Structural states in the Z band of skeletal muscle correlate with states of active and passive tension

In skeletal muscle Z bands, the ends of the thin contractile filaments interdigitate in a tetragonal array of axial filaments held together by periodically cross-connecting Z filaments. Changes in these two sets of filaments are responsible for two distinct structural states observed in cross section, the small-square and basketweave forms. We have examined Z bands and A bands in relaxed, tetanized, stretched, and stretched and tetanized rat soleus muscles by electron microscopy and optical diffraction. In relaxed muscle, the A-band spacing decreases with increasing load and sarcomere length, but the Z lattice remains in the small-square form and the Z spacing changes only slightly. In tetanized muscle at sarcomere lengths up to 2.7 micron, the Z lattice assumes the basketweave form and the Z spacing is increased. The increased Z spacing is not the result of sarcomere shortening. Further, passive tension is not sufficient to cause this change in the Z lattice; active tension is necessary.     

Three-dimensional structure of the Z band in a normal mammalian skeletal muscle

The three-dimensional structure of the vertebrate skeletal muscle Z band reflects its function as the muscle component essential for tension transmission between successive sarcomeres. We have investigated this structure as well as that of the nearby I band in a normal, unstimulated mammalian skeletal muscle by tomographic three- dimensional reconstruction from electron micrograph tilt series of sectioned tissue. The three-dimensional Z band structure consists of interdigitating axial filaments from opposite sarcomeres connected every 18 +/- 12 nm (mean +/- SD) to one to four cross-connecting Z- filaments are observed to meet the axial filaments in a fourfold symmetric arrangement. The substantial variation in the spacing between cross-connecting Z-filament to axial filament connection points suggests that the structure of the Z band is not determined solely by the arrangement of alpha-actinin to actin-binding sites along the axial filament. The cross-connecting filaments bind to or form a "relaxed interconnecting body" halfway between the axial filaments. This filamentous body is parallel to the Z band axial filaments and is observed to play an essential role in generating the small square lattice pattern seen in electron micrographs of unstimulated muscle cross sections. This structure is absent in cross section of the Z band from muscles fixed in rigor or in tetanus, suggesting that the Z band lattice must undergo dynamic rearrangement concomitant with crossbridge binding in the A band.     

The Z-band lattice in skeletal muscle in rigor

Previous work with tetanized and relaxed muscle has shown a correlation between active tension and the structure of the Z-band. This suggests that there is a correlation between the cross-bridge binding in the A-band and the structure of the Z-band. Using electron microscopy and optical diffraction we have examined this correlation in glycerinated muscle in rigor and in unstimulated intact muscle. We have found that the Z-bands of muscles in rigor always show the basketweave form, while those of the unstimulated muscles always show the small square form. The basketweave form found in rigor muscles is similar in form and dimension to that found in tetanized muscle. Thus it appears that the small square form of the Z-band is found in physiological states with little cross-bridge binding and the basketweave form is found in states with a high degree of cross-bridge binding.     

Time-resolved changes in equatorial x-ray diffraction and stiffness during rise of tetanic tension in intact length-clamped single muscle fibers.

We report the first time-resolved x-ray diffraction studies on tetanized intact single muscle fibers of the frog. The 10, 11, 20, 21, 30, and Z equatorial reflections were clearly resolved in the relaxed fiber. The preparation readily withstood 100 1-s duration (0.4-s beam exposure) tetani at 4 degrees C (less than 4% decline of force and no deterioration in the 10, 11 equatorial intensity ratio at rest or during activation). Equatorial intensity changes (10 and 11) and fiber stiffness led tension (t1/2 lead 20 ms at 4 degrees C) during the tetanus rise and lagged during the isometric phase of relaxation. These findings support the existence of a low force cross-bridge state during the rise of tetanic tension and isometric relaxation that is not evident at the tetanus plateau. In "fixed end" tetani lattice expansion occurred with a time course similar to stiffness during the tetanus rise. During relaxation, lattice spacing increased slightly, while the sarcomere length remained isometric, but underwent large changes after the "shoulder" of tension. Under length clamp control, lattice expansion during the tetanus rise was reduced or abolished, and compression (2%) of the lattice was observed. A lattice compression is predicted by certain cross-bridge models of force generation (Schoenberg, M. 1980. Biophys. J. 30:51-68; Schoenberg, M. 1980. Biophys. J. 30:69-78).     

The relation between Z--disk lattice spacing and sarcomere length in sartorius muscle fibres from Hyla cerulea.

Sartorius muscles from the green tree frog Hyla cerulea were set at variety of muscle lengths and fixed for electron microscopy using acrolein followed by osmium tetroxide. The sarcomere length, s, was determined in thick sections using laser-diffraction. The Z-disk lattice spacing, z, was measured in electron micrographs of thin sections from the same muscles. The Z-disk lattice was found to expand as sarcomere length decreased such that the quantity sz2 was constant at 1-05 X 10(6) nm3 for all sarcomere lengths in the range 1-9-2-9 mum. Thus, the sarcomere length dependency of the Z-disk lattice is similar to that of the myosin filament lattice. The density of thin filaments per unit cross section of fibril leaving the Z-disk is less than their density in the A band. Thus, fibrils have a smaller cross section in the I band, leaving more inter-fibrillar space there. This may explain why more mitochondria and lipid droplets are located in the I bands than in the A bands. It is suggested that the Z-disk may contributed to the short range elasticity of muscle fibres.     

Lattice swelling with the selective digestion of elastic components in single-skinned fibers of frog muscle.

Changes in the 1.0 lattice spacing during trypsin (0.25 micrograms/ml) treatment in mechanically skinned single fibers of frog muscle was examined by an x-ray diffraction method at various sarcomere lengths. The resting tension of a relaxed fiber was decreased by trypsin treatment but the stiffness of a rigor fiber was not, suggesting that elastic components were selectively digested. With progression of the digestion, the lattice spacing increased remarkably at longer sarcomere lengths and finally became independent of the sarcomere length. The increase in the lattice spacing was proportional to the decrease in the resting tension. These results support our previous suggestion (Higuchi, H., and Y. Umazume, 1986, Biophys. J., 50:385-389) that the lattice spacing decreases at long lengths due to compressive force exerted by a lateral elastic component that connects thick filaments to an axial elastic component. Consequently, it is unlikely that the decrease in the lattice spacing is determined by a decrease in the repulsive force between thick and thin filaments with stretching a fiber.     

Lattice spacing changes accompanying isometric tension development in intact single muscle fibers.

The myosin lattice spacing of single intact muscle fibers of the frog, Rana temporaria, was studied in Ringer's solution (standard osmolarity 230 mOsm) and hyper- and hypotonic salines (1.4 and 0.8 times standard osmolarity respectively) in the relaxed state, during "fixed end" tetani, and during shortening, using synchrotron radiation. At standard tonicity, a tetanus was associated with an initial brief lattice expansion (and a small amount of sarcomere shortening), followed by a slow compression (unaccompanied by sarcomere length changes). In hypertonic saline (myosin lattice compressed by 8.1%), these spacing changes were suppressed, in hypotonic saline (lattice spacing increased by 7.5%), they were enhanced. During unloaded shortening of activated fibers, a rapid lattice expansion occurred at all tonicities, but became larger as tonicity was reduced. This expansion was caused in part by the change in length of the preparation, but also by a recoil of a stressed radial compliance associated with axial force. The lattice spacing during unloaded shortening was equal to or occasionally greater than predicted for a relaxed fiber at that sarcomere length, indicating that the lattice compression associated with activation is rapidly reversed upon loss of axial force. Lattice recompression occurred upon termination of shortening under standard and hypotonic conditions, but was almost absent under hypertonic conditions. These observations indicate that axial cross-bridge tension is associated with a compressive radial force in intact muscle fibers at full overlap; however, this radial force exhibits a much greater sensitivity to lattice spacing than does the axial force.     

X-ray evidence for radial cross-bridge movement and for the sliding filament model in actively contracting skeletal muscle

Equatorial X-ray diffraction patterns have been studied from muscles at rest, during contraction and in rigor. It is confirmed that the relative intensity ($\text{I}\text{1,0}\text{I}\text{1,1}$) of the two main equatorial reflections depends both on the sarcomere length and on the state of the muscle; in any one state the ratio $\text{I}\text{1,0}\text{I}\text{1,1}$ increases as the sarcomere length of the muscle increases, while at any fixed sarcomere length the ratio is smaller for contracting muscle than for resting muscle and smaller still for rigor muscles. The change of $\text{I}\text{1,0}\text{I}\text{1,1}$ with change of state at constant sarcomere length is interpreted as being due to radial movement of cross-bridges: the average movement during contraction being about 40% of that in rigor.Over the whole range of sarcomere length studied (between 1.8 and 2.7 μm) there was no evidence for any change in lattice spacing when a muscle contracts isometrically.Muscles were studied generating tension after they had shortened actively against a load. The lattice spacings and intensity ratio $\text{I}\text{1,0}\text{I}\text{1,1}$ both changed during active shortening in a way entirely consistent with the sliding filament theory of contraction.     

Effects of the number of actin-bound S1 and axial force on X-ray patterns of intact skeletal muscle

Effects of the number of actin-bound S1 and of axial tension on x-ray patterns from tetanized, intact skeletal muscle fibers were investigated. The muscle relaxant, BDM, reduced tetanic M3 meridional x-ray reflection intensity (I(M3)), M3 spacing (d(M3)), and the equatorial I(11)/I(10) ratio in a manner consistent with a reduction in the fraction of S1 bound to actin rather than by generation of low-force S1-actin isomers. At complete force suppression, I(M3) was 78% of its relaxed value. BDM distorted dynamic I(M3) responses to sinusoidal length oscillations in a manner consistent with an increased cross-bridge contribution to total sarcomere compliance, rather than a changed S1 lever orientation in BDM. When the number of actin-bound S1 was varied by altering myofilament overlap, tetanic I(M3) at low overlap was similar to that in high [BDM] (79% of relaxed I(M3)). Tetanic d(M3) dependence on active tension in overlap experiments differed from that observed with BDM. At high BDM, tetanic d(M3) approached its relaxed value (14.34 nm), whereas tetanic d(M3) at low overlap was 14.50 nm, close to its value at full overlap (14.56 nm). This difference in tetanic d(M3) behavior was explicable by a nonlinear thick filament compliance which is extended by both active and passive tension.     

A comparative study of the supramolecular structure of frog sartorius and dorsal semitendinosus muscle

This report describes a comparative X-ray diffraction study of the supramolecular structure of frog sartorius and semitendinosus muscles. For sarcomere lengths of 2.7 microns and below the X-ray diffraction diagrams of each muscle type are very similar; the only differences being that the diffraction diagram for semitendinosus muscles exhibit the presence of a broad diffraction band or a cluster of diffraction orders at a spacing of ca. 230.0 nm and, also, they lack a periodicity of ca. 102.0 nm. For sarcomere lengths greater than 2.7 microns disruption of the sarcomere from sartorius muscle occurs as seen by the loss of sampling in the diffraction diagram. The semitendinosus muscle can be stretched to much longer lengths (in excess of 3.0 microns) before a loss of sampling is detected. The data also shows that in the case of the semitendinosus muscle for long sarcomere lengths transverse bands of mass are able to move without retaining a defined distance to either the Z or the M lines. This is not observed in the case of the sartorius muscle. Thus, at resolutions between ca. 3.6 microns and 7.50 nm significant ultrastructural differences between these two muscles are apparent. The data suggest that the ability of these mass bands to move may be responsible for the differences in the development of passive tension exhibited by these two muscles.     

Time-resolved equatorial X-ray diffraction studies of skinned muscle fibres during stretch and release.

Equatorial X-ray diffraction patterns were recorded from small bundles of one to three chemically skinned frog sartorius muscle fibres (time resolution 250 microseconds) during rapid stretch and subsequent release. In the relaxed state, the dynamic A-band lattice spacing change as a result of a 2 % step stretch (determined from the positions of the 10 and 11 reflections) resulted in a 21 % increase in lattice volume, while static studies of spacing and sarcomere length indicated than an increase in volume of >/=50 % for the same length change. In rigor, stretch caused a lattice volume decrease which was reversed by a subsequent release. In activated fibres (pCa 4.5) exposed to 10 mM 2,3-butanedione 2-monoxime (BDM), stretch was accompanied by a lattice compression exceeding that of constant volume behaviour, but during tension recovery, compression was partially reversed to leave a net spacing change close to that observed in the relaxed fibre. In the relaxed state, spacing changes were correlated with the amplitude of the length step, while in rigor and BDM states, spacing changes correlated more closely with axial force. This behaviour is explicable in terms of two components of radial force, one due to structural constraints as seen in the relaxed state, and an additional component arising from cross-bridge formation. The ratio of axial to radial force for a single thick filament resulting from a length step was four in rigor and BDM, but close to unity for the relaxed state.     

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 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.     

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.     

Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle

Connectin (also called titin) is a huge, striated muscle protein that binds to thick filaments and links them to the Z-disc. Using an mAb that binds to connectin in the I-band region of the molecule, we studied the behavior of connectin in both relaxed and activated skinned rabbit psoas fibers by immunoelectron microscopy. In relaxed fibers, antibody binding is visualized as two extra striations per sarcomere arranged symmetrically about the M-line. These striations move away from both the nearest Z-disc and the thick filaments when the sarcomere is stretched, confirming the elastic behavior of connectin within the I- band of relaxed sarcomeres as previously observed by several investigators. When the fiber is activated, thick filaments in sarcomeres shorter than 2.8 microns tend to move from the center to the side of the sarcomere. This translocation of thick filaments within the sarcomere is accompanied by movement of the antibody label in the same direction. In that half-sarcomere in which the thick filaments move away from the Z-disc, the spacings between the Z-disc and the antibody and between the antibody and the thick filaments both increase. Conversely, on the side of the sarcomere in which the thick filaments move nearer to the Z-line, these spacings decrease. Regardless of whether I-band spacing is varied by stretch of a relaxed sarcomere or by active sliding of thick filaments within a sarcomere of constant length, the spacings between the Z-line and the antibody and between the antibody and the thick filaments increase with I-band length identically. These results indicate that the connectin filaments remain bound to the thick filaments in active fibers, and that the elastic properties of connectin are unaltered by calcium ions and cross-bridge activity.     

Effects of pressure on equatorial x-ray fiber diffraction from skeletal muscle fibers.

When skeletal muscle fibers are subjected to a hydrostatic pressure of 10 MPa (100 atmospheres), reversible changes in tension occur. Passive tension from relaxed muscle is unaffected, rigor tension rises, and active tension falls. The effects of pressure on muscle structure are unknown: therefore a pressure-resistant cell for x-ray diffraction has been built, and this paper reports the first study of the low-angle equatorial patterns of pressurized relaxed, rigor, and active muscle fibers, with direct comparisons from the same chemically skinned rabbit psoas muscle fibers at 0.1 and 10 MPa. Relaxed and rigor fibers show little change in the intensity of the equatorial reflections when pressurized to 10 MPa, but there is a small, reversible expansion of the lattice of 0.7 and 0.4%, respectively. This shows that the order and stability of the myofilament lattice is undisturbed by this pressure. The rise in rigor tension under pressure is thus probably due to axial shortening of one or more components of the sarcomere. Initial results from active fibers at 0.1 MPa show that when phosphate is added the lattice spacing and equatorial intensities change toward their relaxed values. This indicates cross-bridge detachment, as expected from the reduction in tension that phosphate induces. 10 MPa in the presence of phosphate at 11 degrees C causes tension to fall by a further 12%, but not change is detected in the relative intensity of the reflections, only a small increase in lattice spacing. Thus pressure appears to increase the proportion of attached cross-bridges in a low-force state.     

Sarcomere formation and longitudinal growth in the developing flight muscles of Calliphora

New sarcomere formation and length changes in sarcomeres have been investigated in the sixth dorsal longitudinal flight muscles in puparia and newly-emerged adults of Calliphora vomitoria. The hypotheses are investigated that new sarcomeres are formed during a period of rapid longitudinal growth by either Z band division or by serial addition at the ends of the muscles. At about 3 days and 9 hr after puparium formation, when the muscles are just beginning their longitudinal growth, the Z bands in existing sarcomeres appear to divide throughout the muscles. Calculations indicate that the number of sarcomeres quadruple. By 3 days and 15 hr the final number of sarcomeres is formed. Thereafter length increases in the sarcomeres account for length increases in the muscles. Sarcomere lengthening can account for a 26% increase in muscle length over the course of adult emergence. [¹⁴C] Leucine incorporation into proteins is equally distributed throughout the muscles at 3 days and 9 hr supporting the hypothesis that the new sarcomeres are formed throughout the muscles. [¹⁴C] Adenosine similarly shows no concentration of incorporation. Guide cells at the ends of the muscles appear to be pulling on the muscles. It is suggested that the tension from the guide cells is inducing the Z band division and the length increases of the sarcomeres. If the guide cells are cut, the muscles collapse and no longer increase in length.     

The length–tension curve in muscle depends on lattice spacing

Classic interpretations of the striated muscle length–tension curve focus on how force varies with overlap of thin (actin) and thick (myosin) filaments. New models of sarcomere geometry and experiments with skinned synchronous insect flight muscle suggest that changes in the radial distance between the actin and myosin filaments, the filament lattice spacing, are responsible for between 20% and 50% of the change in force seen between sarcomere lengths of 1.4 and 3.4 µm. Thus, lattice spacing is a significant force regulator, increasing the slope of muscle''s force–length dependence.     

X-ray diffraction observations of chemically skinned frog skeletal muscle processed by an improved method.

Whole frog sartorius muscles can be chemically skinned in approximately 2 h by relaxing solutions containing 0.5% Triton X-100. The intensity and order of the X-ray diffraction pattern from living muscle is largely retained after such skinning, indicating good retention of native structure in fibrils and filaments. Best X-ray results were obtained using a solution with (mM): 75 K acetate; 5 Mg acetate; 5 ATP; 5 EGTA; 15 K phosphate, 2% PVP, pH 7.0. Equatorial X-ray patterns showed that myofibrils swell after detergent skinning, as also observed after mechanical skinning. This swelling could be reversed by adding high molecular weight colloids (PVP or dextran) to the extracting solution. By finding the colloid osmotic pressure needed to restore the in vivo interfilament spacing (3% PVP, 4 X 10(4) mol wt) the swelling pressure was estimated as 35 Torr in a standard KCl-based relaxing solution. The swelling pressure and the extent of swelling were less than acetate replaced chloride as the major anion. Detergent-skinned muscle lost the constant-volume relation between sarcomere length and lattice spacing seen in intact muscle. Changes in A band spacing were paralleled by changes in I and band-Z line spacing at a constant sarcomere length. After detergent skinning, I1,0 rose while I1,1 fell, a change in the relaxing direction. Since raising the calcium ion concentrations from pCa 9 to PCa 6.7 was without effect on equatorial or axial X-ray patterns, we concluded that these intensity changes were not due to calcium-dependent cross-bridge movement but rather to disordering of thin filaments in the A band.     

The T-axial membrane system in striated muscles of the horseshoe crab

Cardiac muscle and skeletal muscles powering walking leg, tailspine and gill movements in Limulus polyphemus were studied by transmission electron microscopy. All muscles examined had extensive invaginations of the sarcolemma at the Z disc and at the lateral margins of the A band. Invaginations at the Z disc were often branched in the transverse and longitudinal planes, but branching was not observed at other locations within a sarcomere. Dyads, and occasional triads, were observed at the A band in all muscles examined.