Journal of Muscle Research and Cell Motility 13, 153-160 (1992)

Effects of 2,3-butanedione monoxime on contraction of frog skeletal muscles: an X-ray diffraction study N . Y A G P ' * , S. T A K E M O R I 2, M . W A T A N A B E

z, K. H O R I U T I 2 " * a n d Y . A M E M I Y A 3

1 Department of Pharmacology, Tohoku University School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai 980, Japan 2 Department of Physiology, The ]ikei University School of Medicine, Minatoku, Tokyo 105, Japan 3 Photon Factory, National Laboratory for High Energy Physics, Tsukuba 203, Japan Received 4 June 1991; revised and accepted 5 August 1991

Summary We studied the effects of BDM (2,3-butanedione monoxime) on the tetanic contraction of frog skeletal muscles using an X-ray diffraction technique. BDM significantly increased the resting equatorial intensity ratio (I~,o/I~,O. In sartorius muscle, 3 mM BDM suppressed tetanic tension by 40-70% whereas the equatorial intensity ratio, which is 2.6 at rest, decreased to 0.75 during tetanus, close to the value in normal contraction (about 0.50). BDM (3 raM) reduced the intensity increase of the 5.1-nm layer-line to 41%, that of the 5.9-nm layer-line to 24%, and the intensity decrease of the second myosin meridional reflection (at 1/21.5 nm -~) at 81%. In overstretched semitendinosus muscle, 3 mM BDM did not significantly reduce the intensity increase of the second actin layer-line during activation, suggesting that enough calcium is released to activate the regulatory system and the regulatory proteins are intact. These results indicate that BDM suppresses tetanic tension by mainly inhibiting actin-myosin interaction, it has a smaller effect on the equatorial reflections and myosin layer-lines than on the actin layer-lines, suggesting that BDM-influenced myosin heads may bind to actin without following the symmetry of the actin helix.

Introduction The contractile force in muscle is developed from interaction between myosin and actin molecules. Although much is known about the biochemical properties of these molecules, it is not yet possible to correlate biochemical steps in the actomyosin ATPase reaction with those of the tension development process. One way to tackle this problem is to use a chemical substance that affects a certain step in the reaction and study how tension development and structural changes are affected. BDM (2,3-butanedione monoxime) is a substance which may be useful for this purpose. Horiuti and colleagues (1988) showed that in frog skeletal muscle BDM reversibly suppresses tension development of both intact and skinned fibres, and ascribed this to its action on the contractile or regulatory muscle proteins. Higuchi and Takemori (1989) made biochemical studies o n the effects of BDM on rabbit contractile proteins and showed that BDM inhibits both heavy meromyosin- and actomyosin- ATPases. Li and colleagues (1985) and West and Stephenson (1989) also found that BDM has an inhibitory effect on cardiac myofibrils. On the other hand, BDM also *To whom correspondence should be addressed. :1:Present address: Department of Physiology, Medical College of Oita, Oita 879-56, Japan. 0142-4319 9

1992 Chapman & Hall

has an inhibitory effect on the excitation-contraction coupling, which is prominent only at high (10mM) concentration in frog muscles (Horiuti et al., 1988; Hui & Maylie, 1988; Maylie & Hui, 1988) but is significant even at low (0.5 mM) concentration in mammalian muscles (Fryer et al., 1988a, b). In the present experiments, to further clarify the mechanisms of action of BDM on the contractile proteins, we made X-ray diffraction studies on frog skeletal muscles under the influence of BDM.

Materials and methods

Specimen preparations In all experiments except those concerned with recording the second actin layer-line, thin (0.6-0.8mm thick) sartorius muscles from frogs (Rana nigromacrata)were used. Frogs were killed by decapitation and the brain and upper spinal cord were destroyed by pithing. In experiments on the effects of BDM on resting equatorial intensities, the sacromere length was adjusted to 1.9-2.0 }.tm by using layer diffraction, and the temperature was 1~ C. In all other experiments, the sarcomere length was .adjusted to 2.4 I,tm and the temperature was 5-6 ~ C. In preliminary experiments, the diffusion of 3 mM BDM into the specimen was checked by observing the decline in twitch tension; a single electrical pulse was applied every 10-20 min and the reduction of the tension response was observed. The

YAGI et al.

154 tension reached a steady level of about 10% of the normal value (tetanic tension was reduced to about 40% at this BDM concentration; see Results) within 30-60 min after transfer of the muscle to a Ringer solution containing 3 mM BDM at 5--6~ C. Therefore, in the experiments described in this paper, specimens were left for at least I h to equilibrate. Similarly, the washout of BDM was monitored using a twitch response as an indicator. After the washout of 3 mM BDM, the tension recovered to its original level in 2-4 h. Therefore, in experiments made at the synchrotron radiation facility, where the machine schedule was tight, no recovery experiments could be made. We did not assume complete reversibility of the action of BDM in the analysis and discussion of the experimental results. To record the second actin layer-line (i.e. the thin filament based second layer-line), semitendinosus muscles from bullfrogs (Rana catesbeiana) were used at a sarcomere length above 4.0 gm, as determined by laser diffraction. The muscle was about 2 mm in diameter. To study the effects of BDM, muscles were soaked in a Ringer solution containing 3 mM BDM for 2 h before X-ray diffraction experiments. A longer incubation time was used to ensure complete diffusion of BDM into the thicker specimen. The muscles were stimulated isometrically by a 2-s train of electrical pulses (20 Hz, field strength of 20 V cm -~) given through a pair of platinum electrodes placed parallel to the muscle. When necessary, the contraction was repeated 20 times at 2-min intervals. The Ringer solution contained 115 mM NaC1, 2.5 mM KC1, 1.8 mM CaC12 and 2 mM HEPES (pH 7.2 adjusted with NaOH at room temperature).

X-ray measurements X-ray experiments were made at the beam line 15A of Photon Factory, Tsukuba, using the camera and the one-dimensional detector described in Amemiya and colleagues (1983) with a specimen-to-detector distance of 230 cm. The ring current was 100-300 mA. In equatorial experiments, the detector was set perpendicular to the muscle axis. The counter covered the region down to 0.011 nm -~ in the meridional direction on each side of the equator. To record the second actin layer-line, the detector was placed parallel to the axis of the muscle, covering the region from 0.22 to 0.25 nm -1 in the lateral direction. Only two quadrants could be measured at the same time. As this layer-line is very weak in patterns from resting muscle (Kress et aL, 1986), the intensity distribution in the resting state was used as a background and subtracted. The intensity in the area from 0.040 to 0.075 nm -~ axially was measured as the intensity of the second layer-line. Axial X-ray diffraction patterns were recorded on Fuji imaging plates. Active patterns were recorded during the latter half of a 2-s tetanus. Imaging plates were read by the image reader described in Amemiya and colleagues (1987). The effect of BDM on the resting equatorial intensity was studied using a rotating-anode X-ray generator (FR, Rigaku-denki, Tokyo, Japan) and a camera with a bent monochromator with a specimen-to-detector distance of 45 cm. Equatorial diffraction patterns were recorded on films and densitometered for quantitative analysis. The exposure time was 10 min.

Analysis of data All data from experiments using a one-dimensional detector were analysed on an engineering workstation (NEWS-821,

Sony, Tokyo, Japan). To quantify the intensity of a reflection, a straight line was drawn by connecting background points on each side of the reflection and the area above the line was measured. No attempt was made to separate the Z-band and (2,0) reflections which overlap the (1, 1) reflection. The intensities of these two reflections, when added together, amount to about 30% and 15-20% of that of the (1, 1) reflection in a resting and active state, respectively (Yu et al., 1985). The centre of gravity of the intensity distribution was taken as the spacing of the reflection. Diffraction patterns on imaging plates and films were analysed using an NEC ACOS-2020 computer at Tohoku University Computer Centre and an image processing system (NEC RSIPS-DT). For analysis of the patterns on imaging plates, the data were normalized by the exposure time and the total intensity of X-rays in the circular area 0.1--0.2 nm -I from the origin. The area of lateral integration was 0--0.014 nm -I for meridional reflections, 0.026-0.076nm -1 for myosin layerlines, and 0.050-0.150nm -~ for the 5.1 and 5.9-nm actin layer-lines.

Biochemical experiments Troponin-C was prepared from rabbit back and leg muscles (rabbit killed by overdose of pentobarbital) and labelled with dansylaziridine as described by Iio and Kondo (1981). Fluorescence measurements were made in 0.1 M KC1, 50 mM HEPES (pH=7.0), 4mMEGTA and 2mMMgC12. Aliquots of 100 mM CaC12 were added to obtain a Ca-titration curve. The pH decreased from 7.00 to 6.80 as the CaC12 was added.

Results

Equatorial reflections during contraction Equatorial reflections were measured to study the effects of BDM on the contractile system. In a normal Ringer solution, the equatorial intensity ratio I~,o/I~a was 2.2 in the resting state and decreased to 0.50 during contraction (Fig. lb). In the presence of 3 mM BDM, the tetanic tension was 41% of the value in control experiments (Fig. la) and the intensity ratio was 2.6 at rest, and 0.76 during contraction. The development of tension was slower in the presence of BDM, and correspondingly the intensity ratio changed more gradually. In a tetanus longer than 2 s, the intensity ratio in the presence of BDM did not decrease further within experimental error (data not shown). The reduction of tension, to 41%, is about twice as large as that found b y Horiuti and colleagues (1988) at 18~ on frog (Rana temporaria) single muscle fibres. However, as BDM is more effective at lower temperatures (Horiuti et al., 1988), the present results may be considered consistent with theirs. The relationship between tetanic tension and intensity ratio was studied by using higher concentrations of BDM (up to 6 mM). The plot (Fig. lc) shows that the relationship is non-linear. The extrapolation of the line fitted to the data in the area where tension is greater than 10% intercepts the ordinate I~,o/Ixa at 1.2. Similar non-linearity was also found when the data were plotted in terms of

155

BDM on skeletal muscle a

the inverse ratio, I u ~Is,o, or the mass transfer (Haselgrove & Huxley, 1973).

3.00 2.50

Equatorial reflections at rest

2.00

Figure lb shows that the intensity ratio is affected by BDM not only during contraction but also in the resting state. To further study this phenomenon, experiments at shorter sarcomere lengths (1.9-2.0 ~tm) and higher BDM concentration (10 mM) were performed using a laboratory X-ray source. The short sarcomere length was employed to increase the intensity of the (1, 1) reflection and obtain a reliable intensity ratio using a weaker X-ray source compared to the synchrotron source. The results are summarized in Table 1. Soaking a muscle in a normal Ringer solution for 2 h did not affect the intensity ratio. However, in a Ringer solution containing 10 mM BDM, the intensity ratio increased from 1.74 to 2.40. The intensity of the (1, O) reflection increased by 11 q- 10% (mean + SEM, n = 7) and that of the (1, 1) reflection decreased by 20 + 9% (n = 7). After washing out the BDM, intensity returned close to the initial value. These results show that BDM does cause an increase in the Ii,o/I1,~ intensity ratio. It is possible that the change in the intensity ratio is caused by the change in the lattice spacing. The spacing of the (1, O) reflection decreases while the muscle is soaked in a Ringer solution (Table 1). A similar phenomenon was observed by Haselgrove and Huxley (1973). Although the extent of shrinkage is greater on average in the presence of 10 mM BDM, shrinkage also takes place in control experiments in which no change in intensity ratio is observed. The shrinkage may be accelerated in the presence of BDM because of the higher osmolarity of the Ringer solution containing BDM. Also, shrinkage continues even after the intensity ratio returns to the control value after the BDM is washed out. Thus it seems unlikely that the change in lattice spacing is the cause of the change in intensity ratio.

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Fig. 1. (a) Typical tension developed by flog (Rana mgromacrata) sartorius muscle in the presence (equilibrated for I h) and absence of 3 mM BDM (upper line) at a sarcomere length of 2.4 ~m and 5--6~ C. (b) Ratio of intensities of the equatorial (1, 0) and (1, 1) reflections during contraction. 9 ratios during a control tetanus; @, those during a test tetanus in the presence of 3 mM BDM. The time resolution was 0.5 s. Bars = SEM of 12 muscles. (c) Relationship between tension and equatorial intensity ratio (I~,o/Iu) during isometric contraction. The tension was varied by using different concentrations of BDM. @, results in the absence of BDM; O, results recorded in the presence of 3 mM BDM; @, obtained with 4.5 mM BDM, | with 6 mM BDM. Points at zero tension represent resting values.

Axial patterns were recorded on Fuji imaging plates. A muscle was tetanically stimulated once to record control tension, and then soaked in a Ringer solution with 3 mM BDM. After I h, a resting pattern with an exposure time of 20 s was recorded (Fig. 2a). This pattern was not significantly different from that taken before soaking in 3 mM BDM. The specimen was then shifted about 3 mm vertically to avoid radiation damage and stimulated for 2 s, and the diffraction pattern was recorded between 1.0 and 2.0 s after the beginning of stimulation using a shutter (Fig. 2b). Stimulation was repeated 20 times and the pattern was accumulated on the plate. In the presence of 3 mM BDM, tetanic tension at the beginning of the series of contractions was 50 _ 4% (n = 7) of the control. In control experiments, tension declined to about 70% of initial tension in the last (20th) contraction, whereas in the presence of 3 mM BDM, it declined to about 80%. Thus

156

YAGI et al.

Table 1. Intensity ratio of equatorial reflections (I~,o/I1,~) and the spacing of the (1,0) reflection (in nm) in resting frog (Rana nigromacrata) skeletal musdes Control

I~,o/I1,~ dl,0 n

10 M BDM

Control

After 2 h

Control

2 hr after BDM

2 h after wash

4 h after wash

1.58 + 0.15 38.1 q- 0.4 5

1.58 + 0.15 37.4 q- 0.4 5

1.74 --}-0.08 37.9 _-4-0.5 12

2.40 + 0.1I 36.5 q- 0.3 12

1.87 + 0.I0 35.7 q- 0.3 7

1.75 + 0.II 35.5 + 0.5 4

Sarcomere length 1.9-2.0 ~tm.;Temperature i ~C. Mean 4- SEM shown. The differencebetween the control Ii.o/I~,~values of the control and the 10 mM BDM experiments is from variation among the specimens used. X-ray exposure 10 min. tension averaged over the series of contractions was 85% of initial tension in the absence and 45% in the presence of BDM, the effect of BDM being to suppress tension by 47%. This decrease in tension in the course of experiments is mainly from fatigue, as exposure of the same part of the specimen was limited to 20 s to reduce radiation damage from the strong X-ray beam. In the present experiments, the muscle developed tension on stimulation and moved sideways. Because small muscles were used, such a movement would cause a large change in the mass of the specimen on the X-ray beam and hence in the intensity of the diffraction pattern. Correction was made by using the total intensity in the circular region at 0.1-O.2nm -I from the origin as a standard. In this region, most of the intensity comes from the background and layer-lines do not contribute much to the total intensity. However, background scattering intensity as well as layer-line intensities are known to change during contraction (Huxley et d., 1982; Lowy & Poulsen, 1987). Thus there is some uncertainty in the measurement of absolute intensities. Of the 12 muscles studied, four required corrections > 10%. The results of these experiments were not significantly different from the other eight.

Thick filament-reNted layer-lines In patterns from relaxed muscles, strong myosin layerlines were observed (Fig. 2a). During normal contraction, the first myosin layer-line decreased in intensity to 29% of its resting value (Fig. 2b, Table 2). In the presence of 3 mM BDM, the first myosin layer-line was still weak during contraction (38% of that at rest). A similar observation was made on the second meridional reflection, whose intensity decreased to 15% and to 31% of the resting value in the presence and absence of BDM, respectively (Table 2). The intensity of the third meridional reflection increases on average during contraction (Table 2; Yagi et at., 1981; Huxley et al., 1982) although there was considerable scatter of data. In the presence of 3 mM BDM, it did not show a clear increase suggesting that the axial arrangement of myosin heads is less regular. As the intensity of this reflection depends on various factors, such as fatigue (Yagi et al., 1981) or sarcomere length changes (Huxley et al., 1982), the implication of the present observation requires a better understanding of the nature of this reflection. The Bragg spacing of the third meridional reflection increases slightly during activation (Huxley & Brown,

Fig. 2. Axial diffraction patterns from frog (Rana nigromacrata) sartorius muscle in 3 mM BDM recorded on a Fuji imaging plate. Temperature 5--6~ C. Sarcomere length 2.4 p.m. (a) Static pattern taken at rest after the preparation had been equilibrated with BDM for I h. Exposure time 20 s. (b) Diffraction pattern during isometric contraction. The following reflections are labelled: (1) first order myosin layer-line at 1/43 nm-% (2) second meridional myosin reflection at 1/21.5 nm-% (3) third meridional myosin reflection at 1/14.3 nm-~; (4) 5.9-nm actin layer-line; (5) 5.1-nm actin layer-line.

BDM on skeletal muscle

157

Table 2. Intensities and spacings of the myosin meridional

reflections and layer-lines, and the 5.9- and 5.1-nm actin layer-lines during contraction in the absence and presence of 3 mM BDM. The intensities are expressed as a ratio of the resting value. The spacing of the third meridional reflections is shown as a relative shift from the resting position

Control

3 mM BDM

First layer-line intensity active/rest)

0.29 _ 0.04

0.38 q- 0.04

Second meridian intensity active/rest)

0.15 + 0.06

0.31 _ 0.06

Third meridian intensity active/rest)

1.27 +o.19

0.91 +o.o6

Third meridian spacing fractional change)

0.014 4- 0.001

0.011 __0.002

5.9 nm layer-line intensity active/rest)

1.21 +0.09

•

5.1 nm layer-line intensity active/rest)

1.87 +0.16

1.36 +0.09

5

7

observed intensity change was not affected by interaction of myosin heads with the thin filament. The muscles were tetanically stimulated 20 times in a normal Ringer solution. Then they were equilibrated in the presence or absence of 3 mM BDM for 2 h before the second series of stimulations was made. An increase in intensity was observed during stimulation (Fig. 3). In the absence of BDM, the increase of intensity in the second series of stimulations was 43% of that in the first series (Fig. 3a). This rather low value is considered to be from fatigue of the muscle caused by repeated stimulation. Another possible cause of the low value is radiation damage caused by the strong X-rays from the storage ring, as the total exposure of the specimen to the beam was up to 400 s. In the presence a

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1967). This was also found in a muscle contracting in the presence of 3 mM BDM (Table 2). Although tension developed is half of that in control experiments, the spacing change is more than half, suggesting that developed tension is not the primary cause of the change in spacing.

Thin filament-related layer-lines Intensities of the layer-lines at 15.9 nm -~ and 15.1 nm -~ in the axial position were studied. Both of these layerlines increased in intensity during contraction, and the increase was reduced in the presence of 3mM BDM (Table 2). The intensity change of the 5.9-nm layer-line is too small to be measured precisely in the presence of BDM. The intensity of the 5.I-nm layer-line showed a marked increase during contraction, and the increase was suppressed to 41% in the presence of BDM. As the tension was also halved in the presence of BDM, the intensity increase of the 5.1-nm layer-line, which is thought to be caused primarily by the binding of myosin heads to the thin filament (Kress et al., 1986), changed roughly in proportion to the developed tension. Second actin layer-line To investigate the effects of BDM on calcium release and the regulatory system, the intensity of the second actin layer-line, at about 1 / 1 8 n m -~ axially, was measured during activation. The intensity of this layer-line is known to be associated with structural changes in the thin filament which take place when calcium binds to troponin (Haselgrove, 1973; Parry & Squire, 1973; Kress et al., 1986). The specimen was stretched until there was no overlap between thick and thin filaments so that the

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Fig. 3. Intensity change of the second actin layer-line during activation of highly stretched frog (Rana catesbeiana) semitendinosus muscle in the absence (a) or presence (b) of 3 mM BDM (equilibrated for 2 h). Temperature 5--6~ C; Muscle stimulated from time 0 to 2 s. O, results of the first series of stimulations; @, second series of stimulations. The time resolution was I s. Intensity during rest (before stimulation) was taken as zero, and the intensity averaged over the whole period of stimulations in the first series of the experiments was taken as 1.00. Bars = SEM (n = 6, for both experiments).

YAGI et al.

158 of 3 mM BDM, the increase observed in the second series of stimulation was about 65"% of that in the first (Fig. 3b), which is at least as big as in the control experiment. These results show that the inhibitory effect of 3 mM BDM is not primarily from its action on the calcium release mechanism of the muscle cell or on the regulatory system of the thin filament, although the large errors in the results do not allow us to exclude the possibility that BDM exerts an effect partly by influencing these mechanisms.

Calcium binding to troponin-C To examine the effect of BDM on calcium binding of troponin-C, fluorescence from dansylaziridine bound to rabbit skeletal troponin-C was measured. This fluorescence reflects the binding of Ca2+ to low affinity binding sites (Johnson et al., 1978). In a control experiment, the fluorescence changed with a mid-point at approximately pCa = 5.6 (Fig. 4) (Johnson et al., 1978; lio & Kondo, 1981). At 10 mM, BDM reduced the fluorescence from dansylaziridine bound to troponin-C by about 10%. This is from chemical interaction between BDM and dansylaziridine as it was also observed with free dansylaziridine in solution. The Ca-titration curve in the presence of 10 mM BDM was very similar to the control curve (Fig. 4), showing that Ca-binding to rabbit skeletal troponin-C in solution is not affected by the presence of 10 mM BDM at 23 ~ C. Discussion

In living frog skeletal muscle, the action of BDM on contractile proteins is the major factor responsible for the supression of isometric tetanic tension at BDM concentrations below 10 mM (Horiuti et al., 1988). However, 50

0 4O

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0 8

7

6

5

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Fig. 4. Fluorescent intensity on an arbitrary scale, on an arbitrary scale, from rabbit skeletal troponin-C labelled with dansylaziridine. Titration was made in the absence (C))and presence (0) of 3 mM BDM at 23~ C. pH 6.98 at pCa = 7.15. It decreased as pCa was increased; at pCa = 6.03, pH was 6.90, and at pCa = 4.12, pH was 6.80. Calcium concentration in the figure was corrected for the change in pH.

BDM has other effects on the function of the sarcolemma and sarcoplasmic reticulum (Fryer et al., 1988a, b; Horiuti et al., 1988; Hui & Maylie 1988; Maylie & Hui, 1988). These effects are important in the suppression of tetanic tension even at 0.5 mM in mammalian skeletal muscles at 16 ~ C or 25 ~ C (Fryer et al., 1988a, b), but only at 10 mM in frog skeletal muscle at 18 ~ C (Horiuti et al., 1988; see also Maylie & Hui, 1988). In the present experiments, although the experimental errors were rather large, the intensity increase of the second actin layer-line was not significantly reduced by 3 mM BDM (Fig. 3). As the intensity of this layer-line depends on structural changes in the thin filament which are caused by the binding of Ca2+ to troponin-C (Haselgrove, 1973; Huxley, 1973; Parry & Squire, 1973; Kress et al., 1986), this result indicates that, under the conditions employed (that is, with frog skeletal muscle at 6 ~ C), enough calcium was released to activate the regulatory system on the thin filament, although the activation may not be complete. Another possible site of action of BDM is the regulatory system on the thin filament. BDM is known to decrease the calcium sensitivity of skinned muscle fibres (Fryer et al., 1988b; Horiuti et al., 1988). In the present study, 10 mM BDM was found not to affect the calcium binding of isolated rabbit skeletal troponin-C at 23 ~ C, suggesting that the reduced calcium sensitivity found in skinned muscle fibres is not a result of the effects on the regulation mechanism of the thin filament. In the present X-ray experiments, reduced calcium release or lowered sensitivity of the regulatory system was not considered the major reason for the reduced tension development, for the following reasons. (1) The concentration of BDM was kept low (3 mM in most experiments) to minimize the influence on the regulatory system. Tetanic tension was reduced to 41%. Experiments of 18 ~ C with 5 mM BDM showed that the amount of released calcium is reduced only slightly although force declined to 60% of control (Horiuti et al., 1988, see also Hui & Maylie, 1988). (2) Care was taken to record patterns only in the latter half of a 2-s tetanus when the tension became steady. As calcium concentration keeps increasing during a tetanus (Blinks et al., 1978; Horiuti et al., 1988), the steady tension showed that the cytoplasmic calcium concentration was high enough to fully activate the regulatory system. (3) The influence of BDM on the second actin layer-line (Fig. 3) seems smaller than that on tetanic tension. The relative intensities of the equatorial (1, 0) and (1, 1) reflections reverse when the muscle contracts. This intensity change is interpreted as indicating the amount of myosin-actin interaction (Haselgrove & Huxley, 1973), although it may not quantitatively reflect the amount of attached myosin heads (Lymn, 1978). The present study shows that tension is not linearly correlated with the intensity ratio (-/1,0/I1,1) of the equatorial reflections. At 40% tension in the presence of 3 mM BDM, the intensity ratio of the equatorial reflections (0.76) is close to that of muscle developing maximal tension (0.50). A comparison

BDM on skeletal muscle with the results of three other studies on partially activated muscles makes this point clear. (1) Yu and colleagues (1979) varied the sustained tension of frog muscles using rapid cooling contracture at various caffeine concentrations. Their preparation was similar to that of the present experiment having 11,o/I1,1 value of 2.5 at rest and, 0.45 during maximal tetanus. At a tension level of 40% of the maximum, the equatorial intensity ratio was found to be 1.1. (2) Brenner and Yu (1985) used single skinned rabbit psoas fibres and measured the intensity ratio at various Ca2+ concentrations. Their Ii,o/I1,1 value was 1.25 at rest, 0.63 during maximal activation and about 0.90 at 40% activation. (3) Matsubara and colleagues (1985) measured the intensity ratio of skinned mouse skeletal muscle at various Ca2+ concentrations. Their results show a non-linear relation between the tension and the intensity ratio. The resting value was 1.78 and it decreased to 0.32 during the maximum contraction. At the 40% tension level, the ratio was about 0.8. Note that the ratio was 2.6 at rest and 0.50 during maximum activation in the present experiment, the value of 0.76 obtained during tetanic stimulation in the presence of 3 mM BDM was lower than in all above experiments for the same level of activation (40%). This suggests that the effect of BDM is not simply a lower number of active crossbridges, and that some myosin heads are situated in the vicinity of the thin filament but are not producing tension normally. BDM increases the I~,o/I~,~ ratio in resting muscle (Table 1). Brenner and colleagues (1986) estimated that 2-10% of the myosin heads are bound to actin in resting rabbit glycerinated fibres at ~t = 170 raM, 5 ~ C. If similar amounts of heads are also bound to actin in a resting intact frog muscle, their detachment may increase the intensity ratio. However, binding of myosin heads to actin in resting frog muscle has not been confirmed. As there are other possible explanations (for example, BDM may influence the binding of soluble proteins to myofilaments (Stewart et al., 1979)), it is premature to conclude that the change in the resting equatorial intensities is from an effect on myosin heads. Myosin layer-lines arise from the helical arrangement of myosin heads around the shaft of the thick filament (Huxley & Brown, 1967). Thus their intensities depend on the number of myosin heads arranged in the helix. The present result shows that the intensity of the first myosin layer-line is less sensitive to the presence of BDM than the tension. During contraction, the intensity around the peak of this layer-line drops to 29% of the resting value in control experiments, and to 32% in the presence of 3 mM BDM (Table 2). Although it is not known how the intensity of this layer-line is related to the number of active crossbridges, Haselgrove (1975) suggested that the intensity may be proportional to the square of the number of myosin heads on the helix. If this is the case and the above values are taken literally, then 54% and 57% of the heads would still be on the helix during

159 tetanic contraction in the absence and presence of 3 ram BDM, respectively. The effect of BDM is rather small considering that the tension was reduced by about 50%, suggesting that in the presence of BDM, there is a population of myosin heads which is not arranged according to the helix but do not contribute to tension development. Intensification of the 5.9-nm layer-line during contraction is thought to result from both structural changes associated with the regulation mechanism and binding of myosin heads to the thin filament (Wakabayashi et al., 1985; Kress et al., 1986; Yagi & Matsubara, 1989). On the other hand, intensification of the 5.1 nm layer-line is mostly from binding of myosin heads (Kress el al., 1986). The present study shows that intensification of the 5.1-nm layer-line is significantly reduced, suggesting that, at a given moment, fewer myosin heads are bound to actin during tetanic contraction in the presence of BDM. Biochemical studies by Higuchi and Takemori (1989) showed that BDM suppresses ATPase of heavy meromyosin both in the presence and absence of actin. This indicates that BDM h a s a n inhibitory effect on myosin heads, although an additional effect on actin is not excluded. The results of the present study on equatorial reflections and myosin layer-lines suggest that the number of myosin heads in the vicinity of the thin filament is not reduced to the same extent as tension. Thus some of the myosin heads located close to the thin filament are not developing tension, or are developing less tension than normal heads. Indeed, Fryer and colleagues (1988b) found that, in a skinned rat skeletal muscle, the decrease of stiffness in BDM-influenced fibres was less than that of the maximal Ca2+-activated force. This may indicate that there are myosin heads which are bound to actin but are not developing tension. On the other hand, the results on the actin layer-lines indicate that the number of bound heads is reduced. To explain these results, it is necessary to assume that these anomalous heads are bound to actin and hence contribute to stiffness, but not bound with a fixed configuration to the actin monomers and hence do not contribute to the reflections from the thin filament.

Acknowledgements We thank the late Dr I. Matsubara and Professor Y. Umazume for their encouragement and valuable advice and Dr H. Higuchi for helpful discussion.

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