Download The passive mechanical properties of the extensor digitorum longus

J Appl Physiol 110: 1656–1663, 2011.
First published March 17, 2011; doi:10.1152/japplphysiol.01425.2010.
The passive mechanical properties of the extensor digitorum longus muscle
are compromised in 2- to 20-mo-old mdx mice
Chady H. Hakim,1 Robert W. Grange,2 and Dongsheng Duan1
1
Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri; and 2Department
of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
Submitted 9 December 2010; accepted in final form 14 March 2011
passive properties; skeletal muscle; muscular dystrophy; dystrophin;
myotendinous junction
is a subsarcolemmal cytoskeletal protein (16). Absence of dystrophin leads to Duchenne muscular dystrophy
(DMD), a severe muscle-wasting disease that affects 1 in 3,500
newborn boys. Dystrophin forms a complex with a series of
transmembrane and cytosolic proteins. Throughout this molecular complex, dystrophin links the subsarcolemmal F-actin
network to the extracellular matrix. It is generally agreed that
the dystrophin complex stabilizes the sarcolemma during force
transmission (reviewed in Ref. 10). In dystrophin-deficient
muscle, the sarcolemma is damaged by muscle contraction
(reviewed in Ref. 13). Eventually, muscle cells undergo necrosis and are replaced by fibrotic and/or adipose tissues.
The resistant force develops against the strain when a muscle
is passively lengthened. This resistant force is referred to as the
passive stress. Since the muscle is not actively contracting, the
DYSTROPHIN
Address for reprint requests and other correspondence: D. Duan, Dept. of
Molecular Microbiology and Immunology, Univ. of Missouri, One Hospital
Dr., Columbia, MO 65212 (e-mail: [email protected]).
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passive stress reflects the inherent (passive) properties of the
muscle constituents such as the extracellular matrix, cytoskeletal proteins, and myofibrils. The passive properties of the
muscle can be further defined by the elasticity (stiffness) and
the viscosity (1, 20). Since fibrosis and inflammation are major
pathological changes in dystrophin-deficient muscle, it is expected that the passive properties of the muscle would be
altered in the absence of dystrophin. In support of this notion,
it has been shown that muscle stiffness is markedly increased
in DMD patients (8, 9). Here, we hypothesize that dystrophindeficient muscle pathology profoundly influences the passive
properties of muscle in mdx mice, a murine model for DMD.
The myotendinous junction (MTJ) is the link between the muscle
and the tendon (27, 29). At the MTJ, fingerlike muscle protrusions
invade into fibrous tendon tissue. The sarcolemma folds extensively
at these fingerlike structures. This effectively increases the junctional
area between muscle and tendon and provides a strong interface for
force transmission. Interestingly, dystrophin is highly enriched at the
MTJ (26). It has been reported that the ultrastructure of the MTJ is
impaired in mdx mice and DMD patients (2, 15, 23). Here we
hypothesize that the strength of the MTJ is weakened in mdx mice.
As a consequence of this weakening, mdx muscle will fail at the MTJ
when it is passively stretched.
In contrast to DMD patients, young mdx mice are mildly
affected. Clinically obvious dystrophic symptoms are only
seen in old mdx mice (4, 7). To test our hypotheses, we applied
passive stretch to an intact extensor digitorum longus (EDL)
muscle-tendon unit in 2-, 6-, 14-, and 20-mo-old male mdx as
well as to age- and sex-matched normal control BL10 mice.
We analyzed the stress-strain profile, hydroxyproline content,
and muscle failure position. In addition, we examined MTJ
structure by electron microscopy (EM).
MATERIALS AND METHODS
Evaluation of the passive properties of the EDL muscle. All animal
experiments were approved by the Animal Care and Use Committee of
the University of Missouri and were in accordance with NIH guidelines.
Dystrophin-deficient mdx mice and normal control BL10 mice were
purchased from the Jackson Laboratory (Bar Harbor, ME). Only male
mice were used in the study. Experimental mice were euthanized by
cervical dislocation at the end of study. Mice were anesthetized via
intraperitoneal injection of a cocktail containing 25 mg/ml ketamine, 2.5
mg/ml xylazine, and 0.5 mg/ml acepromazine at 2.5 ␮l/g body weight.
The age and the body mass were recorded (Table 1). The animal was
positioned on a custom-fabricated Plexiglas dissection board (22.9 cm ⫻
17.8 cm ⫻ 1.3 cm) with a reservoir (12.7 cm ⫻ 7.6 cm ⫻ 0.6 cm) to
collect excess buffer during muscle superfusion. The buffer was removed
from the reservoir through a vacuum line. A Sylgard (World Precision
Instruments, Sarasota, FL) ring (3.8 cm in diameter and 1.3 cm in height)
was glued in the middle of the reservoir to secure the hindlimb. A radiant
heat lamp was used to maintain the body temperature at 37°C. All
8750-7587/11 Copyright © 2011 the American Physiological Society
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Hakim CH, Grange RW, Duan D. The passive mechanical properties of the extensor digitorum longus muscle are compromised in 2- to
20-mo-old mdx mice. J Appl Physiol 110: 1656 –1663, 2011. First
published March 17, 2011; doi:10.1152/japplphysiol.01425.2010.—
Muscle rigidity and myotendinous junction (MTJ) deficiency contribute to immobilization in Duchenne muscular dystrophy (DMD), a
lethal disease caused by the absence of dystrophin. However, little is
known about the muscle passive properties and MTJ strength in a
diseased muscle. Here, we hypothesize that dystrophin-deficient muscle pathology renders skeletal muscle stiffer and MTJ weaker. To test
our hypothesis, we examined the passive properties of an intact
noncontracting muscle-tendon unit in mdx mice, a mouse model for
DMD. The extensor digitorum longus (EDL) muscle-tendon preparations of 2-, 6-, 14-, and 20-mo-old mdx and normal control mice were
strained stepwisely from 110% to 160% of the muscle optimal length.
The stress-strain response and failure position were analyzed. In
support of our hypothesis, the mdx EDL preparation consistently
developed higher stress before muscle failure. Postfailure stresses
decreased dramatically in mdx but not normal preparations. Further,
mdx showed a significantly faster stress relaxation rate. Consistent
with stress-strain assay results, we observed significantly higher
fibrosis in mdx muscle. In 2- and 6-mo-old mdx and 20-mo-old BL10
mice failure occurred within the muscle (2- to 14-mo-old BL10
preparations did not fail). Interestingly, in ⱖ14-mo-old mdx mice the
failure site shifted toward the MTJ. Electron microscopy revealed
substantial MTJ degeneration in aged but not young mdx mice. In
summary, our results suggest that the passive properties of the EDL
muscle and the strength of MTJ are compromised in mdx in an
age-dependent manner. These findings offer new insights in studying
DMD pathogenesis and developing novel therapies.
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PASSIVE PROPERTIES OF THE mdx EDL
Table 1. Characterization of experimental mice
Age, mo
n
Body Weight, g
TA Weight, mg
EDL Weight, mg
2
6
14
20
10
10
16
10
26.94 ⫾ 0.34
32.03 ⫾ 0.57
38.03 ⫾ 0.43
37.44 ⫾ 0.64
47.07 ⫾ 0.61
53.70 ⫾ 1.41
55.70 ⫾ 1.01
50.98 ⫾ 1.51b
2
6
14
20
9
13
22
19
28.53 ⫾ 0.79
35.44 ⫾ 0.42a
34.25 ⫾ 0.60 a
31.12 ⫾ 0.56 a,b
66.06 ⫾ 2.68a
77.40 ⫾ 1.79a
64.36 ⫾ 1.41a
56.61 ⫾ 1.50a,b
BL10
CSA, mm2
Po, mN/mm2
11.97 ⫾ 0.35
13.90 ⫾ 0.77
13.44 ⫾ 0.20
13.00 ⫾ 0.18
1.89 ⫾ 0.06
2.12 ⫾ 0.12
2.06 ⫾ 0.03
2.12 ⫾ 0.03
184.71 ⫾ 5.32
185.38 ⫾ 6.96
201.13 ⫾ 7.43
182.03 ⫾ 4.70
13.62 ⫾ 0.49a
16.73 ⫾ 0.42a
17.24 ⫾ 0.48a
15.95 ⫾ 0.33a,b
2.32 ⫾ 0.08 a
2.57 ⫾ 0.07a
2.61 ⫾ 0.07a
2.42 ⫾ 0.05a,b
147.53 ⫾ 5.39a
138.51 ⫾ 5.67a
121.24 ⫾ 7.29a
134.39 ⫾ 5.90a
mdx
Values are means ⫾ SE; n ⫽ no. of mice. TA, tibialis anterior; EDL, extensor digitorum longus; CSA, cross-sectional area aSignificantly different from that
of the age-matched BL10 controls. bSignificantly different from that of 14-m-old mice of the same strain. Po, normalized tetanic force.
J Appl Physiol • VOL
sodium cacodylate, 130 sucrose and 10 ␤-mercaptoethanol), the EDL
muscle was postfixed in a secondary fixative containing 1% OsO4 in
␤-ME buffer for 2 h. The muscle was washed 3 ⫻ 15 min in distilled
water, dehydrated with acetone, and infiltrated in Epon/Spurr’s resin
for 4 days. Finally, the EDL muscle was cut in the middle and cured
in Epon/Spurr’s resin overnight at 60°C. Longitudinal thick sections
(2 ␮m) were stained with toluidine blue. Longitudinal thin sections
(85 nm) were stained with uranyl acetate and lead citrate. The thin
sections were examined using a JEOL 1400 transmission electron
microscope (Tokyo), and images were captured using a Gatan 895
digital camera (Gatan, Warrendale, PA).
Hydroxyproline quantification. The hydroxyproline content was
measured according to our previously published protocol with modification (5). Briefly, the EDL muscle was gently dissected from the
hindlimb and secured on a Sylgard plate as described above. The
proximal and distal tendons were trimmed away and the muscle was
immediately frozen in liquid nitrogen. The muscle was lyophilized
overnight and the dry mass was determined. The lyophilized muscle
was hydrolyzed in 1 ml 6 N HCl for 3 h at 115°C and then neutralized
with 10 N NaOH to the final pH of ⬃7.5. The hydrolyzed muscle
lysate was then oxidized with chloramine-T and reacted with pdimethylamino-benzaldehyde and 60% perchloric acid. The absorbance was measured at 558 nm and the hydroxyproline concentration
was determined using a standard curve.
Statistical analysis. Data are presented as means ⫾ standard error
of the mean (SE). Statistical significance among multiple groups was
determined by two-way ANOVA followed by Turkey-Kramer post
hoc analysis using the SAS software (SAS Institute, Cary, NC). For
two-group comparison, statistical significance was determined by
Student’s t-test. Difference was considered significant when P ⬍ 0.05.
RESULTS
Body mass, muscle mass, and active muscle force in experimental mice. To compare the passive properties of the EDL
muscle between BL10 and mdx mice, we studied young
(2-mo-old), adult (6-mo-old), old (14-mo-old), and very old
(20-mo-old) male mice. BL10 and mdx mice showed significant differences in the body mass at 6, 14, and 20 mo of age
(Table 1). The muscle mass (TA and EDL) and the EDL
muscle cross-sectional area were significantly higher in mdx
mice at all ages. Very old mdx mice also showed significant
body and muscle wasting compared with that of 14-mo-old
mdx mice (Table 1). As expected, the specific active muscle
forces were significantly reduced in mdx mice (Table 1).
The mdx EDL muscle developed significantly higher passive
stresses at strains of 110 –130% Lo. Compared with that of
age-matched BL10, the mdx EDL muscle generated signif-
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exposed muscles were constantly superfused with Ringer buffer (pH 7.4;
composition in mM: 1.2 NaH2PO4, 1 MgSO4, 4.83 KCl, 137 NaCl, 24
NaHCO3, 2 CaCl2, and 10 glucose).
While viewing through a stereomicroscope (Nikon, Melville, NY),
the skin fascia and connective tissue were peeled off and the tibialis
anterior (TA) muscle was gently removed and its mass was determined (Table 1). The distal and proximal EDL tendons were carefully
exposed and tied with a 4-0 suture (SofSilk USSC Sutures, Norwalk,
CT). The proximal tendon was secured to a 305B dual-mode servomotor transducer (Aurora Scientific, Aurora, ON, Canada), and the
distal tendon was attached to a fixed post. The EDL muscle was
submerged in a 30°C jacketed organ bath containing oxygenated (95%
O2-5% CO2) Ringer buffer. After 10 min equilibration, the EDL
muscle was stimulated at the optimal length (Lo). Active tetanic
muscle force was recorded using the Lab View-based DMC program
(version 3.12, Aurora Scientific) (Table 1). The entire muscle-tendon
preparation was then subjected to a six-step passive stretch protocol.
During these stretches, no electric stimulation was applied to the
muscle. At each step, the EDL muscle was passively strained in the
increment of 10% Lo at the rate of 2 cm/s (see Supplementary Fig. S1,
available with the online version of this article) (17). In a subset group
of 14-mo-old mice, the stretch was applied at a rate of 6 cm/s. The
stress-strain response was recorded using the LabView-based DMC
software (version 3.12) and analyzed by the DMA software (version
3.12), respectively (both from Aurora Scientific). After the final
stretch (160% Lo), the EDL muscle was gently removed and pinned on
a Sylgard plate and a whole mount muscle image was captured with
a Nikon digital camera (Nikon, Melville, NY). At the end of each
experiment, the distal and proximal tendons were removed and the
EDL muscle mass was recorded (Table 1). The muscle cross-sectional
area was calculated based on a muscle density of 1.06 g/cm3 and a
fiber length-to-Lo ratio of 0.44 (6, 19).
The viscous property of the EDL muscle was determined by
studying the stress-relaxation rate when the muscle was stretched and
held at 110% Lo (12). The post-peak stresses were recorded at 0.1, 0.2,
0.5, 1 and 1.5 s. The relaxation rate at each time frame (from the peak
to 0.1 s postpeak, from 0.1 to 0.2 s postpeak, from 0.2 to 0.5 s
postpeak, from 0.5 to 1 s postpeak, and from 1 to 1.5 s postpeak) was
calculated by dividing the difference in the stress with the time
elapsed between two time points.
Electron microscopy (EM). To preserve morphology, a separate set
of muscles was used for the EM studies. The mice were anesthetized
as described above. The EDL muscle and associated tendons were
gently dissected out and pinned down on a Sylgard plate at the distal
and proximal tendons. The EDL muscle was then covered with the
primary fixative (2 % glutaraldehyde and 2% paraformaldehyde in
100 mM sodium cacodylate). Twenty minutes later, the EDL muscle
was moved to a fresh primary fixative and incubated for at least 72 h
at 4°C. After 3 ⫻ 15 min of wash with ␤-ME buffer (in mM: 100
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PASSIVE PROPERTIES OF THE mdx EDL
icantly higher stress when it was stretched to 110% of Lo
(Fig. 1). There also appeared an aging effect in mdx mice. The
differences between two strains were greater in older mice.
Specifically, the stresses generated in 2-, 6-, 14-, and 20-moold mdx mice were 22, 84, 104, and 146% higher, respectively,
than those of the same age BL10 mice at the strain of 110% Lo.
While aging appeared to have nominal influence on the stress
developed prior to the peak stress in BL10 mice, old mdx mice
(14 mo old and 20 mo old) yielded significantly higher stresses
than young mdx mice (2 mo old and 6 mo old) at the strains of
110 and 120% Lo (Fig. 1).
At the strains of 120 and 130% Lo, mdx also generated
significantly higher stresses (Fig. 1). The peak stress was
achieved at the strain of 130% Lo in BL10 mice of all ages as
well as in 2-, 6-, and 14-mo-old mdx mice. For very old (20 mo
old) mdx mice, the numerically highest mean stress value was
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Fig. 1. Age-matched comparison of the extensor digitorum longus (EDL) muscle stress-strain curves in BL10
and mdx mice. A, C, E, and G depict the stress responses
to the strains of 110 –160% of muscle optimal length (Lo)
at the rate of 2 cm/s. *Stresses developed in mdx muscles
significantly different from those of aged-matched BL10
at the same strain. #Strain at which the peak stress was
generated. †Stress developed at 160% of Lo significantly
different from that at 150% of Lo in 20-mo-old BL10
mice. At the end of the passive stretch protocol, macroscopic images of the muscle were obtained. B, D, F, and
H are the representative muscle images at ages of 2, 6, 14,
and 20 mo, respectively. In each panel, the BL10 muscle
image is on the left side and the mdx muscle image is on
the right side. The scale bar (1 mm) applies to all images.
White arrows indicate the position of the muscle failure.
Filled arrowheads indicate residual muscle attached to the
tendon. Open arrowheads indicate the region of the tendon that is completely separated from the muscle.
J Appl Physiol • VOL
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PASSIVE PROPERTIES OF THE mdx EDL
J Appl Physiol • VOL
Fig. 2. Influence of the stretch rate on the stress-strain curve in 14-mo-old
mice. A: change of the stretch rate from 2 to 6 cm/s did not significantly alter
the stress response in BL10 mice. B: at the higher rate (6 cm/s), the stress-strain
curve shifted leftward in mdx mice. *Stress is significantly different from that
at the low rate (2 cm/s).
determined by hydroxyproline quantification (Fig. 4). Compared
with mdx mice, the BL10 mice showed significantly lower hydroxyproline content. Interestingly, there was no significant difference in the hydroxyproline content among 2-, 14-, and 20-moold BL10 mice. The 6-mo-old mdx mice contained ⬃12% more
hydroxyproline than 2-mo-old mdx mice. However, the difference
did not reach statistical significance. Compared with 6-mo-old
mdx mice, the hydroxyproline content was increased by 42% in
14-mo-old mdx mice (P ⫽ 0.0003). The 20-mo-old mdx mice
showed the statistically highest hydroxyproline level (P ⱕ 0.006
compared with other groups). It was 84% higher than that of
6-mo-old mdx mice (Fig. 4).
Shifting of the muscle failure site correlated with structural
changes at the MTJ in aged mdx mice. In 2- and 6-mo-old mdx
mice and 20-mo-old BL10 mice, tearing occurred within the
proximal end of the muscle (Fig. 1). Interestingly, the failure
site shifted toward the proximal MTJ in almost all 14- and
20-mo-old mdx mice. While there were still residual pieces of
muscle attached to the proximal tendon, the far end of the
proximal tendon appeared to have completely separated from
the muscle (Fig. 1F and H, left panels).
To determine whether MTJ insufficiency is responsible for
the failure site shift in aged mdx mice, we examined the MTJ
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at the strain of 130% Lo. However, this was not significantly
different from that at the strain of 120% Lo. Hence, the peak
stress was shifted leftward in very old mdx mice. Interestingly,
the overall peak stress value was not influenced by the age in
either BL10 or mdx. Mdx showed an average peak stress of
644.3 ⫾ 16.7 mN/mm2. This is significantly higher than that of
BL10 (451.5 ⫾ 8.3 mN/mm2, P ⬍ 0.0001) (Fig. 1).
The postpeak stresses dropped rapidly in the mdx but not
BL10 EDL muscle. In mdx mice, the stress declined significantly when the muscle was stretched beyond 130% of Lo. In
2-mo-old mdx mice, a 25% drop was seen from the strain of
130 to 140% of Lo. The magnitude of the drop became less
afterward. In 6- to 20-mo-old mdx mice, a 30 to 60% drop was
observed at each step of strain from 130 to 160% of Lo. In
BL10 mice, a significant stress reduction was only found in the
oldest age group (20 mo old) at the strain of 160% of Lo. For
2-, 6-, and 14-mo-old BL10 mice, the stress stabilized at the
plateau after it attained the peak (Fig. 1).
Consistent with the stress-strain results, macroscopic examination
also revealed different levels of muscle tearing in the stretched mdx
EDL muscle. In 2-mo-old mdx mice, a partial tear was observed at
the proximal end of the muscle (Fig. 1B, right panel). At 6 mo of age,
the separation appeared to have crossed the entire muscle belly
although there were still substantial attachments (Fig. 1D, right
panel). In 14- and 20-mo-old mdx mice, the mdx EDL muscle was
essentially torn apart except for a few residual connections (Fig. 1,F
and H). In contrast, passive stretches resulted in minimal visible
changes in the EDL muscles of 2-, 6-, and 14-mo-old BL10 mice. At
these ages, the muscle remained intact (Fig. 1, B, D, and F). By 20 mo
of age, partial tears similar to those seen in 2-mo-old mdx mice were
observed at the proximal end of the muscle in BL10 mice (Fig. 1H,
left panel).
Increasing the stretch rate resulted in a leftward shift of the
mdx stress-strain relation. To further study the influence of dystrophin deficiency on the elastic property, we evaluated the stressstrain curve at a higher stretch rate in 14-mo-old mice. In 14-mo-old
BL10 mice, the stress-strain curve was minimally altered when the
rate was increased from 2 to 6 cm/s (Fig. 2A). However, we observed
a dramatic leftward shift of the stress-strain curve when the increased
stretch rate was applied to 14-mo-old mdx mice (Fig. 2B). At the
higher rate, the peak stress was achieved at a lower strain. At the
strain of 120% Lo, the stress developed at 6 cm/s was significantly
higher than that at 2 cm/s (Fig. 2B).
The viscous property of the mdx EDL muscle was altered.
The viscous property of the EDL muscle was determined by
measuring the stress-relaxation rate while the EDL muscle was
stretched to and held at 110% Lo (Fig. 3). We calculated the
stress-relaxation rate from the peak stress (time 0) to 0.1 s, 0.1
to 0.2 s, 0.2 to 0.5 s, 0.5 to 1 s, and 1 to 1.5 s for all age groups.
In 2-mo-old mice, the mdx EDL muscle showed slightly but
significantly higher relaxation rate from the peak to 0.1 s.
However, there were no significant difference between mdx
and BL10 thereafter. In 6- to 20-mo-old mice, mdx consistently
showed significantly higher stress-relaxation rate at all time
frames measured. Interestingly, as mice got older, the difference between mdx and BL10 also became greater. For example, from the peak to 0.1 s, the relaxation rate of mdx mice
were 14, 56, 65, and 125% higher than that of BL10 mice at 2,
6, 14, and 20 mo, respectively (Fig. 3B).
The mdx EDL muscle was significantly more fibrotic. The
amount of fibrotic tissue deposition in the EDL muscle was
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PASSIVE PROPERTIES OF THE mdx EDL
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Fig. 3. Age-matched comparison of the viscous property between BL10 and mdx at
110% of Lo. A: absolute stress decay at 0.1,
0.2, 0.5, 1, and 1.5 s after the peak stress
(time 0). A rapid drop is observed within the
first 0.2 s in both BL10 and mdx. B: stress
relaxation rates at different time frames after
the peak stress. *Significantly different from
that of BL10.
ultrastructure by EM. To avoid potential influence of the
stretch protocol on the morphology, a separate set of muscles
was used for the EM studies. We first examined the size and
the shape of the digitlike muscle protrusions. We observed
small and large bundles of protrusions in both BL10 and mdx
mice in all age groups (Fig. 5A; Supplementary Fig. S3).
Interestingly, at the tip of each protrusion some showed deep
digitlike invasions while others were relatively flat. There was
neither an age nor a disease-associated trend (Fig. 5B). Quantification of the invasion depth showed no significant difference between 14-mo-old BL10 and age-matched mdx mice
(Supplementary Fig. S2).
J Appl Physiol • VOL
In 2- to 14-mo-old BL10 MTJ, thin filaments associate laterally
with the sarcolemma. As a result, the membrane at the digitlike
muscle protrusions appeared thick and dense (such as the one
shown for 14-mo-old BL10 MTJ in Fig. 5C, top left panel). While
sarcolemmal myofibril condensation was preserved at the MTJ in
all young mdx mice (Fig. 5C, top right panel, an example of
2-mo-old mdx) and occasionally some old mdx mice (Fig. 5C,
bottom left panel, an example of 14-mo-old mdx), this structural
feature was lost in many MTJs in aged mdx mice (Fig. 5C, bottom
right panel, an example of 14-mo-old mdx). In these cases, thin
filaments were either partially or completely replaced by vacuolelike degenerative structures (Fig. 5C, bottom right panel).
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PASSIVE PROPERTIES OF THE mdx EDL
DISCUSSION
Essentially all teenage DMD patients are wheelchair bound.
Several factors may have contributed to immobilization. These
may include muscle wasting, the loss of contractility, MTJ damage, and increased muscle stiffness. While pathogenic significance of muscle wasting and force loss have been well established, fewer studies have examined muscle passive properties
and the MTJ strength. Herein, we hypothesize that dystrophindeficient muscle pathology may alter muscle elasticity and viscosity and compromise the strength of the MTJ. To test this
hypothesis, we examined the stretch-induced stress responses of
noncontracting EDL muscle in 2-, 6-, 14-, and 20-mo-old male
mdx and BL10 mice. We also quantified muscle fibrosis. We
found that mdx muscle was stiffer and displayed an altered
viscous property. Further, mdx muscle was significantly more
fibrotic. We also observed structural alterations and weakened
connection of the MTJ in 14- and 20-mo-old mdx mice.
Our results corroborated findings reported in DMD patients (8, 9).
Cornu et al. (8, 9) compared muscle stiffness of the knee extensors
and elbow flexors in healthy and DMD boys. They found that DMD
muscles were significantly stiffer than normal muscles. As disease
progresses, muscle stiffness was further increased (8, 9).
Mdx mice are the most widely used animal model in DMD
studies. However, it has been debated whether the passive properties of mdx muscle are altered. Berquin et al. (2a) reported that
2-mo-old mdx EDL muscle generated higher stress than that of
age-matched BL10 mice at a strain of 115% Lo. Unfortunately,
only very few mice of unknown sex were studied (n ⫽ 5 for BL10
and n ⫽ 4 for mdx) and there was no statistical analysis on the
data. Law et al. (17) compared EDL muscle passive stresses in
11-mo-old BL10 (n ⫽ 3) and mdx (n ⫽ 4) mice (sex not
mentioned). The muscle was stretched to the point of complete
tearing at the rate of 2 cm/s. Although the mdx muscle developed
22% higher stress than that of the BL10 EDL muscle at the failure
point, the difference was not significant (17). Bobet et al. (3)
examined passive stress of the EDL muscle in 20- to 21-mo-old
female mdx and BL10 mice. The muscle was stretched in a 20°C
J Appl Physiol • VOL
organ bath at a rate of 10 cm/s for either 1 mm (⬃8% of Lo) or 2.5
mm (⬃20% of Lo). The mdx and BL10 muscles developed
identical stresses when stretched for 1 mm (small stretch). For the
2.5 mm stretch, mdx muscles appeared to develop higher stresses
but these were not statistically different (3). To study whether
dystrophin deficiency affects the passive properties of the EDL
muscles in very young mice, Wolff et al. (32) applied a single (or
a series of single) very mild stretches at the strain of 105% Lo and
the stretch rate of 1.5 Lo/s (⬃0.15 cm/s) in 2-, 3-, 4-, and 5-wk-old
mdx and BL6 mice. Comparison of the pre- and poststretch
tetanic forces suggested that muscle was not damaged by this
stretching protocol. Interestingly, mdx and BL6 mice showed
similar passive properties under this experimental condition (32).
Our study confirmed and extended the preliminary findings
by Berquin et al. (2a).Using a larger sample size, broader age
range, and more comprehensive experimental approach, we
demonstrated that 2- to 20-mo-old male mdx EDL muscles
were significantly stiffer than age- and sex-matched BL10 EDL
muscle. Law et al. (17) showed a similar trend in 11-mo-old
mdx mice. We suspect that the small sample size used by Law
et al. (17) may have limited their statistical power. Results
from Bobet et al. (3) and Wolff et al. (32) are quite interesting.
The differences in the sex (3), age (32), and assay temperature
(3) are some obvious factors that may partially explain the
discrepancy between our results and these reported by Bobet et
al. (3) and Wolff et al. (32). Another important factor is the
conditions used in passive stretch. In our study, the EDL
muscle was stretched with an initial strain of 110% of Lo and
the strain was then increased by an increment of 10% of Lo in
subsequent stretches until it reached 160% of Lo. This allowed
us to evaluate the full spectrum of stress development during
mild, moderate, and extreme length changes. Our data suggest
that the stress developed prior to the peak is directly proportional to the length stretched. Low strain resulted in low stress
(Fig. 1). A similar trend was observed by Bobet et al. (3). The
stresses developed at 2.5 mm stretch were 100% and 40%
higher than those developed at 1 mm stretch in mdx and BL10,
respectively (3). In the study of Wolff et al. (32), a very mild
strain (105% of Lo) was applied at a very low stretch rate
(⬃0.15 cm/s). In our study, 2-mo-old mdx only yielded slightly
higher stress than that of 2-mo-old BL10 at the strain of 110%
of Lo although it is statistically significant (P ⫽ 0.037).
Considering the age effect (the older mdx mice developed
higher stress between the strain of 110 to 130% of Lo) and the
stretch rate effect (the higher rate yielded a higher stress in mdx
mice) (Fig. 1 and 2), the difference between our results and that
of Wolff et al. (32) may likely be due to the difference in the
mouse age and stretch rate used.
Skeletal muscle has both elastic and viscous properties in a
passive state (11, 22). To study viscous properties, we quantified the stress-relaxation rate at a fixed strain length of 110% of
Lo (Fig. 3) (21). Consistently, mdx muscle showed a significantly faster relaxation rate than that of age-matched BL10
muscle (Fig. 3). Further, older mdx muscles relaxed faster than
that of the younger ones (Fig. 3). This result suggests that not
only the elastic property of the mdx EDL muscle is altered;
dystrophin-deficient muscular dystrophy also profoundly influences the viscous property of the muscle.
Massive muscle structural remodeling has been demonstrated in dystrophin-deficient mice. These include myofiber
degeneration/regeneration, a switch of fast-twitch fibers to
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Fig. 4. Quantification of the hydroxyproline content. n ⫽ 7, 7, and 7 for 2-, 14-,
and 20-mo-old BL10 mice; n ⫽ 9, 7, 7, and 7 for 2, 6, 14, and 20-mo-old mdx
mice. *Significantly higher than that of BL10 mice. †Significantly higher than
that of 2- and 6-mo-old mdx mice. ‡Significantly higher than that of 14-mo-old
mdx mice.
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PASSIVE PROPERTIES OF THE mdx EDL
slow type, cytoskeleton alterations, inflammation, and fibrosis
(14, 24, 30, 31). As an initial effort to investigate the mechanism(s) underlying the observed passive property changes in
mdx muscle, we quantified the level of fibrosis (Fig. 4).
Consistent with the increased mdx muscle stiffness, the hydroxyproline content was significantly increased in mdx muscle (Fig. 4). Further, aged mdx muscle was significantly more
fibrotic than young adult mdx muscle (Fig. 4). These data
suggest that muscle fibrosis may have at least partially contributed to the increased stiffness in mdx muscle.
It has been shown that dystrophin is highly enriched at the
MTJ, the major force transmission structure between the muscle and the tendon (26). Previous EM studies in 1-wk-old to
6-mo-old mdx mice showed reduced junctional folding at the
MTJ and the loss of the lateral association of thin filament to
the sarcolemma (17, 18, 25, 28). Results from our passive
stretch assay suggest that in young adult mdx mice, the MTJ
was sufficiently strong to hold the tendon and muscle together
under extreme strain (Fig. 1, B and D). However, as disease
progressed in aged mice, we began to see muscle failure
occurred at the proximal MTJ separating part of the tendon
from the muscle. To understand the structural basis of this
age-associated MTJ weakening, we performed a detailed EM
J Appl Physiol • VOL
study on the EDL muscle freshly isolated from a new set of
muscles. These muscles were directly fixed and embedded for
the EM study. Interestingly, we did not see a consistent pattern
on either the size of muscle protrusion or the depth of MTJ
folding. In both young and old, BL10 and mdx, we found
interfaces that were small or large, shallowly or deeply invaginated (Fig. 5, A and B). Quantification of the folding depth in
14-mo-old mice failed to reveal a statistical difference between
mdx and BL10 (see Supplementary Fig. S2, available with the
online version of this article). In support of our observation,
Miosge et al. (21a) have also shown that there is no difference
at the MTJ folding in 3-mo-old BL10 and mdx mice.
We also examined lateral associations of the thin filament to
the sarcolemma at the MTJ folding (Fig. 5C). Consistent with
previous reports (17, 18, 25, 28), thin filaments condensed to
the muscle cell membrane in BL10 mice irrespective of the age.
Surprisingly, thin filament lateral associations were apparently
intact in 2-mo-old mdx mice (Fig. 5C). Even in some regions in
14-mo-old mdx mice, we still detected thin filament lateral associations similar to those of 14-mo-old BL10 mice (Fig. 5C).
Nevertheless, we observed substantial vacuolar degeneration in
many muscle protrusions in 14-mo-old mdx mice (Fig. 5C). In
these MTJs, thin filaments were either partially or completely
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Fig. 5. Ultrastructure changes at the myotendinous junction (MTJ). A: muscle protrusions
showed variable size. M, muscle; T, tendon.
Scale bar, 2 ␮m. B: representative low-magnification EM photomicrographs showing
variations in the depth of the digitlike folding
at the muscle-tendon interface. M, muscle; T,
tendon. Scale bar, 2 ␮m. C: representative
high-magnification EM photomicrographs of
the myofiber digitlike processings at the MTJ
in 14-mo-old BL10 (top left panel), 2-mo-old
mdx (top right panel), and 14-mo-old mdx
(relatively normal, bottom left panel; degenerated, bottom right panel). Arrow, lateral
condensation of the thin filament at the sarcolemma; arrowhead, vacuolar degeneration.
M, muscle; T, tendon. Scale bar, 200 nm.
PASSIVE PROPERTIES OF THE mdx EDL
ACKNOWLEDGMENTS
We thank Isabella Zaniletti at the Biostatistics Group, Office of Research,
University of Missouri for the help with two-way ANOVA analysis. We thank
Drs. Kerry McDonald and Ron Terjung for helpful discussions and critical
reading of the manuscript. We thank Marianne Abdo, Juveria Nayeem, and
Alexandra Kellogg for technical assistance.
GRANTS
This work was supported by grants from the National Institutes of Health
(NIH) (AR-49419, D. Duan), Muscular Dystrophy Association (D. Duan), and
NIH Training Grant T90-DK-70105 (C. Hakim).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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replaced by vacuole-like structures and the sarcolemmal lining
was disrupted. Essentially, the normal pattern of thin filament
lateral association was lost in these degenerated MTJs. We speculate that these defective MTJ may have failed to hold muscle and
tendon together and resulted in separation of the proximal tendon
from the muscle (Fig. 1, E and F).
Conclusions. Compared with that of the age-matched BL10
control, the mdx EDL muscle was significantly stiffer and the
viscosity was also altered in mdx. Muscle failure was not
observed in ⱕ14-mo-old BL10. In young (2 mo old and 6 mo
old) mdx mice and very old (20 mo old) BL10 mice, muscle
failure was found within the proximal end of the EDL muscle.
However, the failure site shifted toward the proximal MTJ in
old (14 mo old and 20 mo old) mdx mice. Electron microscopic
examination revealed substantial MTJ degeneration in old but not
young mdx mice. Taken together, we have demonstrated for the
first time that the passive properties (viscosity and elasticity) of
mdx muscle were significantly altered. Further, MTJ strength was
weakened in aged mdx mice. These findings will not only help
explain the severe dystrophic phenotype in aged mdx mice, but
may also shed new light on our understanding of DMD clinical
presentation. Future studies are needed to determine whether
novel gene/cell/pharmacological therapies can halt the deterioration of the passive properties and improve function.
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