The effects of fatigue and oxidation on contractile function of intact muscle fibers and myofibrils isolated from the mouse diaphragm.

The goal of this study was to investigate the effects of repetitive stimulation and the oxidant H2O2 on fatigue of diaphragm intact fibers and in myofibrils measured with different Ca2+ concentrations. Intact fibers were isolated from mice diaphragm, and twitch and tetanic contractions (500 ms duration) were performed at different frequencies of stimulation ranging from 15 Hz to 150 Hz to establish a force-frequency relation before and after a fatigue and recovery protocol, without or after a treatment with H2O2. Fatigue was induced with isometric contractions (500 ms, 40 Hz) evoked every 0.8 seconds, with a total of 625 tetani. After the fatigue, the force recovery was followed by invoking tetanic contractions (500 ms, 40 Hz) every 1 min, with a total duration of 30 min. Individual myofibrils were also isolated from the mouse diaphragm and were tested for isometric contractions before and after treatment with H2O2 and NAC. In a second series of experiments, myofibrils were activated at different pCa (pCa = -log10 [Ca2+]), before and after H2O2 treatment. After 15 minutes of H2O2 treatment, the myofibrillar force was decreased to 54 ± 12% of its control, maximal value, and a result that was reversed by NAC treatment. The force was also decreased after myofibrils were treated with H2O2 and activated in pCa ranging between 4.5 and 5.7. These results suggest that fatigue in diaphragm intact fibers and at the myofibrils level is caused partially by oxidation of the contractile proteins that may be responsible for changing the force in various levels of Ca2+ activation.

Force decline during fatigue is due to both a decrease in the force per individual cross-bridge and the number of cross-bridges.

Fatigue occurring during exercise can be defined as the inability to maintain the initial force or power output. As fatigue becomes pronounced, force and maximum velocity of shortening are greatly reduced and force relaxation is prolonged. In principle, force loss during fatigue can result from a decrease in the number of cross-bridges generating force or a decrease of the individual cross-bridge force or to both mechanisms. The present experiments were made to investigate this point in single fibres or small fibre bundles isolated from flexor digitorum brevis (FDB) of C57BL/6 mice at 22-24◦C. During a series of 105 tetanic contractions, we measured force and fibre stiffness by applying small sinusoidal length oscillations at 2.5 or 4 kHz frequency to the activated preparation and measuring the resulting force changes. Stiffness data were corrected for the influence of compliance in series with the cross-bridge ensemble. The results show that the force decline during the first 20 tetani is due to the reduction of force developed by the individual cross-bridges and thereafter as fatigue becomes more severe, the number of cross-bridges decreases. In spite of the force reduction in the early phase of fatigue, there was an increased rate of tetanic force development and relaxation. In the latter stages of fatigue, the rate of force development and relaxation became slower. Thus, the start of fatigue is characterised by decreased cross-bridge force development and as fatigue becomes more marked, the number of cross-bridges decreases. These findings are discussed in the context of the current hypotheses about fatigue mechanisms.

Cross-bridge properties in single intact frog fibers studied by fast stretches.

Cross-bridges properties were measured under different experimental conditions by applying fast stretches to activated skeletal frog muscle fiber to -forcibly detach the cross-bridge ensemble. This allowed to measure the tension needed to detach the cross-bridges, P(c), and the sarcomere elongation at the rupture force, L(c). These two parameters are expected to be correlated with cross-bridges number (P(c)) and their mean extension (L(c)). Conditions investigated were: tetanus rise and plateau under normal Ringer and Ringer containing different BDM -concentrations, hyper (1.4T) and hypotonic (0.8T) solutions, 5 and 14 degrees C temperature. P(c) was linearly correlated with the tension (P) developed by the fibers under all the conditions examined, however the ratio P(c)/P changed depending on conditions being greater at low temperature and higher tonicity. These results indicate that, (a) P(c) can be used as a measure of attached cross-bridge number and (b) the force developed by the individual cross-bridge increases at high temperature and low tonicity. L(c) was not affected by tension developed, however it changed under different conditions, being greater at low temperature and high tonicity. These findings, suggests, in agreement with P(c) data, that cross-bridge extension is smaller at low temperature and high tonicity. By comparing these data with tetanic tension we concluded that potentiation or depression induced on tetanic force by tonicity or temperature changes are entirely accounted for by changes of the force developed by the individual cross-bridge.

Is the cross-bridge stiffness proportional to tension during muscle fiber activation?

The cross-bridge stiffness can be used to estimate the number of S1 that are bound to actin during contraction, which is a critical parameter for elucidating the fundamental mechanism of the myosin motor. At present, the development of active tension and the increase in muscle stiffness due to S1 binding to actin are thought to be linearly related to the number of cross-bridges formed upon activation. The nonlinearity of total stiffness with respect to active force is thought to arise from the contribution of actin and myosin filament stiffness to total sarcomere elasticity. In this work, we reexamined the relation of total stiffness to tension during activation and during exposure to N-benzyl-p-toluene sulphonamide, an inhibitor of cross-bridge formation. In addition to filament and cross-bridge elasticity, our findings are best accounted for by the inclusion of an extra elasticity in parallel with the cross-bridges, which is formed upon activation but is insensitive to the subsequent level of cross-bridge formation. By analyzing the rupture tension of the muscle (an independent measure of cross-bridge formation) at different levels of activation, we found that this additional elasticity could be explained as the stiffness of a population of no-force-generating cross-bridges. These findings call into question the assumption that active force development can be taken as directly proportional to the cross-bridge number.

Reversal of the myosin power stroke induced by fast stretching of intact skeletal muscle fibers.

Force generation and movement in skeletal muscle result from a cyclical interaction of overlapping myosin and actin filaments that permits the free energy of ATP hydrolysis to be converted into mechanical work. The rapid force recovery that occurs after a step release imposed on a muscle is thought to result from a synchronized tilting of myosin lever arms toward a position of lower free energy (the power stroke). We investigated the power stroke mechanism in intact muscle fibers of Rana esculenta using a fast stretch to detach forcibly cross-bridges. Stretches were applied either with or without a conditioning step release. Cross-bridge rupture tension was not significantly influenced by the release, whereas sarcomere elongation at the rupture point increased immediately after the release and returned to the prerelease condition within 15-20 ms, following a slower time course compared to the recovery of tension. These observations suggest that the rupture force of a bridge is unaltered by a conditioning release, but rupture must first be preceded by a power stroke reversal, which restores the prepower stroke state. The sarcomere extension at the rupture point indicates both the extent of this power stroke reversal and the time course of strained bridge replenishment.

Mechanical properties of intact single fibres from wild-type and MLC/mIgf-1 transgenic mouse muscle.

The effects of overexpression of the local form of insulin like growth factor-1 (mIgf-1) on skeletal muscle were investigated by comparing the mechanical properties of single intact fibres from the flexor digitorum brevis of wild-type (WT) and (MLC/mIgf-1) transgenic mice (TG)at 21-24 degrees C. Isolated single fibres were clean enough to measure accurately the sarcomere length. The parameters investigated were: tetanic absolute and specific force, the force-velocity relationship, and the sarcomere length-tension relationship. In addition, we investigated the properties of the "static stiffness", a non-crossbridge Ca(2+)-dependent increase of fibre stiffness previously found in frog muscle. Both average cross-sectional area and tetanic force almost doubled in TG fibres, so that specific force was the same in both preparation: 312 +/- 20 and 344 +/- 34 kN m(-2) in WT and TG fibres, respectively. None of the relative force-velocity parameters was altered by Igf-1 overexpression, however, V(max) (8-10 l(0) s(-1)) was greater than previously reported in whole muscles. The sarcomere length-tension relationship was the same in TG and WT fibres showing the classical shape with a plateau region between 2.28 and 2.52 microm and a linear descending limb. The static stiffness was present in both WT and TG fibres and showed similar characteristics to that of frog skeletal muscle. In contrast to the other parameters, static stiffness in TG fibres was about 24% smaller than in WT fibres suggesting a possible effect of Igf-1 overexpression on its mechanism.

Effect of temperature on cross-bridge properties in intact frog muscle fibers.

It is well known that the force developed by skeletal muscles increases with temperature. Despite the work done on this subject, the mechanism of force potentiation is still debated. Most of the published papers suggest that force enhancement is due to the increase of the individual cross-bridge force. However, reports on skinned fibers and single-molecule experiments suggest that cross-bridge force is temperature independent. The effects of temperature on cross-bridge properties in intact frog fibers were investigated in this study by applying fast stretches at various tension levels (P) on the tetanus rise at 5 degrees C and 14 degrees C to induce cross-bridge detachment. Cross-bridge number was measured from the force (critical force, P(c)) needed to detach the cross-bridge ensemble, and the average cross-bridge strain was calculated from the sarcomere elongation needed to reach P(c) (critical length, L(c)). Our results show that P(c) increased linearly with the force developed at both temperatures, but the P(c)/P ratio was considerably smaller at 14 degrees C. This means that the average force per cross bridge is greater at high temperature. This mechanism accounts for all the tetanic force enhancement. The critical length L(c) was independent of the tension developed at both temperatures but was significantly lower at high temperature suggesting that cross bridges at 14 degrees C are more strained. The increased cross-bridge strain accounts for the greater average force developed.

Characterization of actomyosin bond properties in intact skeletal muscle by force spectroscopy.

Force generation and motion in skeletal muscle result from interaction between actin and myosin myofilaments through the cyclical formation and rupture of the actomyosin bonds, the cross-bridges, in the overlap region of the sarcomeres. Actomyosin bond properties were investigated here in single intact muscle fibers by using dynamic force spectroscopy. The force needed to forcibly detach the cross-bridge ensemble in the half-sarcomere (hs) was measured in a range of stretching velocity between 3.4 x 10(3) nm.hs(-1).s(-1) or 3.3 fiber length per second (l(0)s(-1)) and 6.1 x 10(4) nm.hs(-1).s(-1) or 50 l(0).s(-1) during tetanic force development. The rupture force of the actomyosin bond increased linearly with the logarithm of the loading rate, in agreement with previous experiments on noncovalent single bond and with Bell theory [Bell GI (1978) Science 200:618-627]. The analysis permitted calculation of the actomyosin interaction length, x(beta) and the dissociation rate constant for zero external load, k(0). Mean x(beta) was 1.25 nm, a value similar to that reported for single actomyosin bond under rigor condition. Mean k(0) was 20 s(-1), a value about twice as great as that reported in the literature for isometric force relaxation in the same type of muscle fibers. These experiments show, for the first time, that force spectroscopy can be used to reveal the properties of the individual cross-bridge in intact skeletal muscle fibers.

Crossbridge properties investigated by fast ramp stretching of activated frog muscle fibres.

Very fast ramp stretches at 9.5-33 sarcomere lengths s(-1) (l0 s(-1)) stretching speed, 16-25 nm per half-sarcomere (nm hs(-1)) amplitude were applied to activated intact frog muscle fibres at tetanus plateau, during the tetanus rise, during the isometric phase of relaxation and during isotonic shortening. Stretches produced an almost linear tension increase above the isometric level up to a peak, and fell to a lower value in spite of continued stretching, indicating that the fibre became suddenly very compliant. This suggests that peak tension (critical tension, P(c)) represents the tension at which crossbridges are forcibly detached by the stretch. The ratio of P(c) to the isometric tension at tetanus plateau (P0) was 2.37 +/- 0.12 (S.E.M.). This ratio did not change significantly at lower tension (P) during the tetanus rise but decreased with time during the relaxation and increased with speed during isotonic shortening. At tetanus plateau P(c) occurred when sarcomere elongation attained a critical length (L(c)) of 10.98 +/- 0.13 nm hs(-1), independently of the stretching speed. L(c) remained constant during the tetanus rise but decreased on the relaxation and increased during isotonic shortening. Length-clamp experiments on the relaxation showed that the lower values of P(c)/P ratio and L(c), were both due to the slow sarcomere stretching occurring during this phase. Our data show that P(c) can be used as a measure of crossbridge number, while L(c) is a measure of crossbridge mean extension. Accordingly, for a given tension, crossbridges on the isometric relaxation are fewer than during the rise, develop a greater individual force and have a greater mean extension, while during isotonic shortening crossbridges are in a greater number but develop a smaller individual force and have a smaller extension.