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Low-angle X-ray diffraction studies of living striated muscle during contraction
J. Mol. Biol. (1967) 25, 3135
Low-angle X-Ray Diffraction Studies of Living Striated Muscle during Contraction C,. I’. ELLIOTT.
J. LOWY AND R. M. M~LLMAN~
Medical Research Council Biophysics Research, (in it 26-29 Drury Lane, London, V.C.2~ En$anrl (Received 3 iTmember
7.066)
l’he spacings and intensities of t)ho small-angle equatorial X-ray reflexions from living t,oad sartorius muscle were studied in the resting and contracted state. On contraction, there is a small and nearly constant decrease of 6 to 12 A in the lattice spacing over the whole range of muscle lengths studied and the 1,l nafkxion becomes slightly more intense relative to the 1,0 reflexion. In both distance between actin rc&ing and contracting muscle, the centre-to-centre and myosin filaments depends on sarcomere length, being about SO A larger in t,he short muscles (sarcomere length 2.1 CL) than in the long ones (sarcomere length 3.6 CL). In another set of experiments with the toad sartorills muscle, it was found that on contraction no change occurred in the spacings of the meridional X-rag rt+flcxions from either the nctin or the myosin filaments. We have also observed a tlocrease in intensity of the myosin layer lines (given by the projections on the Illyosin filaments) as first reported by Huxley, Brown & Holmes (1965). By indicating that there is no change in length in the actin or myosin filaments dnring contraction, these results provide general support for t,he sliding filament hypothesis.
1. Introduction There is now a wealth of evidence from structural, chemical and physiological studies which supports the theory that vertebrate skeletal muscle, and probably ot,her types of muscle as well, shorten according to the sliding filament model originally proposed by A. F. Huxley & R. Niedergerke (1954) and H. E. Huxley & J. Hanson (1954). In this model, conversion of chemical into mechanical energy results from interaction between actin and myosin molecules which are organized into separate filaments of limited lengths. On stimulation, a force (of unknown nature) is generated which causes the two kinds of filaments to move relative to one another. During shortening, or when tension is developed at a constant muscle length, no over-all length changes are supposed to occur in either type of filament. But the precise physical mechanism involved in the operation of the sliding filament system is still obscure (see Huxley. 1960; Hanson & Lowy, 1965). In the many experiments which have been designed with the object of testing the sliding filament theory, a variety of methods has been used. But for mainly technical reasons, the met’hod which can give the most direct information about structural changes in the intact living system at the molecular level-X-ray diffraction-has t Present Canada.
aclclress: Department
of Biosciences, 31
Broclr
University,
St. Catherines,
Ontario,
32
Q.
F.
ELT,TOTT,
J.
T,OWsTy
ANTI
T3. %I. 31JT,T,MAN
not yet been extensively used. Only a few attempts have so far been mnde to use this technique in studies of living muscle during contraction. Bernal, during the 1920’s, took high-angle X-ray diffraction pictures of t)hc leg muscle of a living frog, stimldated with an induction motor, but did not observe any changes in the patt’ern (Bernal, 1962). Boehm (1931) studied frog and tortoise muscles, relaxed and when contracting isotonically or isometrically, by high-angle diffraction. He also did not obscrvcx any spacing changes in the patterns. Astbury (1947) could detect no change in the high-angle pattern obtained from a smooth molluscan muscle during (tonic) contrwtion. Later, Huxley (1953) studied the low-angle diffraction patterns of living resting vertebrate skeletal muscle. From this type of muscle, he obtained a series of meridional reflexions, as well as two on the equator. The meridional reflexions are now known to come from two different systems, one within the actin filaments and the other within the myosin filaments (Elliott & Worthington, 1959; Worthington, 1959). Huxley (1953) found that when the resting living muscle was stretched, no change in the axial low-angle diffraction pattern could be detected. Huxley & Hanson (1954) used this observation together with A&bury’s result on the contracting molluscan muscle as supporting evidence for the sliding Lament hypothesis. Elliott (1964) recorded a series of low-angle layer lines, which he attributed to the helical arrangements of projections on the myosin filaments. The presence of such projections had been known for some time from electron micrographs of sectioned muscle (Huxley, 1957,196O). Evidence from other investigations strongly suggests that these projections contain the part of the myosin molecule where ATPase and actin-combining sites are known to be located (Huxley, 1960,1963; Hanson & Lowy, 1965). Huxley (1963), who observed I,0 and 1,l equatorial reflexions from a hexagonal lattice in resting vertebrate skeletal muscle, suggested (1957,196O) that this lattice has myosin filaments at the corners of the unit cell, and actin filaments at the threefold (trigonal) positions, with two actin Laments per unit cell. Electron microscopy of sectioned material confirmed this, and also showed that the filaments are grouped in distinct transverse arrays which overlap in a particular region (A-band). It is in this region that the filaments form a double hexagonal array with the actin filaments in the trigonal positions (Huxley, 1960). Huxley (1963) discovered that when the living resting muscle is stretched, the spacings of the equatorial reflexions change so that the volume of the filament lattice is kept constant. This was confirmed by Elliott, Lowy & Worthington (1963), who showed that the centre-to-centre distance between actin and myosin filaments varied by about 70 A from the long to the short muscle. The very regular array of the actin and myosin filaments is maintained in spite of variations in the interaament separation. Worthington (1961) found that t,his also applies to insect muscle: in swelling experiments, he was able to increase the interfilament distance by as much as 100 A without upsetting the hexagonal organization of the lattice. Perhaps the most interesting question to be answered about muscular contraction concerns the nature of the forces set up between sites on the actin and myosin filaments when the muscle is activated. Several specific hypotheses for a sliding-filament system (e.g. Hanson $ H. E. Huxley, 1955; A. F. Huxley, 1967; Davies, 1963) have suggested that during contraction the projections from the myosin filaments form direct mechanical “cross-links” to the actin filaments. Since in the resting state the
X-RAY
DIFFRACTION
FROM
CONTRACTING
MUSCLE
33
interfilament di&nce varies widely with muscle length, it is very import,ant to know whdhrr this is also the case in contracting muscle. If so, then the projections nlrist inclde a mrrhnnism 60 operat,e over a distmlce which vnrics by ns mnch R,s 70 a.
The experiments to be described here were undertaken with the object of studying possible changes in the equatorial and meridional low-angle X-ray pattern during contraction. The results, which have been published briefly (Elliott, Lowy & Millman, 1965), show that during contraction at any particular muscle length there are no large
changes
either
in the interfilament
spacing,
or in the arrangement
of the molrl-
cules within the actin and myosin filaments. On the other hand, our results confirm those of Huxley et al. (1965), that the intensity of the layer-line reflexions given by the
projections
on
the
myosin
filaments
decreases
markedly
during
contract.iol~.
results provide strong supporting evidence for the sliding filament theory; but equally important at this stage, they clearly dei?ne certain requirements which These
must
be satisfied
by any
specific
contractile
mechanism.
2. Materials and Methods The sartorius of the toad Bufo bufo (L.) was used, since contraction in this muscle is slower than that of frog sartorius muscle by a factor of about two (Hill, 1961). The muscle was dissected whilst immersed in cooled Ringer’s solution of a composition given by Jewel1 & Wilkie (1958). Rest length (I,) was taken as the length of the muscle between the knee tendon and the insertion on the pelvic bone, with the leg straight and held at an angle of about 90” to the body axis. Sarcomere lengths were measured by light diffraction, initially with the “Eriometer” method used by Elliott et al. (1963), and later with a similar but more accurate photographic method which employed a helium-neon gas laser as :L light source (Rome, 1967).
(a) Experimental
proceclure
The muscle was mounted vertically on the X-ray apparatus, and a constant stream of oxygenated Ringer’s solution flowed over it. This solution was refrigerated and recirculated. The muscle temperature was about 5°C. The muscle was stimulated longitudinally through platinum electrodes at the ends. The stimulus was a double condenser pulse (differentiated square wave, time constant of about 0.5 m-set) supplied from an electronic stimulator (Copeland, 1959). The resulting contraction was recorded isometrically with an RCA 6734 transducer in a conventional bridge circuit. The output was differentiated (time constant about 20 set) to avoid long-term base-line drift, and fed to a transistorized switch which controlled E high-speed relay. The switch circuit could be biased so that the relay could close at any pre-set muscle tension from 1 to 50 g. (It was found necessary to place the switch circuit in a refrigerator to eliminate thermal drift in the transistors.) The armature of the relay was extended and carried a small lead shutter which remained in the path of the X-ray beam unless the relay was energized. Thus only when the muscle was stimulated, and developed more than a pre-set amount of tension, were X-rays allowed to reach it. A second pair of contacts on the relay was used to record the opening of the shutter on one beam of a double-beam oscilloscope. The same beam also served to monitor the stimulus applied to the muscle, while the other beam was connected to the output of the RCA 5734 circuit. Thus it was possible to have, throughout each experiment, a continuous record of the time-course and size of the contractions, as well RS of the functioning of the shutt,rI and stimulator (Fig. 1). The whole apparatus was controlled by a series of circular cams operating microswitches and turned by a single spindle. One of the microswitches triggered the stimulus to the muscle, another started the oscilloscope camera and brightened the oscilloscope beams. A third microswitch energized a relay within the X-ray set (Hilger, microfocus model Y 33) which short-circuited the bias resistor in the X-ray tube circuit. This not only made it
34
-
+
FIG. 1. Typical oscilloscope record showing (upper txace) the tension developed and (lower trace) the stimulus artifact (retouched) and shutter movement.
by tho muscle
possible to run the X-ray tube for most of the time at a very low beam current (< 0.1 ma) but also to over-run it during the exposure period and obtain a beam current of 2 to 3 mA for this period. This is 5 to 6 times the normal operating current of the Hilger microfocus tube on 40 p (nominal) point focus. We found that this short overload (about 0.5 set) did not cause damage to the tube, and that in general the X-ray spot was not appreciably de-focused by this overloading. Occasionally the spot was broadened, but not sufficiently to obscure the diffraction pattern (see Plate I). Low-angle X-ray cameras of the basic Franks design (1955) were used as modified by Elliott & Worthington (1963). (b) Equatorial
diffraction
In our experiments on the equatorial diffraction, a pattern was first recorded from the A good pattern could be obtained in 2 hr resting muscle with a 0.5~mu beam current. or less. A pattern from the contracting muscle was then obtained with the shutter relay in position, and set to open the shutter when the tension reached about 5 g. At aboiit 5”C, the contraction duration was about 0.5 sec. This meant that with 5 times the gain in beam current (obtained as described above), and the muscle being stimulated once or twice per min, 48 or 24 hr, respectively, were required to accumulate the necessary 2 hr. exposure. We found that the muscle would give contractions for such periods; in fact it, could continue contracting, still producing about half the initial tension, for up to 72 hr. After the pattern for the contracting muscle had been obtained, another pattern from the resting muscle was recorded with continuous X-ray exposures. We noticed that at the shorter sarcomere lengths there was a tendency for the resting muscles to become slack. Thus the resting pattern was obtained at a somewhat longer length than the contracting pattern. To some extent this could be counteracted by recording the resting pattern between successive contractions, reversing the shutter relay so as to cut off the X-rays when the muscle was stimulated. Even so, the short muscles could be seen to extend after each contraction, so that they were clearly not contracting isometrically (see also Results, section (ii)). (c) Meridional The experiments in which the meridional depended on modifications to the geometry high-power line focus available on the Hilger
diflraction pattern was recorded during contraction of the X-ray camera and made use of the X-ray set (Millman, manuscript in prepara-
X-RAY
DIFFRACTION
FROM
COSTRz\CTING
AfUSCLE
x^i
tionj. At full current saturation, this line focus is less than 100 p wide, and can be loaded with a 12-InA beam current. The modified X-ray camera could register the “59” A actin roflexion, and the “144” Lk and “72” A myosin reflesions in exposures of 1 to 2 hr from resting muscle. We found that the muscle would givo about t,he same total number of contractions when stimulated 6 times as opposed to twice per min, though at 6 times per min it, would cont*ract for about 24 hr only. This, t)ogether with the life-time of the filament under these conditions (about 30 hr) set the limit to the exposures in these exporimpnts.
3. Results (a) Equatorial
diffract ion
(i) Resting muscle It has been shown previously in resting frog and mammalian muscle that the interfilament lattice spacing is greater at shorter muscle lengths and varies so that thrb lattice volume remains constant over a wide rangc of muscle lengths (Huxley, 1953; Elliott et al., 1963). Using toad sartorius muscle, we have confirmed and extended this result (Plate I(a), (c) and (e)). 0 ver a range of sarcomere lengths from 2.1 p (where the two sets of actin filaments just meet half-way along t,he sarcomere) to 3.6 p (where the A- and I-band filaments just cease to overlap), there is an inverse relationship between sarcomere length and the cross-sectional area of the filament lattice (Fig. 2). (The measurements for filament lengths were obtained by Dr Sally Page, personal communication.) In this experiment the sarcomere length (s) was determined by light diffraction; the filament lattice spacing, given by measurements of the 1,O spacing of the hexagonal lattice (d), was determined by X-ray diffraction. Each point in Fig. 2 represents an individual measurement, but in some cases more than one measurement8 was made on a single muscle. The straight line represents the constant volume for the filament lattice, and has been fitted by the method of least squares to the point,s
Sorcomere
FIG.
2. The collstitllt-\,ululne
bebaviour
length
(,c)
of relit iug
toad
sartoriua
musole.
Tho inverse square of d (measured by X-ray diffraction) is plotted against s (measured by light diffraction). ( 0) Early results where s was measured using mercury-green line; ( l ) s measured using the laser. The straight line represents the best least-squares fit to the points, passing through the origin, and is a line corresponding to a constant lattice volume. Each point represents an individual X-ray pattern, but in some cases several patterns were obtained from a single muscle.
36
G. F. ELLIOTT,
J. LOWY
AND
B. 111. MILLMAX
(assuming that the line passes through the origin). This line corresponds to a latticac volume (i.e. 1.155 ri?s) of 3.66 x 10m3p3. For a large number of the muscles used, and in particular the muscles studied during contraction, we did not obtain light diffraction mcasurcments of the sarcomere length. This was primarily because the rather large muscles optimal for obtaining X-ra,y diffraction patterns were usually too thick to give suitable light diffraction patterns. Also, in order to avoid any unnecessary handling of the muscles before the part of the experiments when they were stimulated, light diffraction measurcmcnts were not attempted until after the X-ray patterns had been obtained. By this tinrc (although the muscle could contract normally) the muscles were usually too opaque for these measurements. It was therefore necessary to use a different measure for the length of many of the muscles, We found that the ratio of the over-all muscle length to I,, the length of the muscle in the animal (see Materials and Methods, par. 1) provided a suitable parameter. When l/d2 was plotted against this ratio, a curve similar to Fig. 2 was obtained with an experimental scatter which was not much greater than that obtained when l/d2 was plotted against sarcomere length. The averages from 84 such results on 56 muscles are shown in Fig. 3 (filled circles), together with the standard deviations of the means for each length interval. Except for the points corresponding to shorter muscle lengths, the results indicate a linear relationship between muscle length and the inverse of the cross-sectional area (i.e. constantvolume behaviour). At the shorter lengths, the resting muscle was often observed to be slack, suggesting that the deviation from linearity at lengths below 80% 1, may be explained by this effect (see p. 40). The solid line in Fig. 3 represents the best leastsquares fit to the points for the resting muscle, ignoring the three points for the shortest muscle lengths, because of this slackness. In contrast to the constant-volume line
Muscle length (%l,j:
Wm. 3. The inverse square of d plotted
against muscle 1engLh for Loud awtorius
~~~usole.
(0) Averages from resting muscles over fked length intervals, with the stantlltrd deviatiorl of muscle, each point representing one complete the’ mean in&cated by lines; (0) contracting experiment. Straight line (which does not paas through the origin) is best least-squares fit to the filled circlea, omitting the lowest three points.
Contracted
Resting
(4
(0
(4
haTE I. Equatori& X-ray diffraction pattern t’rou~ toad mid during “isometric“ contraction ((b), (d), (f)). The 1,O and 1,1 reflexions from the hexagonal fila~nent~ where only the 1,0 reflexion is visible. (a), (b): Musck at, 64 ?L t,o film distsnwr: ((a), (d): Different muscle at I,; specimen I IS?{, I,,; sperirnrn to film dintwwe: 6.64 cm. ‘< 33
swtorius
~nusclf:
at rest
((a),
(c). (e))
ttrraj~ aw SWIL, eurept, in (P) and (f). I,; specirnar t,o film distance: 6.63 cm. 6.53 cam. (
t’l.a,rb; 1 I. Equatorial S-ray diffraction pattern from resting toad sartorius muscle, showing I .O itnd I,1 hexagonal latt,icr reflexions and the (faintj) non-litMice reflexiou lying b&wren them. to film distanre: fi.i
PLATE III. Z,, using
Meridional the Hilger point
X-ray focus
(4 PLATE IV.
diffraction pattern from restitlg toad sartorius muscle at about X-ray source. Specimen t,o film distance: 8.92 cm. x 8.2.
(b)
Meridional X-ray difFraction patterns from toad sartorius muscle at rest (a) and during contraction (b). In both patterns, reflexions from the thick (myosin) and thin (actin) filaments cau be seen on the meridian at 144 A, and just off the meridian at 59.5 il, respectively. The layer-lines seen in (a) and only faintly in (b) are from projections on the myosin filaments. (These layer-lines may be seen more easily if the Plate is tilted along the layer-line direction.)
X-RAY
DIFFRACTION
FROM
CONTRACTING
hlUSCLE
31
in Fig. 2, this line does not pass through the origin, and would have to be shift,etl considerably to do so. The reason for this is not clear. Thus, while muscle length provides a reasonable independent variable for comparative studies between resting and contracting muscles, it is not useful for’ studying the basic filament lat’ticc behaviour; here the sarcomere length itself should be used. In their study of frog and of mammalian muscle, Elliott et al. (1963) found that the relative intensities of the 1,0 and 1,l hexagonal lattice reflexions varied with muscle length. They placed the l,O/l,l intensity ratios in a series of categories ranging from 1 to 7 (see their Fig. 1 and Fig. 4 of this paper). In category 1, only the 1,0 reflexion is visible; in category 7, only the 1,l reflexion is visible; the other categories correspond to intermediate conditions. Using the toad muscle, we have examined these ratios in somewhat more detail. For each X-ray picture, the intensity ratio was estimated independently by each of the three authors, and the results averaged to the near& quarter-category. These results, from the same group of resting muscles as used in Fig. 3, are shown in Fig. 4, averaged over standard intervals of d and plotted against l/d2 (with the standard deviations of the means). Since l/d2 is directly proportional t’o the sarcomere length (see Fig. 2) the abscissa of Fig. 4 can also be calibrated in terms of s (from the line in Fig. 2). It appears that the intensity ratio category is directly related to s up to about 3-O TV(intensity ratio category 1); but above this length, no change in the intensity ratio can be detected since only one reflexion is visible.
FIG. 4. The 1,0 : I,1 intensity ratio of toad sartorius muscle plotted against the inverse scluaru of d. (0) Averagesfrom resting muscles over fixed intervals of d, with standard deviations of the means; (0) contracting muscle, each representing an individual experiment. Straight lines fitted by eye only. The abscissa has also been calibrated in terms of sarcomere length from the straight line in Fig. 2. Intensity categories (as def?ned by Elliott et al., 1963) are as follows: Category 1: 1,0 reflexion seen alone. 2: 1,0 reflexion seen with traces of 1,l reflexion. 3: 1,0 more intense than 1,l (both clearly seen). 4: I,0 and I.1 intensities equal.
38
G. F. ELLIOTT,
J. LO\VY
ANU
IS. M. MILLMAS
The same result is obtained if one plot,s the intensity ratio category muscles in which the sarcomere length was measured directly. (ii) Nuscle
contracting
against s for those
at constant length (isometric)
The equatorial X-ray diffraction pattern of isometrically contracting muscle was compared with t,hat from resting muscle (Plate I). To obtain the pattern during contraction, t’he muscle was stimulated and the pattern recorded only while the muscle was holding a tension greater than about 5 g. The muscle was automatically stimulated once or twice per minute for two or three days to give an aggregate exposure of about one hour-just sufficient to record the pattern. Similar patterns from the resting muscle, obtained with a continuous X-ray exposure of about one hour, were recorded before and aft’er each contracting pattern. It was, of course, only possible to obtain one pattern from each muscle. A series of 23 complete experiments on 23 separate muscles covered as large a range of muscle lengths as possible. The 1,0 spacings (d) were measured and l,O/l,l intensityratio categories assigned for each pattern as described above. The results for the contracting muscles are shown in Figs 3 and 4 as open circles. These results can be compared with the results from resting muscles (filled circles). (In Figs 2 and 3, resting muscle data from the same individual muscles as were used for the contracting muscle results could have been plotted, rather than averaged results from the larger series of resting muscles. If this had been done, the curves for the resting muscles in Figs 3 and 4 would be unchanged in form, though because of the smaller number of points there would be greater scatter in these points.) From a comparison of t.he patterns obt,aincd from resting and isometrically contracting muscles, four points emerge: (1) In the contracting muscle, d2 is inversely proportional to muscle length over the xvhole length range studied (Fig. 3). (2) The line relating l/d2 to muscle length in the contracting muscle lies slightly above that for the resting muscle; i.e. for a given muscle length, the interfilament spacing is slightly smaller in the contracting muscle (Fig. 3). (3) In the contracting muscle, the range of values of l/d2 extends to lower values; i.e. larger interfilament spacings were found than in the resting muscles (Fig. 4). (4) The l,O/l,l intensity ratio in contracting muscle appears to be linearly related to l/d2 and thus to s over its whole range; and for any value of l/d2 the intensity ratio is increased by about 314 of a category as compared with the ratio for the resting muscle (Fig. 4). The change in interfilament spacing on contraction was examined in more detail (Fig. 5). Here, the 1,0 spacing (d) from the contracting muscle is compared with that from the resting muscle, measured either before (Fig. 5(a)) or after (Fig. 5(b)) the muscle was stimulated. In both cases the spacing change is uniform (allowing for the very high scatter of these results) up to a value of d of about 385 8. In short muscles with values of d greater than about 385 A, however, cl increases substantially on contraction. An interesting feature of the results at values of d below 385 L%is that when the spacing from contracting muscle is compared wit’h that measured from the resting muscle before contraction (Fig. 5(a)) the spacing change on corkaction is about -10 A. But when compared to that measured from the resting muscle after contraction (Fig. 5(b)) there is no spacing change. This indicates tha,t during a series of
X-R,AY
DIFFRACTION
FROM
CONTRBCTISG
MUSCLE
0
i %
-20
an
c,
0 c
0
e 0 2
0
0 0 0
0
-40 300
350 d,(i)
400
FIG. 5. (a) Difference in d between the contracting (d,) and resting muscle (CE,)plotted against d, (d, measured before the contraction series). (b), as (a), but d, measured a.fter the contraction series. (c) Difference in d, as measured before and after t,he contraction series, plotted as in (a). (For explanation of solid line in (a) see text, p. 40.)
short contractions (twitches) the interfilament spacing at a given muscle length decreases by about 10 A, and that this decrease is not reversed after the series of contractions. Since the muscle length and, presumably, the sarcomere length, is essentially unchanged (except at the short muscle lengths, see below) this indicates that the lattice volume is permanently diminished on contraction. In fact, this decrease in the resting interfilament spacing seems to hold over the whole range of muscle lengths (Fig. 5(c)). It should also be noted that the sarcomere length may decrease slightly on contraction because of the effect of series elasticity at the ends of the muscle. This would mean that the volume decrease may be even greater than suggested above.
40
G. F. ELLIOTT,
.J. LOWY
AND
B. RI. MILLMAN
The results from contracting muscles provide several lines of evidence to support the suggestion made oarlic,r (see p. 36) that the non-linear part, of the l/rE2 : muscle length curve for tlrc rrst,ing muscle (I’i4 g. 3, f&d circ,lcs) was caused by slackness in the muscle. First,, no such non-linearity is seen in the corresponding ollrve for the contracting muscle (Fig. 3, open circles). Second, larger values of d are found in contracting than in resting muscles (see Fig. 4), suggesting that the contract,ing muscles have a length range which extends to shorter sarcomere lengths. Thirdly, the change in spacing on contraction is uniform for values of d up to about 385 h (Fig. 5), which is approximately the highest value for d observed in the resting muscles. The points observed above d = 385 A show an increase in d on contraction, which suggests that t,hcse muscles are actually shortening on contraction. If one assumes that the resting muscle can passively maintain sarcomere lengths corresponding to d = 385 A (2.15 p from Fig. 2) but no shorter, and that on stimulation the muscle will shorten further so as t.o remove any slackness in the muscle fibres, the change predicted in the l,O spacing on contraction is shown by the solid line in Fig. 5(a). It is clear that the experimental results are in good agreement with this prediction. (iii) Additional
re$exions
Frequently, especially when longer X-ray exposures were given, equatorial reflexions in addition to the 1,0 and 1,l hexagonal l&tic reflexions could be seen on the negative. These reflexions were always very faint in comparison with the 1,0 reflexion. Although the 2,0 hexagonal reflexion was often seen, the most commonly observed additional reflexion was one which lay between the 1,0 and 1,l reflexions and could not be indexed on the hexagonal lattice (Plate II). This reflexion was seen at all muscle lengths and in patterns from both relaxed and contJracting muscles. The measurements varied considerably (from 197 to 301 A), and a high variation was also found if the reflexion was expressed in terms of the 1,O hexagonal lattice reflexion (from O-621 to 0.780). In the latter case, however, there was less spread in the results, and the distribution was more uniform, suggesting that, the structure responsible for this reflexion changes its dimensions with muscle length in the same general way as does the filament lattice. It seems possible that this reflexion could come from the square I-filament lattice adjacent to the Z-line, in which case it would appear that this lattice expands in the same general manner as the filament lattice. Electron microscope observations on frog sartorius muscle have shown (M. K. Reedy, personal communication) that the dimensions of the Z-line lattice and adjacent I-filament lattice change (over the range from 200 to 300 A in the I-filament lattice) inversely with sarcomere length (between 3.2 and 1.7 p). (b) Meridional
dilraction
Toad sartorius muscle gives very detailed X-ray diffraction patterns at low angles in the meridional direction (Plate III). There is a series of reflexions from the thick (myosin) filaments which lie on layer lines with a basic periodicity of about 432 A. These layer lines, which were first observed in living frog sartorius muscle by Elliott (1964), show considerable detail in the equatorial direction, and almost certainly come from the projections on the thick filaments seen with the electron microscope by Huxley (1957,1963). In addition to the myosin reflexions, there is a series of meridional and near-meridional reflexions at angles corresponding to about 59 A and greater, which come from the actin filaments (Astbury R: Spark, 1947; Selby & Bear, 1956)
X-RAY
DIFFRACTION
FROM
CONTRACTING
41
MUSCLE
of which only the 59 B near-meridional reflexion is seen in Plate III. In some X-ray photographs, additional reflexions at smaller angles, and particularly an off-meridiona,l reflexion at about 400 8, can be seen. These have been assigned to actin layer-lines (Millman, Elliott & Lowy, 1967). I n some muscles, sharp meridional reflexions arc seen just outside the 3rd and 6th order myosin layer-lines. These probably come from collagen, present in small amounts in this muscle, indexing as the 5th and 10th orders of a basic period of about 670 a (see Worthington & Tomlin, 1955). It has been found that the actin and myosiu meridional spacings do not change when the muscles contract isometrically (Elliott et al., 1965; Huxley et al., 1965). Table 1 shows the averaged spacings from all the myosin and actin reflexions which could be measured on the diffraction patterns of resting and contracting muscles. l’here is no change greater than 0.2% in either the actin or myosin periodicity (Table 1). The myosin results were collected and averaged to give the basic periodicity in the resting and contracting conditions of 432.4 d (137 reflexion measurements from 34 resting muscles) and 432.8 A (39 reflexion measurements from 12 contracting muscles), respectively. TABLEI Layer-line
order
Resting
muscle
Contracting
muscle
Spacings in A (a) Myosin
reflexions 11 10 9 8 7 6 5 4 3 2
Average basic myosin periodicity
30.3 & 0.06t (9)$ 43.5 49.7 54.1 60.1 71.8 86.2 107.5 143.7 217
* f f -& f & 4 & &
0.17 0.87 0.17 0.09 0.20 0.34 0.47 0.39 1.5
-
(7) (4) (6) (3) (27) (17) (17) (28) (19)
44.0 48.8 72.2 86.8 107.8 143.5 218
432.4 + 0.63
(137)
432.8 & 1.43
(39)
59.5 + 0.12 404 & 3.3
(32) (23)
59.3 & 0.22 407 * 11
(10) (3)
& h & * *
0.31 0.69 1.14 0.79 3.7
(1) (1) (9) (4) (6) (11) (7)
(b) Actin reflexions 7 1 t Standard deviation of the mean. $ Number of measurements.
A few experiments were done using a lever and stops, arranged so that the muscle was allowed to shorten under a light load by 1 to 2 mm (5 to 10%) before developing isometric tension. In these experiments the actin and myosin spacings again remained unchanged (within the experimental error of about 2%). Huxley et al. (1965), using frog sartorius muscle, have shown that during isometric contraction the intensity of the myosin layer lines is greatly diminished. We have confirmed this change of intensity using the toad muscle. In Plate IV, X-ray patterns from the same toad muscle at rest and during contraction are shown in which this
42
G. F. ELLIOTT,
J. LOWY
AND
B. M. MILLMAX
decrease in myosin layer-line intensity can be seen. This result indicates a decrease in the order or change in the arrangement of the structure giving rise to the la,yer-lines; i.e. in the projections from the myosin filaments. On the other hand, there does not, appear to be an appreciable change in the intensities of the actin reflexions.
4. Discussion We will first consider the physiological state of the muscles we examined. It could be thought that the parts of the muscle from which reflexions were obtained had been damaged by irradiation, so that they were no longer capable of contraction. This is not so. In the first place, at any particular muscle length, stimulation produces definite and predictable structural effects; namely, a change in the spacings and intensity ratios of the equatorial reflexions, and a decrease in the intensity of the myosin layer-lines. Second, in the muscles which were slack before stimulation, contraction results in a structural change which is the opposite of that occurring at longer lengths. As seen in Fig. 5(a), the lattice spacing decreases on contraction by a fixed amount for starting sarcomere lengths greater than 2.15 TV,corresponding to a d value of 385 A. Assuming that 2.15 p is the shortest sarcomere length which the resting muscle can maintain-and there is good evidence for this (see p. 40)-the extent of the increase in the lattice spacing on contraction when the starting value of d is greater than 385 A (i.e. when sarcomere length is less than 2.15 CL)can be accurately predicted. The agreement of our results with these predictions (Fig. 5(a)) clearly shows that active shortening takes place in the parts of the muscle from which the X-ray diffraction pattern has been obtained. Concerning activation in the muscles used in our experiments, Hill (1949) established that in a twitch of the frog’s sartorius at 0°C the muscle is almost fully activated. In all probability this applies also to our toad muscles, particularly as we applied a double stimulus. There is therefore every reason to believe that the X-ray reflexions we have recorded during contractions were obtained from fully activated, normally functioning muscles. With toad muscle we have conlirmed, and in some respects extended, results obtained with resting frog muscle (Elliott et al., 1963) which showed that the spacings and intensities of the equatorial reflexions change with muscle length. The general implications were discussed in that paper. The following points about the present results may be noted. The sarcomere spacing at rest length (I,) in the toad muscle was found to be 2.9 p as compared with 2.5 p for the frog’s sartorius. The natural position (and use) of the frog’s leg is, however, very different from that of the toad; and it seems quite likely that the rest length we have used as a standard here bears a quite different relationship to the natural operating muscle length in the toad as compared with the frog. We found it impossible to prepare a resting muscle which could maintain a length shorter than that which corresponds to a sarcomere spacing of about 2.15 TV(p. 40 and Fig. 2). In frog muscle this is the spacing at which the actin filaments move into the zone where the myosin filaments have no projections (see Page & Huxley, 1963). A similar sarcomere spacing (2.1 p) was obtained by Gordon, A. F. Huxley & Julian (1966) for the slack length of single fibres from the frog’s semitendinosus muscle. lt is significant that the length of the muscle when slack is the same in whole toad sartorius as in single fibres of frog semitendinosus.
S-RAP
DIFFRACTION
FROM
COKTRACTING
MT’SCLE
43
On cont’raction, the intensity ratio is decreased by about 314 of a category; the 1 ,l reflexion becoming more intense relative to the 1,0 reflexion. This effect might be due either to an increased order in the actin filaments or to a decreasing order in the myosin filaments, or to both these effects together. An explanation in terms of an increased order of the actin filaments over the whole of the I-bands (Elliott et nl., 1965) seems unlikely, because the effect is much the same at all muscle lengths (except for the very long lengths where only the 1,0 reflexion is visible). Rather it appears in that cast that during contraction the ordering of the actin filaments within the hexagonal lattice is improved over a region of definite length (irrespective of sarcomere lcngth)possibly the region where the myosin filaments taper. This implies that in the regions where the myosin filaments taper (about 0.2 p at either end, cf. Huxley, 1957,1963), the actin filaments are not well ordered in the resting muscle. This is supported by the observation that the 1,l reflexion is not seen (i.e. the intensity ratio category is 1) with sarcomere lengths greater than about 3 p in the resting muscle (Fig. 3). If t,he actin filament order were maintained in the overlap regions, the 1,l reflexion might be expected to appear, although any calculation necessarily involves a number of assumptions (see Elliott et al., 1963). An observation that is a,s yet unexplained is the permanent decrease of dl,, following a long series of contractions (Fig. 4(c)). Conceivably this might be the result of changes in pH as a, result of metabolic activity. The fact that changes in pH can bring about changchs in this spacing has recently been demonstrated in glycerol-extracted preparations of rabbit psoas muscle, where a decrease of about 0.7 pH unit reduces d,,, by about 12 a (Rome, manuscript in preparation). Thus our decrease of 6 to 10 ii in might be explained by a drop in pH of about 0.4 pH unit. Studies on the internal 4,o pH of muscle fibres do not show changes of this order during short contra&ions @s&he, 1960). In fat’igued muscle, however, the pH can be lowered by as much as 1 pH unit (see Caldwell, 1958) and this may be t’he cause of the decrease in d,,, that, we have observed. Our studies of meridional patterns show that within the experimental error of 0.2:/, there is no detectable change in the structure within either the actin or myosin filaments during “isometric” contraction. This indicates that there is no over-all length change in these filaments. Cyclical changes could occur if t,heyinvolved only a small fraction of the structure at any one time; these would not be detected by our experiment,s. These results can be fully accommodated by t.he sliding filament hypothesis. However, our results provide strong evidence against models which assume that filaments shorten during contraction (e.g. Podolsky, 1962). If this were so, our experiments would have detected such a change. Even under very stringentI>, external isometric conditions, frog sartorius muscle can still shorten by about 27,, due t,o intrinsic series elasticity (Jewel1 & Wilkie, 1958). In our experiments, tht conditions of mounting the muscles were such that they could probably shorten by at, least 5%. Thus, if contraction is caused by a shortening of either type of filament, this should have been observed in our experiments, in which a change in axial spacing of as lit’tle as 0.20/, could be detected. This conclusion is further strengthened by the experiments where the muscles contracted under isotonic conditions (p. 41). We have identified t.he 400 A layer-line reflexion (see Table 1) as coming from the actin filaments (Millman, Elliott & Lowy, 1967). Our results here indicate that there is no change in this reflexion, and therefore in the long period of the actin filaments, during contraction.
44
G. I?. ELLIOTT,
J. LOWY
ASI)
11. I\I. MILI,!vl~~N
Two basically different kinds of physical explanation can be proposed to account for the operation of a sliding filament mechanism. One is that development of tension and shortening are the result of direct mechanical attachment and detachment of “cross-links” between actin and myosin filaments (see Hanson & H. E. Huxley, 1955; A. F. Huxley, 1957; Davies, 1963). If so, the links between the filaments must be able to act over a distance which varies widely with musclelength. Huxley et al. (1965) have pointed out, however, that the changes in the intensities of hhe myosin layerlines during contraction suggest that the projections on the myosin filaments could undergo quite large configurational changes. A second basic type of sliding filament mechanism requires the operation of longrange forces, perhaps electrostatic forces or electromagnetic resonance forces (see, for example, Spencer & Worthington, 1960). From the results on interfilament spacings in living muscle at rest and during contraction and from the recent work of Rome (manuscript in preparation) on interfilament spacings in glycerol-extracted material, it seems to us that an explanation of this type provides a good working hypothesis. We do not believe, however, that it is possible to decide finally between these two basic types of mechanism until it is known whether the change in order observed in the myosin projections is a cause of the force-producing activity or a consequence of it. We are grateful to Professor Sir John Randall for constant encouragement and to Professor Jean Hanson for useful discussions. We thank Miss B. J. Kimble, Mrs S. Weindling, Miss M. Kinchin, Mr D. Herbert and Mr C. McCarthy for technical assistance and Mr Z. Gabor for help with photographic reproduction. REFERENCES Astbury, W. T. (1947). Proc. Roy. Sot. B, 134, 303. A&bury, W. T. & Spark, L. C. (1947). Biochim. biophys. Acta, 1, 388. Bernal, J. D. (1962). In Fifty Years of X-ray Diffraction, ed. by P. P. Ewdd, Int. Union of Crystallography. Utrecht. Boehm, G. (1931). 2.f. BioZogie, 91, 203. Caldwell, P. (1958). J. Phyeiol. 142, 22. Copeland, K. (1969). Second I.F.M.E. Conference, Paris. Davies, R. E. (1963). Nature, 199, 1068. DistBche, A. (1960). Nature, 187, 1119. Elliott, G. F. (1964). Proc. Roy. Sot. B, 160, 467. Elliott, G. F., Lowy, J. & Milhnan, B. M. (1965). Nature, 206, 1357. Elliott, G. F., Lowy, J. & Worthington, C. R. (1963). J. Mol. Biol. 6, 296. Elliott, G. F. & Worthington, C. R. (1959). J. Physiol. 149, 32 P. Elliott, G. F. & Worthington, C. R. (1963). J. UZtrastructure Ree. 9, 166. Franks, A. (1955). Proc. Phys. Sot. B, 68, 1054. Gordon, A. M., Huxley, A. F. & Julian, F. J. (1966). J. Phyaiol. 184, 170. Hanson, J. & Huxley, H. E. (1955). Symp. Sot. Exp. BioZ. 9, 228. Hanson, J. & Lowy, J. (1963). J. Mol. BioZ. 6, 46. Hanson, J. & Lowy, J. (1965). Brit. Med. Bull. 21, 264. Hill, A. V. (1949). Proc. Roy. Sot. B, 136, 399. Hill, A. V. (1961). J. Physiol. 159, 518. Huxley, A. F. (1957). Prog. Biophys. Biophys. Chene. 7, 255. Huxley, A. F. & Niedergerke, R. (1954). Nature, 173, 971. Huxley, H. E. (1953). Proc. Roy. Sot. B, 141, 59. Huxley, H. E. (1957). J. Biophys. B&hem. Cytol. 3, 631. Huxley, H. E. (1960). In The Cell, ed. by J. Brachet & A. Mirsky, vol. 4. New York: Academic Press.
S-RAY
DIFFRACTION
FltOhf
CONTl
MUSCLE
Huxley, H. E. (1963). J. Mol. Biol. 7, 281. Huxley, H. E., Brown, W. & Holmes, K. C. (1965). Nature, 206, 1358. Huxley, H. E. & Hanson, J. (1954). ib’uture, 173, 973. Jewcll, B. R. & Willrie, D. R. (1958). J. Physiol. 143, 515. Millman, B. M.. Elliott, 0. F. & Lowy, J. (1967). ATuture, 213, 356. Poclolsky, R. J. (1962). E’ed. I’ror. 21, 964. Sclhy, C’. (1. & Bcu, It. S. (1956). .T. Uioph?/s. Uiocho,r. Cl/M. 2, 71. Spenocr, RI. S.z\vorthin$torl, c. l<. (1960). Natwe, 187. 3%. \\Tortjllington, (1. I{. (1959). .I. illol. Uiol. 1, 398. \\‘ort,lli@on, (‘. le. (1961). .7. AioZ. LlioZ. 3, 61s. \\wort.llington, f.‘. I;. & ‘I’utnlin, S. G. (1!)56). 7’w(,. /:c,!/. Srtc. .2, 235, 189.
Low-angle X-Ray Diffraction Studies of Living Striated Muscle during Contraction C,. I’. ELLIOTT.
J. LOWY AND R. M. M~LLMAN~
Medical Research Council Biophysics Research, (in it 26-29 Drury Lane, London, V.C.2~ En$anrl (Received 3 iTmember
7.066)
l’he spacings and intensities of t)ho small-angle equatorial X-ray reflexions from living t,oad sartorius muscle were studied in the resting and contracted state. On contraction, there is a small and nearly constant decrease of 6 to 12 A in the lattice spacing over the whole range of muscle lengths studied and the 1,l nafkxion becomes slightly more intense relative to the 1,0 reflexion. In both distance between actin rc&ing and contracting muscle, the centre-to-centre and myosin filaments depends on sarcomere length, being about SO A larger in t,he short muscles (sarcomere length 2.1 CL) than in the long ones (sarcomere length 3.6 CL). In another set of experiments with the toad sartorills muscle, it was found that on contraction no change occurred in the spacings of the meridional X-rag rt+flcxions from either the nctin or the myosin filaments. We have also observed a tlocrease in intensity of the myosin layer lines (given by the projections on the Illyosin filaments) as first reported by Huxley, Brown & Holmes (1965). By indicating that there is no change in length in the actin or myosin filaments dnring contraction, these results provide general support for t,he sliding filament hypothesis.
1. Introduction There is now a wealth of evidence from structural, chemical and physiological studies which supports the theory that vertebrate skeletal muscle, and probably ot,her types of muscle as well, shorten according to the sliding filament model originally proposed by A. F. Huxley & R. Niedergerke (1954) and H. E. Huxley & J. Hanson (1954). In this model, conversion of chemical into mechanical energy results from interaction between actin and myosin molecules which are organized into separate filaments of limited lengths. On stimulation, a force (of unknown nature) is generated which causes the two kinds of filaments to move relative to one another. During shortening, or when tension is developed at a constant muscle length, no over-all length changes are supposed to occur in either type of filament. But the precise physical mechanism involved in the operation of the sliding filament system is still obscure (see Huxley. 1960; Hanson & Lowy, 1965). In the many experiments which have been designed with the object of testing the sliding filament theory, a variety of methods has been used. But for mainly technical reasons, the met’hod which can give the most direct information about structural changes in the intact living system at the molecular level-X-ray diffraction-has t Present Canada.
aclclress: Department
of Biosciences, 31
Broclr
University,
St. Catherines,
Ontario,
32
Q.
F.
ELT,TOTT,
J.
T,OWsTy
ANTI
T3. %I. 31JT,T,MAN
not yet been extensively used. Only a few attempts have so far been mnde to use this technique in studies of living muscle during contraction. Bernal, during the 1920’s, took high-angle X-ray diffraction pictures of t)hc leg muscle of a living frog, stimldated with an induction motor, but did not observe any changes in the patt’ern (Bernal, 1962). Boehm (1931) studied frog and tortoise muscles, relaxed and when contracting isotonically or isometrically, by high-angle diffraction. He also did not obscrvcx any spacing changes in the patterns. Astbury (1947) could detect no change in the high-angle pattern obtained from a smooth molluscan muscle during (tonic) contrwtion. Later, Huxley (1953) studied the low-angle diffraction patterns of living resting vertebrate skeletal muscle. From this type of muscle, he obtained a series of meridional reflexions, as well as two on the equator. The meridional reflexions are now known to come from two different systems, one within the actin filaments and the other within the myosin filaments (Elliott & Worthington, 1959; Worthington, 1959). Huxley (1953) found that when the resting living muscle was stretched, no change in the axial low-angle diffraction pattern could be detected. Huxley & Hanson (1954) used this observation together with A&bury’s result on the contracting molluscan muscle as supporting evidence for the sliding Lament hypothesis. Elliott (1964) recorded a series of low-angle layer lines, which he attributed to the helical arrangements of projections on the myosin filaments. The presence of such projections had been known for some time from electron micrographs of sectioned muscle (Huxley, 1957,196O). Evidence from other investigations strongly suggests that these projections contain the part of the myosin molecule where ATPase and actin-combining sites are known to be located (Huxley, 1960,1963; Hanson & Lowy, 1965). Huxley (1963), who observed I,0 and 1,l equatorial reflexions from a hexagonal lattice in resting vertebrate skeletal muscle, suggested (1957,196O) that this lattice has myosin filaments at the corners of the unit cell, and actin filaments at the threefold (trigonal) positions, with two actin Laments per unit cell. Electron microscopy of sectioned material confirmed this, and also showed that the filaments are grouped in distinct transverse arrays which overlap in a particular region (A-band). It is in this region that the filaments form a double hexagonal array with the actin filaments in the trigonal positions (Huxley, 1960). Huxley (1963) discovered that when the living resting muscle is stretched, the spacings of the equatorial reflexions change so that the volume of the filament lattice is kept constant. This was confirmed by Elliott, Lowy & Worthington (1963), who showed that the centre-to-centre distance between actin and myosin filaments varied by about 70 A from the long to the short muscle. The very regular array of the actin and myosin filaments is maintained in spite of variations in the interaament separation. Worthington (1961) found that t,his also applies to insect muscle: in swelling experiments, he was able to increase the interfilament distance by as much as 100 A without upsetting the hexagonal organization of the lattice. Perhaps the most interesting question to be answered about muscular contraction concerns the nature of the forces set up between sites on the actin and myosin filaments when the muscle is activated. Several specific hypotheses for a sliding-filament system (e.g. Hanson $ H. E. Huxley, 1955; A. F. Huxley, 1967; Davies, 1963) have suggested that during contraction the projections from the myosin filaments form direct mechanical “cross-links” to the actin filaments. Since in the resting state the
X-RAY
DIFFRACTION
FROM
CONTRACTING
MUSCLE
33
interfilament di&nce varies widely with muscle length, it is very import,ant to know whdhrr this is also the case in contracting muscle. If so, then the projections nlrist inclde a mrrhnnism 60 operat,e over a distmlce which vnrics by ns mnch R,s 70 a.
The experiments to be described here were undertaken with the object of studying possible changes in the equatorial and meridional low-angle X-ray pattern during contraction. The results, which have been published briefly (Elliott, Lowy & Millman, 1965), show that during contraction at any particular muscle length there are no large
changes
either
in the interfilament
spacing,
or in the arrangement
of the molrl-
cules within the actin and myosin filaments. On the other hand, our results confirm those of Huxley et al. (1965), that the intensity of the layer-line reflexions given by the
projections
on
the
myosin
filaments
decreases
markedly
during
contract.iol~.
results provide strong supporting evidence for the sliding filament theory; but equally important at this stage, they clearly dei?ne certain requirements which These
must
be satisfied
by any
specific
contractile
mechanism.
2. Materials and Methods The sartorius of the toad Bufo bufo (L.) was used, since contraction in this muscle is slower than that of frog sartorius muscle by a factor of about two (Hill, 1961). The muscle was dissected whilst immersed in cooled Ringer’s solution of a composition given by Jewel1 & Wilkie (1958). Rest length (I,) was taken as the length of the muscle between the knee tendon and the insertion on the pelvic bone, with the leg straight and held at an angle of about 90” to the body axis. Sarcomere lengths were measured by light diffraction, initially with the “Eriometer” method used by Elliott et al. (1963), and later with a similar but more accurate photographic method which employed a helium-neon gas laser as :L light source (Rome, 1967).
(a) Experimental
proceclure
The muscle was mounted vertically on the X-ray apparatus, and a constant stream of oxygenated Ringer’s solution flowed over it. This solution was refrigerated and recirculated. The muscle temperature was about 5°C. The muscle was stimulated longitudinally through platinum electrodes at the ends. The stimulus was a double condenser pulse (differentiated square wave, time constant of about 0.5 m-set) supplied from an electronic stimulator (Copeland, 1959). The resulting contraction was recorded isometrically with an RCA 6734 transducer in a conventional bridge circuit. The output was differentiated (time constant about 20 set) to avoid long-term base-line drift, and fed to a transistorized switch which controlled E high-speed relay. The switch circuit could be biased so that the relay could close at any pre-set muscle tension from 1 to 50 g. (It was found necessary to place the switch circuit in a refrigerator to eliminate thermal drift in the transistors.) The armature of the relay was extended and carried a small lead shutter which remained in the path of the X-ray beam unless the relay was energized. Thus only when the muscle was stimulated, and developed more than a pre-set amount of tension, were X-rays allowed to reach it. A second pair of contacts on the relay was used to record the opening of the shutter on one beam of a double-beam oscilloscope. The same beam also served to monitor the stimulus applied to the muscle, while the other beam was connected to the output of the RCA 5734 circuit. Thus it was possible to have, throughout each experiment, a continuous record of the time-course and size of the contractions, as well RS of the functioning of the shutt,rI and stimulator (Fig. 1). The whole apparatus was controlled by a series of circular cams operating microswitches and turned by a single spindle. One of the microswitches triggered the stimulus to the muscle, another started the oscilloscope camera and brightened the oscilloscope beams. A third microswitch energized a relay within the X-ray set (Hilger, microfocus model Y 33) which short-circuited the bias resistor in the X-ray tube circuit. This not only made it
34
-
+
FIG. 1. Typical oscilloscope record showing (upper txace) the tension developed and (lower trace) the stimulus artifact (retouched) and shutter movement.
by tho muscle
possible to run the X-ray tube for most of the time at a very low beam current (< 0.1 ma) but also to over-run it during the exposure period and obtain a beam current of 2 to 3 mA for this period. This is 5 to 6 times the normal operating current of the Hilger microfocus tube on 40 p (nominal) point focus. We found that this short overload (about 0.5 set) did not cause damage to the tube, and that in general the X-ray spot was not appreciably de-focused by this overloading. Occasionally the spot was broadened, but not sufficiently to obscure the diffraction pattern (see Plate I). Low-angle X-ray cameras of the basic Franks design (1955) were used as modified by Elliott & Worthington (1963). (b) Equatorial
diffraction
In our experiments on the equatorial diffraction, a pattern was first recorded from the A good pattern could be obtained in 2 hr resting muscle with a 0.5~mu beam current. or less. A pattern from the contracting muscle was then obtained with the shutter relay in position, and set to open the shutter when the tension reached about 5 g. At aboiit 5”C, the contraction duration was about 0.5 sec. This meant that with 5 times the gain in beam current (obtained as described above), and the muscle being stimulated once or twice per min, 48 or 24 hr, respectively, were required to accumulate the necessary 2 hr. exposure. We found that the muscle would give contractions for such periods; in fact it, could continue contracting, still producing about half the initial tension, for up to 72 hr. After the pattern for the contracting muscle had been obtained, another pattern from the resting muscle was recorded with continuous X-ray exposures. We noticed that at the shorter sarcomere lengths there was a tendency for the resting muscles to become slack. Thus the resting pattern was obtained at a somewhat longer length than the contracting pattern. To some extent this could be counteracted by recording the resting pattern between successive contractions, reversing the shutter relay so as to cut off the X-rays when the muscle was stimulated. Even so, the short muscles could be seen to extend after each contraction, so that they were clearly not contracting isometrically (see also Results, section (ii)). (c) Meridional The experiments in which the meridional depended on modifications to the geometry high-power line focus available on the Hilger
diflraction pattern was recorded during contraction of the X-ray camera and made use of the X-ray set (Millman, manuscript in prepara-
X-RAY
DIFFRACTION
FROM
COSTRz\CTING
AfUSCLE
x^i
tionj. At full current saturation, this line focus is less than 100 p wide, and can be loaded with a 12-InA beam current. The modified X-ray camera could register the “59” A actin roflexion, and the “144” Lk and “72” A myosin reflesions in exposures of 1 to 2 hr from resting muscle. We found that the muscle would givo about t,he same total number of contractions when stimulated 6 times as opposed to twice per min, though at 6 times per min it, would cont*ract for about 24 hr only. This, t)ogether with the life-time of the filament under these conditions (about 30 hr) set the limit to the exposures in these exporimpnts.
3. Results (a) Equatorial
diffract ion
(i) Resting muscle It has been shown previously in resting frog and mammalian muscle that the interfilament lattice spacing is greater at shorter muscle lengths and varies so that thrb lattice volume remains constant over a wide rangc of muscle lengths (Huxley, 1953; Elliott et al., 1963). Using toad sartorius muscle, we have confirmed and extended this result (Plate I(a), (c) and (e)). 0 ver a range of sarcomere lengths from 2.1 p (where the two sets of actin filaments just meet half-way along t,he sarcomere) to 3.6 p (where the A- and I-band filaments just cease to overlap), there is an inverse relationship between sarcomere length and the cross-sectional area of the filament lattice (Fig. 2). (The measurements for filament lengths were obtained by Dr Sally Page, personal communication.) In this experiment the sarcomere length (s) was determined by light diffraction; the filament lattice spacing, given by measurements of the 1,O spacing of the hexagonal lattice (d), was determined by X-ray diffraction. Each point in Fig. 2 represents an individual measurement, but in some cases more than one measurement8 was made on a single muscle. The straight line represents the constant volume for the filament lattice, and has been fitted by the method of least squares to the point,s
Sorcomere
FIG.
2. The collstitllt-\,ululne
bebaviour
length
(,c)
of relit iug
toad
sartoriua
musole.
Tho inverse square of d (measured by X-ray diffraction) is plotted against s (measured by light diffraction). ( 0) Early results where s was measured using mercury-green line; ( l ) s measured using the laser. The straight line represents the best least-squares fit to the points, passing through the origin, and is a line corresponding to a constant lattice volume. Each point represents an individual X-ray pattern, but in some cases several patterns were obtained from a single muscle.
36
G. F. ELLIOTT,
J. LOWY
AND
B. 111. MILLMAX
(assuming that the line passes through the origin). This line corresponds to a latticac volume (i.e. 1.155 ri?s) of 3.66 x 10m3p3. For a large number of the muscles used, and in particular the muscles studied during contraction, we did not obtain light diffraction mcasurcments of the sarcomere length. This was primarily because the rather large muscles optimal for obtaining X-ra,y diffraction patterns were usually too thick to give suitable light diffraction patterns. Also, in order to avoid any unnecessary handling of the muscles before the part of the experiments when they were stimulated, light diffraction measurcmcnts were not attempted until after the X-ray patterns had been obtained. By this tinrc (although the muscle could contract normally) the muscles were usually too opaque for these measurements. It was therefore necessary to use a different measure for the length of many of the muscles, We found that the ratio of the over-all muscle length to I,, the length of the muscle in the animal (see Materials and Methods, par. 1) provided a suitable parameter. When l/d2 was plotted against this ratio, a curve similar to Fig. 2 was obtained with an experimental scatter which was not much greater than that obtained when l/d2 was plotted against sarcomere length. The averages from 84 such results on 56 muscles are shown in Fig. 3 (filled circles), together with the standard deviations of the means for each length interval. Except for the points corresponding to shorter muscle lengths, the results indicate a linear relationship between muscle length and the inverse of the cross-sectional area (i.e. constantvolume behaviour). At the shorter lengths, the resting muscle was often observed to be slack, suggesting that the deviation from linearity at lengths below 80% 1, may be explained by this effect (see p. 40). The solid line in Fig. 3 represents the best leastsquares fit to the points for the resting muscle, ignoring the three points for the shortest muscle lengths, because of this slackness. In contrast to the constant-volume line
Muscle length (%l,j:
Wm. 3. The inverse square of d plotted
against muscle 1engLh for Loud awtorius
~~~usole.
(0) Averages from resting muscles over fked length intervals, with the stantlltrd deviatiorl of muscle, each point representing one complete the’ mean in&cated by lines; (0) contracting experiment. Straight line (which does not paas through the origin) is best least-squares fit to the filled circlea, omitting the lowest three points.
Contracted
Resting
(4
(0
(4
haTE I. Equatori& X-ray diffraction pattern t’rou~ toad mid during “isometric“ contraction ((b), (d), (f)). The 1,O and 1,1 reflexions from the hexagonal fila~nent~ where only the 1,0 reflexion is visible. (a), (b): Musck at, 64 ?L t,o film distsnwr: ((a), (d): Different muscle at I,; specimen I IS?{, I,,; sperirnrn to film dintwwe: 6.64 cm. ‘< 33
swtorius
~nusclf:
at rest
((a),
(c). (e))
ttrraj~ aw SWIL, eurept, in (P) and (f). I,; specirnar t,o film distance: 6.63 cm. 6.53 cam. (
t’l.a,rb; 1 I. Equatorial S-ray diffraction pattern from resting toad sartorius muscle, showing I .O itnd I,1 hexagonal latt,icr reflexions and the (faintj) non-litMice reflexiou lying b&wren them. to film distanre: fi.i
PLATE III. Z,, using
Meridional the Hilger point
X-ray focus
(4 PLATE IV.
diffraction pattern from restitlg toad sartorius muscle at about X-ray source. Specimen t,o film distance: 8.92 cm. x 8.2.
(b)
Meridional X-ray difFraction patterns from toad sartorius muscle at rest (a) and during contraction (b). In both patterns, reflexions from the thick (myosin) and thin (actin) filaments cau be seen on the meridian at 144 A, and just off the meridian at 59.5 il, respectively. The layer-lines seen in (a) and only faintly in (b) are from projections on the myosin filaments. (These layer-lines may be seen more easily if the Plate is tilted along the layer-line direction.)
X-RAY
DIFFRACTION
FROM
CONTRACTING
hlUSCLE
31
in Fig. 2, this line does not pass through the origin, and would have to be shift,etl considerably to do so. The reason for this is not clear. Thus, while muscle length provides a reasonable independent variable for comparative studies between resting and contracting muscles, it is not useful for’ studying the basic filament lat’ticc behaviour; here the sarcomere length itself should be used. In their study of frog and of mammalian muscle, Elliott et al. (1963) found that the relative intensities of the 1,0 and 1,l hexagonal lattice reflexions varied with muscle length. They placed the l,O/l,l intensity ratios in a series of categories ranging from 1 to 7 (see their Fig. 1 and Fig. 4 of this paper). In category 1, only the 1,0 reflexion is visible; in category 7, only the 1,l reflexion is visible; the other categories correspond to intermediate conditions. Using the toad muscle, we have examined these ratios in somewhat more detail. For each X-ray picture, the intensity ratio was estimated independently by each of the three authors, and the results averaged to the near& quarter-category. These results, from the same group of resting muscles as used in Fig. 3, are shown in Fig. 4, averaged over standard intervals of d and plotted against l/d2 (with the standard deviations of the means). Since l/d2 is directly proportional t’o the sarcomere length (see Fig. 2) the abscissa of Fig. 4 can also be calibrated in terms of s (from the line in Fig. 2). It appears that the intensity ratio category is directly related to s up to about 3-O TV(intensity ratio category 1); but above this length, no change in the intensity ratio can be detected since only one reflexion is visible.
FIG. 4. The 1,0 : I,1 intensity ratio of toad sartorius muscle plotted against the inverse scluaru of d. (0) Averagesfrom resting muscles over fixed intervals of d, with standard deviations of the means; (0) contracting muscle, each representing an individual experiment. Straight lines fitted by eye only. The abscissa has also been calibrated in terms of sarcomere length from the straight line in Fig. 2. Intensity categories (as def?ned by Elliott et al., 1963) are as follows: Category 1: 1,0 reflexion seen alone. 2: 1,0 reflexion seen with traces of 1,l reflexion. 3: 1,0 more intense than 1,l (both clearly seen). 4: I,0 and I.1 intensities equal.
38
G. F. ELLIOTT,
J. LO\VY
ANU
IS. M. MILLMAS
The same result is obtained if one plot,s the intensity ratio category muscles in which the sarcomere length was measured directly. (ii) Nuscle
contracting
against s for those
at constant length (isometric)
The equatorial X-ray diffraction pattern of isometrically contracting muscle was compared with t,hat from resting muscle (Plate I). To obtain the pattern during contraction, t’he muscle was stimulated and the pattern recorded only while the muscle was holding a tension greater than about 5 g. The muscle was automatically stimulated once or twice per minute for two or three days to give an aggregate exposure of about one hour-just sufficient to record the pattern. Similar patterns from the resting muscle, obtained with a continuous X-ray exposure of about one hour, were recorded before and aft’er each contracting pattern. It was, of course, only possible to obtain one pattern from each muscle. A series of 23 complete experiments on 23 separate muscles covered as large a range of muscle lengths as possible. The 1,0 spacings (d) were measured and l,O/l,l intensityratio categories assigned for each pattern as described above. The results for the contracting muscles are shown in Figs 3 and 4 as open circles. These results can be compared with the results from resting muscles (filled circles). (In Figs 2 and 3, resting muscle data from the same individual muscles as were used for the contracting muscle results could have been plotted, rather than averaged results from the larger series of resting muscles. If this had been done, the curves for the resting muscles in Figs 3 and 4 would be unchanged in form, though because of the smaller number of points there would be greater scatter in these points.) From a comparison of t.he patterns obt,aincd from resting and isometrically contracting muscles, four points emerge: (1) In the contracting muscle, d2 is inversely proportional to muscle length over the xvhole length range studied (Fig. 3). (2) The line relating l/d2 to muscle length in the contracting muscle lies slightly above that for the resting muscle; i.e. for a given muscle length, the interfilament spacing is slightly smaller in the contracting muscle (Fig. 3). (3) In the contracting muscle, the range of values of l/d2 extends to lower values; i.e. larger interfilament spacings were found than in the resting muscles (Fig. 4). (4) The l,O/l,l intensity ratio in contracting muscle appears to be linearly related to l/d2 and thus to s over its whole range; and for any value of l/d2 the intensity ratio is increased by about 314 of a category as compared with the ratio for the resting muscle (Fig. 4). The change in interfilament spacing on contraction was examined in more detail (Fig. 5). Here, the 1,0 spacing (d) from the contracting muscle is compared with that from the resting muscle, measured either before (Fig. 5(a)) or after (Fig. 5(b)) the muscle was stimulated. In both cases the spacing change is uniform (allowing for the very high scatter of these results) up to a value of d of about 385 8. In short muscles with values of d greater than about 385 A, however, cl increases substantially on contraction. An interesting feature of the results at values of d below 385 L%is that when the spacing from contracting muscle is compared wit’h that measured from the resting muscle before contraction (Fig. 5(a)) the spacing change on corkaction is about -10 A. But when compared to that measured from the resting muscle after contraction (Fig. 5(b)) there is no spacing change. This indicates tha,t during a series of
X-R,AY
DIFFRACTION
FROM
CONTRBCTISG
MUSCLE
0
i %
-20
an
c,
0 c
0
e 0 2
0
0 0 0
0
-40 300
350 d,(i)
400
FIG. 5. (a) Difference in d between the contracting (d,) and resting muscle (CE,)plotted against d, (d, measured before the contraction series). (b), as (a), but d, measured a.fter the contraction series. (c) Difference in d, as measured before and after t,he contraction series, plotted as in (a). (For explanation of solid line in (a) see text, p. 40.)
short contractions (twitches) the interfilament spacing at a given muscle length decreases by about 10 A, and that this decrease is not reversed after the series of contractions. Since the muscle length and, presumably, the sarcomere length, is essentially unchanged (except at the short muscle lengths, see below) this indicates that the lattice volume is permanently diminished on contraction. In fact, this decrease in the resting interfilament spacing seems to hold over the whole range of muscle lengths (Fig. 5(c)). It should also be noted that the sarcomere length may decrease slightly on contraction because of the effect of series elasticity at the ends of the muscle. This would mean that the volume decrease may be even greater than suggested above.
40
G. F. ELLIOTT,
.J. LOWY
AND
B. RI. MILLMAN
The results from contracting muscles provide several lines of evidence to support the suggestion made oarlic,r (see p. 36) that the non-linear part, of the l/rE2 : muscle length curve for tlrc rrst,ing muscle (I’i4 g. 3, f&d circ,lcs) was caused by slackness in the muscle. First,, no such non-linearity is seen in the corresponding ollrve for the contracting muscle (Fig. 3, open circles). Second, larger values of d are found in contracting than in resting muscles (see Fig. 4), suggesting that the contract,ing muscles have a length range which extends to shorter sarcomere lengths. Thirdly, the change in spacing on contraction is uniform for values of d up to about 385 h (Fig. 5), which is approximately the highest value for d observed in the resting muscles. The points observed above d = 385 A show an increase in d on contraction, which suggests that t,hcse muscles are actually shortening on contraction. If one assumes that the resting muscle can passively maintain sarcomere lengths corresponding to d = 385 A (2.15 p from Fig. 2) but no shorter, and that on stimulation the muscle will shorten further so as t.o remove any slackness in the muscle fibres, the change predicted in the l,O spacing on contraction is shown by the solid line in Fig. 5(a). It is clear that the experimental results are in good agreement with this prediction. (iii) Additional
re$exions
Frequently, especially when longer X-ray exposures were given, equatorial reflexions in addition to the 1,0 and 1,l hexagonal l&tic reflexions could be seen on the negative. These reflexions were always very faint in comparison with the 1,0 reflexion. Although the 2,0 hexagonal reflexion was often seen, the most commonly observed additional reflexion was one which lay between the 1,0 and 1,l reflexions and could not be indexed on the hexagonal lattice (Plate II). This reflexion was seen at all muscle lengths and in patterns from both relaxed and contJracting muscles. The measurements varied considerably (from 197 to 301 A), and a high variation was also found if the reflexion was expressed in terms of the 1,O hexagonal lattice reflexion (from O-621 to 0.780). In the latter case, however, there was less spread in the results, and the distribution was more uniform, suggesting that, the structure responsible for this reflexion changes its dimensions with muscle length in the same general way as does the filament lattice. It seems possible that this reflexion could come from the square I-filament lattice adjacent to the Z-line, in which case it would appear that this lattice expands in the same general manner as the filament lattice. Electron microscope observations on frog sartorius muscle have shown (M. K. Reedy, personal communication) that the dimensions of the Z-line lattice and adjacent I-filament lattice change (over the range from 200 to 300 A in the I-filament lattice) inversely with sarcomere length (between 3.2 and 1.7 p). (b) Meridional
dilraction
Toad sartorius muscle gives very detailed X-ray diffraction patterns at low angles in the meridional direction (Plate III). There is a series of reflexions from the thick (myosin) filaments which lie on layer lines with a basic periodicity of about 432 A. These layer lines, which were first observed in living frog sartorius muscle by Elliott (1964), show considerable detail in the equatorial direction, and almost certainly come from the projections on the thick filaments seen with the electron microscope by Huxley (1957,1963). In addition to the myosin reflexions, there is a series of meridional and near-meridional reflexions at angles corresponding to about 59 A and greater, which come from the actin filaments (Astbury R: Spark, 1947; Selby & Bear, 1956)
X-RAY
DIFFRACTION
FROM
CONTRACTING
41
MUSCLE
of which only the 59 B near-meridional reflexion is seen in Plate III. In some X-ray photographs, additional reflexions at smaller angles, and particularly an off-meridiona,l reflexion at about 400 8, can be seen. These have been assigned to actin layer-lines (Millman, Elliott & Lowy, 1967). I n some muscles, sharp meridional reflexions arc seen just outside the 3rd and 6th order myosin layer-lines. These probably come from collagen, present in small amounts in this muscle, indexing as the 5th and 10th orders of a basic period of about 670 a (see Worthington & Tomlin, 1955). It has been found that the actin and myosiu meridional spacings do not change when the muscles contract isometrically (Elliott et al., 1965; Huxley et al., 1965). Table 1 shows the averaged spacings from all the myosin and actin reflexions which could be measured on the diffraction patterns of resting and contracting muscles. l’here is no change greater than 0.2% in either the actin or myosin periodicity (Table 1). The myosin results were collected and averaged to give the basic periodicity in the resting and contracting conditions of 432.4 d (137 reflexion measurements from 34 resting muscles) and 432.8 A (39 reflexion measurements from 12 contracting muscles), respectively. TABLEI Layer-line
order
Resting
muscle
Contracting
muscle
Spacings in A (a) Myosin
reflexions 11 10 9 8 7 6 5 4 3 2
Average basic myosin periodicity
30.3 & 0.06t (9)$ 43.5 49.7 54.1 60.1 71.8 86.2 107.5 143.7 217
* f f -& f & 4 & &
0.17 0.87 0.17 0.09 0.20 0.34 0.47 0.39 1.5
-
(7) (4) (6) (3) (27) (17) (17) (28) (19)
44.0 48.8 72.2 86.8 107.8 143.5 218
432.4 + 0.63
(137)
432.8 & 1.43
(39)
59.5 + 0.12 404 & 3.3
(32) (23)
59.3 & 0.22 407 * 11
(10) (3)
& h & * *
0.31 0.69 1.14 0.79 3.7
(1) (1) (9) (4) (6) (11) (7)
(b) Actin reflexions 7 1 t Standard deviation of the mean. $ Number of measurements.
A few experiments were done using a lever and stops, arranged so that the muscle was allowed to shorten under a light load by 1 to 2 mm (5 to 10%) before developing isometric tension. In these experiments the actin and myosin spacings again remained unchanged (within the experimental error of about 2%). Huxley et al. (1965), using frog sartorius muscle, have shown that during isometric contraction the intensity of the myosin layer lines is greatly diminished. We have confirmed this change of intensity using the toad muscle. In Plate IV, X-ray patterns from the same toad muscle at rest and during contraction are shown in which this
42
G. F. ELLIOTT,
J. LOWY
AND
B. M. MILLMAX
decrease in myosin layer-line intensity can be seen. This result indicates a decrease in the order or change in the arrangement of the structure giving rise to the la,yer-lines; i.e. in the projections from the myosin filaments. On the other hand, there does not, appear to be an appreciable change in the intensities of the actin reflexions.
4. Discussion We will first consider the physiological state of the muscles we examined. It could be thought that the parts of the muscle from which reflexions were obtained had been damaged by irradiation, so that they were no longer capable of contraction. This is not so. In the first place, at any particular muscle length, stimulation produces definite and predictable structural effects; namely, a change in the spacings and intensity ratios of the equatorial reflexions, and a decrease in the intensity of the myosin layer-lines. Second, in the muscles which were slack before stimulation, contraction results in a structural change which is the opposite of that occurring at longer lengths. As seen in Fig. 5(a), the lattice spacing decreases on contraction by a fixed amount for starting sarcomere lengths greater than 2.15 TV,corresponding to a d value of 385 A. Assuming that 2.15 p is the shortest sarcomere length which the resting muscle can maintain-and there is good evidence for this (see p. 40)-the extent of the increase in the lattice spacing on contraction when the starting value of d is greater than 385 A (i.e. when sarcomere length is less than 2.15 CL)can be accurately predicted. The agreement of our results with these predictions (Fig. 5(a)) clearly shows that active shortening takes place in the parts of the muscle from which the X-ray diffraction pattern has been obtained. Concerning activation in the muscles used in our experiments, Hill (1949) established that in a twitch of the frog’s sartorius at 0°C the muscle is almost fully activated. In all probability this applies also to our toad muscles, particularly as we applied a double stimulus. There is therefore every reason to believe that the X-ray reflexions we have recorded during contractions were obtained from fully activated, normally functioning muscles. With toad muscle we have conlirmed, and in some respects extended, results obtained with resting frog muscle (Elliott et al., 1963) which showed that the spacings and intensities of the equatorial reflexions change with muscle length. The general implications were discussed in that paper. The following points about the present results may be noted. The sarcomere spacing at rest length (I,) in the toad muscle was found to be 2.9 p as compared with 2.5 p for the frog’s sartorius. The natural position (and use) of the frog’s leg is, however, very different from that of the toad; and it seems quite likely that the rest length we have used as a standard here bears a quite different relationship to the natural operating muscle length in the toad as compared with the frog. We found it impossible to prepare a resting muscle which could maintain a length shorter than that which corresponds to a sarcomere spacing of about 2.15 TV(p. 40 and Fig. 2). In frog muscle this is the spacing at which the actin filaments move into the zone where the myosin filaments have no projections (see Page & Huxley, 1963). A similar sarcomere spacing (2.1 p) was obtained by Gordon, A. F. Huxley & Julian (1966) for the slack length of single fibres from the frog’s semitendinosus muscle. lt is significant that the length of the muscle when slack is the same in whole toad sartorius as in single fibres of frog semitendinosus.
S-RAP
DIFFRACTION
FROM
COKTRACTING
MT’SCLE
43
On cont’raction, the intensity ratio is decreased by about 314 of a category; the 1 ,l reflexion becoming more intense relative to the 1,0 reflexion. This effect might be due either to an increased order in the actin filaments or to a decreasing order in the myosin filaments, or to both these effects together. An explanation in terms of an increased order of the actin filaments over the whole of the I-bands (Elliott et nl., 1965) seems unlikely, because the effect is much the same at all muscle lengths (except for the very long lengths where only the 1,0 reflexion is visible). Rather it appears in that cast that during contraction the ordering of the actin filaments within the hexagonal lattice is improved over a region of definite length (irrespective of sarcomere lcngth)possibly the region where the myosin filaments taper. This implies that in the regions where the myosin filaments taper (about 0.2 p at either end, cf. Huxley, 1957,1963), the actin filaments are not well ordered in the resting muscle. This is supported by the observation that the 1,l reflexion is not seen (i.e. the intensity ratio category is 1) with sarcomere lengths greater than about 3 p in the resting muscle (Fig. 3). If t,he actin filament order were maintained in the overlap regions, the 1,l reflexion might be expected to appear, although any calculation necessarily involves a number of assumptions (see Elliott et al., 1963). An observation that is a,s yet unexplained is the permanent decrease of dl,, following a long series of contractions (Fig. 4(c)). Conceivably this might be the result of changes in pH as a, result of metabolic activity. The fact that changes in pH can bring about changchs in this spacing has recently been demonstrated in glycerol-extracted preparations of rabbit psoas muscle, where a decrease of about 0.7 pH unit reduces d,,, by about 12 a (Rome, manuscript in preparation). Thus our decrease of 6 to 10 ii in might be explained by a drop in pH of about 0.4 pH unit. Studies on the internal 4,o pH of muscle fibres do not show changes of this order during short contra&ions @s&he, 1960). In fat’igued muscle, however, the pH can be lowered by as much as 1 pH unit (see Caldwell, 1958) and this may be t’he cause of the decrease in d,,, that, we have observed. Our studies of meridional patterns show that within the experimental error of 0.2:/, there is no detectable change in the structure within either the actin or myosin filaments during “isometric” contraction. This indicates that there is no over-all length change in these filaments. Cyclical changes could occur if t,heyinvolved only a small fraction of the structure at any one time; these would not be detected by our experiment,s. These results can be fully accommodated by t.he sliding filament hypothesis. However, our results provide strong evidence against models which assume that filaments shorten during contraction (e.g. Podolsky, 1962). If this were so, our experiments would have detected such a change. Even under very stringentI>, external isometric conditions, frog sartorius muscle can still shorten by about 27,, due t,o intrinsic series elasticity (Jewel1 & Wilkie, 1958). In our experiments, tht conditions of mounting the muscles were such that they could probably shorten by at, least 5%. Thus, if contraction is caused by a shortening of either type of filament, this should have been observed in our experiments, in which a change in axial spacing of as lit’tle as 0.20/, could be detected. This conclusion is further strengthened by the experiments where the muscles contracted under isotonic conditions (p. 41). We have identified t.he 400 A layer-line reflexion (see Table 1) as coming from the actin filaments (Millman, Elliott & Lowy, 1967). Our results here indicate that there is no change in this reflexion, and therefore in the long period of the actin filaments, during contraction.
44
G. I?. ELLIOTT,
J. LOWY
ASI)
11. I\I. MILI,!vl~~N
Two basically different kinds of physical explanation can be proposed to account for the operation of a sliding filament mechanism. One is that development of tension and shortening are the result of direct mechanical attachment and detachment of “cross-links” between actin and myosin filaments (see Hanson & H. E. Huxley, 1955; A. F. Huxley, 1957; Davies, 1963). If so, the links between the filaments must be able to act over a distance which varies widely with musclelength. Huxley et al. (1965) have pointed out, however, that the changes in the intensities of hhe myosin layerlines during contraction suggest that the projections on the myosin filaments could undergo quite large configurational changes. A second basic type of sliding filament mechanism requires the operation of longrange forces, perhaps electrostatic forces or electromagnetic resonance forces (see, for example, Spencer & Worthington, 1960). From the results on interfilament spacings in living muscle at rest and during contraction and from the recent work of Rome (manuscript in preparation) on interfilament spacings in glycerol-extracted material, it seems to us that an explanation of this type provides a good working hypothesis. We do not believe, however, that it is possible to decide finally between these two basic types of mechanism until it is known whether the change in order observed in the myosin projections is a cause of the force-producing activity or a consequence of it. We are grateful to Professor Sir John Randall for constant encouragement and to Professor Jean Hanson for useful discussions. We thank Miss B. J. Kimble, Mrs S. Weindling, Miss M. Kinchin, Mr D. Herbert and Mr C. McCarthy for technical assistance and Mr Z. Gabor for help with photographic reproduction. REFERENCES Astbury, W. T. (1947). Proc. Roy. Sot. B, 134, 303. A&bury, W. T. & Spark, L. C. (1947). Biochim. biophys. Acta, 1, 388. Bernal, J. D. (1962). In Fifty Years of X-ray Diffraction, ed. by P. P. Ewdd, Int. Union of Crystallography. Utrecht. Boehm, G. (1931). 2.f. BioZogie, 91, 203. Caldwell, P. (1958). J. Phyeiol. 142, 22. Copeland, K. (1969). Second I.F.M.E. Conference, Paris. Davies, R. E. (1963). Nature, 199, 1068. DistBche, A. (1960). Nature, 187, 1119. Elliott, G. F. (1964). Proc. Roy. Sot. B, 160, 467. Elliott, G. F., Lowy, J. & Milhnan, B. M. (1965). Nature, 206, 1357. Elliott, G. F., Lowy, J. & Worthington, C. R. (1963). J. Mol. Biol. 6, 296. Elliott, G. F. & Worthington, C. R. (1959). J. Physiol. 149, 32 P. Elliott, G. F. & Worthington, C. R. (1963). J. UZtrastructure Ree. 9, 166. Franks, A. (1955). Proc. Phys. Sot. B, 68, 1054. Gordon, A. M., Huxley, A. F. & Julian, F. J. (1966). J. Phyaiol. 184, 170. Hanson, J. & Huxley, H. E. (1955). Symp. Sot. Exp. BioZ. 9, 228. Hanson, J. & Lowy, J. (1963). J. Mol. BioZ. 6, 46. Hanson, J. & Lowy, J. (1965). Brit. Med. Bull. 21, 264. Hill, A. V. (1949). Proc. Roy. Sot. B, 136, 399. Hill, A. V. (1961). J. Physiol. 159, 518. Huxley, A. F. (1957). Prog. Biophys. Biophys. Chene. 7, 255. Huxley, A. F. & Niedergerke, R. (1954). Nature, 173, 971. Huxley, H. E. (1953). Proc. Roy. Sot. B, 141, 59. Huxley, H. E. (1957). J. Biophys. B&hem. Cytol. 3, 631. Huxley, H. E. (1960). In The Cell, ed. by J. Brachet & A. Mirsky, vol. 4. New York: Academic Press.
S-RAY
DIFFRACTION
FltOhf
CONTl
MUSCLE
Huxley, H. E. (1963). J. Mol. Biol. 7, 281. Huxley, H. E., Brown, W. & Holmes, K. C. (1965). Nature, 206, 1358. Huxley, H. E. & Hanson, J. (1954). ib’uture, 173, 973. Jewcll, B. R. & Willrie, D. R. (1958). J. Physiol. 143, 515. Millman, B. M.. Elliott, 0. F. & Lowy, J. (1967). ATuture, 213, 356. Poclolsky, R. J. (1962). E’ed. I’ror. 21, 964. Sclhy, C’. (1. & Bcu, It. S. (1956). .T. Uioph?/s. Uiocho,r. Cl/M. 2, 71. Spenocr, RI. S.z\vorthin$torl, c. l<. (1960). Natwe, 187. 3%. \\Tortjllington, (1. I{. (1959). .I. illol. Uiol. 1, 398. \\‘ort,lli@on, (‘. le. (1961). .7. AioZ. LlioZ. 3, 61s. \\wort.llington, f.‘. I;. & ‘I’utnlin, S. G. (1!)56). 7’w(,. /:c,!/. Srtc. .2, 235, 189.