Aim: 

We study the relation between chemical and mechanical steps of the myosin-actin ATPase cycle in muscle by applying fast mechanics to skinned fibres of rabbit psoas muscle. Here we investigate the relation between release of orthophosphate (Pi) from the myosin motor domain and force generation, by determining the effects of the increase in [Pi] on the stiffness of the half-sarcomere (hs) during transient and steady state conditions of the isometric contraction.

Methods: 

The fibre is Ca⁺⁺-activated at 2.5 mm sarcomere length by using a temperature jump method (from 1 to 12.5 °C), to preserve the ordered sarcomeric structure (Linari et al. Biophys J92:2476, 2007). Stiffness is measured by the force response to step changes in sarcomere length (rise time 110 ms, amplitude range 4 nm per hs).

Results: 

In control conditions (0 Pi), we find that the hs stiffness is 22.8 1.1 kPa/nm and, taking into account of filament compliance, the stiffness of the array of myosin cross-bridges (e) is 40.7 3.7 kPa/nm. Increase in [Pi] (range 3–20 mM) decreases e, and thus the number of myosin cross-bridges, in proportion with the isometric force. During isometric force redevelopment following a period of unloaded shortening, e increases in proportion with force, indicating that there is no significant (>1 ms) delay between stiffness generation and force generation by the cross-bridges.

Conclusions: 

These results, as well as the reduced effect of Pi on the isometric ATPase rate (Potma et al. Biophys J69:2580, 1995), are explained with a kinetic model that provides that, under isometric conditions, the myosin cross-bridges can detach at an early stage of the ATPase cycle, with Pi still bound to their catalytic site, and then rapidly release the hydrolysis products and bind another ATP.

Supported by NIH (R01 AR049033), MiUR and ITB-CNR