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Journal of Muscle Research and Cell Motility 10, 67-84 (1989)
Sarcomere length behaviour along single frog muscle fibres at different lengths during isometric tetani
KEVIN BURTON*, W I L L I A M N. Z A G O T T A * , and R O N A L D J. B A S K I N
Department of Zoology, Graduate Group in Biophysics, University of California at Davis, Davis, California 95616, USA
Received 12 May 1988
Summary
A detailed investigation of sarcomere lengthening and shortening during fixed-end tetani has been made along frog muscle fibres stretched over a large range of sarcomere lengths. A variety of sources of error common in such measurements are quantitated and give an uncertainty in sarcomere length of about 53-62 nm. The difference in sarcomere length calculated from the left and right first orders at rest was 21 nm+ 16 nm and this is suggested to be a measure of 'Bragg artefact'. The laser diffraction measurements showed that the shortening end regions decrease in size during contraction and that the magnitude of shortening is increased at greater fibre extensions. The average length change and sarcomere length of the central and end regions was 0.10 gm (2.85 ~m) and -0.37 ~m (2.66 ~un), respectively. The sarcomere length of the end regions at the end of creep was regularly observed to be <2.1 ~m. An unexpected finding was the occasional observation of striations in the transition zone between lengthening and shortening regions which remained nearly isometric during a period of tension rise during creep. Measurements of diffraction order linewidth do not suggest increased sarcomere length dispersion in these areas. A smooth transition from shortening to lengthening was always observed. Although our data are in general agreement with the models proposed by Morgan, Mochon and Julian (Biophys. J. 39 (1982) 189-96) and Edman and Reggiani (J. Physiol. (Lond.) 351 (1984) 169-98), specific differences which do exist are discussed.
Introduction
The nature of the variability of sarcomere length (SL) along a single fibre at rest and during activity has been investigated by a number of researchers. Huxley and Peachey (1961) observed microscopically a marked shortening of regions near the myotendinous junction during a tetanus in which the length of the fibre was held constant. These observations have been confirmed on a number of occasions (Carlsen et al., 1961; Gordon et al., 1966a; & Julian & Morgan, 1979b). Laser diffraction has also been used to monitor changes in sarcomere length during an isometric tetanus (Cleworth & Edman, 1972; Kawai & Kuntz, 1973; Borejdo & Mason, 1976; ter Keurs et al., 1978). Altringham and Pollack (1984) used this technique to measure lengthening of sarcomeres in central regions of stretched fibres and Lieber and Baskin (1983) followed sarcomere dynamics in selected regions of moderately stretched fibres. Such sarcomere movements have been shown to account for the slow rise of tension during an isometric tetanus (Julian & Morgan 1979a; Lieber & Baskin, 1983). Although controlling the length of a central
segment during an isometric tetanus reduces or virtually eliminates the slow rise (Gordon et al., 1966a; Edman & Reggiani, 1987), some differences in the measured length-tension relationships remain.
If the various phases in the isometric tension record (i.e., fast rise, creep, and plateau/decline) can be explained by a series arrangement of sarcomeres with assumed mechanical characteristics, then these rela- tionships should be amenable to quantitative modell- ing and this has in fact been done (Morgan et al., 1982; Edman & Reggiani, 1984). The detailed predic- tions provided by this type of modelling for the behaviour of various regions along a fibre are best evaluated if analogous empirical data are available. The studies mentioned above have in general only monitored one or a few regions of a fibre or have made measurements over a narrow range of fibre extensions. Recently, this situation has been im- proved by the thorough study of Edman and Reggiani (1984) in which the active length changes of segments demarcated with surface markers were monitored. Of the methods used for measuring
*To whom correspondence should be addressed at: MRC Cell Biophysics Unit, 26-29 Drury Lane, King's College London, London WC2B 5RL, UK *Present address: Department of Neurobiology, Stanford University, Stanford, California 94305, USA
0142-4319/89 $03.00 + 112 �9 1989 Chapman and Hall Ltd.
68 K. BURTON, W. N. ZAGOTTA a n d R. J. BASKIN
sarcomere length, laser diffraction has the advan tage of moni to r ing sa rcomeres t h r o u g h o u t a large por t ion of the i l luminated v o l u m e of a fibre, whe reas microscopy or surface marke r s m eas u re the be- hav iour of an optical section or the fibre surface, respectively. A l t r ingham and Bottinelli (1985) have u sed the diffraction technique in a recent s tudy to moni to r sa rcomere dynamics a long fibres s t re tched to var ious lengths. The accuracy of laser diffraction has, however , been ques t ioned because of 'Bragg artefact ' caused by preferent ia l in tens i ty re inforcement of the diffraction order by dom a i ns of sa rcomeres wi th in the fibre (Rudel & Zite-Ferenczy, 1979a; Huxley, 1980).
The p resen t invest igat ion is an extension of work in our laboratory (Lieber & Baskin, 1983) in which we have used laser light diffraction to m eas u re directly average sa rcomere length in isometrically contract ing single fibres. We have carried out exper iments des igned to obtain an indicat ion of the accuracy and reliability o f the sa rcomere length m e a s u r e m e n t s and these include mon i to r ing bo th left and r ight first order lines dur ing a contract ion and compar ing the behav iou r of the two. The difficulties which m u s t be cons idered w h e n us ing the technique wi th contract- ing fibres are discussed. These include longitudinal m o v e m e n t s , sa rcomere disorder , and Bragg effects. Sarcomere dynamics have been mon i to red at m a n y posi t ions a long any g iven fibre, concentra t ing on the end regions w h e r e spatial gradients of sarcomere length are m o s t prevalent . These data have been obta ined at var ious degrees of s tretch of the fibres. Phases in the sa rcomere length records are correlated wi th those of the tens ion records and these c o m p a r e d wi th the predic t ions of in te rsarcomere dynamics models . Pre l iminary resul ts f rom this s tudy have been pub l i shed (Baskin et al., 1984).
Methods
Fibre preparation and experimental protocol Single fibres were isolated from the lateral head of the tibialis anterior muscle of the grass frog (Rana pipiens). The dissection was carried out in chilled Ringer's solution, composed of (mM): 115NaC1, 2.5KCI, 1.8CAC12, 2.15 Na2HPO4, 0.85NaH2PO4), pH7.0. Two widely spaced fibres were often isolated together from a single muscle. The one which appeared under the dissecting microscope to be in the best condition based on criteria such as transparency of structure, uniformity of diameter and lack of connective tissue or visible points of damage, was then chosen for an experiment. While in the dissecting dish, aluminium foil t-clips (Ford et al., 1977) (0.25mg) were attached to the tendon. The tendon was about 0.4-0.6 mm wide at the clip. The amount of the tendon between the clip and the fibre insertion was particularly important because excess tendon allowed the fibre to move across the laser beam as tension was developed. In these experiments, this distance varied from 0.1 mm to 0.5 mm with an average of
about 0.3mm. The fibre was transferred to the ex- perimental chamber on a moist coverslip. The chamber was viewed from above with a dissecting microscope. The foil clips were mounted on small wire hooks (1 mg) which at one end of the fibre was attached to the arm of a high-speed servomotor (300S, Cambridge Technology,, Watertown, MA, USA) and at the other end to a capicitance-type force transducer. The force transducer-hook assembly had a resonant frequency in air which was adjustable between 4 and 10kHz and a sensitivity of between I and 40mV mg -~ which varied inversely with the resonant frequency. A layer of epoxy or cyanoacrylate glue bonded the hook to the transducer.
A 300ml volume of Ringer's solution was circulated through the chamber (volume 3 ml) at 5 or 13 ml min -1. The temperature of the circulating Ringer's was maintained at 3 to 20~ + 0.2~ in these experiments. Although total tension and the rate of sarcomere length change was increased at higher temperatures, the variation in sarco- mere dynamics with position or fibre extension (see Results) was not observed to be a function of temperature. The chamber, motor and force transducer were mounted on the stage of a Zeiss IM-35 inverted microscope. These three entities were moved as a unit to place any position along the fibre in the path of the laser beam. The length of the fibre was adjustable at either end via micrometers to which were attached the motor and force transducer. The fibre was viewed directly through the 40 • water immersion objective of the microscope. A calibrated eyepiece reticle was used to measure the diameter of the fibre with an accuracy of +1.0 ~m. The laser beam was aligned with the centre of the objective. The length of the fibre was measured by using the eyepiece reticle in the dissecting scope above the chamber. As the ends of the fibres generally taper down to a small diameter and are often difficult to distinguish from the attached tendon, the 'end' point was operationally defined as the halfway point of the tapered portion. The true end was less than a fibre diameter away. The length was set so the fibre was just taut (rest length, SL = 2.0-2.2 ~m) and a tetanus and twitch elicited.
The system used to stimulate the fibre has been previously described (Lieber & Baskin, 1983). Bipolar stimuli were passed between two electrodes made of platinum wire at a voltage typically 1.3 times that necessary to elicit a single twitch. Tetani were produced by stimulating the fibre at a frequency sufficient to produce a fused tension record. The frequency of stimulation varied from 20 Hz to 70 Hz, depending on the temperature of the Ringer's solution. The duration of each pulse was generally 0.5ms, although pulses of a few ms in duration were sometimes used. Rest periods of at least 5 min were given between tetani.
Data acquisition The data acquisition system has in large part been discussed previously (Lieber et al., 1983; Lieber & Baskin, 1983). A 5mW helium-neon laser (model 05-LHP-151; Melles Griot, Irvine, CA) was held in the condenser mount of the inverted microscope from which the laser beam illuminated the fibre and the diffraction pattern passed through the glass bottom of the chamber and into the
Sarcomere length behaviour along fibres dur ing tetani 69
objective (0.75NA). The laser beam had a gaussian intensity profile in cross section with a standard deviation of 200 ~-a. The length of fibre responsible for diffracting the light which passed through the microscope was deter- mined. First, fibres 50-65 ~m in diameter were translated perpendicular to their long axis across the laser beam. The intensity of a diffraction order was measured as a function of the position of the fibre in the beam. When the fibre was over the centre of the laser beam, the intensity was highest. The intensity consistently dropped to near zero as the fibre was moved about 80-90 ~m to either side of centre. Also, if an opaque object such as a tendon was brought into the laser beam, the intensity of the scattered light changed from zero to its maximum within an 80-90 ~m movement of the object. These experiments suggest that a portion of the fibre 160-180 ~'n long provided the major portion of the light which finally passed out of the microscope. Therefore, we assume that a length of fibre approximately 180 ~un long was sampled.
A lower power objective which would have sampled a longer segment of fibre was not used because we wanted to monitor small regions of fibre near the transition zone and ends where the sarcomere length gradient is high. This choice was a compromise between increased spatial resolution and an increase in 'thick grating' effects due to an effectively smaller beamwidth (discussed in Lieber et al., 1984). Sampling over the centre of the beam also has the advantage that the intensity roll-off (8-10%) is not so severe as when sampling over the entire width of the laser beam (about I mm).
In the latter stages of this work, a coverslip was placed on the chamber in order to stabilize the surface of the Ringer's solution and thereby reduce refraction artefact resulting from variation in the angle of incidence of the beam to the fibre. In the case of Ringer's chilled to below the dewpoint, condensation on the coverslip was prevented by a steady stream of air and condensation underneath the chamber was inconsequential because of the water-immersion objective. The diffraction pattern was passed out of the microscope through a video tube and was focused onto a 128-element photodiode array (PDA) (Model 128S, EG&G Reticon, Sunnyvale, CA). The intensity of the laser incident upon the fibre was adjusted with crossed polarizers so that the diffraction intensity was appropriate to the dynamic range of the PDA. The intensity of the zero-th order was adjusted by a linear wedge neutral density filter in order to bring the intensity down to a level comparable to the first order(s). In those experiments in which both first diffrac- tion orders (left and right) were monitored simultaneously, custom-made gradient neutral-density filters of width 1.1 or 1.6 mm were used to filter only the zero-th order without affecting either first order.
The voltage output of the PDA was digitized and sent to the memory of a PDP-11/2 minicomputer (Digital Equip- ment Corporation, Maynard, MA) via a custom Direct Memory Access interface (Lieber et al., 1983). The full 128 elements of the PDA were scanned once every 291 ~s and the period between the start of successive scans sent to the computer was adjustable from 0.291 ms to 149ms. The photodiode array could be sampled up to 378 times in one experimental run. The output of the tension transducer was, in the later half of these experiments, multiplexed into
one of the signal lines which normally carried the signal of one of the elements of the PDA. This analogue signal was then digitized and sent to the computer in the same fashion as the PDA signal. The digitized data was stored on floppy disk for later analysis.
Data analysis The diffraction data was calibrated after each experiment using ruled transmission gratings of grating constants 2.50, 2.77 and 3.33 ~m. The data from the photodiode array were analysed using the equations of Kawai and Kuntz (1973) to calculate the centroid (mean position), linewidth (2 x SD) and area (total intensity) of the intensity distribution of each diffraction order. The spacing between the centroids of the zero-th and first orders was used to calculate sarcomere length. The importance of simultaneously measuring the positions of the first and zero-th orders has been pointed out previously (Klimov & Andreyev, 1982; Lieber & Baskin, 1983). The photodiode elements were arranged along the meridian of the diffraction pattern. Each photodiode element illuminated by diffracted light inte- grated intensity along the equator of the diffraction order such that greater than 95% of the diffraction intensity of an order was monitored. The integration along the equator largely eliminated the effects of fine structure on the diffraction order profile, which can be quite considerable if only the intensity at the meridian is considered (for example, see Tameyasu et al., 1982).
Reliability of sarcomere length measurements Control experiments were carried out to determine the influence of various factors upon the accuracy of our laser diffraction technique. High frequency electrical noise results in an average standard deviation of 2nm in sarcomere length. This value is from 27 records (~378 points each) of SL data from gratings and resting fibres. The range of SL values due to this noise generally varied from 4 to 8 nm. A greater source of difficulty is the accuracy of the 'calibration factor' used to relate the SL to the spacing between orders at the PDA. The variation in the calibration factor determined from the three gratings was described by a standard deviation (averaged over 17 experiments in which this was carefully determined) of 0.42 + 0.27%. This variation would yield an uncertainty of about 15 nm at 2 ~m SL and 24 nm at 3 ~m SL. This variation probably results from variations in the groove spacing of the gratings, the angle of the laser beam to the grating, the angle of the grooves (and therefore diffraction lines) to the PDA, and any angle between the PDA and the meridional plane of the pattern. This uncertainty is nearly constant throughout a tetanus (being only somewhat dependent on SL) and does not appreciably affect temporal comparisons of SL. There is also a slight dependence of the spacing of the orders upon the focus of the microscope (due to its effect on the calibration factor). This value averaged 3 + 2 nm (n = 5) when the focus was changed from a position well above to well below the fibre. The intensity of illumination was regularly adjusted between positions. A relatively large change in the incident intensity resulted in a small change in measured sarcomere length (10 + 4 nm, n = 4).
A concern related to making comparisons between tetani
70 K. BURTON, W. N. Z A G O T T A a n d R. J. BASKIN
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Fig. 1. (A) Sarcomere length records from the same posit ion for three tetani given over a 20 min period. (B) Tension during the per iod when sarcomere length data was acquired. Fibre length, 5.88 mm; i l luminated posit ion, I m m from the end; diameter, 65 ~rn; temperature , 18~
at different posi t ions was the repeatabil i ty of SL behaviour at one posit ion. The repeatabil i ty of the SL data during successive contractions was tested in 56 experiments in ten fibres with intervals be tween contractions varying from 5 min to 2 h 45 min. Each pair of data sets (two tetani in this case) generated an average value (over all the sweeps of the PDA) characterizing the difference for the entire contrac- tion. The mean difference for these repeats was 41 + 46 nm or 1.6 + 1.7% of the mean SL (n = 56). The median value was 29nm (1.1%), reflecting the skewed distribution. An example of good repeatabil i ty is given in Fig. 1. Al though not quantified, the repeatabil i ty of SL at rest was much better than that at the height of a tetanus. The repeatabil i ty of the central por t ions of the fibre was found to be better
than that of the terminal 5--15%. The above sources of uncertainty are included in Table 1.
Sarcomere length versus axial posi t ion was measured by translating the fibre through the beam. This was done by moving the stage so the beam moved along the fibre at approximately constant rate as judged by repeatabili ty of t he data and by watching the fibre movement through the microscope. The coefficient of variation (sD/mean) of resting SL along the central posi t ion of the fibres for 30 experiments averaged 1.1% and this value is in agreement with previous studies (Edman, 1966; Cleworth & Edman, 1972; Paolini et al., 1976). The sarcomere length variation along the fibre increased by 35 + 21% (n = 4) of the original value after a series of tetani. After three or four tetani, the
Table 1. Sources of error in SL measurement .
Uncertainty in SL Error (mean + sD) n Comments
(1) Electrical noise 4 + 1 nm (2) Calibration factor 15 - 24nm (3) Focus 3 + 2 nm (4) Incident intensi ty 10 + 4 nm (5) Left/right order
Difference at rest: 21 + 16 nm (6) and While active: 23 + 20 nm (7) Change in left/right difference
dur ing contraction 21 + 19 nm (8) Beam posi t ion
across fibre 27 + 14 nm 5 " 10nm
(9)
(lO)
Beam posi t ion along fibre: 27 + 38 nm Fibre movement : 74 + 65 ~m Repeatabil i ty 41 + 46 nm
1.6 + 1.7%
27 Twice average SD 17 SL = 2-3 ~m 5 4
103 Absolute values 96 Absolute values
91 Absolute values
7 Entirely across; Average movement
24 Median = 12 nm 24 Median = 53 ~m 56 Median = 29 nm
( 1 . 1 % )
Sarcomere length behaviour along fibres during tetani 71
variation generally remained stable and the sarcomere length behaviour reported below was more repeatable than for the first two or three contractions. A series of tetani was therefore generally elicited before making comparisons among separate contractions.
Results
Comparison of sarcomere length in the two first orders
Groups of sarcomeres of a defined sarcomere length can dominate the intensity of a given order if they are arranged into striations tilted so as to closely satisfy the 'Bragg' condition (Rudel & Zite-Ferenczy, 1979b; Lieber et al., 1984; Burton & Baskin, 1986). The error introduced in laser diffraction measurements of SL due to unequal sampling of sarcomere domains has been a source of concern (Rudel & Zite-Ferenczy, 1979a; Huxley, 1980; Lieber et al., 1984). In order to quantitate the Bragg artefact, experiments were done in which the two first orders were monitored simultaneously at rest and during tetani. In relaxed fibres, the absolute value of the difference between the left and right first orders averaged 21.1 + 16.3 nm (mean + SD) for 103 experiments from 11 fibres (SL, 2-3.4 ~m). For comparisons during tetani, a value for the difference in SL was obtained for each sweep of the PDA and an average of these values was produced for each contraction. The left/right order difference for active fibres averaged 22.6 + 20.0nm (n = 96, 7 fibres).
The accuracy of the measured SL change when a fibre contracts is affected by any change in the sampling of sarcomere domains due to the Bragg artefact. For each contraction, a comparison was made between the left/right order SL difference at rest and while active. The absolute value of the change in the left/fight difference averaged 21.1 + 19.9nm (n = 91, 7 fibres). If the changes in the SL difference were calculated as signed quantities (for example, left-right), the average was near zero. Although the SL measured from the two orders was usually different and there was a small change in this difference during a contraction, the general be- haviour of the two was similar in the majority of records. Fig. 2A-D shows typical records in which the two orders parallel each other. Examples of dissimilar records are given in Fig. 2E and F. The sudden changes observed at the end of some of the records are the result of relaxation.
Effect of fibre movement on measured sarcomere length
The accuracy of temporal comparisons of SL be- haviour during a contraction is affected by movement of the fibre in the beam. Movement perpendicular to the beam is most pronounced in the early stages of the fast rise in tension. The amount of lateral movement was variable, although usually less than
about 30 ~rn. For seven positions on two fibres at rest, the average variation in SL due to laterally moving the fibre completely across the beam was 27 + 14 nm.
In 24 experiments on three fibres, the effects of longitudinal movement along the axis of the fibre during 3.5s tetani were monitored by allowing the fibre to contract and then quickly (0.1-0.25) moving the stage so the position illuminated at rest was once again over the centre of the beam. The average change in SL due to movement was 27 _+ 38 nm (mean + SD; median = 12nm) resulting from movements of 74 + 65 ~m (median = 53 ~m). These sources of error and those from Bragg artefact are included in Table 1.
Total uncertainty in sarcomere length measurement
The sources of error listed in Table I can be combined to give a total uncertainty in measurement of sarcomere length. For relaxed fibres, items 1-5 combine to give an uncertainty at one point in time of approximately 53 nm at 2 ~m to 62 nm at 3 ~m SL. The values for active fibres are nearly the same: 55-64 nm (items 1-4 , 6). The error in comparing SL in active versus relaxed fibres results principally from fibre movement and changes resulting from Bragg artefact. Items I and 7-9 combine to give an uncertainty in the change of SL of about 60 nm (up to 79 nm for very large lateral movements). Item 4 is unchanged during the contraction and small changes in items 2 and 3 may add another 4 -5nm of error. Our results therefore suggest that measurements of SL changes can be made with an accuracy of greater than 0.1 ~m, in contrast to the suggestion of Morgan et al. (1982).
Sarcomere length versus position and fibre length
Sarcomere length behaviour of different regions of fibres was monitored during fixed-end tetani. The fibre to fibre variability resulted in the need to do many experiments and several examples of SL records are provided in the Figures. In fibres stretched beyond rest length, laser diffraction shows that the shorter sarcomeres at the extreme ends consistently shorten while the central sarcomeres lengthen (Fig. 3A, records i, iii), as observed by Lieber and Baskin (1983). Sarcomere length be- haviour was also observed in a 'transition zone' in which the sarcomeres were often nearly isometric after a period of transient SL change (Fig. 3A, record ii). An extreme example of the dichotomy between the centre and end regions is given in Fig. 3B, in which a nearly isometric transition zone is bounded by regions of significant shortening and lengthening.
Records such as those of Fig. 3 were obtained at several positions along fibres. Figure 4 gives exam- ples of families of curves from four fibres in which SL versus position is plotted at rest and twice during the
72 K. BURTON, W. N. Z A G O T T A a n d R. J. BASKIN
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Fig. 2. SL records s imultaneously acquired from both left and right first orders from experiments on six fibres. Fibre length, posit ion i l luminated, d iameter and temperature are for (A) 8.95 mm, 540 inn, 73 ~m, 4 ~ C, respectively; (B) 9.92 mm, 892 wn, 77 I~m, 4 ~ C; (C) 6.54 mm, 668 inn, 60 wn, 3 ~ C; (D) 5.07 mm, 2.5 mm, - , 5 ~ C; (E) 7.58 mm, 309 Inn, 65 pm, 3 ~ C; (F) 6.46 mm, 703 ~m, 65 pm, 5 ~ C. Longi tudinal translation was 40 i~m in (C), 20 Inn in (E) and 35 pm in (F). The sudden change of SL in (B) is due to relaxation.
Sarcomere length behaviour along fibres during tetani 73
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Fig. 3. SL records from two fibres illustrating the behaviour observed in the three general regions of stretched fibres. (A) i, end; ii, transition zone; and iii, central region (5.4, 9 and 27% from the tendon insertion, respectively). Fibre length, 7.37mm at 49 pm diameter; temperature, 17~ (B) 2.7, 26 and 65% positions. Fibre length 8.08mm at a diameter of 46 I~m. Tension is given for the period of SL measurement. Temperature, 15 ~ C.
course of a tetanus. The four fibres had been stretched to different lengths. The diffraction data clearly show a distinct decrease in sarcomere length towards the extreme ends when the fibre is stretched beyond rest length. The difference in SL between the central and end regions increases with stretch of the fibre. At rest length, the end sarcomeres may be longer than those of the central region. Figure 4A shows that when the sarcomere length along the fibre is at or just above rest length, there are shortening regions interspersed with stretching regions along the fibre. As the fibre is stretched, a pattern appears in which small shortening regions at the end give way to a large lengthening central region which generally comprises 70 to 85% (average = 75%) of the f i b r e .
A number of experiments concentrated principally on characterizing the terminal 15% of the fibre in which the largest gradients of sarcomere length are
found. Figure 5 shows records from the end and transition zones of four fibres stretched to different lengths. There was a smooth transition from shorten- ing to stretching regions. When a fibre was at rest length, the sarcomeres usually shortened during the fast rise of tension and then became relatively isometric for the remainder of the tetanus. At longer lengths, a decrease in the size of the end shortening region was often observed as the contraction progres- sed. This result took the form of regions which initially shortened quickly, but subsequently slowed or even showed a reversal of velocity (Fig. 5B; see also Fig. 4). Figure 6 gives data in which sets of records are taken from the same fibre stretched to different lengths. Three sets each of SL versus time records from two fibres are shown. As the fibre is stretched, the proportion of the fibre in which the sarcomeres shorten throughout the contraction decreases. Note that in the case of Fig. 6B, this fibre had a region
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K. BURTON, W. N. ZAGOTTA and R. J. BASKIN
3 ,0~
B 2.9
2 . 8 ~ -
2.7
2.6
2.5
2.4
2. . . . . i . . . . | . . . . | , a = . i . . . . i . . . . i . . . .
0 1 2 3 4 5 8 7
Position ( m r n )
s.2~C
23 I - 2 . 8 . =_ ', ~=
2 . 7
2.6 == (D
2.2
2.1 . . . . ' ' ' ' ' ' . . . . ' . . . . ' . . . . ' . . . . ' . . . . ~ ' ' ~ 0 I 2 3 4 5 6 7 8
Position (ram)
3., O 3.2
3.0
2.8
2 . 6
2.4
2.2
2.0 0 1 2 3 4 5 6 7 8
Position (mm)
Fig . 4. Data from four fibres over a range of sarcomere lengths. SL is plotted for various positions along the fibre at rest (triangles), near the end of the fast tension rise (except for (C)) (squares), and at the end of stimulation (diamonds). The data from each position was acquired from a separate tetanus. The times are 0, 100, 400 ms from (A); 0, 150, 400 ms for (B); 0, 500, 1000 ms for (C); 0, 200, 400 ms for ([3). In (D), the points at the end of the fibre are not given for time = 400 ms due to a loss of intensity. Fibre length, diameter and temperature are in (A) 7.05 mm, 75 tam, 13 ~ C, respectively; (B) 7.37 mm, 49 pan, 17 ~ C; (C) 7.68 ram, 94 I~m, 16 ~ C; (D) 8.08 ram, 46 I~m, 15 ~ C. In (D), the records from the 0.146, 4.6, & 6.69 mm positions are shown in Fig. 3B.
which shor t ened away f rom the end as well as at the end, the two being separa ted by a segment in which the sarcomeres l eng thened . In panel C of Fig. 6B, the st imulus trains were of different durat ions and the relaxation is accompanied by sudden changes in SL ( lengthening at the end and shor ten ing in the centre). Even w h e n tetani were carried out to as much as 5 s,
there were always regions in which shor tening or lengthening occurred t h roughou t the entire te tanus in fibres s t re tched b e y o n d rest length.
For fibres at m e d i u m sarcomere lengths in which we obta ined data f rom bo th the central and end regions, the average ext reme SL changes were for the central region: 0.10 + 0 .065~m (increase) at a
Fig. 5. Sarcomere length records from four fibres stretched to various sarcomere lengths. (A) Fibre at rest length. Positions along fibre (top to bottom records): 3.4, 18, 46, 20 and 3.7%. Fibre length and diameter were 5.4 mm and 73 ~m. (B), (C), (D) The records for each fibre were taken from the end and transition zones with one record from the centre in (B). The relative positions on the fibre and the fibre length and diameter are from the end region (bottom) at 600 ms: (B) 5.7, 7.9, 10.2, 12.8, 14.9, 19,9 and 50%, 6.54mm and 75 I~m; (C) 2, 7, 11, 14 and 17%, 8.12mm and 53 I~m; (D) 1.6, 6, 9.4, 12.9, 15.7 and 18.4% , 10.28 mm and 56 Ixm. The straight line in (C) from the 7% position indicates diffraction intensity was below background. (E), (F), (G), (H) Tension records corresponding to (A)--(D), respectively. Temperatures: (A) 15 ~ C, (B) 3 ~ C, (C) 3 ~ C, (D) 2.5 ~ C.
Sarcomere length behaviour along fibres during tetani 75
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Fig
. 6.
SL
rec
ords
of
the
type
giv
en i
n Fi
g. 5
. (A
) an
d (B
), se
ts o
f re
cord
s fr
om t
wo
fibr
es,
each
at
thre
e di
ffer
ent
leng
ths.
Eac
h po
siti
on i
s in
dica
ted
by t
he
sam
e ty
pe o
f gr
aph
(sol
id,
dott
ed,
and
so o
n) a
t ea
ch l
engt
h. (
A)
The
rel
ativ
e po
siti
ons
and
fibr
e le
ngth
s ar
e (b
otto
m t
o to
p re
cord
s at
600
ms)
1.4
, 2,
2.6
, 3.
3, 4
.6 a
nd 5
0% a
nd 7
.7,
8.1
and
10.3
mm
. D
iam
eter
, 64
~m
at
the
long
est
leng
th;
tem
pera
ture
, 5.
5 ~ C
. (B
) The
pos
itio
ns a
nd f
ibre
len
gths
are
2.4
, 5.
8, 1
7.3,
8.
7, 1
1.4
and
50%
and
7.5
7, 8
.95
and
9.92
mm
. D
iam
eter
, 67
~m
at t
he s
hort
est
leng
th.
At
the
long
est
leng
th,
the
5.8
and
50%
pos
itio
ns w
ere
not
mon
itor
ed
and
the
full
rela
xati
on w
as m
onit
ored
aft
er d
iffe
rent
sti
mul
us d
urat
ions
. T
his
fibr
e sh
owed
a r
egio
n (1
7.3%
) in
the
cen
tral
pos
itio
n w
hich
sho
rten
ed
agai
nst
the
ends
and
the
se r
ecor
ds a
re m
arke
d w
ith
a st
ar.
(C),
(D)
Ten
sion
rec
ords
for
(A
) an
d (B
). P
anel
3 o
f (D
) w
as a
cqui
red
duri
ng t
he e
xper
imen
t at
th
e 11
.4%
pos
itio
n. T
empe
ratu
re,
4~
78
A
o
A
E �9 �9
, - I o
r
0
~ 0 . 0
X
X
|
6 ~
g
oOo �9 mm �9 �9
m =% |@ = @
x x
x
x x x x x @ @
x x x x
x
x x x
I I I I I I I I I
t tO.O 120.0 t30.O 140.0 tSO.O Fibre length (% rest length)
I I
1~0.0
OJ A .
E ~
0 0 r-
===. === r~
.J
0 i
,h
w. A
J
8
- : 4. 4 . | 4. 4. | , 44.,1~,~,
4. 4. 4. 4.
14.4.1 I l 4.1 I I I I
| 4.
|
I I I
~ 0 . 0 ttO.O 120.0 t30.O t40.O 150.0 tSO.O 170.0 Fibre length (% rest length)
Fig. 7. Data from 19 fibres giving differences between the central and end regions. (A) Changes in sarcomere length during tetani for end (x) and centTal ([3) regions as a function of relative fibre length. Results are only given for cases in which data was acquired from both regions. (B) Sarcomere length difference between the central and end regions at rest (+) and during tetani (A) versus relative fibre length. In both (A) and (B) the circled points give the behaviour of one fibre.
position of 43 + 8% from the insertion of the fibre (SL = 2.85 + 0.17~m at rest); and for the end: 0.37 + 0.18~m (decrease) at a position of 3.8 + 3% (SL = 2.66 + 0.14~wn) (n = 22). Figure 7A shows a compilation from 21 fibres of sarcomere shortening in the end regions and sarcomere lengthening in the central region. These data are given as a function of fibre length relative to that at rest length. The scatter in the plot is due to a number of factors: the data is
K. BURTON, W. N. ZAGOTTA and R. J. BASKIN
drawn from many fibres, many of which were not stretched over the full length range, the relative position at the 'end' was not the same for every fibre; and the data points were taken at the end of SL records of variable duration. Nevertheless, the
increase in shortening of the end regions at longer fibre lengths is apparent. A small increase in the (small) magnitude of sarcomere lengthening in the central region is also apparent. Figure 7B shows the difference in SL between the centre and end regions at rest and while active as a function of relative fibre length. It is clear that the SL difference increases with fibre length at rest and during contraction. The sarcomere length difference in the two regions is much greater for the stimulated fibre.
Changes in sarcomere length and tension during a tetanus
A slowing or reversal of shortening was a regular feature of the SL records (see, for example, Fig. 6). The time at which the minimum in sarcomere length occurred was shorter in regions closer to the centre of the fibre (Fig. 8). An initial shoulder in the SL record occurred during the fast rise of tension in 57% of the contractions over the range of sarcomere lengths used here. However, the proportion was near 100% at rest length and decreased to 20-30% at long sarcomere lengths. The shoulder in the shortening records was never observed to occur after the slow rise of tension. A second shoulder in the SL record, either a slowing of shortening or of lengthening, was observed to occur in about 50% of the contractions. This second shoulder was not observed in the absence of creep at short sarcomere lengths.
Several records from the extreme ends of fibres were obtained which measured sarcomere length in the plateau region of the length-tension curve at the end of creep. At this time, within the measurement accuracy, the sarcomere length of these shortening regions was only rarely measured to be above the plateau of the length-tension curve (about 2.25 ~m) and was often observed to shorten well onto the plateau before the end of creep (Figs 6B and 8). The average SL was 2.1 + 0.1 ~m a t the end of creep. This region (at the centre of the laser beam) was on average 3.6% from the end of the fibre.
Although significant shortening could be observed in the end region, the intensity of the order at the extreme ends usually dropped before the end of creep to the level of background scatter. This is due to disorder of striations at the end of a stretched fibre during a tetanus (Huxley, 1980; Julian & Moss, 1980; Lieber & Baskin, 1983; Altringham & Bottinelli, 1985). This disorder has also been shown to make it difficult to measure striation spacing with the light micro- scope (Julian & Morgan, 1979b; Julian & Moss, 1980; Altringham & Pollack, 1984; also see Fig. 4 of Julian et al., 1978).
Sarcomere length behaviour along fibres during tetani 79
l :
== O E
z~0~ A
2,20
ZlO ~ . '%
0 20 40 60 80 100 120 140 ~60 180 200
Time Ires)
A
2,4 ~ " " ~ " ~ " . . . .
2.~ i ' ~ "
k
0 ~o ,~C 60 80 aO0 ~20 ~40 ~eO '~80 ~00
Time {ml~)
E
bO
z7 i" C 2.6 ~
2.5 ~
Z3
2,0 L ~ _ L L , . ~ o . . , . ~ . . . . . ~ .~ ~ _ , . ~ _ ~ L ~ . . ~ . r ~ x . c ~ z _ ~ _ ~ . , . ~ 0 20 40 $0 80 100 ~20 140 ~eO 180 200
Time (ms)
O E @
3.0~- D ~.9~ - 2.8~ 24 Z6~
2.0 L . ~ - ~ J ~ . ~ . . ~ ~ . ~.1. .~.~ ~ , .LL I ~ ~ ~-~..L~ a~. . |, L ~,.I ~ J . , ~ , ~ . J 0 20 40 60 80 I00 ~0 ~0 ~S0 ~80 :~00
Time (ms)
Fig. 8. Comparisons of SL and tension records from the end (bottom trace) and central (top trace) regions of a fibre stretched to four lengths. The records from the central region show a transient shortening at all but the longest fibre lengtl~ (D) and this rapid shortening ceases after the fast rise of tension. The stimulus record is shown above the tension data. (AI The end region shortens to an SL of 2 l~m and then becomes relatively isometric during the plateau of tension. (B) The en( region shortens over the entire 'plateau' of the length-tension curve and stops at just above 21~m SL. This is the time a which the central region ceases stretching and approximately coincides with the end of the slow rise in tension. The end c the period of shortening occurs somewhat later than for the shorter fibre. (C), (D) Shortening in the end region continue until the end of data acquisition and the SL is about 2.I Bm at the end of the slow tension rise. End and central regions at 5. and 23% from insertion, respectively. Fibre lengths: (A) 6,02 ram, (B) 6.61 mm, (C) 7.12mm, (D) 7.27mm. Diameter, 641~ in (A); temperature, 16 ~ C.
On a number of occasions, sarcomere length appeared to remain nearly constant while tension rose during creep. Sarcomere length and tension data from five fibres are given in Fig. 9. In all these cases, the measured SL was relatively isometric during the creep phase in which there was a substantial rise in tension. This behaviour frequently occurred in both first orders and such regions were usually located in the transition zone. These observations include those from regions which translated axially from 5 to 25 l~m, which was significantly less than the sampled portion of the fibre. In order to try and determine whether there was a significant increase in the sarcomere length distribution in the presence of little average change, diffraction order linewidth was monitored
and values calculated for sarcomere length dispe sion. Account was taken of the portion of the wid of the order due to the width of the laser beam a] the angular spread of the order resulting from t finite dimension of the illuminated portion of t fibre. As sarcomere length was nearly constant these regions, changes in calculated sarcomere len I dispersion very nearly followed changes in li width. After an initial change during the fast ris( tension, the SL dispersion often remained relati~ unchanged during and after the slow rise of ten,, (Fig. 9). In some records there was an increasJ dispersion during this period, but there were al like number of experiments which showed a decr (average = -0 .2%).
80 K. BURTON, W. N. ZAGOTTA and R. J. BASKIN
P e
|
E 8
2.72
2.70
2.68
2.66
2.64
2.62
2.60
2.58
2.56
2.54
2.52
A
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0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0
T i m e (ms)
O~
|
E O o
3.OOrB
, , , 2.95 ~ 'r ~ / %~,,,~, r..,~,~,~.:i r.,,,~.;;~,,~;~:,.,.,~ , ..~ ~ , ,.,, ~, i :'(!
29~ 2.85
. . . . . . . . 10.16 2.80 ~-" ..... "v'~"~'~'~"'~'~~""" ..... ~ m N
f,.-
2.75 / /
/
2.70 . . . . ~ . . . . ' . . . . ' . . . . ' . . . . ' . . . . , ~ i . . . . , . . . . , 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
T ime (ms)
2.60
2.55
2.50
i 2.45 2.40
2.35
2.30
2.25
i ~i~ C 2.95
2.90
' ' /;~ ~' " "'"~ t 2.85
. " - ' , ~ ' . " ' . - " : ' i ~ : ' , i ~ " ' ~ " ~ " . . . . ...... ...., ...................... �9 .L..' ~ .... ~ .,. ~ 2.80
0 . 1 9 |
F . ~ / m N i
( ~ 2.70
2.65
200 400 600 800 1000 1200 1 4 0 0 1 6 0 0 1 6 0 0
T i m e (ms)
D .~ ~ ~ ~, .',.,. t ; :~ ,
~ r,~, ,~''' ff "v' "'Y'v: | 4 , ~ D. FV ;,a,",/" V~
f , ~ . . , ~vT " . _ ~ . ~ . . ' " " m N
/ , = , 1 . . . . i . . . . J . . . . f . . . . i . . . . i . . . . I . . . . i . . . . r . . . . t
2'6%.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Time (ms)
2.95 E
2.85
26o_.
2.75
E . . . . . . . 1 0 . 2 3 mN O o f - " g~ 2.7o
2.65
2.60 . . . , . . . . , . . . . , . . . . ,~_L._..., . . . . , . . . . , . . . . , 0 200 400 600 800 1000 1200 1400 1600 1800
Time (ms)
Fig. 9. Sarcomere length dispersion (- - -, top trace), sarcomere length ( ..... , middle), and tension ( ...... bottom) records for regions of five fibres in which the SL is nearly constant during part or all of the creep phase of the tension record. In (A), the tension is indicated by a solid line between the other records. SL data from both the left and right first orders is given in (C) and was also observed in (D) and (E). Sarcomere velocity and time period of measurement: (A) - 1 nm s -1, 110-400 ms; (B) - 2 n m s -I, 120-350ms; (C) 5nm s -1, 140-750ms; (D) 7nm s -1, 100-1000ms; (E) 7nm s -1, 140-640ms. Fibre length, illuminated position, diameter and temperature are in (A) 6.62mm, 3.3mm, 801~m, 8~ (B) 7.13mm, 1.47mm, 62~m, 3.5~ (C) 6.54mm, 900~m, 70~m, 3~ (D) 6.81mm, 262~m, - , 5~ (E) 8.01mm, 622~m, 98~m, 5~ Longitudinal translation was 58 ~m in (B) (late inflection due to repositioning of fibre over beam), essentially zero in (C) and (D), and 23 I~m in (E).
Sarcomere length behaviour along fibres during tetani 81
We looked for evidence that the linewidth is sensitive to sarcomere length dispersion, As there is less striation order and hence less diffraction intensi- ty in the end regions than in the centre, we suspected that dispersion would be greater in the end regions. For the end, transition and central regions, values of SL dispersion from 14 fibres averaged 7.3, 3.6 and 2.4%, respectively. Using a paired comparisons t-test, the differences between the dispersion of the end and transition, and, end and central regions were highly significant (P ~ 0.001) and the difference between the transition and central regions slight (P < 0.1).
Discussion
Accuracy of sarcomere length measurements
Laser diffraction has been used to characterize the average sarcomere length along single muscle fibres over a range of fib.~ extensions at rest and during fixed-end tetani. Because the technique is commonly used to measure sarcomere length and because questions have been raised concerning its accuracy, several experiments were carried out to quantitate a variety of sources of uncertainty in these measure- ments of sarcomere length (Table 1).
The differences in sarcomere length obtained from measurements of the two first orders is a measure of 'Bragg artefact' (Rudel & Zite-Ferenczy, 1979b; Burton & Baskin, 1986). The possibility also exists that the difference in the positions of left and right orders could be due to a tilt of the fibre in the plane formed by the laser beam and the fibre axis (Leung, 1983; Lieber & Baskin, 1983). We feel this is unlikely because care was taken to ensure that the fibre was in focus along its entire length and thus parallel to the objective. As the laser was held in the condenser mount of the microscope and the beam illuminated the centre of the objective, the beam was perpendicu- lar to the objective and thus to the fibre. It could also be that there is a tilt of myofibrils within the fibre.
Bragg artefact is of particular concern because the measured centroid may not be representative of the mean SL of the illuminated portion of the fibre. This would result from certain groups of sarcomeres which dominate the diffraction intensity because they have SL and striation tilt close to satisfying the Bragg equation. These experiments were done to determine whether one diffraction order might initially be 'sampling' one sarcomere population during a con- traction and then switch to another of different SL. In this case, one would expect an apparent change in sarcomere length in one order which is not observed in the other, as reported by Rudel and Zite-Ferenczy (1979a). The majority of our records of left/right order data show that both orders behave similarly in that shortening, lengthening, or a change from shorten-
ing to lengthening in one first order was generally observed in the other. However, there were a few occasions in which there were significant changes in SL which were not seen in both orders. Sudden deviations observed in only one of the orders usually occurred during the fast rise of tension (this was the case in the one example provided by Rudel and Zite-Ferenczy, 1979a). Because of the artefact which can occur due to changes in striation tilt and/or SL, it is essential that conclusions about the sarcomere length behaviour be based on evidence from many experimental records, as in the present study. As populations of sarcomeres may be ,contributing to orders other than the +1st, the value of 21 nm for the variation in the 'sample mean' SL is probably an underestimate.
As discussed by Altringham et al. (1984), the i}lumination of new regions resulting from longitu- d~;.nal movement during the plateau of a tetanus is one of the more significant difficulties of the method; quantitation of changes in SL due to such movement is given in Table 1. Longitudinal movement could contribute, for example, to the apparent sarcomere lengthening regularly observed after a period of shortening. However, such reversal cannot be com- pletely accounted for by movement. For example, the record of Fig. 6A, middle fibre length, fourth record from the end (3.3%), was taken from a region which translated 50 ~tm. From data of the type shown in Fig. 4, such movement would result in a SL increase of about 5 nm at rest. If we assume that the SL gradient along the active fibre is about three times that at rest (Fig. 7B), then the increase in SL would have been about 0.015 ~tm. This is one third the lengthening observed in this region. The proportion of lengthen- ing accounted for by movement is still less in the records from this fibre at the longer length. It should be noted that although the SL gradient is greatest at the ends, the movement is less.
The present results suggest that monitoring one first order provides an accuracy of about 60 nm in the measurement of sarcomere length. However, con- siderable reduction in Bragg artefact can be provided by varying the parameters pertinent to the Bragg equation, that is the angle of diffraction (simul- taneously measuring left and right first, second, and so on, orders), the angle of incidence (Goldman & Simmons, 1984) or the wavelength (Goldman, 1987). Lateral movement during a contraction can be reduced to less than 151~m by careful mounting. Longitudinal movement is inevitable for fibres stretched beyond rest length. A possible solution would be to mask off a region of interest with foil markers so only this region is observed while it translates through a laser beam sufficiently wide to maintain constant illumination.
Laser diffraction measurement of SL does provide
82 K. BURTON, W. N. ZAGOTTA and R. J. BASKIN
certain advantages over the use of segment markers to measure changes in length (Edman & Reggiani, 1984). The laser beam illuminates the entire volume of the fibre and as such the diffraction order samples a large portion of this volume. Markers measure surface events and cannot detect the behaviour of sarcomeres and myofibrils within the fibre. Also, the position of the diffraction order provides a direct measure of sarcomere length changes, as opposed to changes in segment lengths which must be related to measurements of sarcomere length at rest. It is a simple matter to move the laser beam any given distance at will to measure SL. Small movements of the beam along the fibre provide a moving or 'boxcar' average of SL versus position. The above considera- tions lead u s to conclude that our measurements of sarcomere length are sufficiently accurate and sensi- tive to characterize SL behaviour along single fibres at rest and during contraction and we discuss these observations below.
Sarcomere length in fibres at rest and during tetani
The present investigation has used the same techni- que to monitor both shortening and lengthening regions in fibres which have been stretched over a wide range of sarcomere lengths (1.8-3.5 ~m). Our findings are in general agreement with previous studies using light microscopy (Huxley & Peachey, 1961; Carlsen et al., 1961; Julian et al., 1978; Julian & Morgan, 1979b; Altringham & Pollack, 1984). Our results also confirm observations using laser diffrac- tion at about 2.6~m (Lieber & Baskin, 1983) and recently over a wide range of fibre extensions (Altringham & Bottinelli, 1985). Regional movements similar to those reported here were reported in a recent extensive study using surface markers (Edman & Reggiani, 1984) which expanded on earlier marker studies (Gordon et al., 1966a; Julian et al., 1978; Julian & Morgan, 1979a).
Some general aspects of SL behaviour as a function of fibre extension can be described. At lengths where the fibre is slightly stretched (centre SL = 2.3-2.6 ~m), most of the regions in the fibre shorten during the fast rise in tension (Figs 5 and 6). The end regions continue to shorten, although often with diminishing velocity, throughout the creep and plateau phases of the tension record. The central regions tend to stretch slightly during this period. At medium sarcomere lengths (2.6-3 ~n), there is a greater shortening at the ends and stretching of the centre (Fig. 7A). This behaviour is commensurate with the difference in SL between the two regions being greater at rest in stretched fibres (Fig. 7B). Regions near the 'transition zone' (Lieber & Baskin, 1983) which shortened continuously at shorter fibre lengths now often lengthen after an initial shortening (Fig. 6). This change is expected from the observation that the
relative size of the short SL end region decreases with stretch. The smaller size of the shortening region can also account for a constant rate of shortening during a period when the lengthening of the central region is slowing. At long sarcomere lengths (>3 ~m), only a very small region at the end shortens during the tetanus.
Sarcomere length at the end of creep
Julian and Morgan (1979a) suggest that the end of creep and subsequent slow decline of tension would be expected to occur when sarcomeres reach the ascending limb of the length tension curve and begin losing tension-generating capability as they further shorten. This behaviour is also reflected in the model calculations of Morgan et al. (1982; their Figs 3-6). Julian et al. (1978) show one record in which the SL is 2.12~m at the end of creep. The slowing of shortening as the sarcomeres pass over the plateau of the length-tension curve could in this case account for the continued increase in tension during the later stages of creep. Edman and Reggiani (1984), on the other hand, show three records in which sarcomere length is estimated (from the length of a segment) to be about 2.2~'a at the end of creep and a similar value (2.16-2.18~m) also results from their model calculations. In their model, tension ceases to increase as the sarcomeres reach a length at which further shortening no longer increases their isometric tension generating capability~ The small decline in tension after the peak during a tetanus is then reflected in the decrease of the stretching velocity of the central segments, comensurate with the force- velocity relationship. The decline in tension in their model does seem to occur only after the SL of the end segment reaches the ascending limb of the length- tension curve (Edman & Reggiani, 1984; their Fig. 13).
Our data do not clearly agree with either of these two predictions. Cases were analysed for which the measured SL of the ends of fibres was on or near the plateau of the length-tension curve when creep ended. Sarcomere length at the ends of fibres was very rarely measured to be above the plateau at this time. The average value of SL was 2.1 ~m, reflecting the fact that this value ranged both over the entire plateau and below. The data does, therefore, seem to exclude the idea that the shortest sarcomeres are just reaching the plateau of the classic length-tension curve at the end of creep. In fact, since in most cases the shortest sarcomeres at the very ends of the fibre are not actually detected, the length of those sarcomeres may reach the ascending limb of the length-tension curve at the end of creep.
Time course of sarcomere shortening
In our records of sarcomere shortening at the ends of the fibres, the sarcomere length either steadily
Sarcomere length behaviour along fibres during tetani i' i
decreases or the !shortening slows with time. The models of Morgan et al. (1982) and Edman and Reggiani (1984), however, both predict a period of acceleration of shortening during a fixed-end tetanus. The model calculations of Morgan et al. show an initial period of slow shortening which is followed by an acceleration phase to rapid shortening and finally by slowing to isometric behaviour when the sarco- mere length has shortened to the ascending limb of the length-tension curve (SL about 1.6ttm). The behaviour occurs earliest in the end region and gives the impression that a shortening region moves from the end towards the centre of the fibre as the contraction proceeds. Our data do not reflect such a pattern of acceleration of sarcomere shortening. Rather, we observe a decrease in the size of the shortening region as the positions closer to the transition zone begin to shorten more slowly. There is a relatively smooth transition of sarcomere length and velocity from the end to the central positions.
Regions which remain "isometric" while tension changes
One observation which we find surprising is the occasional existence of regions which appear to be isometric while the tension rises considerably during creep. Although the low velocity of lengthening sometimes measured will result in a force elevated above the isometric level appropriate to those sarcomeres, it cannot account for the large change in tension at that velocity. As there is essentially no change in the mean sarcomere length, an increase in tension due to stretching of parallel elastic structures
83
will generally not result. It is conceivable that the laser is simply averaging over regions which are shortening and lengthening during creep. In order to test this, we measured the linewidth of these regions because this quantity is related to sarcomere length dispersion (Kawai & Kuntz, 1973; Paolini et al., 1976). We would expect dispersion to increase in these regions if the shorter sarcomeres were shortening and the longer sarcomeres lengthening. Although there is usually a transient change in linewidth during the fast tension rise, there is no consistent change during or after creep. The assumption that the linewidth is sensitive to differences in SL dispersion is supported by the measurement of significant differences in dispersion between the ends and other regions of the fibres. These measurements do not therefore support the idea of an increased distribution of sarcomere lengths which averages to zero change. However, given the uncertainties in the interpretation of the light diffraction pattern, this possibility cannot be completely excluded. Resolution of this question awaits more precise measurements of sarcomere length in these transition zones in the future.
Acknowledgements
We acknowledge the skilful fibre dissection services of Mrs Quan You Li. This work was supported by a National Science Foundation grant (PCM 83-00048) to R.J.B.
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