Isometric Tetanus Research Articles

МАТЕМАТИЧНЕ МОДЕЛЮВАННЯ ФІЗІОЛОГІЧНОГО ПРОЦЕСУ М’ЯЗОВОГО СКОРОЧЕННЯ

The article discusses improved mathematical models for the physiological process of muscle contraction based on the well-known hypotheses of the functioning of the musculoskeletal system of the human body. In particular, according to the first phenomenological hypothesis of A. Hill, on the basis of rheological models of the components of muscle tissue, a mathematical model was developed for changing the power load of muscle tissue for isometric tetanus and muscle contraction (lengthening) at a constant rate. It has been established that a common drawback of A. Hill's approach is the assumption that the force-speed ratio must be fulfilled instantly after changing the power load. This is inconsistent with the experimental data on the recovery of strength tension after a stepwise change in muscle length. To overcome these disadvantages, A. Huxley's hypothesis was chosen. It is based on the principles of kinetics of the distribution of the binding sites of actin (monomer) with the protein filament (cross bridges). It is assumed that the binding sites on actin are far enough from each other so that only one such binding site is available to each bridge. On the basis of A. Huxley's hypothesis, a mathematical model of muscle tissue strength load was developed, which depends on the distribution function of the number of cross bridges. The results of the comparison of theoretical and experimental studies of the power load on the muscle, based on the developed mathematical models in the form of differential equations, confirmed the adequacy of the use of known theoretical provisions to describe the course of biological processes in muscle tissues.

Thick Filament Mechano-Sensing in Skeletal and Cardiac Muscles: A Common Mechanism Able to Adapt the Energetic Cost of the Contraction to the Task.

A dual regulation of contraction operates in both skeletal and cardiac muscles. The first mechanism, based on Ca2+-dependent structural changes of the regulatory proteins in the thin filament, makes the actin sites available for binding of the myosin motors. The second recruits the myosin heads from the OFF state, in which they are unable to split ATP and bind to actin, in relation to the force during contraction. Comparison of the relevant X-ray diffraction signals marking the state of the thick filament demonstrates that the force feedback that controls the regulatory state of the thick filament works in the same way in skeletal as in cardiac muscles: even if in an isometric tetanus of skeletal muscle force is under the control of the firing frequency of the motor unit, while in a heartbeat force is controlled by the afterload, the stress-sensor switching the motors ON plays the same role in adapting the energetic cost of the contraction to the force. A new aspect of the Frank-Starling law of the heart emerges: independent of the diastolic filling of the ventricle, the number of myosin motors switched ON during systole, and thus the energetic cost of contraction, are tuned to the arterial pressure. Deterioration of the thick-filament regulation mechanism may explain the hyper-contractility related to hypertrophic cardiomyopathy, an inherited heart disease that in 40% of cases is due to mutations in cardiac myosin.

Release of calcium into the myofibrillar space in response to active shortening of striated muscle.

The study was undertaken to explore whether shortening of striated muscle during activity is associated with release of bound Ca2+ into the myofibrillar space as has previously been proposed in order to explain the depressant effect of active shortening. The experiments were carried out on single muscle fibres isolated from the anterior tibialis muscle of Rana temporaria. The fibres were loaded with the calcium sensitive indicator Fluo-3. The fibres, stimulated to produce a partially fused isometric tetanus, were subjected to a shortening ramp or, alternatively, to a stretch ramp during activity while force, fibre length, sarcomere length and the Fluo-3 signal were recorded. A shortening ramp performed during a partially fused tetanus caused an increase in the myofibrillar free calcium concentration and produced, simultaneously, a decrease in active force. The isometric force recovered gradually after the shortening ramp, while the intracellular Ca2+ concentration stayed above the control level during the remainder of the stimulation period. A stretch ramp applied during a partially fused tetanus caused a considerably smaller change in the myofibrillar Ca2+ concentration. The results provide evidence that the myosin cross-bridges interact with the calcium binding sites on the thin filaments during active shortening, causing sustained release of calcium and reduced contractile strength.

Structural Kinetics of the RLC Domain of Myosin during Activation of Skeletal Muscle Fibers by Photolysis of Caged-Calcium

Recent fluorescence polarization studies in skeletal muscle fibers using bifunctional rhodamine (BSR) probes on the C-lobe of the regulatory light chain (RLC) of myosin showed that the steady-state calcium-sensitivity and co-operativity of the orientation change of the RLC C-lobe are higher than those of force (Fusi et al., Nat Commun 2016,doi:10.1038/ncomms13281). We have now investigated the in situ kinetics of the conformational change of the RLC during isometric contraction of a muscle fiber triggered by a rapid (>104 s−1) increase in [Ca2+] following UV-photolysis of caged-calcium (NP-EGTA). We monitored by polarized fluorescence changes in the orientation of the E-helix of a BSR-labeled RLC exchanged into demembranated fibers from rabbit psoas muscle during activation by photolysis of NP-EGTA, under conditions which preserve the physiological resting structure of the thick filament (T=25°C, 5% Dextran, 2.45 µm sarcomere length). After photolysis force increased with a sigmoidal time course similar to that of an isometric tetanus in an intact muscle fiber. The change in the order parameter P2 of the RLC probe, associated with it becoming more perpendicular to the fiber axis, also had a sigmoidal time course. The fractional change in P2 was faster than that of force. The P2 change was 25% complete 20ms after photolysis when activation of the thin filament is already maximal (T=12°C, Fusi et al., PNAS 2014, 111:4626-31) but force had developed to only 10% of its plateau value (T0). The P2 change was essentially complete by ca 70ms after photolysis when force was only 60% T0. These results show that the change in RLC orientation associated with the activation of the myosin motors is slower than thin-filament activation but faster than force generation. Supported by MRC, UK.

Larger lacertid lizard species produce higher than expected iliotibialis muscle power output; the evolution of muscle contractile mechanics with body size in lacertid lizards

Increases in body size can lead to alterations in morphology, physiology, locomotor performance and behaviour of animals. Most studies considering the effects of scaling on muscle performance have studied within-species effects, with few studies considering differences between species. A previous review of published data indicates that maximum muscle-shortening velocity decreases, but that maximum isometric stress does not change, with increased body mass across species of terrestrial animals. However, such previous analyses do not account for the phylogenetic relatedness of the species studied. Our aim was to use phylogenetically informed analysis to determine the effects of body size on isolated iliotibialis muscle performance across 17 species of lacertid lizards. Between one and five individuals were used to obtain mean performance values for each species. We analysed the relationship between each variable and body size, as estimated by snout-vent length (SVL), whilst taking into account the phylogenetic relationships between species. We found that isometric tetanus relaxation time, maximal tetanus stress (force per muscle cross-sectional area) and maximal work loop power output (normalised to muscle mass) all significantly increased with greater SVL. In contrast, fatigue resistance during repeated work loops significantly decreased with SVL and there was no effect of size on tetanus activation time. When we compare our findings with those that would be predicted by dynamic similarity, then as these lacertid species become bigger, there is a greater than expected increase in the normalised muscle power output, probably to counter the larger than expected increase in body mass.

Regional thermal specialisation in a mammal: temperature affects power output of core muscle more than that of peripheral muscle in adult mice (Mus musculus).

In endotherms, such as mammals and birds, internal organs can specialise to function within a narrow thermal range. Consequently, these organs should become more sensitive to changes in body temperature. Yet, organs at the periphery of the body still experience considerable fluctuations in temperature, which could select for lower thermal sensitivity. We hypothesised that the performance of soleus muscle taken from the leg would depend less on temperature than would the performance of diaphragm muscle taken from the body core. Soleus and diaphragm muscles were isolated from mice and subjected to isometric and work-loop studies to analyse mechanical performance at temperatures between 15 and 40°C. Across this thermal range, soleus muscle took longer to generate isometric force and longer to relax, and tended to produce greater normalised maximal force (stress) than did diaphragm muscle. The time required to produce half of maximal force during isometric tetanus and the time required to relax half of maximal force were both more sensitive to temperature in soleus than they were in diaphragm. However, thermal sensitivities of maximal force during isometric tetani were similar for both muscles. Consistent with our hypothesis, power output (the product of speed and force) was greater in magnitude and more thermally sensitive in diaphragm than it was in soleus. Our findings, when combined with previous observations of muscles from regionally endothermic fish, suggest that endothermy influences the thermal sensitivities of power output in core and peripheral muscles.

Nonaffine response of skeletal muscles on the ‘descending limb’

Tetanized muscle myofibrils are often modeled as one-dimensional chains where springs represent half-sarcomeres (HS). The force–length relation for individual HSs (isometric tetanus) is known to have a ‘descending limb’, a segment with an apparently negative stiffness. Despite the potential mechanical instability on the descending limb, the isometric tetanus is usually interpreted as describing an affine deformation. At the same time, active stretching during tetanus around the descending limb is known to produce non-affine sarcomere patterns. In view of this paradox, the question whether the mechanical behavior of a myofibril can be interpreted as a response of a single contractile unit has been a subject of considerable controversy over the last 50 years. In this paper we question the claim that the isometric tetanus describes homogeneous configurations of the HS chain. To distinguish between the multitudes of non-affine equilibrium states available to this mechanical system, we propose to use the concept of a stored mechanical energy. While the notion of energy is natural from a mechanical point of view, physiologists have resisted it so far on the grounds that the contractile elements are active. We discuss how this objection can be overcome and show that the appropriately defined stored energy of a tetanized myofibril with N contractile units has exponentially many local minima. We then argue that the ruggedness of the ensuing energy landscape is responsible for the experimentally observed history dependence and hysteresis in the mechanical response of a tetanized muscle near the descending limb. A nonlocal extension of the chain model, accounting for surrounding tissues, shows that both the ground states and the marginally stable states are fine mixtures of short and long HSs. These mixtures are homogeneous at the macro-scale and inhomogeneous at the micro-scale and we show that the negative overall slope of the step-wise tetanus can coexist with a positive instantaneous stiffness. A salient feature of the nonlocal model is that the variation of the degree of non-uniformity with elongation follows a complete devil’s staircase.

The non‐linear elasticity of the muscle sarcomere and the compliance of myosin motors

Force in striated muscle is due to attachment of the heads of the myosin, the molecular motors extending from the myosin filament, to the actin filament in each half-sarcomere, the functional unit where myosin motors act in parallel. Mechanical and X-ray structural evidence indicates that at the plateau of isometric contraction (force T0), less than half of the elastic strain of the half-sarcomere is due to the strain in the array of myosin motors (s), with the remainder being accounted for by the compliance of filaments acting as linear elastic elements in series with the motor array. Early during the development of isometric force, however, the half-sarcomere compliance has been found to be less than that expected from the linear elastic model assumed above, and this non-linearity may affect the estimate of s. This question is investigated here by applying nanometre-microsecond-resolution mechanics to single intact fibres from frog skeletal muscle at 4 °C, to record the mechanical properties of the half-sarcomere throughout the development of force in isometric contraction. The results are interpreted with mechanical models to estimate the compliance of the myosin motors. Our conclusions are as follows: (i) early during the development of an isometric tetanus, an elastic element is present in parallel with the myosin motors, with a compliance of ∼200 nm MPa(-1) (∼20 times larger than the compliance of the motor array at T0); and (ii) during isometric contraction, s is 1.66 ± 0.05 nm, which is not significantly different from the value estimated with the linear elastic model.

Crossbridge Properties during Fatigue and Recovery in Mouse Skeletal Muscle Fibres

Fatigue occurring during exercise can be defined as the inability to maintain the initial force or power output. We showed recently that fatigue during repetitive tetanic stimulation at 24 °C of fibre bundles dissected from FDB mouse (C57BL/6) muscle, occurs in two phases: an initial one during which individual crossbridge force decreases, and a later phase during which also crossbridge number decrease (Nocella et al.,2011 J Physiol 589).The present experiments were made on the same preparation to compare the fatigue mechanism at 24°C and at physiological temperature of 35 °C and to investigate the mechanism of force recovery from fatigue at 24°C. Fatigue was induced with 105 consecutive isometric tetani evoked every 1.5 s. Force recovery was followed by tetani evoked every 90s until force recovered to 90-100% of pre-fatigue value. Stiffness was measured with small sinusoidal length oscillations at 6.5 kHz. At both temperatures fatigue occurred initially through the decrease of the individual crossbridge force followed by the reduction of crossbridge number. However the initial phase lasted for ∼40 tetani at 35°C and ∼20 tetani at 24°C. This suggests a greater resistance to fatigue of this mechanism at high temperature. In contrast, during the second phase the tension loss was faster at 35°C than at 24°C so that after 105 tetani tension was similar at both temperatures. Force recovery also occurred in two phases. The initial phase lasted from ∼1.5-4 min and recovered 40-90% of tension loss. The second phase lasted for ∼60 min and its amplitude was well correlated with tension decrease during the second phase of fatigue. Thus the mechanism of tension recovery after fatigue seems symmetrical to tension loss during fatigue.

ROS-Mediated Decline in Maximum Ca2+-Activated Force in Rat Skeletal Muscle Fibers following In Vitro and In Vivo Stimulation

We hypothesised that normal skeletal muscle stimulated intensely either in vitro or in situ would exhibit reactive oxygen species (ROS)-mediated contractile apparatus changes common to many pathophysiological conditions. Isolated soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were bubbled with 95% O2 and stimulated in vitro at 31°C to give isometric tetani (50 Hz for 0.5 s every 2 s) until maximum force declined to ≤30%. Skinned superficial slow-twitch fibers from the SOL muscles displayed a large reduction (∼41%) in maximum Ca2+-activated specific force (Fmax), with Ca2+-sensitivity unchanged. Fibers from EDL muscles were less affected. The decrease in Fmax in SOL fibers was evidently due to oxidation effects on cysteine residues because it was reversed if the reducing agent DTT was applied prior to activating the fiber. The GSH∶GSSG ratio was ∼3-fold lower in the cytoplasm of superficial fibers from stimulated muscle compared to control, confirming increased oxidant levels. The presence of Tempol and L-NAME during in vitro stimulation prevented reduction in Fmax. Skinned fibers from SOL muscles stimulated in vivo at 37°C with intact blood supply also displayed reduction in Fmax, though to a much smaller extent (∼12%). Thus, fibers from muscles stimulated even with putatively adequate O2 supply display a reversible oxidation-induced decrease in Fmax without change in Ca2+-sensitivity, consistent with action of peroxynitrite (or possibly superoxide) on cysteine residues of the contractile apparatus. Significantly, the changes closely resemble the contractile deficits observed in a range of pathophysiological conditions. These findings highlight how readily muscle experiences ROS-related deficits, and also point to potential difficulties when defining muscle performance and fatigue.

Theory of muscle contraction mechanism with -cooperative interaction among crossbridges

The power stroke model was criticized and a model was proposed for muscle contraction mechanism (Mitsui, 1999). The proposed model was further developed and calculations based on the model well reproduced major experimental data on the steady filament sliding (Mitsui and Ohshima, 2008) and on the transient phenomena (Mitsui, Takai and Ohshima, 2011). In this review more weight is put on explanation of the basic ideas of the model, especially logical necessity of the model, leaving mathematical details to the above-mentioned papers. A thermodynamic relationship that any models based upon the sliding filament theory should fulfill is derived. The model which fulfills the thermodynamic relationship is constructed on the assumption that a myosin head bound to an actin filament forms a complex with three actin molecules. In shortening muscles, the complex moves along the actin filament changing the partner actin molecules with steps of about 5.5 nm. This process is made possible through cooperative interaction among cross-bridges. The ATP hydrolysis energy is liberated by fraction at each step through chemical reactions between myosin and actin molecules. The cooperativity among crossbridges disappears in length-clamped muscles, in agreement with experimental observations that the cross-bridge produces force independently in the isometric tetanus state. The distance of the head movement per ATP hydrolysis cycle is expected to be about 5.5 nm or a few times of it under the condition of the in vitro single head experiments. Calculation results are surveyed illustrating that they are in good agreement with major experimental observations.

Structural Changes in Myosin Heads and Filaments during Unloaded Shortening and Force Redevelopment

X-ray diffraction patterns were recorded with 5-ms time resolution at the ID02 beamline, ESRF, from single intact muscle fibers of the frog during steady shortening at the maximum velocity V0, imposed at the plateau of an isometric tetanus (T0), and during isometric force redevelopment following such shortening. During the first 20nm/half-sarcomere (hs) of shortening force decreased to near zero and changes in the X-ray pattern were consistent with a working stroke in actin-attached myosin heads followed by net detachment from actin (Piazzesi et al., Cell 131:784, 2007). As V0 shortening continued the M3 meridional reflection (associated with the conformation of the myosin heads) and its second order M6 (associated with the thick filament structure) became more like those recorded at rest. At 110 nm/hs shortening the M3 spacing and its fine structure were the same as at rest, while the M3 intensity, M6 spacing, and intensity of the first myosin layer line from the helical packing of the myosin heads, had recovered about half-way to their resting values, without sign of saturation. Isometric force redevelopment following 110 nm/hs shortening at V0 and the associated structural changes were faster than those at the start of electrical stimulation (Reconditi et al., PNAS, 108:7236, 2011). In both cases the initial force generation involves a small fraction of the myosin heads, whilst the majority are in the resting-like helically ordered conformation on the surface of the thick filament. The relationship between force and structural change is the same in the two cases for forces above 40% T0. The rates of the structural changes at the start of stimulation are limited by the rate of thin filament activation.Supported by MiUR and FIRB - Futuro in ricerca (Italy), MRC (UK), ESRF.

Sarcomere-Length Dependence of the Low Angle X-Ray Pattern from Skeletal Muscle Fibers at Rest and during Isometric Contraction

X-ray patterns were recorded from bundles of 2-3 fibers of R. Esculenta at 4°C at rest and at the plateau of an isometric tetanus (T0) at sarcomere length (SL) 2.0 to 3.6 μm at the ID02 beamline at the European Synchrotron Radiation Facility, Grenoble. The patterns were normalized by the intensity of the 1,0 equatorial reflection at rest at each experimental SL to compensate for variation of diffracting mass with SL and between fibers. The axial diffraction pattern from resting fibers was independent of SL in the range 2.0-2.6 μm; in the range 2.6-3.0 μm the intensity and interference fine structure of the meridional M3 reflection from the axial repeat of the myosin heads along the filaments was constant, but its spacing (SM3) increased. The intensity of the first myosin layer line decreased in this SL range, indicating decreased helical ordering of the myosin heads. The intensity of the 44nm merdional reflection associated with myosin binding protein C was constant up to SL 2.7 μm, but much reduced for SL>2.7 μm. At T0, the M3 reflection intensity was smaller at longer SL, in proportion to the overlap between thick and thin filaments. The interference fine structure of the M3 was independent of SL up to 2.8 μm; at longer SL it varied between preparations and SM3 reduced with increasing SL. The SL-dependence of the M3 reflection at T0 indicates that the detached myosin heads in the non-overlap region of the thick filament are axially disordered compared with actin-attached heads in the overlap region, although the axial center of mass of the detached and attached heads is similar. Supported by Ente Cassa di Risparmio di Firenze, FIRB-Futuro in Ricerca, MRC and ESRF.

The different muscle-energetics during shortening and stretch.

The helical shape of the thin filaments causes their passive counterclockwise rotation during muscle stretch that increases tensile stress and torque at first by unwinding and then by winding up the four anchoring Z-filaments. This means storage of energy in the series elastic Z-filaments and a considerable decrease of the liberated energy of heat and work to (h—wap), where h is the heat energy and wap the stretch energy induced from outside by an apparatus. The steep thin filament helix with an inclination angle of 70° promotes the passive rotation during stretch, but impedes the smooth sliding of shortening by increased friction and production of frictional heat. The frictional heat may be produced by the contact with the myosin cross-bridges: (1) when they passively snap on drilling thin filaments from cleft to cleft over a distance 2 × 2.7 nm = 5.4 nm between the globular actin monomers in one groove, causing stepwise motion; or (2) when they passively cycle from one helical groove to the next (distance 36 nm). The latter causes more heat and may take place on rotating thin filaments without an effective forward drilling (“idle rotation”), e.g., when they produce “unexplained heat” at the beginning of an isometric tetanus. In an Appendix to this paper the different states of muscle are defined. The function of its most important components is described and rotation model and power-stroke model of muscular contraction is compared.

The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle

One of the obstacles to the potential clinical utility of bioengineered skeletal muscle is its limited force generation capacity. Since engineered muscle, unlike most native muscle tissue, is composed of relatively short myofibers, we hypothesized that, its force production and transmission would be profoundly influenced by cell-matrix interactions. To test this hypothesis, we systematically varied the matrix protein type (collagen I/fibrin/Matrigel) and concentration in engineered, hydrogel-based neonatal rat skeletal muscle bundles and assessed the resulting tissue structure, generation of contractile force, and intracellular Ca 2+ handling. After two weeks of culture, the muscle bundles consisted of highly aligned and cross-striated myofibers and exhibited standard force–length and force–frequency relationships achieving tetanus at 40 Hz. The use of 2 mg/ml fibrin (control) yielded isometric tetanus amplitude of 1.4 ± 0.3 mN as compared to 0.9 ± 0.4 mN measured in collagen I-based bundles. Higher fibrin and Matrigel concentrations synergistically yielded further increase in active force generation to 2.8 ± 0.5 mN without significantly affecting passive mechanical properties, tetanus-to-twitch ratio, and twitch kinetics. Optimized matrix composition yielded significant cellular hypertrophy (protein/DNA ratio = 11.4 ± 4.1 vs. 6.5 ± 1.9 μg/μg in control) and a prolonged Ca 2+ transient half-width (Ca 50 = 232.8 ± 33.3 vs. 101.7 ± 19.8 ms). The use of growth-factor-reduced Matrigel, instead of standard Matrigel did not alter the obtained results suggesting enhanced cell-matrix interactions rather than growth factor supplementation as an underlying cause for the measured increase in contractile force. In summary, biomaterial-based manipulation of cell-matrix interactions represents an important target for improving contractile force generation in engineered skeletal muscle.

What limits exercise during high-intensity aerobic exercise?

An important and unresolved issue is the mechanism(s) that lead to task failure during intense aerobic exercise. Marcora and Staiano (2010) have recently reinvestigated this problem using human subjects on a bicycle ergometer. Subjects were asked to exercise at 80% of their maximal aerobic power (242 W) and failed (unable to maintain the pedal frequency above 60 min despite encouragement) after 10.5 min. Under control conditions, their peak power output measured over 5 s and at a pedal frequency of 137 min was 1,075 W. When peak power was measured as soon as possible after the fatiguing event, it was 731 W. Because peak power (731 W) was so much greater than the power which the subjects failed to achieve at the end of the exercise period (242 W) Marcora and Staiano argue that muscle performance was not the main problem, instead the subjects stopped because their ‘perception of effort’ was greater than they were prepared to tolerate. One interpretation problem with these experiments has already been raised by Burnley (2010) who is concerned that the difference in pedal frequency at exhaustion (\60 min) as compared to the peak power (137 min) may compromise the comparison. Here, we raise a second interpretation issue with potential impact on Marcora and Staiano’s conclusion. Our concern is based on the fact that a small degree of muscle recovery can have a large effect on the power output of fatigued muscles. Because of the need to change the mode of the ergometer between the exercise bout and the peak power determination, there was a delay of 3–4 s between these activities in Marcora and Staiano’s study. It is conceivable that this short period was sufficient for a substantial recovery of power between the two activities. Marcora and Staiano argue against this possibility on the grounds that recovery of peak power after high-intensity exercise is relatively slow with a half time of 32 s (Sargeant and Dolan 1987). Whilst this is correct, it is also the case that half the recovery to 100% occurred at 5 s (Fig. 6, Sargeant and Dolan 1987). Thus, we suggest that the subjects’ muscles may have been severely fatigued and that much of the observed difference between the fatigued performance and the peak power may simply be rapidly occurring recovery processes. In further support of our interpretation, we point to experiments on isolated muscle in which substantial recovery from fatigue occurs in a few seconds. For instance, in mouse muscle fibres fatigued by repeated isometric tetani, it is possible for force to recover by 100% in 10 s (Fig. 5, Westerblad and Allen 1991). This large effect is related to the fact that the slope of the force–Ca relation is very steep in fatigued muscles so a small recovery of sarcoplasmic reticulum (SR) Ca release and/or myofibrillar Ca sensitivity leads to a large recovery of force. For instance, with the force–Ca relation being described by a Hill equation with a Hill coefficient of 5, a 10% increase in either tetanic Ca or myofibrillar Ca sensitivity results in a force increase of *40%. In addition, the loss of power during exercise involves a decrease in shortening velocity (Jones et al. 2006), and experiments on single mouse muscle fibres show that this slowing Communicated by Susan Ward.

The influence of muscle length on the fatigue-related reduction in joint range of motion of the human dorsiflexors

The fatigue-related reduction in joint range of motion (ROM) during dynamic contraction tasks may be related to muscle length-dependent alterations in torque and contractile kinetics, but this has not been systematically explored previously. Twelve young men performed a repetitive voluntary muscle shortening contraction task of the dorsiflexors at a contraction load of 30% of maximum voluntary isometric contraction (MVC) torque, until total 40 degrees ROM had decreased by 50% at task failure (POST) to 20 degrees ROM. At both a short (5 degrees dorsiflexion) and long muscle length (35 degrees plantar flexion joint angle relative to a 0 degrees neutral ankle joint position), voluntary activation, MVC torque, and evoked tibialis anterior contractile properties of a 52.8 Hz high-frequency isometric tetanus [peak evoked torque, maximum rate of torque development (MRTD), maximum rate of relaxation (MRR)] were evaluated at baseline (PRE), at POST, and up to 10 min of recovery. At POST, we measured similar fatigue-related reductions in torque (voluntary and evoked) and slowing of contractile kinetics (MRTD and MRR) at both the short and long muscle lengths. Thus, the fatigue-related reduction in ROM could not be explained by length-dependent fatigue. Although torque (voluntary and evoked) at both muscle lengths was depressed and remained blunted throughout the recovery period, this was not related to the rapid recovery of ROM at 0.5 min after task failure. The reduction in ROM, however, was strongly related to the reduction in joint angular velocity (R(2) = 0.80) during the fatiguing task, although additional factors cannot yet be overlooked.

The Isotonic Velocity Transient Following a Sudden Rise in Force Imposed on the Muscle Sarcomere During Unloaded Shortening Reveals a Rate Limiting Step in Detached Myosin Motors

Energy balance studies indicate that ATP splitting by myosin motors during rapid shortening of skeletal muscle is not sufficient to account for the energy (mostly heat) output (Rall et al., J Gen Physiol 68(1), 13, 1976; Homsher et al., J Physiol 321, 423, 1981). We investigated the kinetic step of the myosin ATP-ase cycle related to this phenomenon in single muscle fibers from Rana esculenta (∼2.15 μm sarcomere length, 4°C), by recording the isotonic velocity transient following a force step from zero to the isometric tetanic value (T0). Once the isometric tetanus had developed, the force was first clamped to zero for a range of times from 10 ms to 18 ms (during which the fiber shortened at the maximum velocity by 30 nm hs−1 to 50 nm hs−1) and then raised again to T0 in a stepwise manner (∼120 μs). The elastic lengthening induced by the force step was followed by a transient isotonic lengthening, the size of which ranged from 40 to 60 nm hs−1 depending on the size of the preceding shortening. The lengthening velocity was larger for larger shortening size and progressively decreased to approach the isometric condition with a half-time of 2-3 ms. Similarly, the half-sarcomere stiffness recovered the isometric value e0 from the unloaded shortening value of 0.4 e0 with an exponential time course with τ ∼3 ms. We conclude that during rapid shortening a ∼3 ms-transition between detached states of the myosin motor, likely related to the ATP hydrolysis, becomes rate limiting. Accumulation of motors in the state preceding the hydrolysis step can account for the unexplained energy during rapid shortening. Supported by MIUR (Italy).

Reduced thin filament length in nebulin-knockout skeletal muscle alters isometric contractile properties

Nebulin (NEB) is a large, rod-like protein believed to dictate actin thin filament length in skeletal muscle. NEB gene defects are associated with congenital nemaline myopathy. The functional role of NEB was investigated in gastrocnemius muscles from neonatal wild-type (WT) and NEB knockout (NEB-KO) mice, whose thin filaments have uniformly shorter lengths compared with WT mice. Isometric stress production in NEB-KO skeletal muscle was reduced by 27% compared with WT skeletal muscle on postnatal day 1 and by 92% on postnatal day 7, consistent with functionally severe myopathy. NEB-KO muscle was also more susceptible to a decline in stress production during a bout of 10 cyclic isometric tetani. Length-tension properties in NEB-KO muscle were altered in a manner consistent with reduced thin filament length, with length-tension curves from NEB-KO muscle demonstrating a 7.4% narrower functional range and an optimal length reduced by 0.13 muscle lengths. Expression patterns of myosin heavy chain isoforms and total myosin content did not account for the functional differences between WT and NEB-KO muscle. These data indicate that NEB is essential for active stress production, maintenance of functional integrity during cyclic activation, and length-tension properties consistent with a role in specifying normal thin filament length. Continued analysis of NEB's functional properties will strengthen the understanding of force transmission and thin filament length regulation in skeletal muscle and may provide insights into the molecular processes that give rise to nemaline myopathy.

Structural Changes in the Myosin Motors During Activation of Skeletal Muscle

Structural changes in the myosin motors during the transition from the resting state to the plateau of isometric contraction were investigated by X-ray interference from single fibers of frog skeletal muscle. Isolated intact fibers (2.1μm sarcomere length, 4°C) were mounted vertically at beamline ID2 of the ESRF synchrotron (Grenoble, France) between a loudspeaker motor and a capacitance force transducer. 2D diffraction patterns were collected on a CCD detector 10 m from the preparation with 5 ms time resolution. During the development of the isometric tetanus, the intensity of the M3 reflection, originating from the axial repeat of the myosin motors, first decreases to 30% (at 50 ms) of its resting value, then increases to a steady value 70% of that at rest. The M3 reflection has a major peak with spacing 14.34 nm at rest and two peaks with mean spacing 14.57 nm at the tetanus plateau (Linari et al., Proc. Natl. Acad. Sci. USA 97:7226, 2000). The changes in the fine structure of the M3 reflection during activation were best fit by a structural model in which (1) all thick filaments have the same mean spacing at a given time during activation (2) the number of active motors increases in proportion to the isometric force (Brunello et al., J. Physiol. 577:971 2006), (3) the conformation of the active motors is independent of the level of force and strain in the thick filament.Supported by MiUR Italy, MRC UK, CNISM, EMBO, ESRF, EMBL.