Changes in thick filament length in Limulus striated muscle

Here we describe the change in thick filament length in striated muscle of Limulus, the horseshoe crab. Long thick filaments (4.0 microns) are isolated from living, unstimulated Limulus striated muscle while those isolated from either electrically or K+-stimulated fibers are significantly shorter (3.1 microns) (P less than 0.001). Filaments isolated from muscle glycerinated at long sarcomere lengths are long (4.4 microns) while those isolated from muscle glycerinated at short sarcomere lengths are short (2.9 microns) and the difference is significant (P less than 0.001). Thin filaments are 2.4 microns in length. The shortening of thick filaments is related to the wide range of sarcomere lengths exhibited by Limulus telson striated muscle.     

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.     

Unusual thick and thin filament packing in a crustacean muscle

The proximal accessory flexor (PAF) of the myochordotonal organ (MCO) in the meropodite of crayfish walking legs contains two populations of muscle fibers which are distinguishable by their diameters. The large accessory (LA) fibers are 40-80 micrometer in diam and are similar in ultrastructure to other slow crustacean fibers. The small accessory (SA) fibers are 1-12 micrometer in diam and have a unique myofilament distribution at normal body lengths. There is extensive double overlap of thin filaments at these lengths, and some of them form bundles that may extend the length of the sarcomere. In the middle of the sarcomeres, thick and thin filaments are totally segregated from each other. When the fibers are stretched to lengths beyond double overlap length, the myofilament patterns are conventional. The segregated pattern is reestablished when stretched fibers are allowed to shorten passively. The length-tension relationship of the SA fibers is described by a linear ascending branch, a plateau, and a linear descending branch. The ascending branch encompasses normal body lengths from slack length (Ls) with maximum double overlap to the length at which double overlap ceases (1.8 X Ls). The descending phase is comparable to that of other skeletal muscles. That is, tension decreases in proportion with the reduction in thick-thin filament interdigitation (2 X Ls to 3 X Ls).     

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 sarcomere length-tension relation in skeletal muscle

Tension development during isometric tetani in single fibers of frog semitendinosus muscle occurs in three phases: (a) in initial fast-rise phase; (b) a slow-rise phase; and (c) a plateau, which lasts greater than 10 s. The slow-rise phase has previously been assumed to rise out of a progressive increase of sarcomere length dispersion along the fiber (Gordon et al. 1966. J. Physiol. [Lond.]. 184:143--169;184:170-- 192). Consequently, the "true" tetanic tension has been considered to be the one existing before the onset of the slow-rise phase; this is obtained by extrapolating the slowly rising tension back to the start of the tetanus. In the study by Gordon et al. (1966. J. Physiol. [Lond.] 184:170--192), as well as in the present study, the relation between this extrapolated tension and sarcomere length gave the familiar linear descending limb of the length-tension relation. We tested the assumption that the slow rise of tension was due to a progressive increase in sarcomere length dispersion. During the fast rise, the slow rise, and the plateau of tension, the sarcomere length dispersion at any area along the muscle was less than 4% of the average sarcomere length. Therefore, a progressive increase of sarcomere length dispersion during contraction appears unable to account for the slow rise of tetanic tension. A sarcomere length-tension relation was constructed from the levels of tension and sarcomere length measured during the plateau. Tension was independent of sarcomere length between 1.9 and 2.6 microgram, and declined to 50% maximal at 3.4 microgram. This result is difficult to reconcile with the cross-bridge model of force generation.     

Muscle and sarcomere lengths in the hind limb of the rabbit (Oryctolagus cuniculus) during a galloping stride

Rabbits were filmed galloping, and the length changes of the principal hind limb muscles were determined. Sarcomere lengths were measured in carcasses set by rigor mortis in four of the positions adopted during a stride. These sarcomere lengths were measured by means of a diffraction technique, devised for the purpose, using an ordinary microscope. Expected sarcomere lengths for three of the positions were predicted from that observed in the fourth, together with muscle length changes. A theoretical length-tension curve for rabbit muscle was constructed, using A and I filament lengths, it was shown that when the muscles were active, their sarcomere lengths corresponded to the plateau of the length-tension curve.     

Length-tension relationship of abdominal expiratory muscles: effect of emphysema

The present study examined the active and passive length-tension relationship of the abdominal expiratory muscles in vitro during electrically stimulated contractions. Studies were performed on isolated strips of transverse abdominis and external oblique muscle from nine adult hamsters with normal lung function. The effect of chronic hyperinflation on the two muscles was assessed in eight hamsters with elastase-induced emphysema. In normal animals the maximal active tension per cross-sectional area (Po) was equal in the two muscles. The absolute muscle fiber length at which Po occurred (Lo) was less for the external oblique than the transverse abdominis and the length-tension curve operated at shorter fiber lengths. However, the change in tension produced by an increase or decrease in muscle length expressed in relative terms (i.e., as %Lo) was greater for the transverse abdominis than the external oblique. Mean total lung capacity of emphysematous animals was 198% of control. Po of the transverse abdominis and external oblique were the same in emphysematous and control animals. However, Lo and the length-tension curve of the transverse abdominis occurred at shorter fiber lengths in emphysematous animals because of a reduction in the number of sarcomeres in series along the fiber. The length-tension curve and the number of sarcomeres in the external oblique was the same in emphysematous and control animals. These results in normal animals indicate that the magnitude of the change in active and passive tension produced by a change in muscle length differs in the transverse abdominis and external oblique. Moreover, chronic hyperinflation of the thorax produced by elastase injection alters the length-tension relationships of some but not all the expiratory muscles.     

Model of muscle-tendon interaction during frog semitendinosis fixed-end contractions.

A structural model was developed to explain sarcomere shortening at the expense of tendon lengthening in the frog semitendinosis (ST) muscle-tendon system. The model was based on the data of Lieber et al. [Am. J. Physiol. 261, C86-C92 (1991)], who determined the relationship between the sarcomere length, tendon load (as a fraction of maximum isometric tension) and tendon, bone-tendon junction (BTJ), and aponeurosis strain. The model was generated assuming a finite time-course of cross-bridge attachment [Huxley, Prog. Biophys. 7,255-318 (1957)], an ideal sarcomere length-tension relationship [Gordon et al., J. Physiol. 184, 170-192 (1966)] and an ideal force-velocity relationship [Katz, J. Physiol. 96, 45-64 (1939); Edman, J. Physiol. 291, 143-159 (1979)]. Functionally, sarcomeres operated on three distinct regions of the length-tension curve: (1) regions where the muscle force decreased as sarcomeres shortened (the shallow and steep ascending limbs); (2) regions where the muscle force increased as sarcomeres shortened and there was little passive tension (descending limb, where sarcomere length greater than or equal to 3.0 microns); and (3) regions where the muscle force increased as sarcomeres shortened and there was a significant passive tension (descending limb where sarcomere length greater than 3.0 microns). Using such a physiological model, it was found that the effect of tendon compliance was to 'skew' the sarcomere length-tension curve to the right and to increase the operating range of the muscle-tendon unit.(ABSTRACT TRUNCATED AT 250 WORDS)     

The dynamical stability of isometrically contracting muscle and its relation to transient force response

The length-tension relation for tetanically contracting muscle indicates that for lengths longer than the resting length, the muscle should be dynamically unstable; i.e. some sarcomeres should lengthen and others shorten. This behavior is not observed experimentally. The theoretical behavior of muscle in this respect is determined both by the length-tension curve, and by the response of muscle to rapid changes in its mechanical state. In this paper it is shown that the force generated after a small step change in length is related to the dynamical stability of muscle. By means of a simple model, the behavior of isometrically contracting muscle is predicted based on in vitro mechanical studies and classical control theory. It is found that inhomogeneities in sarcomere length can develop only after many seconds, and that this relative stability is due entirely to the presence of the muscle transients.     

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).     

A quantitative model of intersarcomere dynamics during fixed-end contractions of single frog muscle fibers.

A numerical model of a muscle fiber as 400 sarcomeres, identical except for their initial lengths, was used to simulate fixed-end tetanic contractions of frog single fibers at sarcomere lengths above the optimum. The sarcomeres were represented by a lumped model, constructed from the passive and active sarcomere length-tension curves, the force-velocity curve, and the observed active elasticity of a single frog muscle fiber. An intersarcomere force was included to prevent large disparities in lengths of neighboring sarcomeres. The model duplicated the fast rise, slow creep rise, peak, and slow decline of tension seen in tetanic contractions of stretched living fibers. Decreasing the initial non-uniformity of sarcomere length reduced the rate of rise of tension during the creep phase, but did not decrease the peak tension reached. Limitations of the model, and other processes that might contribute to the shape of the fixed end tetanic tension record are discussed. Taking account of model and experimental results, it is concluded that the distinctive features of the tension records of fixed end tetanic contraction at lengths beyond optimum can be explained by internal motion within the fiber.     

Damage to different motor units from active lengthening of the medial gastrocnemius muscle of the cat.

Slow-twitch motor units in the medial gastrocnemius muscle of the anesthetized cat were found to have an average optimum length for active tension that was 0.8 +/- 0.5 (SE) mm longer than the whole muscle optimum. For fast-twitch units (time to peak < 50 ms), the average optimum was 1.3 +/- 0.3 mm shorter than the whole muscle optimum. After the muscle had been subjected to 10 stretches while maximally activated, beginning at the whole muscle optimum length, the optimum lengths of the 27 fast-twitch motor units shifted significantly further in the direction of longer muscle lengths (mean 4.3 +/- 0.3 mm) than for the eight slow-twitch units (2.1 +/- 0.4 mm). A shift in the muscle's length-tension relation was interpreted as being due to sarcomere disruption. Statistical analysis showed that a motor unit's optimum length for a contraction, relative to the whole muscle optimum, was a better indicator of the unit's susceptibility to damage from active lengthenings than was motor unit type.     

Length-tension relationship of the external anal sphincter muscle: implications for the anal canal function

The length at which a muscle operates in vivo (operational length) and the length at which it generates maximal force (optimal length) may be quite different. We studied active and passive length-tension characteristics of external anal sphincter (EAS) in vivo and in vitro to determine the optimal and operational length of rabbit EAS. For the in vitro studies, rings of EAS (n = 4) were prepared and studied in a muscle bath under isometric conditions. For in vivo studies, female rabbits (n = 19) were anesthetized and anal canal pressure was recorded by use of a sleeve sensor placed in the custom-designed catheter holders of 4.5-, 6-, and 9-mm diameters. Measurements were obtained at rest and during EAS electrical stimulation. Sarcomere length of EAS muscle was measured by laser diffraction technique with no probe and three probes in the anal canal. In vitro studies revealed 2,054 mN/cm(2) active tension at optimal length. In vivo studies revealed a probe size-dependent increase in anal canal pressure and tension. Maximal increase in anal canal tension with stimulation was recorded with the 9-mm probe. Increases in anal canal tension with increase in probe size were completely abolished by pancuronium bromide. EAS muscle sarcomere length without and with 9-mm probe in the anal canal were 2.11 +/- 0.08 and 2.99 +/- 0.07 microm, respectively. Optimal sarcomere length, based on the thin filament length measured by thin filament analysis, is 2.44 +/- 0.10 microm. These data show that the operational length of EAS is significantly shorter than its optimal length. Our findings provide insight into EAS function and we propose the possibility of increasing anal canal pressure by surgical manipulation of the EAS sarcomere length.     

Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments.

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.     

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.     

Whole muscle length-tension relationships are accurately modeled as scaled sarcomeres in rabbit hindlimb muscles

An a priori model of the whole active muscle length-tension relationship was constructed utilizing only myofilament length and serial sarcomere number for rabbit tibialis anterior (TA), extensor digitorum longus (EDL), and extensor digitorum II (EDII) muscles. Passive tension was modeled with a two-element Hill-type model. Experimental length-tension relations were then measured for each of these muscles and compared to predictions. The model was able to accurately capture the active-tension characteristics of experimentally-measured data for all muscles (ICC=0.88 ± 0.03). Despite their varied architecture, no differences in predicted versus experimental correlations were observed among muscles. In addition, the model demonstrated that excursion, quantified by full-width-at-half-maximum (FWHM) of the active length-tension relationship, scaled linearly (slope=0.68) with normalized muscle fiber length. Experimental and theoretical FWHM values agreed well with an intraclass correlation coefficient of 0.99 (p<0.001). In contrast to active tension, the passive tension model deviated from experimentally-measured values and thus, was not an accurate predictor of passive tension (ICC=0.70 ± 0.07). These data demonstrate that modeling muscle as a scaled sarcomere provides accurate active functional but not passive functional predictions for rabbit TA, EDL, and EDII muscles and call into question the need for more complex modeling assumptions often proposed.     

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.     

Rises in whole muscle passive tension of mammalian muscle after eccentric contractions at different lengths.

This is a report of experiments carried out on the medial gastrocnemius muscle of the anesthetized cat, investigating the effects of eccentric contractions carried out at different muscle lengths on the passive and active length-tension relationships. In one series of experiments, the motor supply to the muscle was divided into three approximately equal parts; in the other, whole muscles were used. Fifty eccentric contractions were carried out over different regions of the active length-tension curve for each partial or whole muscle. Active and passive length-tension curves were measured before and after the eccentric contractions. When eccentric contractions were carried out at longer lengths, there was a larger shift of the optimum length for active tension in the direction of longer muscle lengths and a larger fall in peak isometric tension. Passive tension was higher immediately after the eccentric contractions, and if the muscle was left undisturbed for 40 min, it increased further to higher values, particularly after contractions at longer lengths. A series of 20 passive stretches of the same speed and amplitude and covering the same length range as the active stretches, reduced the passive tension which redeveloped over a subsequent 40-min period. It is hypothesized that there are two factors influencing the level of passive tension in a muscle after a series of eccentric contractions. One is injury contractures in damaged muscle fibers tending to raise passive tension; the other is the presence of disrupted sarcomeres in series with still-functioning sarcomeres tending to reduce it.     

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.     

STRUCTURE OF LIMULUS STRIATED MUSCLE : The Contractile Apparatus at Various Sarcomere Lengths

The musculature of the telson of Limulus polyphemus L. consists of three dorsal muscles: the medial and lateral telson levators and the telson abductor, and one large ventral muscle; the telson depressor, which has three major divisions: the dorsal, medioventral, and lateroventral heads. The telson muscles are composed of one type of striated muscle fiber, which has irregularly shaped myofibrils. The sarcomeres are long, with discrete A and I and discontinuous Z bands. M lines are not present. H zones can be identified easily, only in thick (1.0 µm) longitudinal sections or thin cross sections. In lengthened fibers, the Z bands are irregular and the A bands appear very long due to misalignment of constituent thick filaments. As the sarcomeres shorten, the Z lines straighten somewhat and the thick filaments become more aligned within the A band, leading to apparent decrease in A band length. Further A band shortening, seen at sarcomere lengths below 7.4 µm may be a function of conformational changes of the thick filaments, possibly brought about by alterations in the ordering of their paramyosin cores.