Effect of low cytoplasmic [ATP] on excitation-contraction coupling in fast-twitch muscle fibres of the rat

Abstract

In this study we investigated the roles of cytoplasmic ATP as both an energy source and a regulatory molecule in various steps of the excitation-contraction (E-C) coupling process in fast-twitch skeletal muscle fibres of the rat. Using mechanically skinned fibres with functional E-C coupling, it was possible to independently alter cytoplasmic [ATP] and free [Mg2+]. Electrical field stimulation was used to elicit action potentials (APs) within the sealed transverse tubular (T-) system, producing either twitch or tetanic (50 Hz) force responses. Measurements were also made of the amount of Ca2+ released by an AP in different cytoplasmic conditions. The rate of force development and relaxation of the contractile apparatus was measured using rapid step changes in [Ca2+]. Twitch force decreased substantially (∼30%) at 2 mM ATP compared to the level at 8 mM ATP, whereas peak tetanic force only declined by ∼ 10% at 0.5 mM ATP. The rate of force development of the twitch and tetanus was slowed only slightly at [ATP] ≥ 0.5 mM, but was slowed greatly (> 6-fold) at 0.1 mM ATP, the latter being due primarily to slowing of force development by the contractile apparatus. AP-induced Ca2+ release was decreased by ∼10 and 20% at 1 and 0.5 mM ATP, respectively, and by ∼40% by raising the [Mg2+] to 3 mM. Adenosine inhibited Ca2+ release and twitch responses in a manner consistent with its action as a competitive weak agonist for the ATP regulatory site on the ryanodine receptor (RyR). These findings show that (a) ATP is a limiting factor for normal voltage-sensor activation of the RyRs, and (b) large reductions in cytoplasmic [ATP], and concomitant elevation of [Mg2+], substantially inhibit E-C coupling and possibly contribute to muscle fatigue in fast-twitch fibres in some circumstances. © The Physiological Society 2004.