Leonardo Lucii

Skeletal muscle performance determined by modulation of number of myosin motors rather than motor force or stroke size.

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.

The myosin motor in muscle generates a smaller and slower working stroke at higher load

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.

Mechanism of force generation by myosin heads in skeletal muscle

Muscles generate force and shortening in a cyclical interaction between the myosin head domains projecting from the myosin filaments and the adjacent actin filaments. Although many features of the dynamic performance of muscle are determined by the rates of attachment and detachment of myosin and actin, the primary event in force generation is thought to be a conformational change or ‘working stroke’ in the actin-bound myosin head. According to this hypothesis, the working stroke is much faster than attachment or detachment, but can be observed directly in the rapid force transients that follow step displacement of the filaments. Although many studies of the mechanism of muscle contraction have been based on this hypothesis, the alternative view—that the fast force transients are caused by fast components of attachment and detachment —has not been excluded definitively. Here we show that measurements of the axial motions of the myosin heads at ångström resolution by a new X-ray interference technique rule out the rapid attachment/detachment hypothesis, and provide compelling support for the working stroke model of force generation.

The size and the speed of the working stroke of muscle myosin and its dependence on the force

Myosin II is the motor protein that produces force and shortening in muscle by ATP‐driven cyclic interactions of its globular portion, the head, with the actin filament. During each interaction the myosin head undergoes a conformational change, the working stroke, which, depending on the mechanical conditions, can generate a force of several piconewtons or an axial displacement of the actin filament toward the centre of the sarcomere of several nanometres. However, the sizes of the elementary force and length steps and their dependence on the mechanical conditions are still under question. Due to the small fraction of the ATPase cycle time myosin II spends attached to actin, single molecule mechanics failed to produce definitive measurements of the individual events. In intact frog muscle fibres, however, myosin IIs working stroke can be synchronised in the few milliseconds following a step reduction in either force or length superimposed on the isometric contraction. Here we show that with 150 μs force steps it is possible to separate the elastic response from the subsequent early rapid component of filament sliding due to the working stroke in the attached myosin heads. In this way we determine how the size and the speed of the working stroke depend on the clamped force. The relation between mechanical energy and force provides a molecular basis for muscle efficiency and an estimate of the isometric force exerted by a myosin head.

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 tetanised 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 (∼70 %, from 2 to 17 °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 °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

1 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 μm 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. 2 With respect to the value at the isometric tetanus plateau (T0), IM3 was depressed to 0.67 ± 0.04 during steady lengthening at ≈160 nm s−1 (T≈ 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≈ 1.3); under the same conditions stiffness increased to 1.25 ± 0.02 and to 1.12 ± 0.03, respectively. 3 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. 4 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.

Structure‐Function Relation of the Myosin Motor in Striated Muscle

Abstract: Force and shortening in striated muscle are driven by a structural working stroke in the globular portion of the myosin molecules—the myosin head—that cross‐links the myosin‐containing filaments and the actin‐containing filaments. We use time‐resolved X‐ray diffraction in single fibers from frog skeletal muscle to link the conformational changes in the myosin head determined at atomic resolution in crystallographic studies with the kinetic and mechanical features of the molecular motor in the preserved sarcomeric structure. Our approach exploits the improved brightness and collimation of the X‐ray beams of the third generation synchrotrons by using X‐ray interference between the two arrays of myosin heads in each bipolar myosin filament to measure with Å sensitivity the axial motions of myosin heads in situ during the synchronous execution of the working stroke elicited by rapid decreases in length or load imposed during an active isometric contraction. Changes in the intensity and interference‐fine structure of the axial X‐ray reflections following the mechanical perturbation allowed to establish the average conformation of the myosin heads during the active isometric contraction and the extent of tilt during the elastic response and during the subsequent working stroke. The myosin working stroke is 12 nm at low loads, which is consistent with crystallographic studies, while it is smaller and slower at higher loads. The load dependence of the size and speed of the myosin working stroke is the molecular determinant of the macroscopic performance and efficiency of muscle.