Temperature dependence of the force-generating process in single fibres from frog skeletal muscle.

Generation of force and shortening in striated muscle is due to the cyclic interactions of the globular portion (the head) of the myosin molecule, extending from the thick filament, with the actin filament. The work produced in each interaction is due to a conformational change (the working stroke) driven by the hydrolysis of ATP on the catalytic site of the myosin head. However, the precise mechanism and the size of the force and length step generated in one interaction are still under question. Here we reinvestigate the endothermic nature of the force-generating process by precisely determining, in tetanized intact frog muscle fibres under sarcomere length control, the effect of temperature on both isometric force and force response to length changes. We show that raising the temperature: (1) increases the force and the strain of the myosin heads attached in the isometric contraction by the same amount (approximately 70 %, from 2 to 17 degrees C); (2) increases the rate of quick force recovery following small length steps (range between -3 and 2 nm (half-sarcomere)-1) with a Q10 (between 2 and 12 degrees C) of 1.9 (releases) and 2.3 (stretches); (3) does not affect the maximum extent of filament sliding accounted for by the working stroke in the attached heads (10 nm (half-sarcomere)-1). These results indicate that in isometric conditions the structural change leading to force generation in the attached myosin heads can be modulated by temperature at the expense of the structural change responsible for the working stroke that drives filament sliding. The energy stored in the elasticity of the attached myosin heads at the plateau of the isometric tetanus increases with temperature, but even at high temperature this energy is only a fraction of the mechanical energy released by attached heads during filament sliding.

A combined mechanical and X-ray diffraction study of stretch potentiation in single frog muscle fibres.

The nature of the force (T) response during and after steady lengthening has been investigated in tetanized single muscle fibres from Rana temporaria (4 C; 2.15 micrometer sarcomere length) by determining both the intensity of the third order myosin meridional X-ray reflection (IM3) and the stiffness (e) of a selected population of sarcomeres within the fibre. With respect to the value at the isometric tetanus plateau (To), IM3 was depressed to 0.67 +/- 0.04 during steady lengthening at approximately 160 nm s(-1) (T approximately 1.7) and recovered to 0.86 +/- 0.05 during the 250 ms period of after-stretch potentiation following the rapid decay of force at the end of lengthening (T approximately 1.3); under the same conditions stiffness increased to 1.25 +/- 0.02 and to 1.12 +/- 0.03, respectively. After subtraction of the contribution of myofilaments to the half-sarcomere compliance, stiffness measurements indicated that (1) during lengthening the cross-bridge number rises to 1.8 times the original isometric value and the average degree of cross-bridge strain is similar to that induced by the force-generating process in isometric conditions (2.3 nm), and (2) after-stretch potentiation is explained by a residual larger cross-bridge number. Structural data are compatible with mechanical data if the axial dispersion of attached heads is doubled during steady lengthening and recovers half-way towards the original isometric value during after-stretch potentiation.

Conformation of the myosin motor during force generation in skeletal muscle.

Myosin motors drive muscle contraction, cytokinesis and cell locomotion, and members of the myosin superfamily have been implicated in an increasingly diverse range of cell functions. Myosin can displace a bound actin filament several nanometers in a single interaction. Crystallographic studies suggest that this 'working stroke' involves bending of the myosin head between its light chain and catalytic domains. Here we used X-ray fiber diffraction to test the crystallographic model and measure the interdomain bending during force generation in an intact single muscle fiber. The observed bending has two components: an elastic distortion and an active rotation that generates force. The average bend of the force-generating myosin heads in a muscle fiber is intermediate between those in crystal structures with different bound nucleotides, and the C-terminus of the head is displaced by 7 nm along the actin filament axis compared with the in vitro conformation seen in the absence of nucleotide.