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(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

Masataka Sata, MD; Seiryo Sugiura, MD; Hiroshi Yamashita, MD; Shin-ichi Momomura, MD; Takashi Serizawa, MD

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.

	Abstract

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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 K_m 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 K_m 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

	Introduction

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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}

	Methods

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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 P_i 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 P_i (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 K_m and the maximum filament velocity (V_max) with SDs.²⁷ A value of P<.01 was considered to be significant.

	Results

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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).

View this table: [in this window] [in a new window]   Table 1. Estimated CK Activities and Myosin Densities of the Flow Cell

The K-EDTA/ATPase activity of the myosin layers fixed with CK was 0.22±0.03 nmol P_i per minute per flow cell and that without CK was 0.23±0.02 nmol P_i per minute per flow cell. When the measured K-EDTA/ATPase activity of the myosin in solution was used (0.30±0.01 µmol P_i·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=V_max/(1+K_m/[MgATP]), where V is the observed filament velocity, V_max is the velocity at saturating MgATP, and K_m is the apparent Michaelis constant. K_m 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). V_max 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.

View larger version (13K): [in this window] [in a new window]   Figure 1. Graph showing the relation between the MgATP concentration and the filament velocity on the cardiac myosin layer fixed with () 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 K_m 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 V_max (7.6±0.1 versus 7.9±0.1 µm/s).

View larger version (14K): [in this window] [in a new window]   Figure 2. Graph showing the relation between the MgATP concentration and the filament velocity on the skeletal myosin layer fixed with () or without () skeletal CK. Solid lines were least-squares fits to data by the same equation as that of cardiac myosin. For the myosin layer fixed with CK, Km was estimated as 0.059±0.001 mmol/L; Vmax, 7.9±0.1 µm/s; and r2, .96. For that without CK, Km was 0.105±0.001 mmol/L; Vmax, 7.6±0.1 µm/s; and r2, .94.

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.

View larger version (9K): [in this window] [in a new window]   Figure 3. Representative chromatograms of the effluent solutions after the motility assays were performed for 1.5 minutes at 30°C in the presence of 0.5 mmol/L MgATP using cardiac proteins. A, Effluent from the flow cell in which the assay was done on the myosin layer fixed with CK in the buffer containing 5 mmol/L phosphocreatine; B, effluent from the flow cell in which the assay was done on the myosin layer fixed without CK in the buffer containing 5 mmol/L phosphocreatine; and C, effluent from the flow cell in which the assay was done on the myosin layer fixed without CK in the buffer containing 5 mmol/L phospho(enol)pyruvate and 20 IU/mL pyruvate kinase.

View this table: [in this window] [in a new window]   Table 2. [MgATP]/[MgADP] Ratios of the Effluents From the Flow Cell in Which the Assay Was Done for 1.5 Minutes at Various Concentrations of MgATP

Effects of a Lower pH and the Addition of P_i
Fig 4A shows the relation between the MgATP concentration and the filament velocity under acidic conditions (pH 6.7). The K_m 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). V_max 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).

View larger version (12K): [in this window] [in a new window]   Figure 4. A, Graph showing the relation between the MgATP concentration and the filament velocity on the myosin layer fixed with () or without () CK under a lower pH (pH 6.7). Solid lines were least-squares fits to data using the same equation as that used under control conditions (Fig 1). For the myosin layer fixed with CK, Km was estimated as 0.074±0.001 mmol/L; Vmax, 2.0±0.1 µm/s; and r2, .87. For that without CK, Km was 0.200±0.001 mmol/L; Vmax, 1.4±0.1 µm/s; and r2, .89. B, Graph showing the relation between the MgATP concentration and the filament velocity on the myosin layer fixed with () or without () CK in the presence of Pi (10 mmol/L). Solid lines were least-squares fits to data. For the myosin layer fixed with CK, Km was 0.056±0.001 mmol/L; Vmax, 5.3±0.1 µm/s; and r2, .98. For that without CK, Km was 0.091±0.001 mmol/L; Vmax, 4.6±0.1 µm/s; and r2, .93.

Fig 4B shows the relation between the MgATP concentration and the filament velocity in the presence of P_i (10 mmol/L). The K_m 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). V_max 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 P_i 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.

View larger version (14K): [in this window] [in a new window]   Figure 5. Graph showing the effect of the addition of MgADP on the sliding velocity under a constant concentration of MgATP (1 mmol/L). On the myosin layer fixed without CK (), addition of as little as 0.025 mmol/L MgADP reduced the sliding velocity significantly. On the myosin layer fixed with CK (), the sliding velocity became significantly lower only after the addition of 1.0 mmol/L MgADP or more. *P<.01.

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 K_m 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 K_m (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.

View larger version (15K): [in this window] [in a new window]   Figure 6. Graph showing the relation between the MgATP concentration and the filament velocity on the myosin layer fixed without CK in the presence of other ATP-regeneration systems supplied from the assay buffer. Solid lines were least-squares fits to data. In the presence of pyruvate kinase (PK) and phospho(enol)pyruvate in the assay buffer (), 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) is indicated by a broken line.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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 myofibrils^{2 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 31}P 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 K_m significantly without affecting the V_max, whereas cardiac CK changed both K_m and V_max 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 P_i 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 P_i concentration and/or intracellular pH.^{17 19 45} In this study, under acidic conditions, V_max decreased to about one third of that under the control conditions. On the other hand, the addition of P_i did not affect V_max significantly. However, quite recently, we showed that the accumulation of P_i 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 P_i 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.

	Selected Abbreviations and Acronyms

 

CK = creatine kinase DTT = dithiothreitol FDNB = 1-fluoro-2,4-dinitrobenzene HPLC = high-performance liquid chromatography NMR = nuclear magnetic resonance Pi = inorganic phosphate

	Acknowledgments

 
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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