Skeletal muscle can bear a high load at constant length, or shorten rapidly when the load is low. This force-velocity relationship is the primary determinant of muscle performance in vivo. Here we exploited the quasi-crystalline order of myosin II motors in muscle filaments to determine the molecular basis of this relationship by X-ray interference and mechanical measurements on intact single cells. We found that, during muscle shortening at a wide range of velocities, individual myosin motors maintain a force of about 6 pN while pulling an actin filament through a 6 nm stroke, then quickly detach when the motor reaches a critical conformation. Thus we show that the force-velocity relationship is primarily a result of a reduction in the number of motors attached to actin in each filament in proportion to the filament load. These results explain muscle performance and efficiency in terms of the molecular mechanism of the myosin motor.

Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched 'OFF') structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched 'ON') myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart.

SUMMARY1. Steady lengthenings at different velocities (0-025-1.2 ,tm/s per half-sarcomere; temperature 2-5-5°C) were imposed on isolated frog muscle fibres at the isometric tetanus plateau by means of a loudspeaker motor. The lengthening at the sarcomere level was measured by means of a striation follower either in fixed-end or in lengthclamp mode. The force response was measured by a capacitance gauge transducer (resonance frequency 50 kHz). Preparations showing gross non-homogeneity during lengthening were excluded.2. A steady tension was in all cases reached after about 20 nm per half-sarcomere of lengthening. Tension during this steady phase rose with speed of elongation up to 0-25-0-4 #sm/s per half-sarcomere, when tension was 1-9-2 times isometric tetanic force (TO). Further increase in speed produced only very little increase in the steady tension.3. During the transitory phase, before steady tension was reached, the tension rose monotonically if speed of lengthening was less than 0-25-0-3 ,um/s per halfsarcomere; at higher speed the tension rose above the steady level, reaching a peak when extension was 10-14 nm per half-sarcomere, and then fell to the steady level. Tension at the peak continued to rise with speed of lengthening above 0 3 ,um/s per half-sarcomere.4. During the tension rise within the transitory phase of force response the segment elongated at a speed 15-20% lower than that imposed on the whole fibre, as a consequence of-tendon compliance.5. During the steady phase, non-homogeneity of lengthening speed began above a speed of lengthening which varied from fibre to fibre. At speeds below this value, segments elongated at the same speed as that imposed on the fibre.6. Tension responses to large step stretches (up to 12 nm per half-sarcomere), applied at the plateau of isometric tetanus, showed that the instantaneous elasticity of contractile machinery is not responsible for the limit in force attained with highspeed lengthening.7. Instantaneous stiffness was determined during the steady state of force response by superposing small steps (< 15 nm per half-sarcomere) on steady lengthening at different velocities. Stiffness was 10-20% larger during lengthening MS 8214 V. LOMBARDI AND G. PIAZZESI than at the plateau of isometric tetanus and remained practically constant, independent of lengthening velocity, in the range of velocities used.8. The results indicate that steady lengthening of a tetanized fibre induces a crossbridge cycle characterized by fast detachment of the cross-bridge extended beyond a critical level. Reattachment of cross-bridges detached in this way is also very rapid. 9. A model of contraction, including separate steps for cross-bridge attachment, force generation and detachment, was found to be compatible with the experimental force-and stiffness-velocity relations. In the model, detachment of extended crossbridges occurs at an early stage of the cycle. The rate constant of this detachment process increases sharply beyond a critical amount of cross-bridge strain; reattac...

Muscle contraction is driven by the motor protein myosin II, which binds transiently to an actin filament, generates a unitary filament displacement or 'working stroke', then detaches and repeats the cycle. The stroke size has been measured previously using isolated myosin II molecules at low load, with rather variable results, but not at the higher loads that the motor works against during muscle contraction. Here we used a novel X-ray-interference technique to measure the working stroke of myosin II at constant load in an intact muscle cell, preserving the native structure and function of the motor. We show that the stroke is smaller and slower at higher load. The stroke size at low load is likely to be set by a structural limit; at higher loads, the motor detaches from actin before reaching this limit. The load dependence of the myosin II stroke is the primary molecular determinant of the mechanical performance and efficiency of skeletal muscle.