Filament lattice of frog striated muscle. Radial forces, lattice stability, and filament compression in the A-band of relaxed and rigor muscle.

Repulsive pressure in the A-band filament lattice of relaxed frog skeletal muscle has been measured as a function of interfilament spacing using an osmotic shrinking technique. Much improved chemical skinning was obtained when the muscles were equilibrated in the presence of EGTA before skinning. The lattice shrank with increasing external osmotic pressure. At any specific pressure, the lattice spacing in relaxed muscle was smaller than that of muscle in rigor, except at low pressures where the reverse was found. The lattice spacing was the same in the two states at a spacing close to that found in vivo. The data were consistent with an electrostatic repulsion over most of the pressure range. For relaxed muscle, the data lay close to electrostatic pressure curves for a thick filament charge diameter of approximately 26 nm, suggesting that charges stabilizing the lattice are situated about midway along the thick filament projections (HMM-S1). At low pressures, observed spacings were larger than calculated, consistent with the idea that thick filament projections move away from the filament backbone. Under all conditions studied, relaxed and rigor, at short and very long sarcomere lengths, the filament lattice could be modeled by assuming a repulsive electrostatic pressure, a weak attractive pressure, and a radial stiffness of the thick filaments (projections) that differed between relaxed and rigor conditions. Each thick filament projection could be compressed by approximately 5 or 2.6 nm requiring a force of 1.3 or 80 pN for relaxed and rigor conditions respectively.     

The myofilament lattice: studies on isolated fibers. IV. Lattice equilibria in striated muscle.

Accounts of similarities between the thick filament lattice of striated muscle and smectic liquid-crystalline structures have focused upon an equilibrium between electrostatic (repulsive) and van der Waal's (attractive) forces. In living, intact muscle the fiber volume constitutes an additional important parameter which influences the amount of interaxial separation between the filaments. This is demonstrable by comparison of the lattice behavior of living fibers with that of fibers from which the sarcolemma has either been removed or made leaky by glycerination. These comparisons were made mainly by low-angle X-ray diffraction under conditions of changes in sarcomere length, ionic strength or osmolarity, and pH. Single fibers with the sarcolemma removed and glycerinated muscle have lattices which behave in accord with equilibrium liquid-crystalline systems in which the thick filament spacing is determined by the balance between electrostatic and van der Waal's forces. Conversely, osmotic and shortening studies demonstrate that the living, intact muscle has a lattice which behaves in accord with the so-called non-equilibrium (volume-constrained) liquid-crystalline condition in which the interaxial separation between the thick filaments is solely due to the amount of volume available as determined by the Donnan steady-state across the sarcolemma.     

Average electrostatic potential between the filaments in striated muscle and its relation to a simple Donnan potential.

An approximate analytical solution to the Poisson-Boltzmann equation for a cylindrical particle was used to calculate the relationship between the charge on the filaments and the average electrostatic potential. Both thick and then filaments were considered in the muscle lattice with a filament charge ratio of 4 to 1. Comparing this with a similar relationship obtained using simple Donnan theory showed a discrepancy at high charge where the Poisson-Boltzmann equation leads to saturation of the average potential. However, using two separate experiments from the literature, we have shown that at pH 7.0 muscle must not be close to saturation and thus is in a region of the curve where the two approaches agree.     

Donnan potentials from striated muscle liquid crystals. Lattice spacing dependence.

Electrochemical potentials were measured as a function of myofilament packing density in crayfish striated muscle. The A-band striations are supramolecular smectic B1 lattice assemblies of myosin filaments and the I-band striations are nematic liquid crystals of actin filaments. Both A- and I-bands generate potentials derived from the fixed charge that is associated with structural proteins. In the reported experiments, filament packing density was varied by osmotically reducing lattice volume. The electrochemical potentials were measured from the A- and I-bands in the relaxed condition over a range of lattice volumes. From the measurements of relative cross-sectional area, unit-cell volume (obtained by low-angle x-ray diffraction) and previously determined effective linear charge densities (Aldoroty, R.A., N.B. Garty, and E.W. April, 1985, Biophys. J., 47:89-96), Donnan potentials can be predicted for any amount of compression. In the relaxed condition, the predicted Donnan potentials correspond to the measured electrochemical potentials. In the rigor condition, however, a net increase in negative charge associated with the myosin filament is observed. The predictability of the data demonstrates the applicability of Donnan equilibrium theory to the measurement of electrochemical potentials from liquid-crystalline systems. Moreover, the relationship between filament spacing and the Donnan potential is consistent with the concept that surface charge provides the necessary electrostatic force to stabilize the myofilament lattice.     

Lateral forces in the filament lattice of vertebrate striated muscle in the rigor state.

The repulsive pressure between filaments in the lattice of skinned rabbit and frog striated muscle in rigor has been measured as a function of interfilament spacing, using the osmotic pressure generated by solutions of large, uncharged polymeric molecules (dextran and polyvinylpyrrolidone). The pressure/spacing measurements have been compared with theoretically derived curves for electrostatic pressure. In both muscles, the major part of the experimental curves (100-2,000 torr) lies in the same region as the electrostatic pressure curves, providing that a thick filament charge diameter of approximately 30 nm in rabbit and approximately 26 nm in frog is assumed. In chemically skinned or glycerol-extracted rabbit muscle the fit is good; in chemically skinned frog sartorius and semitendinosus muscle the fit is poor, particularly at lower pressures where a greater spacing is observed than expected on theoretical grounds. The charge diameter is much larger than the generally accepted value for thick filament backbone diameter. This may be because electron microscope results have underestimated the amount of filament shrinkage during sample preparation, or because most of the filament charge is located at some distance from the backbone surface, e.g., on HMM-S2. Decreasing the ionic strength of the external solution, changing the pH, and varying the sarcomere length all give pressure/spacing changes similar to those expected from electrostatic pressure calculations. We conclude that over most of the external pressure range studied, repulsive pressure in the lattice is predominantly electrostatic.     

Non-isovolumic behavior of the unit cell of skinned striated muscle fibers.

X-ray diffraction studies indicate that the sarcomeric unit cell of the thick filament lattice of crayfish muscle fibers stripped of the sarcolemma does not shorten isovolumically. This particular behavior can be explained in terms of the variation of negative charge within the A-band with interdigitation of thin filaments. Values for effective charge densities of the filaments are determined and used to calculate the total effective charge within the A-band which is then related to separation between myosin filaments. Correlation between empirical and theoretical values illustrates that interfilament separation in skinned fibers is a linear function of A-band charge.     

Myosin thick filament lability induced by mechanical strain in airway smooth muscle.

Airway smooth muscle adapts to different lengths with functional changes that suggest plastic alterations in the filament lattice. To look for structural changes that might be associated with this plasticity, we studied the relationship between isometric force generation and myosin thick filament density in cell cross sections, measured by electron microscope, after length oscillations applied to the relaxed porcine trachealis muscle. Muscles were stimulated regularly for 12 s every 5 min. Between two stimulations, the muscles were submitted to repeated passive +/- 30% length changes. This caused tetanic force and thick-filament density to fall by 21 and 27%, respectively. However, in subsequent tetani, both force and filament density recovered to preoscillation levels. These findings indicate that thick filaments in airway smooth muscle are labile, depolymerization of the myosin filaments can be induced by mechanical strain, and repolymerization of the thick filaments underlies force recovery after the oscillation. This thick-filament lability would greatly facilitate plastic changes of lattice length and explain why airway smooth muscle is able to function over a large length range.     

Influence of calcium on myosin thick filament formation in intact airway smooth muscle

Myosin thick filaments have been shown tobe structurally labile in intact smooth muscles. Although the mechanismof thick filament assembly/disassembly for purified myosins in solution has been well described, regulation of thick filament formation inintact muscle is still poorly understood. The present study investigates the effect of resting calcium level on thick filament maintenance in intact airway smooth muscle and on thick filament formation during activation. Cross-sectional density of the thick filaments measured electron microscopically showed that the density increased substantially (144%) when the muscle was activated. Theabundance of filamentous myosins in relaxed muscle was calcium sensitive; in the absence of calcium (with EGTA), the filament densitydeceased by 35%. Length oscillation imposed on the muscle underzero-calcium conditions produced no further reduction in the density.Isometric force and filament density recovered fully after reincubationof the muscle in normal physiological saline. The results suggest thatin airway smooth muscle, filamentous myosins exist in equilibrium withmonomeric myosins; muscle activation favors filament formation, and theresting calcium level is crucial for preservation of the filaments inthe relaxed state.

     

Structure of the cross-striated adductor muscle of the scallop

The structure of the cross-striated adductor muscle of the scallop has been studied by electron microscopy and X-ray diffraction using living relaxed, glycerol-extracted (rigor), fixed and dried muscles. The thick filaments are arranged in a hexagonal lattice whose size varies with sarcomere length so as to maintain a constant lattice volume. In the overlap region there are approximately 12 thin filaments about each thick filament and these are arranged in a partially disordered lattice similar to that found in other invertebrate muscles, giving a thin-to-thick filament ratio in this region of 6:1.The thin filaments, which contain actin and tropomyosin, are about 1 μm long and the actin subunits are arranged on a helix of pitch 2 × 38.5 nm. The thick filaments, which contain myosin and paramyosin, are about 1.76 μm long and have a backbone diameter of about 21 nm. We propose that these filaments have a core of paramyosin about 6 nm in diameter, around which the myosin molecules pack. In living relaxed muscle, the projecting myosin heads are symmetrically arranged. The data are consistent with a six-stranded helix, each strand having a pitch of 290 nm. The projections along the strands each correspond to the heads of one or two myosin molecules and occur at alternating intervals of 13 and 16 nm. In rigor muscle these projections move away from the backbone and attach to the thin filaments.In both living and dried muscle, alternate planes of thick filaments are staggered longitudinally relative to each other by about 7.2 nm. This gives rise to a body-centred orthorhombic lattice with a unit cell twice the volume of the basic filament lattice.     

Tension fluctuations in contracting myofibrils and their interpretation

Repulsive pressure has been measured as a function of lattice spacing in gels of tobacco mosaic virus (TMV) and in the filament lattice of vertebrate striated muscle. External pressures up to ten atm have been applied to these lattices by an osmotic stress method. Numerical solutions to the Poisson-Boltzmann equation in hexagonal lattices have been obtained and compared to the TMV and muscle data. The theoretical curves using values for k calculated from the ionic strength give a good fit to experimental data from TMV gels, and an approximate fit to that from the muscle lattice, provided that a charge radius for the muscle thick filaments of approximately 16 nm is assumed. Variations in ionic strength, sarcomere length and state of the muscle give results which agree qualitatively with the theory, though a good fit between experiment and theory in the muscle case will clearly require consideration of other types of forces. We conclude that Poisson-Boltzmann theory can provide a good first approximation to the long-range electrostatic forces operating in such biological gel systems.     

Stepping between Membrane Microdomains

In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P₂〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads.     

Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations

By taking advantage of a lethal phenotype characteristic of Caenorhabditis elegans embryos that fail to move, we have identified 13 genes required for muscle assembly and function and discovered a new lethal class of alleles for three previously known muscle-affecting genes. By staining mutant embryos for myosin and actin we have recognized five distinct classes of genes: mutations in four genes disrupt the assembly of thick and thin filaments into the myofilament lattice as well as the polarized location of these components to the sarcolemma. Mutations in another three genes also disrupt thick and thin filament assembly, but allow proper polarization of lattice components based on the myosin heavy chain isoform that we analyzed. Another two classes of genes are defined by mutations with principal effects on thick or thin filament assembly into the lattice, but not both. The final class includes three genes in which mutations cause relatively minor defects in lattice assembly. Failure of certain mutants to stain with antibodies to specific muscle cell antigens suggest that two genes associated with severe disruptions of myofilament lattice assembly may code for components of the basement membrane and the sarcolemma that are concentrated where dense bodies (Z- line analogs) and M-lines attach to the cell membrane. Similar evidence suggests that one of the genes associated with mild effects on lattice assembly may code for tropomyosin. Many of the newly identified genes are likely to play critical roles in muscle development and function.     

Dependence of thick filament structure in relaxed mammalian skeletal muscle on temperature and interfilament spacing

Contraction of skeletal muscle is regulated by structural changes in both actin-containing thin filaments and myosin-containing thick filaments, but myosin-based regulation is unlikely to be preserved after thick filament isolation, and its structural basis remains poorly characterized. Here, we describe the periodic features of the thick filament structure in situ by high-resolution small-angle x-ray diffraction and interference. We used both relaxed demembranated fibers and resting intact muscle preparations to assess whether thick filament regulation is preserved in demembranated fibers, which have been widely used for previous studies. We show that the thick filaments in both preparations exhibit two closely spaced axial periodicities, 43.1 nm and 45.5 nm, at near-physiological temperature. The shorter periodicity matches that of the myosin helix, and x-ray interference between the two arrays of myosin in the bipolar filament shows that all zones of the filament follow this periodicity. The 45.5-nm repeat has no helical component and originates from myosin layers closer to the filament midpoint associated with the titin super-repeat in that region. Cooling relaxed or resting muscle, which partially mimics the effects of calcium activation on thick filament structure, disrupts the helical order of the myosin motors, and they move out from the filament backbone. Compression of the filament lattice of demembranated fibers by 5% Dextran, which restores interfilament spacing to that in intact muscle, stabilizes the higher-temperature structure. The axial periodicity of the filament backbone increases on cooling, but in lattice-compressed fibers the periodicity of the myosin heads does not follow the extension of the backbone. Thick filament structure in lattice-compressed demembranated fibers at near-physiological temperature is similar to that in intact resting muscle, suggesting that the native structure of the thick filament is largely preserved after demembranation in these conditions, although not in the conditions used for most previous studies with this preparation.     

Filament lattice changes in smooth muscle assessed using birefringence

The long functional range of some types of smooth muscle has been the subject of recent study. It has been proposed that the muscle filament lattice adapts to longer lengths by placing more filaments in series and that lattice plasticity is facilitated by myosin filament evanescence, with filaments dissociating during relaxation and reforming upon activation. Support for these dynamic changes in the filament lattice has been provided partly by changes in contractile parameters at different times in the contraction-relaxation cycle at different lengths. If the changes in contractile parameters result from filament formation and dissociation, these structural changes must occur on the time scale of tension development and relaxation. To assess whether thick-filament formation could account for the contractile changes, we measured birefringence continuously during activation and relaxation and compared these optical changes with the time course of force development and relaxation. Birefringence is a well-known property of muscle; striations in skeletal and cardiac muscle result from the A-bands being anisotropic, i.e., birefringent, and it is now known that this optical property is due to the presence of myosin thick filaments in the A-bands. Thus, the strength of birefringence is expected to represent the density of thick filaments. Here, we describe the principle of the method, the techniques for recording the optical signals, some initial results, and discuss the interpretation of results and some limitations of the method.     

In vivo x-ray diffraction of indirect flight muscle from Drosophila melanogaster

Small-angle x-ray diffraction from isolated muscle preparations is commonly used to obtain time-resolved structural information during contraction. We extended this technique to the thoracic flight muscles of living fruit flies, at rest and during tethered flight. Precise measurements at 1-ms time resolution indicate that the myofilament lattice spacing does not change significantly during oscillatory contraction. This result is consistent with the notion that a net radial force maintains the thick filaments at an equilibrium interfilament spacing of approximately 56 nm throughout the contractile cycle. Transgenic flies with amino-acid substitutions in the conserved phosphorylation site of the myosin regulatory light chain (RLC) exhibit structural abnormalities that can explain their flight impairment. The I(20)/I(10) equatorial intensity ratio of the mutant fly is 35% less than that of wild type, supporting the hypothesis that myosin heads that lack phosphorylated RLC remain close to the thick filament backbone. This new experimental system facilitates investigation of the relation between molecular structure and muscle function in living organisms.     

Effects of hyperosmotic solutions on the filament lattice of intact frog skeletal muscle.

The effect of increasing the osmotic strength of the extracellular solution on the fifament lattice of living frog sartorius and semitendinosus muscle has been studied using low-angle x-ray diffraction to measure the lattice spacing. As the extracellular osmotic strength is increased, the filament lattice shrinks like an osmometer until a minimal spacing between the thick filaments is reached. This minimal spacing varies from 20 to 31 nm, depending on the sarcomere length. Further increase in the osmotic strength produces little further shrinkage. The osmotic shrinkage curve indicates, for both muscles, an osmotically-inactive volume of approximately 30% of the volume in normal Ringer's solution. Shrinkage appears to be independent of temperature and the type of particle used to increase the osmotic strength (glucose, sucrose, small ions). The rate at which osmotic equilibruim is reached depends on muscle size, being slower for greater muscle diameters. Equilibrium spacings are approached exponentially with time constants ranging from 20 to 60 min. Independent of osmotic equilibrium, the lattice tends to shrink slowly by approximately 3% over the first few hours after dissection, probably because of a leakage of K+ ions from inside the muscle cells. This can be partly prevented by using an extracellular solution which contains a higher concentration of K+ ions or which is hypoosmotic. The volume of the muscle filament lattice (1.155d10(2) . S) is constant over a very wide range of sarcomere lengths, and is equal to approximately 3.6 x 10(6) nm3 for a range of amphibian muscle types.     

Compliant realignment of binding sites in muscle: transient behavior and mechanical tuning.

The presence of compliance in the lattice of filaments in muscle raises a number of concerns about how one accounts for force generation in the context of the cross-bridge cycle--binding site motions and coupling between cross-bridges confound more traditional analyses. To explore these issues, we developed a spatially explicit, mechanochemical model of skeletal muscle contraction. With a simple three-state model of the cross-bridge cycle, we used a Monte Carlo simulation to compute the instantaneous balance of forces throughout the filament lattice, accounting for both thin and thick filament distortions in response to cross-bridge forces. This approach is compared to more traditional mass action kinetic models (in the form of coupled partial differential equations) that assume filament inextensibility. We also monitored instantaneous force generation, ATP utilization, and the dynamics of the cross-bridge cycle in simulations of step changes in length and variations in shortening velocity. Three critical results emerge from our analyses: 1) there is a significant realignment of actin-binding sites in response to cross-bridge forces, 2) this realignment recruits additional cross-bridge binding, and 3) we predict mechanical behaviors that are consistent with experimental results for velocity and length transients. Binding site realignment depends on the relative compliance of the filament lattice and cross-bridges, and within the measured range of these parameters, gives rise to a sharply tuned peak for force generation. Such mechanical tuning at the molecular level is the result of mechanical coupling between individual cross-bridges, mediated by thick filament deformations, and the resultant realignment of binding sites on the thin filament.     

The M-band: an elastic web that crosslinks thick filaments in the center of the sarcomere

The sarcomere of striated muscle is an efficient molecular machine, characterized by perfect structural organization of contractile filaments. This order is ensured by the sarcomere cytoskeleton, an important element of which is the M-band, believed to maintain the thick filament lattice. We review here recent progress in understanding the M-band function and its structural organization. We explain how the M-band might reduce the intrinsic instability of thick filaments and help titin to maintain order in the sarcomeres. The M-band molecular structure has been clarified recently by biochemical and biophysical approaches that focused on the properties of the prominent M-band component myomesin. These have shown that antiparallel myomesin dimers might link the thick filaments in the M-band, a role analogous to that of alpha-actinin in the Z-disc. Furthermore, similar to titin, myomesin is a molecular spring with complex visco-elastic properties that can be modified by alternative splicing. M-band protein composition correlates with the expression of titin isoforms and appears to be a reliable marker for biomechanical conditions in contracting muscle. We propose that the M-band is in fact a dynamic structure that monitors the stress appearing in the thick filament lattice during contraction and quickly reorganizes to meet new physiological requirements.     

The myosin filament: IX. Determination of subfilament positions by computer processing of electron micrographs

Computer image processing of electron micrographs has been employed to delineate the position of thick filament subunits in transverse sections of extensively crosslinked vertebrate skeletal muscle. Both back projection and rotational averaging methods indicate the presence of 12 subunits arranged on an approximately hexagonal lattice similar to that proposed by Pepe (1967). The spacing between subunits and the myosin content of the thick filament indicate that these subunits probably contain more than one myosin molecule and are most likely dimers.     

Sarcomere lattice geometry influences cooperative myosin binding in muscle

In muscle, force emerges from myosin binding with actin (forming a cross-bridge). This actomyosin binding depends upon myofilament geometry, kinetics of thin-filament Ca²⁺ activation, and kinetics of cross-bridge cycling. Binding occurs within a compliant network of protein filaments where there is mechanical coupling between myosins along the thick-filament backbone and between actin monomers along the thin filament. Such mechanical coupling precludes using ordinary differential equation models when examining the effects of lattice geometry, kinetics, or compliance on force production. This study uses two stochastically driven, spatially explicit models to predict levels of cross-bridge binding, force, thin-filament Ca²⁺ activation, and ATP utilization. One model incorporates the 2-to-1 ratio of thin to thick filaments of vertebrate striated muscle (multi-filament model), while the other comprises only one thick and one thin filament (two-filament model). Simulations comparing these models show that the multi-filament predictions of force, fractional cross-bridge binding, and cross-bridge turnover are more consistent with published experimental values. Furthermore, the values predicted by the multi-filament model are greater than those values predicted by the two-filament model. These increases are larger than the relative increase of potential inter-filament interactions in the multi-filament model versus the two-filament model. This amplification of coordinated cross-bridge binding and cycling indicates a mechanism of cooperativity that depends on sarcomere lattice geometry, specifically the ratio and arrangement of myofilaments.