J. Phyjsol. (1978), 283, pp. 469-480 With 4 text-figures Printed in Great Britain
469
RELAXATION OF VENTRICULAR CARDIAC MUSCLE
By DIRK L. BRUTSAERT, NORA M. DE CLERCK, MARC A. GOETHALS AD PHILIPPE R. HOUSMANS From the Department of Phy8iolog, University of Antwerp, Antwerp, Belgium
(Received 30 October 1977) SUrMARY
1. The load bearing capacity during relaxation of ventricular cardiac muscle from various animal species was investigated. 2. The effect of load on the time course of relaxation was analysed either by comparing afterloaded contractions against various loads or by imposing abrupt alterations in load (load clamps). 3. In heart muscle from the mammalian species studied relaxation was sensitive to loading conditions, whereas in frog heart muscle relaxation was largely independent of the loading conditions. The mechanical properties of relaxation of cardiac muscle appear, therefore, governed by the interplay of a load-controlled and an activationcontrolled decay mechanism, the relative importance of which differs with species. 4. Load-dependence may be the mechanical expression of the ratio of the number of force generating sites at any time during contraction and relaxation to the load to be carried; this mechanism would predominate in mammalian animal species with a well developed calcium sequestering sarcoplasmic reticulum. Activation-dependence would seem to predominate in animal species, such as frog, in which calcium sequestration appears to be the rate limiting step during relaxation. INTRODUCTION
Relaxation of cardiac muscle is a major determinant of diastolic filling, yet the mechanical properties of the relaxation phase are less well understood than those of the contraction phase. There exists general agreement that relaxation follows active removal of the sarcoplasmic calcium ions. However, the way in which the capacity to bear a load decays when cardiac muscle returns to its initial length or resting tension has hardly been studied. In frog skeletal muscle, this load bearing capacity during relaxation was analysed several years ago (Jewell & Wilkie, 1960). There have also been some studies of mechanical relaxation in cardiac muscle from cat (Parmley & Sonnenblick, 1969; Strauer, 1973; Strobeck, Bahler & Sonnenblick, 1975), dog (Tamiya, Kikkawa, Gunji, Hori & Sakurai, 1977), rat and guinea pig (Meerson & Kapelko, 1975) and frog (Goto, Kimoto, Saito & Wada, 1972). These studies however have given little insight into the intrinsic mechanisms underlying the decay of load bearing capacity. The present study was undertaken to investigate the decay of load bearing capacity of the myocardium in various animal species with known differences in excitation-contraction coupling mechanisms. From this comparative study we
D. L. BRUTSAERT AND OTHERS suggest that the mechanical properties of relaxation of cardiac muscle are governed by the interplay of activation-controlled and load-controlled decay mechanisms, the relative importance of which differs with species. 470
METHODS
Isolated preparations of ventricular myocardium from adult cat (n = 30), rat (n = 8), rabbit (n = 3), pig (n = 1) and frog (n = 15) were used for this study. Papillary muscles were removed from the right ventricles of cats and rabbits and from left ventricles of rats; one thin trabecula was obtained from the right ventricle of an adult pig; in frog, thin longitudinal strips were cut from the ventricle. The criteria used for selection of suitable preparations have been published previously for cat (Brutsaert & Housmans, 1977), rat (Henderson, Brutsaert, Parmley & Sonnenblick, 1969) and frog (Henderson & Brutsaert, 1974). The criteria for selection of the papillary muscles in rabbit were the same as in cat, although the ratio of resting to total force at I.. was always somewhat higher; the muscle characteristics of the pig trabecula at I.. were: length 7-0 mm, cross-sectional area 0-76 mm2, preload 9-8 mN and ratio of resting to total tension 0-28. The muscles were mounted vertically, the lower end being held by a force transducer (compliance 0-3 ,um/mN, resonant frequency 250 Hz in aqueous solution) and the upper tendinous end tied (7-0 braided thread, Deknatel, Surgical Tevdek, Code 103-T) to an electromagnetic lever system (compliance 0-2 1um/mN, equivalent moving mass 155 mg, step response 3 ms). The current through the coil of the electromagnet determined the load on the muscle and was controlled by a current source, which was calibrated for step changes of 0-98 mN and 9-8 mN and could be switched from one level to another by means of two reference voltage sources. A detailed description of force transducer, electromagnetic lever system and the response characteristics of each to abrupt alterations in load has been published previously (Claes & Brutsaert, 1971; Brutsaert & Claes, 1974). The methodological and physiological aspects of studying abrupt alterations of load (the 'load clamp' technique) have also been reported (Brutsaert & Claes, 1974; Brutsaert, 1974). For each contraction, length and force traces were recorded simultaneously and displayed as functions of time on a Storage Display Unit (Tektronix 61 1) and photographed with a Hard Copy Unit (Tektronix 4601). The bathing solution contained (mM): NaCl 118, KCl 4-7, MgSO4 . 7H20 1-2, KH2PO, 1-1, NaHCO3 24, CaCl2.6H20 2-5 and glucose 4-5. The solution was bubbled with a gas mixture of 95 % 02-5 % CO2 and the bath temperature was maintained at 29 'C. Homogenous electrical stimulation (12 beats/min) was obtained by rectangular pulses of 5 ms duration about 10 % above threshold through two platinum electrodes arranged longitudinally along the entire muscle. All experiments were performed with the initial muscle length set to l,, i.e. the length at which active force development was maximal. The muscles were allowed to equilibrate for 3 hr before the actual experiments began. In order to avoid the effects of the loading conditions present in preceding contractions, all test contractions were separated by a series of at least eight equally loaded standardized contractions (Parmley, Brutsaert & Sonnenblick, 1969; Kaufmann, Lab, Hennekes & Krause, 1971; Jewell & Rovell, 1973; Brutsaert, 1974; Brutsaert & Paulus, 1977). The results were shown to be qualitatively similar whatever load was used for the interpolated, standardized contractions if this remained constant throughout the experiment. The results were the same in all preparations from any of the animal species studied, and are illustrated by representative examples. RESULTS
1. Afterloaded contractions A muscle that is allowed to shorten during a twitch loses some of its ability to produce force during the remainder of the contraction. The extent of the deactivation associated with an isotonic contraction varied in different types of cardiac muscle, being most marked in cat, less pronounced in rat, and almost absent in frog. In cat papillary muscle (Fig. 1A) the records of relaxation at different loads were well separated in time; isotonic contractions were always over before the isometric
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Fig. 1. Length (upper) and force (lower) traces of a series of afterloaded isotonic contractions against various loads, i.e. from an isotonic twitch at preload only, then with different afterloads up to a full isometric twitch, in cat papillary muscle (panel A; muscle characteristics at
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Fig. 2. Length (upper) and force (lower) traces of a load clamp at three different times (contractions a, b and c) during a preloaded isotonic contraction in cat (panel A), frog (panel B) and rat (panel C). The control preloaded and afterloaded contractions are also shown. All test contractions were separated by a series of at least eight equally loaded standardized contractions. The characteristics of the three muscles are the same as in Fig. 1. In cat, load clamps which were imposed after the first two thirds of the contraction markedly abbreviated the total duration of the isotonic phase of the twitch (contractions b and c). In frog, the contractions were hardly affected by similar clamps. Again, the responses in rat were intermediate between cat and frog.
473 RELAXATION OF VENTRICULAR CARDIAC MUSCLE control beat; the ability to sustain 'supra-isometric' loads could be demonstrated only in heavily afterloaded contractions; and isotonic lengthening during relaxation, even at the smaller loads, was always followed by an initial abrupt fall of isometric force. Some of these properties, and also the exponential nature of the slow isometric
relaxation phase of afterloaded contractions have already been described for cat (Parmley & Sonnenblick, 1969), rabbit (Brady, 1965), dog (Tamiya et al. 1977) and frog (Goto et al. 1972) cardiac muscle, and for frog (Jewell & Wilkie, 1960), rat (Hill, 1972) and crayfish (Matsumara, 1972) skeletal muscle. By contrast, in frog myocardium the overall duration of all contractions was little affected by loading conditions (Fig. 1 B), with the isometric relaxation phases coinciding over their largest portion. The ability to sustain a load at 'supra-isometric' levels during isotonic lengthening was not limited to contractions at heavy afterloads but was present also at intermediate loads. The load-dependent changes in duration of contraction in rat ventricular cardiac muscle (Fig. 1 C) were intermediate between those seen in cat and those in frog heart muscle. 2. Load clamped contractions In Fig. 2 the load bearing capacity of cardiac muscle was tested at various times during isotonic contraction by imposing small additional loads. In cat (Fig. 2A), load clamps imposed during the first half of the shortening phase (contraction a), only slightly influenced the isotonic relaxation phase and the over all duration of the contraction. Load clamps of the same magnitude at later times (in the second half and especially the last third of the shortening phase) abbreviated the total duration of the isotonic phase of the twitch (contractions b and c). The initial rapid lengthening which accompanied the clamp step itself, was followed by a slower lengthening. Load clamps of the appropriate amplitude and timing were followed by a slower lengthening phase which occurred relatively slowly at first and then more quickly with a concomitant abbreviation of the contraction. In contrast, in frog cardiac muscle (Fig. 2B) relaxation was much less affected by similar load clamps. Immediately after the initial extension accompanying the abrupt increase in load, some delayed extension occurred in late or large clamps. The delayed extension did not consist of a first slow and subsequent fast phase, as typically occurred in cat ventricular myocardium (Housmans & Brutsaert, 1976; Brutsaert & Housmans, 1977); instead, it fused smoothly with the relaxation phase of the normal afterloaded control contraction and the overall duration of the contraction was hardly affected. Again, the mechanical properties of rat ventricular cardiac muscle under the same loading conditions were intermediate between those of cat and frog heart muscle but rather more like the cat. The same principle is illustrated in Fig. 3 where load clamps of increasing magnitude were superimposed at the same time near peak shortening, Again, cat myocardium (Fig. 3A) was markedly affected by these higher loads, rat myocardium (Fig. 3 C) less so, and frog myocardium (Fig. 3B) scarcely so at all except for very large clamp steps or if the experiment was repeated at still later times during relaxation (not shown). The load-dependence of cat and, to a smaller extent, of rat ventricular myocardium, in contrast to the relative load-independence of frog myocardium, was further tested by comparing the effects of loading and unloading steps of the same
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Fig. 3. Length (upper) and force (lower) traces of load clamped contractions with clamp steps of various magnitudes imposed near peak shortening of a preloaded isotonic contraction in cat (panel A), frog (panel B) and rat (panel C). The control preloaded isotonic and the control isometric contractions are also shown. Characteristics of cat papillary muscle at lnxax: length 10-5 mm, cross sectional area 0 49 mm2 and preload 4 9 mN; of frog ventricular strip at lax,: length 7-5 mm, cross sectional area 0-91 mm' and preload 3-92 mN; of rat papillary muscle: see Fig. 1. All test contractions were separated by a series of at least eight equally loaded standardized contractions. In cat myocardium, the contractions were abruptly abbreviated by imposing the higher loads. Rat myocardium was somewhat less sensitive and frog myocardium scarcely so at all.
RELAXATION OF VENTRICULAR CARDIAC MUSCLE
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Fig. 4. Length (upper) and force (lower) traces of a loading (contraction c) and an unloading step (contraction b) of the same magnitude and imposed at the same time during the contraction in cat (panel A; characteristics of muscle: see Fig. 3), frog (panel B; characteristics of strip: see Fig. 1) and rat (panel C; characteristics of muscle: see Fig. 1). Both contractions were clamped to the same afterload. The control afterloaded isotonic contraction (contraction a) to which the muscle was clamped and the control isometric contraction are also shown. All test contractions were separated by a series of at least eight equally loaded standardized contractions. Although the load after the clamp step was the same in contractions a, b and c, the subsequent course of contraction and relaxation was strikingly different in cat papillary muscle, less so in rat myocardium and scarcely so at all in frog myocardium.
D. L. BRUTSAERT AND OTHERS 476 amplitude imposed at the same instant during the contraction. Fig. 4 illustrates both a single unloading step (contraction b) and a single loading step (contraction c). Although the load after the clamp step was the same in contractions a, b and c the subsequent course of contraction and relaxation was strikingly different in cat papillary muscle (Fig. 4A). Again, this difference was less pronounced in rat myocardium (Fig. 4C), and scarcely detectable in frog myocardium (Fig. 4B). The duration of contraction and the course of relaxation were thus very greatly affected by the previous loading history in cat heart muscle, but less so in rat and scarcely at all in frog heart muscle. 3. Influence of inotropic interventions; other species This marked difference between cat and frog ventricular preparations with respect to the mechanical properties of their relaxation was a constant finding. It persisted at different inotropic states (adrenaline 104 M, propranolol 10-7-10-5 M, at extracellular calcium concentrations 1x25, 2-5 and 5-0 mM), different initial muscle lengths (100 % and 90 % imax.), and different temperatures (24, 29 and 36 TC). The experiments with adrenaline and propranolol were performed because of the known relaxing effects of catecholamines on cardiac muscle (Morad & Rolett, 1972). The findings persisted also when preparations were examined before they had fully equilibrated. The somewhat less pronounced load-dependence during relaxation of ventricular myocardium of the rat was also a constant finding in all tissue specimens that were examined. Experiments with preparations from rabbit and pig showed that relaxation behaved in a manner essentially similar to that of cat ventricular myocardium. DISCUSSION
1. Activation- versus load-dependent relaxation Manipulation of loading conditions during relaxation of ventricular heart muscle from different species can influence its course, though not in frog preparations. In frog heart muscle, relaxation was largely independent of load so that it may simply reflect the rate limiting step of calcium sequestration. Similarly, differences of mechanical relaxation of skeletal muscle have been related to differences in calcium sequestration as a possible rate limiting step in relaxation (Hasselbach, 1964; Ebashi & Endo, 1968; Sandow, 1970; Hill, 1972; Briggs, Poland & Solaro, 1977). We have called this type of relaxation 'activation-dependent' relaxation. In heart muscle from the mammalian species studied, however, relaxation was sensitive to loading conditions implying that some other factor may then be rate limiting. We have called this 'load-dependent' relaxation. Load-dependence may be the mechanical expression of the ratio of the number of cross-bridges to the load to be carried at any moment during relaxation. Hence, relaxation of ventricular cardiac muscle in different species appears to be governed to different extents by these two mechanisms. Studies with frog skeletal muscle also showed that relaxation is very sensitive to loading (Jewell & Wilkie, 1960) implying the predominance of a load dependent mechanism for relaxation in this type of muscle too.
RELAXATION OF VENTRICULAR CARDIAC MUSCLE
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2. Nature of activation-dependence Recent observations (Brutsaert, Claes & De Clerck, 1978) showed that the time course of relaxation in isotonic contractions of single, Brij-58 pretreated, ventricular cardiac cells of cat, rabbit and rat is insensitive to load in contrast to the behaviour of intact muscle preparations. These cells resemble intact frog myocardium in this respect. The data could be compatible with a model in which the relative preponderance of activation- and load-dependent mechanism during relaxation would be determined largely by the extent of development, organization and pumping capacity of calcium sequestering systems. With a less well developed sarcoplasmic reticulum in frog (Staley & Benson, 1968), most activating calcium is derived from transsarcolemmal calcium current when the membrane is depolarized (Niedergerke, 1963; Vassort, 1973; Chapman, 1973; Winegrad, 1973; Fabiato & Fabiato, 1977). Relaxation would relate to the disappearance rate of calcium ions, possibly across the sarcolemma (Chapman, 1973; Vassort, 1973; Goto et al. 1972). As in the Brij-58 pretreated cells, relaxation would directly follow the reduction of the sarcoplasmic calcium level, since a sufficient number of crossbridges would still be reformed to make this muscle largely insensitive to load. Several studies have indicated that the amount of released activating calcium in ventricular cardiac muscle of cat would be insufficient to give full activation in normal conditions (Katz, 1970; Bassingthwaighte & Reuter, 1972; Fozzard, 1973). Since, in contrast to frog, the sarcoplasmic reticulum is well developed in adult cat myocardium (Fawcett & McNutt, 1969), the difference between intact cat preparations and Brij-pretreated cells would result from an early efficient sequestration of the relatively small amounts of released calcium by a relatively well developed sarcoplasmic reticulum. With a too low sarcoplasmic calcium level after the first two thirds of the contraction, crossbridges would not recycle further. The observations on rat cardiac muscle also tend to support the proposed model. Rat ventricle has a well developed sarcotubular system (Forssmann & Girardier, 1970) and normal activation appears to be maximal (Henderson et al. 1969; Kelly & Hoffman, 1960; Mainwood & McGuigan, 1975; Fabiato & Fabiato, 1977). One could therefore postulate that even the well developed sarcoplasmic reticulum would not be able to handle efficiently the saturating sarcoplasmic calcium level in rat. A sufficient number of crossbridges would still be reformed at later times than in other mammalian species and allow for load alteration not to affect significantly the remainder of the shortening phase. Only near peak shortening and throughout relaxation would sarcoplasmic calcium be sufficiently lowered for the muscle to become sensitive to load. 3. Nature of load-dependence Vertebrate skeletal muscle (Jewell & Wilkie, 1960; Gordon, Huxley & Julian, 1966; Edman & Nilsson, 1971) and rabbit (Brady, 1965, 1967), cat (Brutsaert, 1974) and in situ perfused dog (Suga & Yamakoshi, 1977) cardiac muscle, that is allowed to shorten during a twitch loses some of its ability to produce force during the remainder of the contraction. This familiar deactivation may be related to the influence of changes in muscle length on the activation of the contractile system (Brutsaert, Claes & Donders, 1972; Jewell, 1977; Julian & Moss, 1976). Although peak
D. L. BRUTSAERT AND OTHERS shortening of isotonic contractions is reached not earlier than peak force of the corresponding isometric twitch, Fig. 1 A showed that the subsequent course of both isotonic and isometric phases of relaxation was greatly shortened. Hence, as was previously noted (Brutsaert & Housmans, 1977, Fig. 3) the reduction in overall twitch duration after shortening is confined mainly to an abbreviation of the relaxation phase. The foregoing discussion raised the possibility that the ratio of the number of crossbridges to total load could be the major determinant of isotonic relaxation of cardiac muscle in species in which the sarcoplasmic calcium had diminished to very low levels already before the onset of relaxation. An abrupt increase in load would decrease this ratio and would make the muscle relax prematurely. Load clamp analysis did indeed reveal a premature isotonic relaxation in cardiac muscle of cat, rabbit and pig, and to a smaller extent of rat; after an initial fast extension at the time of the load clamp, lengthening of the muscle proceeded in a slow and a subsequent faster isotonic extension. This delayed lengthening was proposed previously to reflect load-induced back rotation of crossbridges with eventual detachment and sliding of myofilaments back to their original positions (Housmans & Brutsaert, 1976; Brutsaert & Housmans, 1977). Separation of the decay of myoplasmic calcium and of force potential in the second half of a twitch contraction constitutes an essential assumption for this model to be acceptable as the underlying mechanism for load-dependent relaxation. Such difference between the time courses of 'activation' and force was predicted recently by computer simulation of muscle mechanics (Julian & Moss, 1976). An alternative interpretation could be that the delayed isometric relaxation, as compared to the more rapid isotonic relaxation, could be attributed to an increase of calcium affinity of troponin by high concentration of force-generating actin-myosin complexes in isometric conditions (Weber & Murray, 1973). However, as rightly pointed out by these authors, one must rule out first that the delay does not simply represent the lifetime of a force-generating complex under isometric conditions that inhibit movement of the bridge. Obviously, this latter view would be more in line with our model where in isometric relaxation the decay of force-generating sites is postulated to be rate limiting, whereas in isotonic relaxation load-induced backrotation and detachment would prematurely abbreviate the lifetime of the crossbridges. 478
4. Restoring forces and non-uniformity during relaxation We have ignored so far any load other than the external one and heavy reliance was put on mechanical uniformity of relaxation. Any attempt to reduce the length of a resting muscle fibre below its so called slack length will result in 'restoring forces' that return the fibre to this length (Jewell, 1977); obviously, these forces will add to the 'external' load during relaxation (Parsons & Porter, 1966). Whereas these restoring, or elongating forces may enhance relaxation, other forces, e.g. due to
internal friction, viscosity and inertia, may actually counteract relaxation. However, there seems no obvious reason to assume that the contribution of such forces would differ in the various preparations used in this study. Uniformity of relaxation is another assumption that is less easily dealt with (Cleworth & Edman, 1972; Edman & Flitney, 1977; Krueger & Strobeck, 1977). Lengthening of the various sarcomeres during relaxation may vary much along the length of the fibre or from
479 RELAXATION OF VENTRICULAR CARDIAC MUSCLE one fibre to another in multicellular preparations. Non-uniformity of relaxation may result from spatial inhomogeneity in the distribution of force along the preparation or from temporal inhomogeneity of the decaying activation in various fibres. Yet, again in order to explain the present results major differences of non-uniformity ought to be assumed among these various cardiac preparations. The authors wish to thank A. H. Henderson for his helpful comments. REFERENCES
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HENDERSON, A. H. & BRuTSAERT, D. L. (1974). Force-velocity-length relationship in heart muscle: lack of time-independence during twitch contractions of frog ventricle strips with caffeine. Pfluger8 Arch. 348, 59-64. HENDERSON, A. H., BRuTSAERT, D. L., PARMLEY, W. W. & SONNENBLICK, E. H. (1969). Myocardial mechanics in papillary muscles of the rat and cat. Am. J. Phy8iol. 217, 1273-1279. HILL, D. K. (1972). Resting tension and the form of the twitch of rat skeletal muscle at low temperature. J. Phy8iol. 221, 161-171. HousMAws, PH. R. & BRuTSAERT, D. L. (1976). Three step yielding of load clamped mammalian cardiac muscle. Nature, Lond. 262, 56-58. JEWELL, B. R. (1977). A reexamination of the influence of muscle length on myocardial performance. Circulation Re8. 40, 221-230. JEWELL, B. R. & RovELL, J. M. (1973). Influence of previous mechanical events on the contractility of isolated cat papillary muscle. J. Physiol. 235, 715-740. JEWELL, B. R. & WiT.I, D. R. (1960). The mechanical properties of relaxing muscle. J. Physiol. 152, 30-47. JULIAN, F. J. & Moss, R. L. (1976). The concept of active state in striated muscle. Circulation Res. 38, 53-59. KATz, A. M. (1970). Contractile proteins of the heart. Physiol. Rev. 50, 63-158. KAUFMANN, R. L., LAB, M. J., HEsNN-EKEs, R. & KRAuSE, H. (1971). Feedback interaction of mechanical and electrical events in the isolated mammalian ventricular myocardium (Cat papillary muscle). Pfliuger8 Arch. 324, 100-123. KELLY, J. J. Jr. & HOFFMAN, B. F. (1960). Mechanical activity of rat papillary muscle. Am. J. Phy8iol. 199, 157-162. KRUEGER, J. W. & STROBECK, J. E. (1977). Influence of sarcomere motion on cardiac relaxation. (Abstract, European Journal of Cardiology: Proceedings of the 4th Workshop on Contractile Behaviour of the Heart). MAINWOOD, G. W. & McGmGoAN, J. A. S. (1975). Evidence for inward calcium current in the absence of external sodium in rat myocardium. Experientia 31, 67-69MATSUMARA, M. (1972). Electromechanical coupling in crayfish muscle fibres examined by the voltage clamp method. Jap. J. Phyaiol. 22, 53-69. MEERSON, F. Z. & KAPELKO, V. I. (1975). The significance of the interrelationship between the intensity of the contractile state and the velocity of relaxation in adapting cardiac muscle to function at high work loads. J. mol. cell. Cardiol. 7, 793-806. MORAD, M. & RoLrrE, E. L. (1972). Relaxing effects of catecholamines on mammalian heart. J. Physiol. 224, 537-558. NIEDERGERKE, R. (1963). Movements of calcium in beating ventricles ofthe frog heart. J. Physiol. 167, 551-580. PARMLEY, W. W. & SONNENBLICK, E. H. (1969). Relation between mechanics of contraction and relaxation in mammalian cardiac muscle. Am. J. Phyaiol. 216, 1084-1091. PARMLEY, W. W., BRUTSAERT, D. L. & SONNENBLICK, E. H. (1969). Effects of altered loading on contractile events in isolated cat papillary muscle. Circulation Res. 24, 521-532. PARSONS, C. & PORTER, K. R. (1966). Muscle relaxation: evidence for an intrafibrillar restoring force in vertebrate striated muscle. Science, N.Y. 153, 426-427. SANDOW, A. (1970). Skeletal muscle. A. Rev. Physiol. 32, 87-138. STALEY, N. A. & BENSON, E. S. (1968). The ultrastructure of frog ventricular cardiac muscle and its relationship to mechanisms of excitation-contraction coupling. J. cell Biol. 38, 99-114. STRAUER, B. E. (1973). Force-velocity relations of isotonic relaxation in mammalian heart muscle. Am. J. Physiol. 224, 431-434. STROBECK, J. E., BARtER, A. S. & SONNENBLICK, E. H. (1975). Isotonic relaxation in cardiac muscle. Am. J. Physiol. 229, 646-651. SUGA, H. & YAMAKOSHI, K.-I. (1977). Reduction of the duration of isovolumic relaxation in the ejecting left ventricle of the dog: residual volume clamping. J. Physiol. 267, 63-74. TAMIYA, K., KIKKAWA, S., GUNJI, A., HORI, M. & SAEuim, Y. (1977). Maximum rate of tension fall during isometric relaxation at end-systolic fibre length in canine papillary muscle. Circulation Res. 40, 584-589. VAssoRT, G. (1973). Influence of sodium ions on the regulation of frog myocardial contractility. Pfluger8 Arch. 339, 225-240. WEBER, A. M. & MURRAY, J. M. (1973). Molecular control mechanisms in muscle contraction. Physiol. Rev. 53, 612-673. WINEGRAD, S. (1973). Intracellular calcium binding and release in frog heart. J. gen. Phyaiol. 62, 693-706.
469
RELAXATION OF VENTRICULAR CARDIAC MUSCLE
By DIRK L. BRUTSAERT, NORA M. DE CLERCK, MARC A. GOETHALS AD PHILIPPE R. HOUSMANS From the Department of Phy8iolog, University of Antwerp, Antwerp, Belgium
(Received 30 October 1977) SUrMARY
1. The load bearing capacity during relaxation of ventricular cardiac muscle from various animal species was investigated. 2. The effect of load on the time course of relaxation was analysed either by comparing afterloaded contractions against various loads or by imposing abrupt alterations in load (load clamps). 3. In heart muscle from the mammalian species studied relaxation was sensitive to loading conditions, whereas in frog heart muscle relaxation was largely independent of the loading conditions. The mechanical properties of relaxation of cardiac muscle appear, therefore, governed by the interplay of a load-controlled and an activationcontrolled decay mechanism, the relative importance of which differs with species. 4. Load-dependence may be the mechanical expression of the ratio of the number of force generating sites at any time during contraction and relaxation to the load to be carried; this mechanism would predominate in mammalian animal species with a well developed calcium sequestering sarcoplasmic reticulum. Activation-dependence would seem to predominate in animal species, such as frog, in which calcium sequestration appears to be the rate limiting step during relaxation. INTRODUCTION
Relaxation of cardiac muscle is a major determinant of diastolic filling, yet the mechanical properties of the relaxation phase are less well understood than those of the contraction phase. There exists general agreement that relaxation follows active removal of the sarcoplasmic calcium ions. However, the way in which the capacity to bear a load decays when cardiac muscle returns to its initial length or resting tension has hardly been studied. In frog skeletal muscle, this load bearing capacity during relaxation was analysed several years ago (Jewell & Wilkie, 1960). There have also been some studies of mechanical relaxation in cardiac muscle from cat (Parmley & Sonnenblick, 1969; Strauer, 1973; Strobeck, Bahler & Sonnenblick, 1975), dog (Tamiya, Kikkawa, Gunji, Hori & Sakurai, 1977), rat and guinea pig (Meerson & Kapelko, 1975) and frog (Goto, Kimoto, Saito & Wada, 1972). These studies however have given little insight into the intrinsic mechanisms underlying the decay of load bearing capacity. The present study was undertaken to investigate the decay of load bearing capacity of the myocardium in various animal species with known differences in excitation-contraction coupling mechanisms. From this comparative study we
D. L. BRUTSAERT AND OTHERS suggest that the mechanical properties of relaxation of cardiac muscle are governed by the interplay of activation-controlled and load-controlled decay mechanisms, the relative importance of which differs with species. 470
METHODS
Isolated preparations of ventricular myocardium from adult cat (n = 30), rat (n = 8), rabbit (n = 3), pig (n = 1) and frog (n = 15) were used for this study. Papillary muscles were removed from the right ventricles of cats and rabbits and from left ventricles of rats; one thin trabecula was obtained from the right ventricle of an adult pig; in frog, thin longitudinal strips were cut from the ventricle. The criteria used for selection of suitable preparations have been published previously for cat (Brutsaert & Housmans, 1977), rat (Henderson, Brutsaert, Parmley & Sonnenblick, 1969) and frog (Henderson & Brutsaert, 1974). The criteria for selection of the papillary muscles in rabbit were the same as in cat, although the ratio of resting to total force at I.. was always somewhat higher; the muscle characteristics of the pig trabecula at I.. were: length 7-0 mm, cross-sectional area 0-76 mm2, preload 9-8 mN and ratio of resting to total tension 0-28. The muscles were mounted vertically, the lower end being held by a force transducer (compliance 0-3 ,um/mN, resonant frequency 250 Hz in aqueous solution) and the upper tendinous end tied (7-0 braided thread, Deknatel, Surgical Tevdek, Code 103-T) to an electromagnetic lever system (compliance 0-2 1um/mN, equivalent moving mass 155 mg, step response 3 ms). The current through the coil of the electromagnet determined the load on the muscle and was controlled by a current source, which was calibrated for step changes of 0-98 mN and 9-8 mN and could be switched from one level to another by means of two reference voltage sources. A detailed description of force transducer, electromagnetic lever system and the response characteristics of each to abrupt alterations in load has been published previously (Claes & Brutsaert, 1971; Brutsaert & Claes, 1974). The methodological and physiological aspects of studying abrupt alterations of load (the 'load clamp' technique) have also been reported (Brutsaert & Claes, 1974; Brutsaert, 1974). For each contraction, length and force traces were recorded simultaneously and displayed as functions of time on a Storage Display Unit (Tektronix 61 1) and photographed with a Hard Copy Unit (Tektronix 4601). The bathing solution contained (mM): NaCl 118, KCl 4-7, MgSO4 . 7H20 1-2, KH2PO, 1-1, NaHCO3 24, CaCl2.6H20 2-5 and glucose 4-5. The solution was bubbled with a gas mixture of 95 % 02-5 % CO2 and the bath temperature was maintained at 29 'C. Homogenous electrical stimulation (12 beats/min) was obtained by rectangular pulses of 5 ms duration about 10 % above threshold through two platinum electrodes arranged longitudinally along the entire muscle. All experiments were performed with the initial muscle length set to l,, i.e. the length at which active force development was maximal. The muscles were allowed to equilibrate for 3 hr before the actual experiments began. In order to avoid the effects of the loading conditions present in preceding contractions, all test contractions were separated by a series of at least eight equally loaded standardized contractions (Parmley, Brutsaert & Sonnenblick, 1969; Kaufmann, Lab, Hennekes & Krause, 1971; Jewell & Rovell, 1973; Brutsaert, 1974; Brutsaert & Paulus, 1977). The results were shown to be qualitatively similar whatever load was used for the interpolated, standardized contractions if this remained constant throughout the experiment. The results were the same in all preparations from any of the animal species studied, and are illustrated by representative examples. RESULTS
1. Afterloaded contractions A muscle that is allowed to shorten during a twitch loses some of its ability to produce force during the remainder of the contraction. The extent of the deactivation associated with an isotonic contraction varied in different types of cardiac muscle, being most marked in cat, less pronounced in rat, and almost absent in frog. In cat papillary muscle (Fig. 1A) the records of relaxation at different loads were well separated in time; isotonic contractions were always over before the isometric
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471
D. L. BRUTSAERT AND OTHERS
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Fig. 2. Length (upper) and force (lower) traces of a load clamp at three different times (contractions a, b and c) during a preloaded isotonic contraction in cat (panel A), frog (panel B) and rat (panel C). The control preloaded and afterloaded contractions are also shown. All test contractions were separated by a series of at least eight equally loaded standardized contractions. The characteristics of the three muscles are the same as in Fig. 1. In cat, load clamps which were imposed after the first two thirds of the contraction markedly abbreviated the total duration of the isotonic phase of the twitch (contractions b and c). In frog, the contractions were hardly affected by similar clamps. Again, the responses in rat were intermediate between cat and frog.
473 RELAXATION OF VENTRICULAR CARDIAC MUSCLE control beat; the ability to sustain 'supra-isometric' loads could be demonstrated only in heavily afterloaded contractions; and isotonic lengthening during relaxation, even at the smaller loads, was always followed by an initial abrupt fall of isometric force. Some of these properties, and also the exponential nature of the slow isometric
relaxation phase of afterloaded contractions have already been described for cat (Parmley & Sonnenblick, 1969), rabbit (Brady, 1965), dog (Tamiya et al. 1977) and frog (Goto et al. 1972) cardiac muscle, and for frog (Jewell & Wilkie, 1960), rat (Hill, 1972) and crayfish (Matsumara, 1972) skeletal muscle. By contrast, in frog myocardium the overall duration of all contractions was little affected by loading conditions (Fig. 1 B), with the isometric relaxation phases coinciding over their largest portion. The ability to sustain a load at 'supra-isometric' levels during isotonic lengthening was not limited to contractions at heavy afterloads but was present also at intermediate loads. The load-dependent changes in duration of contraction in rat ventricular cardiac muscle (Fig. 1 C) were intermediate between those seen in cat and those in frog heart muscle. 2. Load clamped contractions In Fig. 2 the load bearing capacity of cardiac muscle was tested at various times during isotonic contraction by imposing small additional loads. In cat (Fig. 2A), load clamps imposed during the first half of the shortening phase (contraction a), only slightly influenced the isotonic relaxation phase and the over all duration of the contraction. Load clamps of the same magnitude at later times (in the second half and especially the last third of the shortening phase) abbreviated the total duration of the isotonic phase of the twitch (contractions b and c). The initial rapid lengthening which accompanied the clamp step itself, was followed by a slower lengthening. Load clamps of the appropriate amplitude and timing were followed by a slower lengthening phase which occurred relatively slowly at first and then more quickly with a concomitant abbreviation of the contraction. In contrast, in frog cardiac muscle (Fig. 2B) relaxation was much less affected by similar load clamps. Immediately after the initial extension accompanying the abrupt increase in load, some delayed extension occurred in late or large clamps. The delayed extension did not consist of a first slow and subsequent fast phase, as typically occurred in cat ventricular myocardium (Housmans & Brutsaert, 1976; Brutsaert & Housmans, 1977); instead, it fused smoothly with the relaxation phase of the normal afterloaded control contraction and the overall duration of the contraction was hardly affected. Again, the mechanical properties of rat ventricular cardiac muscle under the same loading conditions were intermediate between those of cat and frog heart muscle but rather more like the cat. The same principle is illustrated in Fig. 3 where load clamps of increasing magnitude were superimposed at the same time near peak shortening, Again, cat myocardium (Fig. 3A) was markedly affected by these higher loads, rat myocardium (Fig. 3 C) less so, and frog myocardium (Fig. 3B) scarcely so at all except for very large clamp steps or if the experiment was repeated at still later times during relaxation (not shown). The load-dependence of cat and, to a smaller extent, of rat ventricular myocardium, in contrast to the relative load-independence of frog myocardium, was further tested by comparing the effects of loading and unloading steps of the same
D. L. BRUTSAERT AND OTHERS
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Fig. 3. Length (upper) and force (lower) traces of load clamped contractions with clamp steps of various magnitudes imposed near peak shortening of a preloaded isotonic contraction in cat (panel A), frog (panel B) and rat (panel C). The control preloaded isotonic and the control isometric contractions are also shown. Characteristics of cat papillary muscle at lnxax: length 10-5 mm, cross sectional area 0 49 mm2 and preload 4 9 mN; of frog ventricular strip at lax,: length 7-5 mm, cross sectional area 0-91 mm' and preload 3-92 mN; of rat papillary muscle: see Fig. 1. All test contractions were separated by a series of at least eight equally loaded standardized contractions. In cat myocardium, the contractions were abruptly abbreviated by imposing the higher loads. Rat myocardium was somewhat less sensitive and frog myocardium scarcely so at all.
RELAXATION OF VENTRICULAR CARDIAC MUSCLE
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Fig. 4. Length (upper) and force (lower) traces of a loading (contraction c) and an unloading step (contraction b) of the same magnitude and imposed at the same time during the contraction in cat (panel A; characteristics of muscle: see Fig. 3), frog (panel B; characteristics of strip: see Fig. 1) and rat (panel C; characteristics of muscle: see Fig. 1). Both contractions were clamped to the same afterload. The control afterloaded isotonic contraction (contraction a) to which the muscle was clamped and the control isometric contraction are also shown. All test contractions were separated by a series of at least eight equally loaded standardized contractions. Although the load after the clamp step was the same in contractions a, b and c, the subsequent course of contraction and relaxation was strikingly different in cat papillary muscle, less so in rat myocardium and scarcely so at all in frog myocardium.
D. L. BRUTSAERT AND OTHERS 476 amplitude imposed at the same instant during the contraction. Fig. 4 illustrates both a single unloading step (contraction b) and a single loading step (contraction c). Although the load after the clamp step was the same in contractions a, b and c the subsequent course of contraction and relaxation was strikingly different in cat papillary muscle (Fig. 4A). Again, this difference was less pronounced in rat myocardium (Fig. 4C), and scarcely detectable in frog myocardium (Fig. 4B). The duration of contraction and the course of relaxation were thus very greatly affected by the previous loading history in cat heart muscle, but less so in rat and scarcely at all in frog heart muscle. 3. Influence of inotropic interventions; other species This marked difference between cat and frog ventricular preparations with respect to the mechanical properties of their relaxation was a constant finding. It persisted at different inotropic states (adrenaline 104 M, propranolol 10-7-10-5 M, at extracellular calcium concentrations 1x25, 2-5 and 5-0 mM), different initial muscle lengths (100 % and 90 % imax.), and different temperatures (24, 29 and 36 TC). The experiments with adrenaline and propranolol were performed because of the known relaxing effects of catecholamines on cardiac muscle (Morad & Rolett, 1972). The findings persisted also when preparations were examined before they had fully equilibrated. The somewhat less pronounced load-dependence during relaxation of ventricular myocardium of the rat was also a constant finding in all tissue specimens that were examined. Experiments with preparations from rabbit and pig showed that relaxation behaved in a manner essentially similar to that of cat ventricular myocardium. DISCUSSION
1. Activation- versus load-dependent relaxation Manipulation of loading conditions during relaxation of ventricular heart muscle from different species can influence its course, though not in frog preparations. In frog heart muscle, relaxation was largely independent of load so that it may simply reflect the rate limiting step of calcium sequestration. Similarly, differences of mechanical relaxation of skeletal muscle have been related to differences in calcium sequestration as a possible rate limiting step in relaxation (Hasselbach, 1964; Ebashi & Endo, 1968; Sandow, 1970; Hill, 1972; Briggs, Poland & Solaro, 1977). We have called this type of relaxation 'activation-dependent' relaxation. In heart muscle from the mammalian species studied, however, relaxation was sensitive to loading conditions implying that some other factor may then be rate limiting. We have called this 'load-dependent' relaxation. Load-dependence may be the mechanical expression of the ratio of the number of cross-bridges to the load to be carried at any moment during relaxation. Hence, relaxation of ventricular cardiac muscle in different species appears to be governed to different extents by these two mechanisms. Studies with frog skeletal muscle also showed that relaxation is very sensitive to loading (Jewell & Wilkie, 1960) implying the predominance of a load dependent mechanism for relaxation in this type of muscle too.
RELAXATION OF VENTRICULAR CARDIAC MUSCLE
477
2. Nature of activation-dependence Recent observations (Brutsaert, Claes & De Clerck, 1978) showed that the time course of relaxation in isotonic contractions of single, Brij-58 pretreated, ventricular cardiac cells of cat, rabbit and rat is insensitive to load in contrast to the behaviour of intact muscle preparations. These cells resemble intact frog myocardium in this respect. The data could be compatible with a model in which the relative preponderance of activation- and load-dependent mechanism during relaxation would be determined largely by the extent of development, organization and pumping capacity of calcium sequestering systems. With a less well developed sarcoplasmic reticulum in frog (Staley & Benson, 1968), most activating calcium is derived from transsarcolemmal calcium current when the membrane is depolarized (Niedergerke, 1963; Vassort, 1973; Chapman, 1973; Winegrad, 1973; Fabiato & Fabiato, 1977). Relaxation would relate to the disappearance rate of calcium ions, possibly across the sarcolemma (Chapman, 1973; Vassort, 1973; Goto et al. 1972). As in the Brij-58 pretreated cells, relaxation would directly follow the reduction of the sarcoplasmic calcium level, since a sufficient number of crossbridges would still be reformed to make this muscle largely insensitive to load. Several studies have indicated that the amount of released activating calcium in ventricular cardiac muscle of cat would be insufficient to give full activation in normal conditions (Katz, 1970; Bassingthwaighte & Reuter, 1972; Fozzard, 1973). Since, in contrast to frog, the sarcoplasmic reticulum is well developed in adult cat myocardium (Fawcett & McNutt, 1969), the difference between intact cat preparations and Brij-pretreated cells would result from an early efficient sequestration of the relatively small amounts of released calcium by a relatively well developed sarcoplasmic reticulum. With a too low sarcoplasmic calcium level after the first two thirds of the contraction, crossbridges would not recycle further. The observations on rat cardiac muscle also tend to support the proposed model. Rat ventricle has a well developed sarcotubular system (Forssmann & Girardier, 1970) and normal activation appears to be maximal (Henderson et al. 1969; Kelly & Hoffman, 1960; Mainwood & McGuigan, 1975; Fabiato & Fabiato, 1977). One could therefore postulate that even the well developed sarcoplasmic reticulum would not be able to handle efficiently the saturating sarcoplasmic calcium level in rat. A sufficient number of crossbridges would still be reformed at later times than in other mammalian species and allow for load alteration not to affect significantly the remainder of the shortening phase. Only near peak shortening and throughout relaxation would sarcoplasmic calcium be sufficiently lowered for the muscle to become sensitive to load. 3. Nature of load-dependence Vertebrate skeletal muscle (Jewell & Wilkie, 1960; Gordon, Huxley & Julian, 1966; Edman & Nilsson, 1971) and rabbit (Brady, 1965, 1967), cat (Brutsaert, 1974) and in situ perfused dog (Suga & Yamakoshi, 1977) cardiac muscle, that is allowed to shorten during a twitch loses some of its ability to produce force during the remainder of the contraction. This familiar deactivation may be related to the influence of changes in muscle length on the activation of the contractile system (Brutsaert, Claes & Donders, 1972; Jewell, 1977; Julian & Moss, 1976). Although peak
D. L. BRUTSAERT AND OTHERS shortening of isotonic contractions is reached not earlier than peak force of the corresponding isometric twitch, Fig. 1 A showed that the subsequent course of both isotonic and isometric phases of relaxation was greatly shortened. Hence, as was previously noted (Brutsaert & Housmans, 1977, Fig. 3) the reduction in overall twitch duration after shortening is confined mainly to an abbreviation of the relaxation phase. The foregoing discussion raised the possibility that the ratio of the number of crossbridges to total load could be the major determinant of isotonic relaxation of cardiac muscle in species in which the sarcoplasmic calcium had diminished to very low levels already before the onset of relaxation. An abrupt increase in load would decrease this ratio and would make the muscle relax prematurely. Load clamp analysis did indeed reveal a premature isotonic relaxation in cardiac muscle of cat, rabbit and pig, and to a smaller extent of rat; after an initial fast extension at the time of the load clamp, lengthening of the muscle proceeded in a slow and a subsequent faster isotonic extension. This delayed lengthening was proposed previously to reflect load-induced back rotation of crossbridges with eventual detachment and sliding of myofilaments back to their original positions (Housmans & Brutsaert, 1976; Brutsaert & Housmans, 1977). Separation of the decay of myoplasmic calcium and of force potential in the second half of a twitch contraction constitutes an essential assumption for this model to be acceptable as the underlying mechanism for load-dependent relaxation. Such difference between the time courses of 'activation' and force was predicted recently by computer simulation of muscle mechanics (Julian & Moss, 1976). An alternative interpretation could be that the delayed isometric relaxation, as compared to the more rapid isotonic relaxation, could be attributed to an increase of calcium affinity of troponin by high concentration of force-generating actin-myosin complexes in isometric conditions (Weber & Murray, 1973). However, as rightly pointed out by these authors, one must rule out first that the delay does not simply represent the lifetime of a force-generating complex under isometric conditions that inhibit movement of the bridge. Obviously, this latter view would be more in line with our model where in isometric relaxation the decay of force-generating sites is postulated to be rate limiting, whereas in isotonic relaxation load-induced backrotation and detachment would prematurely abbreviate the lifetime of the crossbridges. 478
4. Restoring forces and non-uniformity during relaxation We have ignored so far any load other than the external one and heavy reliance was put on mechanical uniformity of relaxation. Any attempt to reduce the length of a resting muscle fibre below its so called slack length will result in 'restoring forces' that return the fibre to this length (Jewell, 1977); obviously, these forces will add to the 'external' load during relaxation (Parsons & Porter, 1966). Whereas these restoring, or elongating forces may enhance relaxation, other forces, e.g. due to
internal friction, viscosity and inertia, may actually counteract relaxation. However, there seems no obvious reason to assume that the contribution of such forces would differ in the various preparations used in this study. Uniformity of relaxation is another assumption that is less easily dealt with (Cleworth & Edman, 1972; Edman & Flitney, 1977; Krueger & Strobeck, 1977). Lengthening of the various sarcomeres during relaxation may vary much along the length of the fibre or from
479 RELAXATION OF VENTRICULAR CARDIAC MUSCLE one fibre to another in multicellular preparations. Non-uniformity of relaxation may result from spatial inhomogeneity in the distribution of force along the preparation or from temporal inhomogeneity of the decaying activation in various fibres. Yet, again in order to explain the present results major differences of non-uniformity ought to be assumed among these various cardiac preparations. The authors wish to thank A. H. Henderson for his helpful comments. REFERENCES
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