Z/I and A-band lattice spacings in frog skeletal muscle: effects of contraction and osmolarity.

A-band and Z-line/I-band lattice spacings were measured by small-angle X-ray diffraction from relaxed and isometrically-contracting whole frog sartorius muscles with lattice spacings reduced or swollen by changing the osmolarity of the bathing solution. A-band spacing increased by approximately 3% upon isometric contraction at reduced lattice spacings (245-356 mOsm) and decreased by approximately 1% at swollen spacings (172 mOsm), similarly to the behaviour of skinned muscles upon changing from the relaxed state to rigor. The Z/I lattice underwent a significant lattice expansion (3-8%) upon isometric contraction at all osmolarities, in qualitative agreement (but quantitative disagreement) with results from electron microscopy on mammalian skeletal muscle. Lattice areas calculated for the Z/I and A-band lattices indicate a barrel-shaped sarcomere in the resting state, which may provide a partial explanation for how longitudinal forces produced in the A-band can produce a radial expansive force in the Z-line during contraction. The radial component of cross-bridge stiffness was calculated from the A-band data for contracting muscle, using a lattice stability model incorporating structural, osmotic and electrostatic forces. The calculations gave a radial cross-bridge stiffness during contraction of about 9 x 10(5) N m-2, and outward radial force per thick filament in normal Ringer's solution of 6 x 10(-9) N, corresponding to a radial force per cross-bridge of 10(-11) N.

The filament lattice of striated muscle.

The filament lattice of striated muscle is an overlapping hexagonal array of thick and thin filaments within which muscle contraction takes place. Its structure can be studied by electron microscopy or X-ray diffraction. With the latter technique, structural changes can be monitored during contraction and other physiological conditions. The lattice of intact muscle fibers can change size through osmotic swelling or shrinking or by changing the sarcomere length of the muscle. Similarly, muscle fibers that have been chemically or mechanically skinned can be compressed with bathing solutions containing very large inert polymeric molecules. The effects of lattice change on muscle contraction in vertebrate skeletal and cardiac muscle and in invertebrate striated muscle are reviewed. The force developed, the speed of shortening, and stiffness are compared with structural changes occurring within the lattice. Radial forces between the filaments in the lattice, which can include electrostatic, Van der Waals, entropic, structural, and cross bridge, are assessed for their contributions to lattice stability and to the contraction process.

Z-line/I-band and A-band lattices of intact frog sartorius muscle at altered interfilament spacing.

Muscle contraction has long been known to be affected by the osmolarity of the bathing solution. Part of this effect is caused by changes in interfilament spacing in the A-band. We have investigated the variation in spacing of the square lattice of thin filaments within and near the Z-line (the Z-line/I-band or Z-I lattice) in intact frog sartorius muscle over a wide range of osmolarities and compared it with the corresponding changes in the A-band lattice. Both lattices have a lower limit for compression and an upper limit for swelling. The spacing of the Z-I lattice is nearly proportional to that of the A-band, but shows a 2-3% variation at extreme shrinkage or swelling. In normal intact muscle, the osmotically-inactive volume of both lattices is between 20 and 30%. These in vivo measurements of lattice spacing differ significantly from those observed in electron micrographs. With moderate variations in osmolarity, lattice spacing and muscle fibre width show similar behaviour, but at extreme osmolarities, the lattice spacing changes less than the fibre width. An equatorial reflection was observed in intact muscle, previously identified in skinned muscle, which does not index on the A-band and which changes with osmolarity in a manner different from that observed for the A-band and Z-I lattices. This reflection may arise from changes in the ordering of the Z-I lattice or may involve components additional to the thick and thin filaments.

Changes in thick filament structure during compression of the filament lattice in relaxed frog sartorius muscle.

Equatorial X-ray diffraction patterns from relaxed, chemically-skinned frog sartorius muscles under a range of external osmotic pressures from 0 to 290 torr have been analysed and compared to the pattern from the relaxed intact muscle. Lattice spacings and electron density diagrams were determined as a function of external pressure. Reflection intensities, averaged over small ranges of pressures, were determined out to the 4,0 reflection; phases for the first five orders were established as ++--+ over the whole pressure range. As external pressure was increased, lattice spacing decreased, as did full width at half maximum density for both thick and thin filaments. Most of the lattice spacing and thick filament compression occurred at low pressure, whereas thin filaments were compressed proportionally to pressure over the whole pressure range. These conclusions were confirmed by fitting cylindrical models for filament density to the X-ray diffraction patterns. Axially-projected electron density across the A-band filament lattice showed that in relaxed muscle the thick filament projections (myosin heads) are concentrated in regions between adjacent thick filaments, as far as possible from the thin filaments, and they tend to become pushed against the thick filament backbone as the lattice is compressed. Both thick and thin filament axes can be displaced randomly from their lattice positions; on average this displacement is about twice as great for the thin filaments, accounting for their larger projected size as compared to isolated thin filaments and for their apparent decrease in diameter as the lattice is compressed.

Fluid distribution in pork, measured by x-ray diffraction, interference microscopy and centrifugation compared to paleness measured by fiber optics.

Moderately PSE (pale, soft, exudative) and moderately DFD (dark, firm, dry) pork was examined by x-ray diffraction for interfilament separation, by differential interference contrast microscopy for interfiber area, and was centrifuged to measure water holding capacity (WHC). Internal reflectance spectra were measured by fiber optics. For PSE to DFD pork, filament separation ranged from 39 to 48 nm, interfiber area from 42 to 3%, and WHC from 49 to 64%, respectively. The correlation of reflectance with interfilament separation varied considerably with wavelength (reaching r = -.83 at 680 nm, P less than .005). The correlation of reflectance with interfiber area was more uniform across the spectrum (reaching r = .90 at 450 nm, P less than .005), as was the correlation of reflectance with WHC (reaching r = -.80 at 400 nm, P less than .005). At 24 h postmortem, fiber-optic spectrophotometry may be used as a rapid, nondestructive method to predict WHC and potential fluid losses from commercial pork with a moderate range from PSE to DFD. Interfiber area was correlated negatively with filament lattice area and WHC, but no significant correlation was found between filament lattice area and WHC. Filament separation was decreased only slightly by centrifugation. These results indicate that at 24 h postmortem the extra fluid released from PSE pork already has been lost from the myofilament lattice and is awaiting release from compartments downstream such as interfiber and interfascicular spaces.

Orientation of alpha-helical peptides in a lipid bilayer.

Samples of pure lipid (dipalmitoylphosphatidylcholine) and lipid containing short alpha-helical peptides were oriented and examined by X-ray diffraction, together with unoriented samples of pure peptide. X-ray reflections from the bilayer and the alpha-helices showed that the peptides had oriented in the bilayer with their helical axes perpendicular to the surface.

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.

X-ray diffraction measurements of postmortem changes in the myofilament lattice of pork.

Postmortem changes in the lateral spacing between filaments of the longissimus muscle in pork were examined by small-angle x-ray diffraction. Samples that were fixed in glutaraldehyde as soon as they were collected showed a rapid decrease in filament spacing from 1 h to 3 h and then a further, slower decrease to 24 h. Samples that were examined immediately or were kept prior to examination in buffered Ringer's solutions at pH values similar to those expected in the carcass showed a rapid decrease in filament spacing from 1 h to 3 h and then little further change to 24 h. In contrast, samples taken at various times postmortem and stored in Ringer's solutions at pH 7.2 for several hours before examination showed little postmortem change in lattice spacing. Fixed samples showed similar changes to those of unfixed samples, but the lattice spacing always was less in fixed than in unfixed samples. These results support the classic theory that much of the water that may be lost by drip and evaporation from meat originates from the spaces between the filaments. The major factor that caused shrinkage of the filament lattice and loss of water from the fibrils was pH.

Interrod forces in aqueous gels of tobacco mosaic virus.

The lateral separation of virus rod particles of tobacco mosaic virus has been studied as a function of externally applied osmotic pressure using an osmotic stress technique. The results have been used to test the assumption that lattice equilibrium in such gels results from a balance between repulsive (electrostatic) and attractive (van der Waals and osmotic) forces. Results have been obtained at different ionic strengths (0.001 to 1.0 M) and pH's (5.0 to 7.2) and compared with calculated curves for electrostatic nad van der Waals pressure. Under all conditions studied, interrod spacing decreased with increasing applied pressure, the spacings being smaller at higher ionic strengths. Only small differences were seen when the pH was changed. At ionic strengths near 0.1 M, agreement between theory and experiment is good, but the theory appears to underestimate electrostatic forces at high ionic strengths and to underestimate attractive forces at large interrod spacings (low ionic strengths). It is concluded that an electrostatic-van der Waals force balance can explain stability in tobacco mosaic virus gels near physiological conditions and can provide a good first approximation elsewhere.

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.

X-ray diffraction from striated muscles and nerves in normal and dystrophic mice.

The structure of striated muscle (thick and thin filaments, filament lattice, and collagen), peripheral nerve myelin, and tendon collagen were studied in tissues from dystrophic and normal mice using small-angle x-ray diffraction. There were increases in the amount of disorganized tissue in the dystrophic mice, and the time course of the changes was monitored over the first 42 weeks of life. As the dystrophic mice became older, the contractile apparatus of the muscles appeared to atrophy, while the amount of collagen increased. In general, the molecular structure and packing appeared to remain unchanged as the disease progressed, although changes in the relative amounts and the organization of proteins were noted. In both normal and dystrophic mice, the collagen periodicity (65.7 nm) was 2% smaller when detected in muscle tissue compared with that detected in tendon tissue.

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.

Electrostatic forces in muscle and cylindrical gel systems.

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.

An x-ray diffraction study of contracting molluscan smooth muscle.

The living anterior byssus retractor muscle of Mytilus (ABRM), a smooth, "catch" muscle, has been studied by X-ray diffraction while relaxed and while tonically contracted. X-ray reflections were observed from the actin and paramyosin filaments and from the alpha-helical substructure of the paramyosin filaments. No differences in spacings or relative intensities were observed when the relaxed and contracting muscle patterns were compared. This result is consistent with a sliding filament mechanism involving an interaction between actin and paramyosin filaments.