The conformation of myosin head domains in rigor muscle determined by X-ray interference

In the absence of adenosine triphosphate, the head domains of myosin cross-bridges in muscle bind to actin filaments in a rigor conformation that is expected to mimic that following the working stroke during active contraction. We used x-ray interference between the two head arrays in opposite halves of each myosin filament to determine the rigor head conformation in single fibers from frog skeletal muscle. During isometric contraction (force T(0)), the interference effect splits the M3 x-ray reflection from the axial repeat of the heads into two peaks with relative intensity (higher angle/lower angle peak) 0.76. In demembranated fibers in rigor at low force (<0.05 T(0)), the relative intensity was 4.0, showing that the center of mass of the heads had moved 4.5 nm closer to the midpoint of the myosin filament. When rigor fibers were stretched, increasing the force to 0.55 T(0), the heads' center of mass moved back by 1.1-1.6 nm. These motions can be explained by tilting of the light chain domain of the head so that the mean angle between the Cys(707)-Lys(843) vector and the filament axis increases by approximately 36 degrees between isometric contraction and low-force rigor, and decreases by 7-10 degrees when the rigor fiber is stretched to 0.55 T(0).     

Stiffness and fraction of Myosin motors responsible for active force in permeabilized muscle fibers from rabbit psoas

The stiffness of the single myosin motor (epsilon) is determined in skinned fibers from rabbit psoas muscle by both mechanical and thermodynamic approaches. Changes in the elastic strain of the half-sarcomere (hs) are measured by fast mechanics both in rigor, when all myosin heads are attached, and during active contraction, with the isometric force (T0) modulated by changing either [Ca2+] or temperature. The hs compliance is 43.0+/-0.8 nm MPa-1 in isometric contraction at saturating [Ca2+], whereas in rigor it is 28.2+/-1.1 nm MPa-1. The equivalent compliance of myofilaments is 21.0+/-3.3 nm MPa-1. Accordingly, the stiffness of the ensemble of myosin heads attached in the hs is 45.5+/-1.7 kPa nm-1 in isometric contraction at saturating [Ca2+] (e0), and in rigor (er) it rises to 138.9+/-21.2 kPa nm-1. Epsilon, calculated from er and the lattice molecular dimensions, is 1.21+/-0.18 pN nm-1. epsilon estimated, using a thermodynamic approach, from the relation of T0 at saturating [Ca2+] versus the reciprocal of absolute temperature is 1.25+/-0.14 pN nm-1, similar to that estimated for fibers in rigor. Consequently, the ratio e0/er (0.33+/-0.05) can be used to estimate the fraction of attached heads during isometric contraction at saturating [Ca2+]. If the osmotic agent dextran T-500 (4 g/100 ml) is used to reduce the lateral filament spacing of the relaxed fiber to the value before skinning, both e0 and er increase by approximately 40%. Epsilon becomes approximately 1.7 pN nm-1 and the fraction and the force of myosin heads attached in the isometric contraction remain the same as before dextran application. The finding that the fraction of myosin heads attached to actin in an isometric contraction is 0.33 rules out the hypothesis of multiple mechanical cycles per ATP hydrolyzed.     

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.     

The Effect of Myofilament Compliance on Kinetics of Force Generation by Myosin Motors in Muscle

We use the inhibitor of isometric force of skeletal muscle N-benzyl-p-toluene sulfonamide (BTS) to decrease, in a dose dependent way, the number of myosin motors attached to actin during the steady isometric contraction of single fibers from frog skeletal muscle (4°C, 2.1 μm sarcomere length). In this way we can reduce the strain in the myofilament compliance during the isometric tetanus (T₀) from 3.54 nm in the control solution (T_0,NR) to ∼0.5 nm in 1 μM BTS, where T₀ is reduced to ∼0.15 T_0,NR. The quick force recovery after a step release (1-3 nm per half-sarcomere) becomes faster with the increase of BTS concentration and the decrease of T₀. The simulation of quick force recovery with a multistate model of force generation, that adapts Huxley and Simmons model to account for both the high stiffness of the myosin motor (∼3 pN/nm) and the myofilament compliance, shows that the increase in the rate of quick force recovery by BTS is explained by the reduced strain in the myofilaments, consequent to the decrease in half-sarcomere force. The model estimates that i), for the same half-sarcomere release the state transition kinetics in the myosin motor are five times faster in the absence of filament compliance than in the control; and ii), the rate of force recovery from zero to T₀ is ∼6000/s in the absence of filament compliance.     

Compliance of thin filaments in skinned fibers of rabbit skeletal muscle.

The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction.     

Protein osmotic pressure and cross-bridge attachment determine the stiffness of thin filaments in muscle ex vivo

The properties of some models of the actin filament are compared with those of the thin filament in muscle. The greater stiffness of thin filaments ex vivo with respect to F-actin in vitro is attributed to the effect of both protein osmotic pressure and the attached cross-bridges. By comparing the stiffness of thin filaments in vitro and in isometric and rigor muscles the stiffness of thin filaments in relaxed muscle is computed. The upper limit of thin filament stretching is deduced to approach approximately 10 nm microm(-1). It is also calculated that, on stretching by 2.02 nm of the fully non-overlapped thin filament or by 1.59 nm of the thin filament on isometric contraction, the energy released on the hydrolysis of one molecule of ATP is fully used up.     

X-ray diffraction studies of the contractile mechanism in single muscle fibres

The molecular mechanism of muscle contraction was investigated in intact muscle fibres by X-ray diffraction. Changes in the intensities of the axial X-ray reflections produced by imposing rapid changes in fibre length establish the average conformation of the myosin heads during active isometric contraction, and show that the heads tilt during the elastic response to a change in fibre length and during the elementary force generating process: the working stroke. X-ray interference between the two arrays of myosin heads in each filament allows the axial motions of the heads following a sudden drop in force from the isometric level to be measured in situ with unprecedented precision. At low load, the average working stroke is 12 nm, which is consistent with crystallographic studies. The working stroke is smaller and slower at a higher load. The compliance of the actin and myosin filaments was also determined from the change in the axial spacings of the X-ray reflections following a force step, and shown to be responsible for most of the sarcomere compliance. The mechanical properties of the sarcomere depend on both the motor actions of the myosin heads and the compliance of the myosin and actin filaments.     

Effect of inorganic phosphate on the force and number of myosin cross-bridges during the isometric contraction of permeabilized muscle fibers from rabbit psoas

The relation between the chemical and mechanical steps of the myosin-actin ATPase reaction that leads to generation of isometric force in fast skeletal muscle was investigated in demembranated fibers of rabbit psoas muscle by determining the effect of the concentration of inorganic phosphate (Pi) on the stiffness of the half-sarcomere (hs) during transient and steady-state conditions of the isometric contraction (temperature 12°C, sarcomere length 2.5 μm). Changes in the hs strain were measured by imposing length steps or small 4 kHz oscillations on the fibers in control solution (without added Pi) and in solution with 3-20 mM added Pi. At the plateau of the isometric contraction in control solution, the hs stiffness is 22.8 ± 1.1 kPa nm⁻¹. Taking the filament compliance into account, the total stiffness of the array of myosin cross-bridges in the hs (e) is 40.7 ± 3.7 kPa nm⁻¹. An increase in [Pi] decreases the stiffness of the cross-bridge array in proportion to the isometric force, indicating that the force of the cross-bridge remains constant independently of [Pi]. The rate constant of isometric force development after a period of unloaded shortening (r_F) is 23.5 ± 1.0 s⁻¹ in control solution and increases monotonically with [Pi], attaining a maximum value of 48.6 ± 0.9 s⁻¹ at 20 mM [Pi], in agreement with the idea that Pi release is a relatively fast step after force generation by the myosin cross-bridge. During isometric force development at any [Pi], e and thus the number of attached cross-bridges increase in proportion to the force, indicating that, independently of the speed of the process that leads to myosin attachment to actin, there is no significant (>1 ms) delay between generation of stiffness and generation of force by the cross-bridges.     

Non-specific termination of simian virus 40 DNA replication.

Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å.     

Structural features of cross-bridges in isometrically contracting skeletal muscle

Two-dimensional x-ray diffraction was used to investigate structural features of cross-bridges that generate force in isometrically contracting skeletal muscle. Diffraction patterns were recorded from arrays of single, chemically skinned rabbit psoas muscle fibers during isometric force generation, under relaxation, and in rigor. In isometric contraction, a rather prominent intensification of the actin layer lines at 5.9 and 5.1 nm and of the first actin layer line at 37 nm was found compared with those under relaxing conditions. Surprisingly, during isometric contraction, the intensity profile of the 5.9-nm actin layer line was shifted toward the meridian, but the resulting intensity profile was different from that observed in rigor. We particularly addressed the question whether the differences seen between rigor and active contraction might be due to a rigor-like configuration of both myosin heads in the absence of nucleotide (rigor), whereas during active contraction only one head of each myosin molecule is in a rigor-like configuration and the second head is weakly bound. To investigate this question, we created different mixtures of weak binding myosin heads and rigor-like actomyosin complexes by titrating MgATPgammaS at saturating [Ca2+] into arrays of single muscle fibers. The resulting diffraction patterns were different in several respects from patterns recorded under isometric contraction, particularly in the intensity distribution along the 5.9-nm actin layer line. This result indicates that cross-bridges present during isometric force generation are not simply a mixture of weakly bound and single-headed rigor-like complexes but are rather distinctly different from the rigor-like cross-bridge. Experiments with myosin-S1 and truncated S1 (motor domain) support the idea that for a force generating cross-bridge, disorder due to elastic distortion might involve a larger part of the myosin head than for a nucleotide free, rigor cross-bridge.     

Cross-bridge duty cycle in isometric contraction of skeletal myofibrils

During interaction of actin with myosin, cross-bridges impart mechanical impulses to thin filaments resulting in rotations of actin monomers. Impulses are delivered on the average every tc seconds. A cross-bridge spends a fraction of this time (ts) strongly attached to actin, during which it generates force. The "duty cycle" (DC), defined as the fraction of the total cross-bridge cycle that myosin spends attached to actin in a force generating state (ts/ tc), is small for cross-bridges acting against zero load, like freely shortening muscle, and increases as the load rises. Here we report, for the first time, an attempt to measure DC of a single cross-bridge in muscle. A single actin molecule in a half-sarcomere was labeled with fluorescent phalloidin. Its orientation was measured by monitoring intensity of the polarized TIRF images. Actin changed orientation when a cross-bridge bound to it. During isometric contraction, but not during rigor, actin orientation oscillated between two values, corresponding to the actin-bound and actin-free state of the cross-bridge. The average ts and tc were 3.4 and 6 s, respectively. These results suggest that, in isometrically working muscle, cross-bridges spend about half of the cycle time attached to actin. The fact that 1/ tc was much smaller than the ATPase rate suggests that the bulk of the energy of ATP hydrolysis is used for purposes other than performance of mechanical work.     

Cross-bridge attachment during high-speed active shortening of skinned fibers of the rabbit psoas muscle: implications for cross-bridge action during maximum velocity of filament sliding

To characterize the kinetics of cross-bridge attachment to actin during unloaded contraction (maximum velocity of filament sliding), ramp-shaped stretches with different stretch-velocities (2-40,000 nm per half-sarcomere per s) were applied to actively contracting skinned fibers of the rabbit psoas muscle. Apparent fiber stiffness observed during such stretches was plotted versus the speed of the imposed stretch (stiffness-speed relation) to derive the rate constants for cross-bridge dissociation from actin. The stiffness-speed relation obtained for unloaded shortening conditions was shifted by about two orders of magnitude to faster stretch velocities compared to isometric conditions and was almost identical to the stiffness-speed relation observed in the presence of MgATPgammaS at high Ca(2+) concentrations, i.e., under conditions where cross-bridges are weakly attached to the fully Ca(2+) activated thin filaments. These data together with several control experiments suggest that, in contrast to previous assumptions, most of the fiber stiffness observed during high-speed shortening results from weak cross-bridge attachment to actin. The fraction of strongly attached cross-bridges during unloaded shortening appears to be as low as some 1-5% of the fraction present during isometric contraction. This is about an order of magnitude less than previous estimates in which contribution of weak cross-bridge attachment to observed fiber stiffness was not considered. Our findings imply that 1) the interaction distance of strongly attached cross-bridges during high-speed shortening is well within the range consistent with conventional cross-bridge models, i.e., that no repetitive power strokes need to be assumed, and 2) that a significant part of the negative forces that limit the maximum speed of filament sliding might originate from weak cross-bridge interactions with actin.     

Ca-activation and stretch-activation in insect flight muscle

Asynchronous insect flight muscle is specialized for myogenic oscillatory work, but can also produce isometric tetanic contraction. In skinned insect flight muscle fibers from Lethocerus, with sarcomere length monitored by a striation follower, we determined the relation between isometric force (F(0)) at serial increments of [Ca(2+)] and the additional active force recruited at each [Ca(2+)] by a stretch of approximately 12 nm per half-sarcomere (F(SA)). The isometric force-pCa relation shows that 1.5-2 units of pCa are necessary to raise isometric force from its threshold (pCa approximately 6.5) to its maximum (F(0,max)). The amplitude of F(SA) depends only on the preceding baseline level of isometric force, which must reach at least 0.05 F(0,max) to enable stretch-activation. F(SA) rises very steeply to its maximum as F(0) reaches approximately 0.2 F(0,max), then decreases as F(0) increases so as to produce a constant sum (F(0) + F(SA)) = F(max). Thus Ca- and stretch-activation are complementary pathways that trigger a common process of cross-bridge attachment and force production. We suggest that stretch-induced distortion of attached cross-bridges relieves the steric blocking by tropomyosin of additional binding sites on actin, thereby enabling maximum force even at low [Ca(2+)].     

Cross-bridge number, position, and angle in target zones of cryofixed isometrically active insect flight muscle

Electron micrographic tomograms of isometrically active insect flight muscle, freeze substituted after rapid freezing, show binding of single myosin heads at varying angles that is largely restricted to actin target zones every 38.7 nm. To quantify the parameters that govern this pattern, we measured the number and position of attached myosin heads by tracing cross-bridges through the three-dimensional tomogram from their origins on 14.5-nm-spaced shelves along the thick filament to their thin filament attachments in the target zones. The relationship between the probability of cross-bridge formation and axial offset between the shelf and target zone center was well fitted by a Gaussian distribution. One head of each myosin whose origin is close to an actin target zone forms a cross-bridge most of the time. The probability of cross-bridge formation remains high for myosin heads originating within 8 nm axially of the target zone center and is low outside 12 nm. We infer that most target zone cross-bridges are nearly perpendicular to the filaments (60% within 11 degrees ). The results suggest that in isometric contraction, most cross-bridges maintain tension near the beginning of their working stroke at angles near perpendicular to the filament axis. Moreover, in the absence of filament sliding, cross-bridges cannot change tilt angle while attached nor reach other target zones while detached, so may cycle repeatedly on and off the same actin target monomer.     

Radial equilibrium lengths of actomyosin cross-bridges in muscle.

Radial equilibrium lengths of the weakly attached, force-generating, and rigor cross-bridges are determined by recording their resistance to osmotic compression. Radial equilibrium length is the surface-to-surface distance between myosin and actin filaments at which attached cross-bridges are, on average, radially undistorted. We previously proposed that differences in the radial equilibrium length represent differences in the structure of the actomyosin cross-bridge. Until now the radial equilibrium length had only been determined for various strongly attached cross-bridge states and was found to be distinct for each state examined. In the present work, we demonstrate that weakly attached cross-bridges, in spite of their low affinity for actin, also exert elastic forces opposing osmotic compression, and they are characterized by a distinct radial equilibrium length (12.0 nm vs. 10.5 nm for force-generating and 13.0 nm for rigor cross-bridge). This suggests significant differences in the molecular structure of the attached cross-bridges under these conditions, e.g., differences in the shape of the myosin head or in the docking of the myosin to actin. Thus, the present finding supports our earlier conclusion that there is a structural change in the attached cross-bridge associated with the transition from a weakly bound configuration to the force-generating configuration. The implications for imposing spatial constraints on modeling actomyosin interaction in the filament lattice are discussed.     

Force generation and work production by covalently cross-linked actin-myosin cross-bridges in rabbit muscle fibers.

To separate a fraction of the myosin cross-bridges that are attached to the thin filaments and that participate in the mechanical responses, muscle fibers were cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and then immersed in high-salt relaxing solution (HSRS) of 0.6 M ionic strength for detaching the unlinked myosin heads. The mechanical properties and force-generating ability of the cross-linked cross-bridges were tested with step length changes (L-steps) and temperature jumps (T-jumps) from 6-10 degrees C to 30-40 degrees C. After partial cross-linking, when instantaneous stiffness in HSRS was 25-40% of that in rigor, the mechanical behavior of the fibers was similar to that during active contraction. The kinetics of the T-jump-induced tension transients as well as the rate of the fast phase of tension recovery after length steps were close to those in unlinked fibers during activation. Under feedback force control, the T-jump initiated fiber shortening by up to 4 nm/half-sarcomere. Work produced by a cross-linked myosin head after the T-jump was up to 30 x 10(-21) J. When the extent of cross-linking was increased and fiber stiffness in HSRS approached that in rigor, the fibers lost their viscoelastic properties and ability to generate force with a rise in temperature.     

Does thin filament compliance diminish the cross-bridge kinetics? A study in rabbit psoas fibers.

The effect of thin filament compliance on our ability to detect the cross-bridge kinetics was examined. Our experiment is based on the facts that in rabbit psoas the thin filament (1.12 micrometer) is longer than half the thick filament length (0.82 micrometer) and that the thick filament has a central bare zone (0.16 micrometer). Consequently, when sarcomere length is increased from 2.1 to 2.4 micrometer, the same number of cross-bridges is involved in force generation but extra series compliance is introduced in the I-band. Three apparent rate constants (2pia, 2pib, and 2pic) were characterized by sinusoidal analysis at pCa 4.66. Our results demonstrate that 2pia and 2pib increased 13-16% when sarcomere length was increased from 2.0 to 2.5 micrometer, and 2pic decreased slightly (9%). This slight decrease can be explained by compression of the lattice spacing. These observations are at variance with the expectation based on increased series compliance, which predicts that the rate constants will decrease. We also determined compliance of the I-band during rigor. I-band compliance during rigor induction was 35% of sarcomere compliance at sarcomere length 2.4 micrometer, and 24% at sarcomere length 2.1 micrometer. We conclude that the presence of thin filament compliance does not seriously interfere with our ability to detect cross-bridge kinetics using sinusoidal analysis.     

Interplay between passive tension and strong and weak binding cross- bridges in insect indirect flight muscle. A functional dissection by gelsolin-mediated thin filament removal

The interplay between passive and active mechanical properties of indirect flight muscle of the waterbug (Lethocerus) was investigated. A functional dissection of the relative contribution of cross-bridges, actin filaments, and C filaments to tension and stiffness of passive, activated, and rigor fibers was carried out by comparing mechanical properties at different ionic strengths of sarcomeres with and without thin filaments. Selective thin filament removal was accomplished by treatment with the actin-severving protein gelsolin. Thin filament, removal had no effect on passive tension, indicating that the C filament and the actin filament are mechanically independent and that passive tension is developed by the C filament in response to sarcomere stretch. Passive tension increased steeply with sarcomere length until an elastic limit was reached at only 6-7% sarcomere extension, which corresponds to an extension of 350% of the C filament. The passive tension-length relation of insect flight muscle was analyzed using a segmental extension model of passive tension development (Wang, K, R. McCarter, J. Wright, B. Jennate, and R Ramirez-Mitchell. 1991. Proc. Natl. Acad. Sci. USA. 88:7101-7109). Thin filament removal greatly depressed high frequency passive stiffness (2.2 kHz) and eliminated the ionic strength sensitivity of passive stiffness. It is likely that the passive stiffness component that is removed by gelsolin is derived from weak-binding cross-bridges, while the component that remains is derived from the C filament. Our results indicate that a significant number of weak-binding cross-bridges exist in passive insect muscle at room temperature and at an ionic strength of 195 mM. Analysis of rigor muscle indicated that while rigor tension is entirely actin based, rigor stiffness contains a component that resists gelsolin treatment and is therefore likely to be C filament based. Active tension and active stiffness of unextracted fibers were directly proportional to passive tension before activation. Similarly, passive stiffness due to weak bridges also increased linearly with passive tension, up to a limit. These correlations lead us to propose a stress-activation model for insect flight muscle in which passive tension is a prerequisite for the formation of both weak-binding and strong-binding cross-bridges.     

Myosin filament sliding through the Z-disc relates striated muscle fibre structure to function

Striated muscle contraction requires intricate interactions of microstructures. The classic textbook assumption that myosin filaments are compressed at the meshed Z-disc during striated muscle fibre contraction conflicts with experimental evidence. For example, myosin filaments are too stiff to be compressed sufficiently by the muscular force, and, unlike compressed springs, the muscle fibres do not restore their resting length after contractions to short lengths. Further, the dependence of a fibre''s maximum contraction velocity on sarcomere length is unexplained to date. In this paper, we present a structurally consistent model of sarcomere contraction that reconciles these findings with the well-accepted sliding filament and crossbridge theories. The few required model parameters are taken from the literature or obtained from reasoning based on structural arguments. In our model, the transition from hexagonal to tetragonal actin filament arrangement near the Z-disc together with a thoughtful titin arrangement enables myosin filament sliding through the Z-disc. This sliding leads to swivelled crossbridges in the adjacent half-sarcomere that dampen contraction. With no fitting of parameters required, the model predicts straightforwardly the fibre''s entire force–length behaviour and the dependence of the maximum contraction velocity on sarcomere length. Our model enables a structurally and functionally consistent view of the contractile machinery of the striated fibre with possible implications for muscle diseases and evolution.     

Recent X-ray Diffraction and Electron Microscope Studies of Striated Muscle

The sliding filament model for muscular contraction supposes that an appropriately directed force is developed between the actin and myosin filaments by some process in which the cross-bridges are involved. The cross-bridges between the filaments are believed to represent the parts of the myosin molecules which possess the active sites for ATPase activity and actin-binding ability, and project out sidewise from the backbone of the thick filaments. The arrangement of the cross-bridges is now being studied by improved low-angle X-ray diffraction techniques, which show that in a resting muscle, they are arranged approximately but not exactly in a helical pattern, and that there are other structural features of the thick filaments which give rise to additional long periodicities shown up by the X-ray diagram. The actin filaments also contain helically arranged subunits, and both the subunit repeat and the helical repeat are different from those in the myosin filaments. Diffraction diagrams can be obtained from muscles in rigor (when permanent attachment of the cross-bridges to the actin subunits takes place) and now, taking advantage of the great increase in the speed of recording, from actively contracting muscles. These show that changes in the arrangement of the cross-bridges are produced under both these conditions and are no doubt associated in contraction with the development of force. Thus configurational changes of the myosin component in muscle have been demonstrated: these take place without any significant over-all change in the length of the filaments.