Effects of inorganic phosphate analogues on stiffness and unloaded shortening of skinned muscle fibres from rabbit.

Abstract

SUMMARY1. We examined the effects of aluminofluoride (AIF,) and orthovanadate (Vi), tightly binding analogues of orthophosphate (Pi), on the mechanical properties of glycerinated fibres from rabbit psoas muscle. Maximum Ca2+-activated force, stiffness, and unloaded shortening velocity (Vus) were measured under conditions of steady-state inhibition (up to 1 mm of inhibitor) and during the recovery from inhibition.2. Stiffness was measured using either step or sinusoidal (1 kHz) changes in fibre length. Sarcomere length was monitored continuously by helium-neon laser diffraction during maximum Ca2+ activation. Stiffness was determined from the changes in sarcomere length and the corresponding changes in force. Vus was measured using the slack test method.3. AlFX and Vi each reversibly inhibited force, stiffness and Vus. Actively cycling cross-bridges were required for reversal of these inhibitory effects. Recovery from inhibition by AlFx was 3-to 4-fold slower than that following removal of Vi.4. At various degrees of inhibition, AlFx and Vi both inhibited steady-state isometric force more than either VuK1 or stiffness. For both AlFx and Vi, the relatively greater inhibition of force over stiffness persisted during recovery from steady-state inhibition. We interpret these results to indicate that the cross-bridges with AlFx or Vi bound are analogous to those which occur early in the cross-bridge cycle.

Myosin regulatory light chain phosphorylation inhibits shortening velocities of skeletal muscle fibers in the presence of the myosin inhibitor blebbistatin

“…In the great majority of conditions where tension is inhibited, velocity is also inhibited (Dantzig and Goldman 1985;Chase et al 1993;Seow et al 1997;Regnier et al 1999;Franks-Skiba et al 2007). The inhibition of velocity has been hypothesized to be the result of an internal load imposed by nonworking myosin heads.…”

The catch state of mollusc catch muscle is established during activation:experiments on skinned fibre preparations of the anterior byssus retractor muscle of Mytilus edulis L. using the myosin inhibitors orthovanadate and blebbistatin

“…This is also an important hint for the presence of catch linkages, because a similar increase is usually observed when catch is induced by either Ca 2+ removal after Ca 2+ activation in skinned preparations (Pfitzer and Rüegg, 1982;Höpflinger et al, 2006) or removal of acetylcholine in intact preparations (Pfitzer and Rüegg, 1982;Sugi et al, 1999). In skinned skeletal muscle preparations [frog (Brozovich et al, 1988), rabbit psoas (Martyn and Gordon, 1992;Chase et al, 1993)] it was found that application of phosphate or orthovanadate increases the population of attached but non-force generating cross-bridges. This cross-bridge population is detectable only with very rapid length changes (<1·ms) at low temperature (5-13°C) indicating rapid detachments and reattachments (Chase et al, 1993;Dantzig and Goldman, 1985).…”

“…V i is thought to bind to the force-generating myosin heads with bound ADP (AM.ADP state) and induce an isomerisation to a stable non-or low-force-generating complex (Dantzig and Goldman, 1985;Chase et al, 1993). At a concentration of 10·mmol·l -1 the force depression by V i is saturated in skinned ABRM preparations .…”

“…In skinned skeletal muscle preparations [frog (Brozovich et al, 1988), rabbit psoas (Martyn and Gordon, 1992;Chase et al, 1993)] it was found that application of phosphate or orthovanadate increases the population of attached but non-force generating cross-bridges. This cross-bridge population is detectable only with very rapid length changes (<1·ms) at low temperature (5-13°C) indicating rapid detachments and reattachments (Chase et al, 1993;Dantzig and Goldman, 1985). Thus, this cross-bridge population markedly differs from long-lasting catch linkages.…”

“…Therefore, in the present study we substantially extended the approach of Butler et al (Butler et al, 2006) by using maximally Ca 2+ -activated skinned ABRM preparations and by including rapid changes of the fibre length: as in the work of Butler et al (Butler et al, 2006), we inhibited the active cycling cross-bridges by specific myosin inhibitors during Ca 2+ activation. Orthovanadate (Herzig et al, 1981;Dantzig and Goldman, 1985;Chase et al, 1993;Jaworowski et al, 1999;Galler et al, 2005) and blebbistatin (Kovacs et al, 2004;Butler et al, 2006) were chosen as myosin inhibitors. The force remaining after myosin inhibition was investigated by addressing the following questions.…”