Electron Microscopic Recording of the Power and Recovery Strokes of Individual Myosin Heads Coupled with ATP Hydrolysis: Facts and Implications.

The most straightforward way to get information on the performance of individual myosin heads producing muscle contraction may be to record their movement, coupled with ATP hydrolysis, electron-microscopically using the gas environmental chamber (EC). The EC enables us to visualize and record ATP-induced myosin head movement in hydrated skeletal muscle myosin filaments. When actin filaments are absent, myosin heads fluctuate around a definite neutral position, so that their time-averaged mean position remains unchanged.

Electron microscopic recording of myosin head power stroke in hydrated myosin filaments.

Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures.

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber.

Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position.

Direct demonstration of the cross-bridge recovery stroke in muscle thick filaments in aqueous solution by using the hydration chamber.

Despite >50 years of research work since the discovery of sliding filament mechanism in muscle contraction, structural details of the coupling of cyclic cross-bridge movement to ATP hydrolysis are not yet fully understood. An example would be whether lever arm tilting on the myosin filament backbone will occur in the absence of actin. The most direct way to elucidate such movement is to record ATP-induced cross-bridge movement in hydrated thick filaments.

High mechanical efficiency of the cross-bridge powerstroke in skeletal muscle.

We were interested to estimate the maximum mechanical efficiency with which chemical energy derived from ATP hydrolysis is converted into mechanical work by individual cross-bridges when they perform their powerstroke synchronously. Glycerinated rabbit psoas muscle fibres, containing ATP molecules almost equal in number to the cross-bridges within the fibre, were activated to shorten under various afterloads by laser-flash photolysis of caged Ca(2+). In these conditions, almost all the cross-bridges are in the state where the ATP is hydrolyzed but the products have not yet been released from the cross-bridge (M-ADP-P(i)) immediately before activation, and can hydrolyze only one ATP molecule during the flash-induced mechanical response.

Electron microscopic evidence for the thick filament interconnections associated with the catch state in the anterior byssal retractor muscle of Mytilus edulis.

The anterior byssal retractor muscle (ABRM) of a bivalve mollusc Mytilus edulis is known to exhibit catch state, i.e. a prolonged tonic contraction maintained with very little energy expenditure.