normal in that
they lack dystrophin and it is tempting to ascribe the
differences in contractile properties to the absence of this
protein, but there are alternative explanations. rndx
muscles contain populations of degenerating and regenerating fibres and it is possible that the altered contractile properties of the whole muscle reflect the function of
these abnormal fibres. It has been known for a number of
years that excessive isometric exercise [21] or exercise
involving stretch in man [S-101 and in mice [22] results in
a relatively greater loss of force at low frequencies of stimulation (low-frequency fatigue) that has similarities with
the unusual shape of the force-frequency curve of control, undamaged, rndx mouse muscle. Closer inspection,
however, reveals that the similarities are more apparent
than real. The loss of low-frequency force seen in
damaged normal muscles was primarily due to a loss of
amplitude of the twitch. There was no significant change
in the twitch duration or TP, suggesting that there was a
reduced activation, possibly a smaller release of calcium
for each action potential [23], with no change in the
kinetics of calcium release or re-uptake. With the undamaged rndx the loss of force at low frequencies (Fig. 3b)
was a consequence of a twitch of shorter duration while
the amplitude was relatively well maintained. It would
appear, therefore, that the reduced force at low
frequencies of stimulation in the mdx muscles is unlikely
to have been due to the presence of otherwise normal
fibres which had been damaged by exercise. Dystrophic
muscles also contain a population of regenerating fibres,
but it is unlikely that these can account for the unusual
force-frequency relationship of rndx muscle. Six and 12
days after damaging exercise there was evidence of
regeneration in the damaged C57 muscles (Fig. lg),but at
this time the C57 muscles were generating normal forces
at 40 Hz, while for the mdx muscles there was relatively
more force at low frequencies of stimulation (Fig. 8).
Y
Fig. 8. Changes in the 40 HzllOO Hz force ratio of mdx (I)
and
C57 ( 0 ) mouse TA muscles after exercise. Broken lines represent
mean values for control muscles. n =4-6 muscles for each data point.
Statistical significance: *P < 0.05 for the difference between mdx
exercised and control muscles; t P (0.05 for the difference between C57
exercised and control muscles. Values are meansksm.
I
0
2
4
6
8
10
12
Time after exercise (days)
Fig. 9. Maximum tetanic forces of mdx (I)
and C57 ( 0 ) mouse
TA muscles after exercise expressed as a percentage of that of
contralateral muscles. n=4-6 muscles for each data point. Values are
means ri- SEN.
animals. Coulton et al. [7] reported that maximal isometric force of the soleus muscle of adult m& mice was
similar to that of control mice, whereas Dangain &
Vrbovi [4] found that the TA of rndx mice developed
greater tensions than normal. These findings suggest an
element of muscle specificity in the strength development
of nzdx mice.
Although the cause of muscle fibre hypertrophy in
dystrophic muscle is not known, a common suggestion is
that, as the number of viable fibres decreases, there may
be a work-induced stimulus to the remaining fibres to
undergo hypertrophy and fibre-splitting [20]. Such a
mechanism might explain why a muscle maintains a
strength close to the normal value, but it cannot account
for muscles which are stronger than expected.
Contractile properties and susceptibility t o damage of mdx mouse muscle
235
Previous studies have found that dystrophic muscle is
more fatigue-resistant than normal [24, 251. In the fatigue
test we have used, the mdx TA muscles proved to be more
resistant to fatigue than those from normal mice. The
protocol involved repetitive stimulation at 40 Hz, which
generates different proportions of maximal force in the
normal and rndx muscles. Consequently, the greater
fatigue resistance of the dystrophic muscle might have
been because the muscle was less fully activated, and the
test less metabolically demanding, than that for normal
muscle. It is not clear whether this explanation can also
account for the differences seen by previous workers
studying human Duchenne muscular dystrophic muscles
and mouse dystrophic muscles.
Although stronger than normal, the rndx muscles were
not as strong as might be expected from their size and
cross-sectional area. When the force data was normalized
for muscle cross-sectional area, the mdx TA was only able
to generate 70% of normal force. Thus mdx muscles have
a significantly reduced ‘specific muscle tension’ compared
with normal muscle. The reduced force-generating
capacity of rndx muscle is probably not the result of fat or
fibrous tissue infiltration (see above). The presence of
areas of segmental fibre degeneration, although each only
affecting a small portion of the total fibre length, may
result in substantial loss of force production, since the
active contractile portions of these fibres will lack
contiguity. Secondly, fibre-splitting may have deleterious
effects on force generation. Schmalbruch [26] suggested
that fibre splitting arises from incomplete fusion of
myotubes within the basement membrane during regeneration, resulting in the formation of two or more
daughter fibres. These can branch off and reconnect to
the original fibre further along its length or to neighbouring muscle fibres. This process would result in a reduction
in the number of myofibrils which contract in parallel.
Furthermore, Bradley [27] has postulated that necrotic
fibre segments and split fibres of small diameter may
present a conduction block to the muscle action potential,
leading to incomplete activation of fibres.
found that more fibres stained positively for IgG (which
the authors suggest represents activation of the complement cascade and is thus a precursor to fibre necrosis) in
the rndx than normal TA muscle after 60 min. Hutter et al.
[30], however, found comparatively minor abnormalities
in the physical characteristics of the rndx surface
membrane, and McArdle et al. [31] found slightly less
efflux of creatine kinase from isolated mdx as compared
with normal mouse extensor digitorum longus muscles
after stimulation under anaerobic conditions. These
workers also stretched the muscles while stimulating and,
again, found no difference between normal and mdx
muscles. There is, therefore, conflicting evidence as to
whether the absence of dystrophin renders the dystrophic
muscle more susceptible to damage. One difficulty with
the studies of McArdle et al. [31] and of Weller et al. [29]
is that, although they looked at eccentric exercise, they
did so for only a matter of hours after the initial insult,
although it is well known that the main effects, and
certainly the muscle fibre necrosis, occur several days
after the exercise. We have followed events for up to 12
days after the exercise, allowing us to monitor both the
phases of degeneration and regeneration. The results
were unequivocal: the mak muscles responded to the
exercise in the same way as did the normal C57 muscles,
showing a similar loss of force and a similar time course of
both degeneration and recovery (Fig. 9).
Our results suggest that the unusual contractile
properties and greater fatigue resistance of mdx muscles
are not due to the presence of damaged or regenerating
fibres. It is possible, therefore, that dystrophin may have
some role to play in modulating force production in
normal muscles, although we have no suggestions as to
how it may do this. Our second conclusion is that the
absence of dystrophin does not make rndx muscles more
susceptible to stretch-induced injury, and this argues
against a role for dystrophin as a structural component
strenthening the surface membrane against mechanical
disruption.
Susceptibility to damage
ACKNOWLEDGMENTS
In its histological appearance, the rndx muscle (Fig. 16)
has many similarities to damaged normal muscle,
containing both degenerating fibres similar to those seen
3 days after exercise in C57 muscles (Fig. l e ) and regenerating fibres as seen 6 days after exercise (Fig. lg). A
similar observation in human muscle was the basis for the
suggestion of Edwards et al. [l11 concerning the initiation
of the dystrophic process, and now provides the basis for
one hypothesis concerning the role of dystrophin in
muscle fibres.
Recently, there have been two reports of an increased
sensitivity of dystrophic muscle to mechanical stress.
Menke & Jockush [28] reported that isolated mdx mouse
muscle fibres were more susceptible to osmotic shock
than normal muscle fibres. Weller et al. [29] investigated
IgG staining of muscle fibres in mdx mice after shortening
and lengthening contractions of the TA muscle; they
P.S. is a Wellcome Prize Scholar. We are grateful to the
Muscular Dystrophy Group of Great Britain for support.
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