EDITORIAL FOCUS
Myosin molecular motor dysfunction in dystrophic
mouse diaphragm
Catherine
Coirault1,
Francine
Lambert1,
Sylvain
Marchand-Adam1,
Pierre
Attal2,
Denis
Chemla3, and
Yves
Lecarpentier3
1 Institut National de la
Santé et de la Recherche Médicale U451-LOA-Ensta-Ecole
Polytechnique, 91761 Palaiseau Cedex; and Services
2 d'ORL and
3 d'Explorations
Fonctionnelles Cardiovasculaires et Respiratoires, Centre
Hospitalier et Universitaire de Bicêtre, Assistance
Publique-Hôpitaux de Paris, 94275 Le Kremlin-Bicêtre,
France
 |
ABSTRACT |
Cross-bridge properties and myosin heavy chain (MHC) composition
were investigated in isolated diaphragm from 6-mo-old control (n = 12) and
mdx
(n = 12) mice. Compared with control,
peak tetanic tension fell by 50% in
mdx mice
(P < 0.001). The total number of
cross bridges per square millimeter
(×109), the elementary
force per cross bridge, and the peak mechanical efficiency were lower
in mdx than in control mice (each
P < 0.001). The duration of the
cycle and the rate constant for cross-bridge detachment were
significantly lower in mdx than in
control mice. In the overall population, there was a linear
relationship between peak tetanic tension and either total number of
cross bridges per square millimeter or elementary force per cross
bridge (r = 0.996 and
r = 0.667, respectively, each
P < 0.001). The
mdx mice presented a higher proportion
of type IIA MHC (P < 0.001) than
control mice and a reduction in type IIX MHC
(P < 0.001) and slow
myosin isoforms (P < 0.01) compared
with control mice. We concluded that, in
mdx mice, impaired diaphragm strength
was associated with qualitative and quantitative changes in myosin molecular motors. It is proposed that reduced force generated per cross
bridge contributed to diaphragm weakness in
mdx mice.
mdx mice; cross bridge; muscle
efficiency; skeletal muscle
 |
INTRODUCTION |
THE MDX mouse is a widely used animal
model of human Duchenne muscular dystrophy, since a point mutation in
the dystrophin gene results in lack of dystrophin (11). In skeletal
muscle, this cytoskeletal protein forms a mechanical link between the submembranous network of cytoskeletal actin, a complex of
membrane-bound glycoproteins and the extracellular matrix (32).
Disruption of this linkage is presumed to cause sarcolemmal
instability, stress-induced rupture of the sarcolemma, and/or excessive
Ca2+ influx through altered ion
channels (8, 32). This results in a progressive replacement of
contractile muscle fibers by fat or connective tissue that is generally
thought to account for severe muscle atrophy and weakness (24). Recent
studies, however, have shown that the decrease in diaphragm strength in
mdx mice is more pronounced than that
predicted by the fibrosis area (18, 22). This suggests that, in
addition to a reduction in contractile tissue, qualitative alterations
of the contractile apparatus may also contribute to diaphragm weakness.
However, the extent to which diaphragm mechanical changes in
mdx mice reflect alterations in the
molecular mechanics of myosin remains to be determined.
In striated muscles, cross bridges represent the molecular motors of
force generation. According to the most widely accepted theory of
contraction (12), cross bridges act as independent force generators.
Therefore, muscle force depends on the elementary force produced per
cross bridge and the total number of cross bridges formed (12, 13). The
aim of our study was to determine the number, kinetics, and single
force of cross bridges in diaphragm from
mdx mice. Huxley's equations (12)
were used to calculate the single force of cross bridges, total number
of cross bridges, rate constant for attachment and detachment, peak
mechanical efficiency, and total duration of the cross-bridge cycle (5,
16, 17) in control and mdx mouse
diaphragm. We also studied the myosin isoform composition in control
and mdx diaphragm. We tested two hypotheses: 1) that changes in
diaphragm tension in dystrophic mice were related to modifications in
the total number of cross bridges and/or in the amount of force
generated per cross bridge and 2)
that changes in the kinetics of the cross-bridge cycle were associated
with changes in myosin isoform expression.
Glossary
Lo |
Optimal initial muscle length
|
s |
Resting sarcomere length at
Lo
|
Po |
Maximum isometric tension
|
Vmax |
Maximum shortening velocity
|
x |
Instantaneous movement of the myosin head relative to actin (0 x h)
|
h |
Step size of the cross bridge
|
l |
Distance between two actin sites
|
f1 |
Maximum value of the rate constant for cross-bridge attachment at
x = 0
|
g1 |
Maximum value of the rate constant for cross-bridge detachment at
x = 0
|
g2 |
Maximum value of the rate constant for cross-bridge detachment at
x h
|
m |
Cross-bridge number per mm2
(× 109) at
Po
|
 |
Elementary force per cross bridge
|
Effmax |
Peak mechanical efficiency
|
MHC |
Myosin heavy chain
|
 |
MATERIALS AND METHODS |
Experimental Protocol
Experiments were conducted on 6-mo-old male
mdx mice
(n = 12) and age-matched
control mice (C57BL/10ScSn, n = 12)
obtained from Jackson Laboratory (Bar Harbor, ME). Care of the animals conformed to the Helsinki Convention. After anesthesia with
pentobarbital sodium (40 mg/kg body wt ip), the animals were subjected
first to a laparotomy and then to a thoracotomy. A strip of the ventral part of the costal diaphragm was carefully dissected out from the
muscle in situ. The insertions on the central tendon and ribs were kept
intact. The diaphragm strip was rapidly mounted in a tissue chamber
containing Krebs-Henseleit solution (in mM): 118 NaCl, 24 NaHCO3, 4.7 KCl, 1.2 MgSO4 · 7 H2O, 1.1 KH2PO4,
2.5 CaCl2 · 6 H2O, and 4.5 glucose. The solution
was bubbled with 95% O2-5% CO2 and maintained at 26°C and
pH 7.4. The costal end of the muscle strip was held in a stationary
clip at the bottom of the chamber; the central tendon end was
maintained with a second clip, attached to an electromagnetic
force-transducer device. After a 15-min equilibration period the muscle
was supramaximally stimulated via two platinum electrodes arranged
longitudinally on either side of the muscle. A force-frequency curve
was determined by stimulating muscle strips at 25, 33, 50, 75, 100, and
200 Hz (train duration 300 ms, 10/min). Maximum isometric tension
(Po) was generally achieved at a
stimulation frequency of 100 Hz. Experiments were carried out at
Lo, i.e., the
initial resting length corresponding to the apex of the initial
length-active tension curve. At the end of the experiment, the
cross-sectional area (in mm2)
was calculated from the ratio of muscle weight to muscle length at
Lo, with the
assumption of a muscle density of 1. The electromagnetic lever system
has been described elsewhere (5, 16, 17). Lo
was 6.8 ± 0.4 and 7.1 ± 0.3 mm in
control and mdx diaphragm muscle
strips, respectively (not significant).
Mechanical Parameters
Force-velocity relationship.
The peak velocity (V) of 10 afterloaded contractions was plotted against the isotonic load level
normalized per cross-sectional area (P), obtained by successive load
increments from zero load up to the isometric tension.
Po, i.e., peak force normalized
per cross-sectional area, was measured from the fully isometric
contraction (in mN/mm2). Maximum
unloaded shortening velocity
(Vmax, in
Lo/s) was
measured from the contraction abruptly clamped to zero load just after stimulus. The experimental P-V
relationship was fitted according to Hill's equation (10): (P + a)(V + b) = (cPmax + a)b, where
a and
b
are the asymptotes of the hyperbola, as determined by multilinear
regression and the least-squares method, and
cPmax is the calculated Po at
V = 0. The curvature G of the P-V relationship is
equal to cPmax/a.
Cross-bridge number and kinetics.
Huxley's equations were used to calculate the rate of total energy
release (E, in
mW/mm2), the isotonic tension
(PHux, in
mN/mm2), and the rate of
mechanical energy
(WM, in
mW/mm2) as a function of
V (12).
E is given as
|
(1)
|
where
ms/2 is the number of cross bridges
per square millimeter at Po (12),
s is the resting sarcomere length at
Lo,
f1 is the maximum
value of the rate constant for cross-bridge attachment, and
g1 and
g2 (in
Eq. 2) are the peak values of the
rate constants for cross-bridge detachment (12). The
instantaneous movement (x) of the
myosin head relative to actin varies from 0 to
h, the step size of the cross bridge,
which is defined by the translocation distance of the actin filament
per ATP hydrolysis and produced by the swing of the myosin head (6,
13); f1 and
g1 correspond to
x = 0, and
g2 corresponds to
x
h (12);
e is the free energy required to split
one ATP molecule (6, 12, 13), l is the distance between two actin sites, and
= (f1 + g1)h/2 = b (12). For reasons of equation
dimensions,
was multiplied by s/2
compared with the initial hypothesis (12). Consequently, calculations of f1,
g1, and
g2 were divided
by s/2 compared with those previously detailed (5, 16, 17) and are given by the following
equations
|
(2)
|
|
(3)
|
|
(4)
|
The
maximum value of total energy release
(Emax) occurs
at Vmax. The
minimum value of the rate of total energy release
(E0, in
mW/mm2) occurs in isometric
conditions; E0 is
equal to a × b (12, 30) and is also given by the
following equation
|
(5)
|
The
maximum turnover rate of myosin ATPase per site in isometric conditions
(kcat, in
s
1) is
E0/(ems/2)
(12)
|
(6)
|
The
total duration of the time cycle (tc) is
tc = 1/kcat. PHux
is given by the following equation (12)
|
(7)
|
where
w is the mechanical work of a single
cross bridge. The elementary force per single cross bridge in isometric
conditions (
, in pN) is
= PHux max/(ms/2)
|
(8)
|
The rate of mechanical work (WM) is
WM = PHux · V.
At any given load, the mechanical efficiency (Eff) of the muscle is
defined as the ratio of
WM to
E (12): Eff = WM/E,
and Effmax is the maximum value of Eff.
The accuracy and reliability of Huxley's parameters depend on how well
the experimental data can be fitted to Huxley's equations. The
validity of each of the mathematical fits was checked as previously recommended (16).
Values of Huxley's equation constants.
Stroke sizes ranging from 5 to 11 nm have been determined by using
optical tweezers (7, 19). These values correspond to the
three-dimensional structure of the myosin head (23) and support a
one-to-one coupling for ATP energy transduction, which is inherent to
Huxley's theory (6, 12, 13). In our study we chose a stroke size value
(h) of 11 nm;
l = 36 nm (30). The free energy
required to split one ATP molecule per contraction site
(e) is 5.1 × 10
20 J. Because
w is
0.75e (12),
w = 3.8 × 10
20 J.
Myosin Electrophoresis
Preparations of crude myosin were obtained from the ventral part of the
costal diaphragm, as previously described (5). Electrophoresis was
performed in a Bio-Rad Mini-Protean II Dual Slab Cell electrophoresis
system for 32 h at 4°C and 70 V (constant voltage). Long-duration
electrophoresis was performed to discriminate between developmental
(i.e., embryonic and neonatal) and adult heavy chains (1, 14). Control
electrophoresis showing developmental myosin heavy chains (MHCs) from
newborn mouse (1 day postnatal) skeletal muscles was also performed.
MHCs were separated in dissociating conditions with 0.75 mM SDS-PAGE
minigel electrophoresis (20). The stacking gel was composed of 4%
acrylamide (2.67% bisacrylamide), 70 mM Tris (pH 6.8), 30% glycerol,
4 mM EDTA, and 0.1% SDS. The composition of the separating gel was 8%
acrylamide (1% bisacrylamide), 0.2 M Tris, pH 8.8, 0.1 M glycine, and
0.4% SDS. Separate upper and lower running buffers were used. The
upper running buffer consisted of 0.1 M Tris (base), 150 mM glycine,
11.5 mM 2-mercaptoethanol, and 0.1% SDS. The lower running buffer
consisted of 50 mM Tris (base), 75 mM glycine, and 0.05% SDS. Both
buffers were prepared shortly before use and cooled at 4°C. Gels
were stained with 0.2% Coomassie blue, 50% ethanol, and 10% acetic
acid and destained with 5% ethanol and 5% acetic acid. The different
MHC isoforms were quantified by one-dimensional densitometry (model
GS-690, Bio-Rad, Hercules, CA) with Macintosh software for Bio-Rad's
image analysis system. The amount of each isoform was determined by the
area of each peak. Data are expressed as percentages of the area of
each peak divided by the sum of the areas of all peaks. It
is widely considered that these methods provide reliable, reproducible data (1, 14). It has been suggested that an MHC band is detectable when
present in ~1% of the sample (3). Identification of the specific
myosin isoform bands was based on previous studies (1, 14, 20).
Statistical Analysis
Values are means ± SE. After ANOVA, comparisons between groups were
performed using Student's unpaired
t-test. All comparisons were
two-tailed, and P < 0.05 was
considered statistically significant.
 |
RESULTS |
Contractile Performance of the Diaphragm
Mechanical parameters of the diaphragm are presented in Fig.
1.
Po was markedly depressed in
mdx mice compared with age-matched controls (50% difference between mdx
and control, P < 0.001). In
addition, there was an ~30% reduction in
Vmax in the
mdx mice (P < 0.001).

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Fig. 1.
Mechanical parameters in control (C) and
mdx mouse diaphragm.
Po, peak isometric force
normalized per cross-sectional area;
Vmax, maximum
unloaded shortening velocity;
Lo, optimal
muscle length at which active tension is maximum. Values are means ± SE (n = 12 in each group).
P < 0.001 compared with
controls.
|
|
Diaphragm Cross-Bridge Mechanics
The mdx mouse diaphragm exhibited an
~48% reduction in the total number of cross bridges per square
millimeter compared with controls (P < 0.001; Fig. 2). Moreover,
was
significantly lower in mdx than in
control mice (5% difference between
mdx and control, P < 0.001; Fig. 2).
Cross-bridge kinetics in control and
mdx mice are presented in Fig.
3. The total duration of the cross bridge cycle (tc) was
significantly shorter in mdx than in
control mice (P < 0.05; Fig. 3).
There was no difference in
f1 between groups (Fig. 3). In mdx mice,
g2 was
significantly lower than in control mice
(P < 0.001; Fig. 3). Conversely,
g1 was
significantly higher in mdx than in
control mice (P < 0.05; Fig. 3). The
G curvature of the force-velocity hyperbola was
significantly lower in mdx than in
control mice (9.5 ± 0.7 vs. 6.4 ± 0.3, P < 0.001). Moreover, Effmax and the relative tension at
which Effmax occurred were significantly lower in mdx than in
control mice (P < 0.001 and P < 0.05, respectively; Fig.
4).

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Fig. 2.
Total number of cross bridges (m)
and elementary force per cross bridge ( ) in control and
mdx mouse diaphragm. Values are means ± SE (n = 12 in each group).
P < 0.001 compared with
controls.
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Fig. 3.
Cross-bridge kinetics in control and
mdx mouse diaphragm.
tc, Total
duration of cross-bridge cycle;
f1, peak value
for rate constant for cross-bridge attachment;
g1 and
g2, peak values
for rate constants of detachment. Values are means ± SE
(n = 12 in each group).
* P < 0.05;
P < 0.001 compared with
controls.
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Fig. 4.
Energetics in control and mdx mouse
diaphragm: peak mechanical efficiency
(Effmax) and percentage of
tension (P) at which Effmax
occurred. Values are means ± SE (n = 12 in each group). * P < 0.05; P < 0.001 compared with controls.
|
|
Relationships Between Parameters
In the overall population,
Po was linked to
the total number of cross bridges and
. Indeed, there was a strong
linear relationship between Po and
the total number of cross bridges per square millimeter (P < 0.001; Fig.
5A): the
greater the number of cross bridges, the higher was
Po. There was also a linear
relationship between Po and
(P < 0.001; Fig.
5B). However, when separate
correlations for mdx and control mice
were performed, there was no longer any correlation between
Po and
.

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Fig. 5.
Relationship between
Po and
m or . In control and
mdx mouse diaphragm
together, Po = 9.0m 1.8 (r = 0.996, P < 0.001); in addition,
Po = 76.4 631.2 (r = 0.667, P < 0.001). Within
groups, there was a close linear relationship between
Po and
m in control
(Po = 8.8m + 1.1, r = 0.981, P < 0.001) and
mdx mice
(Po = 8.5m + 1.0, r = 0.999, P < 0.001).
Conversely, there was no correlation between
Po and within groups.
|
|
Myosin Isoform Composition
The electrophoretic separation of the different MHC isoforms present in
normal and mdx mouse diaphragm is
shown in Fig. 6. The
mdx diaphragm contained a greater
amount of type IIA MHC than controls (60 ± 1 vs. 37 ± 1%,
P < 0.001), whereas the proportion of type IIX MHC was significantly lower (36 ± 2 vs. 55 ± 2, P < 0.001). In addition, there was a
lower proportion of slow myosin isoforms in
mdx than in control mice (3 ± 1 vs. 5 ± 1%, P < 0.01). Type IIB myosin isoforms, which represented 3 ± 1% of MHC
in control diaphragm, represented only 1 ± 1% of MHC in
mdx mouse diaphragm (P < 0.01). None of the adult
diaphragm muscles demonstrated reinduction of developmental MHC
isoforms (Fig. 6). In the overall population, there was a negative
correlation between the proportion of type IIA MHC and both
Vmax and
Po
(r =
0.83 and
r =
0.82, respectively), whereas the proportion of type IIX MHC was positively correlated to
Vmax and
Po
(r = 0.87 and
r = 0.73, respectively). However, when
separate correlations for mdx and
control mice were performed, there was no correlation between the
proportion of type IIA or IIX MHC isoforms and either
Vmax or
Po.

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Fig. 6.
Typical electrophoretic profiles of myosin heavy chain (MHC) in control
and mdx mouse diaphragm. MHC isoforms
were also obtained from newborn (NB) mouse skeletal muscles.
Developmental MHC band (Dev) does not migrate at same position as type
IIX MHC band, as previously reported (1, 14). Developmental MHC
isoforms were not observed in diaphragm from 6-mo-old
mdx mouse.
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|
 |
DISCUSSION |
We demonstrated that, in mdx mice,
diaphragm dystrophy was associated with functional and quantitative
changes in myosin molecular motors. Reduced diaphragm strength was
associated with a decrease in the number of cross bridges generating
contractile force and also in the elementary force generated per
actomyosin interaction. In mdx mouse
diaphragm, there were changes in the cross-bridge kinetics and a
reduction in peak mechanical efficiency. Molecular myosin dysfunction
was associated with changes in myosin isoform pattern, mainly
characterized by a shift from the type IIX to the type IIA MHC isoform.
Relevance of the Experimental Model
In our study, cross-bridge kinetics and
were calculated from
mechanical data in isolated diaphragm muscle by using Huxley's classic
equations (5, 12, 16, 17). In muscle strips, series compliance and
muscle fiber heterogeneity may affect mechanical properties and
cross-bridge cycling kinetics. Given that impaired muscle compliance
has been reported in mdx diaphragm
(24), we cannot totally exclude the possibility that the small
difference in the calculated unitary force per cross bridge may be due
entirely to the differences in the passive properties of the tissue. It is important to note that experiments designed to apply the principles of Huxley's theory were performed on isolated frog sartorius muscles, and not on isolated fibers (12). The equations can therefore be applied
to multicellular preparations such as diaphragm muscle strips. The
question arises as to whether Huxley's model can be applied to muscles
with heterogeneous MHC isoform composition. In his paper
(12), Huxley demonstrates that his equations accurately fit the
force-velocity characteristics shown by Hill in frog sartorius muscle
(10). The model thus accurately fits the mechanical properties of a
muscle that is composed of different MHCs. In heterogeneous muscle the
force-velocity characteristics are thought to reflect the relative
contribution of each fiber type (30). Likewise, according to Huxley's
equations (12), cross-bridge characteristics are thought to reflect the
average value of the myosin molecular motors.
In normal skeletal muscle, novel methodologies such as optical tweezers
(7, 19, 21) and glass needle techniques (31) have enabled direct
measurement of cross-bridge unitary force. In these studies the single
force produced by a unitary myosin head was found to vary from 1 to 9 pN. The cross-bridge single force reported in our study was of the same
order of magnitude as that previously measured in skeletal muscle
myosins (7, 19, 31).
Force and Number of Cross Bridges
According to the theory of muscle contraction (12, 13),
Po is related to
m ×
. Our results showed that
diaphragm from mdx mice exhibited an
~50% reduction in m, a finding in
agreement with the decrease in viable contractile tissue reported in
mdx diaphragm (18, 22, 24). Moreover,
we found that the elementary force produced per myosin head was lower
in diaphragm from mdx mice than from
controls (Fig. 2). Although moderate (~5%), the difference in force
produced per cross-bridge interaction between control and
mdx mouse diaphragm was highly
significant. Moreover, linear relationships between muscle tension and
either
or m suggested that
decreases in m and
accounted for
the impaired respiratory muscle function in the dystrophic mouse (Fig.
5). This result is in good agreement with the proposal that
"viable" fibers from mdx mouse
diaphragm have an impaired capacity to produce force (18, 22).
Theoretically, reduced elementary force produced per myosin head may
result from molecular modifications of the myosin head structure and/or
from functional alterations in the actomyosin interaction (26). It has
been suggested that different myosin isoforms differ substantially in
their force-generating capacities (4, 9, 22). In 3-mo-old
mdx mouse diaphragm, a transitory expression of embryonic myosin isoforms has been found, which might
contribute to the weaker force observed (22). However, in 6-mo-old
mdx mouse diaphragm, developmental MHC
isoforms were not observed (Fig. 6), as previously reported in older
mdx mouse diaphragm (22).
Alternatively, one can hypothesize that changes in the relative
proportion of adult myosin isoforms observed in mdx mouse diaphragm may help explain
changes in cross-bridge force. Compared with controls, 6-mo-old
mdx diaphragm exhibited an increase in
type IIA MHC and a corresponding decrease in type IIX MHC, with small
changes in the fast-to-slow MHC ratio. In skeletal muscle the amount of
force generated by slow myosin isoform has been reported to be higher
than that produced by fast myosin isoforms (4). Conversely, no
significant difference in isometric tension has been found among fast
fiber types (4). Therefore, it is unlikely that changes in the relative
proportion of myosin isoforms would account for the reduced force per
cross bridge in the mdx mouse, even if
a substantial number of fibers expressed varying amounts of type IIA + type IIX myosins in mdx diaphragm.
Cross-Bridge Kinetics and Reduced Muscle Efficiency
In our study, Effmax was lower in
mdx than in control diaphragm, and the
percentage of total tension at peak efficiency was shifted toward lower
levels of load (Fig. 4). Similar findings have been reported in rabbit
diaphragm during congestive heart failure (16). These two abnormalities
may place the mdx mouse diaphragm in
disadvantageous energetic conditions, particularly during a breathing
effort. Moreover,
tc was
significantly shorter in mdx than in
control mouse diaphragm (Fig. 3). If it is assumed that one molecule of
ATP is hydrolyzed per cross-bridge cycle (6, 12, 13), then the overall
cycle of ATP splitting takes place more rapidly in
mdx than in control mouse diaphragm.
Accordingly, the rate of myosin ATPase activity per cross bridge (i.e.,
the reciprocal of
tc) was
expected to be higher in mdx mouse
diaphragm, consistent with the decreased
Effmax (16, 30). Reduced
efficiency of skeletal muscles and a shorter time cycle have generally
been explained by a reduced percentage of slow-twitch type I fibers and
an increased percentage of fast-twitch type II fibers (30). In
mdx mice, there was a slight but
significant decrease in the proportion of slow myosin isoforms (3%
compared with 5% in controls). However, it is unlikely that this 2%
decrease was an important factor in explaining the reduction in
Effmax and
tc. Moreover, because of the lower actomyosin ATPase activity of the type IIA MHC
isoform (25), the shift from the type IIX to the type IIA MHC isoform
in mdx mice would be expected to
prolong tc,
thereby increasing Effmax. It is
therefore unlikely that changes in the relative proportion of myosin
isoforms in the mdx mouse would account for the reduction in
Effmax and
tc.
Our results showed that
Vmax was
significantly lower in mdx than in
control mouse diaphragm.
Vmax is thought
to be governed by the rate constant of cross-bridge dissociation at the
end of the working stroke
(g2) (12). In accordance
with this theory, we found a significantly lower
g2; i.e., the
time for cross-bridge dissociation was higher in
mdx than in control mice. Several
studies have identified a correlation between
Vmax and either
maximum actin-activated myosin ATPase activity (2) or the myosin
isoform composition (3, 27). With the diversity of fast-twitch fiber subtypes taken into account, other studies have also found a
correlation between
Vmax and the
relative expression of fast myosin isoforms (3, 15). However, no
significant differences in shortening velocity between type IIA and
type IIX fibers have been reported (3). It is thus unlikely that the
higher proportion of type IIA MHC and the lower proportion of type IIX
MHC can explain the reduced
Vmax in
mdx mouse diaphragm. Different
hypotheses have been proposed to explain changes in velocity of
movement in skeletal muscle fibers. It has been suggested that myosin
light chain isoforms have a role in determining
Vmax (3, 27).
Thus we cannot exclude the possibility that changes occurred in myosin
light chain isoform composition and that these changes contributed to
the reduction in shortening velocity in
mdx mouse diaphragm. Alternatively, it
has been proposed that distinct molecular mechanisms regulate actin-activated ATPase activity and the velocity of filament movement (26, 29), so that changes in actin-activated ATP hydrolysis can occur
without significant alteration in the velocity of filament movement.
Such mechanisms may play a role in dissociating changes in shortening
velocity and actin-activated ATP hydrolysis.
Our study has certain limitations, which need to be discussed. When all
cross bridges generating contractile force were taken into account, we
found that the elementary peak force generated per myosin interaction
was lower in mdx than in control mice. Given the nonhomogeneity in mdx mouse
diaphragm, it is possible that some cross bridges were mildly affected
and thus generated normal or subnormal force per cross-bridge
interaction, whereas others were more severely affected and thus
generated very low force per cross-bridge interaction. Because the
calculated
reflected a mean value based on all the cross bridges,
it is possible that this mean value was higher than the elementary
force produced by the more severely affected cross bridges. Absence of
dystrophin is the primary defect in Duchenne muscular dystrophy and
diaphragm from mdx mice (11, 32).
Although the precise mechanism whereby lack of dystrophin causes muscle
degeneration is unknown, it is widely thought that the absence of
dystrophin directly or indirectly results in sarcolemma instability,
increased intracellular free Ca2+,
enhanced net degradation of muscle proteins, and cell necrosis (28).
Our results indicated that myosin motor dysfunction also occurred in
mdx mouse diaphragm and may, at least
in part, account for diaphragm weakness. However, the nature and train
of events that lead to myosin motor dysfunction remain to be elucidated.
In conclusion, in mdx mouse diaphragm,
the decline in the total number of cross bridges was associated with a
reduction in the elementary force produced per actomyosin interaction.
Changes in MHC isoforms may help explain some, but not all, of the
modifications in the kinetics of myosin molecular motors. Further
studies are needed to determine whether functional and/or structural
abnormalities are involved in molecular motor dysfunction in diaphragm
from mdx mice.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. Coirault,
INSERM 451-LOA-Ecole Polytechnique, Batterie de l'Yvette, 91761 Palaiseau Cedex, France (E-mail:
coirault{at}enstay.ensta.fr).
Received 30 November 1998; accepted in final form 5 August 1999.
 |
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