Two attached non-rigor crossbridge forms in insect flight muscle

We have performed thin-section electron microscopy on muscle fibers fixed in different mechanically monitored states, in order to identify structural changes in myosin crossbridges associated with force production and maintenance. Tension and stiffness of fibers from glycerinated Lethocerus flight muscle were monitored during a sequence of conditions using AMPPNP and then AMPPNP plus increasing concentrations of ethylene glycol, which brought fibers through a graded sequence from rigor relaxation. Two intermediate crossbridge forms distinct from the rigor or relaxed forms were observed. The first was produced by AMPPNP at 20 degrees C, which reduced isometric tension 60 to 70% below rigor level without reducing rigor stiffness. Electron microscopy of these fibers showed that, in spite of the drop in tension, no obvious change from the 45 degrees crossbridge angle characteristic of rigor occurred. However, the thick filament ends of the crossbridges were altered from their rigor positions, so that they now marked a 14.5 nm repeat, and formed four separate origins at each crossbridge level. The bridges were also less slewed and bent than rigor bridges, as seen in transverse sections. The second crossbridge form was seen in glycol-AMPPNP at 4 degrees C, just below the glycol concentration that produced mechanical relaxation. These fibers retained 90% of rigor stiffness at 40 Hz oscillation, but would not bear sustained tension. Stiffness was also high in the presence of calcium at room temperature under similar conditions. Electron microscopy showed crossbridges projecting from the thick filaments at an angle that centered around 90 degrees, rather than the 45 degree angle familiar from rigor. This coupling of relaxed appearance with persistent stiffness suggests that the 90 degree form may represent a weakly attached crossbridge state like that proposed to precede force development in current models of the crossbridge power stroke.     

An analysis of the possible source of contractile forces in striated muscle

A thermodynamic analysis, based upon current understanding of the molecular structure and chemistry of striated muscle, defines the conditions under which contractile forces may be generated and external work done. Under these conditions tension is generated by crossbridges acting individually and asynchronously, so that tension is proportional to the extent of overlap of actin and myosin filaments. Estimation of the mean isometric tension per crossbridge, in a simple case, gives a value comparable to that observed experimentally. The role of ATP hydrolysis in the generation of tension and performance of work is an indirect one. Isometric tension is maintained at the expense of an enhanced rate of hydrolysis of ATP, and for isotonic contraction the rate of ATP hydrolysis increases with increasing speed of contraction. The mean tension during isotonic contraction varies inversely with the speed of contraction.     

Behaviour of the crossbridges in stretched or compressed muscle fibres

The maximum chord of the myosin heads is comparable to the closest surface-to-surface spacing between the myofilaments in a muscle at the slack length. Therefore, when the sarcomere length increases or when the fibre is compressed, the surface-to-surface myofilament spacing becomes lower than the head long axis. We conclude that, in stretched or compressed fibres, some crossbridges cannot attach, owing to steric hindrance. When the amount of compression is limited, this hindrance may be overcome by a tilting of the heads in the plane perpendicular to the filament axes; in this case, there is no consequence as concerns the crossbridge properties. In highly compressed fibres, the crossbridges become progressively hindered and all the crossbridges are hindered for an axis-to-axis spacing representing about 60% of the spacing observed under zero external osmotic pressure. In this case, both the isometric tension and the ATPase activity of the fibre are zero. In fibres stretched up to 3.77 microns (sarcomere length corresponding to the disappearance of the overlap between the thick and the thin filaments), the ratio of hindered crossbridges over the functional crossbridges may be estimated at about 55%. In stretched fibres, a noticeable proportion of crossbridges are sterically hindered and the crossbridges performance (e.g. constants of attachment and detachment) depends on filament spacing, i.e. on sarcomere length. Therefore, we think it is probably impossible to consider the crossbridges as independent force converters, since this idea requires that the crossbridge properties are independent of sarcomere length. In this connection, all the experiments performed on osmotically compressed fibres are of major importance for the understanding of the true mechanisms of muscle contraction.     

A theory of contraction and a mathematical model of striated muscle

A theory of contraction and an associated model of striated muscle are presented, based on the assumption that chemical energy is being converted into electrical energy which, in turn, is being converted into mechanical energy and heat.The model, set up for the frog sartorius muscle, is able to predict the “rowing” motion of the cross-bridges, the force-velocity relation, the tension-length curve, the isometric force, all energy rates (heat and work rates), the metabolic rates and all known features of the stretched, stimulated muscle (no ATP-splitting, stretching tension higher than isometric tension, etc.). It also offers an alternative explanation for Hill's thermoelastic effect. The significance of Hill's force-velocity equation in the context of this theory is also discussed in detail.     

Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin

F-actin in a glycerinated muscle fiber was specifically labeled with fluorescent phalloidin-(fluorescein isothiocyanate) FITC complex at 1:1 molar ratio. Binding of phalloidin-FITC to F-actin affected neither contraction of the fiber nor its regulation by Ca2+. Comparison of polarized fluorescence from phalloidin-FITC bound to F-actin in the relaxed state, rigor, and during isometric contraction of the fiber revealed that the changes in polarization accompanying activation are quantitatively as well as qualitatively different from those accompanying transition of the fiber from the relaxed state to rigor. The extent of the changes of polarized fluorescence during isometric contraction increased with decreasing ionic strength, in parallel with increase in isometric tension. On the other hand, polarized fluorescence was not affected by addition of ADP or by stretching of the fiber in rigor solution. It is concluded from these observations that conformational changes in F-actin are involved in the process of active tension development.     

Formation of ATP-insensitive weakly-binding crossbridges in single rabbit psoas fibers by treatment with phenylmaleimide or para-phenylenedimaleimide.

Chaen et al. (1986. J. Biol. Chem. 261:13632-13636) showed that treatment of relaxed single muscle fibers with para-phenylenedimaleimide (pPDM) results in inhibition of a fiber's ability to generate active force and a diminished ATPase activity. They postulated that the inhibition of force production was due to pPDM's ability to prevent crossbridges from participating in the normal ATP hydrolysis cycle. We find that the crossbridges produced by pPDM treatment of relaxed muscle cannot bind strongly to the actin filaments in rigor, but do bind weakly to the actin filaments in the presence and also absence of ATP. After pPDM treatment, fiber stiffness, as measured using ramp stretches of varying duration, is ATP-insensitive and identical to that of untreated relaxed fibers (both at high [165 mM] and low [40 mM] ionic strength). These results suggest that the pPDM-treated crossbridges, in both the presence and absence of ATP, are locked in a state that resembles the weakly-binding myosin ATP state of normal crossbridges. Their resemblance to the ATP-crossbridges of relaxed untreated fibers is quite strong; both bind to actin about equally tightly and have similar attachment and detachment rate constants. We also found that crossbridges are locked in a weakly-binding state after treatment with N-phenylmaleimide (NPM). In muscle fibers, this method of producing weakly-binding crossbridges appears preferable to pPDM treatment because, unlike treatment with pPDM, it does not increase the fiber's resting tension and stiffness and it does not disrupt the titin band seen on SDS-PAGE.     

Cross-bridge behavior in rigor muscle

Properties of the rigor state in muscle can be explained by a simple cross-bridge model, of the type which has been suggested for active muscle, in which detachment of cross-bridges by ATP is excluded. Two attached cross-bridge states, with distinct force vs. distortion relationships, are required, in addition to a detached state, but the attached cross-bridge states in rigor muscle appear to differ significantly from the attached cross-bridge states in active muscle. The stability of the rigor force maintained in muscle under isometric conditions does not require exceptional stability of the attached cross-bridges, if the positions in which attachment of cross-bridges is allowed are limited so that the attachment of cross-bridges in positions which have minimum free energy is excluded. This explanation of the stability of the rigor state may also be applicable to the maintenance of stable rigor waves on flagella.     

Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium

Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)     

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".     

The sliding filament model of muscle contraction. III. Stability analysis and sinusoidal perturbations

A theoretical model of the energy transducing part of a single sarcomere, which has previously accounted for most of the steady state energetic and dynamical properties of striated muscle, is subjected to stability analysis. The steady states of isotonic and isometric contraction turn out to be stable and the magnitudes of the characteristic roots allow accurate reduction of the original seven state theory to a four state theory. Both versions possess a complex pair of roots giving a damped oscillatory character to the contraction velocity, the tension generated and the populations of the crossbridge states.     

Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges

As a first step toward freeze-trapping and 3-D modeling of the very rapid load-induced structural responses of active myosin heads, we explored the conformational range of longer lasting force-dependent changes in rigor crossbridges of insect flight muscle (IFM). Rigor IFM fibers were slam-frozen after ramp stretch (1000 ms) of 1-2% and freeze-substituted. Tomograms were calculated from tilt series of 30 nm longitudinal sections of Araldite-embedded fibers. Modified procedures of alignment and correspondence analysis grouped self-similar crossbridge forms into 16 class averages with 4.5 nm resolution, revealing actin protomers and myosin S2 segments of some crossbridges for the first time in muscle thin sections. Acto-S1 atomic models manually fitted to crossbridge density required a range of lever arm adjustments to match variably distorted rigor crossbridges. Some lever arms were unchanged compared with low tension rigor, while others were bent and displaced M-ward by up to 4.5 nm. The average displacement was 1.6 +/- 1.0 nm. "Map back" images that replaced each unaveraged 39 nm crossbridge motif by its class average showed an ordered mix of distorted and unaltered crossbridges distributed along the 116 nm repeat that reflects differences in rigor myosin head loading even before stretch.     

Load-dependent kinetics of force production by smooth muscle myosin measured with optical tweezers

Muscle contraction is driven by the cyclical interaction of myosin with actin, coupled with ATP hydrolysis. Myosin attaches to actin, forming a crossbridge that produces force and movement as it tilts or rocks into subsequent bound states before finally detaching. It has been hypothesized that the kinetics of one or more of these mechanical transitions are dependent on load, allowing muscle to shorten quickly under low load, but to sustain tension economically, with slowly cycling crossbridges under high load conditions. The idea that muscle biochemistry depends on mechanical output is termed the 'Fenn effect'. However, the molecular details of how load affects the kinetics of a single crossbridge are unknown. Here, we describe a new technique based on optical tweezers to rapidly apply force to a single smooth muscle myosin crossbridge. The crossbridge produced movement in two phases that contribute 4 nm + 2 nm of displacement. Duration of the first phase depended in an exponential manner on the amplitude of applied load. Duration of the second phase was much less affected by load, but was significantly shorter at high ATP concentration. The effect of load on the lifetime of the bound crossbridge is to prolong binding when load is high, but to accelerate release when load is low or negative.     

Structural changes in muscle crossbridges accompanying force generation

We have investigated the structure of the crossbridges in muscles rapidly frozen while relaxed, in rigor, and at various times after activation from rigor by flash photolysis of caged ATP. We used Fourier analysis of images of cross sections to obtain an average view of the muscle structure, and correspondence analysis to extract information about individual crossbridge shapes. The crossbridge structure changes dramatically between relaxed, rigor, and with time after ATP release. In relaxed muscle, most crossbridges are detached. In rigor, all are attached and have a characteristic asymmetric shape that shows strong left-handed curvature when viewed from the M-line towards the Z-line. Immediately after ATP release, before significant force has developed (20 ms) the homogeneous rigor population is replaced by a much more diverse collection of crossbridge shapes. Over the next few hundred milliseconds, the proportion of attached crossbridges changes little, but the distribution of the crossbridges among different structural classes continues to evolve. Some forms of attached crossbridge (presumably weakly attached) increase at early times when tension is low. The proportion of several other attached non-rigor crossbridge shapes increases in parallel with the development of active tension. The results lend strong support to models of muscle contraction that have attributed force generation to structural changes in attached crossbridges.     

The effect of sarcomere length and stretching on the rate of ATP splitting in glycerinated rabbit psoas muscle fibers.

The effect of sarcomere length and stretching on the tension and the rate of ATP splitting was studied using small fiber bundles from glycerinated rabbit psoas muscle. The rate of ATP slitting was determined by measuring ADP production, while the tension development in response to a contracting solution (at pCa 5.3) was recorded in the same preparation. The isometric tension developed by the preparation decreased when the sarcomere length was increased. The decrease of tension development was accompanied by a decrease in the rate of ATP splitting. If a preparation exerting steady isometric tension was stretched by 5--10% at a velocity of 0.1 mm/s, the rate of ATP splitting was increased after stretching, while the steady isometric tension attained after stretching was also higher than the initial value. The extent of the excess ATP splitting caused by stretching decreased with increasing sarcomere length. These results suggest that the rate of the interaction cycle between actin and myosin molecules may increase as a result of stretching.     

Activation of skinned trabeculae of the guinea pig induced by laser photolysis of caged ATP.

The kinetics of force production in chemically skinned trabeculae from the guinea pig were studied by laser photolysis of caged ATP in the presence of Ca2+. Preincubation of the tissue during rigor with the enzyme apyrase was used to reduce the population of MgADP-bound cross-bridges (Martin and Barsotti, 1994). In untreated tissue, tension remained constant or dipped slightly below the rigor level immediately after ATP release, before increasing to the maximum measured in pCa 4.5 and 5 mM MgATP. The in-phase component stiffness, which is a measure of cross-bridge attachment, exhibited a large decrease before increasing to 55% of that measured in rigor. Neither the rate of the decline nor of the rise in tension was sensitive to the concentration of photolytically released ATP. The rate of the decline in stiffness was found to be dependent on [ATP]: 1.8 x 10(4) M-1/s-1, a value more than four times higher than that previously measured in similar experiments in the absence of Ca2+. The rate of tension development averaged 14.9 +/- 2.5 s-1. Preincubation with apyrase altered the mechanical characteristics of the early phase of the contraction. The rate and amplitude of the initial drop in both tension and stiffness after caged ATP photolysis increased and became dependent on [ATP]. The second-order rate constants measured for the initial drop in tension and stiffness were 8.4 x 10(4) M-1 s-1 and 1.5 x 10(5) M-1 s-1. These rates are more than two times faster than those previously measured in the absence of Ca2+. The effects of apyrase incubation on the time course of tension and stiffness were consistent with the hypothesis that during rigor, skinned trabeculae retain a significant population of MgADP-bound cross-bridges. These in turn act to attenuate the initial drop in tension after caged ATP photolysis and slow the apparent rate of rigor cross-bridge detachment. The results also show that Ca2+ increases the rate of cross-bridge detachment in both untreated and apyrase-treated tissue, but the effect is larger in untreated tissue. This suggests that in cardiac muscle Ca2+ modulates the rate of cross-bridge detachment.     

On the molecular dynamics of the chemo-mechanical conversion in muscle contraction

The molecular dynamics of energy conversion by the actomyosin system in muscle contraction is studied by comparing two different types of model on the motion of crossbridge on thin filament. The motion is associated with a transition between two stable states in Huxley and Simmons' model while in Shimizu et al.'s model with a transition from an unstable to a stable state. The rate of the transition, which is proportional to the velocity of shortening of muscle in steady state, is calculated by representing the motion of crossbridge by that of a Brownian particle moving on a one-dimensional linear potential. In the case of the Huxley-Simmons model the energy conversion process is essentially a thermal one and the velocity of shortening depends sharply on the number of crossbridges on muscular filament, which is proportional to the overlapping length between thin and thick filaments. On the other hand, in the case of the Shimizu model the energy conversion process is a deterministic one which means that muscle is able to shorten smoothly and that the velocity of shortening is almost independent of the overlapping length. Experimental observations by Gordon et al. are consistent with the latter model.     

Spin-label study of actin-myosin-nucleotide interactions in contracting glycerinated muscle fibers

This paper presents the results of simultaneous measurements of the electron paramagnetic resonance signal of spin-label bound to myosin cross-bridges and the mechanical response of glycerol-treated rabbit psoas fibers under isometric contraction. No observable change has been detected in vitro in the local motion of spin-label bound to myosin-ATP with conventional electron paramagnetic resonance techniques when F-actin is added, even under conditions where more than 30% of the myosin is expected to be in an attached state. In contrast, a clear change in the spin-label mobility is observed when cross-bridges are attached to thin filaments. Similar spectra are also observed when cross-bridges are in the rigor state or in an attached state in the presence of 5′-adenylyl imidodiphosphate in place of ATP. A good proportionality is found between the change in the electron paramagnetic resonance signal and the tension when substrate concentration is varied under conditions where no appreciable amount of rigor complex is present. Thus, by assuming 0 and 100% attachment in the relaxed and rigor states, respectively, the extent of cross-bridge attachment can be estimated; it is about 80% at a relatively low ATP concentration where the maximum tension is observed, while it is about 35% in the millimolar range of ATP concentration. A consistent explanation can be given for the spectra obtained both in solution and in the fiber, provided that two distinct states, the preactive and active states, exist in cross-bridges attached to thin filaments. The contribution of intermediate complexes to the force generation is discussed. The effect of Ca²⁺ control on cross-bridge attachment is also studied at various concentrations of substrate.     

Equilibrium muscle crossbridge behavior: The interaction of myosin crossbridges with actin

The interaction of myosin crossbridges with actin under equilibrium conditions is reviewed. Similarities and differences between the weakly- and strongly-binding interactions of myosin crossbridges with actin filaments are discussed. A precise, narrow definition of weakly- binding crossbridges is given. It is postulated that the fundamental interaction of crossbridges with actin is that the crossbridge heads are mobile after attachment in the first case but not in the second. It is argued that because the weakly-binding crossbridge heads are mobile after attachment, the heads appear to function independently of each other. The lack of head mobility in attached strongly-binding crossbridges makes the strongly-binding crossbridge heads appear to act cooperatively. This model of the strongly-binding crossbridge gives an explanation for two important and otherwise unexplained observations. It explains why the rate constant of force decay after a small stretch is a sigmoidal function of nucleotide analogue concentration, and why, in the presence of analogues or in rigor, the rate constant of force decay after a small stretch is often significantly slower than the rate constant for myosin subfragment-1 detachment from actin in solution. The model of the weakly-binding crossbridge accurately describes the behavior of the myosin·ATP crossbridge.     

Resolution of three structural states of spin-labeled myosin in contracting muscle.

We have used electron paramagnetic resonance (EPR) spectroscopy to detect ATP- and calcium-induced changes in the structure of spin-labeled myosin heads in glycerinated rabbit psoas muscle fibers in key physiological states. The probe was a nitroxide iodoacetamide derivative attached selectively to myosin SH1 (Cys 707), the conventional EPR spectra of which have been shown to resolve several conformational states of the myosin ATPase cycle, on the basis of nanosecond rotational motion within the protein. Spectra were acquired in rigor and during the steady-state phases of relaxation and isometric contraction. Spectral components corresponding to specific conformational states and biochemical intermediates were detected and assigned by reference to EPR spectra of trapped kinetic intermediates. In the absence of ATP, all of the myosin heads were rigidly attached to the thin filament, and only a single conformation was detected, in which there was no sub-microsecond probe motion. In relaxation, the EPR spectrum resolved two conformations of the myosin head that are distinct from rigor. These structural states were virtually identical to those observed previously for isolated myosin and were assigned to the populations of the M*.ATP and M**.ADP.Pi states. During isometric contraction, the EPR spectrum resolves the same two conformations observed in relaxation, plus a small fraction (20-30%) of heads in the oriented actin-bound conformation that is observed in rigor. This rigor-like component is a calcium-dependent, actin-bound state that may represent force-generating cross-bridges. As the spin label is located near the nucleotide-binding pocket in a region proposed to be pivotal for large-scale force-generating structural changes in myosin, we propose that the observed spectroscopic changes indicate directly the key steps in energy transduction in the molecular motor of contracting muscle.     

Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle

Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross-bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179-192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.