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. On application of ATP, myosin heads are found to move away from, but not towards, the bare region, indicating that myosin heads perform a recovery stroke (average amplitude, 6 nm). After exhaustion of ATP, myosin heads return to their neutral position. In the actin⁻myosin filament mixture, myosin heads form rigor actin myosin linkages, and on application of ATP, they perform a power stroke by stretching adjacent elastic structures because of a limited amount of applied ATP ≤ 10 µM. The average amplitude of the power stroke is 3.3 nm and 2.5 nm at the distal and the proximal regions of the myosin head catalytic domain (CAD), respectively. The power stroke amplitude increases appreciably at low ionic strength, which is known to enhance Ca2+-activated force in muscle. In both the power and recovery strokes, myosin heads return to their neutral position after exhaustion of ATP.

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. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

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. Using the hydration chamber, with which biological specimens can be kept in an aqueous environment in an electron microscope, we have succeeded in recording ATP-induced cross-bridge movement in hydrated thick filaments consisting of rabbit skeletal muscle myosin, with gold position markers attached to the cross-bridges. The position of individual cross-bridges did not change appreciably with time in the absence of ATP, indicating stability of time-averaged cross-bridge mean position. On application of ATP, individual cross-bridges moved nearly parallel to the filament long axis. The amplitude of the ATP-induced cross-bridge movement showed a peak at 5-7.5 nm. At both sides of the filament bare region, across which the cross-bridge polarity was reversed, the cross-bridges were found to move away from, but not toward, the bare region. Application of ADP produced no appreciable cross-bridge movement. Because ATP reacts rapidly with the cross-bridges (M) to form complex (M x ADP x Pi) with an average lifetime >10 s, the observed cross-bridge movement is associated with reaction, M + ATP --> M x ADP x Pi. The cross-bridges were observed to return to their initial position after exhaustion of ATP. These results constitute direct demonstration of the cross-bridge recovery stroke.

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. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed.

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. Power output records of the fibres following activation indicated that the cross-bridges actually started their powerstroke almost synchronously. The amount of ATP utilized at 1 s after activation was estimated from the amount of isometric force developed after interruption of fibre shortening, while the amount of work done was calculated by multiplying the amount of afterload by the distance of fibre shortening. A conservative estimation of the maximum mechanical efficiency at a load of 0.5-0.6 P(o) was 0.7, suggesting that the actual maximum mechanical efficiency of cross-bridge powerstrokes may be close to unity.

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. Two different hypotheses have been put forward concerning the catch state; one assumes actin-myosin linkages between the thick and thin filaments that dissociate extremely slowly (linkage hypothesis), while the other postulates a load-bearing structure other than actin-myosin linkages (parallel hypothesis). We explored the possible load-bearing structure responsible for the catch state by examining the arrangement of the thick and thin filaments within the ABRM fibers, using techniques of quick freezing and freeze substitution. No thick filament aggregation was observed in the cross-section of the fibers quickly frozen not only in the relaxed and actively contracting states but also in the catch state. The thick filaments were, however, occasionally interconnected with each other either directly or by distinct projections in all the three states studied. The proportion of the interconnected thick filaments relative to the total thick filaments in a given cross-sectional area was much larger in the catch state than in the relaxed and actively contracting states, providing evidence that the thick filament interconnection is responsible for the catch state.