Twitch and tetanic tension during culture of mature Xenopus laevis single muscle fibres

Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twitch and tetanic force characteristics, number of sarcomeres in series and fibre cross-section. Fibres dissected from m. iliofibularis (n = 10) were kept in culture at a fibre mean sarcomere length of 2.3 microm in a culture medium without serum. Twitch and tetanic tension were determined daily. Before and after culture the number of sarcomeres was determined by laser diffraction and fibre cross-sectional area (CSA) was determined by microscopy. For five fibres twitch tension increased during culture and tetanic tension was stable for periods varying from 8 to 14 days ('stable fibres'), after which fibres were removed from culture for analysis. Fibre CSA and the number of sarcomeres in series remained constant during culture. Five other fibres showed a substantial reduction in twitch and tetanic tension within the first five days of culture ('unstable fibres'). After 7-9 days of culture, three of these fibres died. For two of the unstable fibres, after the substantial force reduction, twitch and tetanic tension increased again. Finally at day 14 and 18 of culture, respectively, the tensions attained values higher than their original values. For stable fibres, twitch contraction time, twitch half-relaxation time and tetanus 10%-relaxation time increased during culture. For unstable fibres these parameters fluctuated. For all fibres the stimulus threshold fluctuated during the first two days, and then remained constant, even for the fibres that were cultured for at least two weeks. It is concluded that the present culture system for mature muscle fibres allows long-term studies within a well-defined medium. Unfortunately, initial tetanic and twitch force are poor predictors of the long-term behaviour of the fibres.     

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 effect of muscle fatigue on the isometric contractile characteristics of skeletal muscle in the cat

The rise time of an isometric twitch, the tetanic tension, the twitch tetanus ratio, the frequency-tension relationship, and the height of the MUAP (motor unit action potential) were measured in fast twitch (medial gastrocnemius) and slow twitch (soleus) muscles of the cat immediately before, in the middle, and immediately after fatiguing isometric contractions at tensions of 30, 50 and 80% of each muscle's initial strength (tetanic tension recorded from the unfatigued muscle). Although the twitch-tetanus ratio was always less for the soleus than for the medial gastrocnemius muscles, the twitch-tetanus ratio for any one muscle was constant throughout the duration of fatiguing isometric contractions at any of the tensions examined. In contrast, the twitch tension and tetanic tension of the muscles were both less after the contractions, the largest reduction occurring for both muscles during contractions sustained at the lowest isometric tensions. The time to peak tension of an isometric twitch was prolonged for both muscles following the contractions. This was associated with a corresponding shift in the frequency tension relationship such that at the point of muscular fatigue, the muscles tetanized at lower frequencies of stimulation than did the unfatigued muscle. In contrast, the amplitude of the MUAP showed only a modest reduction throughout the duration of the fatiguing contractions.     

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.     

A comparison of quantitative ultrastructural and contractile characteristics of muscle fibre types of the perch,Perca fluviatilis L.

Summary Quantitative ultrastructural and physiological parameters were investigated in three types of muscle fibres ofPerca fluviatilis: white fibres from the m. levator operculi anterior, pink (intermediate) fibres of the m. hyohyoideus and deep red fibres of the m. levator operculi anterior. Times to peak tension and half relaxation times of isometric twitches increased in the mentioned order. The extent of contact between the T system and the sarcoplasmic reticulum and the relative volume and surface area of the terminal cisternae showed an inverse relation with the time to peak tension of the twitch. The maximal isometric tetanic force per unit cross section area was similar for all three investigated types. The inverse relation between the time to peak tension of the twitch and the relative length of contact between T system and SR is in agreement with data obtained for fast- and slow twitch muscle fibres of the carp,Cyprinus carpio L.Abbreviations LOPA musculus levator operculi anterior - HH musculus hyohyoideus - SR Sarcoplasmic reticulum     

Earliest mechanical evidence of cross-bridge activity after stimulation of single skeletal muscle fibers.

The stiffness of single fibers from frog skeletal muscle was measured by the application of small 2-kHz sinusoidal length oscillations during twitch and tetanic contractions at a range of initial sarcomere lengths. The earliest mechanical signs of activation were a fall in tension (latency relaxation) and a rise in stiffness. The earliest stiffness increase and the earliest tension fall occurred simultaneously at all sarcomere lengths. This suggests a cross-bridge origin for the latency relaxation. The lead of stiffness over tension seen during the rise of tension was substantially established during the latent period. Reducing the size of the twitch by reducing calcium release with D-600 (methoxyverapamil) reduced the latency relaxation and the stiffness development during latency much less than it reduced the twitch tension. For very small twitches the peak of the stiffness response occurred during the latent period and the times of onset of both latency relaxation and stiffness rise were delayed, but remained coincident. This suggests a strong connection between the latency relaxation and the rise of stiffness during the latent period, whereas the connection between these events and positive tension generation appears to be less strong.     

The rate of changes in tension within fused tetani of single motor units in rat medial gastrocnemius muscle.

The time course of fused tetani of three main types of motor units: slow (S), fast resistant (FR) and fast fatigable (FF) was studied in the rat medial gastrocnemius. The rate of tension generation and of the relaxation within a tetanus was measured under isometric conditions. These measurements were performed at three points during both the contraction and relaxation: the beginning, the middle and the end of the phase of changes in tension. Significant differences were found in the rate of tension changes between fast and slow units. Comparison of FF and FR units showed less pronounced differences in their rates of the contraction and the relaxation. Moreover, slow units showed significantly greater slowing of both the contraction and relaxation within a tetanus in relation to the speed of their twitch when compared to fast motor units. The rate of changes in tetanic tension correlated to twitch time parameters and to tension generated during twitch or tetanus. The results point out that the well known difference in the speed of twitch contraction between fast and slow units is also visible in their fused tetani.     

Mechanical properties of denervated amphibian muscle

Denervated amphibian muscle does not show the prolongation of action potential found in mammalian denervated muscle. It was, therefore, predicted that denervated amphibian muscle would not show prolongation of the mechanical twitch. The sartorius muscles in one leg of toads--Xenopus borealis--were denervated for 140-268 days. Isometric twitch time to peak, time to half relaxation and twitch/tetanus ratio were not changed following denervation, confirming our prediction. Twitch tension decreased to 68% and tetanic tension decreased to 75% of control values. The maximum velocity of unloaded shortening (muscle length/s) was also unchanged.     

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.     

Structure and function of skeletal muscle in zebrafish early larvae

Zebrafish muscles were examined at an early developmental stage (larvae 5-7 d). Using aluminum clips, preparations (approximately 1.5 mm length, 150 microm diameter) were mounted for force registration and small angle x-ray diffraction. Sarcomeres were oriented mainly in parallel with the preparation long axis. Electrical stimulation elicited fast and reproducible single twitch contractions. Length-force relations showed an optimal sarcomere length of 2.15 microm. X-ray diffraction revealed clear equatorial 1.1/1.0 reflections, showing that myofilaments are predominantly arranged along the preparation long axis. In contrast, reflections from older (2 mo) zebrafish showed two main filament orientations each at an approximately 25 degrees angle relative to the preparation long axis. Electrical stimulation of larvae muscles increased the 1.1/1.0 intensity ratio, reflecting mass transfer to thin filaments during contraction. The apparent lattice volume was 3.42 x 10(-3) microm(3), which is smaller than that of mammalian striated muscle and more similar to that of frog muscles. The relation between force and stimulation frequency showed fusion of responses at a comparatively high frequency (approximately 186 Hz), reflecting a fast muscle phenotype. Inhibition of fast myosin with N-benzyl-p-toluene sulphonamide (BTS) showed that the later phase of the tetanus was less affected than the initial peak. This suggests that, although the main contractile phenotype is fast, slow twitch fibers can contribute to sustained contraction. A fatigue stimulation protocol with repeated 220 ms/186 Hz tetani showed that tetanic force decreased to 50% at a train rate of 0.1 s(-1). In conclusion, zebrafish larvae muscles can be examined in vitro using mechanical and x-ray methods. The muscles and myofilaments are mainly orientated in parallel with the larvae long axis and exhibit a significant fast contractile component. Sustained contractions can also involve a small contribution from slower muscle types.     

Mechanical properties of muscles from Xenopus borealis following maintenance in organ culture

The mechanical properties of sartorii muscles from the toad Xenopus borealis were studied in organ cultures lasting up to 26 days. Isometric twitch and tetanic tensions diminished in force reaching half their initial values after 15 days in culture. In contrast, twitch time to peak time to half relaxation, twitch-tetanus ratio and muscle weight-body weight ratio, were unchanged. Maximum isotonic shortening velocity (Vmax) declined, with a half time similar to that of the isometric force. The fall in isometric tension is probably due to a breakdown of activation in some fibres. The change in Vmax could be due to a loss of functional sarcomeres in series with the tendons.     

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.     

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 peripheral nerve allograft in the primate immunosuppressed with Cyclosporin A: II. Functional evaluation of reinnervated muscle.

Isometric contractile function was evaluated in primates receiving peripheral nerve allografts and autografts. Twelve adult male cynomolgus monkeys received both sural nerve allografts and autografts to the ulnar nerve in opposite forearms. Half the animals received Cyclosporin A (CsA) immunosuppression (25 mg/kg per day); the remaining animals received placebo. One year following nerve engraftment, isometric contractile muscle function was evaluated in reinnervated abductor digiti quinti and intact abductor pollicis brevis muscles. Maximal twitch tension (Pt), tetanic tension (P(o)), time to peak tension (tpt), rate of rise of twitch tension (DP/dt), and muscle fatigue were evaluated at optimal muscle length (L(o)). All reinnervated muscles distal to nerve autografts and allografts in both Cyclosporin A-immunosuppressed and placebo-treated animals generated equivalent maximal twitch tension, tetanic tension, and time to peak tension, with no significant difference between groups (p > 0.05 by ANOVA). There was a tendency toward increased muscle fatiguability in Cyclosporin A-treated animals (p > 0.05). However, the rate of rise of twitch tension was significantly faster in the reinnervated and intact muscles of Cyclosporin A-treated primates (p < 0.05). Evidence of excellent functional reinnervation across nerve allografts and autografts similar to that seen in histologic and electrophysiologic studies was noted. Cyclosporin A immunosuppression did not significantly enhance recovery of muscle function distal to nerve allografts in this model.     

Fatigue and contraction of slow and fast muscles in hypokinetic/hypodynamic rats

This investigation examined the effects of hypokinesia/hypodynamia (H/H) on fatigability and contractile properties of rat soleus (S) and gastrocnemius (G) muscles. Whole-body suspension for 1 wk was used to eliminate hindlimb load-bearing functions and simultaneously permit voluntary isotonic contractions. Train stimulations (45/min, 16 min) resulted in significantly (P less than 0.05) faster rates of fatigue to lower asymptotes in G from H/H rats. Fatigue in the S was minimal at this stimulation frequency and differences between H/H and control animals were not significant. Contractile properties (twitch and tetanic) were measured before and after train stimulations. H/H suspension resulted in an increased twitch tension in G. However, H/H did not change train or tetanic tensions per gram or other G contractile properties. Peak twitch, train, and tetanic tensions, time to peak tension, one-half relaxation time, and twitch and tetanic peak rates of tension development and decline were unchanged by H/H in S muscles. These results indicate that 1 wk of H/H-induced muscle atrophy significantly increases fatigability in G but does not effect contractile properties of fast-twitch (G) or slow-twitch (S) muscles.     

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).     

Modulation of passive force in single skeletal muscle fibres

In this study, we investigated the effects of activation and stretch on the passive force-sarcomere length relationship in skeletal muscle. Single fibres from the lumbrical muscle of frogs were placed at varying sarcomere lengths on the descending limb of the force-sarcomere length relationship, and tetanic contractions, active stretches and passive stretches (amplitudes of ca 10% of fibre length at a speed of 40% fibre length/s) were performed. The passive forces following stretch of an activated fibre were higher than the forces measured after isometric contractions or after stretches of a passive fibre at the corresponding sarcomere length. This effect was more pronounced at increased sarcomere lengths, and the passive force-sarcomere length relationship following active stretch was shifted upwards on the force axis compared with the corresponding relationship obtained following isometric contractions or passive stretches. These results provide strong evidence for an increase in passive force that is mediated by a length-dependent combination of stretch and activation, while activation or stretch alone does not produce this effect. Based on these results and recently published findings of the effects of Ca2+ on titin stiffness, we propose that the observed increase in passive force is caused by the molecular spring titin.     

Isometric contractile properties and instantaneous stiffness of amphibian skeletal muscle in the temperature range from 0 to 20 degrees C

The isometric contractile properties of frog (Rana pipiens) and toad (Bufo bufo) sartorii have been studied over the temperature range from 0 to 20 degrees C. The isometric twitch tension was found to vary considerably between these two species and between muscles in the same species. Between 0 and 4 degrees C there was very little change in maximum isometric twitch tension. Between 4 and 12 degrees C several muscles from frog or toad showed a potentiation of twitch tension whereas others showed a decline. Over this temperature range the toad sartorii consistently demonstrated a greater potentiation. By 12 degrees C a steady decline in twitch tension in both muscles was seen as the temperature range the toad sartorii consistently demonstrated a greater potentiation. By 12 degrees C a steady decline in twitch tension in both muscles was seen as the temperature approached 20 degrees C. The maximum isometric tetanic tension recorded between 18 and 20 degrees C increased fractionally to an average of 1.504 +/- 0.029 (n = 4) for frog sartorii and to 1.377 +/- 0.008 (n = 5) for toad sartorii. The time to peak twitch tension and the half-relaxation time decreased markedly with an increase in temperature. Moreover, the half-relaxation time was reduced by a greater proportion than the time to peak twitch tension. Measurements of instantaneous stiffness by controlled velocity releases from the plateau of isometric tetani revealed that the large increase in isometric tetanus tension as the muscle was warmed was not accompanied by a corresponding increase in the total number of active cross-bridges. The possibility that a decreased availability of intracellular Ca2+ ions at the contractile sites contributing to the fall of isometric twitch tension at elevated temperatures is discussed. The possibility exists that at elevated temperatures a change inthe intrinsic contractile ability of the muscle occurs which produces an increased tension per cross-bridge.     

Effects of high-pressure acclimatization on silver eel (Anguilla anguilla, L.) slow muscle contraction

As the spawning migration of the eel is supposed to correspond to a long swimming activity at depth, patterns of slow red muscle contraction have been investigated in European silver eel (Anguilla anguilla L.) exposed for 3 weeks to 10.1 MPa hydrostatic pressure. The results show that pressure-acclimated eels (male and female) show a three-fold decrease in maximum isometric stress of twitch and tetanic contractions while time to peak force, time from peak force to 90% relaxation and ratio of twitch tension to tetanic tension remain unchanged. The observed modifications in slow red muscle mechanical properties do not impede the spawning migration of the eel and are possibly partially compensated by an improvement in the efficiency of oxidative phosphorylation. Effects of changes in membrane fluidity are also discussed.     

Thermal adaptations in lizard muscle function

Summary This study was undertaken to investigate thermal adaptations in muscle contractile properties in closely-related lizards with different preferred body temperatures (PBT). The species examined all belong to theSphenomorphus group of Australian skinks (Scincidae: Lygosominae). Preferred body temperatures are conservative at the generic level as follows:Ctenotus, 35°C;Sphenomorphus, 30°C;Eremiascincus, 25°C. Contractile properties of the fast glycolytic portion of the iliofibularis muscle were measured. Translational adaptations are evident in several isometric factors, including tetanic tension (Po), twitch tension (Pt), twitch time to peak tension (TPT), and twitch half-relaxation time (1/2 RT). Capacity adaptations are not evident in rates of tetanic tension development (dPo/dt) or in maximal velocities of isotonic shortening (V _max). Rotational adaptations are not evident in any contractile properties. Thermal limits on upper response temperatures are about 5°C warmer inCtenotus than in the more cryophilic species, indicative of resistance adaptation in muscle performance. Despite these adaptive shifts, there is little indication that muscle functional capacities are optimized or equalized at PBT in these lizards.Abbreviations FG fast glycolytic - IF iliofibularis muscle - PBT preferred body temperature - Po tetanic tension - Pt twitch tension - 1/2RT twitch half relaxation time - TPT twitch time to peak tension