Tension in frog single muscle fibers while shortening actively and passively at velocities near Vu.

Experiments were undertaken to determine the contribution of passive tension to total tension during rapid shortening in a stimulated muscle fiber. Results were obtained by applying shortening movements at constant velocities slightly less than Vu (the velocity of unloaded shortening) to intact twitch fibers isolated from the frog (Rana temporaria). The tension maintained by unstimulated fibers during such shortening movements ("dynamic passive tension") from moderately long lengths was greater than zero but much less than the passive tension measured under static conditions ("static passive tension") at the same lengths. Fibers maximally activated by electrical stimulation and then shortened at the same velocity over the same range of average sarcomere lengths maintained tension that was greater than zero but less than the dynamic passive tension. For average sarcomere lengths up to approximately 3.1 microns, the dynamic passive tension appeared to be substantially abolished by activation. The onset of the apparent disappearance of dynamic passive tension was studied by initiating the stimulation and the shortening movement simultaneously. The resulting tension response exhibited a latency relaxation that was increased in amplitude compared with the isometric case, followed by a brief tension rise, giving way to a steady tension level equal to that expected if stimulation had been initiated well before the release. These changes are qualitatively explained in terms of the establishment of a steady state distribution of deformations of attached cross-bridges.     

A non-cross-bridge stiffness in activated frog muscle fibers

Force responses to fast ramp stretches of various amplitude and velocity, applied during tetanic contractions, were measured in single intact fibers from frog tibialis anterior muscle. Experiments were performed at 14 degrees C at approximately 2.1 microm sarcomere length on fibers bathed in Ringer's solution containing various concentrations of 2,3-butanedione monoxime (BDM) to greatly reduce the isometric tension. The fast tension transient produced by the stretch was followed by a period, lasting until relaxation, during which the tension remained constant to a value that greatly exceeded the isometric tension. The excess of tension was termed "static tension," and the ratio between the force and the accompanying sarcomere length change was termed "static stiffness." The static stiffness was independent of the active tension developed by the fiber, and independent of stretch amplitude and stretching velocity in the whole range tested; it increased with sarcomere length in the range 2.1-2.8 microm, to decrease again at longer lengths. Static stiffness increased well ahead of tension during the tetanus rise, and fell ahead of tension during relaxation. These results suggest that activation increased the stiffness of some sarcomeric structure(s) outside the cross-bridges.     

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.     

Sarcomere lengthening and tension drop in the latent period of isolated frog skeletal muscle fibers

A laser diffraction technique has been developed for registering small changes in sarcomere length. The technique is capable of resolving changes as small as 0.2 A in isolated frog skeletal muscle fibers. The small sarcomere lengthening that accompanies the drop in tension in the latent period of contraction was investigated. We suggest this lengthening be named latency elongation (LE). The LE is present in a completely slack fiber and must, therefore, be caused by a forcible lengthening process. Furthermore, the LE is dependent on the existence of an overlap between thin and tick filaments. The rate of elongation and the time interval between stimulation and maximum elongation may vary along the fiber. The maximum elongation was 3-5 A per sarcomere. At any instant the drop in tension is a product of the sum of sarcomere lengthenings along the fiber and the slope stiffness of the series elasticity. The latency relaxation (LR) could be registered in the sarcomere length range from 2.2 mum to 3.6-3.7 mum. The amplitude went through a sharp maximum at 3.0-3.1 mum. In the sarcomere length range from 2.2 to 2.8 mum the delay from onset to maximum LR was nearly proportional to the distance from the Z-line to the overlap zone. A working hypothesis is presented. It is suggested that the LE is caused by a lengthening of the thin filaments.     

Tension relaxation after stretch in resting mammalian muscle fibers: stretch activation at physiological temperatures.

Tension responses to ramp stretches of 1-3% Lo (fiber length) in amplitude were examined in resting muscle fibers of the rat at temperatures ranging from 10 degrees C to 36 degrees C. Experiments were done using bundles of approximately 10 intact fibers isolated from the extensor digitorum longus (a fast muscle) and the soleus (a slow muscle). At low temperatures (below approximately 20 degrees C), the tension response consisted of an initial rise to a peak during the ramp followed by a complex tension decay to a plateau level; the tension decay occurred at approximately constant sarcomere length. The tension decay after a standard stretch at approximately 3-4.Lo/s contained a fast, an intermediate, and a (small amplitude) slow component, which at 10 degrees C (sarcomere length approximately 2.5 microns) were approximately 2000.s-1, approximately 150.s-1, and approximately 25.s-1 for fast fibers and approximately 2000.s-1, approximately 70.s-1 and approximately 8.s-1 for slow fibers, respectively. The fast component may represent the decay of interfilamentary viscous resistance, and the intermediate component may be due to viscoelasticity in the gap (titin, connectin) filament. The two- to threefold fast-slow muscle difference in the rate of passive tension relaxation (in the intermediate and the slow components) compares with previously reported differences in the speed of their active contractions; this suggests that "passive viscoelasticity" is appropriately matched to contraction speed in different muscle fiber types. At approximately 35 degrees C, the fast and intermediate components of tension relaxation were followed by a delayed tension rise at approximately 10.s-1 (fast fibers) and 2.5.s-1 (slow fibers); the delayed tension rise was accompanied by sarcomere shortening. BDM (5-10 mM) reduced the active twitch and tetanic tension responses and the delayed tension rise at 35 degrees C; the results indicate stretch sensitive activation in mammalian sarcomeres at physiological temperatures.     

The Effect of Bathing Solution Tonicity on Resting Tension in Frog Muscle Fibers

Resting tension and short-range elastic properties of isolated twitch muscle fibers of the frog have been studied while bathed by solutions of different tonicities. Resting tension in isotonic solution at 2.3-µm sarcomere spacing averaged 0.46 mN·mm^-2 and was proportional to the fiber cross-section area. Hypertonic solutions, containing 0.1–0.5 mM tetracaine to block contracture tension, caused a small sustained tension increase, which was proportional to the fiber cross-section area and which reached 0.9 mN·mm^-2 at two times normal tonicity (2T). Further increases in tonicity caused little increase in tension. Hypotonic solutions decreased tension. Thus, tension at 2.3 µm is a continuous, direct function of tonicity. The dependence of tension on tonicity lessened at greater sarcomere lengths. At 3.2 µm either a very small rise or, in some fibers, a fall in tension resulted from an increase in tonicity. Hypertonic solutions also decreased the tension of extended sarcolemma preparations. In constant-speed stretch experiments the elastic modulus, calculated from the initial part of the stretch response, rose steeply with tonicity over the whole range investigated (1–2.5T). The results show that tension and stiffness of the short-range elastic component do not increase in parallel in hypertonic solutions.     

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).     

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.     

Electrically evoked contractions of the triceps surae during and following 21 days of voluntary leg immobilization

The effects of 21 days voluntary leg (plaster) immobilization on the mechanical properties of the triceps surae have been studied in 11 young female subjects, mean age 19.4 years. The results show that during the period of immobilization the mean time to peak tension (TPT) and half relaxation time (1/2RT) and tension (Pt) of the maximal twitch increased significantly (p less than 0.001) but the effects were short lived. Maximal tension and contraction times of the twitch recovered within 2-14 days following the removal of the plaster cast. The electrically evoked tetanic tensions at 10 Hz and 20 Hz did not change significantly (P greater than 0.1) during immobilization, but the 50 Hz tetanic tension (Po50) and maximal voluntary contraction (MVC) were reduced (p less than 0.05). The fall in Po50 and MVC was associated with 10% decrease in the estimated muscle (plus bone) cross-sectional area. The relative (%) change in Po50 and MVC following immobilization was related to the initial physiological status (as indicated by the response of the triceps surae to a standard fatigue test prior to immobilization) of the muscle. The rate of rise and recovery fall of the tetanus were slightly but significantly (p less than 0.01) reduced on day 7 of immobilization, but thereafter remained constant. The isokinetic properties of the triceps surae as reflected in the measured torque/velocity relation of the muscle in 4 subjects did not change significantly if account was taken of the slight degree of atrophy present following immobilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The Pattern of Activation in the Sartorius Muscle of the Frog

The development of isometric twitch tension has been compared with the redevelopment of isometric tension in the fully active frog sartorius muscle following release. At 0°C the rate of rise of isometric twitch tension is the same as that for the muscle in the fully active state at the same tension but not until about 40 msec. after the stimulus and then only for a few milliseconds. The rates of rise of tension in the twitch and in the redevelopment of tension in the fully active muscle appear to be nearly the same at low tensions. Substitution of nitrate for chloride in the Ringer's solution bathing the muscle retards the development of tension during the early part of the contraction phase of the twitch and the effect reaches a maximum within 3 minutes after changing the solutions. These observations have been discussed in connection with some possible patterns of activation and the hypothesis has been advanced that the rate of activation of a sarcomere is determined mainly by the rate at which the transverse component of the link between excitation and contraction is propagated inwards from the periphery to the center of the fiber. This hypothesis has been discussed in relation to others concerning the nature of excitation-contraction coupling.     

Light diffraction studies of sarcomere dynamics in single skeletal muscle fibers.

A position-sensitive optical diffractometer has been used to examine the diffraction spectra produced by single skeletal muscle fibers during twitch and tetanic contraction. First-order diffraction lines were computer-analyzed for mean sarcomere length, line intensity, and percent dispersion in sarcomere length. Line intensity was observed to decrease rapidly by about 60 percent during a twitch, with an exponential recovery to resting intensity persisting well beyond cessation of sarcomere shortening; recovery was particularly prolonged at zero myofilament overlap. A number of single fibers at initial lengths from 2.5 to 3.5 MICRON EXHIBITED a splitting of the first-order line into two or more components during relaxation, with components merging back into a single peak by 200 ms after stimulation. This splitting reflects the asynchronous nature of myofibrillar relaxation within a single fiber. During tetanus, the dispersion decreased by more than 10 percent from onset to plateau, implying a gradual stabilization of sarcomeres.     

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.     

Phase transition in force during ramp stretches of skeletal muscle.

Active glycerinated rabbit psoas fibers were stretched at constant velocity (0.1-3.0 lengths/s) under sarcomere length control. As observed by previous investigators, force rose in two phases: an initial rapid increase over a small stretch (phase I), and a slower, more modest rise over the remainder of the stretch (phase II). The transition between the two phases occurred at a critical stretch (LC) of 7.7 +/- 0.1 nm/half-sarcomere that is independent of velocity. The force at critical stretch (PC) increased with velocity up to 1 length/s, then was constant at 3.26 +/- 0.06 times isometric force. The decay of the force response to a small step stretch was much faster during stretch than in isometric fibers. The addition of 3 mM vanadate reduced isometric tension to 0.08 +/- 0.01 times control isometric tension (P0), but only reduced PC to 0.82 +/- 0.06 times P0, demonstrating that prepowerstroke states contribute to force rise during stretch. The data can be explained by a model in which actin-attached cross-bridges in a prepowerstroke state are stretched into regions of high force and detach very rapidly when stretched beyond this region. The prepowerstroke state acts as a mechanical rectifier, producing large forces during stretch but small forces during shortening.     

The variation of characteristics of twitch and tetanic contractions with sarcomere length in isolated muscle fibres of the frog.

The relation between sarcomere length, tension and time course of tension development in twitch and tetanic contractions at 20 degrees C was determined for isolated fibres from the semitendinosus muscle of the frog (Rana esculenta). In twenty fibres at about 2.15 micron sarcomere length, the peak twitch tension, the maximum tetanic tension and the twitch/tetanus ratio ranged, respectively, from 0.22 to 1.6 kg/cm2, from 2.13 o 3.96 kg/cm2 an from 0.07 to 0.53. The peak twitch tension was found to be: i) directly correlated with the twitch/tetanus ratio and the time to the peak of the first derivative of the twitch tension, ii) inversely correlated with the time to the peak of the first derivative of tetanic tension. No significant correlation was found between the maximal tetanic tension and the peak twitch tension or the twitch/tetanus ratio. Peak twitch tension and twitch/tetanus ratio were not correlated with the fibre cross-sectional area which ranged from 1.052 to 6,283 micron2. Sarcomere length-tension curves for twitch and tetanic isometric contractions at 20 degrees C were determined in twelve fibres. Increases in sarcomere length from about 2.15 to 2.85 micron produced, depending on the peak twitch tension or the twitch/tetanus ratio at about 2.15 micron, either decrease and no change or increase in peak twitch tension, but constantly enhanced the twitch/tetanus ratio and the degree of this potentiation was inversely correlated with the twitch/tetanus ratio at 2.15 micron. Increase in sarcomere length above 2.15 micron did not alter the course of the early development of twitch and tetanic tensions, reduced considerably the variation in peak twitch tension and twitch/tetanus ratio, without altering that of tetanic tension and swamped the correlation between the peak twitch tension and the time to peak of the differentiated twitch tension. However, the peak twitch tension at about 2.85 micron resulted to be directly correlated with the peak twitch tension at about 2.15 micron and in addition the relative length-dependent change in the time of the peak of the first derivative of the twitch tension resulted to be directly correlated with the relative length-dependent change in the peak twitch tension. It is concluded that both the duration of the active state and the rate factors of activation contribute to the determining of the large variation in peak twitch tension at about 2.15 micron, whereas the length-dependent increase in twitch/tetanus ratio appears to be mainly determined by prolongation of the active state duration.     

The motor units of cat medial gastrocnemius: electrical and mechanical properties as a function of muscle length.

The effects of changing muscle length on the mechanical properties of 89 motor units from adult cat medial gastrocnemius have been studied in eight experiments. Few differences were found between the effects of length on tetanic tension, twitch tension, twitch-tetanus ratio, twitch contraction time, twitch half relaxation time, rate of force development and electrical activity for fast contracting (twitch contraction time less than or equal to 45 msec) and slowly contracting (greater than 45 msec) units. Those differences that did appear did not persist when these two groups were matched by tetanic tension. It is concluded that the biophysical mechanisms responsible for the changes in mechanical and electrical properties with length must be similar for fast and slow twitch units and not related to potential differences in their muscle fiber type. The effects of changing muscle length on the mechanical properties of the eight whole muscles suggest that changes in force output with length are of minor importance during normal movements as the muscle is found to be electrically active over a relatively narrow range of lengths close to the optimum length for tetanus of the whole muscle. The very shortest muscle lengths at which there is only minimal force development are not used in natural movements, while the declining limb of the length tension curve is at muscle lengths beyond the maximum in situ length.     

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.     

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.     

Energetics of activation in frog skeletal muscle at sarcomere lengths beyond myofilament overlap.

Experiments were designed to gain information about the effects of extremely long sarcomere lengths (greater than 3.8 microns) on muscle activation. The amount of energy liberated in an isometric twitch by muscles stretched to sarcomere lengths where myofilament overlap is vanishingly small (greater than 3.6 microns) is thought to be an indirect measure of the Ca2+ cycled during contraction. The effects of altering sarcomere length from 3.8 to 4.3 microns on the amount of Ca2+ cycled was measured using twitch energy liberation as an indicator of the Ca2+ cycled. Twitch energy liberation decreased by approximately 20% over this sarcomere length region, suggesting that the amount of Ca2+ released by a single action potential is not altered dramatically when a muscle is stretched to extreme lengths.