Back to the future: cross-bridge working stroke in muscle fibres measured using force steps

In the 45 years since A. F. Huxley (1957) first proposed a quantitative model of how the myosin cross-bridges in muscle act to produce shortening, it has proved difficult to determine precise values for the amount of filament movement generated by one cross-bridge during one interaction with an actin filament (i.e. the cross-bridge working stroke) and the load dependence of the working stroke. These are important parameters because they characterise the basic process underlying muscle contraction. A paper in this issue of The Journal of Physiology (Piazzesi et al. 2002) fills an important gap, describing the load dependence of cross-bridge filament movement, and does so by returning to a type of experiment first attempted more than 40 years ago.

Perhaps the most well known experiments directed at understanding the contraction mechanism are those in which fast, small amplitude steps in length were applied to a contracting frog muscle fibre (Huxley & Simmons, 1971). The characteristic pattern of changes in force output after a step decrease in length (Fig. 1) were used as the basis for the Huxley-Simmons cross-bridge model. Of particular relevance to this commentary, Huxley & Simmons (1971) proposed that the initial, rapid force redevelopment (Phase b, Fig. 1) reflected force arising from cross-bridges that were attached prior to the step and which, without detaching, rapidly recommenced generating force after the length step. When the applied length step was greater than ≈14 nm per half-sarcomere, there was no rapid force redevelopment (Ford et al. 1977), consistent with the idea that tilting or rotating of an attached cross-bridge can only accommodate filament displacements less than 14 nm. About 6 nm of the 14 nm length step are taken up by shortening of compliant structures in series with attached cross-bridges, leaving ≈8 nm attributable to the cross-bridge working stroke.

Figure 1. Illustration of force response to step decrease in fibre length.

A fast, small (< 0.5 % fibre length) decrease in length (top trace) applied to a muscle fibre contracting isometrically (force output, P₀) causes an immediate drop in force output (a), simultaneous with the length step. Force partially redevelops over several milliseconds (b), then is almost constant for a short period (c), before the onset of a much slower force redevelopment (d). These have been interpreted as follows (Huxley, 2000). Phase (a), instantaneous shortening of the elastic components of the fibre (within both cross-bridges and contractile filaments); Phase (b), force redevelopment by cross-bridges attached prior to the length step; Phase (c), basis unclear; Phase (d), consistent with cross-bridges detaching and reattaching further along the actin filament.

Two aspects of length step experiments are, at least conceptually, not ideal. First, after a length step the muscle is isometric and, in the absence of filament sliding, data directly relating working stroke amplitude to the load opposing shortening cannot be obtained. Second, when force output varies, as during the transient response to a length step, the length of any elastic structures in series with attached cross-bridges will also alter so that, although the length of a fibre or fibre segment is constant after the length step, the relative position of attached cross-bridges and their attachment sites will alter. If the underlying cross-bridge processes are strain dependent, then compliance can potentially alter the kinetics, and perhaps magnitude, of the force transient. Recent measurements have shown that non-cross-bridge compliance is considerably greater than supposed by Huxley & Simmons (for references, see Piazessi et al. 2002). Thus, it is important to check the length step data against data from a type of experiment that is free of these uncertainties. Such an experiment exists and is that in which step changes in force, rather than length, are used.

Podolsky (1960) developed the force step technique before the length step technique was developed. If the force opposing shortening in a contracting fibre is decreased abruptly from above the isometric force to below it, then the fibre shortens. Shortening velocity approaches its final steady value via a series of oscillations (e.g. Fig. 1B, Piazzesi et al. 2002). Cross-bridges attached before the perturbation can go through their working stroke after the force step because the fibre is shortening. Because force is constant, the shortening is unaffected by series compliance and thus reflects filament movement by only the cross-bridges. Neither Podolsky (1960) nor Huxley and his colleagues (Armstrong et al. 1966) were able to produce force steps that were sufficiently fast or stable to clearly distinguish between the initial rapid shortening, attributable to elastic structures, and the subsequent velocity response attributable to attached cross-bridges.

Piazzesi et al. (2002) resurrected the force step technique but were able to generate force steps that were more than an order of magnitude quicker than their predecessors and much more stable. The force steps illustrated in their paper (Fig. 1C, Piazessi et al. 2002) are quite superb. This improvement was achieved by using a force transducer with resonant frequency two orders of magnitude higher than the earlier researchers and a faster, more stable, servo motor (V. Lombardi, personal communication).

Their results show that cross-bridge working stroke does vary with load, between ≈4 nm when shortening against heavy loads and ≈8 nm when the load was very light. Furthermore, their force step data are almost identical to earlier length step data, supporting the idea that both techniques are probing the same phenomenon (Huxley, 2000) and reinforcing the conclusions of the earlier work.

Recent work on the amplitude of the cross-bridge working stroke has been dominated by experiments using isolated proteins and optical traps to hold molecules and then measuring forces and displacements resulting from interactions of a single cross-bridge with an actin filament. This isolates the fundamental contractile event, albeit in the absence of the highly ordered filament structure that is so prominent in muscle. Estimates of working stroke of skeletal muscle myosin from various experiments of this type have ranged from 5 nm to over 15 nm (for a short review, see Mehta et al. 1999). It is of interest to see whether these data become more consistent as techniques are refined and whether data are obtained that are compatible with cross-bridge properties determined from muscle fibres.