The basic contractile unit of striated muscle, the sarcomere, has a very high structural homogeneity. The basic mechanism of contraction appears the same for sarcomeric myosin (Myosin II) of all species. However, contractile and energetic properties of skeletal muscle fibres vary very widely from species to species and, within the same species, from muscle fibre to muscle fibre. In the last 20 years it has become clear that many myofibrillar proteins exist in several isoforms. Isoforms give to structurally homogeneous sarcomeres a large molecular heterogeneity both through species and within species. Investigations searching for the bases of the very high functional heterogeneity and plasticity of skeletal muscle have mostly focused on myosin heavy chain (MHC) isoforms.

MHC isoform function has been mainly studied by concomitant analysis of contractile and energetic properties and of MHC content of single skinned muscle fibres from small mammals and humans (Bottinelli & Reggiani, 2000). Single muscle fibres containing different MHC isoforms were found to have mostly different contractile and energetic properties with some relevant exception. Single muscle fibres containing MHC-I had 10-fold lower maximum shortening velocity (V_o), maximum power, and rate of tension raise, and 3- to 4-fold lower optimal velocity, ATPase activity and tension cost than single muscle fibres containing MHC-IIB. MHC-IIA- and MHC-IIX-containing fibres had intermediate properties. Specific tension (Po/CSA) and thermodynamic efficiency represent exception to the isoform based variability: Po/CSA was only 40 % lower in MHC-I-containing fibres than in the other fibre types and thermodynamic efficiency was very similar in fibres containing different MHC isoforms.

Notwithstanding the progress in the understanding of skeletal muscle heterogeneity and plasticity, several open questions remain. It is still unclear whether MHC isoforms are the only determinant of skeletal muscle functional heterogeneity both within and through species. Moreover, very little is known about the mechanisms at the basis of functional diversity of skeletal myosin isoforms.

To clarify whether the differential expression of myosin isoforms can fully account for V_o differences among skeletal muscle fibres, in vitro motility assays (IVMA) were used to study the velocity of sliding of unregulated actin filaments (V_f) on different MHC isoforms. In such IVMA any difference in the velocity of sliding of actin solely depends on the myosin isoforms loaded in the flow chamber. Sarcomere organization is in fact lost, all other myofibrillar proteins are absent, and actin is the same for all assays. With the aim to relate V_f of a given myosin isoform and V_o of the parent fibres over a large range of velocities and in several species, ten MHC isoforms from four species were analysed: mouse (MHC-I and -IIB), rat (MHC-I and -IIB), rabbit (MHC-I, -IIX and -IIB) and human (MHC-I, -IIA and -IIX). A linear relation was found between V_f and V_o through species. As in different species, myofibrillar protein isoforms and not only myosin isoforms are different, the results strongly support the view that variability in V_o among single fibres mostly depends on MHC isoform content.

The sequence of events in the acto-myosin cross-bridge cycle is thought to be the same for all myosin II forms. To generate different values of V_o, myosin isoforms might differ either in the amount of displacement determined by a single acto-myosin interaction (step size) or in the kinetics of such interaction. To clarify whether the kinetics of acto-myosin interaction varies among myosin isoforms, acto-myosin interaction was studied in solution in a new flash-photolysis light-scattering apparatus (Weiss et al. 2001), developed in Mike Geeves’s laboratory at the University of Canterbury, UK. Such apparatus enabled the analysis of the ATP-induced acto-myosin dissociation (apparent second-order rate constant, K₁k₊₂) on 1Ð2 µg of myosin and of affinity of ADP for acto-myosin (K_AD) on ~5 µg of myosin. The tiny amount of pure myosin isoforms required for analysis were obtained by extraction from single fibres. All four MHC isoforms from rat (MHC-I, -IIA, -IIX and -IIB) were studied. Consistently with the increase in V_o in the order type I, IIA, IIX, IIB fibres, K₁k₊₂ got faster whereas the affinity for ADP weakened (K_AD increased) in the isoform order I, IIA, IIX, IIB. Both K₁k₊₂ and K_AD linearly correlated with V_o of the parent fibres. Therefore, further analysis was required to identify which of the two parameters was rate-limiting of the whole process. The rate of ADP release (k_-AD) was calculated from K_AD and compared with K₁k₊₂. It was suggested that k_-AD, being 4- to 8-fold slower than K₁k₊₂, is more likely to be the rate limiting step of V_o. To clarify whether differences in the rate of ADP release of myosin isoforms were sufficient to account for differences in V_o of single fibres, the minimum value of the rate constant of the event limiting shortening velocity (K_min) was calculated for each fibre type from V_o values. k_min was determined according to Siemankowski et al. (1985) (k_min = V_o (sarcomere length) (step size)^-1). k_min agreed within a factor of 2 with k_-AD. The latter result confirms that k_-AD is the rate-limiting step of V_o, and strongly suggest that kinetics differences among skeletal myosin isoforms, namely a difference in ADP release rate, is the main determinant of V_o variability among skeletal muscle fibres.

All procedures accord with current local guidelines and the Declaration of Helsinki.