Viscoelasticity of the sarcomere matrix of skeletal muscles. The titin-myosin composite filament is a dual-stage molecular spring.

The mechanical roles of sarcomere-associated cytoskeletal lattices were investigated by studying the resting tension-sarcomere length curves of mechanically skinned rabbit psoas muscle fibers over a wide range of sarcomere strain. Correlative immunoelectron microscopy of the elastic titin filaments of the endosarcomeric lattice revealed biphasic extensibility behaviors and provided a structural interpretation of the multiphasic tension-length curves. We propose that the reversible change of contour length of the extensible segment of titin between the Z line and the end of thick filaments underlies the exponential rise of resting tension. At and beyond an elastic limit near 3.8 microns, a portion of the anchored titin segment that adheres to thick filaments is released from the distal ends of thick filament. This increase in extensible length of titin results in a net length increase in the unstrained extensible segment, thereby lowering the stiffness of the fiber, lengthening the slack sarcomere length, and shifting the yield point in postyield sarcomeres. Thus, the titin-myosin composite filament behaves as a dual-stage molecular spring, consisting of an elastic connector segment for normal response and a longer latent segment that is recruited at and beyond the elastic limit of the sarcomere. Exosarcomeric intermediate filaments contribute to resting tension only above 4.5 microns. We conclude that the interlinked endo- and exosarcomeric lattices are both viscoelastic force-bearing elements. These distinct cytoskeletal lattices appear to operate over two ranges of sarcomere strains and collectively enable myofibrils to respond viscoelastically over a broad range of sarcomere and fiber lengths.     

Passive force generation and titin isoforms in mammalian skeletal muscle.

When relaxed striated muscle cells are stretched, a resting tension is produced which is thought to arise from stretching long, elastic filaments composed of titin (also called connectin). Here, I show that single skinned rabbit soleus muscle fibers produce resting tension that is several-fold lower than that found in rabbit psoas fibers. At sarcomere lengths where the slope of the resting tension-sarcomere length relation is low, electron microscopy of skinned fibers indicates that thick filaments move from the center to the side of the sarcomere during prolonged activation. As sarcomeres are stretched and the resting tension sarcomere length relation becomes steeper, this movement is decreased. The sarcomere length range over which thick filament movement decreases is higher in soleus than in psoas fibers, paralleling the different lengths at which the slope of the resting tension-sarcomere length relations increase. These results indicate that the large differences in resting tension between single psoas and soleus fibers are due to different tensions exerted by the elastic elements linking the end of each thick filament to the nearest Z-disc, i.e., the titin filaments. Quantitative gel electrophoresis of proteins from single muscle fibers excludes the possibility that resting tension is less in soleus than in psoas fibers simply because they have fewer titin filaments. A small difference in the electrophoretic mobility of titin between psoas and soleus fibers suggests the alternate possibility that mammalian muscle cells use at least two titin isoforms with differing elastic properties to produce variations in resting tension.     

The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments

Electron microscopy was used to study the positional stability of thick filaments in isometrically contracting skinned rabbit psoas muscle as a function of sarcomere length at 7 degrees C. After calcium activation at a sarcomere length of 2.6 micron, where resting stiffness is low, sarcomeres become nonuniform in length. The dispersion in sarcomere length is complete by the time maximum tension is reached. A-bands generally move from their central position and continue moving toward one of the Z-discs after tension has reached a plateau at its maximum level. The lengths of the thick and thin filaments remain constant during this movement. The extent of A-band movement during contraction depends on the final length of the individual sarcomere. After prolonged activation, all sarcomeres between 1.9 and 2.5 micron long exhibit A-bands that are adjacent to a Z-disc, with no intervening I-band. Sarcomeres 2.6 or 2.7 micron long exhibit a partial movement of A-bands. At longer sarcomere lengths, where the resting stiffness exceeds the slope of the active tension-length relation, the A-bands remain perfectly centered during contraction. Sarcomere symmetry and length uniformity are restored upon relaxation. These results indicate that the central position of the thick filaments in the resting sarcomere becomes unstable upon activation. In addition, they provide evidence that the elastic titin filaments, which join thick filaments to Z-discs, produce almost all of the resting tension in skinned rabbit psoas fibers and act to resist the movement of thick filaments away from the center of the sarcomere during contraction.     

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.     

Lattice shrinkage with increasing resting tension in stretched, single skinned fibers of frog muscle.

The 1,0 lattice spacing d1,0 in chemically and mechanically skinned single fibers of frog muscle was measured at various sarcomere lengths, L, in the range from L = 2.1 to 6.0 microns by an x-ray diffraction method. In chemically skinned fibers, d1,0 decreased with a similar slope to that of mechanically skinned fibers up to L congruent to 3 microns, but beyond this point d1,0 steeply decreased with further stretching. This steep decrease in d1,0 could be ascribed mainly to an increase in the compressing force associated with the longitudinal extension of a remnant of the sarcolemma. In mechanically skinned fibers, the gradual decrease in d1,0 continued beyond filament overlap (L greater than or equal to 3.5 microns) and was highly proportional to a resting tension. This decrease in d1,0 at L greater than or equal to 3.5 microns could be ascribed to an increase in the force exerted by lateral elastic components, which is proportional to the longitudinal resting tension. A conceptual model is proposed of a network structure of elastic components in a sarcomere.     

Stiffness of glycerinated rabbit psoas fibers in the rigor state. Filament-overlap relation

The stiffness of glycerinated rabbit psoas fibers in the rigor state was measured at various sarcomere lengths in order to determine the distribution of the sarcomere compliance between the cross-bridge and other structures. The stiffness was determined by measuring the tension increment at one end of a fiber segment while stretching the other end of the fiber. The contribution of the end compliance to the rigor segments was checked both by laser diffractometry of the sarcomere length change and by measuring the length dependence of the Young's modulus; the contribution was found to be small. The stiffness in the rigor state was constant at sarcomere lengths of 2.4 microns or less; at greater sarcomere lengths the stiffness, when corrected for the contribution of resting stiffness, scaled with the amount of overlap between the thick and thin filaments. These results suggest that the source of the sarcomere compliance of the rigor fiber at the full overlapping of filaments is mostly the cross-bridge compliance.     

Changes in the lateral filament spacing of skinned muscle fibres when cross-bridges attach

When a skinned fibre prepared from frog skeletal muscle goes from the relaxed to the rigor state at a sarcomere length of about 2.2 μm, the 1, 0 transverse spacing of the filament lattice, measured by X-ray diffraction, decreases by about 11%. In measurements at various sarcomere lengths, the decrease in the spacing was approximately proportional to the degree of overlap between the thick and thin filaments. This suggests that the shrinkage of the lattice is caused by a lateral force produced by cross-bridges. In order to estimate the magnitude of the lateral force, the decrease of spacing between relaxed and rigor states was compared with the shrinkage caused osmotically by adding a high molecular weight polymer, polyvinylpyrrolidone, to the bathing solution. The results indicate that the lateral force produced per unit length of thick filament in the overlap zone is of the same order of magnitude as the axially directed force produced during maximum isometric contraction (10^?10 to 10^?9 N/μm).Experiments in the presence of a high concentration of polyvinylpyrrolidone (100 g/l) show that when the lattice spacing is decreased osmotically beyond a certain value, the lateral force produced when the fibre goes into rigor changes its direction, causing the lattice to swell. This result can be explained by assuming that there is an optimum interfilament spacing at which the cross-bridges produce no lateral force. At other spacings, the lateral force tends to displace the filament lattice toward that optimum value.     

Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin

Muscle needs an elastic framework to maintain its mechanical stability. Removal of thin filaments in rabbit skeletal muscle with plasma gelsolin has revealed the essential features of elastic filaments. The selective removal of thin filaments was confirmed by staining with phalloidin-rhodamine for fluorescence microscopy, examination of arrowhead formation with myosin subfragment 1 by electron microscopy, and analysis by SDS-PAGE. Thin section electron microscopy revealed the elastic fine filaments (approximately 4 nm in diameter) connecting thick filaments and the Z line. After removal of thin filaments, both rigor stiffness and active tension generation were lost, but the resting tension remained. These observations indicate that the thin filament-free fibers maintain a framework composed of the serial connections of thick filaments, the elastic filaments, and the Z line, which gives passive elasticity to the contractile system of skeletal muscle. The resting tension that remained in the thin filament-free fibers was decreased by mild trypsin treatment. The only protein component that was digested in parallel with the decrease in the resting tension and the disappearance of the elastic filaments was alpha-connectin (also called titin 1), which was transformed from the alpha to the beta form (from titin 1 to 2, respectively). Thus, we conclude that the main protein component of the elastic filaments is alpha-connectin (titin 1).     

Mechanical Properties of the Sarcolemma and Myoplasm in Frog Muscle as a Function of Sarcomere Length

The elastimeter method was applied to the single muscle fiber of the frog semitendinosus to obtain the elastic moduli of the sarcolemma and myoplasm, as well as their relative contributions to resting fiber tension at different extensions. A bleb which was sucked into a flat-mouthed pipette at the fiber surface separated into an external sarcolemmal membrane and a thick inner myoplasmic region. Measurements showed that the sarcolemma does not contribute to intact fiber tension at sarcomere lengths below 3 µ. It was estimated that the sarcolemma contributed on the order of 10% to intact fiber tension at sarcomere lengths between 3 and 3.75 µ, and more so with further extension. Between these sarcomere lengths, the sarcolemma can be linearly extended and has a longitudinal elastic modulus of 5 x 10⁶ dyne/cm² (assuming a thickness of 0.1 µ). Resistance to deformation of the inner bleb region is due to myoplasmic elasticity. The myoplasmic elastic modulus was estimated by use of a model and was used to predict a fiber length-tension curve which agreed approximately with observations.     

Thick-filament strain and interfilament spacing in passive muscle: effect of titin-based passive tension

We studied the effect of titin-based passive tension on sarcomere structure by simultaneously measuring passive tension and low-angle x-ray diffraction patterns on passive fiber bundles from rabbit skinned psoas muscle. We used a stretch-hold-release protocol with measurement of x-ray diffraction patterns at various passive tension levels during the hold phase before and after passive stress relaxation. Measurements were performed in relaxing solution without and with dextran T-500 to compress the lattice toward physiological levels. The myofilament lattice spacing was measured in the A-band (d_1,0) and Z-disk (d_Z) regions of the sarcomere. The axial spacing of the thick-filament backbone was determined from the sixth myosin meridional reflection (M6) and the equilibrium positions of myosin heads from the fourth myosin layer line peak position and the I_1,1/I_1,0 intensity ratio. Total passive tension was measured during the x-ray experiments, and a differential extraction technique was used to determine the relations between collagen- and titin-based passive tension and sarcomere length. Within the employed range of sarcomere lengths (∼2.2–3.4 μm), titin accounted for >80% of passive tension. X-ray results indicate that titin compresses both the A-band and Z-disk lattice spacing with viscoelastic behavior when fibers are swollen after skinning, and elastic behavior when the lattice is reduced with dextran. Titin also increases the axial thick-filament spacing, M6, in an elastic manner in both the presence and absence of dextran. No changes were detected in either I_1,1/I_1,0 or the position of peaks on the fourth myosin layer line during passive stress relaxation. Passive tension and M6 measurements were converted to thick-filament compliance, yielding a value of ∼85 m/N, which is several-fold larger than the thick-filament compliance determined by others during the tetanic tension plateau of activated intact muscle. This difference can be explained by the fact that thick filaments are more compliant at low tension (passive muscle) than at high tension (tetanic tension). The implications of our findings are discussed.     

Resting Sarcomere Length-Tension Relation in Living Frog Heart

The sarcomere pattern and tension of isolated resting frog atrial trabeculae were continuously monitored. In the absence of any resting tension the sarcomere lengths varied with the diameter of the trabeculae. In over 75 % of the trabeculae the value exceeded 2.05 µm, the estimated in vivo length of the thin filaments, and it was never less than 1.89 µm. When the trabeculae were stretched the increase in length of the central undamaged portion could be completely accounted for by an increase in sarcomere length. The width of the A band was constant only at sarcomere lengths between 2.3 and 2.6 µm it decreased at smaller and increased at larger sarcomere lengths. A group of spontaneously active cells stretched the sarcomeres in cells in series to longer lengths than could be produced by passive tension applied to the ends of the trabeculae, but they did not influence the sarcomeres of adjacent cells. It is proposed that the connective tissue is a major factor in determining sarcomere length and that there are interactions between thick and thin filaments in resting muscles.     

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.     

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.     

Thick filament movement and isometric tension in activated skeletal muscle.

Thick filaments can move from the center of the sarcomere to the Z-disc while the isometric tension remains stable in skinned rabbit psoas fibers activated for several minutes (Horowits and Podolsky, 1987). Using the active and resting tension-length relations and the force-velocity relation, we calculated the time course and mechanical consequences of thick filament movement in the presence and absence of the elastic titin filaments, which link the ends of the thick filaments to the Z-discs and give rise to the resting tension. The calculated time course of thick filament movement exhibits a lag phase, during which the velocity and extent of movement are extremely small. This lag phase is dependent only on the properties of the cross-bridges and the initial position of the thick filament. The time course of thick filament movement in skinned rabbit psoas fibers at 7 degrees C is well fit assuming a small initial thick filament displacement away from the center of the sarcomere; this leads to a lag of approximately 80 s before any significant thick filament movement occurs. In the model incorporating titin filaments, this lag is followed by a phase of slow, steady motion during which isometric tension is stable. The model excluding titin filaments predicts a phase of acceleration accompanied by a 50% decrease in tension. The observed time course of movement and tension are consistent with the model incorporating titin filaments. The long lag phase suggests that in vivo, significant movement of thick filaments is unlikely to occur during a single contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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 effect of mepyramine on the development of morphine tolerance and physical dependence in mice.

Latency relaxation (LR) as well as resting tension and twitch tension of frog toe muscles are studied in an isotonic solution (= 1 T) and in solutions made hypotonic by leaving out the appropriate amounts of NaCl and KCl (0.54 T and 0.76 T). In hypotonic solutions there is an increase in peak twitch tension as well as a decrease in the depth of the LR: the resting tension is increased at sarcomere lengths which are greater than 2.8 μm and is decreased at sarcomere lengths which are less than this value. The behaviour of twitch tension is discussed with respect to the influence of the sarcoplasmic ionic strength on the interaction between the contractile filaments. Concerning the decrease in both the LR and the resting tension, it is assumed that these effects are induced osmotically, the tension of the membranes of the longitudinal sarcoplasmic reticulum being the particular parameter which is influenced.     

Disassembly kinetics of thick filaments in rabbit skeletal muscle fibers. Effects of ionic strength, Ca2+ concentration, pH, temperature, and cross-bridges on the stability of thick filament structure.

The kinetics of dissociation from both ends of thick filaments in a muscle fiber was investigated by an optical diffraction method. The dissociation velocity of thick filaments at a sarcomere length of 2.75 microns increased with increasing the KCl concentration (from 60 mM to 0.5 M), increasing the pH value (from 6.2 to 8.0) or decreasing the temperature (from 25 to 5 degrees C) in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed the dissociation velocity markedly at shorter sarcomere lengths. The dissociation velocity, v, decreased as thick filaments became shorter, and v = -db/dt = vo exp (alpha b), where b is the length of the thick filament at time t and vo and alpha are constants. The vo value was largely dependent on the KCl concentration but the alpha value was not. The stiffness of a muscle fiber decreased nearly in proportion to the decrease of overlap between thick and thin filaments induced by the dissociation of thick filaments. This indicates that cross-bridges are uniformly distributed and contribute independently to the stiffness of a muscle fiber during the dissociation of thick filaments.     

A mechanism for sarcomere breathing: volume change and advective flow within the myofilament lattice

During muscle contraction, myosin motors anchored to thick filaments bind to and slide actin thin filaments. These motors rely on energy derived from ATP, supplied, in part, by diffusion from the sarcoplasm to the interior of the lattice of actin and myosin filaments. The radial spacing of filaments in this lattice may change or remain constant during contraction. If the lattice is isovolumetric, it must expand when the muscle shortens. If, however, the spacing is constant or has a different pattern of axial and radial motion, then the lattice changes volume during contraction, driving fluid motion and assisting in the transport of molecules between the contractile lattice and the surrounding intracellular space. We first create an advective-diffusive-reaction flow model and show that the flow into and out of the sarcomere lattice would be significant in the absence of lattice expansion. Advective transport coupled to diffusion has the potential to substantially enhance metabolite exchange within the crowded sarcomere. Using time-resolved x-ray diffraction of contracting muscle, we next show that the contractile lattice is neither isovolumetric nor constant in spacing. Instead, lattice spacing is time varying, depends on activation, and can manifest as an effective time-varying Poisson ratio. The resulting fluid flow in the sarcomere lattice of synchronous insect flight muscles is even greater than expected for constant lattice spacing conditions. Lattice spacing depends on a variety of factors that produce radial force, including cross-bridges, titin-like molecules, and other structural proteins. Volume change and advective transport varies with the phase of muscle stimulation during periodic contraction but remains significant at all conditions. Although varying in magnitude, advective transport will occur in all cases in which the sarcomere is not isovolumetric. Akin to “breathing,” advective-diffusive transport in sarcomeres is sufficient to promote metabolite exchange and may play a role in the regulation of contraction itself.     

Passive and active tension in single cardiac myofibrils.

Single myofibrils were isolated from chemically skinned rabbit heart and mounted in an apparatus described previously (Fearn et al., 1993; Linke et al., 1993). We measured the passive length-tension relation and active isometric force, both normalized to cross sectional area. Myofibrillar cross sectional area was calculated based on measurements of myofibril diameter from both phase-contrast images and electron micrographs. Passive tension values up to sarcomere lengths of approximately 2.2 microns were similar to those reported in larger cardiac muscle specimens. Thus, the element responsible for most, if not all, passive force of cardiac muscle at physiological sarcomere lengths appears to reside within the myofibrils. Above 2.2 microns, passive tension continued to rise, but not as steeply as reported in multicellular preparations. Apparently, structures other than the myofibrils become increasingly important in determining the magnitude of passive tension at these stretched lengths. Knowing the myofibrillar component of passive tension allowed us to infer the stress-strain relation of titin, the polypeptide thought to support passive force in the sarcomere. The elastic modulus of titin is 3.5 x 10(6) dyn cm-2, a value similar to that reported for elastin. Maximum active isometric tension in the single myofibril at sarcomere lengths of 2.1-2.3 microns was 145 +/- 35 mN/mm2 (mean +/- SD; n = 15). This value is comparable with that measured in fixed-end contractions of larger cardiac specimens, when the amount of nonmyofibrillar space in those preparations is considered. However, it is about 4 times lower than the maximum active tension previously measured in single skeletal myofibrils under similar conditions (Bartoo et al., 1993).