Milrinone inhibits contractility in skinned skeletal muscle
fibers
C. Y.
Seow,
L.
Morishita, and
B. H.
Bressler
Departments of Anatomy and Pharmacology and Therapeutics, University
of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
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ABSTRACT |
Direct action of the cardiotonic bipyridine milrinone on the
cross bridges of single fibers of skinned rabbit skeletal muscle was
investigated. At 10°C and pH 7.0, milrinone reduced isometric tension in a logarithmically concentration-dependent manner, with a
55% reduction in force at 0.6 mM. Milrinone also reduced
Ca2+ sensitivity of skinned fibers
in terms of force production; the shift in the force-pCa curve
indicated a change in the pCa value at 50% maximal force from 6.10 to
5.94. The unloaded velocity of shortening was reduced by 18% in the
presence of 0.6 mM milrinone. Parts of the transient tension response
to step change in length were altered by milrinone, so that the test
and control transients could not be superimposed. The results indicate
that milrinone interferes with the cross-bridge cycle and possibly
detains cross bridges in low-force states. The results also suggest
that the positive inotropic effect of milrinone on cardiac muscle is
probably not due to the drug's direct action on the muscle cross
bridges. The specific and reversible action of the bipyridine on muscle cross bridges makes it a potentially useful tool for probing the chemomechanical cross-bridge cycle.
cardiotonic bipyridine; force-pCa relations; tension transients; shortening velocity
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INTRODUCTION |
MILRINONE
(1,6-dihydro-2-methyl-6-oxo-3,4'-bipyridine-5-carbonitrile) and
the related bipyridine amrinone,
5-amino-(3,4'-bipyridine)-6(1H)-one, are positive cardiac inotropic agents with vasodilator properties (1,
2). The cardiovascular effects are thought to be partly due to the
bipyridines' ability to inhibit phosphodiesterase III and therefore
increase cAMP level (2, 6, 8). Other mechanisms that increase
Ca2+ influx into myocardium are
also believed to be involved (1). It has been shown that direct binding
of amrinone on skeletal muscle cross bridges potentiates isometric
force (4, 12, 13). The bipyridine's inotropic property, however, is
probably not due to the compound's direct interaction with the
contractile proteins, because the binding also results in decreased
shortening velocity and hence decreased power output of the muscle (4). One interesting observation made in these studies is that amrinone appears to be able to detain cross bridges in high-force states in the
ATPase cycle. Very few compounds are known to have this property. The
bipyridines are therefore potentially useful tools for studying the
chemomechanics of the actomyosin cross-bridge interaction. Direct
effects of milrinone on muscle cross-bridge cycle have not been
studied. One objective of the present study is to determine whether
milrinone's direct action on the cross bridges could contribute to the
inotropic effect of the drug. Another objective of this study is to
evaluate milrinone as a potential probe for investigating the
chemomechanics of muscle cross-bridge cycle.
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METHODS |
Muscle preparation.
Adult New Zealand White rabbits weighing 2-3 kg were killed in a
carbon monoxide chamber. Bundles of muscle fibers were then dissected
from the psoas muscle, skinned as previously described (17). Skinned
single fibers were dissected from a bundle. The ends of the fiber
segment were fixed with glutaraldehyde and held by aluminum clips.
Solutions.
All solutions contain 56 g/l dextran T-70, sufficient KCl to bring the
ionic strength to 200 mM, sufficient
Mg2+ to yield a free ion
concentration of 1 mM, and 10 mM PIPES to buffer the solution to pH
7.0. Dithiothreitol (0.5 mM) was also added to the solutions. The
ATP-regenerating system was provided by 20 mM creatine phosphate and
the endogenous phosphocreatine kinase. Activating solution contained
Ca2+ buffered to the desired pCa
with 5 mM EGTA. Relaxing solution contained 5 mM EGTA, and a rinse
solution of 0.1 mM EGTA was used to lower the EGTA concentration
immediately before each activation. Skinning solution was the same as
the relaxing solution except that the free
Mg2+ concentration was 0.1 mM, and
the fiber was briefly (5 min) exposed to 1% Triton X-100 immediately
before the experiment. Milrinone (purchased from Sigma, St. Louis, MO)
was made into a 60 mM stock solution by dissolving the chemical into
150 mM HCl solution. The milrinone stock was later diluted to desired
concentrations in relaxing, rinse, and activating solutions. The same
amount of HCl (without milrinone) was added to the corresponding
control solutions, and the pH of both control and test solutions was
then adjusted back to 7.0 with KOH.
Experimental procedure.
The clipped fibers were attached to a force transducer and servomotor
horizontally in a trough containing experiment solutions. A circulating
coolant underneath the trough maintained the temperature of the
solution at 10°C. The photoelectric force transducer had a
resonance frequency of 18 kHz, a sensitivity of 10 mV/mg, and a
signal-to-noise ratio of ~20. The transducer was of the same type
used in experiments by Seow and Ford (17). The servomotor (Cambridge
Technology model 308) was modified to achieve a faster step speed. The
modification involved replacing the aluminum motor arm with a much
lighter carbon fiber arm strengthened by a thin layer of epoxy. The
step duration of the modified motor was 160 µs for steps as large as
100 µm. Sarcomere length of resting fiber was determined with the use
of a videocamera mounted on an inverting microscope. Displaying of the
images and calculation of the sarcomere spacing were facilitated by the
OPTIMAS computer program (Optimas). The resting sarcomere length was
set to 2.6 µm. The isometrically contracted sarcomere length was
~2.5 µm. The sarcomere spacing was checked after each contraction,
and, if the spacing changed significantly, the fiber was discarded.
Test and control conditions were always studied in pairs. Fibers were
relaxed after each activation. Between contractions, several minutes
were allowed for the complete diffusion of milrinone. For tension
transient experiments, a length step was applied to the fiber at the
plateau of contraction. A large step release was applied at the end of
the transient for two purposes: one to make the fiber go slack, thus
setting zero baseline for the force transducer, and the other to
determine maximal fiber shortening velocity with the "slack
test." The size of the large end steps was varied to allow the fiber
to take up the slack after a brief period of time, and the time of
tension upstroke was determined. A plot of the step size vs. time
required to take up the slack gave maximal velocity of shortening.
Hill's equation was used to fit the force-pCa curves:
y = a/[1 + 10(x
b) · n], where y was the normalized force
value, x was the pCa value, and a, b,
and n were fitting constants that represented the maximal force, the
pCa value corresponding to 50% of maximal force
(pCa50), and the Hill's
coefficient, respectively.
High-speed and high-resolution recording was achieved through the use
of a Tektronix TDS 420 digitizing oscilloscope. The maximal sampling
rate used to record the T1 and
T2 phase of a tension transient
was 40 µs/sample.
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RESULTS |
Milrinone reduced Ca2+-activated
(pCa 4.5) isometric force of skinned fibers in a logarithmically
concentration-dependent manner, as shown in the plot of
force vs. the logarithm of milrinone concentration (Fig.
1). The milrinone concentration ranged from
0.05 to 1.2 mM, and a linear relation was observed within the range.

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Fig. 1.
Semilog plot of isometric force vs. milrinone concentration (mM) of
skinned fibers at 10°C and pCa 4.5. Means ± SE bars are
plotted. A straight line is fitted to data through linear regression.
Numbers on data points indicate number of fibers used.
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In addition to reducing isometric force, milrinone also reduced
Ca2+ sensitivity of the fiber in
terms of force generation, as shown in the right shift of the force-pCa
curve (Fig. 2). Solid symbols are the
controls, solid circles representing data obtained before addition of
milrinone and solid triangles representing data obtained after removal
of milrinone; open circles represent data obtained with 0.6 mM of
milrinone. The pCa50 points were
6.12 ± 0.030 (SE) and 6.08 ± 0.028 for controls before and
after addition of milrinone, respectively. The
pCa50 value for 0.6 mM milrinone
was 5.94 ± 0.025. Values were obtained from nonlinear curve fitting
of the Hill equation described in
METHODS. The Hill's coefficients were 3.01 ± 0.54 for data obtained before addition of milrinone, 2.80 ± 0.43 after removal of milrinone, and 3.39 ± 0.56 with
milrinone. The goodness of fit
(r2) for the
three curves was >0.97, and the shift due to milrinone was
significant (P < 0.05).

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Fig. 2.
Isometric force vs. pCa curve of skinned fibers. Data are fitted with
3-parameter Hill's equation as described in text. , Initial
controls before addition of milrinone; , final control after removal
of milrinone; , 0.6 mM milrinone. Means ± SE are plotted. Number
of fibers used per data point ranges from 4 to 9.
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Maximal shortening velocity was estimated using the slack test. Large
length steps were applied to the fiber making the fiber go slack, and
the duration for the fiber shortening under zero load was determined by
marking the moment the step was applied and the moment when force
deviated from zero, as shown in Fig. 3A. The
slack durations,
t1 and
t2,
corresponded to two step releases. The size of the step release and the
slack duration were then plotted in Fig.
3B for all fibers used in the
experiment. A linear regression was used to determine the slope of the
line fitted to the data. The slope represents unloaded shortening
velocity. The y-intersects for control
(0 milrinone) and test (0.6 mM milrinone) were 16.1 ± 16.9 (SD) and
14.2 ± 15.6 nm/half-sarcomere, respectively, and they were not
different statistically. The slopes (maximal shortening velocities,
nm · half-sarcomere
1 · ms
1)
were, however, significantly different
(P < 0.05), and they were 3.53 ± 0.42 and 2.90 ± 0.32, respectively, for 0 and 0.6 mM milrinone.

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Fig. 3.
A: example of step releases in a slack
test. Both steps are control steps (0 milrinone). Sizes of length steps
are 89.9 and 194.2 nm/half-sarcomere, and corresponding slack
durations, t1
and t2, are
23.5 and 54.4 ms, respectively. B:
t vs. step release size. ,
Controls; , tests (0.6 mM milrinone). Straight lines are fitted
through data with linear regression. Slope of lines indicates
shortening velocity under 0 load.
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A tension transient was obtained by applying a rapid length step to a
maximally activated fiber. Figure 4,
A and
B, shows three length steps for
control and test, respectively, one stretch and two releases, that were
used to obtain the tension transients shown in Fig. 4,
C and
D. The transients were first described by Huxley and Simmons (10). The extreme tension reached during the step
is termed T1; the rapid tension
recovery after the step is termed
T2; the pause or sometimes
reversal of tension recovery after
T2 is termed
T3; and the slow recovery toward
the prestep tension level is termed
T4. The traces were averages of
nine traces from nine fibers. Data averaging was used to reduce noise
and simplify data presentation. High step speed was essential for these
experiments because tension recovery after the step was mostly over
after 2 ms, as evidenced in Fig. 4, C
and D. The length steps applied to the
fibers in these experiments were completed in ~200 µs, as shown in
Fig. 4A. In Fig. 4, tension traces
were normalized to their respective isometric force level; therefore, the control and test traces superimposed at both the isometric and zero
force levels. Because the step sizes were the same for both control and
test, the greater change in force after the length step observed under
test condition (0.6 mM milrinone) indicated that the relative stiffness
was higher in the presence of milrinone. This is more clearly
illustrated in Fig. 5. Relative stiffness was represented by the slope of the relative
T1 curve. There was a slight bent
in the T1 curves, the slopes were
greater in stretch than in release, and this was more pronounced for
test (milrinone) than control. The relative stiffness increased
significantly (P < 0.05) by 12.0 ± 2.0% (SE) due to 0.6 mM milrinone in stretch (Fig. 5). With
release, the increase in relative stiffness was not significant. The
T2 force recovery under the test
condition paralleled the control, although within the recording period
the relative force never recovered to the control level during the T3 and
T4 phases of the transient
response (Fig. 4). The force deficit was ~5% of isometric force. The
time course of the phases in the transients was not identical in test
and control conditions. For example, there was a more pronounced
tension reversal after a step release in the
T3 phase in the presence of
milrinone (Fig. 4). The rate of T2
force recovery was step size dependent, slowest in stretch and fastest
in the largest release, characteristic of tension transients first
described by Huxley and Simmons (10).

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Fig. 4.
Averaged records (n = 9) for tension
transients. A: averaged control length
records. B: averaged test (0.6 mM
milrinone) length records. Three steps, one stretch and two releases,
are shown in both A and
B. Only a small portion of length
records are displayed to show details during step.
C: superimposed tension transients of
control (thick traces) and test (thin trace). Three pairs of transients
correspond to 3 length steps shown in
A and
B. Test transients are normalized to
control isometric tension (Po).
D: traces are the same as in
C, except time scale.
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Fig. 5.
Curves of relative tension reached during step
(T1/T0)
for control ( ) and test (0.6 mM milrinone; ). Means ± SE are
plotted.
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DISCUSSION |
Direct action of milrinone on skeletal muscle cross bridges has
resulted in reduced isometric force and shortening velocity of the
fiber. It is therefore unlikely that the positive inotropic effect of
the drug on intact cardiac muscle is due to direct interaction of the
bipyridine with the cross bridges.
Ca2+ handling and other aspects of
the excitation-contraction coupling of the muscle are likely targets
for milrinone's contractility-enhancing action (14). Although cardiac
muscle cross bridges are similar to those of skeletal muscle, it is not
known that they will respond to milrinone intervention in exactly the
same way. A conclusion regarding milrinone's direct action on cardiac
cross bridges has to be derived from experiments on cardiac muscle.
Milrinone's specific interaction with skeletal muscle cross bridge
makes it a useful tool for probing the cross-bridge cycle. The
bipyridine has many similar properties as inorganic phosphate and
2,3-butanedione monoxime (BDM) in terms of inhibiting force generation
in skinned skeletal muscle fibers (15, 18). Both phosphate and BDM
detain cross bridges in low-force states. Energy for cross-bridge power
stroke is derived from the hydrolysis of MgATP. The free energy of
MgATP hydrolysis decreases with the logarithm of metabolite
concentration (15). The observation that milrinone inhibits isometric
force in a logarithmically dose-dependent manner (Fig. 1) suggests that
the drug may interfere with the cross-bridge cycle by decreasing the
free energy of MgATP hydrolysis. A more intuitive interpretation is
that milrinone binds to a cross bridge in a low-force state and
prevents the bridge from going through the force-generating power
stroke. Due to the stochastic nature of the cross-bridge transition
(9), detaining a bridge before the phosphate-releasing step would have
a similar effect as raising the phosphate concentration and preventing
phosphate release.
A possible mechanism for the positive inotropic effect of milrinone
could be that the drug increases
Ca2+ sensitivity of the actomyosin
interaction. The present finding that milrinone reduces
Ca2+ sensitivity of the skinned
fiber in terms of force generation (Fig. 2) excludes that mechanism. It
is not clear how milrinone decreases the
Ca2+ sensitivity, but it is
possible that the drug interferes with the troponin complex.
The 18% decrease in unloaded shortening velocity (Fig.
3B) in the presence of 0.6 mM
milrinone supports the interpretation that milrinone detains a fraction
of the cross bridges from going through a certain transition. These
detained bridges act as internal loads and slow the shortening velocity
in much the same way as H+ and BDM
molecules do in skinned fibers (16, 18).
Tension transients obtained in the presence of milrinone have been
scaled to match the isometric tension of the control. This normalization procedure implicitly assumes that the force recovery after a length step is the sum of stochastic responses of individual bridges. If milrinone merely reduces the number of active cross bridges
and has no effect on the transitions of the cross-bridge cycle, one
would expect the normalized transients to be parallel to each other.
Figure 4 shows that this is not the case and that milrinone appears to
interfere with the transitions among the attached states. The results
can best be explained by a model in which some of the bridges respond
to the step length change normally, whereas others are affected by the
presence of milrinone. If a drug intervention detains bridges in
low-force states, the isometric force is mainly attributed to attached
bridges that have moved beyond the inhibited transition. These bridges
are assumed to be able to respond normally. This could explain the time
course of the T2 tension recovery
in the first few milliseconds, which is relatively unaltered by
milrinone (Fig. 4); i.e., the traces are largely parallel to each
other. This suggests that the site of intervention of milrinone is
probably not the power stroke itself. Note that, as tension response
progresses, some of the normally responding bridges will move to
detained states, whereas detained bridges will move past the inhibited
transition. T3 and
T4 phases of recovery therefore
provide information on the subsequent movements of the bridges after
the initial power stroke. The more prominent
T3 tension reversal (negative
slope) after a step release suggests that milrinone facilitates
detachment of the bridges after the power stroke. The inhibitory effect
of milrinone on shortening velocity is therefore probably not due to
the drug's inhibition of cross-bridge detachment after power stroke,
as with ADP (17), but instead an inhibition of transition between
low-force states before power stroke.
Stiffness of muscle fiber is related to the number of attached cross
bridges (5, 7). More recently, it has been found that the nonoverlap
portion of the thin filaments contribute significantly to the fiber
compliance (3, 11, 19). Stiffness of the fiber is therefore not a
straightforward function of the number of attached cross bridges.
Because of the lack of sarcomere length control in this study and also
the lack of knowledge about the stress-strain relations of the
myofilaments in our preparation, the relationship between fiber
stiffness and the number of attached cross bridges cannot be described
accurately. Therefore no attempt is made to use relative stiffness
change as an indicator of shifting of bridges between high- and
low-force states.
In the presence of milrinone, the fiber appears to be stiffer during
stretch than during release. We have previously identified a low-force
state in the cross-bridge cycle that possesses such a characteristic.
High ionic strength appears to cause accumulation of bridges in a
low-force state from which the bridges can easily detach after a step
release (16) but not a stretch. This explains the fact that high ionic
strength inhibits isometric force but not shortening velocity. The
existence of such a state has been predicted by A. F. Huxley (9) to
accommodate the finding that energy consumption rate decreases when the
muscle is shortening near its maximal velocity. The detachment from
this state of initial attachment prevents the bridges from going
through the power stroke and hydrolyzing ATP. The present results
indicate that milrinone causes accumulation of bridges in the initial
state of attachment from which the bridges can detach easily without
impeding shortening velocity. The inhibitory effect of milrinone on
velocity further suggests that accumulation of the bridges must occur
in other low-force states as well. This also explains why milrinone
causes a much greater force decrease (55%) than velocity decrease
(18%). Our previous finding (16, 18) of a comparable force inhibition by H+ and BDM accompanies a much
greater depression of velocity.
Milrinone and its closely related analog amrinone are both positive
cardiac inotropic agents. The small variation in the structure of these
bipyridines, however, has led to a large difference in the way the
compounds interact with the muscle cross bridges. Amrinone has been
found to increase isometric force and drastically decrease shortening
velocity (4; also our own unpublished observation), whereas milrinone
in the same preparation causes a decrease in force and slight decrease
in velocity. It appears that amrinone detains muscle cross bridges in a
high-force state and that milrinone detains them in a low-force state.
Understanding the nature of the interactions of these molecules with
the actomyosin complex will undoubtedly yield insights into the
transitions in a cross-bridge cycle.
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FOOTNOTES |
Address for reprint requests: C. Y. Seow, Dept. Pharmacology and
Therapeutics, University of British Columbia, 2176 Health Sciences
Mall, Vancouver, BC, Canada V6T 1Z3.
Received 2 September 1997; accepted in final form 5 February 1998.
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REFERENCES |
1.
Alousi, A. A., and D. C. Johnson.
Pharmacology of the bipyridines: amrinone and milrinone.
Circulation 73, Suppl. III: III-10-III-24, 1986.
2.
Alousi, A. A.,
G. P. Stankus,
J. C. Stuart,
and
L. H. Walton.
Characterization of the cardiotonic effects of milrinone, a new and potent cardiac bipyridine, on isolated tissues from several animal species.
J. Cardiovasc. Pharmacol.
5:
804-811,
1983[Medline].
3.
Bagni, M. A.,
G. Cecchi,
F. Colomo,
and
C. Poggesi.
Tension and stiffness of frog muscle fibres at full filament overlap.
J. Muscle Res. Cell Motil.
11:
371-377,
1990[Medline].
4.
Bottinelli, R.,
V. Cappelli,
S. E. J. N. Morner,
and
C. Reggiani.
Effects of amrinone on shortening velocity and force development in skinned skeletal muscle fibres.
J. Muscle Res. Cell Motil.
14:
110-120,
1993[Medline].
5.
Bressler, B. H., and N. F. Clinch. The
compliance of contracting skeletal muscle. J. Physiol.
(Lond.) 237: 477-493.
6.
Endoh, M.,
S. Yamashita,
and
N. Taira.
Positive inotropic effect of amrinone in relation to cyclic nucleotide metabolism in the canine ventricular muscle.
J. Pharmacol. Exp. Ther.
221:
775-783,
1982[Abstract].
7.
Ford, L. E.,
A. F. Huxley,
and
R. M. Simmons.
The relation between stiffness and filament overlap in stimulated frog muscle fibres.
J. Physiol. (Lond.)
311:
219-249,
1981[Abstract].
8.
Honerjager, P.,
M. Schafer-Korting,
and
M. Reiter.
Involvement of cyclic AMP in the direct inotropic action of amrinone: biochemical and function evidence.
Naunyn Schmiedebergs Arch. Pharmacol.
318:
112-120,
1981[Medline].
9.
Huxley, A. F.
Reflections on Muscle. The Sherrington Lecture XIV. Liverpool, UK: Liverpool Univ. Press, 1980.
10.
Huxley, A. F.,
and
R. M. Simmons.
Proposed mechanism of force generation in striated muscle.
Nature
233:
533-538,
1971[Medline].
11.
Huxley, H. E.,
A. Stewart,
H. Sosa,
and
T. Irving.
X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle.
Biophys. J.
67:
2411-2421,
1994[Abstract].
12.
Mansson, A.,
and
K. A. P. Edman.
Effects of amrinone on the contractile behaviour of frog striated muscle fibres.
Acta Physiol. Scand.
125:
481-493,
1985[Medline].
13.
Mansson, A.,
J. Morner,
and
K. A. P. Edman.
Effects of amrinone on twitch, tetanus and shortening kinetics in mammalian skeletal muscle.
Acta Physiol. Scand.
136:
37-45,
1989[Medline].
14.
Morgan, J. P., J. K. Gwathmey, T. T. DeFeo, and K. G. Morgan. The effects of amrinone
and related drugs on intracellular calcium in isolated mammalian
cardiac and vascular smooth muscle.
Circulation 73, Suppl. III: III-65-III-77, 1986.
15.
Pate, E.,
and
R. Cooke.
Addition of phosphate to active muscle fibers probes actomyosin states within the powerstroke.
Pflügers Arch.
414:
73-81,
1989[Medline].
16.
Seow, C. Y.,
and
L. E. Ford.
High ionic strength and low pH detain activated skinned rabbit skeletal muscle crossbridges in a low force state.
J. Gen. Physiol.
101:
487-511,
1993[Abstract].
17.
Seow, C. Y.,
and
L. E. Ford.
Exchange of ATP for ADP on high-force cross-bridges of skinned rabbit muscle fibers.
Biophys. J.
72:
2719-2735,
1997[Abstract].
18.
Seow, C. Y.,
S. G. Shroff,
and
L. E. Ford.
Detachment of low force bridges contributes to the rapid tension transients of skinned rabbit skeletal muscle fibres.
J. Physiol. (Lond.)
501:
149-164,
1997[Abstract].
19.
Wakabayashi, K.,
Y. Sugimoto,
H. Tanaka,
Y. Ueno,
Y. Takezawa,
and
Y. Ameniya.
X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.
Biophys. J.
67:
2422-2435,
1994[Abstract].
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