Mechanical Control of the Rising Phase of Contraction of Frog Skeletal and Cardiac Muscle EMIL BOZLER From the Department of Physiology,Ohio State University, Columbus, Ohio 43210
ABSTRACT The effect of shortening on contractile activity was studied in experiments in which shortening during the rising phase of an isotonic contraction was suddenly stopped. At the same muscle length and the same time after stimulation the rise in tension was much faster, if preceded by shortening, than during an isometric contraction, demonstrating an increase in contractile activity. In this experiment the rate of tension rise determined in various phases of contraction was proportional to the rate of isotonic shortening at the same time after stimulation. Therefore, the time course of the isotonic rising phase could be derived from the tension rise after shortening. The rate of isotonic shortening was found to be unrelated to the tension generated at various lengths and to correspond closely to the activation process induced by shortening. The length response explains differences between isotonic and isometric contractions with regard to energy release (Fenn effect) and time relations. These results extend previous work which showed that shortening during later phases of a twitch prolongs, while lengthening abbreviates contraction. Thus the length responses, which have been called shortening activation and lengthening deactivation, control activity throughout an isotonic twitch. INTRODUCTION
In the frog heart a d i m i n u t i o n o f the load d u r i n g an isotonic twitch prolongs and an increase in load abbreviates, twitch (2). These o p p o s i n g effects have been called, respectively, s h o r t e n i n g activation a n d l e n g t h e n i n g deactivation. T h e same results were obtained for frog skeletal a n d m a m m a l i a n cardiac muscle (unpublished). However, mechanical interventions c h a n g e d the duration o f activity only if applied d u r i n g the late part o f the rising phase and d u r i n g relaxation. In the work presented here we e x a m i n e d the question o f whether changes in length influence contractile activity also d u r i n g the earlier part o f the twitch. For this p u r p o s e , s h o r t e n i n g in a contraction which started isotonically was suddenly s t o p p e d and the subsequent rise in tension was c o m p a r e d with that d u r i n g an isometric contraction at the same muscle length and the same time after stimulation. T h e rate o f tension rise was used as a measure o f the state o f activity o f the muscle. T h e s e a n d other experiments showed that s h o r t e n i n g increases not only the duration, but also the strength o f contractile activity. THE JOURNAL OF GENERAL PHYSIOLOGY ' VOLUME 7 0 , 1 9 7 7 " p a g e s 6 9 7 - 7 0 5
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This result is in s h a r p conflict with generally accepted views, according to which s h o r t e n i n g as well as l e n g t h e n i n g has a relaxing effect (see r e f e r e n c e 7, p. 172). This contention is based mainly on two tyl~q,s Of observations. It was f o u n d that after release d u r i n g the rising phase o f an isometric twitch, tension did not rise as high as d u r i n g an isometric control twitch at the s h o r t e r length (6, 8, 11), an effect that has been i n t e r p r e t e d as a w e a k e n i n g of contractile activity. H o w e v e r , it seems that a c o m p l e t e recovery o f tension could hardly be e x p e c t e d a f t e r the loss o f mechanical e n e r g y d u r i n g s h o r t e n i n g , because the muscle would have to m a k e u p for this loss at a time w h e n activity was diminishing in a n o r m a l twitch. Moreover, records o f this type o f e x p e r i m e n t (8, 12) show that the tension rise after the release was faster a n d larger than in a control twitch d u r i n g the s a m e phase o f the twitch, observations which a r g u e m o r e for activation t h a n the reverse. A n o t h e r a r g u m e n t for a relaxing action o f s h o r t e n i n g is based on the observation o f Jewell a n d Wilkie (10) that the isometric phase o f relaxation o f an a f t e r l o a d e d twitch starts earlier and is faster than d u r i n g an isometric twitch, the m o r e so, the larger the previous shortening. It has b e e n a s s u m e d (4-8, 11) that these effects are d u e to the previous shortening. H o w e v e r , it has b e e n shown by Jewell a n d Wilkie that acceleration o f relaxation is due not to s h o r t e n i n g but to the elongation o f the muscle d u r i n g the isotonic phase o f relaxation. T h e early b e g i n n i n g o f the isometric phase o f relaxation also is not due to the previous s h o r t e n i n g p e r se. It is fully e x p l a i n e d by the rapidity o f isotonic relaxation. This i n t e r p r e t a t i o n is c o n f i r m e d by the fact that the b e g i n n i n g is actually delayed with large preloads, as shown by g r a p h s o f previous a u t h o r s (4, 12). This is so because isometric relaxation then begins b e f o r e the rapid phase o f isotonic relaxation. As pointed out previously (2), the early onset and acceleration o f isometric relaxation d u r i n g a f t e r l o a d e d twitches can be explained as being due to l e n g t h e n i n g deactivation. T h e mistaken conclusion that these p h e n o m e n a show deactivation by s h o r t e n i n g is due to failure to recognize that transient length changes involve two changes in opposite directions, which have to be considered separately. In work on skeletal a n d cardiac muscle it has been shown that the two changes p r o d u c e s e p a r a t e a n d opposite responses which may partly cancel each o t h e r and that the direction o f the r e s p o n s e is determ i n e d by the last o f the length changes ( u n p u b l i s h e d observations). T h e r e f o r e , the effect o f transient s h o r t e n i n g is due not to s h o r t e n i n g but to l e n g t h e n i n g . MATERIALS
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METHODS
The sartorius of very small frogs and rings of the frog ventricle, dissected as described previously (1), were used. The muscles were attached vertically by a thin copper wire to a lever above the muscle. The lever was 7 cm long and was made of thin aluminum sheet. For stimulation Pt electrodes were used, the cathode being placed near the middle of the muscle for the sartorius, at one end for the ventricle. The lever was attached to a torque motor, as described previously (2). The motor was improved by diminishing the mass of the coil by more than 95%. The force acting on the muscle was monitored by recording the current through the torque motor. The force could be changed during a contraction by a digital pulse generator (W-P Instruments). Movements of the lever reached 90% deflection in 10 ms or less, depending on
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the state of the muscle. For recording movements a photoelectric transducer, or a displacement transducer based on absorption of Eddy current (Kaman Sciences Corp., Colorado Springs, Colo.), was used. Both types of transducers were highly linear. To record isometrically, a rod attached to a force transducer was placed below the muscle lever near the attachment of the muscle. The compliance of the system was 5/~m/g. For recording, a storage oscilloscope and a Polaroid camera were used. In the experiments reported the starting load or resting tension was varied between 5% and 30% of maximum twitch tension without changing the nature of the results. To obtain uniform twitches, supermaximal stimuli were applied twice a minute. Tetani were produced by stimulating for 0.3 s at a frequency of 25 Hz. Their magnitude usually dropped less than 1% in 10 min if they were repeated once a minute. The magnitude of the effects reported, except for the staircase, was very uniform. Each type of experiment was carried out with more than 10 preparations and repeated several times with each muscle. The physiological solution used contained in millimoles per liter: NaCI 113; KCI 2.5; CaCi2 1.5; Na-acetate 2; Na-phosphate buffer (pH 7.2)2. It was bubbled with 02. The temperature was I°C. RESULTS
Isotonic-Isometric Contractions Experiments designed to study the question o f whether a length c h a n g e d u r i n g the rising phase o f a contraction alters contractile activity are based on the following consideration. I f such an alteration is p r o d u c e d a n d if it persists after s h o r t e n i n g is stopped, the steepness o f tension rise should be different from that d u r i n g a purely isometric contraction, p r o v i d e d the c o m p a r i s o n is made at the same length and the same time after stimulation. T h e following e x p e r i m e n t was designed to study this question. A rod c o n n e c t e d to a force t r a n s d u c e r was placed below the lever a n d adjusted so that the load was fully s u p p o r t e d without stretching the muscle. An isometric contraction was then recorded. Before each o f the following contractions the load was increased, thereby e x t e n d i n g the muscle and allowing the muscle to shorten various distances before the contraction became isometric. S h o r t e n i n g always s t o p p e d at the same length. Changes in length and tension were r e c o r d e d . Fig. 1 clearly shows that the tension rise was steeper, if p r e c e d e d by shortening, than was the c o r r e s p o n d i n g phase o f an isometric contraction o f cardiac and skeletal muscle. In the graphs the rates o f tension rise with and without previous s h o r t e n i n g and isotonic s h o r t e n i n g were plotted against time. In some phases o f contraction the tension rise after s h o r t e n i n g was several times faster than d u r i n g an isometric contraction at the same length a n d the same time after stimulation. It is also i m p o r t a n t that the rate o f tension rise was increased d u r i n g the whole rising phase, showing that the activating effect o f s h o r t e n i n g had a long duration. T h e r e were characteristic differences, between different types o f muscles in the effects o f s t o p p i n g shortening. D u r i n g twitches o f the sartorius the rate o f tension rise after s h o r t e n i n g was the same as d u r i n g a large part o f the rising phase (Fig. 1 B). D u r i n g a tetanus the rate was constant at first, but later became larger (Fig. 1 D). I n the ventricle the rate first increased then decreased. T h e s e differences are significant because they are related to differences in the
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t i m e c o u r s e o f isotonic c o n t r a c t i o n . T h e first p a r t o f t h e r i s i n g p h a s e was Ss h a p e d i n t h e v e n t r i c l e . I n t h e s a r t o r i u s , s h o r t e n i n g b e c a m e l i n e a r 50 ms a f t e r the s t i m u l u s d u r i n g a twitch, b u t t h e l i n e a r r a n g e v a r i e d f r o m 50% to 80% o f total s h o r t e n i n g i n d i f f e r e n t m u s c l e s . D u r i n g r e p e t i t i v e s t i m u l a t i o n t h e slope o f the r i s i n g p h a s e was exactly t h e s a m e as d u r i n g a twitch a n d , at s m a l l loads, the
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FIGURE 1. Isotonic-isometric contractions in which the load varied but shortening stopped at the same length. In records A, C, and E several contractions were superimposed. Upper family of curves: length. T o p line is length at which shortening stopped. Lower family: tension developed after shortening stopped; on extreme left, a purely isometric contraction. A, twitches of sartorius; loads ranged from 80 to 700g force (gf) cm -2. C, brief tetani of sartorius, loads from 60 to 450 gf, cm -2. E, twitches of ventricle, loads from 0.6 to 4 g. Calibration for A and C on left, tension in 1 kgf-cm-2; for E, 5 gf. Calibration on right, length in millimeters. Graphs B, D, and F were obtained from records on left. Circles: rate of tension rise after shortening stopped. Triangles: rate of tension rise d u r i n g isometric contraction at the same time after stimulation. I n t e r r u p t e d line: isotonic shortening at intermediate load. Ordinate of graphs on left: rate of tension rise in kgf-cm-2.s -1 for B and D; gf.s -t for F; on right: length in mm. Time in seconds.
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linear range then was further extended. H o w e v e r , at large loads the rate o f shortening later accelerated until about 80% o f maximal shortening was reached, as illustrated in Figs. 1 and 2. T h e fact that the slope o f the rising phase is not increased by repetitive stimulation confirms the generally accepted view that the a m o u n t o f Ca released in skeletal muscle fibers saturates the contractile mechanism. This argues against the assumption that shortening activation is due to release o f Ca. mm
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FIGURE 2. Isotonic-isometric contractions in which load was constant and shortening was stopped at various lengths. A, sartorius; 11 twitches superimposed. Upper and lower half are records of length and tension, respectively. Tracings on left represent purely isotonic and isometric twitches. Calibration on left, tension in 1 kgf. cm-~; on right, length in mm. B, C, D: graphs obtained by plotting slope of tension rise after shortening various distances (circles), the ratio of this slope and that of isotonic contraction at the same times (triangles), and length (interrupted line) against time. B, based on A; C, brief tetani of sartorius; D, twitches of ventricle.
In the e x p e r i m e n t illustrated in Fig. 1 the comparison between the rate o f tension rise after shortening and the time course o f isotonic contraction was complicated ,by the fact that the load was different in successive twitches. T h e r e f o r e , in another type o f e x p e r i m e n t the rod attached to the force transducer was at first adjusted as in the previous experiment and an isometric contraction was recorded, but before each subsequent contraction the rod was lowered while the load remained u n c h a n g e d , so that again the muscle could
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shorten various distances b e f o r e the contraction became isometric (Fig. 2). T o d e t e r m i n e the relation between the rate o f tension rise after s h o r t e n i n g and the rate o f isotonic shortening, the ratio of these values was plotted against time in the graphs o f Fig. 2. Within the limits o f accuracy o f the m e a s u r e m e n t s this ratio was constant in the ventricle d u r i n g the whole, and in the sartorius d u r i n g most, o f the rising phase. T h u s the rate o f tension rise after s h o r t e n i n g was generally proportional to the previous rate o f shortening. In a n o t h e r modification o f these e x p e r i m e n t s , contractions were almost entirely isometric but isotonic shortening was p e r m i t t e d in various phases for a very short time. S h o r t e n i n g by less than 0.5% accelerated the subsequent rise in tension. T h e magnitude o f shortening activation can also be expressed quantitatively by adding the tension which would have been p r o d u c e d u n d e r isometric conditions d u r i n g the period o f shortening to the tension p r o d u c e d later after m o v e m e n t was stopped and c o m p a r i n g this sum with the tension d e v e l o p e d in an isometric contraction. This sum was 45-53% larger in twitches o f the sartorius, 21-30% larger in the ventricle, than the peak tension d u r i n g isometric twitches if d e t e r m i n e d shortly after the point o f inflexion o f the isometric twitch.
Effect of Passive Changes in Length By changing the load d u r i n g a twitch the effect o f s h o r t e n i n g as well as lengthening can be d e t e r m i n e d . In the e x p e r i m e n t illustrated in Fig. 3, three twitches o f the ventricle were superimposed: an experimental twitch d u r i n g which the load was increased or decreased, and twitches at the low and high loads. In this way the s h o r t e n i n g after the change in load could be c o m p a r e d with that d u r i n g a control at the same load d u r i n g the same time. After diminishing the load, s h o r t e n i n g was at first faster than d u r i n g the control with the lower load, but became later more nearly parallel. T h e a m o u n t o f shortening after the change in load was 15% larger than in the control at the same load and d u r i n g the same time. An increase in load had the opposite effect. T h e s e observations confirm that shortening increases contractile activity and show, f u r t h e r m o r e , that lengthening has the opposite effect. In addition to these effects, diminution of the load late d u r i n g the rising phase p r o l o n g e d , and increase o f the load d u r i n g this phase abbreviated the twitch as r e p o r t e d previously (2).
Staircase Ritchie and Wilkie (13) previously observed that the staircase effect in the sartorius is larger for auxotonic than isometric twitches. T h i s d i f f e r e n c e was still larger if isotonic and isometric twitches were c o m p a r e d . After a rest period o f 1 h the size and duration o f successive isotonic twitches sometimes increased m o r e than 50%, but the slope o f the rising phase r e m a i n e d exactly constant in the sartorius (Fig. 4). This is i m p o r t a n t because it shows that the increase in response was entirely due to a prolongation o f activity. In cardiac muscle, on the contrary, the rising phase became steeper u n d e r the same conditions. This difference u n d o u b t e d l y reflects the fact that d u r i n g the staircase internal Ca increases in the heart (14), not in the skeletal muscle (15).
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DISCUSSION
Role of Length Responses in Muscle Mechanics T h e results described above show that after a period o f s h o r t e n i n g u n d e r otherwise identical conditions, the rise o f tension is faster t h a n in a purely isometric contraction. This is not a m o m e n t a r y effect, but lasts d u r i n g the whole rising phase. Evidence has also been p r e s e n t e d that passive extension d u r i n g the rising phase o f an isotonic twitch slows s h o r t e n i n g a n d that passive
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FIGURE 3. Effect of passive length changes during rising phase of isotonic twitch of frog ventricle. In each record three twitches superimposed, an experimental twitch, during which load was changed from 1.2 to 0.8 g (left) and from 1.2 to 1.6 g (right), and control twitches at the small and large loads. Calibration: length in millimeters. Time marks every 0.1 s. Sortorius Frog v e n t r i c l e
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s h o r t e n i n g has the o p p o s i t e effect. T h e s e results are an extension o f previous studies, which have shown that passive s h o r t e n i n g d u r i n g later phases o f a twitch prolongs, a n d passive extension abbreviates, the isotonic twitch of cardiac a n d skeletal muscles ( r e f e r e n c e 2 a n d u n p u b l i s h e d observations). T h e s e responses, which have b e e n called, respectively, s h o r t e n i n g activation a n d l e n g t h e n i n g deactivation, evidently control mechanical activity t h r o u g h o u t the isotonic twitch. T h e length responses described explain the large differences in the time course o f the isotonic a n d isometric twitches. T h a t these differences are not simply d u e to physical conditions is evident f r o m the observations on the
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staircase effect described above, which show that activity during the rising phase lasts longer in the isotonic than in the isometric twitch. T h e prolongation of activity must be assumed to be caused by shortening activation, which acts as positive feedback during the rising phase. Lengthening deactivating similarly produces positive feedback during relaxation and therefore explains the rapidity of isotonic relaxation. T h e discovery of the length responses may clarify other aspects of muscle mechanics. Jewell and Wilkie (9) have calculated the rise in tension from the force-velocity and load-extension curves on the basis of a model in which the contractile elements are in series with an elastic element. It was found that the rise in tension during a tetanic contraction was much slower than predicted from the model. This discrepancy can be explained by shortening activation which comes into play in the measurement of shortening velocity. This factor also explains that the rise in tension is faster after release than at the beginning of the isometric contraction. T h e velocity of isotonic shortening and the rate o f tension rise after shortening directly depend on the strength of contractile activity. T h e r e f o r e , it seems reasonable to expect that these values, determined at the same time, are proportional to each other in different phases of a contraction. It has been shown above that this is true for most parts of the rising phase (Fig. 2). T h e r e f o r e , the time course of isotonic shortening can be determined by integration from tension records such as those in Fig. 2 A. Specifically, the observation that in the sartorius the rate o f tension rise after shortening is constant at the beginning of a twitch explains the linearity of shortening during this time. During tetani, shortening may accelerate after a linear start. This corresponds to the increase in the rate of tension rise after shortening in the second half of the rising phase (Fig. 2 C). Finally, in cardiac muscle this rate first rises, then falls, in agreement with the S shape o f the rising phase (Fig. 2 D). T h e linearity of the rising phase of the isotonic twitch of the sartorius raises an important question. T h e linear range may extend over 15% of muscle length during a twitch, and more than 20% during a tetanus. This range must be expected to include part of the ascending and descending branches of the active length-tension diagram, an assumption which has been verified experimentally. T h e r e f o r e , overlap of filaments is not important in the control of isotonic shortening; the most important factor evidently is shortening activation.
Significance of Length Responsesfor Energetics T he finding that at the same length and the same time after stimulation tension rises much faster in an isotonic-isometric contraction than during a purely isometric contraction can only signify that metabolic activity is increased by shortening. This conclusion agrees well with the energetics of contraction, specifically, the well-known difference in heat production between isotonic and isometric twitches and, more generally, with shortening heat. In fact, shortening activation and shortening heat should be considered to be two aspects of the same phenomenon.
Nature of Length Responses Length responses have been demonstrated most directly during contractures of
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c a r d i a c muscle (1, 3). U n d e r a p p r o p r i a t e c o n d i t i o n s s h o r t e n i n g activation a n d l e n g t h e n i n g d e a c t i v a t i o n can be o b s e r v e d u n d e r these c o n d i t i o n s , b u t slow r e s p o n s e s o f a d i f f e r e n t c h a r a c t e r can also b e o b t a i n e d . It is p a r t i c u l a r l y i m p o r t a n t t h a t e x t e n s i o n a n d s h o r t e n i n g always p r o d u c e o p p o s i t e effects as in n o r m a l muscles. T h i s r e s u l t , the delay in l e n g t h e n i n g d e a c t i v a t i o n , a n d the l o n g d u r a t i o n o f all t h e effects o b s e r v e d so far p r e c l u d e a s i m p l e m e c h a n i c a l e x p l a n a t i o n o f t h e l e n g t h r e s p o n s e s . It is also significant t h a t such r e s p o n s e s can be o b t a i n e d in muscles i m m e r s e d in isosmotic p o t a s s i u m solutions a n d , t h e r e f o r e , m u s t be a s s u m e d to be d u e to an i n t r a c e l l u l a r m e c h a n i s m . As p o i n t e d o u t a b o v e , it is unlikely t h a t t h e s h o r t e n i n g activation is d u e to release o f Ca. This work was supported by a grant from the Central Ohio Chapter of the American Heart Association.
Received for publication 1 April 1977. REFERENCES 1. BOZLER, E. 1972. Feedback in the contractile mechanism of the frog heart. J. Gen. Physiol. 60:239-247. 2. BOZLER, E. 1975. Mechanical control of the time-course of contraction of the frog heart. J. Gen. Physiol. 65:329-344. 3. BOZLER, E., and J. F. DZLAnAYES. 1973. Mechanical and electrical oscillations in cardiac muscle of the turtle. J. Gen. Physiol. 62:523-538. 4. BRADY, A. J. 1965. Time and displacement dependence of cardiac contractility: problems in defining the active state and force velocity relations. Fed. Proc. 24:1410-1420. 5. BRADY, A . J . 1972. Mechanics of the myocardium. In The Mammalian Myocardium. G. A. Langer and A. J. Brady, editors. John Wiley & Sons, New York. 6. BRXDEN, K. L., and N. R. ALPZRT. 1972. The effect of shortening on the time course of active state decay. J. Gen. Physiol. 60:202-220. 7. BRUTSAERT,D. L. 1974. The force velocity-length-time interaction of cardiac muscle. Physiological Basis of Starling's Law of the Heart. Ciba Foundation, London. 8. EDMAN, K. A. P. 1975. Mechanical deactivation induced by active shortening in isolated muscle fibers of the frog. J. Physiol. (Lond.). 246:255-275. 9..JEwzLL, B. R., and D. R. WILKIE. 1958. An analysis of the mechanical components in muscle. J. Physiol. (Lond.). 143:513-540. 10. JZWELL, B. R., and D. R. WILgIE. 1960. The mechanical properties of relaxing muscle. J. Physiol. (Lond.). 152:30-47. 11. JuuAN, F. J., and R. L. Moss. 1976. The concept of active state in striated muscle. Circ. Res. 38:53-59. 12. KAUF~tANN, R. L., R. M. BAYER, and C. HA~NASCH. 1972. Autoregulation of contractility in the myocardial cell. Pfluegers Arch. Eur. J. Physiol. 332:96-116. 13. RITCI-IIE,J. M., and D. R. WILKI~. 1955. The effect of previous stimulation on the active state of muscle. J. Physiol. (Lond.). 130:488-496. 14. SANDS,S. D., and S. WINECRAD. 1970. Treppe and total calcium content of the frog ventricle. Am. J. Physiol. 218:908-910. 15. WINEGRAD, S. 1970. The intracellular site of Ca activation of contraction in frog skeletal muscle.J. Gen. Physiol. 55:77-88.
ABSTRACT The effect of shortening on contractile activity was studied in experiments in which shortening during the rising phase of an isotonic contraction was suddenly stopped. At the same muscle length and the same time after stimulation the rise in tension was much faster, if preceded by shortening, than during an isometric contraction, demonstrating an increase in contractile activity. In this experiment the rate of tension rise determined in various phases of contraction was proportional to the rate of isotonic shortening at the same time after stimulation. Therefore, the time course of the isotonic rising phase could be derived from the tension rise after shortening. The rate of isotonic shortening was found to be unrelated to the tension generated at various lengths and to correspond closely to the activation process induced by shortening. The length response explains differences between isotonic and isometric contractions with regard to energy release (Fenn effect) and time relations. These results extend previous work which showed that shortening during later phases of a twitch prolongs, while lengthening abbreviates contraction. Thus the length responses, which have been called shortening activation and lengthening deactivation, control activity throughout an isotonic twitch. INTRODUCTION
In the frog heart a d i m i n u t i o n o f the load d u r i n g an isotonic twitch prolongs and an increase in load abbreviates, twitch (2). These o p p o s i n g effects have been called, respectively, s h o r t e n i n g activation a n d l e n g t h e n i n g deactivation. T h e same results were obtained for frog skeletal a n d m a m m a l i a n cardiac muscle (unpublished). However, mechanical interventions c h a n g e d the duration o f activity only if applied d u r i n g the late part o f the rising phase and d u r i n g relaxation. In the work presented here we e x a m i n e d the question o f whether changes in length influence contractile activity also d u r i n g the earlier part o f the twitch. For this p u r p o s e , s h o r t e n i n g in a contraction which started isotonically was suddenly s t o p p e d and the subsequent rise in tension was c o m p a r e d with that d u r i n g an isometric contraction at the same muscle length and the same time after stimulation. T h e rate o f tension rise was used as a measure o f the state o f activity o f the muscle. T h e s e a n d other experiments showed that s h o r t e n i n g increases not only the duration, but also the strength o f contractile activity. THE JOURNAL OF GENERAL PHYSIOLOGY ' VOLUME 7 0 , 1 9 7 7 " p a g e s 6 9 7 - 7 0 5
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This result is in s h a r p conflict with generally accepted views, according to which s h o r t e n i n g as well as l e n g t h e n i n g has a relaxing effect (see r e f e r e n c e 7, p. 172). This contention is based mainly on two tyl~q,s Of observations. It was f o u n d that after release d u r i n g the rising phase o f an isometric twitch, tension did not rise as high as d u r i n g an isometric control twitch at the s h o r t e r length (6, 8, 11), an effect that has been i n t e r p r e t e d as a w e a k e n i n g of contractile activity. H o w e v e r , it seems that a c o m p l e t e recovery o f tension could hardly be e x p e c t e d a f t e r the loss o f mechanical e n e r g y d u r i n g s h o r t e n i n g , because the muscle would have to m a k e u p for this loss at a time w h e n activity was diminishing in a n o r m a l twitch. Moreover, records o f this type o f e x p e r i m e n t (8, 12) show that the tension rise after the release was faster a n d larger than in a control twitch d u r i n g the s a m e phase o f the twitch, observations which a r g u e m o r e for activation t h a n the reverse. A n o t h e r a r g u m e n t for a relaxing action o f s h o r t e n i n g is based on the observation o f Jewell a n d Wilkie (10) that the isometric phase o f relaxation o f an a f t e r l o a d e d twitch starts earlier and is faster than d u r i n g an isometric twitch, the m o r e so, the larger the previous shortening. It has b e e n a s s u m e d (4-8, 11) that these effects are d u e to the previous shortening. H o w e v e r , it has b e e n shown by Jewell a n d Wilkie that acceleration o f relaxation is due not to s h o r t e n i n g but to the elongation o f the muscle d u r i n g the isotonic phase o f relaxation. T h e early b e g i n n i n g o f the isometric phase o f relaxation also is not due to the previous s h o r t e n i n g p e r se. It is fully e x p l a i n e d by the rapidity o f isotonic relaxation. This i n t e r p r e t a t i o n is c o n f i r m e d by the fact that the b e g i n n i n g is actually delayed with large preloads, as shown by g r a p h s o f previous a u t h o r s (4, 12). This is so because isometric relaxation then begins b e f o r e the rapid phase o f isotonic relaxation. As pointed out previously (2), the early onset and acceleration o f isometric relaxation d u r i n g a f t e r l o a d e d twitches can be explained as being due to l e n g t h e n i n g deactivation. T h e mistaken conclusion that these p h e n o m e n a show deactivation by s h o r t e n i n g is due to failure to recognize that transient length changes involve two changes in opposite directions, which have to be considered separately. In work on skeletal a n d cardiac muscle it has been shown that the two changes p r o d u c e s e p a r a t e a n d opposite responses which may partly cancel each o t h e r and that the direction o f the r e s p o n s e is determ i n e d by the last o f the length changes ( u n p u b l i s h e d observations). T h e r e f o r e , the effect o f transient s h o r t e n i n g is due not to s h o r t e n i n g but to l e n g t h e n i n g . MATERIALS
AND
METHODS
The sartorius of very small frogs and rings of the frog ventricle, dissected as described previously (1), were used. The muscles were attached vertically by a thin copper wire to a lever above the muscle. The lever was 7 cm long and was made of thin aluminum sheet. For stimulation Pt electrodes were used, the cathode being placed near the middle of the muscle for the sartorius, at one end for the ventricle. The lever was attached to a torque motor, as described previously (2). The motor was improved by diminishing the mass of the coil by more than 95%. The force acting on the muscle was monitored by recording the current through the torque motor. The force could be changed during a contraction by a digital pulse generator (W-P Instruments). Movements of the lever reached 90% deflection in 10 ms or less, depending on
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the state of the muscle. For recording movements a photoelectric transducer, or a displacement transducer based on absorption of Eddy current (Kaman Sciences Corp., Colorado Springs, Colo.), was used. Both types of transducers were highly linear. To record isometrically, a rod attached to a force transducer was placed below the muscle lever near the attachment of the muscle. The compliance of the system was 5/~m/g. For recording, a storage oscilloscope and a Polaroid camera were used. In the experiments reported the starting load or resting tension was varied between 5% and 30% of maximum twitch tension without changing the nature of the results. To obtain uniform twitches, supermaximal stimuli were applied twice a minute. Tetani were produced by stimulating for 0.3 s at a frequency of 25 Hz. Their magnitude usually dropped less than 1% in 10 min if they were repeated once a minute. The magnitude of the effects reported, except for the staircase, was very uniform. Each type of experiment was carried out with more than 10 preparations and repeated several times with each muscle. The physiological solution used contained in millimoles per liter: NaCI 113; KCI 2.5; CaCi2 1.5; Na-acetate 2; Na-phosphate buffer (pH 7.2)2. It was bubbled with 02. The temperature was I°C. RESULTS
Isotonic-Isometric Contractions Experiments designed to study the question o f whether a length c h a n g e d u r i n g the rising phase o f a contraction alters contractile activity are based on the following consideration. I f such an alteration is p r o d u c e d a n d if it persists after s h o r t e n i n g is stopped, the steepness o f tension rise should be different from that d u r i n g a purely isometric contraction, p r o v i d e d the c o m p a r i s o n is made at the same length and the same time after stimulation. T h e following e x p e r i m e n t was designed to study this question. A rod c o n n e c t e d to a force t r a n s d u c e r was placed below the lever a n d adjusted so that the load was fully s u p p o r t e d without stretching the muscle. An isometric contraction was then recorded. Before each o f the following contractions the load was increased, thereby e x t e n d i n g the muscle and allowing the muscle to shorten various distances before the contraction became isometric. S h o r t e n i n g always s t o p p e d at the same length. Changes in length and tension were r e c o r d e d . Fig. 1 clearly shows that the tension rise was steeper, if p r e c e d e d by shortening, than was the c o r r e s p o n d i n g phase o f an isometric contraction o f cardiac and skeletal muscle. In the graphs the rates o f tension rise with and without previous s h o r t e n i n g and isotonic s h o r t e n i n g were plotted against time. In some phases o f contraction the tension rise after s h o r t e n i n g was several times faster than d u r i n g an isometric contraction at the same length a n d the same time after stimulation. It is also i m p o r t a n t that the rate o f tension rise was increased d u r i n g the whole rising phase, showing that the activating effect o f s h o r t e n i n g had a long duration. T h e r e were characteristic differences, between different types o f muscles in the effects o f s t o p p i n g shortening. D u r i n g twitches o f the sartorius the rate o f tension rise after s h o r t e n i n g was the same as d u r i n g a large part o f the rising phase (Fig. 1 B). D u r i n g a tetanus the rate was constant at first, but later became larger (Fig. 1 D). I n the ventricle the rate first increased then decreased. T h e s e differences are significant because they are related to differences in the
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t i m e c o u r s e o f isotonic c o n t r a c t i o n . T h e first p a r t o f t h e r i s i n g p h a s e was Ss h a p e d i n t h e v e n t r i c l e . I n t h e s a r t o r i u s , s h o r t e n i n g b e c a m e l i n e a r 50 ms a f t e r the s t i m u l u s d u r i n g a twitch, b u t t h e l i n e a r r a n g e v a r i e d f r o m 50% to 80% o f total s h o r t e n i n g i n d i f f e r e n t m u s c l e s . D u r i n g r e p e t i t i v e s t i m u l a t i o n t h e slope o f the r i s i n g p h a s e was exactly t h e s a m e as d u r i n g a twitch a n d , at s m a l l loads, the
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FIGURE 1. Isotonic-isometric contractions in which the load varied but shortening stopped at the same length. In records A, C, and E several contractions were superimposed. Upper family of curves: length. T o p line is length at which shortening stopped. Lower family: tension developed after shortening stopped; on extreme left, a purely isometric contraction. A, twitches of sartorius; loads ranged from 80 to 700g force (gf) cm -2. C, brief tetani of sartorius, loads from 60 to 450 gf, cm -2. E, twitches of ventricle, loads from 0.6 to 4 g. Calibration for A and C on left, tension in 1 kgf-cm-2; for E, 5 gf. Calibration on right, length in millimeters. Graphs B, D, and F were obtained from records on left. Circles: rate of tension rise after shortening stopped. Triangles: rate of tension rise d u r i n g isometric contraction at the same time after stimulation. I n t e r r u p t e d line: isotonic shortening at intermediate load. Ordinate of graphs on left: rate of tension rise in kgf-cm-2.s -1 for B and D; gf.s -t for F; on right: length in mm. Time in seconds.
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linear range then was further extended. H o w e v e r , at large loads the rate o f shortening later accelerated until about 80% o f maximal shortening was reached, as illustrated in Figs. 1 and 2. T h e fact that the slope o f the rising phase is not increased by repetitive stimulation confirms the generally accepted view that the a m o u n t o f Ca released in skeletal muscle fibers saturates the contractile mechanism. This argues against the assumption that shortening activation is due to release o f Ca. mm
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FIGURE 2. Isotonic-isometric contractions in which load was constant and shortening was stopped at various lengths. A, sartorius; 11 twitches superimposed. Upper and lower half are records of length and tension, respectively. Tracings on left represent purely isotonic and isometric twitches. Calibration on left, tension in 1 kgf. cm-~; on right, length in mm. B, C, D: graphs obtained by plotting slope of tension rise after shortening various distances (circles), the ratio of this slope and that of isotonic contraction at the same times (triangles), and length (interrupted line) against time. B, based on A; C, brief tetani of sartorius; D, twitches of ventricle.
In the e x p e r i m e n t illustrated in Fig. 1 the comparison between the rate o f tension rise after shortening and the time course o f isotonic contraction was complicated ,by the fact that the load was different in successive twitches. T h e r e f o r e , in another type o f e x p e r i m e n t the rod attached to the force transducer was at first adjusted as in the previous experiment and an isometric contraction was recorded, but before each subsequent contraction the rod was lowered while the load remained u n c h a n g e d , so that again the muscle could
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shorten various distances b e f o r e the contraction became isometric (Fig. 2). T o d e t e r m i n e the relation between the rate o f tension rise after s h o r t e n i n g and the rate o f isotonic shortening, the ratio of these values was plotted against time in the graphs o f Fig. 2. Within the limits o f accuracy o f the m e a s u r e m e n t s this ratio was constant in the ventricle d u r i n g the whole, and in the sartorius d u r i n g most, o f the rising phase. T h u s the rate o f tension rise after s h o r t e n i n g was generally proportional to the previous rate o f shortening. In a n o t h e r modification o f these e x p e r i m e n t s , contractions were almost entirely isometric but isotonic shortening was p e r m i t t e d in various phases for a very short time. S h o r t e n i n g by less than 0.5% accelerated the subsequent rise in tension. T h e magnitude o f shortening activation can also be expressed quantitatively by adding the tension which would have been p r o d u c e d u n d e r isometric conditions d u r i n g the period o f shortening to the tension p r o d u c e d later after m o v e m e n t was stopped and c o m p a r i n g this sum with the tension d e v e l o p e d in an isometric contraction. This sum was 45-53% larger in twitches o f the sartorius, 21-30% larger in the ventricle, than the peak tension d u r i n g isometric twitches if d e t e r m i n e d shortly after the point o f inflexion o f the isometric twitch.
Effect of Passive Changes in Length By changing the load d u r i n g a twitch the effect o f s h o r t e n i n g as well as lengthening can be d e t e r m i n e d . In the e x p e r i m e n t illustrated in Fig. 3, three twitches o f the ventricle were superimposed: an experimental twitch d u r i n g which the load was increased or decreased, and twitches at the low and high loads. In this way the s h o r t e n i n g after the change in load could be c o m p a r e d with that d u r i n g a control at the same load d u r i n g the same time. After diminishing the load, s h o r t e n i n g was at first faster than d u r i n g the control with the lower load, but became later more nearly parallel. T h e a m o u n t o f shortening after the change in load was 15% larger than in the control at the same load and d u r i n g the same time. An increase in load had the opposite effect. T h e s e observations confirm that shortening increases contractile activity and show, f u r t h e r m o r e , that lengthening has the opposite effect. In addition to these effects, diminution of the load late d u r i n g the rising phase p r o l o n g e d , and increase o f the load d u r i n g this phase abbreviated the twitch as r e p o r t e d previously (2).
Staircase Ritchie and Wilkie (13) previously observed that the staircase effect in the sartorius is larger for auxotonic than isometric twitches. T h i s d i f f e r e n c e was still larger if isotonic and isometric twitches were c o m p a r e d . After a rest period o f 1 h the size and duration o f successive isotonic twitches sometimes increased m o r e than 50%, but the slope o f the rising phase r e m a i n e d exactly constant in the sartorius (Fig. 4). This is i m p o r t a n t because it shows that the increase in response was entirely due to a prolongation o f activity. In cardiac muscle, on the contrary, the rising phase became steeper u n d e r the same conditions. This difference u n d o u b t e d l y reflects the fact that d u r i n g the staircase internal Ca increases in the heart (14), not in the skeletal muscle (15).
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DISCUSSION
Role of Length Responses in Muscle Mechanics T h e results described above show that after a period o f s h o r t e n i n g u n d e r otherwise identical conditions, the rise o f tension is faster t h a n in a purely isometric contraction. This is not a m o m e n t a r y effect, but lasts d u r i n g the whole rising phase. Evidence has also been p r e s e n t e d that passive extension d u r i n g the rising phase o f an isotonic twitch slows s h o r t e n i n g a n d that passive
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FIGURE 3. Effect of passive length changes during rising phase of isotonic twitch of frog ventricle. In each record three twitches superimposed, an experimental twitch, during which load was changed from 1.2 to 0.8 g (left) and from 1.2 to 1.6 g (right), and control twitches at the small and large loads. Calibration: length in millimeters. Time marks every 0.1 s. Sortorius Frog v e n t r i c l e
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s h o r t e n i n g has the o p p o s i t e effect. T h e s e results are an extension o f previous studies, which have shown that passive s h o r t e n i n g d u r i n g later phases o f a twitch prolongs, a n d passive extension abbreviates, the isotonic twitch of cardiac a n d skeletal muscles ( r e f e r e n c e 2 a n d u n p u b l i s h e d observations). T h e s e responses, which have b e e n called, respectively, s h o r t e n i n g activation a n d l e n g t h e n i n g deactivation, evidently control mechanical activity t h r o u g h o u t the isotonic twitch. T h e length responses described explain the large differences in the time course o f the isotonic a n d isometric twitches. T h a t these differences are not simply d u e to physical conditions is evident f r o m the observations on the
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staircase effect described above, which show that activity during the rising phase lasts longer in the isotonic than in the isometric twitch. T h e prolongation of activity must be assumed to be caused by shortening activation, which acts as positive feedback during the rising phase. Lengthening deactivating similarly produces positive feedback during relaxation and therefore explains the rapidity of isotonic relaxation. T h e discovery of the length responses may clarify other aspects of muscle mechanics. Jewell and Wilkie (9) have calculated the rise in tension from the force-velocity and load-extension curves on the basis of a model in which the contractile elements are in series with an elastic element. It was found that the rise in tension during a tetanic contraction was much slower than predicted from the model. This discrepancy can be explained by shortening activation which comes into play in the measurement of shortening velocity. This factor also explains that the rise in tension is faster after release than at the beginning of the isometric contraction. T h e velocity of isotonic shortening and the rate o f tension rise after shortening directly depend on the strength of contractile activity. T h e r e f o r e , it seems reasonable to expect that these values, determined at the same time, are proportional to each other in different phases of a contraction. It has been shown above that this is true for most parts of the rising phase (Fig. 2). T h e r e f o r e , the time course of isotonic shortening can be determined by integration from tension records such as those in Fig. 2 A. Specifically, the observation that in the sartorius the rate o f tension rise after shortening is constant at the beginning of a twitch explains the linearity of shortening during this time. During tetani, shortening may accelerate after a linear start. This corresponds to the increase in the rate of tension rise after shortening in the second half of the rising phase (Fig. 2 C). Finally, in cardiac muscle this rate first rises, then falls, in agreement with the S shape o f the rising phase (Fig. 2 D). T h e linearity of the rising phase of the isotonic twitch of the sartorius raises an important question. T h e linear range may extend over 15% of muscle length during a twitch, and more than 20% during a tetanus. This range must be expected to include part of the ascending and descending branches of the active length-tension diagram, an assumption which has been verified experimentally. T h e r e f o r e , overlap of filaments is not important in the control of isotonic shortening; the most important factor evidently is shortening activation.
Significance of Length Responsesfor Energetics T he finding that at the same length and the same time after stimulation tension rises much faster in an isotonic-isometric contraction than during a purely isometric contraction can only signify that metabolic activity is increased by shortening. This conclusion agrees well with the energetics of contraction, specifically, the well-known difference in heat production between isotonic and isometric twitches and, more generally, with shortening heat. In fact, shortening activation and shortening heat should be considered to be two aspects of the same phenomenon.
Nature of Length Responses Length responses have been demonstrated most directly during contractures of
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c a r d i a c muscle (1, 3). U n d e r a p p r o p r i a t e c o n d i t i o n s s h o r t e n i n g activation a n d l e n g t h e n i n g d e a c t i v a t i o n can be o b s e r v e d u n d e r these c o n d i t i o n s , b u t slow r e s p o n s e s o f a d i f f e r e n t c h a r a c t e r can also b e o b t a i n e d . It is p a r t i c u l a r l y i m p o r t a n t t h a t e x t e n s i o n a n d s h o r t e n i n g always p r o d u c e o p p o s i t e effects as in n o r m a l muscles. T h i s r e s u l t , the delay in l e n g t h e n i n g d e a c t i v a t i o n , a n d the l o n g d u r a t i o n o f all t h e effects o b s e r v e d so far p r e c l u d e a s i m p l e m e c h a n i c a l e x p l a n a t i o n o f t h e l e n g t h r e s p o n s e s . It is also significant t h a t such r e s p o n s e s can be o b t a i n e d in muscles i m m e r s e d in isosmotic p o t a s s i u m solutions a n d , t h e r e f o r e , m u s t be a s s u m e d to be d u e to an i n t r a c e l l u l a r m e c h a n i s m . As p o i n t e d o u t a b o v e , it is unlikely t h a t t h e s h o r t e n i n g activation is d u e to release o f Ca. This work was supported by a grant from the Central Ohio Chapter of the American Heart Association.
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