Cardiotonic bipyridine amrinone slows myosin-induced actin filament sliding at saturating [MgATP]

Abstract

Previously reported effects of amrinone on skeletal muscle function suggest that the drug reduces the rate constant of myosin cross-bridge dissociation. We have used the in vitro motility assay to further elucidate the mechanism underlying this effect and to aid these studies a new, improved, filament tracking software was developed in the Matlab™ environment. The experiments were carried out at 30°C using heavy meromyosin from fast rabbit muscle and rhodamine–phalloidin labeled actin filaments. A slowing effect of amrinone on filament sliding velocity at 1mM MgATP was observed at drug concentrations >0.3 mM. This effect showed signs of saturation at the highest drug concentrations (1–2 mM) that could be readily tested. The sliding velocity exhibited hyperbolic dependence on [MgATP] with a V _max of 7.2 ± 0.9 μm/s and a K _M of 0.18 ± 0.02 mM. Amrinone (1 mM) reduced V _max by 32 ± 5% (P < 0.01) and K _M by 42 ± 8% (P < 0.05; n = 4). These results are accounted for in the most straightforward way by a model where amrinone acts directly on the actomyosin system and reduces the rate constant of MgADP release. Such a well-defined effect on the myosin cross-bridge cycle makes the drug a potentially useful pharmacological tool for further studies of myosin function both in vitro and in the ordered filament array of a living muscle fiber.