Hidehiro Tanaka

X-ray diffraction and electron microscopy fromLethocerus flight muscle partially relaxed by adenylylimidodiphosphate and ethylene glycol

The low-angle X-ray diffraction pattern from Lethocerus flight muscle fibres was recorded in rigor or under two conditions that modify crossbridge structure and behaviour, aqueous adenylylimidodiphosphate (AMPPNP) and AMPPNP + calcium in an ethylene glycol-water mixture. The effects on the 38.7 nm layer-line peaks (hk.6) of the diffraction patterns were studied in detail. In aqueous AMPPNP at room temperature, a condition in which rigor tension drops to half without loss of stiffness, the peaks remained nearly as intense as in rigor except for the 10.6, which dropped to half. In 20% (v/v) ethylene glycol-AMPPNP + 100 microM-Ca2+ at 23 degrees C (gly + pnp + Ca), a condition which removed muscle tension but left stiffness close to the rigor value, the 10.6 and 11.6 peaks greatly decreased but the 31.6 remained relatively high. The 14.5 nm meridional peak (00.16) became stronger on addition of AMPPNP and again on adding glycol + calcium. Considered in terms of constructively interfering filaments and crossbridges, the X-ray data indicated a transfer of diffracting crossbridge mass towards the thick filament as relaxation proceeds. We compared the X-ray diffraction patterns and crossbridge structure seen with electron microscopy (EM) under the same chemical conditions. EM and X-ray observations were mutually quite consistent overall. However, X-ray data indicated that more crossbridge mass was stereospecifically related to actin before fixation in the partially relaxed state (gly + pnp + Ca) than was suggested by the disordered crossbridge profiles seen by EM. We conclude that myosin heads at the start of the power stroke may both be closely related to their thick filament origins and form actin-determined attachments to the thin filament.

Time-resolved X-ray diffraction studies of frog skeletal muscle isometrically twitched by two successive stimuli using synchrotron radiation

In order to clarify the delay between muscular structural changes and mechanical responses, the intensity changes of the equatorial and myosin layer-line reflections were studied by a time-resolved X-ray diffraction technique using synchrotron radiation. The muscle was stimulated at 12-13 degrees C by two successive stimuli at an interval (80-100 ms) during which the second twitch started while tension was still being exerted by the muscle. At the first twitch, the intensity changes of the 1.0 and 1.1 equatorial reflections reached 65 and 200% of the resting values, and further changes to 55 and 220% were seen at the second twitch, respectively. Although the second twitch decreased not only the time to peak tension but also that to the maximum intensity changes of the equatorial reflections (in both cases, about 15 ms), the delay (about 20 ms) between the intensity changes and the development of tension at the first twitch were still observed at the second twitch. On the other hand, the intensities of the 42.9 nm off-meridional and the 21.5 nm meridional myosin reflections decreased at the first twitch to the levels found when a muscle was isometrically tetanized, and no further decrease in their intensities was observed at the second twitch. These results indicate that a certain period of time is necessary for myosin heads to contribute to tension development after their arrival in the vicinity of the thin filaments during contraction.

The First Thin Filament Layer Line Decreases in Intensity During an Isometric Contraction of Frog Skeletal Muscle

During isometric contraction/activation of full-overlap and non-overlap live frog skeletal muscles, the intensity of the first thin filament layer line at the axial spacing of approximately 1/37 nm-1, when separated from the partially overlapping first thick filament layer-line at approximately 1/43 nm-1, remained unchanged in the inner radial region (0.02-0.08 nm-1) where a large intensity increase is observed in the rigor state. The intensity decreased in the outer radial region (0.08-0.18 nm-1) where this layer line is expected to peak in the resting state. The intensity decrease in the outer region became larger with increasing filament overlap; on activation of the non-overlap muscle, it was about half that of the full-overlap muscle. Thus the first thin filament layer line decreases in intensity and any indication of the rigor-like intensification is not observed at all during contraction. This intensity decrease can be attributed to the same structural changes giving rise to the intensity increase of the second thin filament layer line. The results indicate that the configuration of the myosin heads interacting with actin during contraction differs significantly from that of the rigor state. Four-fold rotational symmetry of the thin filament structure as a whole becomes more pronounced during isometric contraction of the overlap muscle.

Factors Affecting the Equatorial X-ray Diffraction Pattern from Contracting Frog Skeletal Muscle

Changes in the equatorial X-ray diffraction pattern from tetanized frog sartorius muscles (Rana catesbiana ) were studied by use of time-resolved data collection technique (time resolution, 0.5 sec) to give information about the dynamic properties of the cross-bridges. No significant changes in the intensity ratio of two equatorial reflections (I1,0/I1,1) were observed when isometrically contracting muscles were slowly stretched by 5-6%, in spite of marked force changes. The intensity ratio also showed no significant changes when the load on isometrically contracting muscles was suddenly increased from Po to 1.2-1.5 Po to produce isotonic muscle lengthening. Closer examination of the data indicated that a small decrease in the value of I1,1 was caused by both slow stretch and isotonic lengthening. Because of the scatter of experimental plots in I1,0, the effect of small change in I1,1 on the intensity ratio fell within the range of accuracy of measurement. It is suggested that no marked changes in myosin head orientation or in the number of the cross-bridges in the vicinity of the thin filaments take place in response to slow stretches or isotonic lengthening, and that the decreased regularity of the filament lattice may produce the change in I1,1.

Changes in the X-ray diffraction pattern from rigor muscles by application of external length changes.

The effect of external force on the X-ray pattern from frog muscles in rigor was studied by a time-resolved diffraction technique. When sinusoidal length changes (1.5-3% of the muscle length, 5 Hz) were applied to the muscle, the 14.3 nm intensity decreased during the releasing phase and increased during the stretching phase. The intensity ratio of the equatorial 1.0 and 1.1 reflections did not change, nor were there any appreciable intensity changes in the 5.9 nm and 5.1 nm reflections during the length change. Experiments were also done with the relaxed muscles and no change was seen in any reflection, indicating that the rigor linkages are needed to produce the 14.3 nm intensity change. Thus the distinct effect of the length change was detected only on the 14.3 nm reflection. These results suggest no large conformational changes are induced in both the distal part of the myosin head attached to actin and the actin filament during the oscillation. It is therefore most probable that the proximal portion of myosin heads including S-2 contributes to the intensity change in response to the length change (see, also ref. 21). When the muscle was stretched beyond the filament overlap, the 14.3 nm intensity change was suppressed to less than 50% of that of the slack length. It was also found that the tension change delayed the intensity change during the length oscillation. However, this delay of the tension change as observed in the muscle at the slack length was lacking in the overstretched muscle, indicating that the 14.3 nm intensity change may arise partly from a portion other than the crossbridges.

Effect of stretch on the equatorial X-ray diffraction pattern from frog skeletal muscle in rigor.

Changes in the equatorial X-ray diffraction pattern during stretch of frog sartorius muscle in rigor were studied. No significant changes in I1,0/I1,1 were observed by stretching the rigor muscles. The value of I1,1 was, however, found to decrease reversibly by stretch, while the value of I1,0 was so small that its changes by stretch fell within the range of accuracy of measurement.

Time-resolved synchrotron X-ray diffraction studies of a single frog skeletal muscle fiber: Time courses of intensity changes of the equatorial reflections and intracellular Ca2+ transients

Time-resolved X-ray equatorial diffraction studies on a single frog skeletal muscle fiber were performed with a 10 ms time resolution using synchrotron radiation in order to compare the time courses of the molecular changes of contractile proteins and the intracellular Ca2+ transient during an isometric twitch contraction at 2.7 degrees C. Measurements of the Ca2+ transient using aequorin as an intracellular Ca2+ indicator were conducted separately just before and after the X-ray experiments under very similar experimental conditions. The results, which showed a similar time course of tension to that observed in the X-ray experiment, were compared with the aequorin light signal, tension and the intensity changes of the 1,0 and 1,1 equatorial reflections. No appreciable change in both reflection spacings indicated that the effect of internal shortening of the muscle was minimized during contraction. The intensity change of the equatorial reflections generally occurred after the aequorin light signal. In the rising phase, the time course of increase in the 1,1 intensity paralleled that of the rise of the light signal and the intensity peak occurred 20-30 ms after the peak of the light signal. The decrease in the 1,0 intensity showed a time course similar to that of tension and the intensity minimum roughly coincided with the tension peak, coming at 80-90 ms and about 60 ms after the peaks of the light signal and the 1,1 intensity change, respectively. In the relaxation phase, the 1,1 intensity seemed to fall rapidly just before the tension peak and then returned to the original level in parallel with the decay of tension. The 1,0 intensity returned more slowly than the tension relaxation. Thus, the change of the 1,1 intensity was faster than that of the 1,0 intensity in both the rising and relaxation phases. When the measured aequorin light signal was corrected for the kinetic delay of the aequorin reaction with a first-order rate constant of either 50 or 17 s-1, the peak of the corrected light signal preceded that of the measured one by approx. 30 ms. Thus, the peak of the Ca2+ transient appeared earlier than the peaks of the 1,1 and 1,0 intensity changes by 50-60 and 110-120 ms, respectively. The time lag between the extent of structural change and the Ca2+ transient is discussed in relation to the double-headed attachment of a cross-bridge to actin.

The effect of hypertonic solutions on contracture tension and volume in Mytilus smooth muscle

Abstract 1. 1. The effect of hypertonic solutions (1.6 times the normal osmolarity) on ACh-and K-con-tractures and the fibre volume in the anterior byssal retractor muscle of Mytilus edulis was studied. 2. 2. The contracture tension was reduced by 20–50% in the NaCl-hypertonic solution, and almost eliminated in the sucrose-and glycerol-hypertonic solutions. 3. 3. The muscle weight decreased by less than 3% in the NaCl-hypertonic solution, and by 8–20% in the sucrose-and glycerol-hypertonic solutions. 4. 4. It is suggested that the osmotic shrinkage of the fibres is the main cause of the reduction of contracture tension rather than the increased ionic strength in the myoplasm.

Sinusoidal Length Change Study of Muscle Contraction and Self-Induced Translation Model of Myosin Motion

By using synchrotron radiation, it was observed that X-ray diffraction pattern changed markedly when sinusoidal length changes were applied to isometrically contracting frog skeletal muscle [1]. We proposed a model for the filament sliding mechanism in order to explain the obtained data as well as various other data. Basic idea of the model was given briefly in a report [2], Here we discuss the model in more detail and present experimental data which are in agreement with what the model predicts.