Isometric and isotonic contractions in airway smooth muscle1 N. L. STEPHENS AND W. VANNIEKERK Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV CHICAGO on 11/16/14 For personal use only.
Depmrtnaent of Plzysiology, University of Manirobu, Winrzipeg, Man., Cunada R3E OW3
Received November 15, 1976 STEPHENS,N. L., and VANNIEKERK.W. 1977. Isometric and isotonic contractions in airway smooth muscle. Can. J. Physiol. Pharmacol. 55, 833-838. Canine tracheal smooth muscle was used as an in vitro model of smooth muscle in intrapulmonary airways to determine whether active tension curves derived from isometric and isotonic muscles are similar, and thus resemble striated muscle in this respect. Isometric, isotonic after-loaded, and isotonic free-loaded contractions elicited at different lengths and loads, were analysed. The data demonstrate that length-tension (L-T) diagrams are diEerent in these various types of contractions for electrically and carbachol driven tracheal smooth muscles strips. In general, at any given length active tension is less in isotonic and free-loaded modes of contraction as compared with isometric. We conclude that the ability to actively develop tension at a given length in airway smooth muscle depends on the mode of contraction. STEPHENS,N. L. et VAN NIEKERK,W. 1977. Isometric and isotonic contractions in airway smooth muscle. Can. J. Physiol. Pharmacol. 55, 833-838. Nous avons utilisk le muscle lisse canin de trachke comrne modtle in vitro de muscle lisse des voies akriennes pour determiner si les courbes de tension actives dresskes B partir des expkriences sur les muscles isorn6triques et isotoniques sont semblables et ressemblent donc, sous ce rapport, aux muscles striCs. Les contractions isomCtriques et isotoniques aprks charge ou sans charge pour diffkrentes longueurs et differentes charges sont analyskes. Les risultants montrent que les diagrammes tension-longueur (L-T) sont diffkrents dans ces diffkrents types de contractions pour les bandes de muscle lisse stimulkes Clectriquement ou par le carbachol. En gknkral, a n'importe quelle longueur, la tension active est moins Blevke dans les contractions isotoniques et les contractions sans charge par rapport aux isomktriques. Nous en concluons que la capacitk de divelopper une tension active A une longueur donnke dans le muscle lisse des voies akriennes dkpend du made de contraction. [Traduit par le journal]
In vascular smooth muscle Peiper et al. (1 973) found that active L-T curves derived from isometric and free-loaded contractions were not identical. They did not study afterloaded contractions. Dobrin ( 1973 ), Peterson and Paul (1974), and Lowy and Mulvany (1973) have shown that in different types of smooth muscle L T curves derived from isometric and nonisometric contractions do not coincide. Uvelius ( 1976) has shown similar results in longitudinal smooth muscle from rabbit urinary bladder. In isolated frog skeletal muscle the curves have been reported by Abbot and Wilkie (1953) to be the same, though Rosenblueth et al. (1958) find they ABBREVIATIONS".-T, length-tension; Po, maximum isometric tetanic tcnsion. 'Supported by grants from the Medical Research Council of Canada and the Canadian Tuberculosis and Respiratory Disease Association.
differ in in situ skeletal muscles. While work by Taylor and Rude1 (1970), Schoenberg and Podalski (1 972), and Edman ( 1966) has done much to further our understanding of the L-T curves of skeletal muscles, it has not been directed at elucidating the cause of the differences in tension development by muscles contracting in different modes. Undoubtedly the mechanisms which they describe of inactivation or deactivation, at short muscle lengths, must be involved. Deleze ( 1961 ) also detects differences in isolated frog skeletal muscle. In cardiac muscle, (Taylor 1970), too, there are discrepancies between isotonic and isometric curves. Jewel1 (1977) in a recent review has discussed the various mechanisms involved in the influence exerted by muscle length on myocardial performance. Fabiato and Fabiato (1975), Krueger and Pollack (1975), and Julian and Sollins (1975) have shown how
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CAN. J. PHYSIOL. PHARMACOL. VOL. 55, 11977
length-dependent inactivation or deactivation mechanisms, or the development of internal loads, could influence the Frank-Starling law of the heart. Again, however, they do not address themselves specifically to the differences in &-T curves obtaincd from isometric and isotonic contractions. Thus there appear to be strong grounds for believing that similar differences may cxist in tracheal smooth muscle. For this reason E-T curves were derived from isometric and isotonic contractions in canine tracheal sstnooth muscle. The latter mode included both after-loaded and free-loaded contractions. Materials and Methods
rntmscle was set at I,, and on stimulation shortened to different Iengths as determined by the position of the stop. The distance shortened was recorded by the lower gauge as a change in tension which with the aid of the spring constant enabled computation of this distance. Once the shortening muscle was arrested by the stop, the upper gauge recorded the isornctric tension which subsequently developed. TO record isotonic free-loaded contractions the inmuscle was stretched to different rest Iengths by applying free Isads; it then shortened to different lengths depending upon the loads. The shortening was recorded by the quasi-isotonic springs. These loads were applied by simply stretching the spring out until it devclsped a tension equivalent to the desired Ioad. The spring has the additional advantage that it tends to keep the tension stable even though the muscle may lengthen. It must be noted that the amount of lengthening. even at the higher load, was less than 3% of the muscle length and it stabiIized very quickly. In any given experiment a muscIe randerwent contraction in one of the three isotonic tetanic modes described and a paired isometric tetanic mode. One thus obtained two curves on each muscle strip. A11 points on the two curves were obtained according to a strictly random procedure. Thus for example, elicitation of isometric tension at 1, may be followed by elicitation of an after-loaded contraction a t Isw Ioad and so on. In this way, when analysing the mean results s f a number of experiments, differences between the cumcs would not be ascribed to the fast that one curve wits elicited later in time (when the muscle had perhaps deteriorated) such as could occur if they were elicited in a nonrandom serial manner. All the isometric curves obtained from the different experiments were pooled to provide the mean isometric tetanic L-T ctlwes, after statistically testing to show that they were not different.
Mongrel dogs weighing 5 to 85 kg were anesthetized with an intravenous injection of 35 n ~ gpentobarbital/ kg body weight. The cervical part of the trachea was removed and rectangular strips of tracheal smooth muscle, I X 0.1 X 0.1 cm, were nlounted in a bath of continuously circulating Krebs-Ringer bicarbonate solution with a P O , of 6W mmWg, Pro, 40 rnmHg, pH 7.40, and temperature of 37°C as described by Stephens e6 fa/. ( 1969). Supramaximal electrical stirnulation was efTected from a 60-cycle AC source. The muscle was then tetanically stimulated for 10-s periods at 5-min intervals throughout the duration of the experiment. Experimental observations were fitted into this regime; muscle length and force were continuously monitored. This programme of regular stimulation yielded very stable muscle performance, the B, not changing by more than 15% over the course of 5 h. Experiments in which a change greater than this mcurred were rejected. After-loaded isotonic curves were obtained similar to those previously reported ( I 1) . Using a preload and a suitable stop the muscle was set at that length at In Fig. 1 are shown idealized tracings taken which isometric tension development is maximum and from an experiment, of isometric tetanic tenwhich is termed 1,; then various after loads were applied. On stimulation the muscle first devel~ped tension isometrically tantil a tension equivalent to the total applied load was developed; thereafter it short9 ened isotonically. The shortening was measured by a IWMFTRlC TRACE AT lo 6 highly compliant quasi-isotonic spring with a linear P,,=amg compliance of 1 cm/g over a 20-cm range. Over this range the spring displayed no hysteresis. Inertia effects were ignored because of the small masses of the springs and the low contractile velocity of the muscle. The inherent elasticity of the spring prevented any contraction from bcing truly isotonic. However, even for 10 the greatest shortenings, deviations from isotonic were rnrn less than 5%. After-loaded curves were also elicited at lSOTBWlC T R X E 6 several lengths other than I,. FREE- L W = 5 0 b In the 'reverse' procedure the contraction pathway TIME, 8 was the reverse of that just described. In this procedure FIG.1. Isometric and isotonic tetanic tension traces. the muscle was mounted, with a suitable system of stops between the two Grass FT.03 force gauges, whose HP, is the load which equals the resting tension of the compliances were 1 p m per gram applied force. The muscle at lo.
835
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STEPHENS AND VAN NIEKERK
sion as a function of time alone and of isotonic shortening versus time under two different free loads, the lighter being 4% of Po and the heavier 50% of Po. As a rule we measure Po at the point shown in thc uppermost trace, since thereafter, tension falls. The contraction time was 5 s. In shortening however the length change is not completed until after 12 to 16 s, i.e., the contraction time is considerably longer. Based a n the way we measure tension developed and distance shortened, it is evident that effector substance depletion could effect the Batter mare, and this was borne in mind in evaluating our results and designing experiments. In studying isotonic after-loaded and freeloaded contractions in any given muscle, isometric curves were always elicited. Cuwe 4 of Fig. 2 was obtained by pooling the active tension isometric curves so obtained. The justification for pooling was that statistical analysis demonstrated these cun7eswere not significantly different. In Fig. 2, mean isotonic free-loaded L-T curves are also shown. The
LENGTH
resting tension, active tension and total tension curves are labelled 1, 2, and 3. It can be seen that the two active tension curves (Nos. 2 and 4) are similar in general configuration, but Po in the free loaded is much less than in the isometric. In the isotonic curves, shortening is much greater at lower loads (up to 20% P o ) , which is not unexpected. Thereafter, it decreases rapidly. For the isometric curve the derived decrease in shortening is less. For skeletal muscle there is said to be almost no decrease in distance shortened until a free load approaching P , is applied (No. 4). In Fig. 3 are shown the results of afterloaded experiments carried out at different initial lengths. These curves strongly resemble those of Rasenblueth et al. ( 1 958) for twitches of cat soleus, wherein after-loaded curves were obtained with the muscle held at different lengths by different preloads as in our experimental protocol. The differences they saw between the is~metricand isot~nicafter-loaded curves are the same as we see. The curves elicited at 100% lo, 100%~lo, and 120% 6, appear to be nearly parallel to each other and
- % lo
FIG.2. Relationship of initial muscle length to shortening in free-loaded isotonic tetanic contractions at different given loads. The active tension curve ( 2 ) is derived by subtracting resting tension (curve 1 ) from total tension (curve 3 ) . Curves 1, 2, and 3 represent mean curves ( N = 12) for isotonic free-loaded experiments. Curve 1 is the resting tension curve, curve 2 the active tension, and curve 3 the total tension. Curve 4 is the mean active tension (N = 28) obtained from all the isometric experiments conducted. Vertical standard error bars are shown only for the two active tension curves ( 3 and 4 ) . The horizontal lines running from c~irve1 to curve 3 represent the shortening (in millimetres) the muscle is capable of at the various after loads shown. The loads range from 10% Po to 120% Po.
LENGTH
- % 10
FIG,3. Mean length - total tension curves. The isometric (curve 1 ) and the isotonic after-loaded (curve 4 ) at I , were obtained randomly as described in Materials and Methods. After-loaded curves were then obtained at 80, 90, 110, and 120% of I, (curves 2, 3, 5 , and 6 respectively), loads again being applied randomly. Whenever the after load imposed upon the muscle was less than the resting tension value for that length (as in curves at 110 and 120% of I,) an isotonic free-loaded situation developed, which accounts for the incompleteness of curves 5 and 6. Curve 1 was derived from seven experiments, curve 2 from four, curve 3 from four, curve 4 from seven, curve 5 from five, and curve 6 from one.
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CAW. J. PIWSIQL. BMARMACQL.
VOE.
55. 1977
LENGTH
FIG.4. Mean length - total tension curves involving different modes of contraction. Tensions shown in curves 1, 2, 3, and 4 have been obtained completely randomly. Curve 1 represents isotonic free-loaded experiments ( N = 12); curve 2 isotonic after-loaded ( N = 1 1 ) r, curve 3 'reverse9 after-loaded ( N = 51, and curve 4 isometric ( N = 2 8 ) .
so the difference between any two is almost the same. Mean total tension-versus-length curves for the isometric, the after-loaded, the 'reverseprocedure,' and the free-loaded preparations are shown in Fig. 4. At all lengths, the afterloaded (curve 2) and the so-called keverse' curve (curve 3 ) are found to be not significantly different. The f re-loaded curve differs from the after-loaded ones at lengths greater than 60% of lo. All comparisons were made using Duncan's new multiple range test (Steel and Torrie 1960) with a = 0.05. The test used the error mean square derived from a factorial analysis of variance with unequal replications per cell, applied to the raw data for a11 the four curves. One of the possible reasons for the differences between the isornetric and the isotonic curves could be that during an electrical stimulus, which stimulates the trachcaIis by releasing acetylcholine from the nerve endings in the tissue, there is a progressive diminution in the amount of available effector. Hence contractions with longer contraction times would be adversely affected. Stcphens and Kroeger (1970) have shown that atropine blocks 90% of the mechanical response to electrical stimulation. To test whether the differences seen between the isometric and isotonic curves were due to reduced availability of effector during electrical
- qb 4
FIG.5. Mean L-T curves for carbachol-treated muscles. Total tensions are a percentage of the maximum active tension obtained at 1, using carbachol. Asterisks ( * ) denote significant differences in mean tensions at 60-9096 lo for the two curves in which muscles were activated by carbachol. A paired t-test with n = 0.05 was used. Curves 1 and 2 ( N = 7 for each) were obtained by carbachol activation, curve 1 being isometric and curve 2 isotonic free loaded. Curve 3 ( N = 7 ) was obtained by electrical stimulation and was isometric. Short vertical bars represent standard errors.
stimulation of the isotonic contractions, the experiments were repeated using carbachol 10-" M as an agonist. In Fig. 5, carbachol-treated, mean isometric and isotonic after-loaded curves are shown. The after-loaded curve was elicited with the muscle at I,. Tensions, or distances shortened, were elicited after the application of carbachol. It took about 3 min before the final tension or length was achieved. The agonist was then washed out until tlae resting tension returned to normal, and then the muscle was stretched to a new rest length or a new after load was applied. The electrically elicited pooled isometric curve which was shown in Fig. 4 is shown here also. It can be seen that carbachol does stimulate the muscle more strongly as judged by the fact that the carbachol curves lie to the left of the electrical curve. However, between 45 % and 100% I,, the carbachol elicited isometric and isotonic after-loaded curves still show statisticalIy significant differences. The important consideration is that even the full stimulation obtained by supramaximal doses of carbachol does not abolish the difference between the two curves. Hence the difference between these two modes of contraction is not solely due to depletion of acetylcholine in the case of the
STEPHENS AND VAN NIEKERK
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after-loaded contraction. Below 4 5 % of 1, the difference between the curves is in the opposite direction.
Discussion Abbot and Wilkie (1953) have suggested that L-T curves in frog skeletal muscle, derived from isometric contractions or from isotonic after-loaded ones, coincide; on the other hand, Deleze ( 1961 ) Rnds differences between the two curves for cardiac muscle. Rosenblueth et al. ( 1958 ) elicited isometric and isotonic curves in cat lower limb muscles, in situ, and noted well-marked differences. Their explanation, based on quick-stretch experiments, is that in isotonic contraction the muscle does not contract fully. They further state that thc more time a muscle spends in an isotonic phase (for example in the second part of an after-loaded contraction) the more does it deviate from the isometric curve. Finally they show that asynchronous contractions of different parts of the muscle cannot account for the discrepancy between the curves. Our findings in tracheal smooth muscle are almost exactly like those of Rosenblueth et nl. (1958). Since we used platinum plate electrodes with a mass discharge it is unlikely that parts of the muscle contracted asynchronously. Though we have no experimental data to support it we feel that Rosenblueth's explanation of incomplete contraction in the isotonic mode correctly explains the difference between isometric and isotonic curves. The longer lasting isotonic contraction is more likely to be affected. Lowy and Mulvany (1973) found differences between isometric and isotonic after-loaded contractions. They feel this may be related to the "inability of the tnenia coli to sustain isotonic contraction." Although they do not say so it is possible the inability stems from a depletion of neurotransmitter. Depletion of acetylcholine thus seemed a good candidate, but the persistence of the statisticaIly significant difference in muscles treated with carbachol indicates that this is not the entire explanation. Whether distortion of fibres during isotonic shortening leads to impaired contraction needs to be studied. It does not appear to be the major factor in tracheal smooth muscle. In this muscle, which can super-contract, the maxi-
837
mum difference between the two types of curves should have been seen at the lowest lengths, whereas it seems to occur at intermediate lengths. Peterson and Paul (1974) found in bovine mesenteric vein that active shortening from lengths greater than 1, results in irreversible losses in contractility. They ascribe this to a possible failure of the actin and myosin Rlaments to interdigitate properly. Presumably, the greater the length from which a free-loaded contraction started, the more would be the discrepancy between the isometric and the isotonic contractions. Since our major deficit is at intermediate muscle lengths this could not be the entire explanation. There are tu70important factors to be taken into consideration in evaluating our findings. The first has to do with the implicit assumption that muscle length values are some index of average sarcomere values. Though sarcomeres still await discovery in smooth muscle, some evidence that they may exist has recently been adduced by Bagby and Pepe (1977). If the hypothetical sarcomere length varied much along the length of the fibre or from one fibre to another in a multicellular preparation, then it would be unwise to attempt any detailed analysis of L-T data. We have no data to answer this criticism. The second factor relates to the influence of injured tissues, e.g. at the knot region, on the L-T relationships. Uvelius (1976) has obtained evidence from the longitudinal muscle of the rabbit urinary bladder that while this may be a factor it does not account for the total amount of the difference between isometric and isotonic L-T curves. He did this by placing three small silver markers along the muscle bundle. He then analyzed L-T relationships for each of the four segments. While some segments were apparently stronger than others, nevertheless in all segments differences between the isometric and isotonic L-T curves persisted. Some explanation is called for to explain the biphasic relationship between the isometric and isotonic curves of Fig. 5. Between 45 and 100% of 1, the two curves elicited by carbachol show the expected relationship, the isometric curve lying to the left of the isotonic. However, below 45% the two curves diverge with the isometric curve now lying to the right of the isotonic. A
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CAN. J. PHYSIOL. PHARMACOL.
tentative explanation is that at these short lengths the smooth muscle is slack and for the isometric contraction is relatively thick. This may result in some diffusion limitation to carbachsl and hence complete stimulation of all the muscle fibres may not result. In the corresponding after-loaded isotonic contraction the muscle spends a considerable part of its contraction time at lengths in the neighbsurhood sf lo. During this time it takes up all the carbachol it needs and hence can continue contracting fully even when approaching shorter lengths. ABBOT,B. C., and WIEKIE.D. R. 1953. The relation between velocity of shortening and the tensionlength curve of skeletal muscle. J. Physiol. 120, 214223. BAGBY,R. M., and PEPE, I?. A. 1977. Striations in antimyosin-stained isolated adult smooth muscle cells. Fed PBOC.Fed. Am. Soc. Exp. Biol. 36, 602. DELEZE,B. B. 1961. The mechanical properties of the semitendinosus muscle at lengths greater than its length in the body. J. Physiol. (London), 158, 154164. DonRrW, P. B. 1973. Isometric and isobaric contraction of carotid arterial smooth muscle. Am. J. Physiol. 225(3), 659-663. EDMAN, K. A, P. 1966. The relation between sarcomere length and active tension in isolated semitendinosus fibres of the frog. J. Physiot. (London), 683, 40741 7. FABIATO, A., and FABIATO, F. 1975. Dependence of the contractile activation of skinned cardiac muscle cells on the sarcomere length. Nature (London), 256, 54-56. JEWELL,B. It. 1977. A reexamination of the influence of myocardial length on n~yocardialperformance. Circ. Res. 40, 221-230. JULIAN,I?. J., and SOLLINS,M. R. 1975. Sarcomere length-tension relations in living rat papillary muscle. Circ. Res. 37,299-388.
VOL. 55. 19'9%
KRUEGER, J. W., and POLLACK, G. H. 1975. Myocardial sarcomere dynamics during isometric contractions. J. Physiol. (London), 251,627-643. LOWY, J., and MULVANY,M. J. 1973. Mechanical properties sf guinca pig taenia coli mt~scles.Acta Physiol. Scand. 88. 123-136. PEIPER.U., SCHMIDT, E., LAVEN,R., and GRHEBEL, k. 1973. Length-tension relationships in resting and activated vascular smooth muscle fibres. Pfluegers Arch. 340, 113-1 22. PETERSON,J. W., and PAUL,R. J. 1974. Effects of initial length and active shortening on vascular smooth muscle contractility. Am. J. Physiol. 227(5), 1010-1024. ROSEWBLUETN, A., ALANIS,J., and R u n ~ o ,R. 1958. A comparative study of the isometric and isotonic contractions of striated muscles. Arch. Int. Physiol. Biochem. 66,330-353. SCHOENBERG, M., and PODQLSKY, I%. J. 1972. Lengthforce relation of calcium activated muscle fibres. Science, 6 76, 52-54. STEEL,It. G . D., and TORRIE,J. H. 1960. Analysis of variance I: The one-way classification. In Principles and procedures of statistics. McGraw-Hill, New York. pp. 99-13 1. STEPHENS, PI%. k., and MWOEGEW, E. A. 1970. Effect of hypoxia on airway smooth muscle mechanics and electrophysiology. J. AppI. Physiol. 28(5), 630-635. STEPHENS, N. L.. KROEGER, E. A., and MEHTA,J. A. 1969. Force-velocity characteristics of respiratory airway smooth muscle. J. Appl. Physiol. 26, 685692. TAYLOR, R. R. 1970. Active length-tension relations compared in isometric, after-loaded and isotonic contractions of cat papillary muscle. Circ, Rcs. 26, 279-288. TAYLOR, S, R., and R ~ B E LR. , 1970. Striated muscle fibres; inactivation of contraction induced by shortening. Science, 667, 882-884. UVELIUS,B. 1976. Isometric and isotonic length-tension relations and variations in cell length in longitudinal smooth muscle from rabbit urinary bladder. Acta Physiol. Scand. 97, 1-12.
Depmrtnaent of Plzysiology, University of Manirobu, Winrzipeg, Man., Cunada R3E OW3
Received November 15, 1976 STEPHENS,N. L., and VANNIEKERK.W. 1977. Isometric and isotonic contractions in airway smooth muscle. Can. J. Physiol. Pharmacol. 55, 833-838. Canine tracheal smooth muscle was used as an in vitro model of smooth muscle in intrapulmonary airways to determine whether active tension curves derived from isometric and isotonic muscles are similar, and thus resemble striated muscle in this respect. Isometric, isotonic after-loaded, and isotonic free-loaded contractions elicited at different lengths and loads, were analysed. The data demonstrate that length-tension (L-T) diagrams are diEerent in these various types of contractions for electrically and carbachol driven tracheal smooth muscles strips. In general, at any given length active tension is less in isotonic and free-loaded modes of contraction as compared with isometric. We conclude that the ability to actively develop tension at a given length in airway smooth muscle depends on the mode of contraction. STEPHENS,N. L. et VAN NIEKERK,W. 1977. Isometric and isotonic contractions in airway smooth muscle. Can. J. Physiol. Pharmacol. 55, 833-838. Nous avons utilisk le muscle lisse canin de trachke comrne modtle in vitro de muscle lisse des voies akriennes pour determiner si les courbes de tension actives dresskes B partir des expkriences sur les muscles isorn6triques et isotoniques sont semblables et ressemblent donc, sous ce rapport, aux muscles striCs. Les contractions isomCtriques et isotoniques aprks charge ou sans charge pour diffkrentes longueurs et differentes charges sont analyskes. Les risultants montrent que les diagrammes tension-longueur (L-T) sont diffkrents dans ces diffkrents types de contractions pour les bandes de muscle lisse stimulkes Clectriquement ou par le carbachol. En gknkral, a n'importe quelle longueur, la tension active est moins Blevke dans les contractions isotoniques et les contractions sans charge par rapport aux isomktriques. Nous en concluons que la capacitk de divelopper une tension active A une longueur donnke dans le muscle lisse des voies akriennes dkpend du made de contraction. [Traduit par le journal]
In vascular smooth muscle Peiper et al. (1 973) found that active L-T curves derived from isometric and free-loaded contractions were not identical. They did not study afterloaded contractions. Dobrin ( 1973 ), Peterson and Paul (1974), and Lowy and Mulvany (1973) have shown that in different types of smooth muscle L T curves derived from isometric and nonisometric contractions do not coincide. Uvelius ( 1976) has shown similar results in longitudinal smooth muscle from rabbit urinary bladder. In isolated frog skeletal muscle the curves have been reported by Abbot and Wilkie (1953) to be the same, though Rosenblueth et al. (1958) find they ABBREVIATIONS".-T, length-tension; Po, maximum isometric tetanic tcnsion. 'Supported by grants from the Medical Research Council of Canada and the Canadian Tuberculosis and Respiratory Disease Association.
differ in in situ skeletal muscles. While work by Taylor and Rude1 (1970), Schoenberg and Podalski (1 972), and Edman ( 1966) has done much to further our understanding of the L-T curves of skeletal muscles, it has not been directed at elucidating the cause of the differences in tension development by muscles contracting in different modes. Undoubtedly the mechanisms which they describe of inactivation or deactivation, at short muscle lengths, must be involved. Deleze ( 1961 ) also detects differences in isolated frog skeletal muscle. In cardiac muscle, (Taylor 1970), too, there are discrepancies between isotonic and isometric curves. Jewel1 (1977) in a recent review has discussed the various mechanisms involved in the influence exerted by muscle length on myocardial performance. Fabiato and Fabiato (1975), Krueger and Pollack (1975), and Julian and Sollins (1975) have shown how
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CAN. J. PHYSIOL. PHARMACOL. VOL. 55, 11977
length-dependent inactivation or deactivation mechanisms, or the development of internal loads, could influence the Frank-Starling law of the heart. Again, however, they do not address themselves specifically to the differences in &-T curves obtaincd from isometric and isotonic contractions. Thus there appear to be strong grounds for believing that similar differences may cxist in tracheal smooth muscle. For this reason E-T curves were derived from isometric and isotonic contractions in canine tracheal sstnooth muscle. The latter mode included both after-loaded and free-loaded contractions. Materials and Methods
rntmscle was set at I,, and on stimulation shortened to different Iengths as determined by the position of the stop. The distance shortened was recorded by the lower gauge as a change in tension which with the aid of the spring constant enabled computation of this distance. Once the shortening muscle was arrested by the stop, the upper gauge recorded the isornctric tension which subsequently developed. TO record isotonic free-loaded contractions the inmuscle was stretched to different rest Iengths by applying free Isads; it then shortened to different lengths depending upon the loads. The shortening was recorded by the quasi-isotonic springs. These loads were applied by simply stretching the spring out until it devclsped a tension equivalent to the desired Ioad. The spring has the additional advantage that it tends to keep the tension stable even though the muscle may lengthen. It must be noted that the amount of lengthening. even at the higher load, was less than 3% of the muscle length and it stabiIized very quickly. In any given experiment a muscIe randerwent contraction in one of the three isotonic tetanic modes described and a paired isometric tetanic mode. One thus obtained two curves on each muscle strip. A11 points on the two curves were obtained according to a strictly random procedure. Thus for example, elicitation of isometric tension at 1, may be followed by elicitation of an after-loaded contraction a t Isw Ioad and so on. In this way, when analysing the mean results s f a number of experiments, differences between the cumcs would not be ascribed to the fast that one curve wits elicited later in time (when the muscle had perhaps deteriorated) such as could occur if they were elicited in a nonrandom serial manner. All the isometric curves obtained from the different experiments were pooled to provide the mean isometric tetanic L-T ctlwes, after statistically testing to show that they were not different.
Mongrel dogs weighing 5 to 85 kg were anesthetized with an intravenous injection of 35 n ~ gpentobarbital/ kg body weight. The cervical part of the trachea was removed and rectangular strips of tracheal smooth muscle, I X 0.1 X 0.1 cm, were nlounted in a bath of continuously circulating Krebs-Ringer bicarbonate solution with a P O , of 6W mmWg, Pro, 40 rnmHg, pH 7.40, and temperature of 37°C as described by Stephens e6 fa/. ( 1969). Supramaximal electrical stirnulation was efTected from a 60-cycle AC source. The muscle was then tetanically stimulated for 10-s periods at 5-min intervals throughout the duration of the experiment. Experimental observations were fitted into this regime; muscle length and force were continuously monitored. This programme of regular stimulation yielded very stable muscle performance, the B, not changing by more than 15% over the course of 5 h. Experiments in which a change greater than this mcurred were rejected. After-loaded isotonic curves were obtained similar to those previously reported ( I 1) . Using a preload and a suitable stop the muscle was set at that length at In Fig. 1 are shown idealized tracings taken which isometric tension development is maximum and from an experiment, of isometric tetanic tenwhich is termed 1,; then various after loads were applied. On stimulation the muscle first devel~ped tension isometrically tantil a tension equivalent to the total applied load was developed; thereafter it short9 ened isotonically. The shortening was measured by a IWMFTRlC TRACE AT lo 6 highly compliant quasi-isotonic spring with a linear P,,=amg compliance of 1 cm/g over a 20-cm range. Over this range the spring displayed no hysteresis. Inertia effects were ignored because of the small masses of the springs and the low contractile velocity of the muscle. The inherent elasticity of the spring prevented any contraction from bcing truly isotonic. However, even for 10 the greatest shortenings, deviations from isotonic were rnrn less than 5%. After-loaded curves were also elicited at lSOTBWlC T R X E 6 several lengths other than I,. FREE- L W = 5 0 b In the 'reverse' procedure the contraction pathway TIME, 8 was the reverse of that just described. In this procedure FIG.1. Isometric and isotonic tetanic tension traces. the muscle was mounted, with a suitable system of stops between the two Grass FT.03 force gauges, whose HP, is the load which equals the resting tension of the compliances were 1 p m per gram applied force. The muscle at lo.
835
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STEPHENS AND VAN NIEKERK
sion as a function of time alone and of isotonic shortening versus time under two different free loads, the lighter being 4% of Po and the heavier 50% of Po. As a rule we measure Po at the point shown in thc uppermost trace, since thereafter, tension falls. The contraction time was 5 s. In shortening however the length change is not completed until after 12 to 16 s, i.e., the contraction time is considerably longer. Based a n the way we measure tension developed and distance shortened, it is evident that effector substance depletion could effect the Batter mare, and this was borne in mind in evaluating our results and designing experiments. In studying isotonic after-loaded and freeloaded contractions in any given muscle, isometric curves were always elicited. Cuwe 4 of Fig. 2 was obtained by pooling the active tension isometric curves so obtained. The justification for pooling was that statistical analysis demonstrated these cun7eswere not significantly different. In Fig. 2, mean isotonic free-loaded L-T curves are also shown. The
LENGTH
resting tension, active tension and total tension curves are labelled 1, 2, and 3. It can be seen that the two active tension curves (Nos. 2 and 4) are similar in general configuration, but Po in the free loaded is much less than in the isometric. In the isotonic curves, shortening is much greater at lower loads (up to 20% P o ) , which is not unexpected. Thereafter, it decreases rapidly. For the isometric curve the derived decrease in shortening is less. For skeletal muscle there is said to be almost no decrease in distance shortened until a free load approaching P , is applied (No. 4). In Fig. 3 are shown the results of afterloaded experiments carried out at different initial lengths. These curves strongly resemble those of Rasenblueth et al. ( 1 958) for twitches of cat soleus, wherein after-loaded curves were obtained with the muscle held at different lengths by different preloads as in our experimental protocol. The differences they saw between the is~metricand isot~nicafter-loaded curves are the same as we see. The curves elicited at 100% lo, 100%~lo, and 120% 6, appear to be nearly parallel to each other and
- % lo
FIG.2. Relationship of initial muscle length to shortening in free-loaded isotonic tetanic contractions at different given loads. The active tension curve ( 2 ) is derived by subtracting resting tension (curve 1 ) from total tension (curve 3 ) . Curves 1, 2, and 3 represent mean curves ( N = 12) for isotonic free-loaded experiments. Curve 1 is the resting tension curve, curve 2 the active tension, and curve 3 the total tension. Curve 4 is the mean active tension (N = 28) obtained from all the isometric experiments conducted. Vertical standard error bars are shown only for the two active tension curves ( 3 and 4 ) . The horizontal lines running from c~irve1 to curve 3 represent the shortening (in millimetres) the muscle is capable of at the various after loads shown. The loads range from 10% Po to 120% Po.
LENGTH
- % 10
FIG,3. Mean length - total tension curves. The isometric (curve 1 ) and the isotonic after-loaded (curve 4 ) at I , were obtained randomly as described in Materials and Methods. After-loaded curves were then obtained at 80, 90, 110, and 120% of I, (curves 2, 3, 5 , and 6 respectively), loads again being applied randomly. Whenever the after load imposed upon the muscle was less than the resting tension value for that length (as in curves at 110 and 120% of I,) an isotonic free-loaded situation developed, which accounts for the incompleteness of curves 5 and 6. Curve 1 was derived from seven experiments, curve 2 from four, curve 3 from four, curve 4 from seven, curve 5 from five, and curve 6 from one.
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55. 1977
LENGTH
FIG.4. Mean length - total tension curves involving different modes of contraction. Tensions shown in curves 1, 2, 3, and 4 have been obtained completely randomly. Curve 1 represents isotonic free-loaded experiments ( N = 12); curve 2 isotonic after-loaded ( N = 1 1 ) r, curve 3 'reverse9 after-loaded ( N = 51, and curve 4 isometric ( N = 2 8 ) .
so the difference between any two is almost the same. Mean total tension-versus-length curves for the isometric, the after-loaded, the 'reverseprocedure,' and the free-loaded preparations are shown in Fig. 4. At all lengths, the afterloaded (curve 2) and the so-called keverse' curve (curve 3 ) are found to be not significantly different. The f re-loaded curve differs from the after-loaded ones at lengths greater than 60% of lo. All comparisons were made using Duncan's new multiple range test (Steel and Torrie 1960) with a = 0.05. The test used the error mean square derived from a factorial analysis of variance with unequal replications per cell, applied to the raw data for a11 the four curves. One of the possible reasons for the differences between the isornetric and the isotonic curves could be that during an electrical stimulus, which stimulates the trachcaIis by releasing acetylcholine from the nerve endings in the tissue, there is a progressive diminution in the amount of available effector. Hence contractions with longer contraction times would be adversely affected. Stcphens and Kroeger (1970) have shown that atropine blocks 90% of the mechanical response to electrical stimulation. To test whether the differences seen between the isometric and isotonic curves were due to reduced availability of effector during electrical
- qb 4
FIG.5. Mean L-T curves for carbachol-treated muscles. Total tensions are a percentage of the maximum active tension obtained at 1, using carbachol. Asterisks ( * ) denote significant differences in mean tensions at 60-9096 lo for the two curves in which muscles were activated by carbachol. A paired t-test with n = 0.05 was used. Curves 1 and 2 ( N = 7 for each) were obtained by carbachol activation, curve 1 being isometric and curve 2 isotonic free loaded. Curve 3 ( N = 7 ) was obtained by electrical stimulation and was isometric. Short vertical bars represent standard errors.
stimulation of the isotonic contractions, the experiments were repeated using carbachol 10-" M as an agonist. In Fig. 5, carbachol-treated, mean isometric and isotonic after-loaded curves are shown. The after-loaded curve was elicited with the muscle at I,. Tensions, or distances shortened, were elicited after the application of carbachol. It took about 3 min before the final tension or length was achieved. The agonist was then washed out until tlae resting tension returned to normal, and then the muscle was stretched to a new rest length or a new after load was applied. The electrically elicited pooled isometric curve which was shown in Fig. 4 is shown here also. It can be seen that carbachol does stimulate the muscle more strongly as judged by the fact that the carbachol curves lie to the left of the electrical curve. However, between 45 % and 100% I,, the carbachol elicited isometric and isotonic after-loaded curves still show statisticalIy significant differences. The important consideration is that even the full stimulation obtained by supramaximal doses of carbachol does not abolish the difference between the two curves. Hence the difference between these two modes of contraction is not solely due to depletion of acetylcholine in the case of the
STEPHENS AND VAN NIEKERK
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after-loaded contraction. Below 4 5 % of 1, the difference between the curves is in the opposite direction.
Discussion Abbot and Wilkie (1953) have suggested that L-T curves in frog skeletal muscle, derived from isometric contractions or from isotonic after-loaded ones, coincide; on the other hand, Deleze ( 1961 ) Rnds differences between the two curves for cardiac muscle. Rosenblueth et al. ( 1958 ) elicited isometric and isotonic curves in cat lower limb muscles, in situ, and noted well-marked differences. Their explanation, based on quick-stretch experiments, is that in isotonic contraction the muscle does not contract fully. They further state that thc more time a muscle spends in an isotonic phase (for example in the second part of an after-loaded contraction) the more does it deviate from the isometric curve. Finally they show that asynchronous contractions of different parts of the muscle cannot account for the discrepancy between the curves. Our findings in tracheal smooth muscle are almost exactly like those of Rosenblueth et nl. (1958). Since we used platinum plate electrodes with a mass discharge it is unlikely that parts of the muscle contracted asynchronously. Though we have no experimental data to support it we feel that Rosenblueth's explanation of incomplete contraction in the isotonic mode correctly explains the difference between isometric and isotonic curves. The longer lasting isotonic contraction is more likely to be affected. Lowy and Mulvany (1973) found differences between isometric and isotonic after-loaded contractions. They feel this may be related to the "inability of the tnenia coli to sustain isotonic contraction." Although they do not say so it is possible the inability stems from a depletion of neurotransmitter. Depletion of acetylcholine thus seemed a good candidate, but the persistence of the statisticaIly significant difference in muscles treated with carbachol indicates that this is not the entire explanation. Whether distortion of fibres during isotonic shortening leads to impaired contraction needs to be studied. It does not appear to be the major factor in tracheal smooth muscle. In this muscle, which can super-contract, the maxi-
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mum difference between the two types of curves should have been seen at the lowest lengths, whereas it seems to occur at intermediate lengths. Peterson and Paul (1974) found in bovine mesenteric vein that active shortening from lengths greater than 1, results in irreversible losses in contractility. They ascribe this to a possible failure of the actin and myosin Rlaments to interdigitate properly. Presumably, the greater the length from which a free-loaded contraction started, the more would be the discrepancy between the isometric and the isotonic contractions. Since our major deficit is at intermediate muscle lengths this could not be the entire explanation. There are tu70important factors to be taken into consideration in evaluating our findings. The first has to do with the implicit assumption that muscle length values are some index of average sarcomere values. Though sarcomeres still await discovery in smooth muscle, some evidence that they may exist has recently been adduced by Bagby and Pepe (1977). If the hypothetical sarcomere length varied much along the length of the fibre or from one fibre to another in a multicellular preparation, then it would be unwise to attempt any detailed analysis of L-T data. We have no data to answer this criticism. The second factor relates to the influence of injured tissues, e.g. at the knot region, on the L-T relationships. Uvelius (1976) has obtained evidence from the longitudinal muscle of the rabbit urinary bladder that while this may be a factor it does not account for the total amount of the difference between isometric and isotonic L-T curves. He did this by placing three small silver markers along the muscle bundle. He then analyzed L-T relationships for each of the four segments. While some segments were apparently stronger than others, nevertheless in all segments differences between the isometric and isotonic L-T curves persisted. Some explanation is called for to explain the biphasic relationship between the isometric and isotonic curves of Fig. 5. Between 45 and 100% of 1, the two curves elicited by carbachol show the expected relationship, the isometric curve lying to the left of the isotonic. However, below 45% the two curves diverge with the isometric curve now lying to the right of the isotonic. A
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CAN. J. PHYSIOL. PHARMACOL.
tentative explanation is that at these short lengths the smooth muscle is slack and for the isometric contraction is relatively thick. This may result in some diffusion limitation to carbachsl and hence complete stimulation of all the muscle fibres may not result. In the corresponding after-loaded isotonic contraction the muscle spends a considerable part of its contraction time at lengths in the neighbsurhood sf lo. During this time it takes up all the carbachol it needs and hence can continue contracting fully even when approaching shorter lengths. ABBOT,B. C., and WIEKIE.D. R. 1953. The relation between velocity of shortening and the tensionlength curve of skeletal muscle. J. Physiol. 120, 214223. BAGBY,R. M., and PEPE, I?. A. 1977. Striations in antimyosin-stained isolated adult smooth muscle cells. Fed PBOC.Fed. Am. Soc. Exp. Biol. 36, 602. DELEZE,B. B. 1961. The mechanical properties of the semitendinosus muscle at lengths greater than its length in the body. J. Physiol. (London), 158, 154164. DonRrW, P. B. 1973. Isometric and isobaric contraction of carotid arterial smooth muscle. Am. J. Physiol. 225(3), 659-663. EDMAN, K. A, P. 1966. The relation between sarcomere length and active tension in isolated semitendinosus fibres of the frog. J. Physiot. (London), 683, 40741 7. FABIATO, A., and FABIATO, F. 1975. Dependence of the contractile activation of skinned cardiac muscle cells on the sarcomere length. Nature (London), 256, 54-56. JEWELL,B. It. 1977. A reexamination of the influence of myocardial length on n~yocardialperformance. Circ. Res. 40, 221-230. JULIAN,I?. J., and SOLLINS,M. R. 1975. Sarcomere length-tension relations in living rat papillary muscle. Circ. Res. 37,299-388.
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KRUEGER, J. W., and POLLACK, G. H. 1975. Myocardial sarcomere dynamics during isometric contractions. J. Physiol. (London), 251,627-643. LOWY, J., and MULVANY,M. J. 1973. Mechanical properties sf guinca pig taenia coli mt~scles.Acta Physiol. Scand. 88. 123-136. PEIPER.U., SCHMIDT, E., LAVEN,R., and GRHEBEL, k. 1973. Length-tension relationships in resting and activated vascular smooth muscle fibres. Pfluegers Arch. 340, 113-1 22. PETERSON,J. W., and PAUL,R. J. 1974. Effects of initial length and active shortening on vascular smooth muscle contractility. Am. J. Physiol. 227(5), 1010-1024. ROSEWBLUETN, A., ALANIS,J., and R u n ~ o ,R. 1958. A comparative study of the isometric and isotonic contractions of striated muscles. Arch. Int. Physiol. Biochem. 66,330-353. SCHOENBERG, M., and PODQLSKY, I%. J. 1972. Lengthforce relation of calcium activated muscle fibres. Science, 6 76, 52-54. STEEL,It. G . D., and TORRIE,J. H. 1960. Analysis of variance I: The one-way classification. In Principles and procedures of statistics. McGraw-Hill, New York. pp. 99-13 1. STEPHENS, PI%. k., and MWOEGEW, E. A. 1970. Effect of hypoxia on airway smooth muscle mechanics and electrophysiology. J. AppI. Physiol. 28(5), 630-635. STEPHENS, N. L.. KROEGER, E. A., and MEHTA,J. A. 1969. Force-velocity characteristics of respiratory airway smooth muscle. J. Appl. Physiol. 26, 685692. TAYLOR, R. R. 1970. Active length-tension relations compared in isometric, after-loaded and isotonic contractions of cat papillary muscle. Circ, Rcs. 26, 279-288. TAYLOR, S, R., and R ~ B E LR. , 1970. Striated muscle fibres; inactivation of contraction induced by shortening. Science, 667, 882-884. UVELIUS,B. 1976. Isometric and isotonic length-tension relations and variations in cell length in longitudinal smooth muscle from rabbit urinary bladder. Acta Physiol. Scand. 97, 1-12.
