Role of Nebulin on Actomyosin Interaction Studied in situ in Demembranated Skeletal Muscle Fibers from Newborn Mice

Abstract

The effects of absence of nebulin, an actin filament associated protein, on mechanical and kinetic properties of Ca2+-activated, chemically skinned, psoas fibers were investigated comparing mechanical performance of fibers from 1-day-old wildtype (wt) mice and 1-day-old nebulin deficient (nebulin−/−) mice. With fast mechanics (Linari et al. Biophys J 92:2476, 2007) on fiber bundles (sarcomere length 2.5μm, temperature 13 °C) we determined i) the relation between isometric force, stiffness and Ca2+ concentration; and ii) the unloaded shortening velocity and the power output at different loads at saturating Ca2+ (pCa, 4.50). Actin filament length in psoas fibers is not affected by the absence of nebulin, as proven by immunofluorescence imaging. Our results show a reduction in isometric force in the absence of nebulin without changes in the Ca2+ sensitivity of the contractile system. Stiffness measurements accompanied by analysis of the compliance of the half-sarcomere indicate that the reduction in isometric force is due to a proportional reduction in the number of myosin motors attached to actin without change in the average force of the motor. In addition, the absence of nebulin increases the unloaded shortening velocity by 63%, while decreases the maximum power output by 80%. These results indicate that the absence of nebulin induces a decrease of the rate of attachment of the myosin motors to actin and an increase of the rate of detachment of negatively strained motors under zero load, revealing a direct role for nebulin in stabilizing the actomyosin interaction. Supported by NIH and MiUR.