Radial stiffness characteristics of the overlap regions of sarcomeres in isolated skeletal myofibrils in pre-force generating state.

We have studied the stiffness of myofilament lattice in sarcomeres in the pre-force generating state, which was realized by a relaxing reagent, BDM (butane dione monoxime). First, the radial stiffness for the overlap regions of sarcomeres of isolated single myofibrils was estimated from the resulting decreases in diameter by osmotic pressure applied with the addition of Dextran. Then, the radial stiffness was also estimated from force-distance curve measurements with AFM technology. The radial stiffness for the overlap regions thus obtained was composed of a soft and a rigid component. The soft component visco-elastically changed in a characteristic fashion depending on the physiological conditions of myofibrils, suggesting that it comes from cross-bridge structures. BDM treatments significantly affected the soft radial component of contracting myofibrils depending on the approach velocity of cantilever: It was nearly equal to that in the contracting state at high approach velocity, whereas as low as that in the relaxing state at low approach velocity. However, comparable BDM treatments greatly suppressed the force production and the axial stiffness in contracting glycerinated muscle fibers and also the sliding velocity of actin filaments in the in vitro motility assay. Considering that BDM shifts the cross-bridge population from force generating to pre-force generating states in contracting muscle, the obtained results strongly suggest that cross-bridges in the pre-force generating state are visco-elastically attached to the thin filaments in such a binding manner that the axial stiffness is low but the radial stiffness significantly high similar to that in force generating state.

Tension Recovery following Ramp-Shaped Release in High-Ca and Low-Ca Rigor Muscle Fibers: Evidence for the Dynamic State of AMADP Myosin Heads in the Absence of ATP.

During muscle contraction, myosin heads (M) bound to actin (A) perform power stroke associated with reaction, AMADPPi → AM + ADP + Pi. In this scheme, A • M is believed to be a high-affinity complex after removal of ATP. Biochemical studies on extracted protein samples show that, in the AM complex, actin-binding sites are located at both sides of junctional peptide between 50K and 20K segments of myosin heavy chain. Recently, we found that a monoclonal antibody (IgG) to the junctional peptide had no effect on both in vitro actin-myosin sliding and skinned muscle fiber contraction, though it covers the actin-binding sites on myosin. It follows from this that, during muscle contraction, myosin heads do not pass through the static rigor AM configuration, determined biochemically and electron microscopically using extracted protein samples. To study the nature of AM and AMADP myosin heads, actually existing in muscle, we examined mechanical responses to ramp-shaped releases (0.5% of Lo, complete in 5ms) in single skinned rabbit psoas muscle fibers in high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states. The fibers exhibited initial elastic tension drop and subsequent small but definite tension recovery to a steady level. The tension recovery was present over many minutes in high-Ca rigor fibers, while it tended to decrease quickly in low-Ca rigor fibers. EDTA (10mM, with MgCl2 removed) had no appreciable effect on the tension recovery in high-Ca rigor fibers, while it completely eliminated the tension recovery in low-Ca rigor fibers. These results suggest that the AMADP myosin heads in rigor muscle have long lifetimes and dynamic properties, which show up as the tension recovery following applied release. Possible AM linkage structure in muscle is discussed in connection with the X-ray diffraction pattern from contracting muscle, which is intermediate between resting and rigor muscles.

Definite differences between in vitro actin-myosin sliding and muscle contraction as revealed using antibodies to myosin head.

Muscle contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.

Enhancement of force generated by individual myosin heads in skinned rabbit psoas muscle fibers at low ionic strength.

Although evidence has been presented that, at low ionic strength, myosin heads in relaxed skeletal muscle fibers form linkages with actin filaments, the effect of low ionic strength on contraction characteristics of Ca(2+)-activated muscle fibers has not yet been studied in detail. To give information about the mechanism of muscle contraction, we have examined the effect of low ionic strength on the mechanical properties and the contraction characteristics of skinned rabbit psoas muscle fibers in both relaxed and maximally Ca(2+)-activated states. By progressively decreasing KCl concentration from 125 mM to 0 mM (corresponding to a decrease in ionic strength µ from 170 mM to 50 mM), relaxed fibers showed changes in mechanical response to sinusoidal length changes and ramp stretches, which are consistent with the idea of actin-myosin linkage formation at low ionic strength. In maximally Ca(2+)-activated fibers, on the other hand, the maximum isometric force increased about twofold by reducing KCl concentration from 125 to 0 mM. Unexpectedly, determination of the force-velocity curves indicated that, the maximum unloaded shortening velocity Vmax, remained unchanged at low ionic strength. This finding indicates that the actin-myosin linkages, which has been detected in relaxed fibers at low ionic strength, are broken quickly on Ca(2+) activation, so that the linkages in relaxed fibers no longer provide any internal resistance against fiber shortening. The force-velocity curves, obtained at various levels of steady Ca(2+)-activated isometric force, were found to be identical if they are normalized with respect to the maximum isometric force. The MgATPase activity of muscle fibers during isometric force generation was found not to change appreciably at low ionic strength despite the two-fold increase in Ca(2+)-activated isometric force. These results can be explained in terms of enhancement of force generated by individual myosin heads, but not by any changes in kinetic properties of cyclic actin-myosin interaction.

Usefulness of electromagnetic induction type of force transducer and actuator for myofibril mechanics.

A high performance device for measuring force and length change during myofibril contraction is fabricated. The principle of a device depends on the law of electromagnetic induction. Homogenized myofibrils were attached between two wires exposed in the uniform magnetic field by silicon adhesive under an inverted microscope. The purpose of this study is to examine performance whether the electromagnetic induction type of device actually works. Sensitivity and time resolution of force transducer was 50nN and 1ms respectively. Working displacement and time resolution of actuator as length transducer was 1-20 µm and 1.2 ms. We confirmed the performance of the device by showing appropriate force response to changes in length during myofibrils contraction, and possibility of application of the device to myofibril mechanics is discussed.

Direct demonstration of the cross-bridge recovery stroke in muscle thick filaments in aqueous solution by using the hydration chamber.

Despite >50 years of research work since the discovery of sliding filament mechanism in muscle contraction, structural details of the coupling of cyclic cross-bridge movement to ATP hydrolysis are not yet fully understood. An example would be whether lever arm tilting on the myosin filament backbone will occur in the absence of actin. The most direct way to elucidate such movement is to record ATP-induced cross-bridge movement in hydrated thick filaments. Using the hydration chamber, with which biological specimens can be kept in an aqueous environment in an electron microscope, we have succeeded in recording ATP-induced cross-bridge movement in hydrated thick filaments consisting of rabbit skeletal muscle myosin, with gold position markers attached to the cross-bridges. The position of individual cross-bridges did not change appreciably with time in the absence of ATP, indicating stability of time-averaged cross-bridge mean position. On application of ATP, individual cross-bridges moved nearly parallel to the filament long axis. The amplitude of the ATP-induced cross-bridge movement showed a peak at 5-7.5 nm. At both sides of the filament bare region, across which the cross-bridge polarity was reversed, the cross-bridges were found to move away from, but not toward, the bare region. Application of ADP produced no appreciable cross-bridge movement. Because ATP reacts rapidly with the cross-bridges (M) to form complex (M x ADP x Pi) with an average lifetime >10 s, the observed cross-bridge movement is associated with reaction, M + ATP --> M x ADP x Pi. The cross-bridges were observed to return to their initial position after exhaustion of ATP. These results constitute direct demonstration of the cross-bridge recovery stroke.

Measurement of muscle fiber stiffness during stretch with two continuous different velocities in skeletal muscle fibers.

Ca2+ activated single glycerinated muscle fibers were stretched by 3% of its slack length Lo with moderate velocities ranging from 0.015 to 0.15Lo, and muscle fiber stiffness during and after stretch was measured by applying sinusoidal vibrations (peak-to-peak amplitude, 0.1% of Lo, 2kHz). During a single stretch, the fiber was first stretched by 1.5% with a velocity, and then it was further stretched by 1.5% with another velocity. It is suggested that, during stretch, a considerable fraction of cross-bridges slow down their cycling rate and increase their force-generating ability.

Structural alterations of thin actin filaments in muscle contraction by synchrotron X-ray fiber diffraction.

Strong evidence has been accumulated that the conformational changes of the thin actin filaments are occurring and playing an important role in the entire process of muscle contraction. The conformational changes and the mechanical properties of the thin actin filaments we have found by X-ray fiber diffraction on skeletal muscle contraction are explored. Recent studies on the conformational changes of regulatory proteins bound to actin filaments upon activation and in the force generation process are also described. Finally, the roles of structural alterations and dynamics of the actin filaments are discussed in conjunction with the regulation mechanism and the force generation mechanism.

Axial dispositions and conformations of myosin crossbridges along thick filaments in relaxed and contracting states of vertebrate striated muscles by X-ray fiber diffraction.

X-ray diffraction patterns from live vertebrate striated muscles were analyzed to elucidate the detailed structural models of the myosin crown arrangement and the axial disposition of two-headed myosin crossbridges along the thick filaments in the relaxed and contracting states. The modeling studies were based upon the previous notion that individual myosin filaments had a mixed structure with two regions, a "regular" and a "perturbed". In the relaxed state the distributions and sizes of the regular and perturbed regions on myosin filaments, each having its own axial periodicity for the arrangement of crossbridge crowns within the basic period, were similar to those reported previously. A new finding was that in the contracting state, this mixed structure was maintained but the length of each region, the periodicities of the crowns and the axial disposition of two heads of a crossbridge were altered. The perturbed regions of the crossbridge repeat shifted towards the Z-bands in the sarcomere without changing the lengths found in the relaxed state, but in which the intervals between three successive crowns within the basic period became closer to the regular 14.5-nm repeat in the contracting state. In high resolution modeling for a myosin head, the two heads of a crossbridge were axially tilted in opposite directions along the three-fold helical tracks of myosin filaments and their axial orientations were different from each other in perturbed and regular regions in both states. Under relaxing conditions, one head of a double-headed crossbridge pair appeared to be in close proximity to another head in a pair at the adjacent crown level in the axial direction in the regular region. In the perturbed region this contact between heads occurred only on the narrower inter-crown levels. During contraction, one head of a crossbridge oriented more perpendicular to the fiber axis and the partner head flared axially. Several factors that significantly influence the intensities of the myosin based-meridional reflections and their relative contributions are discussed.

Effect of deuterium oxide on contraction characteristics and ATPase activity in glycerinated single rabbit skeletal muscle fibers.

We studied the effect of deuterium oxide (D(2)O) on contraction characteristics and ATPase activity of single glycerinated muscle fibers of rabbit psoas. D(2)O increased the maximum isometric force P(0) by about 20%, while the force versus stiffness relation did not change appreciably. The maximum shortening velocity under zero load V(max) did not change appreciably in D(2)O, so that the force-velocity (P-V) curve was scaled depending on the value of P(0). The Mg-ATPase activity of the fibers during generation of steady isometric force P(0) was reduced by about 50% in D(2)O. Based on the Huxley contraction model, these results can be accounted for in terms of D(2)O-induced changes in the rate constants f(1) and g(1) for making and breaking actin-myosin linkages in the isometric condition, in such a way that f(1)/(f(1)+g(1)) increases by about 20%, while (f(1)+g(1)) remains unchanged. The D(2)O effect at the molecular level is discussed in connection with biochemical studies on actomyosin ATPase.

Electrical and mechanical properties and mode of innervation in scorpionfish sound-producing muscle fibres.

To obtain information about the neural mechanism underlying sound production in teleost fish, we studied the electrical and mechanical properties and mode of innervation in the swimbladder muscle (SBM) fibres of scorpionfish Sebastiscus marmoratus. Action potentials of the SBM fibres in response to direct electrical stimulation neither exhibited overshoot nor propagated along the fibre. Stimulation of the motor nerve, however, uniformly evoked action potentials along the fibre. When neuromuscular transmission was blocked by curare, motor nerve stimulation uniformly evoked endplate potentials along the fibre. These results indicate that action potentials propagate along the nerve branches but not along the SBM fibre membrane. In accordance with the above results, histochemical studies showed that motor nerve branches run along the SBM fibres to form many endplates with cholinesterase activity, indicating multiterminal innervation. The SBM consisted of about 600 fibres, while its motor nerve contained about 100 axons, giving an innervation ratio of about 1:6. Like mammalian fast muscle fibres, the SBM fibres exhibited a low succinic dehydrogenase activity and a high ATPase activity. These results are discussed in connection with the function of the SBM fibres in producing sound.

Marked load-bearing ability of Mytilus smooth muscle in both active and catch states as revealed by quick increases in load.

The anterior byssal retractor muscle (ABRM) of the bivalve Mytilus edulis shows a prolonged tonic contraction, called the catch state. To investigate the catch mechanism, details of which still remain obscure, we studied the mechanical responses of ABRM fibres to quick increases in load applied during maximum active isometric force (P(0)) generation and during the catch state. The mechanical response consisted of three components: (1) initial extension of the series elastic component (SEC), (2) early isotonic fibre lengthening with decreasing velocity, and (3) late steady isotonic fibre lengthening. The ABRM fibres could bear extremely large loads up to 10-15P(0) for more than 30-60 s, while being lengthened extremely slowly. If, on the other hand, quick increases in load were applied during the early isometric force development, the ABRM fibres were lengthened rapidly ('give') under loads of 1.5-2P(0). These findings might possibly be explained by two independent systems acting in parallel with each other; one is the actomyosin system producing active shortening and active force generation, while the other is the load-bearing system responsible for the extremely marked load-bearing ability as well as the maintenance of the catch state.

Role of Ca2+ in determining the rate of tension development and relaxation in rat skinned myocardium.

To clarify the roles of Ca2+ and crossbridge kinetics in determining the cardiac contraction profile, we analyzed the rate of tension development following nitrophenyl-EGTA photolysis and the rate of relaxation following diazo-2 photolysis in the absence and presence of phosphate (Pi, 5 mM) in rat skinned ventricular trabeculae. The rate of tension development was fitted with a single exponential function. The rate constant (kc) increased not only with an increase in prephotolysis tension (initial activation level) under the same postphotolysis tension (final Ca2+ level), but also with an increase in postphotolysis tension under the same prephotolysis tension. Pi increased kc, though decreased both the prephotolysis and postphotolysis tension greatly. The rate of relaxation was fitted with a double-exponential function. The rate constants of both initial rapid phase (kr1), which was higher than kc, and subsequent slow (kr2) relaxation were almost independent of either the prephotolysis tension or the postphotolysis tension (i.e. the extent of relaxation). Pi increased both kr1 and kr2 by about twofold. These results apparently contradicting both the "steric blocking model" and the "kinetic model", and can be explained in terms of the changes in number of tension-generating crossbridges through the Ca2+-dependent cooperative thin filament activation/inactivation associated with the Pi-modulated changes in number of tension-generating crossbridges. The thin filament activation kinetics seems to be slower than its inactivation.