Contraction of myofibrils in the presence of antibodies to myosin subfragment 2.

Abstract

In a muscle-based version of in vitro motility assays, the unloaded shortening velocity of rabbit skeletal myofibrils has been determined in the presence and absence of affinity-column-purified polyclonal antibodies directed against the subfragment-2 region of myosin. Contraction was initiated by photohydrolysis of caged ATP and the time dependence of shortening was monitored by an inverted microscope equipped with a video camera. Antibody-treated myofibrils undergo unloaded shortening in a fast phase with initial rates and half-times comparable to control (untreated) myofibrils, despite a marked reduction in the isometric force of skinned muscle fibers in the presence of the antibodies. In antibodytreated myofibrils, this process is followed by a much slower phase of contraction, terminating in elongated structures with well-defined sarcomere spacings (-1 ,um) in contrast to the supercontracted globular state of control myofibrils. These results suggest that although the unloaded shortening of myofibrils (and in vitro motility of actin filaments over immobilized myosin heads) can be powered by myosin heads, the subfragment-2 region as well as the myosin head contributes to force production in actively contracting muscle.The classical rotating cross-bridge model proposes that the contractile force in activated muscle originates from a structural transition within the myosin head [subfragment-1 (S-1) subunit] while it is attached to actin (1, 2). The helix-coil model proposes that melting within subfragment-2 (S-2; the a-helical tail segment of heavy meromyosin) is triggered and generates force when the actin-attached cross-bridge swings away from the thick filament surface (3, 4). In earlier work (5), it was shown that purified anti-S-2 antibody can markedly depress the isometric tension generated by skinned psoas fibers. The reduction in tension (to about 7% ofcontrol fibers) occurs in the absence of any direct effect on the ability of S-1 to undergo cyclic interaction with actin as measured by the ATPase of antibody-treated fibers. One interpretation of this experiment is that S-2 contributes to force production in vivo, possibly through a helix-coil transition in the S-2 hinge domain of the myosin molecule (for review, see ref. 6). However, it seems clear from recent in vitro model studies that S-1 alone, energized by ATP, can produce force (7) and slide actin filaments at speeds approaching those obtained with muscle fibers under no-load conditions (8,9). One possibility to reconcile these observations is that the force generated by S-1 in the model systems is sufficient to produce sliding motion but not sufficient for the expression of the full isometric force developed in working muscle. To test this explanation, we have measured the relative unloaded sliding velocity of overlapping thin and thick filaments in myofibrils, the intact basic structural units of skeletal muscle, in the presence and absence of polyclonal antibodies directed against the S-2 region of myosin. In these experiments, we have...

Evidence for the Essential Role of Myosin Subfragment-2 in Muscle Contraction: Functional Communication between Myosin Head and Subfragment-2

“…-activated isometric force and Mg ATPase activity, measured by NADH fluorescence, in skinned vertebrate muscle fibers. In agreement with the report of Harringon [12], the antibody reduced isometric force in a time-dependent manner, while Mg ATPase activity remained unchanged even when isometric force was reduced to zero [13]. As shown in Figure 11, Muscle fiber stiffness, measured by applying small sinusoidal vibrations (amplitude, 0.1% of fiber slack length) decreased with decreasing isometric force, reaching zero when isometric force was reduced to zero.…”

“…Helical and random coil structures of the S-2 hinge region are expressed as straight and zig-zag shapes, respectively [7]. -activated myofibrillar shortening [11,12]. In 1992, he asked me to work together using his anti-S-2 antibody, and we made the experiments to be described later with unexpected results.…”

Contraction characteristics and ATPase activity of skeletal muscle fibers in the presence of antibody to myosin subfragment 2.

“…In support of this hypothesis, polyclonal anti-S-2 antibody has been shown to reduce Ca2+-activated isometric force in glycerinated skeletal muscle fibers, while ATPase activity of the fibers and the initial unloaded shortening velocity of isolated myofibrils undergo little change (9,10). More recently, it has been shown that, in the presence of antibody directed against a 20-amino acid peptide segment within the hinge region of cardiac myosin, movement of actin filaments in an in vitro motility assay is suppressed, while ATPase activity of myofibrils and purified S-1 remained unchanged (11).…”

Unfolding/refolding studies of the myosin rod

“…Such melting would be expected to give rise to a reduction in the overall length of the molecule, and this is consistent with electron-microscope observations of the effect of temperature on the length of the myosin tail (Walker and Trinick, 1986;Walzthony et al, 1986). This transition could therefore be an important force-generating event in muscle contraction and the subfragment-2 region as well as the myosin head would contribute to force production in actively contracting muscle (Harrington et al, 1990;Sugi et al, 1992).It has recently been proposed that the myosin rod contains six independent domains (Privalov, 1982 ;Lopez-Lacomba et al, 1989;Bertazzon and Tsong, 1990). The Cterminus of the heavy chain has been shown to be unfolded and mobile , and to play a crucial role in myosin assembly Kalbitzer et al (1991) have indicated that the two chains are not in exact register but are slightly staggered.…”

“…Such melting would be expected to give rise to a reduction in the overall length of the molecule, and this is consistent with electron-microscope observations of the effect of temperature on the length of the myosin tail (Walker and Trinick, 1986;Walzthony et al, 1986). This transition could therefore be an important force-generating event in muscle contraction and the subfragment-2 region as well as the myosin head would contribute to force production in actively contracting muscle (Harrington et al, 1990;Sugi et al, 1992).…”