379
J. Physiol. (1979), 293, pp. 379-392 With I plate and 8 text-ftgurea Printed in Great Britain
THE EFFECT ON TENSION OF NON-UNIFORM DISTRIBUTION OF LENGTH CHANGES APPLIED TO FROG MUSCLE FIBRES
BY F. J. JULIAN AND D. L. MORGAN From the Department of Muscle Research, Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA.02114, U.S.A.
(Received 31 October 1978) SUMMARY
1. The stability of sarcomere lengths along single frog twitch fibres was examined, during lengthening and shortening, using a spot follower apparatus to monitor or control the length of a central segment. 2. During active shortening from sarcomere lengths beyond 2-2 jtm the end sarcomeres shortened dramatically, while much of the fibre did not shorten at all. It is proposed that this is the cause of the tension failing to recover, after the shortening ceased, to the value of isometric tension at the shorter length. 3. During active lengthening from sarcomere lengths beyond 2-2 j#m, nonuniformity of stretch was seen, with the middle stretching more than the ends. Some maintained extra tension after stretch above that appropriate to the longer length was found, as were consistent changes in internal movement, and in the shape of the tension record during relaxation. 4. Measurements of stiffness during and after a lengthening suggest that no increased activation is involved. Observation of internal movement during the raised tension after a lengthening contradicts theories involving 'locked on' bridges. 5. From these and other observations, an explanation for the extra tension in terms of non-uniformity of sarcomeres is proposed. The explanation is in accord with that previously suggested for the creep phase of tension rise seen at these lengths. INTRODUCTION
The question of sarcomere length stability under fixed-end conditions at long lengths has been discussed for some years (Hill, 1953), and considered in some detail in the preceding paper (Julian & Morgan, 1979). It is also of some interest to consider the effects of lengthening or shortening a fibre on the degree of uniformity of sarcomeres. A non-uniform distribution of sarcomere lengths, once established, is likely to last for the duration of a contraction, and possibly to persist to some extent even during relaxation, as suggested, for example, by the effects of previous stimulation on creep (Gordon, Huxley & Julian 1966a, Fig. 1; Julian & Morgan, 1979). Consequently, intersarcomere dynamics during changes in muscle length and the resulting sarcomere length distributions must be considered as a possible explanation for effects of motion on tension which are maintained for the duration of the tetanus. 0022-3751/79/4570-0812 $01.50 © 1979 The Physiological Society
F. J. JULIAN AND D. L. MORGAN 380 The experiments reported here are concerned with observation of sarcomere non-uniformities during lengthening and shortening of tetanically stimulated frog twitch fibres, using the spot follower apparatus described previously (Julian & Morgan, 1979) to monitor, and where appropriate, control the length of a central segment. METHODS
The experimental procedure used was the same as that described in the preceding paper (Julian & Morgan, 1979), some fibres being used in both investigations. The motor was used in these experiments to execute ramp and hold movements by following a ramp generator signal. The ramp generator was an analogue device with a control on which was set the voltage representing the length being controlled, i.e. either muscle length or marker spacing. Ramp amplitude and slope controls were thus calibrated in per cent muscle length and muscle lengths/sec respectively. Our convention was to set the just-taut length on the control so that all amplitudes are quoted as a percentage of just -taut length. These can be approximately converted to nm per half-sarcomere by multiplying by 11, i.e. 5 % just-taut length is 55 nm per half-sarcomere. The lamp generator consisted basically of an integrator and two comparators. When the output of the integrator reached the desired limit, up or down, the appropriate comparator became part of an additional feed-back loop which kept the output at a constant value. This gave a non-drifting, transient-free ramp and hold wave form. A similar device has been described more fully by Morgan (1976, pp. 138-142). In some experiments, a small high frequency sinusoidal vibration was imposed, to enable measurement of muscle stiffness. In these experiments, records were taken in pairs, one with and one without vibration. Electronic subtraction of the records in the digital oscilloscope served the two purposes of checking that vibration did not affect the response to the other movement, and of providing a signal containing just the vibration component of tension, which could then be magnified and measured accurately and simply on a cycle-to-cycle basis.
RESULTS
Shortening The shortenings used were generally quite large and slow to maximize the maintained effects while ensuring that a reasonably large tension was maintained throughout the transient. A typical result is shown in Fig. 1 A. The marker spacing record during active shortening is trace c in which the following phases can be observed. During the initial fixed-end stage of the contraction the central segment of the fibre begins to lengthen slowly, typical of a fixed-end contraction at long length. When the fibre shortening began, the marker spacing initially decreased approximately proportionately, but the rate of central shortening rapidly decreased, and over a considerable proportion of the shortening time the central segment was in fact lengthening. At the end of muscle shortening, the initial slow lengthening of the central segment resumed, and only after the 'shoulder' in the relaxation phase of the tension record did the central segment shorten to the length appropriate to the shorter muscle length. Thus, during active shortening, the movement is seen to be extremely non-uniformly distributed, with the ends absorbing most of the movement. The most noticeable feature of the tension record, which has been reported before (Deleze, 1961; Abbott & Aubert, 1952) and is shown in Fig. 2A, is that the tension after the completion of the shortening remains less than that attained
SARCOMERE DYNAMICS IN STRETCH AND RELEASE 381 during a fixed-end contraction at the short length. Also interesting is the humped shape of the tension trace during the shortening, even though the mean sarcomere length (averaged over the whole fibre) is still 2-5 rum at the end of the movement, i.e. well beyond the plateau of the length tension relationship (Gordon, Huxley a
C
2 mm 150 nm ,4 mN 1 sec
A
B
Fig. 1. The effect of shortening. A, from sarcomere length of 2-8-2-5 j#m; trace a is the motor record with thickening indicating stimulation, b is the marker separation when the shortening was done passively, c is the marker separation when the muscle was stimulated during shortening, and d the tension record during active shortening. The tip of the motor arm moved 1 mm at 1 mm/sec. Calibration for marker spacing is in nm/half-sarcomere. B, from sarcomere length 2-25-1-95 ,sm, together with isometric contractions at initial and final lengths. Upper records are motor with thickening indicating stimulation, lower records tension. A little depression of tension is apparent, although the traces are still converging at the end of stimulation. 13 0C, stimulation frequency 35 sec'. a
t S
b
c
0.5 mN
i~~~~~~sec
d
Fig. 2. Interruption of stimulation after shortening. Trace a is the tension record from an isometric contraction at sarcomere length = 2-4 /m. In the other traces, the contraction was begun at sarcomere length = 2-8 /sm, and the fibre was then shortened to 2-4 gm in 1-5 sec. When the tension was nearly steady, the stimulation was stopped for a variable time, and then resumed. Trace c with a short interruption had a final tension near b with no interruption. Trace d, with a longer pause, recovered to approximately the same tension as a. 4 'C, stimulation frequency 20 sec-.
& Julian, 1966b). When this experiment was repeated with shortening around the plateau as shown in Fig. 1 B, the tension deficiency, though still observable, was much reduced, and the hump in the tension record was absent. Abbot & Aubert's (1952) experiment of interrupting the tetanus during the period after the shortening was complete, was repeated with similar results and some extra observations as shown in Fig. 2. If the tension was only allowed to fall
F. J. JULIAN AND D. L. MORGAN 382 as far as the shoulder before the stimulation was recommenced, the tension stayed depressed. However, if the sarcomere length redistribution which follows the shoulder was allowed to proceed during the interruption, the tension recovered to the value appropriate to the new length. Direct microscopic observation of the ends was performed both visually and photographically, and some results are shown in P1. 1. While passive, the striation pattern was distinct and nearly uniform right to the end of the fibre (P1. 1A). During isometric contraction, the usual shortening and loss of order in a small region around the end was seen (P1. 1B) (Huxley & Peachey, 1961; Julian, Sollins & Moss, 1978). When the fibre was observed during the shortening, a progressive 'crumpling' of the ends could be seen, the region of shortened sarcomeres enlarging, until late in the movement the whole field of the microscope was taken up by shortened sarcomeres as in P1. 1C. Accurate measurements of sarcomere length were made difficult by the disorder, but such measurements as were possible did confirm the conclusions drawn from visual observation. Diffraction techniques were not used, as only ordered sarcomeres produce clear diffraction patterns, and sarcomeres as disordered as some of those seen here could be expected to give little more than random scatter. Thus, a diffraction pattern would give primarily the mean spacing of the ordered sarcomeres within the beam, with a much lower weighting factor on the severely shortened and disordered ones, merely because of their disorder. Attempts to force the central segment to shorten by using marker spacing control were unsuccessful, the motor arm moving so far that the markers translated out of the microscope field of view quite early in the shortening, leading to loss of control and possible damage to the fibre. It became apparent from these experiments that the amount of end movement required to shorten the central segment slowly was extremely large. Lengthening
Lengthening movements were usually faster than the shortenings, in order to keep the tetani of a reasonable length while still allowing time for completion of the transient response. The marker spacing records again show non-uniform distribution of the movement, though not as dramatically as for shortening. Fig. 3A shows the main features usually observed. When the end movement was applied before the stimulation began, the central segment stretched proportionally and showed a normal slow lengthening throughout the subsequent tetanus. An end movement of the same size applied during the tetanus produced a significantly and consistently larger increase in marker spacing. Over a large number of records, the extra movement averaged 15 %. During the decay of tension after the imposed lengthening ceased the marker spacing increased only very slowly, much more slowly than during a fixed-end contraction. As the transient ended, the markers began to move apart again, and finally attained a stretch rate comparable to or even greater than that of the fixed-end contraction at the same time. Thus, during active lengthening it appears that most of the fibre is stretched, but that small portions away from the centre lengthen less than a proportional amount. The tension traces of Fig. 3A are most noticeable for the maintenance of tension
SARCOMERE DYNAMICS IN STRETCH AND RELEASE 3383 following an active stretch at a level higher than that attained during a fixedend tetanus at the final length, even after the transient has passed. This is the 'permanent' extra tension after stretch noted by many workers (Abbott & Aubert, 1952; Hill, 1977; Edman, Elzinga & Noble, 1976, 1978a, b). 88 nm 2 mN
2 sec
a
b
A
C
~~~~~~~C
Fig. 3. A, typical result with marker spacing records. For traces a (marker spacing) and c (tension) the muscle was lengthened by 5 % just-taut length in 170 msec before stimulation commenced. For traces b (marker separation) and d (tension) the same lengthening was applied during the tetanus. 4 0C, stimulation frequency 16 sec-, initial sarcomere length = 2-5 jam. B, the effect of stretch amplitude. Stretches of 3, 5 and 7 % just-taut length were applied before and during the tetanus. The stretches before stimulation produced a decreasing tension with increasing length, but the stretches during stimulation all produced the same final tension. 4 0C, stimulation frequency 16 sec-', initial sarcomere length = 2*5 #sm, ramp speed = 0 3 muscle lengths/sec. C, effect of speed of lengthening. Two tension records of actively lengthening a fibre by 7 % just-taut length at speeds of 0.1 and 0-8 muscle lengths/sec. Eventually the tensions are equal. 4 0C, stimulation frequency 16 sec-", initial sarcomere length = 2*5 1um. The calibration bars apply to all parts of the Figure and for the marker spacing records are in nm/half-sarcomere, lengthening up.
There are also significant and consistent differences in the tension records during relaxation. The 'shoulder' generally occurred later if the muscle had been stretched actively, and the whole decay phase was slower. As the shoulder and subsequent relaxation is known to be associated with redistribution of sarcomere lengths (Huxley & Simmons, 1972; Julian & Morgan, 1979), differences in relaxation are suggestive of different sarcomere length distributions at the end of the tetanus. The effect of amplitude of stretch on the final tension (not extra tension) was investigated and found to be negligible over the range studied. This is shown in Fig. 3B where progressively larger stretches before stimulation give progressively smaller final tensions (the descending limb of the length-tension curve) but all the stretches during the tetanus led eventually to the same tension. Similarly, an eightfold change in velocity of lengthening is shown not to effect the final tension (Fig. 3C). It was also found generally, as shown in these Figures, that speed or amplitude of stretch did not greatly alter the shape of the decay phase of the tension records. The initial length of the muscle is already known to effect the extra tension (Hill,
F. J. JULIAN AND D. L. MORGAN 384 1977; Edman et al. 1976, 1978a, b) and our findings in this regard are shown in Figs. 4, 5). At short lengths (sarcomere length < 20 gtm), the final steady tension after a stretch was very close to the fixed-end tension at the new length, i.e. the 'extra' tension was due only to the ascending limb of the length-tension relationship. When the pull was within the plateau region (2-0 < sarcomere length < 2-2 jtm) 2 mN
b
b
2 sec
A A
C 8
C
Fig. 4. The effect of fibre length. Tension records for no stretch (a in each case), stretch active (b), and stretch before stimulation commenced (c). A, 7 % stretch from sarcomere length = 1I7 4um; B, 7 % stretch from sarcomere length = 2 0 4Am; C, 7 % stretch from sarcomere length = 2-4 jam. Extra tension and modification of relaxation are only apparent at sarcomere lengths beyond the plateau. 4 'C, stimulation frequency 20 sec-.
Fig. 5. Stretch at longer lengths. Tension and marker spacing records for a 3 % stretch from sarcomere length = 3 0 /m. This illustrates the method of 'stretch before stimulation' used to account for passive tension. Tension trace b (active stretch) shows what appears to be a well maintained plateau after stretch, but comparison shows that creep in the isometric at the long-length record generates almost as much tension if the tetanus is continued long enough. Calibration for marker spacing is in nm/half-&arcomere, and lengthening is upwards. 11 0C, stimulation frequency 25 sec-1.
there was no extra tension. Provided that the stimulation was continued long enough, the tension returned to the fixed-end tension. In the range of moderately long lengths beyond the plateau but without significant passive tension (2.2 < sarcomere length < 2.9 #um), the tension after the pull was greater than the expected value, that developed by a fixed-end contraction at the final length, but always very close to that developed in a fixed-end contraction at the initial length. Thus, in
385 SARCOMERE DYNAMICS IN STRETCH AND RELEASE one sense, the fibre did not actually develop extra tension after the stretch, it merely failed to decrease its tension to the value given by the descending limb of the length-tension curve for isometric muscles. At very long lengths, several factors complicate the results. Passive tension must be subtracted, but the exact method is uncertain. The usual approach is to subtract the difference between final passive tensions at the initial and final lengths. However, stretching a passive muscle at a, rate similar to that used for the active muscle produces more tension than this difference, and the extra tension persists over a time period longer than that used for tetani. If the active response is simply additive to the passive response, then this passive response to stretch should be subtracted from the active response to accurately correct for passive tension. The use of the spot follower shows that non-uniform stretching occurs at these lengths also (Fig. 5), so even the amplitude of the passive stretch which should be subtracted is uncertain, depending on whether the parallel elasticity is in parallel with the whole muscle or each sarcomere, or some combination of both. Even use of marker spacing control does not remove this problem. Furthermore, the fixed-end tension continues to rise
(creep) for quite some time. The approach which we took to these problems and the results found are shown in Fig. 5. To account for passive tension, in the fixedend contraction at the longer length, or 'stretch before stimulating' contraction, the muscle fibre was brought to the longer length immediately before commencing stimulation using the same end movement as was used in the active stretching. The stimulation was continued until the creep phase ended, and then tensions were compared. As can be seen, the extra tension under these conditions is quite small, certainly no more significant than at the moderately long lengths. Note also the reduction of velocity of stretch after the active pull as compared to the isometric tetanus, and the prolongation of the slow linear phase of tension decay, as in Fig. 3A. At or below the plateau then, the results are in accord with the length-tension curve. At moderately long lengths, there is a definite lack of reduction of tension, while at very long lengths, the experiment is more complicated, but the amount of extra tension appears to be no greater than at moderately long lengths. For this reason, investigation concentrated on the region between the plateau and the length at which passive tension became significant. Interruption of the tetanus for various time intervals after the transient response produced results typified by Fig. 6. After a short interruption, terminated before the shoulder of the tension record, the tension was not reduced significantly below its raised value, but after a longer interruption, allowing significant sarcomere length redistribution, it returned to nearer the tension of the fixed-end tetanus at the long length. It should be remembered that the shoulder signifies only the beginning of restribution, which is often not completed until the tension has decreased to near zero, so an extremely sharp return from distorted to normal sarcomere length distribution should not be expected. Nevertheless, these results are suggestive of a significant role for sarcomere length non-uniformities in producing the extra tension. Stiffness measurement. It is conceivable that some process of activation by stretch (Pringle, 1978) could be acting to increase the number of available sites for cross13
PHY 293
386 F. J. JULIAN AND D. L. MORGAN bridge attachment, and that these sites remain available while the tension is maintained, but revert to being unavailable when the tension is allowed to fall. In order to test this suggestion, the stiffness of a muscle during stimulation and lengthening was measured by means of sinusoidal vibration as shown in Fig. 7, and plotted against tension in Fig. 8. It can be seen that the stiffness rose almost linearly with 1 mN
1 sec
a~~~~~~~~~~~~~~~~
Fig. 6. Interruption of stimulation after stretch. This is the equivalent experiment to Fig. 2. Trace a is the tension record for an isometric contraction at the short length, b is isometric at the long length. The normal stretch during stimulation record, c, shows the usual elevated tension and characteristic changes in decay phase. A short pause in stimulation, d, leads to recovery of the higher tension, but after a longer interruption, e, the tension is nearer that of b. 4 00, stimulation frequency 15 see-1, initial sarcomere length = 2-5 /tm, 7 % stretch at 0 3 muscle lengths/sec.
the rise of tension when stimulation commenced, but that throughout the lengthening and the decay of transient tension the stiffness stayed substantially constant, suggesting that the number of cross-bridges did not change significantly, or at least not proportionally to the tension. Photographic observations of end segments were made in an attempt to complement the evidence from marker spacing observations. Somewhat less lengthening of the end regions was seen when the stretch was applied to a stimulated fibre than when it was applied to a passive fibre. However, the most striking result was the sharp reduction in the shortening rate of the ends brought about by a stretch. During the decay of the transient, the progressive collapse of ends seen in a long length contraction (Julian et al. 1978; Julian & Morgan, 1979) was almost totally halted, so that at a time corresponding to the end of the tension transient, a fibre contracting with fixed ends had much shorter end sarcomeres when compared to one which had been lengthened actively. Marker spacing control was used in some experiments and the results were consistent with those using end-controlled movements. The following points were noted. The end movement required to produce a given change in marker spacing was less for an active than for a passive fibre. The rate of end movement required to keep the marker spacing constant during the tension transient following a stretch was much less than that required during an isometric tetanus. The final tension was as predicted by the length-tension curve at muscle lengths on or below the plateau.
SARCOMERE DYNAMICS IN STRETCH AND RELEASE
387
1 mN
0-25 mN 1 sec
-He
And He~-
Fig. 7. Stiffness measurement. A pull, 5 % in 100 msec from sarcomere length = 2-5 /im, trace a, was repeated b with a 2*5 Aim 2-5 kHz vibration superimposed. The difference between the two records was found by digital subtraction, multiplied by a factor of four, plotted as trace c and measured to provide a measure of stiffness. The frequency visible in the records is a beat frequency between the vibration and sampling rates. Improved time resolution would be obtained by recording only part of the tetanus at a higher sampling rate. 4 'C, stimulation frequency 20 sec-'.
b_:f
Tension P0 Fig. 8. Stiffness results. The plot shows stiffness as a function of tension while passive (point a), during the isometric rise of tension to point b, during a stretch, peaking at point c, and during the decay back to the isometric tension. It can be seen that the stiffness rises almost linearly with tension during the rise of tetanus, but is essentially constant during the stretch and decay. 4 0C, stimulation frequency 20 sec', 7 % stretch from 2-5J sm. Same experiment as shown in Fig. 7.
13-2
388 F. J. JULIAN AND D. L. MORGAN The use of marker spacing control produced even slower decay of the tension transient than end control. This meant that very long tetani were needed to enable accurate comparison. Quite apart from the usual problems of fatigue, prolonged segment-clamped tetani are difficult to carry out accurately as there is a tendency for the tension to rise slowly, particularly during the later stages of a long tetanus (see Discussion). Within these limitations the extra tension did appear to be reduced by the use of marker control rather than end control. However, the uncertainties were such that end control was preferred. DISCUSSION
Shortening It seems quite clear that during slow end shortening of a single fibre at moderately long lengths, sarcomere shortening is extremely non-uniform. This can be accounted for in the following way. In the fixed-end contraction, the end sarcomeres are shortening, and slowly stretching the rest of the fibre. When the end is moved to shorten the muscle, the discontinuity of the force-velocity curve about the point where the velocity changes sign means that a given fall in tension (the same all along the muscle) will be accompanied by a greater change of shortening velocity in sarcomeres that were previously shortening than in those that were previously lengthening, i.e. the shortening sarcomeres will increase their shortening velocity more than lengthening sarcomeres will decrease their lengthening velocity. Consequently, the shortening sarcomeres will shorten much faster, while the lengthening sarcomeres will not shorten at all. Of course, this shortening of the end sarcomeres initially increases their tension generating capacity. This continues until they shorten over the plateau of the sarcomere length-tension curve which is probably the cause of the 'hump' in the tension record of Fig. 1A, and the corresponding curvature of the marker spacing record. The converse experiment, of allowing isotonic shortening from long lengths, was also carried out by D6ekze (1961) with the finding that the final muscle length for a given load depended on the initial length. This is almost certainly due to a similar mechanism, as was suggested by A. F. Huxley (1964, p. 135). The observation of the shortened region spreading by incorporation of adjacent sarcomeres, and indeed the commonly seen gradation from long to short sarcomeres, both suggest the existence of some forces which act to prevent marked disparities of sarcomere length between adjacent sarcomeres. At very long lengths distributed passive elasticity could be sufficient, but another explanation must be found at shorter lengths. If the constant volume constraint on the filament lattice (H. E. Huxley, 1953) holds true on an individual sarcomere scale, then the resistance of the fibre to sudden changes in diameter may be sufficient. Another possible explanation could be based upon the helical sarcomere structure described by Peachey & Eisenberg (1978). These experiments also provide insight into some of the difficulties encountered in length clamping the central segment of a fibre at these sarcomere lengths. It is apparent that moving the end of the fibre is not a very effective way of shortening the central segment, yet it is the only means available. If a 'clamped' tetanus is
SARCOMERE DYNAMICS IN STRETCH AND RELEASE 389 extended for a long time, large end movements will be necessary, and eventually the collapsed region will extend into the segment being clamped, making it nonuniform and defeating the purpose of the clamp, which is to control the length of a uniform central segment. It could be argued that the depression of tension after a shortening presents a problem for the sarcomere length non-uniformity explanation of the creep phase of tension rise (Julian et al. 1978). This explanation assumes that a sarcomere shortening from a long length does increase its capacity to generate tension in accordance with the length-tension curve. From the present work, it is clear that the observation of depressed tension after active shortening does not contradict that assumption, but is simply due to the fact that shortening the over-all muscle fibre does not shorten the sarcomeres uniformly, and may not shorten many of them at all. Furthermore, this non-uniform shortening behaviour is entirely in accord with the sarcomere length non-uniformity explanation of creep.
Lengthening When an active muscle is forcibly lengthened at moderate speed, it shows a short range of stiffness and then 'yields' (Fig. 3B). Assuming that the 'yield point' is a constant fraction of the isometric tension as sarcomere length is varied (Flitney & Hirst, 1978, Fig. 9), the observed non-uniformity of active stretch can be predicted. The shorter, shortening sarcomeres at the ends would be expected to have a higher isometric tension, and hence higher yield point than the rest of the fibre. Consequently, they would tend to resist lengthening, at the expense of the rest of the fibre. The unusual stability of sarcomere lengths after the stretch is probably due to all sarcomeres having 'stretched' cross-bridges, i.e. each cross-bridge exerting more tension than it would if no change of sarcomere length had taken place since it attached. Consequently, all sarcomeres are able to bear the high tension without lengthening, at least temporarily. However, none are able to shorten, since any sarcomere which shortened would need to detach and re-attach cross-bridges, which would decrease the tension. If this explanation is correct the decay rate of the transient reflects the rate at which the stretched cross-bridges detach and reattach with the 'normal' tension in the near complete absence of internal motion. This is not inconsistent with the detachment rate suggested by the slope of the linear phase of tension relaxation in a segment length-clamped fibre as discussed in the previous paper. The question still to be answered is why does the tension following a stretch return to that level appropriate to the original length? The present work has shown that the sarcomere length distribution after active stretch is different from that which exists if the stretch was delivered before activation, so the tensions should be expected to be different. The unexpected finding is that the tension is so close to the fixed-end tension at the original length and so independent of factors such as amplitude and speed of stretch. The final tension is apparently determined primarily by some factor which is not changed by the lengthening. The obvious suggestion is the length and tension generating capacity of those sarcomeres which did not lengthen during the stretch, presumably the ones near the ends which were already
F. J. JULIAN AND D. L. MORGAN shortening. This suggestion would postulate that the tension during a fixed-end tetanus is strongly influenced by the distribution of sarcomere lengths in these end regions. Then the tension reached is determined by the number of sarcomeres which shorten during the contraction, the velocity with which they can shorten, the distance through which they can shorten before reaching the plateau, and possibly the rate at which the collapsed region can incorporate new sarcomeres. The rest of the fibre then stretches under the imposed load, the initial sarcomere length having only a small effect on the stretching speed, due to the steepness of the force-velocity curve for slow lengthening. All the above mentioned factors depend on the initial distribution of sarcomere lengths and, to the extent that the stretch is not shared by these end sarcomeres, remain unaffected by the stretch. Ideally, this proposal should be testable by photomicroscopy, but there are practical difficulties. However, considerable indirect supporting evidence is available. Interruption of a tetanus only abolishes the effect if the decay of tension is allowed to proceed beyond the shoulder, where length redistribution begins to occur. Extra permanent tension is only found beyond the plateau where instability is to be expected and creep is observed in the marker records of fixed-end contractions. The behaviour of the markers, stationary during the raised tension of the transient, and then separating during the sustained lower tension, is opposite to what would be expected if the changes in separation were a passive consequence of the tension rather than being directly involved in tension generation. The explanation offered is consistent with that for creep. Explanations of the raised tension after stretch based on sarcomere length nonuniformity have been rejected by some (Hill, 1977) on the basis of observations, at variance with ours, and those of Edman et al. (1978b), Fig. 6A, that maintained extra tension could be produced in the plateau region. We believe that these discrepancies arise from failure to observe some of the precautions mentioned under Results, particularly failure to wait until a really permanent tension is reached (e.g. Edman et al. 1976, Fig. 1 A) and failure to wait for creep to finish before measuring the tension of a fixed-end tetanus (e.g. Hill (1977, Fig. 1A) where the tension before the stretch is taken as the fixed-end tension, and Edman et al. (1978a) where the fixed-end tension is still rising at the latest time shown). Other differences, particularly at longer lengths may arise from the use of the steady-state rather than transient/passive tension when comparing active tensions. The finding that stiffness does not increase during or after the stretch argues against explanations involving extra bridges. Such explanations would also have difficulty accounting for the interruption of stimulation results. The resumed gradual separation of markers during the maintained tension is incompatible with explanations based on 'locked on' bridges, which could only exist while the sarcomeres were isometric (Hill, 1977). General It is clear from these experiments that instability of sarcomere lengths does exist on the descending limb of the length-tension curve, and that the force-velocity curve, particularly during stretch, is responsible for the heavily damped nature of the instability. It is apparent both from experiment and from reasoning that movement of the end of the fibre accentuates the non-uniformities, irrespective of the
390
SARCOMERE DYNAMICS IN STRETCH AND RELEASE 391 direction of the movement. The sarcomere length dynamics which result from this damped instability are both complex and important. Furthermore, it is important to realize that these intersarcomere dynamics can affect the tension considerably and under a wide range of conditions. This work was supported by the following grants: an N.I.H. research grant, HL-16606, from the National Heart, Lung and Blood Institute, and grants from the American Heart Association, No. 77-616, and the Muscular Dystrophy Association.
REFERENCES ABBOT, B. C. & AUBERT, X. M. (1952). The force exerted by active striated muscle during and after change in length. J. Phygiol. 117, 77-86. DE'LtZE, J. B. (1961). The mechanical properties of the semitendinosus muscle at lengths greater than its length in the body. J. Physiol. 158, 154-164. EDMAN, K. A. P., ELZINGA, G. & NOBLE, M. I. M. (1976). Force enhancement induced by stretch of contracting single isolated muscle fibres of the frog. J. Physiol. 258, 95P. EDMAN, K. A. P., ELZINGA, G. & NOBLE, M. I. M. (1978a). Further characterization of the enhancement of force by stretch during activity in single muscle fibres of the frog. J. Phygiol. 280, 35P. EDMAN, K. A. P., ELZINGA, G. & NOBLE, M. I. M. (1978b). Enhancement of mechanical performance by stretch during tetanic contractions of vertebrate skeletal muscle fibres.
J. Physiol. 281, 139-155. FLITNEY, F. W. & HrRST, D. G. (1978). Crossbridge detachment and sarcomere 'give' during stretch of active frog's muscle. J. Phygiol. 276, 449-465. GORDON, A. M., HUXLEY, A. F. & JULIAN, F. J. (1966a). Tension development in highly stretched vertebrate muscle fibres. J. Phygiol. 184, 143-169. GORDON, A. M., HUXLEY, A. F. & JULIAN, F. J. (1966b). The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J. Physiol. 184, 170-192. HILL, A. V. (1953). The mechanics of active muscle. Proc. R. Soc. B 141, 104-117. HILL, L. (1977). A-band length, striation spacing and tension change on stretch of active muscle. J. Physiol. 266, 677-685. HUXLEY, A. F. (1964). Muscle. A. Rev. Physiol. 26, 131-152. HUXLEY, A. F. & PEACHEY, L. D. (1961). The maximum length for contraction in vertebrate striated muscle. J. Physiol. 156, 150-165. HUXLEY, A. F. & SIMMONs, R. M. (1972). Mechanical transients and the origin of muscular force. Cold Spring Harb. Symp. quant. Biol. 37, 669-680. HUXLEY, H. E. (1953). X-rays analysis and the problem of muscle. Proc. R. Soc. B 141, 59-62. JULIAN, F. J., SOuTINs, M. R. & Moss, R. L. (1978). Sarcomere length non-uniformity in relation to tetanic responses of stretched skeletal muscle fibres. Proc. R. Soc. B 200, 109-116. JULIAN, F. J. & MORGAN, D. L. (1979) Intersarcomere dynamics during fixed-end tetanic contractions of frog muscle fibres. J. Physiol. 293, 365-378. MORGAN, D. L. (1976). Some equipment and experiments in muscle mechanics. Ph.D. thesis, Monash University, Clayton 3168, Australia. PEACHEY, L. D. & EISENBERG, B. R. (1978). Helicoids in the T system and striations of frog skeletal muscle fibres seen by high voltage electron micriscopy. Biophys. J. 22, 145-154. PRINGLE, J. W. S. (1978). Stretch activation of muscle: function and mechanism. Proc. R. Soc. B 201, 107-130.
392
F. J. JULIAN AND D. L. MORGAN EXPLANATION OF PLATE
PLATE I Changes in the striation pattern near the end of a fibre. All photographs were taken at a muscle length corresponding to a mean passive sarcomere length of 2-4 Ium. In A the muscle is passive, B was taken late in a fixed-end contraction, and C was taken after the fibre had been stimulated for 1 sec at a muscle length corresponding to 2-8 Aim sarcomere length, then shortened over 1-5 sec to the same length as A and B. 4 0C, stimulation frequency 20 sec'. Total length = 0-56 mm.
The Journal
cqf Physiology,
F. J. JULIAN A-M) D. L. MORGAN(
Vol. 293
Plate 1
(Facing p. 392())
J. Physiol. (1979), 293, pp. 379-392 With I plate and 8 text-ftgurea Printed in Great Britain
THE EFFECT ON TENSION OF NON-UNIFORM DISTRIBUTION OF LENGTH CHANGES APPLIED TO FROG MUSCLE FIBRES
BY F. J. JULIAN AND D. L. MORGAN From the Department of Muscle Research, Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA.02114, U.S.A.
(Received 31 October 1978) SUMMARY
1. The stability of sarcomere lengths along single frog twitch fibres was examined, during lengthening and shortening, using a spot follower apparatus to monitor or control the length of a central segment. 2. During active shortening from sarcomere lengths beyond 2-2 jtm the end sarcomeres shortened dramatically, while much of the fibre did not shorten at all. It is proposed that this is the cause of the tension failing to recover, after the shortening ceased, to the value of isometric tension at the shorter length. 3. During active lengthening from sarcomere lengths beyond 2-2 j#m, nonuniformity of stretch was seen, with the middle stretching more than the ends. Some maintained extra tension after stretch above that appropriate to the longer length was found, as were consistent changes in internal movement, and in the shape of the tension record during relaxation. 4. Measurements of stiffness during and after a lengthening suggest that no increased activation is involved. Observation of internal movement during the raised tension after a lengthening contradicts theories involving 'locked on' bridges. 5. From these and other observations, an explanation for the extra tension in terms of non-uniformity of sarcomeres is proposed. The explanation is in accord with that previously suggested for the creep phase of tension rise seen at these lengths. INTRODUCTION
The question of sarcomere length stability under fixed-end conditions at long lengths has been discussed for some years (Hill, 1953), and considered in some detail in the preceding paper (Julian & Morgan, 1979). It is also of some interest to consider the effects of lengthening or shortening a fibre on the degree of uniformity of sarcomeres. A non-uniform distribution of sarcomere lengths, once established, is likely to last for the duration of a contraction, and possibly to persist to some extent even during relaxation, as suggested, for example, by the effects of previous stimulation on creep (Gordon, Huxley & Julian 1966a, Fig. 1; Julian & Morgan, 1979). Consequently, intersarcomere dynamics during changes in muscle length and the resulting sarcomere length distributions must be considered as a possible explanation for effects of motion on tension which are maintained for the duration of the tetanus. 0022-3751/79/4570-0812 $01.50 © 1979 The Physiological Society
F. J. JULIAN AND D. L. MORGAN 380 The experiments reported here are concerned with observation of sarcomere non-uniformities during lengthening and shortening of tetanically stimulated frog twitch fibres, using the spot follower apparatus described previously (Julian & Morgan, 1979) to monitor, and where appropriate, control the length of a central segment. METHODS
The experimental procedure used was the same as that described in the preceding paper (Julian & Morgan, 1979), some fibres being used in both investigations. The motor was used in these experiments to execute ramp and hold movements by following a ramp generator signal. The ramp generator was an analogue device with a control on which was set the voltage representing the length being controlled, i.e. either muscle length or marker spacing. Ramp amplitude and slope controls were thus calibrated in per cent muscle length and muscle lengths/sec respectively. Our convention was to set the just-taut length on the control so that all amplitudes are quoted as a percentage of just -taut length. These can be approximately converted to nm per half-sarcomere by multiplying by 11, i.e. 5 % just-taut length is 55 nm per half-sarcomere. The lamp generator consisted basically of an integrator and two comparators. When the output of the integrator reached the desired limit, up or down, the appropriate comparator became part of an additional feed-back loop which kept the output at a constant value. This gave a non-drifting, transient-free ramp and hold wave form. A similar device has been described more fully by Morgan (1976, pp. 138-142). In some experiments, a small high frequency sinusoidal vibration was imposed, to enable measurement of muscle stiffness. In these experiments, records were taken in pairs, one with and one without vibration. Electronic subtraction of the records in the digital oscilloscope served the two purposes of checking that vibration did not affect the response to the other movement, and of providing a signal containing just the vibration component of tension, which could then be magnified and measured accurately and simply on a cycle-to-cycle basis.
RESULTS
Shortening The shortenings used were generally quite large and slow to maximize the maintained effects while ensuring that a reasonably large tension was maintained throughout the transient. A typical result is shown in Fig. 1 A. The marker spacing record during active shortening is trace c in which the following phases can be observed. During the initial fixed-end stage of the contraction the central segment of the fibre begins to lengthen slowly, typical of a fixed-end contraction at long length. When the fibre shortening began, the marker spacing initially decreased approximately proportionately, but the rate of central shortening rapidly decreased, and over a considerable proportion of the shortening time the central segment was in fact lengthening. At the end of muscle shortening, the initial slow lengthening of the central segment resumed, and only after the 'shoulder' in the relaxation phase of the tension record did the central segment shorten to the length appropriate to the shorter muscle length. Thus, during active shortening, the movement is seen to be extremely non-uniformly distributed, with the ends absorbing most of the movement. The most noticeable feature of the tension record, which has been reported before (Deleze, 1961; Abbott & Aubert, 1952) and is shown in Fig. 2A, is that the tension after the completion of the shortening remains less than that attained
SARCOMERE DYNAMICS IN STRETCH AND RELEASE 381 during a fixed-end contraction at the short length. Also interesting is the humped shape of the tension trace during the shortening, even though the mean sarcomere length (averaged over the whole fibre) is still 2-5 rum at the end of the movement, i.e. well beyond the plateau of the length tension relationship (Gordon, Huxley a
C
2 mm 150 nm ,4 mN 1 sec
A
B
Fig. 1. The effect of shortening. A, from sarcomere length of 2-8-2-5 j#m; trace a is the motor record with thickening indicating stimulation, b is the marker separation when the shortening was done passively, c is the marker separation when the muscle was stimulated during shortening, and d the tension record during active shortening. The tip of the motor arm moved 1 mm at 1 mm/sec. Calibration for marker spacing is in nm/half-sarcomere. B, from sarcomere length 2-25-1-95 ,sm, together with isometric contractions at initial and final lengths. Upper records are motor with thickening indicating stimulation, lower records tension. A little depression of tension is apparent, although the traces are still converging at the end of stimulation. 13 0C, stimulation frequency 35 sec'. a
t S
b
c
0.5 mN
i~~~~~~sec
d
Fig. 2. Interruption of stimulation after shortening. Trace a is the tension record from an isometric contraction at sarcomere length = 2-4 /m. In the other traces, the contraction was begun at sarcomere length = 2-8 /sm, and the fibre was then shortened to 2-4 gm in 1-5 sec. When the tension was nearly steady, the stimulation was stopped for a variable time, and then resumed. Trace c with a short interruption had a final tension near b with no interruption. Trace d, with a longer pause, recovered to approximately the same tension as a. 4 'C, stimulation frequency 20 sec-.
& Julian, 1966b). When this experiment was repeated with shortening around the plateau as shown in Fig. 1 B, the tension deficiency, though still observable, was much reduced, and the hump in the tension record was absent. Abbot & Aubert's (1952) experiment of interrupting the tetanus during the period after the shortening was complete, was repeated with similar results and some extra observations as shown in Fig. 2. If the tension was only allowed to fall
F. J. JULIAN AND D. L. MORGAN 382 as far as the shoulder before the stimulation was recommenced, the tension stayed depressed. However, if the sarcomere length redistribution which follows the shoulder was allowed to proceed during the interruption, the tension recovered to the value appropriate to the new length. Direct microscopic observation of the ends was performed both visually and photographically, and some results are shown in P1. 1. While passive, the striation pattern was distinct and nearly uniform right to the end of the fibre (P1. 1A). During isometric contraction, the usual shortening and loss of order in a small region around the end was seen (P1. 1B) (Huxley & Peachey, 1961; Julian, Sollins & Moss, 1978). When the fibre was observed during the shortening, a progressive 'crumpling' of the ends could be seen, the region of shortened sarcomeres enlarging, until late in the movement the whole field of the microscope was taken up by shortened sarcomeres as in P1. 1C. Accurate measurements of sarcomere length were made difficult by the disorder, but such measurements as were possible did confirm the conclusions drawn from visual observation. Diffraction techniques were not used, as only ordered sarcomeres produce clear diffraction patterns, and sarcomeres as disordered as some of those seen here could be expected to give little more than random scatter. Thus, a diffraction pattern would give primarily the mean spacing of the ordered sarcomeres within the beam, with a much lower weighting factor on the severely shortened and disordered ones, merely because of their disorder. Attempts to force the central segment to shorten by using marker spacing control were unsuccessful, the motor arm moving so far that the markers translated out of the microscope field of view quite early in the shortening, leading to loss of control and possible damage to the fibre. It became apparent from these experiments that the amount of end movement required to shorten the central segment slowly was extremely large. Lengthening
Lengthening movements were usually faster than the shortenings, in order to keep the tetani of a reasonable length while still allowing time for completion of the transient response. The marker spacing records again show non-uniform distribution of the movement, though not as dramatically as for shortening. Fig. 3A shows the main features usually observed. When the end movement was applied before the stimulation began, the central segment stretched proportionally and showed a normal slow lengthening throughout the subsequent tetanus. An end movement of the same size applied during the tetanus produced a significantly and consistently larger increase in marker spacing. Over a large number of records, the extra movement averaged 15 %. During the decay of tension after the imposed lengthening ceased the marker spacing increased only very slowly, much more slowly than during a fixed-end contraction. As the transient ended, the markers began to move apart again, and finally attained a stretch rate comparable to or even greater than that of the fixed-end contraction at the same time. Thus, during active lengthening it appears that most of the fibre is stretched, but that small portions away from the centre lengthen less than a proportional amount. The tension traces of Fig. 3A are most noticeable for the maintenance of tension
SARCOMERE DYNAMICS IN STRETCH AND RELEASE 3383 following an active stretch at a level higher than that attained during a fixedend tetanus at the final length, even after the transient has passed. This is the 'permanent' extra tension after stretch noted by many workers (Abbott & Aubert, 1952; Hill, 1977; Edman, Elzinga & Noble, 1976, 1978a, b). 88 nm 2 mN
2 sec
a
b
A
C
~~~~~~~C
Fig. 3. A, typical result with marker spacing records. For traces a (marker spacing) and c (tension) the muscle was lengthened by 5 % just-taut length in 170 msec before stimulation commenced. For traces b (marker separation) and d (tension) the same lengthening was applied during the tetanus. 4 0C, stimulation frequency 16 sec-, initial sarcomere length = 2-5 jam. B, the effect of stretch amplitude. Stretches of 3, 5 and 7 % just-taut length were applied before and during the tetanus. The stretches before stimulation produced a decreasing tension with increasing length, but the stretches during stimulation all produced the same final tension. 4 0C, stimulation frequency 16 sec-', initial sarcomere length = 2*5 #sm, ramp speed = 0 3 muscle lengths/sec. C, effect of speed of lengthening. Two tension records of actively lengthening a fibre by 7 % just-taut length at speeds of 0.1 and 0-8 muscle lengths/sec. Eventually the tensions are equal. 4 0C, stimulation frequency 16 sec-", initial sarcomere length = 2*5 1um. The calibration bars apply to all parts of the Figure and for the marker spacing records are in nm/half-sarcomere, lengthening up.
There are also significant and consistent differences in the tension records during relaxation. The 'shoulder' generally occurred later if the muscle had been stretched actively, and the whole decay phase was slower. As the shoulder and subsequent relaxation is known to be associated with redistribution of sarcomere lengths (Huxley & Simmons, 1972; Julian & Morgan, 1979), differences in relaxation are suggestive of different sarcomere length distributions at the end of the tetanus. The effect of amplitude of stretch on the final tension (not extra tension) was investigated and found to be negligible over the range studied. This is shown in Fig. 3B where progressively larger stretches before stimulation give progressively smaller final tensions (the descending limb of the length-tension curve) but all the stretches during the tetanus led eventually to the same tension. Similarly, an eightfold change in velocity of lengthening is shown not to effect the final tension (Fig. 3C). It was also found generally, as shown in these Figures, that speed or amplitude of stretch did not greatly alter the shape of the decay phase of the tension records. The initial length of the muscle is already known to effect the extra tension (Hill,
F. J. JULIAN AND D. L. MORGAN 384 1977; Edman et al. 1976, 1978a, b) and our findings in this regard are shown in Figs. 4, 5). At short lengths (sarcomere length < 20 gtm), the final steady tension after a stretch was very close to the fixed-end tension at the new length, i.e. the 'extra' tension was due only to the ascending limb of the length-tension relationship. When the pull was within the plateau region (2-0 < sarcomere length < 2-2 jtm) 2 mN
b
b
2 sec
A A
C 8
C
Fig. 4. The effect of fibre length. Tension records for no stretch (a in each case), stretch active (b), and stretch before stimulation commenced (c). A, 7 % stretch from sarcomere length = 1I7 4um; B, 7 % stretch from sarcomere length = 2 0 4Am; C, 7 % stretch from sarcomere length = 2-4 jam. Extra tension and modification of relaxation are only apparent at sarcomere lengths beyond the plateau. 4 'C, stimulation frequency 20 sec-.
Fig. 5. Stretch at longer lengths. Tension and marker spacing records for a 3 % stretch from sarcomere length = 3 0 /m. This illustrates the method of 'stretch before stimulation' used to account for passive tension. Tension trace b (active stretch) shows what appears to be a well maintained plateau after stretch, but comparison shows that creep in the isometric at the long-length record generates almost as much tension if the tetanus is continued long enough. Calibration for marker spacing is in nm/half-&arcomere, and lengthening is upwards. 11 0C, stimulation frequency 25 sec-1.
there was no extra tension. Provided that the stimulation was continued long enough, the tension returned to the fixed-end tension. In the range of moderately long lengths beyond the plateau but without significant passive tension (2.2 < sarcomere length < 2.9 #um), the tension after the pull was greater than the expected value, that developed by a fixed-end contraction at the final length, but always very close to that developed in a fixed-end contraction at the initial length. Thus, in
385 SARCOMERE DYNAMICS IN STRETCH AND RELEASE one sense, the fibre did not actually develop extra tension after the stretch, it merely failed to decrease its tension to the value given by the descending limb of the length-tension curve for isometric muscles. At very long lengths, several factors complicate the results. Passive tension must be subtracted, but the exact method is uncertain. The usual approach is to subtract the difference between final passive tensions at the initial and final lengths. However, stretching a passive muscle at a, rate similar to that used for the active muscle produces more tension than this difference, and the extra tension persists over a time period longer than that used for tetani. If the active response is simply additive to the passive response, then this passive response to stretch should be subtracted from the active response to accurately correct for passive tension. The use of the spot follower shows that non-uniform stretching occurs at these lengths also (Fig. 5), so even the amplitude of the passive stretch which should be subtracted is uncertain, depending on whether the parallel elasticity is in parallel with the whole muscle or each sarcomere, or some combination of both. Even use of marker spacing control does not remove this problem. Furthermore, the fixed-end tension continues to rise
(creep) for quite some time. The approach which we took to these problems and the results found are shown in Fig. 5. To account for passive tension, in the fixedend contraction at the longer length, or 'stretch before stimulating' contraction, the muscle fibre was brought to the longer length immediately before commencing stimulation using the same end movement as was used in the active stretching. The stimulation was continued until the creep phase ended, and then tensions were compared. As can be seen, the extra tension under these conditions is quite small, certainly no more significant than at the moderately long lengths. Note also the reduction of velocity of stretch after the active pull as compared to the isometric tetanus, and the prolongation of the slow linear phase of tension decay, as in Fig. 3A. At or below the plateau then, the results are in accord with the length-tension curve. At moderately long lengths, there is a definite lack of reduction of tension, while at very long lengths, the experiment is more complicated, but the amount of extra tension appears to be no greater than at moderately long lengths. For this reason, investigation concentrated on the region between the plateau and the length at which passive tension became significant. Interruption of the tetanus for various time intervals after the transient response produced results typified by Fig. 6. After a short interruption, terminated before the shoulder of the tension record, the tension was not reduced significantly below its raised value, but after a longer interruption, allowing significant sarcomere length redistribution, it returned to nearer the tension of the fixed-end tetanus at the long length. It should be remembered that the shoulder signifies only the beginning of restribution, which is often not completed until the tension has decreased to near zero, so an extremely sharp return from distorted to normal sarcomere length distribution should not be expected. Nevertheless, these results are suggestive of a significant role for sarcomere length non-uniformities in producing the extra tension. Stiffness measurement. It is conceivable that some process of activation by stretch (Pringle, 1978) could be acting to increase the number of available sites for cross13
PHY 293
386 F. J. JULIAN AND D. L. MORGAN bridge attachment, and that these sites remain available while the tension is maintained, but revert to being unavailable when the tension is allowed to fall. In order to test this suggestion, the stiffness of a muscle during stimulation and lengthening was measured by means of sinusoidal vibration as shown in Fig. 7, and plotted against tension in Fig. 8. It can be seen that the stiffness rose almost linearly with 1 mN
1 sec
a~~~~~~~~~~~~~~~~
Fig. 6. Interruption of stimulation after stretch. This is the equivalent experiment to Fig. 2. Trace a is the tension record for an isometric contraction at the short length, b is isometric at the long length. The normal stretch during stimulation record, c, shows the usual elevated tension and characteristic changes in decay phase. A short pause in stimulation, d, leads to recovery of the higher tension, but after a longer interruption, e, the tension is nearer that of b. 4 00, stimulation frequency 15 see-1, initial sarcomere length = 2-5 /tm, 7 % stretch at 0 3 muscle lengths/sec.
the rise of tension when stimulation commenced, but that throughout the lengthening and the decay of transient tension the stiffness stayed substantially constant, suggesting that the number of cross-bridges did not change significantly, or at least not proportionally to the tension. Photographic observations of end segments were made in an attempt to complement the evidence from marker spacing observations. Somewhat less lengthening of the end regions was seen when the stretch was applied to a stimulated fibre than when it was applied to a passive fibre. However, the most striking result was the sharp reduction in the shortening rate of the ends brought about by a stretch. During the decay of the transient, the progressive collapse of ends seen in a long length contraction (Julian et al. 1978; Julian & Morgan, 1979) was almost totally halted, so that at a time corresponding to the end of the tension transient, a fibre contracting with fixed ends had much shorter end sarcomeres when compared to one which had been lengthened actively. Marker spacing control was used in some experiments and the results were consistent with those using end-controlled movements. The following points were noted. The end movement required to produce a given change in marker spacing was less for an active than for a passive fibre. The rate of end movement required to keep the marker spacing constant during the tension transient following a stretch was much less than that required during an isometric tetanus. The final tension was as predicted by the length-tension curve at muscle lengths on or below the plateau.
SARCOMERE DYNAMICS IN STRETCH AND RELEASE
387
1 mN
0-25 mN 1 sec
-He
And He~-
Fig. 7. Stiffness measurement. A pull, 5 % in 100 msec from sarcomere length = 2-5 /im, trace a, was repeated b with a 2*5 Aim 2-5 kHz vibration superimposed. The difference between the two records was found by digital subtraction, multiplied by a factor of four, plotted as trace c and measured to provide a measure of stiffness. The frequency visible in the records is a beat frequency between the vibration and sampling rates. Improved time resolution would be obtained by recording only part of the tetanus at a higher sampling rate. 4 'C, stimulation frequency 20 sec-'.
b_:f
Tension P0 Fig. 8. Stiffness results. The plot shows stiffness as a function of tension while passive (point a), during the isometric rise of tension to point b, during a stretch, peaking at point c, and during the decay back to the isometric tension. It can be seen that the stiffness rises almost linearly with tension during the rise of tetanus, but is essentially constant during the stretch and decay. 4 0C, stimulation frequency 20 sec', 7 % stretch from 2-5J sm. Same experiment as shown in Fig. 7.
13-2
388 F. J. JULIAN AND D. L. MORGAN The use of marker spacing control produced even slower decay of the tension transient than end control. This meant that very long tetani were needed to enable accurate comparison. Quite apart from the usual problems of fatigue, prolonged segment-clamped tetani are difficult to carry out accurately as there is a tendency for the tension to rise slowly, particularly during the later stages of a long tetanus (see Discussion). Within these limitations the extra tension did appear to be reduced by the use of marker control rather than end control. However, the uncertainties were such that end control was preferred. DISCUSSION
Shortening It seems quite clear that during slow end shortening of a single fibre at moderately long lengths, sarcomere shortening is extremely non-uniform. This can be accounted for in the following way. In the fixed-end contraction, the end sarcomeres are shortening, and slowly stretching the rest of the fibre. When the end is moved to shorten the muscle, the discontinuity of the force-velocity curve about the point where the velocity changes sign means that a given fall in tension (the same all along the muscle) will be accompanied by a greater change of shortening velocity in sarcomeres that were previously shortening than in those that were previously lengthening, i.e. the shortening sarcomeres will increase their shortening velocity more than lengthening sarcomeres will decrease their lengthening velocity. Consequently, the shortening sarcomeres will shorten much faster, while the lengthening sarcomeres will not shorten at all. Of course, this shortening of the end sarcomeres initially increases their tension generating capacity. This continues until they shorten over the plateau of the sarcomere length-tension curve which is probably the cause of the 'hump' in the tension record of Fig. 1A, and the corresponding curvature of the marker spacing record. The converse experiment, of allowing isotonic shortening from long lengths, was also carried out by D6ekze (1961) with the finding that the final muscle length for a given load depended on the initial length. This is almost certainly due to a similar mechanism, as was suggested by A. F. Huxley (1964, p. 135). The observation of the shortened region spreading by incorporation of adjacent sarcomeres, and indeed the commonly seen gradation from long to short sarcomeres, both suggest the existence of some forces which act to prevent marked disparities of sarcomere length between adjacent sarcomeres. At very long lengths distributed passive elasticity could be sufficient, but another explanation must be found at shorter lengths. If the constant volume constraint on the filament lattice (H. E. Huxley, 1953) holds true on an individual sarcomere scale, then the resistance of the fibre to sudden changes in diameter may be sufficient. Another possible explanation could be based upon the helical sarcomere structure described by Peachey & Eisenberg (1978). These experiments also provide insight into some of the difficulties encountered in length clamping the central segment of a fibre at these sarcomere lengths. It is apparent that moving the end of the fibre is not a very effective way of shortening the central segment, yet it is the only means available. If a 'clamped' tetanus is
SARCOMERE DYNAMICS IN STRETCH AND RELEASE 389 extended for a long time, large end movements will be necessary, and eventually the collapsed region will extend into the segment being clamped, making it nonuniform and defeating the purpose of the clamp, which is to control the length of a uniform central segment. It could be argued that the depression of tension after a shortening presents a problem for the sarcomere length non-uniformity explanation of the creep phase of tension rise (Julian et al. 1978). This explanation assumes that a sarcomere shortening from a long length does increase its capacity to generate tension in accordance with the length-tension curve. From the present work, it is clear that the observation of depressed tension after active shortening does not contradict that assumption, but is simply due to the fact that shortening the over-all muscle fibre does not shorten the sarcomeres uniformly, and may not shorten many of them at all. Furthermore, this non-uniform shortening behaviour is entirely in accord with the sarcomere length non-uniformity explanation of creep.
Lengthening When an active muscle is forcibly lengthened at moderate speed, it shows a short range of stiffness and then 'yields' (Fig. 3B). Assuming that the 'yield point' is a constant fraction of the isometric tension as sarcomere length is varied (Flitney & Hirst, 1978, Fig. 9), the observed non-uniformity of active stretch can be predicted. The shorter, shortening sarcomeres at the ends would be expected to have a higher isometric tension, and hence higher yield point than the rest of the fibre. Consequently, they would tend to resist lengthening, at the expense of the rest of the fibre. The unusual stability of sarcomere lengths after the stretch is probably due to all sarcomeres having 'stretched' cross-bridges, i.e. each cross-bridge exerting more tension than it would if no change of sarcomere length had taken place since it attached. Consequently, all sarcomeres are able to bear the high tension without lengthening, at least temporarily. However, none are able to shorten, since any sarcomere which shortened would need to detach and re-attach cross-bridges, which would decrease the tension. If this explanation is correct the decay rate of the transient reflects the rate at which the stretched cross-bridges detach and reattach with the 'normal' tension in the near complete absence of internal motion. This is not inconsistent with the detachment rate suggested by the slope of the linear phase of tension relaxation in a segment length-clamped fibre as discussed in the previous paper. The question still to be answered is why does the tension following a stretch return to that level appropriate to the original length? The present work has shown that the sarcomere length distribution after active stretch is different from that which exists if the stretch was delivered before activation, so the tensions should be expected to be different. The unexpected finding is that the tension is so close to the fixed-end tension at the original length and so independent of factors such as amplitude and speed of stretch. The final tension is apparently determined primarily by some factor which is not changed by the lengthening. The obvious suggestion is the length and tension generating capacity of those sarcomeres which did not lengthen during the stretch, presumably the ones near the ends which were already
F. J. JULIAN AND D. L. MORGAN shortening. This suggestion would postulate that the tension during a fixed-end tetanus is strongly influenced by the distribution of sarcomere lengths in these end regions. Then the tension reached is determined by the number of sarcomeres which shorten during the contraction, the velocity with which they can shorten, the distance through which they can shorten before reaching the plateau, and possibly the rate at which the collapsed region can incorporate new sarcomeres. The rest of the fibre then stretches under the imposed load, the initial sarcomere length having only a small effect on the stretching speed, due to the steepness of the force-velocity curve for slow lengthening. All the above mentioned factors depend on the initial distribution of sarcomere lengths and, to the extent that the stretch is not shared by these end sarcomeres, remain unaffected by the stretch. Ideally, this proposal should be testable by photomicroscopy, but there are practical difficulties. However, considerable indirect supporting evidence is available. Interruption of a tetanus only abolishes the effect if the decay of tension is allowed to proceed beyond the shoulder, where length redistribution begins to occur. Extra permanent tension is only found beyond the plateau where instability is to be expected and creep is observed in the marker records of fixed-end contractions. The behaviour of the markers, stationary during the raised tension of the transient, and then separating during the sustained lower tension, is opposite to what would be expected if the changes in separation were a passive consequence of the tension rather than being directly involved in tension generation. The explanation offered is consistent with that for creep. Explanations of the raised tension after stretch based on sarcomere length nonuniformity have been rejected by some (Hill, 1977) on the basis of observations, at variance with ours, and those of Edman et al. (1978b), Fig. 6A, that maintained extra tension could be produced in the plateau region. We believe that these discrepancies arise from failure to observe some of the precautions mentioned under Results, particularly failure to wait until a really permanent tension is reached (e.g. Edman et al. 1976, Fig. 1 A) and failure to wait for creep to finish before measuring the tension of a fixed-end tetanus (e.g. Hill (1977, Fig. 1A) where the tension before the stretch is taken as the fixed-end tension, and Edman et al. (1978a) where the fixed-end tension is still rising at the latest time shown). Other differences, particularly at longer lengths may arise from the use of the steady-state rather than transient/passive tension when comparing active tensions. The finding that stiffness does not increase during or after the stretch argues against explanations involving extra bridges. Such explanations would also have difficulty accounting for the interruption of stimulation results. The resumed gradual separation of markers during the maintained tension is incompatible with explanations based on 'locked on' bridges, which could only exist while the sarcomeres were isometric (Hill, 1977). General It is clear from these experiments that instability of sarcomere lengths does exist on the descending limb of the length-tension curve, and that the force-velocity curve, particularly during stretch, is responsible for the heavily damped nature of the instability. It is apparent both from experiment and from reasoning that movement of the end of the fibre accentuates the non-uniformities, irrespective of the
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SARCOMERE DYNAMICS IN STRETCH AND RELEASE 391 direction of the movement. The sarcomere length dynamics which result from this damped instability are both complex and important. Furthermore, it is important to realize that these intersarcomere dynamics can affect the tension considerably and under a wide range of conditions. This work was supported by the following grants: an N.I.H. research grant, HL-16606, from the National Heart, Lung and Blood Institute, and grants from the American Heart Association, No. 77-616, and the Muscular Dystrophy Association.
REFERENCES ABBOT, B. C. & AUBERT, X. M. (1952). The force exerted by active striated muscle during and after change in length. J. Phygiol. 117, 77-86. DE'LtZE, J. B. (1961). The mechanical properties of the semitendinosus muscle at lengths greater than its length in the body. J. Physiol. 158, 154-164. EDMAN, K. A. P., ELZINGA, G. & NOBLE, M. I. M. (1976). Force enhancement induced by stretch of contracting single isolated muscle fibres of the frog. J. Physiol. 258, 95P. EDMAN, K. A. P., ELZINGA, G. & NOBLE, M. I. M. (1978a). Further characterization of the enhancement of force by stretch during activity in single muscle fibres of the frog. J. Phygiol. 280, 35P. EDMAN, K. A. P., ELZINGA, G. & NOBLE, M. I. M. (1978b). Enhancement of mechanical performance by stretch during tetanic contractions of vertebrate skeletal muscle fibres.
J. Physiol. 281, 139-155. FLITNEY, F. W. & HrRST, D. G. (1978). Crossbridge detachment and sarcomere 'give' during stretch of active frog's muscle. J. Phygiol. 276, 449-465. GORDON, A. M., HUXLEY, A. F. & JULIAN, F. J. (1966a). Tension development in highly stretched vertebrate muscle fibres. J. Phygiol. 184, 143-169. GORDON, A. M., HUXLEY, A. F. & JULIAN, F. J. (1966b). The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J. Physiol. 184, 170-192. HILL, A. V. (1953). The mechanics of active muscle. Proc. R. Soc. B 141, 104-117. HILL, L. (1977). A-band length, striation spacing and tension change on stretch of active muscle. J. Physiol. 266, 677-685. HUXLEY, A. F. (1964). Muscle. A. Rev. Physiol. 26, 131-152. HUXLEY, A. F. & PEACHEY, L. D. (1961). The maximum length for contraction in vertebrate striated muscle. J. Physiol. 156, 150-165. HUXLEY, A. F. & SIMMONs, R. M. (1972). Mechanical transients and the origin of muscular force. Cold Spring Harb. Symp. quant. Biol. 37, 669-680. HUXLEY, H. E. (1953). X-rays analysis and the problem of muscle. Proc. R. Soc. B 141, 59-62. JULIAN, F. J., SOuTINs, M. R. & Moss, R. L. (1978). Sarcomere length non-uniformity in relation to tetanic responses of stretched skeletal muscle fibres. Proc. R. Soc. B 200, 109-116. JULIAN, F. J. & MORGAN, D. L. (1979) Intersarcomere dynamics during fixed-end tetanic contractions of frog muscle fibres. J. Physiol. 293, 365-378. MORGAN, D. L. (1976). Some equipment and experiments in muscle mechanics. Ph.D. thesis, Monash University, Clayton 3168, Australia. PEACHEY, L. D. & EISENBERG, B. R. (1978). Helicoids in the T system and striations of frog skeletal muscle fibres seen by high voltage electron micriscopy. Biophys. J. 22, 145-154. PRINGLE, J. W. S. (1978). Stretch activation of muscle: function and mechanism. Proc. R. Soc. B 201, 107-130.
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F. J. JULIAN AND D. L. MORGAN EXPLANATION OF PLATE
PLATE I Changes in the striation pattern near the end of a fibre. All photographs were taken at a muscle length corresponding to a mean passive sarcomere length of 2-4 Ium. In A the muscle is passive, B was taken late in a fixed-end contraction, and C was taken after the fibre had been stimulated for 1 sec at a muscle length corresponding to 2-8 Aim sarcomere length, then shortened over 1-5 sec to the same length as A and B. 4 0C, stimulation frequency 20 sec'. Total length = 0-56 mm.
The Journal
cqf Physiology,
F. J. JULIAN A-M) D. L. MORGAN(
Vol. 293
Plate 1
(Facing p. 392())
