Basic properties of ATP-induced myosin head movement in hydrated myosin filaments, studied using the gas environmental chamber.

Although more than 50 years have passed since the monumental discovery of Huxley and Hanson that muscle contraction results from relative sliding between actin and myosin filaments, coupled with ATP hydrolysis, the mechanism underlying the filament sliding still remains to be a mystery. It is generally believed that the myofilament sliding is caused by cyclic attachment-detachment between myosin heads in myosin filaments and myosin-binding sites in actin filaments. Attempts to prove the myosin head movement using techniques of X-ray diffraction and chemical probes attached to myosin heads have failed to obtain clear results because of the asynchronous nature of myosin head movement. Using the gas environmental chamber (EC) attached to an electron microscope, we succeeded in recording myosin head movement in hydrated myosin filaments, coupled with ATP hydrolysis with the following results: (1)In the absence of actin filaments, myosin heads fluctuate around a definite neutral position, so that their time-averaged position remains unchanged; (2) On ATP application, myosin heads bind with ATP to be in the charged-up state, M-ADP-Pi, and perform a recovery stroke in the direction away from the myosin filament central bare zone and stay in the post-recovery stroke position; (3) In the actin-myosin filament mixture, myosin heads form rigor linkages with actin, and bind with applied ATP to be in the charged-up state, M-ADP-Pi, and perform a power stroke in the direction towards the myosin filament bare zone, while releasing ADP and Pi to stay in the post-power stroke position; (4) In both recovery and power strokes, myosin heads in the non charged-up state return to the neutral position. These results indicate that the charged-up myosin heads decide their direction of movement without being guided by actin filaments.

Effect of deuterium oxide on actomyosin motility in vitro.

Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D(2)O) and water (H(2)O) to examine the effect of D(2)O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D(2)O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H(2)O assay solution, but the maximum velocity (4.1+/-0.5 microm/s, n=11) at pD 8.5 in D(2)O was about 60% of that (7.1+/-1.1 microm/s, n=11) at pH 8.5 in H(2)O. The K(m) values of 95 and 80 microM and V(max) values of 3.2 and 5.1 microm/s for the D(2)O and H(2)O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis-Menten equation. The K(m) value of actin-activated Mg-ATPase activity of myosin subfragment 1 (S1) was decreased from 50 microM [actin] in H(2)O to 33 microM [actin] in D(2)O without any significant changes in V(max) (9.4 s(-1) in D(2)O and 9.3 s(-1) in H(2)O). The rate constants of ADP release from the acto-S1-ADP complex measured by the stopped flow method were 361+/-26 s(-1) (n=27) in D(2)O and 512+/-39 s(-1) (n=27) in H(2)O at 6 degrees C. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D(2)O results from a slowing of the release of ADP from the actomyosin-ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D(2)O.

The three-state model for the elementary process of energy conversion in muscle.

A three-state model for the elementary process of energy conversion in striated muscle is analysed; in two of the three states, the crossbridge is attached to an actin site, while the third is a detached state. This model accounts for the mechanical properties of steady shortening and lengthening processes as well as those of isometric and isotonic transient processes in a quantitative way. Moreover, qualitative agreement is obtained for the total energy liberation from muscle. Biochemical properties are also computed for transient processes. Comparisions are made with other models with "three states".

Different cardiac myosin isoforms exhibit equal force-generating ability in vitro.

We measured forces generated by myosin molecules and a single actin filament using an optical trap system. The force per unit length of actin filament did not differ significantly between cardiac myosin isoforms. V1 and V3. This indicates that the ability to generate force is equal between V1 and V3, despite their difference in the unloaded sliding velocity past actin.

Force-velocity relations of rat cardiac myosin isozymes sliding on algal cell actin cables in vitro.

The difference in kinetic properties between two myosin isozymes (V1 and V3) in rat ventricular myocardium was studied by determining the steady-state force-velocity (P-V) relations in the ATP-dependent movement of V1 and V3-coated polystyrene beads on actin cables of giant algal cells mounted on a centrifuge microscope. The maximum unloaded velocity of bead movement was larger for V1 than for V3. The velocity of bead movement decreased with increasing external load applied by the centrifuge microscope, and eventually reached zero when the load was equal to the maximum isometric force (P0) generated by the myosin heads. The maximum isometric force P0 was less than 10 pN, and did not differ significantly between V1 and V3. The P-V curves consisted of a hyperbolic part in the low force range and a non-hyperbolic part in the high force range. The critical force above which the curve deviated from the hyperbola was much smaller for V1 than for V3. An analysis using a model with an extremely small number of myosin heads involved in the bead movement suggested a marked difference in kinetic properties between V1 and V3.

Substrate-concentration dependence of contraction parameters in glycerinated insect flight muscle fibers from Lethocerus derollei.

The contraction characteristics of the dorsal longitudinal muscle of Lethocerus derollei were investigated by applying small sinusoidal length changes (+/- 1% of resting length) to glycerinated muscle bundles and studying the effect of varying the frequency from 0.1 to 10 Hz and the concentration of MgATP from 35 microM to 2.3 mM. The maximum work done by the muscle per cycle increased as the MgATP concentration was decreased from 2.3 mM to 52 microM. Between 52 and 35 microM, the maximum work suddenly changed from a positive to a negative value. The optimal frequency for maximal work shifted from low to high values with increase in the MgATP concentration. As the temperature was increased, the optimal work frequency in 2.3 mM MgATP solution shifted to a higher value. As the MgATP concentration was increased, the optimal frequency for maximal power increased. The maximal value of the power was an increasing function of the MgATP concentration, reaching a plateau above 52 microM MgATP. The muscle stiffness was a decreasing function of the MgATP concentration, and above 52 microM MgATP it reached a minimum of about 22% of that in the rigor solution. These results are discussed in relation to the crossbridge kinetics.

Fluorescence properties and contraction characteristics of ANM (N-(1-anilinonaphthyl-4)maleimide)-labeled rabbit psoas muscle fibers.

Fluorescence spectra of ANM-labeled, glycerinated rabbit psoas muscle fibers were recorded in relaxed, contracted, and rigor states. SDS polyacrylamide gel electrophoresis of the ANM-labeled muscle fibers indicated that proteins labeled with ANM were myosin heavy chain, C protein, and actin. In a relaxed state in the presence of ATP, myosin heavy chain was mainly labeled. During the transition from rigor to the relaxed or contracted state, there was a blue shift (about 5 nm) of the ANM emission spectrum. Similar experiments with FAM (N-(3-fluoranthyl)-maleimide)-labeled muscle fibers showed that these fluorescence changes were not artifacts due to the movement of muscle fibers. The fibers labeled in the ATP relaxing solution showed a marked decrease in both isometric force and unloaded shortening velocity (Vo), while in the fibers labeled in the rigor solution isometric tension was not markedly suppressed, though Vo decreased to the same extent as in the fibers labeled in the ATP relaxing solution. Fluorescence spectra of ANM-labeled HMM in different states were also measured. A fluorescence enhancement and a blue shift (about 5 nm) of the emission maximum were observed in HMM + MgATP or HMM + MgATP + F-actin in comparison with HMM + F-actin. These results suggest that the fluorescence spectra of the ANM-labeled muscle fibers reflect their conformational changes between the rigor state (in the absence of MgATP) and the relaxed or contracted state (in the presence of MgATP).

Substrate-concentration of dependences of tension, shortening velocity and ATPase activity of glycerinated single muscle fibers.

Tension P0 and ATPase activity J0 of glycerinated single muscle fibers under isometric concentration as well as the velocity V0 of unloaded shortening were measured as a function of substrate concentration [S]. The stiffness of fibers with sinusoidal length changes at 1 kHz was used as a qualitative measure of the amount of rigor complex. P0 is an increasing function of [S] at low substrate concentrations and has a broad maximum at about 10-40 micrometers MgATP. In this concentration range, 10-40 micrometers, V0 still has a very small value. Then it increases and finally reaches at a plateau at about 1 mM MgATP. J0 increases as P0 does. However, it reaches at a saturated level at about the same concentration as V0. Either 0.5 mM 8-BrATP or 1 mM PPi was added to the substrate solutions to reduce the amount of rigor complex at low substrate concentrations. The addition of PPi of 8-BrATP decreases P0 dominantly at low concentrations of substrate and shifts the maximum to about 100 micrometers MgATP. 8-BrATP considerably increases V0 at low substrate concentrations while V0 is decreased by added PPi. The temperature coefficients, Q10 values were obtained for P0, J0, and V0. The values are essentially constant, 2.1-2.4, in the cases of P0 and J0, and about the same values were found for V0 at very low substrate concentrations. However, they become about 3.3 in the concentration range from 34 micrometers and 2.3 mM. The P-V relation was obtained at 11 micrometers and 2.3 micrometers MgATP. The normalized P-V relation at 11 micrometers was unchanged when 8-BrATP was added. The results are discussed in connection with the mechanism of actomyosin-ATPase activity as well as that of the elementary cycle of the motive force generation.

Lower activation energy for sliding of F-actin on a less thermostable isoform of carp myosin.

We have examined the temperature-dependence of sliding velocity of fluorescent F-actin on myosins isolated from 10 degrees C- and 30 degrees C-acclimated carp. Activation energies for the sliding of F-actin were 63 and 111 kJ/mol for the 10 degrees C- and 30 degrees C-acclimated carp myosins, respectively. Arrhenius plots of the sliding velocity from 10 degrees C- and 30 degrees C-acclimated carp myosin were shown to intersect at high temperature (about 30 degrees C). The thermostability estimated by measuring the Ca(2-)-ATPase activity was less for myosin from 10 degrees C- than 30 degrees C-acclimated carp. We suggest that a less thermostable structure in cold-acclimated carp myosin results in a reduced activation energy for the contractile process, which allows the F-actin to slide fast even at low temperatures.

Effect of lateral forces on the movement of myosin-coated beads on actin cables studied using a centrifuge microscope.

We developed an in vitro motility assay system, in which myosin-coated polystyrene beads were made to slide on actin filament arrays (actin cables) in giant algal cells and subjected to centrifugal forces, which were parallel to the direction of bead movement to serve as external loads on actin-myosin sliding (Oiwa et al. (1990) Proc Natl Acad Sci USA 87: 7893-7897), and succeeded in determining the steady-state force-velocity relation of ATP-dependent actin-myosin sliding. To give further information about the properties of actin-myosin sliding, we have applied centrifugal forces, in parallel with the plane of actin-myosin sliding but at right angles with the direction of bead movement, and have found that such "lateral" centrifugal forces reduced the velocity of bead movement. In addition, we have also found that the velocity of bead movement is reduced more markedly with lateral forces applied from the left side of the bead ("left" lateral forces) than those applied from the right side of the bead ("right" lateral forces). These results are discussed in connection with the direction of sliding force generated by the myosin heads on the bead which interact with the right-handed double helix of actin monomers constituting actin filaments.

Cooperative interactions of myosin two heads in muscle force generation.

To investigate the possibility of cooperative interactions between the two myosin heads in muscle contraction, Ca2+-activated force development, K+-EDTA- and Mg2+-ATPase activities, muscle fiber stiffness, and the velocity of unloaded shortening were measured on partially p-PDM treated glycerinated muscle fibers, which contained a mixture of myosin molecules with zero, one and two of their heads inactivated. It was found that the magnitude of the Ca2+-activated isometric force development was proportional to the square of both K+-EDTA- and Mg2+-ATPase activities and also to the square of muscle fiber stiffness. If the two myosin heads in the glycerinated fibers are assumed to react independently with p-PDM, the above results strongly suggest that (i) each myosin molecule in the thick filaments can generate force only when its two heads do not react with p-PDM, (ii) muscle fiber stiffness is determined by the total number of native heads, and (iii) there is no cooperative interaction between the two myosin heads in catalyzing ATP hydrolysis.

Dependence of the work done by ATP-induced actin-myosin sliding on the initial baseline force: its implications for kinetic properties of myosin heads in muscle contraction.

The properties of the ATP-dependent actin-myosin sliding responsible for muscle contraction was studied using an in vitro force-movement assay system, in which a myosin-coated glass microneedle was made to slide on actin filament arrays (actin cables) in the giant algal cell with iontophoretic application of ATP. With a constant amount of ATP application, the amount of work done by the actin-myosin sliding increased with increasing baseline force from zero to 0.4-0.6 Po, and then decreased with further increasing baseline force, thus giving a bell-shaped work versus baseline force relation. The result that the maximum actin-myosin sliding velocity did not change appreciably with increasing baseline force up to 0.4-0.6 Po implies, together with the limited number of myosin heads involved, that (1) the rate of power output of actin-myosin sliding is determined primarily by the amount of external load rather than the velocity of actin-myosin sliding, and (2) the bell shaped work versus baseline force relation (and also the hyperbolic force-velocity relation) results from the kinetic properties of individual myosin head rather than the change in the number of myosin heads involved.

Evanescent excitation microscopy. Its application to the study of single molecular process kinetics of actomyosin motor.

Evanescent field was generated on the stage of an inverted microscope upon an incidence of 532 nm Nd-YAG laser beam on interface between aqueous solution and fused silica glass. Thick filaments isolated from Mytilus edulis were adsorbed to the glass surface and nanomolar concentration of adenosine triphosphate (ATP) labeled with rhodamine was allowed to interact with thick filaments. The fluorescence from the surface was observed by triple-view microscopy at video rate. There were many fluorescent spots at the interface, which we identified as individual fluorescent ATP molecules. We found that the fluorescence from those spots was polarized. Fluorescence intensity of individual spots fluctuated considerably. We interpret the latter observation as a result of change in the orientation of emission dipole of the fluorescent ATP analog.

ATP-induced axial movement of myosin heads in living thick filaments recorded with a gas environmental chamber attached to the electron microscope.

Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of 'living' muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by approximately 20 nm along the filament axis, while no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Since ATP reacts rapidly with the myosin head (M) to form the complex (M.ADP.Pi) having average lifetime of > 10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP-->M.ADP.Pi. This work will open a new research field to study dynamic structural changes of individual biomolecules which are kept in 'living' state in an electron microscope.

Measurement of ATP turnover during shortening and lengthening of rabbit psoas myofibrils using a fluorescent ATP analog.

In order to study ATP turnover during shortening and lengthening of rabbit psoas myofibrils, we have used fluorescence microscopy in which the displacement of a fluorescent nucleotide analog, 2'(3')-O-[N-[2-[[Cy3] amido] ethyl] carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP) bound to cross-bridge on flash photolysis of caged ATP was measured [Chaen et al. (1997) Biophys. J. 73, 2033-2042]. In the previous paper, we reported that when a myofibril was imposed to shorten with a constant velocity by a piezo-electric actuator, the nucleotide displacement rate constant initially increased to 0.7 s-1 with increasing shortening velocity and then declined with a further increase in shortening velocity. The rate constant during lengthening measured in the present experiment was found to be not significantly affected. These results suggest that the cross-bridge kinetics show a asymmetrical dependence on the mechanical strain in the cross-bridges, namely, the rate constants are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening.

Distinct kinetic properties of cardiac myosin isoforms revealed by in vitro studies.

To clarify the physiological significance of myosin isoform redistribution in cardiac adaptation process, we compared the kinetic property of the two cardiac myosin isoforms using in vitro motility assay techniques. Cardiac myosin isoforms V1 and V3 were obtained from ventricular muscle of young rats and hypothyroid rats respectively. On each of these myosin isoforms fixed on a glass coverslip, fluorescently labeled actin filaments were made to slide in the presence of ATP. To measure the force generated by actomyosin interaction, a small latex bead was attached to the barbed end of an actin filament and the bead was captured by the laser optical trap installed in a microscope. The force was determined from the distance between the bead and the trap positions under either auxotonic or isometric conditions. The time-averaged force generated by multiple cross-bridges did not differ significantly between the two isoforms. On the other hand, the unitary force measurement revealed the same level of amplitude but a longer duration for V3 isoform. The same level of time-averaged force is in agreement with not only our previous finding but the results of maximum force measurement in muscle preparations. The difference in kinetic characteristics of the two isoforms could account for the difference in economy of force development and the basis for cardiac adaptation mechanism.

Kinetic properties of the ATP-dependent actin-myosin sliding as revealed by the force-movement assay system with a centrifuge microscope.

To study the kinetic properties of the ATP-dependent actin-myosin sliding responsible for muscle contraction, we developed an in vitro force-movement assay system, in which centrifugal forces were applied to myosin-coated polystyrene beads sliding along actin cables of giant algal cells in the presence of ATP. Under constant centrifugal forces directed opposite to the bead movement ("positive" loads), the beads moved with constant velocities. The steady-state force-velocity (P-V) curve thus obtained was double-hyperbolic in shape, being analogous to the P-V curve of single muscle fibers. Under constant centrifugal forces in the direction of the bead movement ("negative" loads), on the other hand, the beads also moved with constant velocities. Unexpectedly, the velocity of bead movement did not increase with increasing negative loads, but decreased markedly (by 20-60%). We also studied the effect of centrifugal forces at right angles with actin cables on the bead movement.

Measurement of work done by ATP-induced sliding between rabbit muscle myosin and algal cell actin cables in vitro.

1. The basic properties of the ATP-dependent actin-myosin interaction responsible for muscle contraction were studied using an in vitro force-movement assay system, in which a glass microneedle coated with rabbit skeletal muscle myosin was made to slide on the actin filament arrays (actin cables) in the internodal cell of an alga Nitellopsis obtusa with ionophoretic application of ATP. 2. In response to an ATP current pulse (intensity, 5-85 nA; duration, 0.5-10 s), the myosin-coated needle moved for a distance and eventually stopped, indicating reformation of rigor actin-myosin linkages to prevent elastic recoil of the bent needle. A subsequent ATP current pulse again produced the needle movement starting from the baseline force attained by the preceding needle movement. 3. With a constant amount of ATP application, the amount of work done by the ATP-induced actin-myosin sliding first increased with increasing baseline force from zero to 0.4-0.6P0, and then decreased with further increasing baseline force, thus giving a bell-shaped work versus baseline force relation. 4. With increasing amount of ATP application, the amount of work done by the actin-myosin sliding increased more steeply as the baseline force was increased from zero to 0.4-0.6P0. 5. These results are discussed in connection with the basic properties of the actin-myosin sliding in muscle contraction.