M. Angela Bagni

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

Very fast ramp stretches at 9.5–33 sarcomere lengths s−1 (l0s−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, Pc) represents the tension at which crossbridges are forcibly detached by the stretch. The ratio of Pc 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 Pc occurred when sarcomere elongation attained a critical length (Lc) of 10.98 ± 0.13 nm hs−1, independently of the stretching speed. Lc 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 Pc/P ratio and Lc, were both due to the slow sarcomere stretching occurring during this phase. Our data show that Pc can be used as a measure of crossbridge number, while Lc 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.

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 × 103 nm·hs−1·s−1 or 3.3 fiber length per second (l0s−1) and 6.1 × 104 nm·hs−1·s−1 or 50 l0·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, xβ and the dissociation rate constant for zero external load, k0. Mean xβ was 1.25 nm, a value similar to that reported for single actomyosin bond under rigor condition. Mean k0 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.

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.

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

Non‐technical summary  Prolonged muscle activity leads to a reduction of mechanical power and force output which is commonly indicated as muscular fatigue. The development of fatigue during repetitive stimulation of a skeletal muscle consists of an initial phase during which force decreases by 10–15%. This is followed by a second phase where force remains almost constant and finally a phase during which force drops precipitously to low levels. We show here that the initial fall in force is due to a reduction of the force generated by the individual molecular force generator, the cross‐bridge, whereas in subsequent phases the force decrease is caused by a reduction in the number of molecular force generators. These results increase our understanding of muscular fatigue mechanisms.

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.

Force Response to Stretches in Activated Frog Muscle Fibres at Low Tension

It is well known that tension development in skeletal muscle fibres upon electrical activation is preceded by an increase of muscle stiffness that begins during the latent period and continues throughout the whole tension rise in both twitch or tetanic contractions (Bressler and Clinch, 1974; Cecchi et al., 1982; Ford et al., 1986). At moderate and high tensions, fibre stiffness increase is essentially due to crossbridge attachment. However, as shown previously (Bagni et al., 1994; Bagni et al., 2002), at zero tension during the latent period and at very low tension during force development, a substantial contribution to fibre stiffness cames from some (unknown) sarcomere structure(s), outside the crossbridges, whose stiffness increases upon stimulation. The presence of this stiffness was inferred from the force response to stretches (and hold) applied to a single muscle fibre during force generation. It was found that the fast force transient produced by the stretch was followed by a period during which the tension settled to a consistent level greater than the isometric tension at the time of the stretch, until relaxation or until the fibre returned to the original length at end of the stretch. Because of this characteristic the excess of tension respect to isometric was referred to as static tension while the ratio between static tension and stretch amplitude, measured at sarcomere level, was termed static stiffness. Experiments made on tetanic contractions in Ringer containing 1-6 mM of 2,3-butanedione monoxime (BDM), an agent which strongly inhibits crossbridge formation (Horiuti et al., 1988; Bagni et al., 1992) without altering static stiffness (Bagni et al., 1994), showed that the structure responsible for static stiffness behaves with Hookean elasticity located in parallel with the crossbridges (Bagni et al., 2002). Interestingly, in both twitch or tetanic contractions, static stiffness development followed a characteristic time course distinct from that of tension and roughly similar to that of internal calcium concentration.

Use of Sinusoidal Length Oscillations to Detect Myosin Conformation by Time- Resolved X-Ray Diffraction

In contrast to many other theories of muscle contraction, the proposal of H.E. Huxley (Huxley, 1969), that force generation by actomyosin systems results from an active tilting of the S1 moiety of myosin, defined a structural change as the central event of the force generation process. As a result, a large range of techniques for probing structural changes in myosin have been applied to the study of contraction, but have yielded only equivocal support for the tilting S1 theory (Yanagida, 1981; Cooke et al., 1982; Tanner et al., 1992). Uniquely, because of the highly ordered structure of striated muscles, X-ray diffraction provides a powerful probe of structural events in this tissue, and the enormous advances over the last 40 years in both detector technology and intensity of X-ray sources available have permitted time-resolved X-ray diffraction studies of intact, working muscle cells to advance from the time domain of hours to that of microseconds.

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.

Static Stiffness in Slow and Fast Mouse Muscle Fibers Expressing Different Titin Isoforms

We showed previously (Bagni et al., 2002) that most of the increase of muscle fiber stiffness during the early phases of a tetanic contraction is due to a non-crossbridge sarcomere component whose stiffness (called static stiffness) increases after stimulation with a time course very similar to the internal Ca2+ concentration. This led us to speculate that Ca2+ concentration, in addition to promote crossbridge formation, could also leads to a stiffening of a sarcomere structure, identified with the titin filament, either directly or through a titin-actin interaction leading to the observed sarcomere stiffness increase. According to this hypothesis, it is expected that static stiffness has different properties in muscles expressing titin with different mechanical properties. Therefore we compared the static stiffness values in soleus and EDL adult mouse muscles, which express titin isoforms with long and short PEVK segment, respectively. Considering that Ca2+ binding to E-rich motifs in the PEVK segment increases its bending rigidity, the higher proportion of these motifs in EDL compared to soleus is expected to lead to a greater static stiffness in EDL. Our results showed that in agreement with the titin hypothesis, the static stiffness measured in single fibers at 25°C was more than five times greater in EDL than in soleus and about two times greater than previously reported on FDB muscle. The static stiffness time course in EDL was about the same as in FDB but slightly faster than in soleus, and it became much faster at 35°C in both EDL and soleus similarly to tension time course. These results are in agreement with the idea that static stiffness depends on the increment of titin stiffness due to the interaction between Ca2+ and E-rich motifs in PEVK segment.