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

Abstract

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 crossbridges, 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 ؋ 10 3 nm⅐hs ؊1 ⅐s ؊1 or 3.3 fiber length per second (l0s ؊1 ) and 6. cross-bridges ͉ rupture force ͉ detachment rate constant ͉ fast stretches A tomic force microscopy (1-4) and force spectroscopy (5, 6) have been used to reveal the properties of the weak noncovalent bond between receptor molecules and their ligands, and in particular dynamic force spectroscopy has been used to define in detail the properties of the single biotin-avidin bond (1-3, 7). As regarding molecular motors, the optical tweezers technique has allowed the investigation of the properties of an individual actomyosin bond and to measure the force and unitary step displacement generated (8-10). The properties of the single actomyosin bond under rigor conditions were also investigated by measuring the dependence of the bond lifetime on the applied load (11). It was found that bond lifetime was strongly reduced when a load was applied to the bond, in good quantitative agreement with the prediction of Bell theory (12). These results on single actomyosin interaction represent a great step forward in our understanding of molecular mechanism of force generation in muscle; however, they were generally obtained under experimental conditions that differ from those encountered by the same motors in native preparation. In living skeletal muscle, myosin heads (the molecular motors) are distributed over the myosin filament in a very regular fashion, and both actin and myosin filaments are arranged in a quasicrystalline lattice that maintains a precise geometrical relationship between myosin heads (or fraction S1 of myosin molecule) and the active sites regularly distributed along the actin filament (13). This remarkable order is lost when experiments are made on single molecules, and this may affect the bond properties and force developed. In addition, at physiological ionic strength, actin filaments tend to detach from myosin heads, and it is therefore necessary to perform the above experiments at low ionic strength. However, it is known that molecular motor performance strongly depends on ionic strength (14). The force generated is also strongly affected by myosin S1 orientation, which is not easily controlled in single-molecule experiments (14). This means that the force measured by the recording apparatus, usually along the actin filament axis, may not represent accurately the real capability of the motors when working in a regularly ordered array. The same is true in experiments in which an external load is applied to the single bond, because it is usually d...

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

“…Most of our data were obtained on the tetanus rise at a tension level between 0.2 and 0.6 P 0 at which fibre damage was very much reduced. Lowering the tension was also important to reduce crossbridge strain non‐uniformity (Colombini et al 2007 b ). To determine the crossbridge rupture force, fast ramp test stretches (duration between 0.4 ms and 0.9 ms and 16–25 nm per half‐sarcomere (nm hs −1 ) amplitude, corresponding to stretching velocity of 16.9 and 59.5 fibre lengths per second ( l 0 s −1 )) were applied to one end of the activated fibre while force response was measured at the other end.…”

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

“…Measurements at 5°C and 14°C were made on the same fiber in a group of 12 fibers at resting sarcomere length of about 2.1 m. Given the relative small stretches used, no change in effective overlap between myofilaments occurred during the experiments. Further details of measurements, including the correction for the first fast phase of the force response due to fiber inertia, can be found in our previous papers (1,3 Figure 1A shows the well-known potentiating effect of temperature on tetanic tension. In this fiber, tension increased by 27% as the temperature was raised from 5°C to 14°C.…”

The lifetime of the actomyosin complex in vitro under load corresponding to stretch of contracting muscle

“…It has also been shown that during stretch a majority of actin-bound myosin heads are attached non-stereospecifically or weakly (Ferenczi et al 2014). The rate of forced detachment of myosin heads during fast stretch of muscle fibers was shown to increase with the stretching force (Colombini et al 2007), in agreement with Bell's theory (Bell 1978).…”