(Circulation. 1996;93:310-317.)
© 1996 American Heart Association, Inc.
Articles |
Coupling Between Myosin ATPase Cycle and Creatine Kinase Cycle Facilitates Cardiac Actomyosin Sliding In Vitro
A Clue to Mechanical Dysfunction During Myocardial Ischemia
From the Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
Correspondence to Masataka Sata, MD, Department of Physiology, University of Massachusetts Medical Center, 55 Lake Ave N, Worcester, MA 01655-0127. E-mail mxs125@po.cwru.edu.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background There is much evidence to support the favorable effects of the phosphocreatine shuttle on myocardial contraction and relaxation. However, experiments in which cardiac muscle fiber or myofibril was used have not elucidated its precise mechanism.
Methods and Results Active movements of fluorescently labeled actin filaments on a cardiac myosin layer coimmobilized with creatine kinase (CK) onto a nitrocellulose-coated glass coverslip were studied under various concentrations of adenine nucleotides. At a constant phosphocreatine concentration (5 mmol/L, pH 7.1), the relation of sliding velocity to MgATP concentration followed Michaelis-Menten kinetics. The apparent Km was significantly smaller in the presence of CK (0.041±0.001 mmol/L) than in the absence of CK (0.080±0.001 mmol/L), indicating that coattached CK facilitated the propelling of actin filaments by the myosin ATPase. This phenomenon was also seen under acidic conditions (pH 6.7) as well as in the presence of inorganic phosphate (10 mmol/L). At a constant MgATP concentration (1 mmol/L), the inhibitory effect of MgADP on the actin-myosin interaction was weaker in the presence of CK than in the absence of CK. Another ATP-regenerating system, pyruvate kinase and phospho(enol)pyruvate, while maintaining a low ratio of [MgADP] to [MgATP], did not reduce the Km value (0.156±0.001 mmol/L), suggesting that the effect of coattached CK was not achieved only by prevention of MgADP accumulation.
Conclusions Coupling between the ATPase cycle and the CK cycle may serve not only to maintain the ATP concentration within the myofibril but also to provide optimal conditions for cardiac actomyosin interaction. Consideration of this coupling will offer a clue to elucidating the systolic or diastolic dysfunction during myocardial ischemia or reperfusion.
Key Words: creatine kinase • mechanics • ischemia
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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In myocytes, ATP, the essential fuel for contraction, is produced mainly at mitochondria by the oxidative phosphorylation process. Instead of being used in its original form, high-energy phosphate in ATP is transferred to phosphocreatine, which diffuses to the myofibril and is utilized for the in situ resynthesis of ATP (phosphocreatine shuttle).1 2 3 4 The only recognized role of this phosphocreatine shuttle has been as a source for maintaining the ATP concentration within the myofibril at a sufficient level.1 2 3 4 5 Besides this, another favorable effect of the phosphocreatine shuttle has been proposed, based on the observation that ATP regenerated by endogenous CK was more effective for cardiac systolic and/or diastolic function than exogenously applied ATP.6 7 8 Two mechanisms could explain this favorable effect of the phosphocreatine shuttle. First, ADP released by myosin ATPase must be recycled by CK continuously to prevent its depressive effect on muscle contraction.9 10 Second, the filament lattice in the sarcomere serves as a molecular sieve so that the exogenous ATP may have limited access to the ATPase site of myosin. In other words, a small amount of adenine nucleotide must be compartmented within the myofibrils and recycled continuously by means of the phosphocreatine shuttle.2 11
To further elucidate these points, precise measurement and/or control of adenine nucleotide concentrations in the close proximity of actomyosin sites would be necessary. This, however, is very difficult in intact or skinned muscle preparations. To overcome this problem, we applied a recently developed in vitro motility assay technique to dissect mechanical properties of actomyosin interaction under well-defined conditions.12 13 14 15 16 We coimmobilized cardiac myosin and CK on a coverslip and investigated the movement of fluorescently labeled actin filaments. Results in this study will demonstrate that the functional coupling between the myosin ATPase cycle and the CK cycle provides optimal conditions for actin-myosin interaction. Consideration of this coupling would provide a clue to the mechanical dysfunction during myocardial ischemia or reperfusion in which the heart loses its systolic and diastolic function at a high concentration of intracellular ATP.17 18 19 20
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Myosin Preparation
Cardiac myosin was obtained from 4-week-old male Wistar rats, whose ventricular myocytes are known to contain only the V1 type of myosin.12 21 Rats were anesthetized with an injection of sodium pentobarbital (40 to 50 mg/kg body wt IV), and the hearts were rapidly excised. All procedures described below were performed at 4°C. The hearts were washed in PBS (10 mmol/L sodium phosphate buffer, 0.9% NaCl, pH 7.2). Atria and right ventricles were trimmed. Because the rat hearts were small, left ventricles from 10 hearts were pooled for myosin extraction. The heart muscle was homogenized in Tris–maleic acid buffer [20 mmol/L tris(hydroxymethyl)aminomethane–maleic acid, 1 mmol/L EDTA, pH 7.0] and was centrifuged (SCR 20B centrifuge, Hitachi) at 1000g for 15 minutes. The pellet was extracted with 3 vol Guba-Straub solution (0.3 mol/L KCl, 100 mmol/L KH2PO4, 50 mmol/L K2HPO4, 1 mmol/L ATP, 5 µg/mL leupeptin, 5 mmol/L DTT, 1 mmol/L EDTA, pH 6.5) for 10 minutes. After the extract was centrifuged at 11 000g for 15 minutes, the supernatant was collected and 14 vol ice-cold distilled water was added to precipitate the myosin. After 2 hours had passed, the myosin was collected by centrifugation at 11 000g for 15 minutes. The myosin was again dissolved in a high-ionic-strength solution (0.6 mol/L KCl, 10 mmol/L Tris-HCl, 5 mmol/L DTT, pH 7.5), and the trace amount of actin was removed by centrifugation (L8M centrifuge, Beckman Instruments, Inc) at 120 000g for 2.5 hours. Skeletal myosin was prepared from back muscle of adult Japanese White rabbits in the same way as the cardiac myosin. Protein concentration was measured according to the method of Lowry et al.22
Coattachment of Myosin and CK on a Glass
Coverslip
Cardiac myosin was diluted to 0.8 mg/mL with a
high-ionic-strength buffer (0.6 mol/L KCl, 50 mmol/L Tris-HCl,
pH 7.5) and mixed with CK from bovine heart muscle (Sigma Chemical Co)
in a final concentration of 800 IU/mL. A simple myosin solution (0.8
mg/mL) without CK was also prepared. Sixty microliters of these myosin
preparations was applied on a nitrocellulose-coated coverslip
(30x30 mm, Matsunami Co) and then covered by another smaller coverslip
(18x18 mm) to create a fluid-filled flow cell as described
previously.12 14 After a 15-minute incubation on ice,
120
µL of BSA solution (0.5 mg/mL BSA, 30 mmol/L KCl, 20 mmol/L HEPES, pH
7.5) was applied to the flow cell to wash out unbound myosin and CK and
to coat the exposed nitrocellulose surface. Skeletal muscle myosin was
also fixed on the coverslip with or without CK from rabbit skeletal
muscle in the same way as the cardiac myosin.
Estimation of Amount of CK and Myosin Molecules Bound to the
Coverslip
The amount of CK fixed to the coverslip was estimated by
measuring the ATP generation from the ADP and phosphocreatine in the
flow cell. The flow cell was filled with 60 µL of
MgADP/phosphocreatine solution (25 mmol/L KCl, 6 mmol/L
MgCl2, 25 mmol/L HEPES, 1 mmol/L EGTA, 1 mmol/L DTT,
2 mmol/L ADP, 5 mmol/L phosphocreatine, pH 7.1). After a 3-minute
incubation at 30°C, the reaction was stopped by perfusion of the flow
cell with 300 µL of cold distilled water containing 1%
FDNB.5 Effluent from the flow cell was collected to
measure the amount of ATP generated.
The density of myosin molecules fixed to the coverslip was estimated by comparing the myosin K-EDTA/ATPase activity in the flow cell with that in the solution.15 23 It was assumed that the immobilized myosin and the myosin in the solution had the same K-EDTA/ATPase activity.15 23 24 The ATPase reaction was started by filling the flow cell with 60 µL of high-salt EDTA buffer (0.5 mol/L KCl, 4 mmol/L EDTA, 0.5 mmol/L ATP, 1 mmol/L DTT, 25 mmol/L imidazole, pH 7.4). After a 7-minute incubation at 25°C, the reaction was stopped by perfusion of the flow cell with 300 µL of cold distilled water containing 5% trichloroacetic acid. The effluent was collected to measure the amount of ADP released by ATP hydrolysis. The K-EDTA/ATPase activity of the myosin in the solution was measured in the same high-salt EDTA buffer at 25°C.
In Vitro Motility Assay
We used the method described by Kron
and Spudich14
with some modifications.12 Briefly, cardiac actin was
prepared from an acetone powder of rat cardiac muscle by the method of
Spudich and Watt25 and incubated at 4°C overnight with a
molar excess of rhodamine-phalloidin (Molecular Probes, Inc). Actin
filaments thus prepared were suspended in the assay buffer (25 mmol/L
KCl, 6 mmol/L MgCl2, 25 mmol/L HEPES, 1 mmol/L EGTA,
1% 2-mercaptoethanol, 4.5 mg/mL glucose, 216 µg/mL glucose oxidase,
36 µg/mL catalase, 5 mmol/L phosphocreatine, pH 7.1) containing
various concentrations of adenine nucleotides and
introduced onto the coverslip coated with cardiac myosin. Then, 120
µL of the assay buffer was perfused to wash out unbound actin
filaments. Active slidings of fluorescently labeled actin
filaments at 30°C were observed with an inverted fluorescence
microscope (TMD-EF2, Nikon) equipped with a x100 oil-immersion
objective lens (numerical aperture, 1.3; Zeiss Neofluor), a 100 W
super-high-pressure mercury lamp, and a rhodamine filter set.
The fluorescent image of the filament was observed via a highly
sensitive silicon intensifier target camera (C2400-08,
Hamamatsu-Photonics) and was recorded with a video recorder
(BR-S601M, JVC). Velocity was measured during a replay of the videotape
recording 1 to 2 minutes after the coverslip had been placed on
the temperature-controlled microscope stage. Each video frame was
digitized at a rate of five frames per second into a 320x240–pixel
array by a video grabber card (Video Charger, Intel Inc) installed in a
personal computer (PC9821Bp, NEC). The investigator, using a mouse,
located the leading edge of an actin filament in successive snapshots,
allowing the computer to calculate the mean velocity of the filament
from the distance moved and the time elapsed. Concentrations of the
adenine nucleotides in the assay buffer were also
analyzed with the collected effluents after the flow cell was
perfused with 300 µL of cold distilled water containing 5%
trichloroacetic acid 1.5 minutes after the assay had been
initiated.
The same assay was also performed with actin, myosin, and CK from rabbit skeletal muscle.
Analysis of the Effluents Collected From the Flow
Cell
Concentrations of adenine nucleotides in the
effluents collected from the flow cell were measured by HPLC as
described by Sellevold et al.26 Each sample (10 µL) was
placed on a reverse-phase column (4.6x250 mm, STR ORS-H, Shimadzu
Inc) and eluted at a flow rate of 1 mL/min with a buffer containing 215
mmol/L KH2PO4, 2.3 mmol/L
tetrabutylammonium hydrogen sulfate, and 3.5% acetonitrile, pH 6.25.
The column effluent was analyzed with a spectrophotometer
(SPD6A, Shimadzu Inc) at 260 nm, and quantification was performed on
each peak area.
Experimental Protocol
Motility assays were performed on the
myosin layers fixed with
CK as well as on the myosin layer fixed without CK while the
composition of the assay buffer was changed (30°C). First, to
investigate the MgATP dependence of the sliding velocity, the
concentration of MgATP was changed from 0 to 2 mmol/L in the presence
of constant phosphocreatine (5 mmol/L) with cardiac myosin, actin, and
CK (control condition, pH 7.1). Next, the same experiment was repeated
with skeletal myosin, actin, and CK. Second, to investigate the effect
of acidosis or accumulation of Pi on the MgATP dependence
of the filament movement, the same experiments were performed at a
lower pH (pH 6.7) or in the presence of Pi (10 mmol/L).
Third, to investigate the effect of MgADP on the sliding velocity, the
experiment was repeated on the cardiac myosin layer fixed with or
without CK under various MgADP concentrations between 0 and 2 mmol/L
while the MgATP concentration was fixed at 1 mmol/L. Fourth, to further
characterize the effect of coattached CK, the MgATP dependence of the
filament movement was investigated on the myosin layer fixed without CK
in the presence of other ATP-regenerating systems supplied from the
supernatant. For this purpose, 5 mmol/L phospho(enol)pyruvate and 20
IU/mL pyruvate kinase (Sigma Chemical Co) as well as 5 mmol/L
phosphocreatine and 20 IU/mL CK in the assay buffer were used.
Statistical Analysis
The mean velocity under each
experimental condition was
calculated from the velocities of 30 to 40 actin filaments. All data
are presented as mean±SD. Student's t test was
used for statistical comparison of mean velocities. The relation
between the actin velocity and the ATP concentration was
analyzed with a computed nonlinear least-squares
curve-fitting program, which estimated the apparent
Km and the maximum filament velocity
(Vmax) with SDs.27 A value of
P<.01 was considered to be significant.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Densities of CK and Myosin Fixed on the Coverslip
Three minutes after perfusion with the MgADP/phosphocreatine solution, the effluent from the flow cell in which cardiac myosin had been fixed with CK contained a certain amount of ATP and reduced ADP, whereas there was no detectable ATP in the effluent from the flow cell without CK. This demonstrated that CK was coattached with myosin onto the coverslip with its catalytic activity preserved. The mean CK activity of the flow cell with CK was calculated as 0.028±0.001 IU (Table 1
). On the other hand, when CK
alone was applied onto the nitrocellulose-coated coverslip, the
flow cell did not show any significant CK activity, suggesting that the
CK that was bound in the presence of myosin was in fact bound directly
to the myosin, rather than just adjacent to the myosin on the
nitrocellulose (data not shown).
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The K-EDTA/ATPase activity of the
myosin layers fixed with CK was
0.22±0.03 nmol Pi per minute per flow cell and that
without CK was 0.23±0.02 nmol Pi per minute per flow cell.
When the measured K-EDTA/ATPase activity of the myosin in solution was
used (0.30±0.01 µmol
Pi·mg-1·min-1), the
density of the myosin layer fixed with CK was 2.61±0.20
ng/mm2 and that without CK was 2.70±0.24
ng/mm2 (Table 1
). This indicated that there was no
significant difference in the amount of myosin molecules attached onto
the coverslip whether myosin was applied with or without CK.
Effect of Coexisting CK
Fig 1 shows the
sliding velocities of
the actin filaments on the cardiac myosin layer fixed with or without
CK when the MgATP concentration of the assay buffer was changed from 0
to 2 mmol/L in the presence of constant phosphocreatine (5 mmol/L). For
both myosin layers, the relation of sliding velocity (V) to MgATP
concentration ([MgATP]) followed Michaelis-Menten kinetics. The
solid
lines represent least-squares fits to the equation
V=Vmax/(1+Km/[MgATP]),
where V is the observed filament velocity, Vmax is the
velocity at saturating MgATP, and Km is
the apparent Michaelis constant. Km of
the myosin layer fixed with CK (0.041±0.001 mmol/L) was significantly
smaller than that without CK (0.080±0.001 mmol/L). Vmax of
the myosin layer with CK (5.9±0.1 µm/s) was significantly higher
than that without CK (4.8±0.1 µm/s), demonstrating that coattached
CK facilitated the propelling of the actin filaments by the myosin
ATPase.
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) or without (
) cardiac CK. Solid lines were
least-squares fits to data by use of the equation
V=Vmax/(1+Km/[MgATP]),
where V is observed velocity, Vmax is the filament velocity
at saturating MgATP, and Km is the
apparent Michaelis constant for MgATP. For the myosin layer fixed with
CK, Km was estimated as 0.041±0.001
mmol/L; Vmax, 5.9±0.1 µm/s; and
r2, .98. For that without CK,
Km was 0.080±0.001 mmol/L;
Vmax, 4.8±0.1 µm/s; and
r2, .97.
In the same experiment using myosin, actin, and CK from rabbit
skeletal
muscle (Fig 2), the MgATP-dependent
Km value was also significantly reduced
in the presence of CK (0.105±0.001 to 0.059±0.001 mmol/L),
whereas
there was no significant difference in the Vmax (7.6±0.1
versus 7.9±0.1 µm/s).
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To further analyze the effect of
coexisting CK, concentrations
of the adenine nucleotides in the effluent from the flow
cell were analyzed by HPLC (Fig 3). At any
initial MgATP concentration (1, 0.5, 0.1, or 0.05 mmol/L), the
[MgADP]/[MgATP] ratio was kept very small in the presence
of CK
(Table 2). However, the MgADP concentration became
rather high in the absence of CK, corresponding to the ATP hydrolysis
to propel the actin filaments.
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Effects of a Lower pH and the Addition of Pi
Fig
4A shows the relation between the MgATP
concentration and the filament velocity under acidic conditions (pH
6.7). The Km value was significantly
smaller in the presence of coattached CK (0.074±0.001 mmol/L) than in
the absence of CK (0.200±0.001 mmol/L). Vmax was
significantly higher in the presence of CK (2.0±0.2 µm/s) than in
the absence of CK (1.4±0.1 µm/s). For both myosin layers, the
maximum sliding velocity decreased to about one third of those under
the control conditions (pH 7.1).
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Fig 4B shows the
relation between the MgATP concentration and the
filament velocity in the presence of Pi (10 mmol/L). The
Km value was significantly smaller in the
presence of CK (0.056±0.001 mmol/L) than in the absence of CK
(0.091±0.001 mmol/L). Vmax was significantly higher in the
presence of CK (5.3±0.1 µm/s) than in the absence of CK
(4.6±0.1
µm/s). For both myosin layers, the maximum sliding velocity was not
significantly different from those under control conditions, confirming
the results of our previous study that the addition of Pi
did not change the unloaded actomyosin sliding
velocity.28
Effect of MgADP
The sliding velocity of the actin filament
was also dependent on
the MgADP concentration in the assay buffer. At a constant
concentration of MgATP (1 mmol/L), the addition of MgADP decreased the
sliding velocity in a dose-dependent manner (Fig 5).
In the absence of CK, the addition of as little as 0.025 mmol/L MgADP
reduced the sliding velocity significantly, whereas in the presence of
CK, the sliding velocities became significantly slower only after the
addition of 1.0 mmol/L MgADP or more, demonstrating that the
inhibitory effect of MgADP on the actin-myosin
interaction was weaker in the presence of bound CK than in the absence
of CK.
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Comparison With Other ATP-Regenerating Systems Supplied From
the Supernatant
To determine whether the effect of CK on myosin ATPase
was
merely due to its MgADP-lowering action, the MgATP dependence of the
sliding velocity on the myosin layer fixed without CK was investigated
in the presence of pyruvate kinase/phospho(enol)pyruvate in the
assay buffer (Fig 6). This ATP-regenerating
system did not reduce the Km value
(0.156±0.001 mmol/L), while analysis of the assay buffer
showed that the [MgADP]/[MgATP] ratio was kept very low
(Fig 3,
Table 2). On the contrary, another ATP-regenerating system
supplied
from the supernatant of the myosin layer (ie, from CK and
phosphocreatine in the assay buffer) significantly reduced the
MgATP-dependent Km (0.046±0.001 mmol/L).
These results suggested that the facilitation of myosin ATPase by the
coattached CK was not attained only by preventing MgADP accumulation,
although it is not possible to determine either whether CK in solution
was as effective as CK bound to myosin or whether CK was bound to the
myosin during the time course of the assay.
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), Km was
0.156±0.001 mmol/L; Vmax, 3.3±0.1 µm/s; and
r2, .99. In the presence of CK and
phosphocreatine in the assay buffer (
),
Km was 0.046±0.001 mmol/L;
Vmax, 5.3±0.1 µm/s; and
r2, .99. The similar relation in
the absence of any ATP-regeneration system (indicated by closed circles
in Fig 1 |
Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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In this study, we showed the favorable effect of coexisting CK on the actin-myosin interaction. The use of the in vitro motility assay technique enabled us to precisely evaluate the environment directly surrounding the ATP-consuming actomyosin molecules, thus giving us an insight into the underlying mechanisms. On the other hand, we should be careful in interpreting our results because they were influenced to some extent by the different molecular arrangement in our system from that in the in vivo situation.
Compartmentation of Adenine
Nucleotides
There is some evidence that only a small amount of ATP is
compartmented within myofibrils2 11 and continuously
regenerated by CK and phosphocreatine. Isolated frog sartorius muscles
whose endogenous CK had been completely inhibited by
preincubation with FDNB were not able to maintain normal isometric
tension for more than three contractions, whereas intact muscle could
contract over 100 times.5 A similar effect of FDNB was
also observed in cardiac muscle.6 For skinned muscle
preparations, phosphocreatine rather than MgATP in the bathing solution
was necessary to prevent deleterious rigor bond formation during
contraction.6 7 Actually, Geisbuhler et
al11
showed that myofibrils contained only 5.7% of total intracellular
adenine nucleotides in isolated adult rat cardiac
cells.
Spatial and Functional Coupling Between CK Cycle and Myosin
ATPase Cycle
There is much evidence to support the existence of the
coupling
between the CK cycle and the myosin ATPase cycle. Experiments with
cardiac muscle fiber or myofibril demonstrated that MgATP regenerated
by endogenous CK had preferred access to the myosin ATPase
in comparison with exogenously applied
MgATP.6 29 30
Ventura-Clapier and Vassort demonstrated that endogenous
MM-type CK was able to ensure maximal efficiency of myosin ATPase to
produce relaxation of rigor tension in chemically skinned rat
ventricular papillary muscle.6 Krause and
Jacobus30 showed specific enhancement of the cardiac
myofibrillar ATPase by bound CK. On the other hand, immunohistochemical
studies showed that MM-type CK was located in the M line of the
sarcomere in cardiac and skeletal myofibrils, indicating that CK was in
close proximity to myosin ATPase and generated ATP for use of myosin
ATPase in situ.4 31
From these observations, a tight coupling between the CK cycle and the myosin ATPase cycle has been proposed.6 29 30 However, the mechanisms underlying the facilitating effects of this coupling on actin-myosin interaction have not been fully understood, probably due to the experimental preparations used in these studies. These preparations preserved the lattice formed by myosin and actin filaments in the sarcomere, which created a small compartment surrounding the ATP-consuming contractile units, resulting in a different environment from the freely diffusible space around it. Going into this isolated space is the key to clarifying the mechanism.
In Vitro Motility Assay
In this study, we adopted the in
vitro motility assay, in which
only purified proteins were used to study the mechanical interaction
between actin and myosin. With this technique, we could get rid of the
diffusion barrier and obtain a direct view into the microenvironment
surrounding the contractile apparatus and CK. We tried to
coimmobilize myosin and CK on the
nitrocellulose-coated coverslip. The flow cell, into which myosin
had been introduced with CK, showed considerable CK activity even after
perfusion with BSA solution to wash out unbound proteins, indicating
that CK was fixed on the coverslip with myosin molecules with its
catalytic activity preserved. Because CK has been shown to be
specifically bound to the myosin
molecule,32 33 34 CK may
have adhered to myosin molecules that were firmly fixed to the
coverslip by the hydrophobic bond.14 15 Although the
molecular arrangement composed in the flow cell could differ from that
in intact muscle, the following two points were confirmed by
analyses of the effluents from the flow cells. There was no
significant difference in the amount of myosin molecules on the
coverslip between the myosin layers fixed with and without CK. Also, CK
was not bound to myosin molecules in a way that would inhibit its
catalytic property.
Possible Mechanism of the Functional Coupling
The underlying
mechanism of facilitating effects exerted by this
coupling could be explained as follows. In the generally accepted view,
myosin molecules cyclically interact with actin filaments either weakly
or strongly while hydrolyzing ATP.35 The binding of ATP
and the release of its hydrolysis products are coupled with
transition between different binding states. ADP release couples with
power stroke and is thought to be the rate-limiting step in the
actomyosin interaction.36 As shown in the experiment with
chemically skinned rabbit psoas muscle fiber,9 the
accumulation of ADP in the proximity of myosin would inhibit the ADP
dissociation, resulting in the slowdown of the actomyosin sliding. In
fact, in this study, the addition of MgADP depressed the actin filament
velocity in a dose-dependent manner under a constant concentration
of MgATP, in agreement with the previous findings in skeletal
actomyosin S-1 ATPase activity in solution.37 38 If
CK is
located very close to myosin heads, ADP bound to the active site can be
transformed into ATP quickly during contraction, making the
cross-bridge detach and get into the next cycle
simultaneously. In this way, CK and phosphocreatine would
provide optimal conditions for the myosin ATPase to propel
actin filaments, compared with the situation in which ATP alone is
applied exogenously.
Furthermore, experiments using other ATP-regenerating systems in solution would give us another standpoint from which to characterize the facilitating effect of coattached CK. Pyruvate kinase and phospho(enol)pyruvate did not facilitate the myosin ATPase, although analysis of the assay buffer showed that the [MgADP]/[MgATP] ratio was kept very small. This finding suggested that the effect of CK was attained not only by the prevention of ADP accumulation around the active site of the myosin head but also by some other mechanisms specific to the CK bound to myosin.
Difference Between Cardiac and Skeletal Myosin
CK is known to
exist in four isoenzymes: MM, MB, BB, and
mitochondrial CK. There is some difference in the distribution of CK
isozymes between cardiac and skeletal muscles.2 Cardiac
myofibril contains primarily the MM form with a small amount of the MB
and BB forms, whereas the MM form is the only isozyme in skeletal
myofibril.2 39 31P NMR studies in the
brain,
heart, and skeletal muscle of the living rat demonstrated that the
velocity of the CK reaction was regulated by total tissue enzyme
activity and concentrations of substrates in a manner that was
independent of isozyme distribution.39 In this study,
skeletal CK decreased the apparent Km
significantly without affecting the Vmax, whereas
cardiac CK changed both Km and
Vmax significantly. Further study will be needed to
elucidate the relation between the CK isozyme composition and the
property of facilitating myosin ATPase.
In this study, the maximum sliding velocity of skeletal myosin was only slightly higher than that of cardiac myosin, whereas many studies have shown several differences in myosin ATPase activity and/or shortening velocity between cardiac and skeletal myosins.40 41 However, we used cardiac V1 isomyosin from 4-week-old rats, whose ATPase activity and shortening velocity are known to be several times higher than those of V3 isomyosin.12 21 Our results are in agreement with previous data about the ATPase activity of V1 cardiac and skeletal myosins.42 43
Implications for Mechanical Dysfunction in Ischemic
Myocardium
Consideration of this functional coupling between the
myosin
ATPase cycle and the CK cycle may further our understanding of the
mechanism of systolic and diastolic dysfunctions of
the heart during ischemia or reperfusion. Since ATP depletion
and/or ADP accumulation were thought to basically lead to the
mechanical dysfunction, numerous workers have extensively investigated
the relation between the intracellular ATP concentration and the
cardiac function by measuring high-energy phosphate content in
frozen, pulverized tissue samples17 or in living animal
hearts by use of NMR techniques (31P
NMR).18 19 These studies, however, failed to show a
significant correlation between cardiac function and total content of
intracellular ATP or ADP.17 18 19 An
ischemic heart
lost its systolic and diastolic activity abruptly
with considerable depletion of phosphocreatine, while the intracellular
ATP concentration remained relatively
high.17 18 19 20 These
researchers measured only the average intracellular ATP or ADP content
and did not offer any information about the ATP or ADP concentration
directly surrounding the cardiac myosin. However, it is the
concentration of ATP within the myofibril, not the total intracellular
ATP content, that should have an important meaning for the actomyosin
interaction. With a decline in phosphocreatine, the ADP/ATP ratio
within myofibril would easily become so high that the cardiac
systolic and diastolic functions could be impaired
due to the increment of the proportion of the cross-bridges in the
strong binding state.10
Furthermore, in consideration of the small compartmented ATP pool regenerated continuously by CK in situ, we could also interpret the results of the former studies about the stunned myocardium from a different viewpoint. Krause8 concluded that the depression of pressure development in stunned hearts was not due to a defect in myofilament function, on the basis of the observation that global myocardial stunning had no effect on the myofibrillar Ca2+-sensitive ATPase activity and CK kinetics. On the other hand, Otsu et al44 demonstrated that myocardial ischemia resulted in a dissociation of CK molecules from the thick filament, which might lead to a defect of the phosphocreatine shuttle after reperfusion. Taken together, mechanical dysfunction observed in the stunned myocardium may be explained, at least in part, by a defect in the energy support system, not by the contractile machine itself.
Finally, our results under acidic conditions or in the presence of Pi gave us further insight into the mechanical dysfunction during ischemia. It is well recognized that altered contractile performance during ischemia may be related to changes in Pi concentration and/or intracellular pH.17 19 45 In this study, under acidic conditions, Vmax decreased to about one third of that under the control conditions. On the other hand, the addition of Pi did not affect Vmax significantly. However, quite recently, we showed that the accumulation of Pi severely decreases the Ca2+ sensitivity of the reconstituted thin filament without changing the sliding velocity in vitro.28 In addition to the defect of the phosphocreatine shuttle, acidosis and/or the accumulation of Pi must contribute to the pathophysiology of the mechanical dysfunction during myocardial ischemia. Further study will be needed to clarify the effect of these factors, which should take place together in vivo.
In summary, we showed that the functional coupling between the myosin ATPase cycle and the CK cycle facilitates cardiac actomyosin sliding. Consideration of the phosphocreatine shuttle and the small compartmentation of adenine nucleotides will provide a novel clue to the mechanical dysfunction during myocardial ischemia or reperfusion.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by a grant-in-aid for Scientific Research on Priority Areas (Differentiation and Regulation of Cardiac Cell) and grant B-0645285 from the Ministry of Education, Science, and Culture of Japan.
Received June 26, 1995; revision received August 10, 1995; accepted September 11, 1995.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Bessman SP, Geiger PJ. Transport of energy in muscle. Science. 1981;211:448-452.
2. Bessman SP, Carpenter CL. The creatine-creatine phosphate energy shuttle. Annu Rev Biochem. 1985;54:831-862. [Medline] [Order article via Infotrieve]
3.
Rossi AM, Eppenberger HM, Volpe P, Cotrufo R,
Wallimann T. Muscle-type creatine kinase is specifically
bound to sarcoplasmic reticulum and can support Ca2+ uptake
and regulate local ATP/ADP ratios. J Biol
Chem. 1990;265:5258-5266.
4. Sharov VG, Saks VA, Smirnov VN, Chazov EI. An electron microscopic histochemical investigation of the localization of creatine phosphokinase in heart cell. Biochim Biophys Acta. 1977;468:495-501. [Medline] [Order article via Infotrieve]
5.
Infante AA, Davies RE. The effect of
2,4-dinitrofluorobenzene on the activity of striated muscle.
J Biol Chem. 1965;240:3996-4001.
6. Ventura-Clapier R, Vassort G. Role of myofibrillar creatine kinase in the relaxation of rigor tension in skinned cardiac muscle. Pflugers Arch. 1985;404:157-161. [Medline] [Order article via Infotrieve]
7.
Maughan DW, Low ES, Alpert NR. Isometric force
development, isotonic shortening and elasticity measurements from
Ca2+-activated ventricular muscle of
the guinea pig. J Gen Physiol. 1978;71:431-451.
8.
Krause SM. Effect of global myocardial stunning
on Ca2+-sensitive myofibrillar ATPase activity and creatine
kinase kinetics. Am J Physiol. 1990;259:H813-H819.
9.
Schoenberg M, Eisenberg E. ADP binding to
myosin cross-bridges and its effect on the cross-bridge
detachment rate constants. J Gen Physiol. 1987;89:905-920.
10.
Yamashita H., Sata M, Sugiura S, Momomura S, Serizawa
T, Iizuka M. ADP inhibits the sliding velocity of
fluorescent actin filaments on cardiac and skeletal
myosin. Circ Res. 1994;74:1027-1033.
11.
Geisbuhler T, Altschuld RA, Trewyn RW, Ansel AZ, Lamka
K, Brierley GP. Adenine nucleotide
metabolism and compartmentalization in isolated adult rat
heart cells. Circ Res. 1984;54:536-546.
12.
Sata M, Sugiura S, Yamashita H, Momomura S, Seizawa
T. Dynamic interaction between cardiac myosin isoforms modifies
velocity of actomyosin sliding in vitro. Circ
Res. 1993;73:696-704.
13.
Homsher E, Wang F, Sellers JR. Factors affecting
movement of F-actin filaments propelled by skeletal heavy
meromyosin. Am J Physiol. 1992;262:C714-C723.
14.
Kron SJ, Spudich JA. Fluorescent actin
filaments move on myosin fixed to a glass surface. Proc
Natl Acad Sci U S A. 1986;83:6272-6276.
15. Uyeda TQP, Kron SJ, Spudich JA. Myosin step size: estimation from slow sliding movement of actin over low densities of heavy meromyosin. J Mol Biol. 1990;214:699-710. [Medline] [Order article via Infotrieve]
16.
Warshaw DM, Desrosiers JM, Work SS, Trybus KM.
Smooth muscle myosin cross-bridge interactions modulate actin
filament sliding velocity in vitro. J Cell
Biol. 1990;111:453-463.
17.
Momomura S, Ingwall JS, Parker A, Sahagian P, Ferguson
JJ, Grossman W. The relationships of high energy phosphates,
tissue pH, and regional blood flow to diastolic
distensibility in the ischemic dog heart.
Circ Res. 1985;57:822-835.
18.
Bittl JA, Balaschi JA, Ingwall JS. Contractile
failure and high-energy phosphate turnover during hypoxia:
31P-NMR surface coil studies in living rat.
Circ Res. 1987;60:871-878.
19. Kusuoka H, Porterfield JK, Weisman HF, Weisfeldt ML, Marban E. Pathophysiology and pathogenesis of stunned myocardium. J Clin Invest. 1987;79:950-961.
20. Gudbjarnason S, Mathes P, Ravens KA. Functional compartmentation of ATP and creatine phosphate in heart muscle. J Mol Cell Cardiol. 1970;1:325-329. [Medline] [Order article via Infotrieve]
21.
Schwartz K, Lompre AM, Bouveret P, Wisnewsky C, Whalen
RG. Comparison of rat cardiac myosin at fetal stages in young
animals and in hypothyroid adults. J Biol
Chem. 1982;257:14412-14418.
22.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.
Protein measurement with the Folin phenol reagent.
J Biol Chem. 1951;193:265-275.
23.
Toyoshima YY, Kron SJ, Spudich JA. The myosin
step size: measurement of the unit displacement per ATP hydrolyzed in
an in vitro assay. Proc Natl Acad Sci U S A. 1990;87:7130-7134.
24.
Harris DE, Warshaw DM. Smooth and skeletal
muscle myosin both exhibit low duty cycles at zero load in
vitro. J Biol Chem.. 1993;268:14764-14768.
25.
Spudich JA, Watt S. The regulation of rabbit
skeletal muscle contraction. J Biol Chem. 1971;246:4866-4871.
26. Sellevold OF, Jynge MP, Aarstad K. High performance liquid chromatography: a rapid isocratic method for determination of creatine compounds and adenine nucleotides in myocardial tissue. J Mol Cell Cardiol. 1986;18:517-527. [Medline] [Order article via Infotrieve]
27. Lutz RA, Bull C, Rodbard D. Computer analysis of enzyme-substrate-inhibitor kinetic data with automatic model selection using IBM-PC compatible microcomputers. Enzyme. 1986;36:197-206.[Medline] [Order article via Infotrieve]
28.
Sata M, Sugiura S, Yamashita H, Fujita H, Momomura S,
Serizawa T. MCI-154 increases Ca2+ sensitivity of
reconstituted thin filament. Circ Res. 1995;76:626-633.
29. Bessman SP, Yang WCT. Intimate coupling of creatine phosphokinase and myofibrillar adenosinetriphosphatase. Biochem Biophys Res Commun. 1980;96:1414-1420. [Medline] [Order article via Infotrieve]
30.
Krause SM, Jacobus WE. Specific enhancement of
the cardiac myofibrillar ATPase by bound creatine kinase.
J Biol Chem. 1992;267:2480-2486.
31. Wegmann G, Zanolla E, Eppenberger HM, Wallimann T. In situ compartmentation of creatine kinase in intact sarcomeric muscle. J Muscle Res Cell Motil. 1992;13:420-435. [Medline] [Order article via Infotrieve]
32. Herasymowych OS, Mani RS, Kay CM, Bradley RD, Scraba DG. Ultrastructure studies on the binding of creatine kinase and the 165,000 molecular weight component to the M-band of muscle. J Mol Biol. 1980;136:193-198. [Medline] [Order article via Infotrieve]
33. Houk TW, Putnam SV. Location of the creatine phosphokinase binding site of myosin. Biochem Biophys Res Commun. 1978;55:1271-1277.
34.
Ventura-Clapier R, Saks VA, Vassort G, Lauer C,
Elizarova GV. Reversible MM-creatine kinase binding to cardiac
myofibrils. Am J Physiol. 1987;253:C444-C455.
35. Lymn RW, Taylor EW. Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry. 1971;10:4617-4624. [Medline] [Order article via Infotrieve]
36.
Siemankowski R, Wiseman MO, White HD. ADP
dissociation from actomyosin subfragment 1 is sufficiently slow to
limit the unloaded shortening velocity in vertebrate muscle.
Proc Natl Acad Sci U S A. 1985;82:658-662.
37.
Biosca JA, Greene LE, Eisenberg E. Binding of
ADP and ATP analogs to cross-linked and non-cross-linked
acto S-1. J Biol Chem. 1986;261:9793-9800.
38. Sleep J, Glyn H. Inhibition of myofibrillar and actomyosin subfragment 1 adenosinetriphosphatase by adenosine 5'-diphosphate, pyrophosphate, and adenyl-5'-yl imidodiphosphate. Biochemistry. 1986;25:1149-1154. [Medline] [Order article via Infotrieve]
39. Bittl JA, DeLayre J, Ingwall JS. Rate equation for creatine kinase predicts the in vivo reaction velocity: 31P-NMR surface coil studies in brain, heart, and skeletal muscle of the living rat. Biochemistry. 1987;26:6083-6090. [Medline] [Order article via Infotrieve]
40.
Stein LA, White MP. Biochemical kinetics of
porcine cardiac subfragment-1. Circ Res. 1987;60:39-49.
41.
Stone DB, Mendelson RA, Botts J, Cheung PH. EPR
and fluorescence depolarization studies on bovine cardiac
myosin. Circ Res. 1981;49:677-684.
42.
Bagchi N, Brown TR, Schneider DS, Banerjee SK.
Effect of amiodarone on rat heart myosin isoenzymes.
Circ Res. 1987;60:621-625.
43. Lim MS, Walsh MP. The effects of caldesmon on the ATPase activities of rabbit skeletal muscle myosin. Biochem J. 1986;238:523-530. [Medline] [Order article via Infotrieve]
44.
Otsu N, Yamaguchi I, Komatsu E, Miyazawa K.
Changes in creatine kinase M localization in acute ischemic
myocardial cells. Circ Res. 1993;73:935-942.
45.
Kentish JC. The effects of inorganic phosphate
and creatine phosphate on force production in skinned muscles
from rat ventricle. J Physiol. 1986;370:585-604.
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