The 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.

SUMMARY1. A mouse toe muscle was chemically skinned with saponin and the 1,0 spacing of the hexagonal myofilament lattice at a sarcomere length of 2-5 Itm was measured with the X-ray-diffraction method. In the relaxed state, the 1,0 spacing was 40-8 nm.2. When the muscle was maximally activated at pCa 4 4, the spacing decreased to 38-4 nm. During contractions at lower calcium concentrations, the spacing decreased less. In rigor, the spacing decreased to almost the same extent as during maximum contraction, although the rigor tension was only 8 O of the maximum tension.3. When the spacing in relaxed muscle had been adjusted osmotically to about 38 nm, activation caused no further decrease in the spacing.4. The results support the view that the force responsible for the lattice shrinkage during contraction is produced by cross-bridges displaced from their optimum lateral positions.

Indirect immunofluorescence microscopy of highly stretched skinned frog semitendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCI, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCI2, and 1 mM EGTA) and with 0.6 M KI. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.

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.

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