Yoshiki Umazume

Mechanism of action of 2, 3-butanedione 2-monoxime on contraction of frog skeletal muscle fibres.

SummaryThe mechanism of the inhibitory effect of 2, 3-butanedione 2-monoxime (BDM) on contraction of frog skeletal muscles was studied using skinned fibres and aequorin-injected intact fibres. The tension development of skinned fibres directly activated with calcium was strongly inhibited by BDM. This agent also had effects on the sarcoplasmic reticulum in the skinned preparations, suppressing the calcium pump function and enhancing the activity of the ‘calcium-induced calcium release’ mechanism. In electrically stimulated intact fibres, although BDM slightly suppressed the elevation of the intracellular calcium ion concentration, this effect was so weak that it would not explain the strong inhibitory effect of the agent on the tension development by the intact fibres. It was concluded that the tension reducing effect of BDM on intact fibres was due mainly to its direct action on the contractile system. The mode of this action of BDM was further examined with skinned fibres in view of its effects on the maximum shortening speed and isometric tension in low MgATP environments.

Lattice shrinkage with increasing resting tension in stretched, single skinned fibers of frog muscle

The 1,0 lattice spacing d1,0 in chemically and mechanically skinned single fibers of frog muscle was measured at various sarcomere lengths, L, in the range from L = 2.1 to 6.0 microns by an x-ray diffraction method. In chemically skinned fibers, d1,0 decreased with a similar slope to that of mechanically skinned fibers up to L congruent to 3 microns, but beyond this point d1,0 steeply decreased with further stretching. This steep decrease in d1,0 could be ascribed mainly to an increase in the compressing force associated with the longitudinal extension of a remnant of the sarcolemma. In mechanically skinned fibers, the gradual decrease in d1,0 continued beyond filament overlap (L greater than or equal to 3.5 microns) and was highly proportional to a resting tension. This decrease in d1,0 at L greater than or equal to 3.5 microns could be ascribed to an increase in the force exerted by lateral elastic components, which is proportional to the longitudinal resting tension. A conceptual model is proposed of a network structure of elastic components in a sarcomere.

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments

SummaryThe localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 μm remained at about 0.8 μm from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 μm. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 μm of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.

Deterioration induced by physiological concentration of calcium ions in skinned muscle fibres

SummaryThe deteriorating effect of μm order of Ca2+ on skinned frog skeletal muscle fibres was studied from the view point of the digestion of proteins by calcium-activated neutral protease (CANP). Tension developed in solutions containing no MgATP (rigor solution) decreased irreversibly with the addition of Ca2+ in quantities of more than 0.1 μm. Low temperature was seen to suppress (Q10>4), and neutral pH to maximize, this decrease in tension. In rigor solution containing Ca2+, SDS electrophoresis indicated that a 95 k dalton component (α-actinin) was released from the fibre; electron micrography showed the disappearance of Z-lines. These results suggest that one of the causes for decrease in rigor tension is the proteolytic activity of CANP, and its inhibitors were shown to be quite useful in experiments on skinned fibre.

Localization of the parallel elastic components in frog skinned muscle fibers studied by the dissociation of the A- and I-bands

Localization of the parallel elastic components (PECs) in skinned muscle fibers was investigated by analyzing the change of the resting tension, which accompanies the dissociation of the A- and I-bands. The A-band was dissociated from both ends by increasing the concentration of KCl under relaxing conditions (0.09-0.54 M KCl, 4.0 mM MgATP, 1.0 mM Mg2+, 4.0 mM EGTA, pH 6.0-9.0, 20 degrees C). At sarcomere lengths greater than or equal to 3.5 microns, the length of the A-band was estimated by comparing the intensity of the first-order optical diffraction line with the results of model calculations. These results were supported by differential-interference microscopy and sodium dodecyl sulfate gel electrophoresis. It was shown that the resting tension decreased nearly in proportion to the residual length of the A-band. At sarcomere lengths less than or equal to 4.0 microns, the resting tension after the dissociation of the A-band was lowered to less than 10% of the initial value. On the other hand, at sarcomere lengths greater than or equal to 5.0 microns the resting tension after the dissociation of the A-band still showed approximately 35% of the initial value and did not change even after the I-band was dissociated by a solution containing KI. From these results, we propose that most of the PECs contributing to resting tension bind almost uniformly to the A-band and there are also PECs connecting Z-lines.

Width and lattice spacing in radially compressed frog skinned muscle fibres at various pH values, magnesium ion concentrations and ionic strengths.

SummaryThe width (D) and the 1,0 lattice spacing (d1,0) at various ionic compositions of mechanically skinned single fibres (from semitendinosus muscle ofRana catesbeiana) were measured at various concentrations of polyvinyl pyrrolidone (PVP K-30,Mn=40000) from 0 to 6% at 20° C. In a standard relaxing solution (4mm MgATP2−, 1mm Mg2+, 4mm EGTA, ionic strength 150mm and pH 7),d1,0 decreased exponentially as the PVP concentration increased:d1,0), was 41.3 ± 0.4 (mean ±s.d.) nm at 0% PVP and 32.9 ± 0.4 nm at 6% PVP.D was proportional tod1,0 except at very low PVP Concentrations, I.e. at 1% PVP,D decreased by 7%, whereasd1,0 decreased by only 3%. At 0% PVP,D andd1,0 decreased when either pH or ionic strength (Γ/2) was lowered. At 6% PVP,D andd1,0 decreased with lowered pH or increased [Mg2+], but was independent of Γ/2. The radial stiffness, or degree of resistance to the changes ofD agaïnst the compressing force, increased considerably atd1,0 < 35nm in a standard relaxing solution, but not at pH 5.5 or 30mm [Mg2+]. These effects of pH, [Mg2+] and Γ/2 onD ord1,0 and on the radial stiffness can be explained by the modification of the properties of the elastic element and the hinge between subfragment-1 and -2 and/or the hinge between subfragment-2 and light meromyosin.

Optical diffraction study of muscle fibers: II. Electro-optical properties of muscle fibers

When an electric field is applied along the fiber axis, the intensities of all observable optical diffraction lines of skeletal muscle fibers increase. This electro-optical effect was extensively studied and it was confirmed that the effect is due to the interaction between electric dipole moments of thin filaments and the applied field. From the present study on the intensity modulation due to applied field in sinusoidal and square forms, we confirmed that (1) the thin filament is a semiflexible rod, (2) the second order mode of the bending motion of thin filaments contributes to the electro-optical effect of muscle fibers at higher frequencies of a sinusodidal field or shorter durations of a square field, (3) the induced moment has no appreciable effect, and (4) the estimated value of the flexural rigidity of thin filaments strongly depends on the concentrations of free calcium ions in the myofibrillar space.

Myosin heads contact with thin filaments in compressed relaxed skinned fibres of frog skeletal muscle.

SummaryWhen skinned skeletal muscle fibres with rest sarcomere length (L=2.5 μm) are compressed by the addition of various concentrations ([PVP]) of polyvinylpyrrolidone, the relation between the 1,0 spacing (d) of thick filament lattice and [PVP] has been known to break at d of around 35 nm, resulting in a steeper slope of the relationship at d > 35 nm. To clarify the cause of this, X-ray diffraction and crosslinking experiments were carried out. Thed versus [PVP] relationship of stretched fibres (L=3.5μm) breaks at a d of around 29 nm. The difference between these characteristic d values, 35-29=6 nm, is close to the diameter of thin filaments (8 nm). The crosslinking efficiency of formaldehyde between myosin heads and thin filament surface, measured by radial stiffness increase, was found to begin to markedly increase when the relaxed fibre with rest L was compressed to a d of nearly 35 nm. In addition to these results, the dversus [PVP] relationship obtained in rigor and in high [Mg2+] (30mM) relaxing solutions, and the crosslinking efficiency seen in high [Mg2+] solutions supported our previous hypothesis that in normal relaxing solution (containing 1mM Mg2+) the probability of myosin heads coming into contact with the thin filament surface abruptly increases at d near 35 nm in fibres with rest L.

Protruding masticatory (superfast) myosin heads from staggered thick filaments of dog jaw muscle revealed by X-ray diffraction

To characterize the structure of jaw muscle fibres expressing masticatory (superfast) myosin, X-ray diffraction patterns of glycerinated fibres of dog masseter were compared with those of dog tibialis anterior in the relaxed state. Meridional reflections of masseter fibres were laterally broad, indicating that myosin filaments are staggered along the filament axis. Compared with tibialis anterior fibres, the peak of the first myosin layer line of masseter fibres was lower in intensity and shifted towards the meridian, while lattice spacings were larger at a similar sarcomere length. These suggest that the myosin heads of masticatory fibres are mobile, and tend to protrude from the filament shaft towards actin filaments. Lowering temperature or treating with N-phenylmaleimide shifted the peak of the first myosin layer line of tibialis anterior fibres towards the meridian and the resulting profile resembled that of masseter fibres. This suggests that the protruding mobile heads in the non-treated masticatory fibres are in the ATP-bound state. The increased population of weakly binding cross-bridges may contribute towards the high specific force of masticatory fibres during contraction. Electron micrographs confirmed the staggered alignment of thick filaments along the filament axis within sarcomeres of masticatory fibres, a feature that may confer efficient force development over a wide range of the sarcomere lengths.

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.