Kinetic differences at the single molecule level account for the functional diversity of rabbit cardiac myosin isoforms.

Myosin is a mechanoenzyme that generates force and motion by cyclically interacting with actin in a reaction driven by the hydrolysis of ATP. Type II myosins, including cardiac, skeletal and smooth muscle myosin, are dimers. Each myosin monomer is structurally defined by a myosin heavy chain (MHC) and two non-covalently bound light chains (Weeds & Lowey, 1971). The MHC contains a globular catalytic domain that possesses the hydrolytic and motor function of myosin. In the heart, both α- and β-MHC isoforms exist (Hoh et al. 1979). Expression of these isoforms is species dependent and both developmentally and hormonally regulated (Everett et al. 1984; Lompre et al. 1984). In smaller adult mammals (e.g. mouse and rat), α-MHC expression predominates (Hamilton & Ianuzzo, 1991) and myosin molecules associate as αα-MHC homodimers known as V1 cardiac myosin. V3 cardiac myosin, a ββ-MHC homodimer, predominates in larger mammals (Hamilton & Ianuzzo, 1991) and humans (Mercadier et al. 1983). Certain pathological conditions, such as pressure-overload hypertrophy or hyperthyroidism, can change the pattern of isoform expression (Litten et al. 1982; Nagai et al. 1987). In the rabbit, pressure-overload hypertrophy promotes an increase in β-MHC expression (up to > 95 %) whereas treatment with L-thyroxine shifts the expression to predominantly α-MHC (> 99 %) (Litten et al. 1982). Reports of pathologically induced changes in MHC isoform expression in human ventricular myocardium, at both the mRNA and protein level, are conflicting and difficult to reconcile (Scheir & Adelstein, 1982; Mercadier et al. 1983; Alousi et al. 1990; Lowes et al. 1997; Nakao et al. 1997). However, recent reports (Lowes et al. 1997; Nakao et al. 1997) suggest that a significant downregulation of α-MHC mRNA expression may indeed be of some consequence to the failing human heart. Changes in cardiac myosin isoform expression should have functional consequences in the myocardium due to the hydrolytic and mechanical differences between V1 and V3 myosin. For example, both the Ca2+-stimulated and actin-activated ATPase activity of V1 myosin are approximately twice that of V3 myosin (Pope et al. 1980; VanBuren et al. 1995). In addition, fibre preparations containing V1 myosin have faster maximal velocities of unloaded shortening than fibres containing V3 myosin (Maughan et al. 1974; Cappelli et al. 1989). Although differences in peak twitch tension are not apparent, the energetic economy and tension-time integral for preparations containing predominantly V1 myosin are less than that of preparations with predominantly V3 myosin (Alpert & Mulieri, 1982; Holubarsch et al. 1985; Hasenfuss et al. 1991). These mechanical differences between V1 and V3 myosin may reflect inherent differences between these myosins' force- and motion-generating capacities. In an in vitro motility assay, V1 cardiac myosin propels actin filaments at velocities (vmax) 2-3 times faster than V3 myosin (Harris et al. 1994; Suguira et al. 1995; VanBuren et al. 1995), while generating half the average isometric force (Favg) (VanBuren et al. 1995). At the single molecule level, vmax is related to the unitary step displacement (d) and step duration (ton) of myosin, under unloaded conditions, as follows (Huxley, 1990): vmax≈ (d/ton). Therefore, differences in d and/or ton may account for differences in vmax for V1 and V3 myosin. Favg is defined by myosin's unitary force (F) and duty cycle (f), with f being the fraction of the cross-bridge cycle time during which myosin is strongly bound to actin under isometric conditions as follows (VanBuren et al. 1994): Favg=Ff. Thus differences in Favg between V1 and V3 myosin can be accounted for by changes in F and/or duty cycle (VanBuren et al. 1995). In both cases, differences in vmax and Favg most probably reflect inherent differences in these myosins' molecular mechanics (i.e. d and F) and/or kinetics of the cross-bridge cycle (i.e. ton and f). To address these possibilities, we have used a dual laser trap to directly measure the molecular parameters d, F and ton. Our results suggest that differences between the V1 and V3 myosin isoforms are solely kinetic. Furthermore, we propose a mechanism to account for the kinetic differences that is related to the rate of MgADP release from the myosin molecule.