Giulia Benelli

Crossbridge properties during force enhancement by slow stretching in single intact frog muscle fibres.

The mechanism of force enhancement during lengthening was investigated on single frog muscle fibres by using fast stretches to measure the rupture tension of the crossbridge ensemble. Fast stretches were applied to one end of the activated fibre and force responses were measured at the other. Sarcomere length was measured by a striation follower device. Fast stretching induced a linear increase of tension that reached a peak and fell before the end of the stretch indicating that a sudden increase of fibre compliance occurred due to forced crossbridge detachment induced by the fast loading. The peak tension (critical tension, Pc) and the sarcomere length needed to reach Pc (critical length, Lc) were measured at various tensions during the isometric tetanus rise and during force enhancement by slow lengthening. The data showed that Pc was proportional to the tension generated by the fibre under both isometric and slow lengthening conditions. However, for a given tension increase, Pc was 6.5 times greater during isometric than during lengthening conditions. Isometric critical length was 13.04 ± 0.17 nm per half‐sarcomere (nm hs−1) independently of tension. During slow lengthening critical length fell as the force enhancement increased. For 90% enhancement, Lc reduced to 8.19 ± 0.039 nm hs−1. Assuming that the rupture force of the individual crossbridge is constant, these data indicate that the increase of crossbridge number during lengthening accounts for only 15.4% of the total force enhancement. The remaining 84.6% is accounted for by the increased mean strain of the crossbridges.

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.

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.