Pre-Power-Stroke Cross-Bridges Contribute to Force Transients during Imposed Shortening in Isolated Muscle Fibers

Fabio C Minozzo,Lennart Hilbert,Dilson E Rassier,Beata G Vertessy

doi:10.1371/journal.pone.0029356

Fabio C Minozzo, Lennart Hilbert + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0029356

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Journal: PloS one	Publication Date: Jan 5, 2012
Citations: 39	License type: CC BY 4.0

Affiliation: McGill University, Christie (Canada)

Abstract

When skeletal muscles are activated and mechanically shortened, the force that is produced by the muscle fibers decreases in two phases, marked by two changes in slope (P1 and P2) that happen at specific lengths (L1 and L2). We tested the hypothesis that these force transients are determined by the amount of myosin cross-bridges attached to actin and by changes in cross-bridge strain due to a changing fraction of cross-bridges in the pre-power-stroke state. Three separate experiments were performed, using skinned muscle fibers that were isolated and subsequently (i) activated at different Ca2+ concentrations (pCa2+ 4.5, 5.0, 5.5, 6.0) (n = 13), (ii) activated in the presence of blebbistatin (n = 16), and (iii) activated in the presence of blebbistatin at varying velocities (n = 5). In all experiments, a ramp shortening was imposed (amplitude 10%Lo, velocity 1 Lo•sarcomere length (SL)•s−1), from an initial SL of 2.5 µm (except by the third group, in which velocities ranged from 0.125 to 2.0 Lo•s−1). The values of P1, P2, L1, and L2 did not change with Ca2+ concentrations. Blebbistatin decreased P1, and it did not alter P2, L1, and L2. We developed a mathematical cross-bridge model comprising a load-dependent power-stroke transition and a pre-power-stroke cross-bridge state. The P1 and P2 critical points as well as the critical lengths L1 and L2 were explained qualitatively by the model, and the effects of blebbistatin inhibition on P1 were also predicted. Furthermore, the results of the model suggest that the mechanism by which blebbistatin inhibits force is by interfering with the closing of the myosin upper binding cleft, biasing cross-bridges into a pre-power-stroke state.

Highlights

A long-standing scientific challenge resides in the explanation of how characteristics of the molecular actin-myosin interaction give rise to macroscopically observed phenomena in striated muscles, and how conditions imposed on macroscopic scales affect actin-myosin kinetics
Length ramps performed at constant velocities are commonly used for studying the molecular mechanisms of muscle contraction [1,2,3,4,5], and show force responses that are qualitatively similar to early studies that used step shortening: 1) the force decreases in proportion to shortening, 2) the force decrease becomes less rapid, 3) the force decrease becomes even slower, 4) and the force shows an asymptotic approach to a lowered but constant steady state
Model development We developed a cross-bridge model consisting of two major elements: (1) an active molecular contractile element, consisting of cross-bridges capable of contraction and (2) a passive element with linear elasticity, which is placed between the active contractile element and the external apparatus which controls fiber length

Summary

Introduction

A long-standing scientific challenge resides in the explanation of how characteristics of the molecular actin-myosin interaction give rise to macroscopically observed phenomena in striated muscles, and how conditions imposed on macroscopic scales affect actin-myosin kinetics. Length ramps performed at constant velocities are commonly used for studying the molecular mechanisms of muscle contraction [1,2,3,4,5], and show force responses that are qualitatively similar to early studies that used step shortening: 1) the force decreases in proportion to shortening, 2) the force decrease becomes less rapid, 3) the force decrease becomes even slower, 4) and the force shows an asymptotic approach to a lowered but constant steady state. While phase 1 in force traces is commonly associated with a purely elastic response, the behaviour during phase 2 is attributed to a repartitioning of cross-bridges from the pre to the post-power stroke state, due to an acceleration of the power-stroke step under conditions of lowered mechanical load on myosin cycling [1,3,5,6]

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