Comparison of Unitary Displacements and Forces Between 2 Cardiac Myosin Isoforms by the Optical Trap Technique

Molecular Basis for Cardiac Adaptation

From The Second Department of Internal Medicine, School of Medicine, University of Tokyo (S.S., N.K., H.F., H.Y., S.M., M.O.), and the Department of Physiology, Teikyo University, School of Medicine (S.C., H.S.), Tokyo, Japan.

Correspondence to Seiryo Sugiura, MD, The 2nd Department of Internal Medicine, School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail Sugiura-2IM{at}h.u.-tokyo.ac.jp

	Abstract

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Abstract—To provide information on the mechanism of cardiac adaptation at the molecular level, we compared the unitary displacements and forces between the 2 rat cardiac myosin isoforms, V₁ and V₃. A fluorescently labeled actin filament, with a polystyrene bead attached, was caught by an optical trap and brought close to a glass surface sparsely coated with either of the 2 isoforms, so that the actin-myosin interaction took place in the presence of a low concentration of ATP (0.5 µmol/L). Discrete displacement events were recorded with a low trap stiffness (0.03 to 0.06 pN/nm). Frequency distribution of the amplitude of the displacements consisted of 2 gaussian curves with peaks at 9 to 10 and 18 to 20 nm for both V₁ and V₃, suggesting that 9 to 10 nm is the unitary displacement for both isoforms. The duration of the displacement events was longer for V₃ than for V₁. On the other hand, discrete force transients were recorded with a high trap stiffness (2.1 pN/nm), and their amplitude showed a broad distribution with mean values between 1 and 2 pN for V₁ and V₃. The durations of the force transients were also longer for V₃ than for V₁. These results indicate that both the unitary displacements and forces are similar in amplitude but different in duration between the 2 cardiac myosin isoforms, being consistent with the reports that the tension cost is higher in muscles consisting mainly of V₁ than those consisting mainly of V₃.

Key Words: cardiac myosin • unitary displacement • unitary force • laser optical trap

	Introduction

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Mammalian ventricular muscle myosin is divided into 3 different isoforms, V₁, V₂, and V₃.¹ Actin-activated Mg-ATPase activity is highest for V₁ and lowest for V₃. Contractile properties of these isoforms have been studied in muscle preparations, in which myosin isoform composition was modified either hormonally² or by imposing overload.^{3 4} All these studies indicated that the maximum unloaded shortening velocity (V_max) of muscle preparations correlated well with their V₁ isoform content, suggesting the fast crossbridge cycling rate in this isoform. In these experiments with muscle preparations, however, it was difficult to preclude the possible influence of concomitant changes of other cellular components on V_max. In addition, it was difficult to estimate the number of crossbridges generating contractile force.

In vitro motility assay systems are effective in studying the kinetics of the ATP-dependent interaction between purified actin and myosin molecules. In accordance with the results for V_max in muscle preparations, the unloaded velocity of actin-myosin sliding in vitro has been shown to be much faster for V₁ than for V₃ isoforms.^{5 6} Using the centrifuge microscope, with which constant centrifugal forces are applied as loads on in vitro actin-myosin sliding, we showed that the shape of force-velocity curves was markedly different between V₁ and V₃ isoforms, reflecting their different interaction kinetics with actin.⁷ Recent development of the laser optical trap technique has made it possible to study mechanical events generated by a single myosin molecule as it splits ATP and interacts with actin.^{8 9 10}

In the present study, we used this technique to measure unitary displacements and forces generated by the 2 cardiac myosin isoforms. It will be shown that the unitary mechanical events generated by V₁ and V₃ isoforms are similar in amplitude but different in duration.

	Materials and Methods

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Proteins
The V₁ isoform was obtained from ventricular muscle of 3-week-old male Wistar rats, whose ventricular muscle consists predominantly of the V₁-type myosin isoform.¹¹ The V₃ isoform was obtained from ventricular muscles of 12-week-old male Wistar rats, after inducing hypothyroidism by adding 6.1 µmol/L 1-methyl-2-mercaptoimidazole (M8506, Sigma Chemical Co) to drinking water for 12 weeks (average dose, 15 mg/d). The animals were anesthetized with diethyl ether, and the hearts were excised rapidly. Myosin samples were prepared from ventricular muscles by the method of Katz et al¹² with some modifications. All procedures were carried out at 4°C in the presence of 5 mmol/L dithiothreitol and 10.8 µmol/L leupeptin (L9783, Sigma). At the final stage, actin was removed from the sample by centrifugation (100 000g for 3 hours). Actin was prepared from rabbit back muscle by the method of Spudich and Watt.¹³ Plasma gelsolin was prepared from bovine plasma by the method of Kurokawa et al.¹⁴

In Vitro Motility Assay System
Fluorescently labeled actin filaments were prepared by the method of Kron and Spudich¹⁵ with some modifications. Briefly, G-actin was incubated at 4°C overnight with a molar excess of rhodamine-phalloidin (Molecular Probes Inc) in a solution containing 25 mmol/L KCl, 6 mmol/L MgCl₂, 25 mmol/L HEPES, and 1 mmol/L EGTA to obtain fluorescently labeled actin filaments. Carboxylated polystyrene beads (diameter, 1 µm; Polyscience), to which gelsolin had been cross-linked with 1-ethyl-2-(3-dimethylaminopropyl)-carbodiimide, were mixed with the actin filaments to attach the beads to the barbed end of the actin filaments.¹⁶

The myosin sample was diluted to 0.5 to 1.0 µg/mL in a high ionic strength buffer (0.6 mol/L KCl and 50 mmol/L Tris-HCl, pH 7.5) and applied to the nitrocellulose-coated surface of a glass coverslip (60x30 mm, Matsunami Co), so that the myosin molecules were sparsely bound on the coverslip. The myosin-bound coverslip was covered with another coverslip (18x18 mm), separated from the former by 100 µm with silicon grease, to form a flow cell. The bead-attached actin filaments were suspended in a motility buffer containing 25 mmol/L KCl, 6 mmol/L MgCl₂, 0.5 µmol/L ATP, 1 mmol/L EDTA, 50 mmol/L imidazole, 25 mmol/L Tris-HCl, and 0.2% methylcellulose (pH 7.5) and introduced into the flow cell.

Laser Optical Trap
The flow cell was mounted on the stage of an inverted microscope (Axiovert, Zeiss) equipped with bright-field and epifluorescence illumination. The bead-bound actin filaments were observed with an oil immersion objective (x100; numerical aperture, 1.3; Zeiss). A bead bound only to a single actin filament (length, 3 to 5 µm) was selected and captured in the optical trap. Figure 1 is a schematic diagram of the laser optical trap system. The beam of a Nd-YAG laser (1047 nm, 1 W, Amoco Laser) was introduced into the back aperture of the objective lens to form an optical trap in the flow cell. The position of the optical trap was controlled both horizontally and vertically with a pair of piezoelectrically driven mirrors (resonance frequency, 9 kHz; Physik Instrument) in the beam path. Trap stiffness was estimated from brownian motion for an actin-free bead¹⁷ to be 0.03 to 0.06 pN/nm. The trapped bead was held close to the myosin-coated glass surface to allow the actin filament to interact with the surface-bound myosin molecules. Bead movement within the trap was recorded by projecting the bright-field image of the bead onto a quadrant photodiode detector (Hamamatsu Photonics), the output of which indicated the bead position along parallel to and perpendicular to the actin filament long axis with nanometer resolution.

View larger version (20K): [in this window] [in a new window]   Figure 1. Schematic diagram of the laser optical trap system. A fluorescently labeled actin filament (AF) with a polystyrene bead (Pb) attached to its barbed end is made to interact with a single or a few myosin molecules sparsely bound to a glass surface, while the bead is captured in the optical trap in the flow cell (F), mounted on the mechanical stage (S) of a microscope. Direction of ATP-dependent actin-myosin sliding is indicated by an arrow. Bright-field illumination with a xenon lamp (Xe) is used to project the bead image onto a quadrant photodiode detector (QD). Two piezoelectric mirrors (PM) rapidly deflect the beam of Nd-YAG laser (thick solid line) before it enters into the back aperture of the objective lens (O) to change the trap position. Epifluorescence illumination with a mercury lamp (Hg) is used to observe the fluorescent actin filament with an silicone-intensified camera (SIT). Other optics in the system are a condenser (C), 2 mirrors (M), and 3 dichroic filters (D). The output from the quadrant detector is fed to the feedback circuit (FC) driving the 2 PMs.

To record isometric forces produced by the actin-myosin interaction, the output of the quadrant photodiode detector was fed to a feedback circuit, which in turn controlled the 2 piezoelectrically driven mirrors to rapidly displace the trap position, so that the bead was held stationary in position. The feedback signals driving the mirrors changed linearly with the trap position changes and were calibrated by the method of Finer et al,⁸ as shown in Figure 2. When a constant viscous force was applied to the trapped bead by moving the microscope stage in a triangular fashion, the bead position changed in a square-shaped manner. When the feedback loop was closed, the trap position changed in a square-shaped manner, while the bead position was kept stationary. Thus, isometric forces were measured as a linear function of the trap displacement.^{8 9 10} With this feedback system, the trap stiffness was increased to 2.1 pN/nm.

View larger version (16K): [in this window] [in a new window]   Figure 2. Responses of the optical trap system to triangular movements of the microscope stage position, on which the flow cell is mounted, with the feedback loop opened (left) and with the feedback loop closed (right). Left, The bead position shows square-shaped changes due to alternating constant viscous drag forces produced by the stage movement. Right, The trap position shows square-shaped changes, while the bead position remains stationary.

Data Collection and Analysis
With the low myosin surface density used, a bead, captured in the low stiffness trap, showed discrete displacement events in the direction parallel to the long axis of the actin filament but not in the direction perpendicular to it. When the feedback loop was closed to increase the trap stiffness, discrete changes of the trap position, ie, force transients, were observed along the same axis. Mechanical events produced by the actin-myosin interaction were scored in the records by eye according to criteria similar to those of Finer et al⁸: (1) the events should be clearly distinct and isolated from other fluctuations above the baseline brownian motion (>5 nm for displacement, >0.5 pN for force); (2) the events should exhibit a rapid rise to a peak and a rapid fall to the baseline; and (3) the baseline brownian motion should be the same before and after the event. In addition, the events <15 milliseconds in duration were not scored to eliminate noise due to thermal vibrations.¹⁸

Both the displacement and the force signals were stored in a data recorder (RD-120TE, TEAC) and analyzed off-line with Laboratory View software (National Instruments) on a personal computer. To estimate the mean duration of unitary events, a single exponential was fit to frequency histograms of the event duration by nonlinear curve-fitting software (Igor, Move Metrics). Data were expressed as mean±SD. A 2-tailed unpaired Student t test was used to determine significant difference between group means and fit parameters. A value of P<0.05 was considered significant.

	Results

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Unitary Displacements
Figure 3 shows examples of displacement records obtained from V₁ and V₃ isoforms under a low trap stiffness. The records consisted of discrete displacement events that fulfilled the criteria stated in "Materials and Methods" and the baseline brownian motion. In the present study, we observed only discrete displacements above the baseline level ("forward" displacements) but did not observe any displacements below the baseline level ("reverse" displacements). Frequency histograms of the amplitude of displacement events for V₁ and V₃ isoforms are shown in Figure 4. Displacements with amplitudes 5 nm were not scored, because their amplitudes were similar to those of brownian motion. Since both histograms exhibited a broad distribution with 2 peaks, a few gaussian curves were fitted to the frequency distribution, on the assumption that the amplitude distribution is built up statistically of the unitary distance of ATP-dependent actin-myosin sliding. In accordance with this assumption, the amplitude distribution was composed of 2 gaussian curves with peaks at 8 to 9 nm and 16 to 18 nm in both V₁ and V₃ isoforms. This may be taken to indicate that the amplitude of unitary displacement is the same in both V₁ and V₃, being 8 to 9 nm. Meanwhile, the mean amplitude of all the displacement events was 15.3±14.6 nm (n=147) for V₁ and 14.9±6.7 nm (n=227) for V₃, being not significantly different between V₁ and V₃.

View larger version (18K): [in this window] [in a new window]   Figure 3. Examples of displacement records obtained from V1 and V3 isoforms under a low trap stiffness. Vertical deflections in the records indicate movements of the trapped bead in the direction parallel to the actin filament long axis.

View larger version (31K): [in this window] [in a new window]   Figure 4. Frequency histograms showing distribution of the amplitude of the displacement in V1 and V3 isoforms. Displacements with amplitudes 5 nm were not scored. Dashed curves represent distributions of gaussian components; solid curves show reconstructed distributions of the gaussian components.

As shown in Figure 5, histograms of the duration of displacement events for V₁ and V₃ isoforms exhibited a broad distribution and were well fitted to single exponential curves, being consistent with the idea that a first-order kinetic process limits detachment of myosin molecules from actin. The mean duration of displacement events, estimated from the exponential fit of the duration distribution,¹⁹ was 204.7±5.1 milliseconds (n=147) for V₁ and 282.7±19.8 milliseconds (n=227) for V₃, indicating that the duration of displacement events was longer in V₃ than in V₁ (P<0.01).

View larger version (18K): [in this window] [in a new window]   Figure 5. Distributions of the duration of the displacement events in V1 and V3 isoforms. Solid curves are single exponential fits to the distributions of durations.

Unitary Forces
Examples of force records obtained from V₁ and V₃ isoforms under a high trap stiffness with the feedback loop closed are shown in Figure 6. As with the displacement events, we observed force transients only above the baseline brownian motion (positive forces) but did not observe those below the baseline level (negative forces). Figure 7 shows the histograms of distribution of the amplitude of force transients for V₁ and V₃. Force transients 0.5 pN in amplitude were not scored. Although the amplitude showed a broad distribution, only 1 peak was evident for both V₁ and V₃. The mean amplitude of force transients was 1.2±0.6 pN (n=267) for V₁ and 1.6±0.9 pN (n=457) for V₃, being not significantly different between V₁ and V₃. With respect to force transients produced by V₃, we obtained a few records in which force transients exhibited 2 distinct amplitudes, one (3.2 to 3.8 pN) being approximately double the other (1.6 to 1.9 nm) (Figure 8).

View larger version (23K): [in this window] [in a new window]   Figure 6. Examples of force transient records obtained for V1 and V3 isoforms under a high trap stiffness with feedback. Vertical deflections in the record indicate movements of the trap position, representing force transients.

View larger version (34K): [in this window] [in a new window]   Figure 7. Frequency histograms showing distribution of the amplitude of the force transients in V1 and V3 isoforms. Force transients 0.5 pN were not scored.

View larger version (15K): [in this window] [in a new window]   Figure 8. Force transient record obtained from V3 isoform, in which the force transients exhibited 2 distinct amplitudes with a ratio of 2 (high force, 4 pN; low force, 2 pN). Bar=5 pN.

As shown in Figure 9, frequency distributions of the duration of force transients also showed broad distributions and were fitted to single exponentials. The mean duration of force transients, estimated from the exponential fit, was 332.7±14.0 milliseconds (n=267) for V₁ and 488.1±82.4 milliseconds (n=457) for V₃, indicating that the duration of force transients was longer in V₃ than in V₁ isoforms (P<0.01).

View larger version (23K): [in this window] [in a new window]   Figure 9. Distribution of the duration of the force transients in V1 and V3 isoforms. Solid curves are single exponential fits to the distribution of durations.

	Discussion

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Using the optical trap technique to capture an actin filament–bound bead in the flow cell, we have recorded displacements and forces produced by myosin molecules of 2 different cardiac myosin isoforms interacting with the actin filament. Discrete mechanical events were recorded by sparsely distributing the myosin molecules on a glass surface and by lowering ATP concentration to 0.5 µmol/L.^{8 9 10 16} In the present study, we used a single trap to hold the actin filament–bound bead, as with the experiments of Miyata et al.¹⁶ With the single-trap method, the whole actin filament cannot be held horizontally (Figure 1), and this filament geometry would lead to an underestimate with respect to the amplitude of mechanical events.²⁰ Meanwhile, the single-trap method enables us to use gelsolin to attach the bead to the filament barbed end, thus eliminating the end compliance at the bead-filament junction. With the double-trap method, 2 beads should be attached to both ends of the actin filament with NEM-inactivated myosin, and this caused end compliance at both ends of the actin filament, which would act to underestimate the amplitude of mechanical events.²¹ In both methods, the degree of underestimation would not be very large.

Amplitude of Unitary Displacements
In the present study, we have scored by eye the displacement events under a low trap stiffness according to the criteria of Finer et al.⁸ In both V₁ and V₃ isoforms, the frequency distribution of the amplitude of the displacement events consisted of 2 gaussian curves with peaks at 8 to 9 and 16 to 18 nm, respectively (Figure 4). This may be taken to indicate that the amplitude of the unitary displacement was 8 to 9 nm in both V₁ and V₃ isoforms. The unitary displacement amplitude of 9 to 10 nm fell within the range of the corresponding values (7 to 11 nm) measured by the optical trap technique for skeletal and smooth muscle myosins^{8 10 16} and was also approximately the same as the values determined by an assay system using a skeletal muscle myosin–coated microneedle sliding on the actin filament (10 nm)²² and a system using a microneedle-bound actin filament sliding along a skeletal muscle myosin–myosin rod cofilament (up to 17 nm).²³

Contrary to the reports cited above, Molloy et al⁹ analyzed their optical trap data on the assumption that bead brownian motion shows a gaussian distribution extending over ±30 nm under a low trap stiffness and that myosin displaces the actin filament by a fixed distance whenever the displacement action happens during the ±30-nm motions. On this basis, they estimated the unitary actin-myosin sliding as the shift of the gaussian distribution of bead motion during the period when the actin-myosin interaction is likely to take place and obtained a value of 3 to 4 nm, being much smaller than the values mentioned above. Although their analysis was unique in not measuring by eye, it has the following problems: (1) their data fitted gaussian curves only when the myosin subfragment-1 was used; (2) their judgment of the period of actin-myosin interaction was made by eye and was therefore somewhat arbitrary; (3) the gaussian distribution of the bead brownian motion could be shifted by any other static linkages other than actin-myosin molecules; (4) their assumption of constant unitary displacement at any time during the brownian motion contradicted the reports that the amplitude of the unitary displacement was a function of the trap stiffness²⁴; and (5) their analysis gave no information about the duration of the displacement events.

Recently, Sugi et al²⁵ have succeeded in recording the ATP-induced myosin head movement in living thick filament by using the gas environmental chamber with which biological specimens were kept in a wet state in an electron microscope. They have shown that the myosin heads move parallel to the filament long axis with an amplitude of 20 nm. Although this myosin head movement takes place in the absence of the actin filament and does not necessarily correspond with the unitary displacement at the present stage, the value at 20 nm seems to be consistent with the reported values of the unitary displacement, if random orientation of myosin molecules in the in vitro motility assay systems and the factors leading to underestimation of the unitary displacement are taken into consideration.

Amplitude of Unitary Forces
The amplitude of the force transients recorded under a high trap stiffness with feedback also showed a broad distribution in both V₁ and V₃ isoforms (Figure 7). The average amplitude of the force transients (1.2 pN for V₁ and 1.6 pN for V₃) did not differ significantly between the 2 isoforms. These values also fell within the broad range of reported values concerning the amplitude of the unitary forces from 1.2 to 6 pN in skeletal and smooth muscle myosins.^{8 9 10 23} The wide variation of the unitary force amplitude may result from incorrect orientation of myosin molecules in the motility assay systems²³ and also from uncertainty concerning the actual number of myosin molecules involved in the force generation, a problem inherent to the motility assay experiments. In this connection, the "quantal" appearance of the force transient records with V₃ (Figure 8) might have arisen from the cooperative action between the 2 heads of a myosin molecule, which was correctly oriented with respect to the actin filament long axis; if the 2 heads of the myosin molecule are assumed to operate in an "arm-over-arm" mechanism, the lower force level may be produced by 1 head interacting with actin, and the higher force level may be due to 2 heads of a myosin molecule, both interacting with actin to produce force in an "additive" manner. Much more experimental work is needed to further investigate this interesting mechanism.

Duration of Unitary Displacements and Forces
Although the amplitude of the displacements and forces did not differ between the 2 cardiac myosin isoforms, the duration of the displacements and forces was longer in V₃ than in V₁ isoforms (Figures 5 and 9). The average duration of the unitary displacements (under a low trap stiffness) was 204.7 milliseconds for V₁ and 282.7 milliseconds for V₃; the average duration of the unitary forces (under a high trap stiffness) was 332.7 milliseconds for V₁ and 488.1 milliseconds for V₃. The average duration of the mechanical events, determined by the exponential fit of its frequency distribution, has been taken as a measure of the rate constant for the process terminating the event,⁸ ie, detachment of myosin from actin. On this basis, the longer average event duration in V₃ than in V₁ is consistent with the result that the ATPase activity in solution is higher with the V₁ sample than with the V₃ sample²⁶ and also with the result that the unloaded velocity of ATP-dependent actin-myosin sliding is higher for V₁ than for V₃.^{5 11} Furthermore, it is of interest that in both V₁ and V₃, the average event duration under a high trap stiffness was nearly twice larger than that under a low trap stiffness, probably reflecting strain-dependent (or load-dependent) changes in the rate constant for detachment of myosin from actin. These results suggest an interesting possibility that the efficiency of conversion of chemical energy of ATP hydrolysis into mechanical work in individual myosin molecules changes in a load-dependent manner.

Implications for the Cardiac Mechanoenergetics
Muscle isometric force is the sum of time-averaged forces produced by each myosin molecule in muscle. According to Shibata et al,²⁷ the maximum isometric force per unit area did not differ between ventricular papillary muscles obtained from hyperthyroid rabbit (containing predominantly the V₁ isoform) and control rabbit (predominantly the V₃ isoform). In accordance with this result, de Tombe et al²⁸ compared the maximal force generated by right ventricular trabeculae from euthyroid rats (V₁) and hypothyroid rats (V₃) to find no significant difference between them. On the other hand, however, we can also find studies reporting greater force-generating ability of cardiac muscle containing V₃ compared with V₁ isoforms.^{29 30} Similarly, contradictory results have been reported on the time-averaged force using in vitro motility assay techniques, ie, V₃>V₁^{6 31} and V₃=V₁.³²

On the basis of the 2-state model, the time-averaged force (Fave) can be related to the unitary force (f) as follows: Fave=fx(duty ratio), where duty ratio is the ratio between the "on time" and total ATPase cycle time under isometric conditions. Because, as mentioned above, the shorter duration of unitary force for the V₁ isoform implies a faster ATPase rate (shorter ATPase cycle time) of this isoform, similar duty ratio and time-averaged force would be expected for V₁ and V₃ isoforms. This view is consistent with our previous finding.³² Importantly, however, to maintain a similar level of time-averaged force, the V₁ isoform must consume more ATP (less economy), which has been suggested by muscle heat measurement as one of the bases for the cardiac adaptation process.³³

To summarize, we have compared the unitary displacement and force produced by V₁ and V₃ myosin isoform molecules with the optical trap technique and have shown that the average duration of mechanical events is longer for V₃ than for V₁. This implies that to maintain the same level of time-averaged force, a V₁ molecule should produce unitary forces more frequently than does a V₃ molecule to result in a higher tension cost for V₁ than for V₃.

	Acknowledgments

 
This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (Differentiation and Regulation of Cardiac Cells) and a Grant-in-Aid for Scientific Research (C-08670767) from the Ministry of Education, Science, and Culture of Japan.

Received December 22, 1997; accepted March 19, 1998.

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