108. Time.Resolved X.Ray Diffraction from Frog Skeletal Muscle during an Isotonic Twitch under a Small Load

Transcription

1 No. 9] Proc. Japan Acad., 54, Ser. B (1978) Time.Resolved X.Ray Diffraction from Frog Skeletal Muscle during an Isotonic Twitch under a Small Load By Haruo SUGI,*> Yoshiyuki AMEMIYA,**> and Hiroo HASHIzUME**> (Communicated by Yasuji KATSUxi, M. J. A., Nov. 13, 1978) When vertebrate skeletal muscle contracts isometrically, the intensity of 1,0 equatorial reflection (Il,o) decreases while that of the 1,1 reflection (I1,1) increases.l>'2) This has been interpreted as being due to the movement of the cross-bridges from the vicinity of the thick filaments towards the thin filaments.2>-4) Though, in the sliding filament model of A. F. Huxley,5~ the number of the cross-bridges attached to the thin filaments at any one moment is less the greater the shortening velocity, Podolsky et al. have reported that the intensity ratio 11,0/11,1 is almost the same in both isometric and isotonic contractions, claiming that the number of attached cross-bridges may not differ significantly between isometric and isotonic conditions. Since their work seems not conclusive because of the difficulty in repeating many tetanic contractions, the above problem still remains to be clarified. The present experiments were undertaken to study the time course of change in the intensity ratio 11,0/11,1 during isotonic shortening under a small load by means of a time-resolved X-ray diffraction technique, to give information about the kinetic properties of the cross-bridges responsible for muscle contraction. The sartorius muscle dissected from the bullfrog Rana catesbiana was mounted in an experimental chamber with two Mylar windows and a multi-electrode assembly ; the pelvic end was clamped while the tibial end was connected to an isotonic lever, the movement of which was sensed with a differential transformer. The initial sarcomere length was adjusted to pm by the diffraction pattern of He-Ne laser light. The muscle was continuously perf used with oxygenated Ringer solution (2-4 C), and stimulated with a single supramaximal current pulse to produce an after-loaded twitch under a small load of 2-3% of the maximum isometric tension P0. The low angle equatorial X-ray diffraction patterns (specimen to focus distance, 36 cm) during the isotonic twitch were recorded with a linear position-sensitive *' Department of Physiology, School of Medicine., Teikyo University, Itabashi-ku, Tokyo. **' Engineering Research Institute, Faculty of Engineering, Tokyo University, Bunkyo-ku, Tokyo.

2 560 H. SUGI, Y. AMEMIYA, and H. HASHIZUME [Vol. 54(B), proportional counter, the output of which was fed into a data collection system.7>,s) The period of the isotonic twitch was divided into 40 phases of 25 msec duration, and the data in each phase were stored in the corresponding separate memory segment of the data collection system to be displayed on a chart recorder for analysis (Fig. 4). To obtain reasonable photon statistics for each phase, the twitches (and the data collection) were repeated times at 10 sec intervals. An example of the time course of change in the intensity ratio h,o/h,l during the isotonic twitch (initial sarcomere length, 2.4 pm) is shown in Fig. 1. Similar results were obtained on four other preparations with the initial sarcomere length of 2.4 pm, and also on two other preparations with the initial sarcomere length of 2.3 and 2.2 pm respectively. In all cases, the intensity ratio started to decrease on stimulation, reached a minimum value of within the first 20-30% of the shortening phase, and maintained this value until the beginning of the relaxation phase. Gradual recovery of the intensity ratio to the resting value was seen during the relaxation phase. During the recovery phase, the intensity ratio appeared to exhibit oscillatory changes which need further investigation. Though the extent of shortening was reduced by about 30% at the end of each experiment, the duration of the shortening phase remained almost Fig. 1. Time course of change in the intensity ratio h,o/i1,1 during an isotonic twitch of a frog sartorius muscle under a small load (0.025 Po). Twitches were repeated 120 times. The initial sarcomere length was 2.4 1am. Curves a and b show the first and the 120th twitches respectively. The degree of shortening is expressed in percentage of the initial muscle length. Note that the duration of the contraction phase did not change appreciably, while the contraction height decreased to some extent. The X-ray generator was a rotating anode type (Rigaku Denki, RU-200PL) with a fine focus operated with a Cu target at 40 KV and 30 ma.

3 No. 9] X-Ray Diffraction of Shortening Muscle 561 unchanged in all the preparations examined. The time course of change in the intensity ratio was also examined during an isometric twitch on four preparations (sarcomere length, 2.4pm) with the tibial end connected to a strain gauge. The extent of internal shortening of muscle fibres against the tendons and the recording system during an isometric twitch or a tetanus at low temperatures was estimated by measuring the compliance of the tendinous part and the recording system, and was found to be less than 1.5% of muscle length. As shown in Fig. 2, the intensity ratio decreased to a minimum value of during the rising phase of isometric tension, and started to return to the resting value after the beginning of relaxation.9~'1 ~ In both isotonic and isometric twitches, the decrease in the intensity ratio resulted from both a decrease in the 1,0 intensity and an increase in the 1,1 intensity.2>>6>>10> Fig. 2. Time course of change in the intensity ratio 11,0/11, during an isometric twitch. Twitches were repeated 120 times. The initial sarcomere length was 2.4 pm. Curves a and b show the tension changes of the first and the 120th twitches respectively. Since the muscle shortens by 10-20% during isotonic twitches, the effect of sarcomere length on the intensity ratio was also examined during isometric tetani. As shown in Fig. 3, the intensity ratio decreased with decreasing sarcomere length in agreement with previous reports,2~,11~ though the dependence of the intensity ratio on sarcomere length was much less marked.6~ Thus, the result that the minimum value of the intensity ratio attained during an isotonic twitch (Fig. 1) is definitely larger than that attained during an isometric twitch (Fig. 2) is opposite to the effect expected from the relation between the intensity ratio and sarcomere length, because both

4 562 H. SUGI, Y. AMEMIYA, and H. HASHIZUME [Vol. 54(B), Fig. 3. The intensity ratio h,0/h,1 for isometrically tetanized muscles plotted as a function of sarcomere length. The data points were obtained from different preparations. Each preparation was tetanized ten times with 1.5 sec train of pulses at 20 Hz with an interval of 1-2 min, so that the total exposure time during the period of steady isometric tension was 10 sec or more. The extent of decrease of isometric tension after repeated tetani was 10-15%. The line was fitted to the data points by the method of least squares. types of twitch started at the same sarcomere length. Then, if it is assumed that the minimum value of the maintained intensity ratio serves as a measure of the number of attached cross-bridges in both isometric and isotonic conditions, the present results imply that the number of attached cross-bridges is about 50% smaller during isotonic shortening under a small load than during isometric contraction,2),10) being consistent with the Huxley model5> but not with the model of Podolsky et a1.12>,13) Though the above view is also compatible with the fact that the stiffness of isotonically contracting muscle fibres decreases with decreasing isotonic load,14> it seems necessary to explain the reason why the intensity ratio remains nearly constant while the thin filaments slide into the hexagonal lattice of the thick filaments, increasing the X-ray scattering mass associated with the 1,1 lattice plane. A possible explanation for this may be that, during contraction, the regularity of the thick filament lattice increases as the thin filaments slide into it, thus increasing the intensity of the 1,0 reflection to cancel the increase in the intensity of the 1,1 reflection due to shortening. The relatively small dependence of the intensity ratio during isometric tetani on the sarcomere length (Fig. 3) may also be accounted for on this basis. Much more experimental work is needed to make this point clear.

5 No. 9] X-Ray Diffraction of Shortening Muscle 563 A question arises whether the steady state of the contractile system attained during the isotonic twitch as indicated by the period of steady 11,0/11,1 (Fig. 1) is identical with that attained during an isotonic tetanus. In this connection, it has been known that, at low temperatures, the shortening phase of an isotonic twitch is mostly superposable on that of an isotonic tetanus, and the force-velocity relation obtained from isotonic twitches is the same as those obtained from after-loaded isotonic tetani or isotonic releases.15~,16> In the isometric condition, the steady.. 11,0/11,1 is known to be the same in both Fig. 4. Constancy of the filament-lattice volume during isotonic shortening. A: A series of consecutive equatorial X-ray diffraction patterns from a muscle (initial sarcomere length, 2.3 rcm) during an isotonic twitch under a small load (0.03 P0). The whole period of twitch was divided into consecutive 25 msec phases, and the diffraction pattern was recorded for each phase. Note that, in addition to their intensity changes, the position of both the 1,0 and the 1,1 reflections changes due to the change in the spacing between the myofilaments. B : Inverse square of the 1,0 lattice spacing (d) as a function of sarcomere length during the shortening phase of an isotonic twitch shown in A. The regression line was drawn by the method of least squares.

6 564 H. SUGI, Y. AMEMIYA, and H. HASHIZUME [Vol. 54(B), twitches and tetani at low temperatures.8~'10) These results suggest that, at least at low temperatures, the contractile system may reach almost the same steady state during both twitches and tetani, though further study with tetanic contractions is needed. Fig. 4A shows consecutive diffraction patterns during the course of an isotonic twitch. The change in the spacing between the myofilaments during the isotonic shortening could be estimated by measuring the position of the 1,0 reflection. In Fig. 4B, the inverse square of the 1,0 lattice spacing is plotted against the corresponding sarcomere length, assuming that the sarcomere length change takes place in parallel with the muscle length change. The regression line starts from the origin (within the experimental error), indicating that the filament-lattice volume may remain constant during the isotonic shortening. Since the lattice volume is known to be constant when the sarcomere length of intact resting muscle is varied1f,17) the above result shows that the isovolumic behaviour of the filament-lattice exists in both resting and actively contracting muscles. We wish to thank Prof. K. Kohra and Prof. S. Ebashi for their encouragement. References 1) Elliott, G. F., Lowy, J., and Millman, B. M.: J. Mol. Biol., 25, (1967). 2) Haselgrove, J. C., and Huxley, H. E.: Ibid., 77, (1973). 3) Huxley, H. E.: Ibid., 37, (1968). 4) Huxley, H. E., and Brown, W.: Ibid., 30, (1967). 5) Huxley, A. F.: Progr. Biophys., 7, (1957). 6) Podolsky, R. J., St. Onge, R., Yu, L., and Lymn, R. W.: Proc. Natl. Acad. Sci. U. S. A., 73, (1976). 7) Hashizume, H., Mase, K., Amemiya, Y., and Kohra, K.: Nucl. Instr. Meth. (1978) (in press). 8) Sugi, H., Amemiya, Y., and Hashizume, H.: Proc. Japan Acad., 53B, (1977). 9) Huxley, H. E.: 5th Int. Biophys. Cong., Copenhagen, S53 (1975). 10) Matsubara, I., and Yagi, H.: J. Physiol., 27$, (1978). 11) Elliott, G. F., Lowy, J., and Worthington, C. R.: J. Mol. Biol., 6, (1963). 12) Podolsky, R. J., Nolan, A. C., and Zavaler, S. A.: Proc. Natl. Acad., Sci. U. S. A., 64, (1969). 13) Podolsky, R. J., and Nolan, A. C.: Cold Spring Harb. Symp. Quant. Biol., 37, (1973). 14) Julian, F. J., and Sollins, M. R.: J. Gen. Physiol., 66, (1975). 15) Buchthal, F., and Kaiser, E.: Dan Biol. Medd., 21(7), (1951). 16) Jewell, B. R., and Wilkie, D. R.: J. Physiol.,,143, (1958). 17) Huxley, H. E.: Proc. R. Soc., ser. B,,141, (1953).