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(Circulation Research. 1995;77:439-444.)
© 1995 American Heart Association, Inc.

Articles

Cardiac V₁ And V₃ Myosins Differ in Their Hydrolytic and Mechanical Activities In Vitro

Peter VanBuren, David E. Harris, Norman R. Alpert, David M. Warshaw

From the Departments of Molecular Physiology and Biophysics (D.E.H., N.R.A., D.M.W.) and Cardiology (P.V.B.), University of Vermont, Burlington.

Correspondence to Dr David M. Warshaw, University of Vermont, Department of Molecular Physiology and Biophysics, Given Medical Bldg, D-205, Burlington, VT 05405. E-mail warshaw@salus.med.uvm.edu.

	Abstract

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Abstract The two mammalian cardiac myosin heavy chain isoforms, and ß, have 93% amino acid homology, but hearts expressing these myosins exhibit marked differences in their mechanical activities. To further understand the function of these cardiac myosins as molecular motors, we compared the ability of these myosins to hydrolyze ATP and to both translocate actin filaments and generate force in an in vitro motility assay. V₁ myosin has twice the actin-activated ATPase activity and three times the actin filament sliding velocity when compared with V₃ myosin. In contrast, the force-generating ability of these myosins is quite different when the total force produced by a small population of myosin molecules (>50) is examined. V₁ myosin produces only one half the average cross-bridge force of V₃ myosin. With discrete areas of primary structural heterogeneity known to exist between and ß heavy chains, the differences we report in the hydrolytic and mechanical activities of the motors are explored in the context of potential structural and kinetic differences between the V₁ and V₃ myosins.

Key Words: cardiac myosin • -myosin heavy chain • ß-myosin heavy chain • molecular motor • motility assay

	Introduction

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Cardiac myosin is a hexameric mechanoenzyme comprising two heavy chains, each having a pair of noncovalently bound light chains. The myosin motor generates force and motion through its cyclic interaction with actin, with the energy to power this process derived from the hydrolysis of ATP by myosin. Two cardiac myosin heavy chain isoforms ( and ß) are expressed in the myocardium of adult laboratory mammals. Myosins containing only the or ß heavy chains are referred to as V₁ and V₃ cardiac myosin, respectively. The proportion of V₁ and V₃ isoforms expressed in the adult rabbit can vary dramatically, depending on the age of the animal or the type of cardiovascular and/or hormonal stress to which the animal is exposed. In the healthy adult rabbit, the ventricular myosin is 70% V₃. Experimental hyperthyroidism induces a shift to the V₁ isoform, whereas experimental hypothyroidism and pressure-overload cardiac hypertrophy¹ induce a shift to the V₃ isoform. Associated with these isoform shifts are dramatic alterations in cardiac function, as evidenced by changes in the maximum unloaded shortening velocity² and ATP consumption by the contractile machinery, generating isometric force in isolated cardiac tissue.³ Can these alterations to the mechanics of the tissue be attributed to inherent differences between the V₁ and V₃ myosin motors?

Earlier in vitro studies of isolated V₁ and V₃ myosin indicate that both the actin-activated and calcium-stimulated myosin ATPases for V₁ myosin are approximately three times greater than for V₃ myosin.^{1 4} Single actin filament velocities over pure populations of V₁ and V₃ myosins in an in vitro motility assay show similar differences.⁴ These data suggest that the faster maximum shortening velocity for predominantly V₁ cardiac tissue reflects inherent differences in the hydrolytic and motion-generating activity of this myosin isoform compared with the V₃ isoform. Regarding force generation, Hasenfuss et al³ estimate that the economy of ATP utilization for isometric force production (ie, cross-bridge tension-time integral per ATP) by V₃ myosin is twice that for predominantly V₁-containing tissue. If this difference can be attributed to the myosin motor, then V₃ myosin may generate greater force per unit time than V₁ myosin. We previously addressed this possibility through an indirect approach using the in vitro motility assay.⁴ In this earlier study, an estimate of the force-generating ability of V₁ and V₃ was predicted by a model that could account for the observed actin filament velocities over mixtures of V₁ and V₃ myosin. The basic assumption of the model was that the two myosin isoforms mechanically interact to determine the resultant actin filament velocity.⁴ When this model is used, the V₃ isoform has been predicted to generate average cross-bridge force (ie, cross-bridge force averaged over time [F_avg]) as much as three times greater than that generated by V₁ myosin.

In the present study, we directly determined whether V₃ myosin generates greater F_avg when compared with V₁ myosin. Our approach was to measure the isometric force exerted from as few as 50 myosin molecules pulling on a single actin filament attached to an ultracompliant glass microneedle.^{5 6} In addition, actin-activated myosin ATPase activities and actin filament sliding velocities were measured for both the V₁ and V₃ isoforms to determine how the kinetics of the cross-bridge cycle might differ for the two isoforms and thus account for differences in the mechanical performance of these myosins.

	Materials and Methods

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Contractile Protein Isolation and Preparation
V₁ and V₃ cardiac myosins were obtained by inducing either hyperthyroidism or hypothyroidism, respectively, in 8-week-old rabbits (weight, 1.5 to 1.7 kg). Hyperthyroidism was induced with daily intramuscular injections of levothyroxine (0.2 mg/kg) for 2 weeks, whereas hypothyroidism was induced by placing propylthiouracil in the drinking water (0.8 mg/mL) for 3 weeks. At the end of the above treatment periods, the rabbits were killed, and the left ventricles from three rabbits were isolated and pooled for myosin purification.

Myosin purification was essentially as described by Shiverick et al,⁷ with the following exception. During purification, all solutions contained the following protease inhibitors: N-p-tosyl-L-lysine chloromethyl ketone (5 mg/L), pepstatin (5 mg/L), leupeptin (10 mg/L), tosylamidophenylethyl chloromethyl ketone (3 mg/L), benzamidine (50 mg/L), and phenylmethylsulfonyl fluoride (0.5 mmol/L), with the addition of dithiothreitol (2 mmol/L) and EDTA (0.5 mmol/L). The purification protocol lasted 2 days and was conducted at 4°C. On completion of the isolation, the purified myosin was stored overnight at -20°C in 0.3 mol/L KCl, 0.02 mol/L Tris-HCl, 0.5 mmol/L sodium azide, 10 mmol/L dithiothreitol, and 50% glycerol, with all of the above protease inhibitors added. All studies using this myosin were conducted the following day.

V₁ and V₃ myosin content was determined via pyrophosphate gel electrophoresis and computer densitometry. After the treatment protocol described above, rabbit hearts treated with levothyroxine contained 100% V₁ myosin (Table). In contrast, rabbit hearts treated with propylthiouracil contained 93% V₃ and 7% V₁ myosin (Table). The V₂ content was very small (<4%), consisting of a heterodimer of - and ß-myosin. For myosin isoenzyme content estimates, one half of the V₂ peak was added to each of the V₁ and V₃ myosin peaks.

View this table: [in this window] [in a new window]   Table 1. Myosin Isoform Content, Force, Velocity, and ATPase

Actin was isolated from chicken pectoralis muscle as previously described.⁸ Before performing the in vitro motility assay, actin filaments were incubated overnight at 4°C in the fluorescent label tetramethylrhodamine-phalloidin, as previously described.⁹

Actin-Activated Myosin ATPase Activity
Actin-activated myosin ATPase rates were determined essentially as previously described.⁴ Briefly, myosin was diluted to 0.1 mg/mL in the following (mmol/L): KCl 25, MgCl₂ 4, imidazole 10 (pH 7.0), and EGTA 1 at 30°C. Actin was added to a final concentration of 5, 10, 20, and 30 µmol/L, and the reaction was started with 2 mmol/L ATP. Aliquots of the reaction mixture were sampled at times from 5 to 60 minutes, and the reaction stopped with SDS. The rate of inorganic phosphate liberation per myosin head was determined colorimetrically.¹⁰ These rates were used for Lineweaver-Burk plot analysis to determine the maximum actin-activated myosin ATPase rate.

Myosin Head Density on Motility Surface
The number of myosin heads on the nitrocellulose-coated glass surface was determined by comparing the NH₄-EDTA ATPase activity of myosins adhered to the coverslip with the activity of known amounts of myosin in solution as described previously.¹¹ Knowing the number of myosin molecules adhered to the surface and the dimensions of the motility surface, we could then estimate the surface density of biochemically active heads. For the 250 µg/mL myosin loading used in the present study, the motility surface was saturated with a density of 2068 myosin heads per square micrometer. This density was not different from previous density estimates for similar loading of either smooth or skeletal muscle myosin.¹¹ Given this similarity in surface densities for the various myosins, it appears that the binding of myosin to the nitrocellulose surface is independent of myosin isoform. Therefore, any mechanical differences that are observed for the V₁ and V₃ cardiac myosin isoforms cannot be attributed to different surface binding characteristics for the two myosins.

Force Measurements
The measurement of isometric force in the in vitro motility assay has been described previously.^{5 6} In brief, monomeric myosin (250 µg/mL) was adhered to a nitrocellulose-coated coverslip, and the coverslip was placed on the stage of an inverted microscope. A single fluorescently labeled actin filament was attached by means of N-ethylmaleimide skeletal muscle myosin to the tip of an ultracompliant calibrated (50 to 200 nm/pN) glass microneedle. The microneedle and attached actin filament were then brought within 4 µm of the myosin-coated surface, and the actin filament was allowed to engage the myosin surface. The pulling of the actin filament by myosin across the surface caused the microneedle to bend, until the maximum steady state isometric force exerted by the myosin population was balanced by the opposing force of the microneedle. The video image of the microneedle and actin filament were recorded on videotape and digitized by computer for later analysis. By use of these digital images, the microneedle deflection (ie, force) and actin filament length in contact with the surface were measured. Given the high surface density of myosin, the maximum steady state isometric force (Fig 1) was normalized to the length of actin in contact with the myosin surface. This normalization was required since a longer actin filament could interact with a greater number of myosin molecules.

View larger version (9K): [in this window] [in a new window]   Figure 1. Time course of force generation by V1 cardiac myosin. The myosin population produced a maximum steady state isometric force for at least 0.6 seconds. The maximum isometric force value was determined by averaging force over this steady state period, and this value was used to construct the maximum force vs actin filament length plot in Fig 3. Note that at peak force for this experiment, the actin filament breaks, and the microneedle returns to its previous zero-force baseline, which serves as a control against baseline drift. The microneedle deflection (ie, force) was determined at a 0.2-second interval from digitized video images.

Freely moving actin filament velocities were calculated from digitized video images via computer.¹² Actin filament velocities are reported as the mean±SD for at least 36 filaments. The motility assay was performed in low-salt (25 mmol/L KCl) assay buffer at 30°C.⁵

Defining Cross-bridge Mechanical Parameters
The in vitro motility assay provides a unique opportunity to record both myosin force and motion generation at a molecular level. Therefore, it is necessary to define the mechanical parameters that now can be determined for a single molecular motor. Force generation can be viewed as a simple two-state cycle¹³ (see Fig 2) in which the myosin motor is first detached from actin and then attaches during its power stroke to generate its unitary force (F_uni). The fraction of the cycle in which the cross-bridge is attached and generates force is defined as its duty cycle (f). Although these parameters can be obtained from a single myosin molecule,^{14 15} the studies using the microneedle assay presented here are limited to resolving the maximum force produced by 50 myosin molecules (see "Results"). Therefore, knowing the myosin surface density and the maximum force per unit length actin (see "Results"), we can only estimate a cross-bridge force that is effectively averaged over the entire cross-bridge cycle (F_avg), which is also the product of F_uni and f (ie, F_avg=F_uni · f; see Fig 2).

View larger version (18K): [in this window] [in a new window]   Figure 2. Illustration of two-state cross-bridge cycle. During the hydrolysis of a single ATP molecule (cross-bridge cycle), the myosin motor is either attached and strongly bound to actin, generating its unitary force (Funi), or detached (ie, weakly bound) and not generating any force (top). The duty cycle (f) is indicated as a fraction of the cross-bridge cycle. The average cross-bridge force (Favg) per cycle is calculated as the product of Funi and f. Two examples are presented to illustrate how a twofold increase in either Funi (middle) or f (bottom) can result in a twofold increase in Favg.

	Results

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Actomyosin ATPase Activity and Actin Filament Velocities
The solution actomyosin ATPase activity per myosin head for V₁ myosin (5.7 s^-1) was nearly twice that of V₃ myosin (3.0 s^-1, Table), as previously reported by this laboratory.⁴ Given these ATPase data, it was not surprising that the velocity of freely moving actin filaments over a myosin-coated surface showed significant differences for the two myosin isoforms. V₁ myosin demonstrated a threefold faster actin filament sliding velocity (4.63 µm/s) than V₃ myosin (1.55 µm/s, Table), confirming our previous estimates⁴ and those of others.¹⁶

Force Measurements
The time course of force production by a population of myosin molecules interacting with a single actin filament was determined by monitoring the deflection of an ultracompliant glass microneedle to which the actin filament was attached (see "Materials and Methods"; Fig 1). To compare the force-generating capacity for V₁ and V₃ myosins, the maximum steady state isometric force for the population was normalized to the length of actin in contact with the myosin surface (see Fig 3). The slope of this relation for V₃ myosin (14.4 pN/µm actin length) is approximately twice that for V₁ myosin (7.7 pN/µm actin length) (see Table). Knowing the surface density of myosin and assuming a 10-nm reach for the myosin head,¹¹ 50 myosin heads can interact with a 1-µm length of actin. Dividing this number of heads into the slopes of the linear regressions, we estimate F_avg to be 0.30 pN for V₃ myosin and 0.15 pN for V₁ myosin. These F_avg estimates are in the range of values reported previously for skeletal muscle myosin^{5 6} by use of a similar technique. There is no doubt that these values underestimate the true F_avg, given the recent 3- to 5-pN estimates for the skeletal muscle myosin F_uni.^{14 15} However, this obvious underestimate can be reconciled given that (1) the random surface orientation of myosin will prevent at least 50% of the myosin molecules from productively interacting with actin¹⁵ and (2) the duty cycle for isometrically contracting muscle may be as low as 20%.¹⁴ When only these two factors are considered, our F_avg estimates suggest F_uni values as high as 3 pN, in agreement with direct single myosin molecule measurements.^{14 15} Since there are no apparent reasons to assume that V₁ and V₃ myosins differ in their surface binding characteristics (see "Materials and Methods"), it appears that the difference in the force-generating capacity of these myosins is an inherent molecular property.

View larger version (19K): [in this window] [in a new window]   Figure 3. Maximal isometric steady state force vs actin filament length in contact with myosin-coated surface for V1 myosin () and V3 myosin (). The data were fitted by linear regression (solid lines), with the 95% confidence limits for the regression indicated by the dashed lines. The slopes of the regressions are reported in the Table.

	Discussion

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In the present study, we have shown dramatic differences between V₁ and V₃ myosin in both their hydrolytic and mechanical activities. Although the actomyosin ATPase activity of V₁ myosin and actin filament velocity are two to three times greater than that of V₃ myosin, its average force-generating ability is only half that of V₃ myosin. Since no change in the myosin light chains is reported to occur with isoform shifts between the V₁ and V₃ myosins,^{1 2} the observed differences in motor performance within this simplified contractile system model can be directly attributed to the innate differences between the and ß heavy chains.

Relation Between Hydrolytic and Mechanical Activities
Given the dual role of myosin as a hydrolytic enzyme and a molecular motor, it may be reasonable to assume that these two functions are interdependent, as proposed by A.F. Huxley.¹⁷ To understand the possible relation between ATPase and motor activities, it is necessary to briefly review the actomyosin cross-bridge cycle both from a biochemical and mechanical viewpoint.

The hydrolysis of MgATP by myosin is a multistep enzymatic cycle in which the various intermediate states are defined by myosin being either weakly or strongly bound to actin.¹⁸ The hydrolysis step is believed to occur while the myosin is weakly bound to actin (ie, when the myosin rapidly attaches and detaches from actin). The rate-limiting step for the entire ATPase cycle in solution is the transition from the weak to strong binding state. From a mechanical viewpoint, this simplified cycle can be described by two states¹³ in which the myosin motor is either detached from actin or attached and generating its F_uni and/or displacement (see Fig 2). The fraction of the cycle in which the cross-bridge is attached and generating force is defined as its duty cycle (f). Finally, F_avg per cycle, which we can estimate in our assay, is the product of F_uni and f (ie, F_avg=F_uni · f).

If we adopt this simple model, as originally proposed by A.F. Huxley¹³ in 1957, actin filament velocity and F_avg are dependent on the rate of transition between the attached and detached states. Furthermore, these transition rates are dependent on the strain within the cross-bridge. Based on this model, actin filament velocity, which is a reasonable analogue for the unloaded shortening velocity of a muscle, is dependent on the detachment rate while the cross-bridge is negatively strained (ie, g₂ in the 1957 Huxley model). Past correlations between the maximum shortening velocity of a muscle and its solution actomyosin ATPase activity¹⁹ do not necessarily imply that these processes share a common rate-limiting step, especially since the actomyosin ATPase measurements are performed in solution in which the cross-bridge is under little or no strain. Indeed, the order-of-magnitude difference between the K_m for ATP of the actomyosin ATPase in solution and actin filament velocity in the motility assay argues that the rates of these processes are controlled by different steps in the cycle.^{9 20 21} In contrast to both the ATPase rate and actin filament velocity, F_avg is dependent on both the rates of attachment (f₁) and detachment (g₁) while the cross-bridge is positively strained (ie, [f₁/(f₁+g₁)] in the 1957 Huxley model; note that this relation defines the cross-bridge duty cycle f as described above).

The fact that V₃ myosin has a slower actin filament velocity but greater average force-generating capacity than V₁ myosin may suggest that these differences are the result of a common mechanism. For example, as described above, both velocity and force are dependent on the cross-bridge detachment rate (albeit under different conditions of cross-bridge strain). If the detachment process is slowed under all cross-bridge strains in V₃ myosin, then this might account for (1) the slower actin filament velocity, since a greater number of negatively strained cross-bridges would exert an internal load to the positive force- and motion-generating population of cross-bridges,¹³ and (2) greater isometric force production, since a slower detachment rate would allow a positively strained cross-bridge to spend a greater proportion of its cycle generating F_uni (ie, greater f value; see Fig 2, bottom).¹³

It should be noted that slower actin filament velocities are not always associated with higher values of F_avg. Both V₁ and V₃ myosin have slower actin filament velocities than skeletal muscle myosin, but neither generates greater F_avg.^{4 5} These exceptions stress that in addition to differences in the kinetics, as described above, alterations may also occur to the inherent mechanical capacity of the motor (ie, F_uni), which is equally as important in determining the ability of the motor to generate force and motion²² (see F_avg above and Fig 2, middle). If such changes in the inherent mechanical capacities of the myosin and/or kinetics of the cross-bridge cycle occur, do they relate to differences in the structure of the V₁ and V₃ myosins?

Myosin Structure and Function
The crystallographic structure for skeletal muscle myosin subfragment-1 (S1) indicates that the myosin head consists of a globular motor/catalytic domain from which extends an 85-Å light chain–stabilized -helical neck region.²³ Since this three-dimensional structure is dependent on the primary amino acid sequence of myosin, it is interesting that the amino acid sequences for the and ß heavy chains are 93% identical and 95% similar.²⁴ When comparing the and ß sequences for S1, the amino acid differences are clustered to four distinct regions. Three of these clusters reside within functionally important domains of the myosin head: (1) ATP binding pocket, (2) actin-binding domain, and (3) neck region.²⁴ Given that the myosin S1 is both hydrolytically and mechanically competent, these natural amino acid substitutions most likely dictate the differences in the V₁ and V₃ motor and hydrolytic activities.

The importance of these domains to motor performance has recently been demonstrated in studies in which biochemical and molecular biological perturbations to these regions have resulted in dramatic alterations to the hydrolytic and mechanical properties of the myosin. For example, Uyeda et al²⁵ genetically engineered chimeras of the slime mold Dictyostelium myosin in which muscle myosin–specific amino acid sequences were inserted between two highly conserved sequences in the actin-binding domain. Two of the substitutions were from the cardiac - and ß-myosin heavy chains. These substitutions resulted in ATP hydrolysis rates for the Dictyostelium chimera that correlated with the rate of the donor myosin. Therefore, this region, although distant from the ATP-binding pocket, clearly influences hydrolysis and thus could contribute to the difference in the V₁ and V₃ hydrolysis rates. It was interesting that no such effect was observed for actin filament velocity in the motility assay.²⁵ However, this may not be surprising, since the rate-limiting steps for ATP hydrolysis (ie, weak to strong binding transition) and maximum actin filament velocity (ie, detachment of negatively strained cross-bridges), as mentioned above, are believed to be different. Since force production is also dependent on the weak-to-strong transition (ie, attachment rate f₁), it may be possible that chimera force would have changed in a manner that correlated with the force-generating capacity of the myosin from which the insert was obtained. Force measurements on such mutant myosins using the techniques described in the present study would be necessary to determine to what extent differences in the actin-binding domain contribute to the enhanced force-generating capacity of the V₃ myosin compared with V₁ myosin.

The S1 neck region may also play an important role in the force and motion generation of myosin. On the basis of the crystal structure, Rayment et al²⁶ proposed that the neck region acts as a lever to transmit the mechanical action of the motor domain. Consistent with this view are motility assay data, which have demonstrated reduced actin filament velocities by use of myosins with presumably shorter neck lengths. A shorter neck length was produced by either light chain removal^{27 28} or genetic deletion of the Dictyostelium myosin II neck region that binds the regulatory light chain.²⁹ We have recently extended these studies by measuring force from light chain–deficient skeletal muscle myosins in the motility assay.²² The results of these studies suggest that removal of the essential light chain can have profound effects on myosin force production. Given that one of the amino acid clusters that differ for the V₁ and V₃ myosin is localized to the heavy chain site that binds the essential light chain, it may be possible that this amino acid cluster contributes to differences in both velocity and force for the two cardiac myosin isoforms.

Conclusions
The present study confirms that a V₁ to V₃ myosin isoform shift contributes to the observed alterations to cardiac performance after cardiovascular stress and that changes in performance reflect inherent differences at the level of the myosin motor. However, the intact muscle possesses a far more complex contractile system in which the interaction of myosin with actin can be modulated greatly by the presence of thin-filament–based regulatory proteins. In fact, there is significant evidence that alterations to this regulatory system occur with hemodynamic stress: the myofibrillar ATPase of failing human hearts is depressed when compared with nonfailing controls,^{30 31 32 33 34 35 36} whereas the calcium-activated myosin ATPase is unchanged.^{31 32 33 34} These results suggest that changes at the thin-filament level may modulate cross-bridge kinetics. This could be the result of troponin or tropomyosin isoform shifts as suggested by Anderson et al,³⁷ who reported that shifts in troponin T isoforms occurred within the heart during maturation of the rabbit. Therefore, even though the V₁ and V₃ myosins most likely contribute to the overall performance of the heart, a second level of modulation in the performance of the motor may exist through the influence of thin-filament regulatory proteins.

	Acknowledgments

 
This study was supported by funds from the National Institutes of Health (HL-45161 to Dr Warshaw, HL-28001 to Dr Alpert, and NRSA HL-08787 to Dr Harris).We would like to thank Jill Martin, Ileen Morgan, and Lisa Akins for the animal and myosin preparations and Dr Martin LeWinter for his financial support to Dr VanBuren during his tenure in the lab as a Cardiology Fellow.

Received January 24, 1995; accepted April 24, 1995.

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