The working stroke elicited in the myosin motors by a sudden drop in force from the isometric plateau (T₀) is at least 5 times larger than the average strain (s) in the motors at T₀ (<=2 nm) (Reconditi et al., Nature 428:578, 2004). s <= 2 nm can be explained by either 20% of the attached motors undergoing stochastic 10 nm lever arm tilting, while the remaining 80% are biased to the beginning of the working stroke, or by attached motors with a narrower distribution of lever arm angles that only reach the end of the working stroke under low load. With a stiffness of the myosin motor (e) as high as 3 pN nm^-1, (Decostre et al. PNAS USA 102:13927, 2005; Piazzesi et al. Cell 131:784, 2007), a 10 nm isometric working stroke is excluded on thermodynamic grounds. To further test the motor stiffness, 4 kHz small length oscillations were used to determine the sarcomere stiffness-force relation during the development of isometric force (when the thick filament extends by 1.5%) and during the force redevelopment following 2.5% unloaded shortening (when the thick filament maintains an extension similar to that at T₀). The results confirm the ~3 pN nm^-1 motor stiffness and the linearity of the filament elasticity and demonstrate that that the non-linearity in the sarcomere stiffness-force relation for forces < 0.3 T₀ originates from the transient presence of an elastic component in parallel with force generating myosin motors.

Supported by MIUR and Ente Cassa di Risparmio di Firenze (Italy).