THE PHYSIOLOGY OF STRIATED MUSCLE

fir. J. Anatsth. (1980), 52, 111 THE PHYSIOLOGY OF STRIATED MUSCLE K. FLOYD The purpose of this article is to present current views on the physiology of striated muscle, and in particular on the mechanism of contraction and the way in which the contractile machinery is activated. The preparation which has been most extensively studied is the isolated living muscle fibre of the frog, and this fact has governed the choice of the experimental evidence cited. The plan of the article is to give a brief outline of structure, a description of the processes involved in activation, and an account of the development of ideas about the mechanism of contraction, starting from the sliding filament theory and A. F. Huxley's 1957 model. Myolibrrt,''Light / I-fiarx)! Structural outline. When viewed under a light microscope, a striated muscle fibre can be seen to consist of a number of myofibrils, each of which is made up of alternate light and dark bands. In an unstained or living fibre these bands, which result from materials of different refractive index, can be made visible by the use of polarized light or interference microscopy. It is now known that the dark or A-band consists of thick filaments of the protein myosin, while the light or I-band consists of thin filaments of the protein actin, together with the proteins tropomyosin and troponin which perform an important regulatory role. The main structural features are shown diagrammatically in figure 1. In addition to the contractile proteins, striated muscle contains two internal membrane systems, the sarcoplasmic reticulum and the transverse tubules. The transverse tubules are invaginations of the surface membrane and run into the core of the muscle fibre. In contrast, the sarcoplasmic reticulum is composed of a longitudinal network of tubules which run into sacs or terminal dstemae where the sarcoplasmic reticulum comes into close proximity to the transverse tubules. The regions of conjunction appear as regularly spaced triads in longitudinal sections of muscle (a triad is a group of three "vesicles", consisting of a pair of terminal cisternae K. FLOYD, B.SC., M.SC, PH.D., Department of Physiology, Worsley Medical and Dental Building, University of Leeds, Leeds LS2 9NQ. 0007-0912/80/020111-11 $01.00 Sarcolemma FIG. 1. Schematic diagram of the structure of frog skeletal muscle. Each muscle fibre (30-150 im in diameter) is composed of a number of myofibrils (approx. 1 im in diameter), and the light and dark bands in each myofibril are normally aligned to give each fibre its striped appearance. The thick filaments are about 1.6 psn in length and the thin filaments (including the Z-line) are between 1.95 and 2.0 (im in length. Structural details of the triads are illustrated in figure 2. Adapted from Ebashi, Endo and Ohtsuki (1969). on either side of a transverse tubule). In frog muscle, triads are found at the level of the Z-line (fig. 2A), whereas in mammalian musde they are situated at the level of the A-I boundary (fig. 2B). ACTIVATION OF THE CONTRACTILE MECHANISM Activation of vertebrate skeletal musde is brought about by depolarization of the surface membrane, which results in the rdease of calcium ions from intracellular stores in the sarcoplasmic reticulum. The stages in this process will be summarized briefly and then described in more detail. The action potential at the surface membrane is propagated radially into the core of the musde fibre via the transverse tubular system. The depolarization of the transverse tubules results in the rdease of caldum ions from the adjacent terminal cisternae of the sarcoplasmic reticulum. The caldum ions diffuse into the interior of the myofibrils, where they O Maanillan Journals Ltd 1980

BRITISH JOURNAL OF ANAESTHESIA 112 ^:# fj*.«f > < FIG. 2. (A) : Frog (Rana pipiens) sartorius muscle in longitudinal section showing triads at the level of the Z-line (Z). I-A marks the ends of the thick filaments. Ferritin particles (which demonstrate continuity with the extracellular fluid) can be seen in the transverse tubules (T) but not in the sarcoplasmic reticulum (SR). Scale: 0.2 (xm (derived from magnification given in original text). Peachey and Schild (1968) by permission of the Journal of Physiology, (B): Cat superior rectus muscle in longitudinal section showing triads at the level of the A-I boundary. Scale: 1 im. Floyd (unpublished), (c) Frog (Rana pipiens) sartorius muscle in longitudinal section showing a single triad in tangent view. Note the periodically repeating feet, and a region (S) where the sarcoplasmic reticulum appears scalloped. The asterisk indicates an area of the sarcoplasmic reticulum where the contents appear particularly regular. Scale: 0.1 im (derived from magnification given in original text). Franzini-Armstrong (1970) by permission of the Rockefeller University Press. combine with the regulatory protein troponin. This removes an inhibitory influence exerted by tropomyosin on actin which can then interact with myosin to produce contraction of the muscle. Excitation. Muscle fibres are electrically excitable cells similar in their essential features to nerve cells, and the first stage in activation normally involves an action potential which will arise when the potential difference across the cell membrane is reduced from its resting value of 90 mv (inside negative) to about 50 mv. Such a depolarization normally occurs at the motor endplate as a result of the arrival of an action potential at the motor nerve terminal, and the action potential generated in the muscle fibre membrane propagates in both directions to the ends of the fibre. It is the change in membrane potential itself which is the trigger to contraction, and the action potential at the surface membrane is followed by the sequence of events described previously. If a muscle fibre is activated by direct depolarization in a solution containing a high concentration of potassium, a contracture occurs in which tension is produced for a few seconds, followed by spontaneous relaxation despite the fact that the membrane remains depolarized. Hodgkin and Horowicz (1960) found that the initial development of tension was related to membrane potential by a steep sigmoid curve with a threshold at about 50 mv. In addition, they showed that the restoration of the ability to contract after spontaneous relaxation required repolarization of the membrane for a certain period of time (repriming). Thus, both activation and repriming appear to be controlled by the membrane potential. t' FIG. 3. Local activation of a twitch fibre from the semitendinosus muscle of Rana temporaria. Polarized light, compensated so that the A-bands appear dark. The fibre is immersed in, and the pipette is also filled with, Ringer's solution. Left-hand pictures before, and right-hand pictures during, an electric pulse applied to the pipette making the interior of its tip go roughly 100 mv negative relative to the surrounding fluid, for a period of about 0.5 s. In the upper pictures, the pipette is applied to an A-band, and there is no response. In the lower pictures, the pipette is applied to an I-band, and during the pulse that I-band (and only that I-band) shortens. Huxley and Taylor (1958), by permission of the Journal of Physiology.

ACTIVATION AND CONTRACTION 113 Inward spread of activation. The existence of a specific inward-conducting mechanism located at the level of the Z-line in frog twitch fibres was elegantly demonstrated by Huxley and Taylor (1958) (fig. 3). The structure responsible for this inward spread of activation has since been shown to be the transverse tubular system. A full account of the association of inward spread of activation with the transverse tubules has been given by Huxley (1971). The electrical conduction in the transverse tubules has been shown to be a regenerative process (Costantin, 1970,1975) qualitatively similar to that in the surface membrane. In addition, it is now known that the action potential in the transverse tubules is necessary for normal contraction of frog muscle at 20 C, but is less important at low temperatures (Bastian and Nakajima, 1974). The explanation for this is that at low temperatures the duration of the action potential is increased, and the prolonged depolarization of the surface membrane increases the effectiveness of purely passive spread of depolarization along the transverse tubules. Coupling between transverse tubules and sarcoplasmic reticulum. The dependence of contracture tension on membrane potential has already been mentioned. If the action potential of an intact twitch fibre is blocked chemically, contractile activation can be produced by graded depolarization of the surface membrane, and contractile force is found to increase with the amplitude of membrane depolarization. Because the applied surface depolarization is attenuated along the transverse tubules under these circumstances, it is important to exclude the possibility that the observed increases in force are the result solely of progressive recruitment of more axially located myofibrils. Costantin and Taylor (1973) have shown that recruitment of myofibrils does occur with increasing depolarization, but they were also able to demonstrate a further gradation of force of contraction with increasing depolarization in contractions in which all myofibrils in the cross-section of the fibre were known to be activated. Thus, assuming that contractile force reflects the quantity of calcium released from the sarcoplasmic reticulum, the amount of calcium released during the activation process appears to be regulated by the transmembrane potential across the transverse tubules. The membranes of the transverse tubules and sarcoplasmic reticulum are brought into close apposition in the triads by numerous small, electrondense projections or "feet" of the terminal cisternae (fig. 2c), but there is believed to be no electrical continuity between the two membranes. How, then> does the depolarization of transverse tubules result in the release of calcium ions from the terminal cisternae? In their quantitative description of the membrane currents in the squid giant axon, Hodgkin and Huxley (1952) suggested that the dependence of sodium and potassium conductance on membrane potential might arise from the effect of the electric field on the distribution or orientation of charged particles within the membrane. The charge movement within the membrane is accompanied by a membrane current (gating current) which is thought to reflect internal rearrangements. Charge movements are detected in voltage clamp experiments on preparations in which ionic currents are abolished by channel-blocking agents and solutions of impermeant ions, and movement may be prevented by the use of hypertonic solutions. To illustrate the sort of procedures involved, it may be helpful to consider a particular experiment which investigates the idea that a charge movement associated with the voltage-dependent release of calcium may be reversed (remobilized) during the repriming period. The fibre was first depolarized to mobilize the "activation" charge; a comparison was then made between the membrane currents for two identical depolarizing voltage steps, one of which was preceded by a brief hyperpolarization, and the other by a repriming hyperpolarization which lasted for several seconds. The difference between the recorded currents thus detects the charge that is remobilized by the repriming hyperpolarization. Adrian (1978) has discussed the difficulties of estimation and interpretation of charge movements in muscle fibre membranes, but he tentatively identifies charge movements related to the sodium channel, the potassium channel, and to contraction activation. However, the events which underlie the contraction activation charge movements are at present unknown, and the attractive idea that the charge movements directly regulate the release of calcium ions by affecting long molecules which interconnect the membranes of the two internal membrane systems via the "feet" of the terminal cisternae must remain speculative. Changes in intracellular calcium concentration. It is now well established that contractile activation is achieved by the release of calcium ions from the sarcoplasmic reticulum. The presence of accumulations of calcium in the sarcoplasmic reticulum, and in the terminal cisternae, has been established by

114 BRITISH JOURNAL OF ANAESTHESIA electron microscopy of preparations involving precipitation of calcium oxalate or autoradiography of calcium-45. Experiments on "skinned" muscle fibres, in which the surface membrane has been removed mechanically or chemically so that the bathing solution has direct access to the contractile proteins, have shown that the threshold concentration of calcium for initiating contraction is about 10~ 7 mol litre" 1, and mammum activation is obtained at 10~~* mol litre" 1. In the absence of calcium ions, the regulatory proteins, troponin and tropomyosin, exert an inhibitory effect on actin so that actin-myosin interaction is prevented. When calcium is released it binds to one of the troponin subunits, and the resulting changes in the conformation of the regulatory proteins remove the inhibition, enabling contraction to occur (Ebashi and Endo, 1968; Ebashi, Endo and Ohtsuki, 1969). Recent reviews by Sandow (1970), Fuchs (1974), Ebashi (1976), Endo (1977) and Caputo (1978) deal extensively with aspects of calcium release induced in skinned fibres, and this aspect will not be pursued further. A rather different approach is to monitor changes in intracellular calcium concentration during normal contractions of living muscle. A promising advance in the measurement of changes in cytoplasmic calcium concentration has involved the intracellular injection of the calcium-sensitive bioluminescent protein aequorin, and results of such experiments in amphibian muscle have been reported by Bunks, Riidel and Taylor (1978). They point out that limitations on the quantitative interpretation of the aequorin response at present preclude any conclusions about possible differences in the degree of activation in twitches and tetani. However, these limitations need not apply in circumstances where the geometrical relations among sites of calcium release, binding and sequestration are likely to be constant, such as during the series of twitches shown in figure 4, where changes in the aequorin response are believed to reflect changes in calcium release and uptake. Thus the decrease in light intensity reflects a progressive decrease in the amount of calcium released in successive twitches, and the simultaneous progressive slowing of decay of the aequorin response probably reflects a gradual slowing of the sequestration of calcium by the sarcoplasmic reticulum. The influence of fibre length on luminescent and mechanical responses was also investigated by Blinks, Riidel and Taylor (1978) who found that the aequorin response during tetani at 50 s -1 (15 C) was mayimni at sarcomere lengths of about 2.4 [un and decreased considerably at longer sarcomere lengths. A similar result was obtained from experiments on single twitches, except that the maximal aequorin responses in different fibres occurred at a range of sarcomere Force FIG. 4. Staircase phenomenon in successive isometric twitches. Single fibre from the tibialis anterior muscle of Xenopus laevis; striation spacing 2.1 im; temperature 15 C. Computeraveraged records of light (aequorin response) and force from 10 trains of 16 twitches during stimulation of the fibre at 5 s~*. The lower panels show high-speed tracings of the first and last contractions in the series. The points above the light trace indicate rate constants for decay of aequorin luminescence in successive twitches. Blinks, Rudel and Taylor (1978), by permission of the Journal of Physiology.

ACTIVATION AND CONTRACTION 115 lengths from 2.4 to 3.0 [isa. These results suggest that less calcium is released at long sarcomere lengths. The mechanism of this reduction of calcium release is unknown, but could be caused by reversible distortion of the junctional region between the transverse tubules and terminal cisternae. The aequorin technique is obviously of major importance in the study of calcium transients in living fibres and improvements in precision of measurement and accuracy of localization are awaited with much interest. The implications of the results reported in the preceding paragraphs are far reaching. For example, measurements of the heat production associated with activation of muscle have assumed that the activation process was independent of length. Also, the changes in calcium concentration during repetitive stimulation suggest the need for a re-evaluation of the contribution of activation processes to maintenance heat (the steady production of heat during an isometric tetanus). These questions are considered, together with a detailed description of the steps that may contribute to the energetics of activation, in the reviews by Curtin and Woledge (1978) and Homsher and Kean (1978). The lack of correlation between light output from aequorin and contractile force during the plateau of tetanic contractions could arise if the sarcoplasmic calcium concentration is greater than that required to saturate the regulatory proteins, thereby producing maximal activation of the contractile system. However, it is known that the mayirmim force generated in potassium contractures is appreciably greater than in tetani. Also, the maximum force developed by single fibres in tetani at optimum length can be increased by caffeine, nitrate or zinc ions, all of which apparently increase the tetanic aequorin response. It is to be hoped that these contradictions can be resolved soon. THE CONTRACTILE RESPONSE Mechanism of contraction Length changes in striated muscle, whether active or passive, take place by a relative sliding motion between two sets of interdigitating filaments (fig. 1) which remain virtually unchanged in length. The sliding filament theory is now accepted as the structural basis of muscular contraction, but within its framework there are many possible mechanisms 100-? 80 ".1 C, 60 o 40 20 w striation spacing FIG. 5. Isometric tetanus tension developed by isolated fibres from the semitendinosus muscle of Rana umporaria at various lengths. Ordinate: tension increase above resting tension, as a percentage of the value in the plateau (2.05-2.20 \isn). Abscissa: striation spacing ( im). Points compiled from the results of Gordon, Huxley and Julian (1966b). Huxley (1971), by permission of the Royal Society.

116 BRITISH JOURNAL OF ANAESTHESIA with the region of overlap of the thin filaments with that part of the thick filament which carried projections. In experiments using isotonic tetani, with a microscope to measure sarcomere length and detect shortening, Huxley and Peachey (1961) found that there was no shortening when the striaticn spacing exceeded a critical value of 3.52 y.m, which is reasonably close to the sarcomere length at which the thick and thin filaments no longer overlap. The interpretation of tension records frqm fibres at stretched lengths, where tension may continue to increase slowly for several seconds, has been questioned by Ter Keurs, Iwazumi and Pollack (1970). They measured the steady tension reached 200 mi SOOms 42 (im after several seconds in contractions of stretched FIG. 6. Isometric tension developed by isolated single muscle fibres and found that this was not proportional fibresfromthe tibialis anterior muscle of Rana pipiens. The to filament overlap at sarcomere lengths between top trace in each part is the output of the spot follower 2.2 [im and 3.65 ^m. This has led them to question apparatus which indicates changes in marker separation the cross-bridge model of force generation. In (lengthening up) for the force records obtained at full overlap (2.13 nm and 2.21 im; the corresponding marker contrast, Julian, Sollins and Moss (1978) (fig. 6) separations were 1.85 mm and 1.90 mm respectively). At re-investigated the length-tension relation and have each sarcomere length (/,) a twitch and a tetanus were fully confirmed the proportionality of tension and recorded: in (A) tetani, and (B) twitches, the temperature filament overlap in stretched muscle fibres. The was 0 C; in (c) tetani, and (D) twitches, the temperature difference in results obtained is probably related to was 20 C. The bottom trace is a record of the stimulus pattern. Julian, Sollins and Moss (1978), by permission of differences in the techniques and procedures used, but a satisfactory explanation has not yet been the Royal Society. provided. for the origin of the contractile force and some of Another feature of contraction resulting from these are discussed in the reviews by Huxley (1974) independent force generators is that the maximum and Simmons and Jewell (1974). speed of shortening (under zero external load) should The idea that a relative force between thick and be independent of the amount of overlap, provided thin filaments might be generated at each of a series there are no length-dependent internal forces of independent sites in the overlap zone was first put forward by Huxley and Niedergerke (1954). It follows that tetanic tension should be proportional to the number of sites at which tension is exerted on each thin filament and hence to the amount of overlap f]myo»ln l t between thick and thin filaments. The determination of the isometric length-pension relation is complicated Actm by non-uniformity in sarcomere length along a filament stretched fibre which arises because the ends do not Equilibrium position ^ < ^ of H site i elongate as much as the middle. A consequence of this is that during an isometric tetanus at long lengths FIG. 7. A. F. Huxley's model of a thick filament projection. the ends of the fibre shorten and stretch the middle The parts of thefilamentsshown are in the right-hand half of part, causing a slow increase in tension. Gordon, an A-band, so that the actinfilamentis attached to a Z-line Huxley and Julian (1966a, b) overcame this problem which is out of the picture to the right. The arrows give the direction of the relative motion between thefilamentswhen by using a photoelectric servo device to keep constant the muscle shortens. The "side-piece" M, elastically conthe length of a part of the isolated fibre within which nected to the thick filament, is assumed capable of binding the striation spacing was nearly uniform. The length- to A sites (only one shown) on the thin filament. Attachtension relation obtained (fig. 5) showed a linear ment occurs spontaneously but may be reversible; detachoccurs principally by a process which involves the decrease in tension at sarcomere lengths between ment hydrolysis of an ATP molecule. Huxley (1957) by per2.2 (im and 3.65 ysn which corresponded closely mission of Pcrgnmon Press Ltd.

ACTIVATION AND CONTRACTION 117 opposing shortening. Gordon, Huxley and Julian (1966b) found that the speed of active shortening under light loads did not change appreciably with the amount of overlap at striation spacings greater than 2 y.m, and Edinan (1979) has shown that velocity of shortening under zero load is approximately constant for sarcomere length between 1.7 and 2.7 [isn. A. F. Huxley's 1957 theory. A hypothesis for the mechanism of contraction was put forward by A. F. Huxley in 1957. Many of the details are probably incorrect, such as the structural features of the model, the method of force production and the chemical assumptions, but the mathematical treatment is independent of the original assumptions and represents a class of models based on the idea of cross-bridges as independent force generators. The general outline of the theory is that cross-bridges operate in a cycle consisting of attachment with moderate rate constant, exertion of force, and detachment with high rate constant after sliding motion of the filaments sufficient to bring the force near to zero. An essential feature is that the probabilities of attachment and detachment of the cross-bridges are determined by their position. The essentials of the model are illustrated in figure 7. The contractile element shown is in the right-hand half of an A-band, so the actin filament is attached to a Z-line which is out of the picture to the right. During shortening, therefore, the actin filament moves to the left relative to the myosin filament. The distance of A, the active site on the thin filament, from 0, the equilibrium position of the M site on the thick filament, is denoted by x (which is positive when A is to the right of 0). The "side-piece" M oscillates over a range of ± 10 run about its equilibrium position 0 as a result of thermal agitation. If an A site happens to be within range there is a chance that attachment of M with A may occur, but in order to provide directionality the combination can occur only to the right of 0 \x positive) so that the tension in the elastic element is in the direction to cause the muscle to shorten. The AM combination moves towards 0 as the muscle shortens; there is a small chance that the link will be broken, but the probability increases greatly when the AM link passes 0 (and x becomes negative) and detachment occurs, thereby preventing the tension in the elastic link (which has now become negative a force tending to extend the muscle) from hindering the shortening of the muscle. At high rates of shortening a large proportion of the links will not be broken in time to prevent a considerable resistance being developed in this way; the speed of shortening of an unloaded muscle reaches its limit when this resistance just equals the force produced by the links to the right of 0. Because the maximum velocity of shortening depends on the balance of these forces, but not on their magnitude, it follows that maximum velocity should be independent of the number of sites that are active and should therefore be independent of the degree of activation of the muscle. Edman (1979) has presented data from experiments on single muscle fibres which show that the velocity of shortening under zero load is unaffected by conditions which substantially alter contractile force and thus strongly support the idea that maximum shortening velocity is independent of the number of myosin bridges that are able to interact with the rhin filament. The absolute value of ma-simnm velocity of shortening depends mainly on the value chosen for the rate of detachment during shortening; a high value reduces the proportion of attached bridges which can reach large negative values of x and consequently a higher shortening velocity can occur before the net force decreases to zero. The magnitude of the value of the rate of attachment mainly influences the number of bridges attached and in consequence affects the shape of the force-velocity relations (Simmons and Jewell, 1974). In his mathematical development of the theory, Huxley chose values for the rates of attachment and detachment which gave the best fit with the mechanical and energetic data then available (Hill, 1938). Mention has already been made of the need for further investigation of activation and maintenance heat (Curtin and Woledge, 1978; Homsher and Kean, 1978). In addition, the report by Allen (1978) that aequorin light emission decreases during shortening indicates a change in some facet of the activation process which has not yet been included in evaluation of the energetic cost of shortening. Another group of experimental findings which are not accounted for by the 1957 theory are the "transient" responses that can be observed when either the load on a muscle fibre or its length is suddenly changed. In this type of experiment, when a sudden small reduction of length is imposed during an isometric contraction, the ensuing tension changes show four phases (fig. 8): (1) an instantaneous decrease of tension, (2) an early tension recovery which lasts only a few milliseconds, (3) a reduction or reversal of the rate of tension recovery, and (4) a gradual recovery of tension with asymptotic approach to the isometric tension. Phase (4) resembles the final part of the increase in tension at the start of the tetanus and the 12

118 BRITISH JOURNAL OF ANAESTHESIA ilfsarcomere 110 kn m" J B A 8-4.5 110 kn m' 2-9 0.1s FIG. 8. (A) : U.v. record to show the sequence of events during a contraction. Initially the servo is controlled from the motor-position signal. At A, tension reaches a preset value, and is then held at that value by the servo so that the fibre shortens isotonically until B, when the sarcomere length reaches its preset value. The servo now holds sarcomere length constant at this value, and the contraction is truly isometric until E, apart from the shortening step (9 nm per half sarcomere) imposed at C, and local elongation during relaxation at D. The servo is permitted to return to tension control at E. Sarcomere length 2.2 \an; temperature 1.0 "C. (B): U.V. records of tension during tetani with step reduction of length of 4.5 nm and 9 nm per half-sarcomere. Same experiment as (A). The four phases of the tension transient are marked on the left-hand trace. The broken line on the 9-nm record is the tangent to the tension trace at the point of inflexion during phase (3); its intersection with the initial decrease of tension is taken as T, (see fig. 9). Compiled from Ford, Huxley and Simmons (1977), by permission of the Journal of Physiology. predominant process is presumably attachment of cross-bridges at the new fibre length. Phase (3) could be accounted for by a predominance of detachment over attachment, but this requires a modification to the 1957 theory in respect of the way in which the probabilities of attachment and detachment vary with the position of the cross-bridge (Huxley and Simmons, 1973). The early events which constitute phases (1) and (2) of the tension transients are shown in figure 9. In the tension trace there is an initial step at the time of the imposed length change, after which the tension recovers, in a few milliseconds, part of the way towards the isometric value. The relations between the length change and tension for a series of steps of different amplitude are also shown in figure 9. The curve TyJT 0 shows the extreme tension reached during the step change in length, and is a measure of the instantaneous stiflhess of the fibre. The upward concavity is largely a result of the rapid onset of the early tension recovery, so the true intercept is probably between 4 and 5 nm, and the relation practically linear. In contrast, the early tension recovery, TJT 0 is highly non-linear; recovery is almost complete in releases (and stretches) in which the length change is only a few nanometres, but becomes progressively less complete in the larger releases so that the curve is concave downwards with a slope somewhat less than that of the TJT 0 curve. This suggests that the structures responsible for the tension recovery are

ACTIVATION AND CONTRACTION 119 1.6- - I 5 nm/half-sircomere 1.4- - 12- -12-9 -6-3 Step amplitude per hilf-urcomere (nm) FIG. 9. Curves of 7\ (extreme tension) and 7", (tension approached during early recovery phase) in length-controlled steps of various amplitudes, both expressed as fractions of T o, the isometric tension immediately before the step. Circles and vertical lines refer to means and ranges respectively of four series of records from one fibre; triangles are the fourth run in the series from records shown in figure 12 of the paper. Sarcomere length 2.2 im; temperature 2.5 C. Inset shows the initial change in tension on a fast time-base for a 3-nm length change in the same fibre, and has been labelled to indicate r M 7\ and T,. Compiled from Ford, Huxley and Simmons (1977), by permission of the Journal of Physiology. not just passive visco-elastic elements but are the tension generators themselves, that is the crossbridges. The displacement of the main part of the TJT 0 curve some 6 nm to the left of the TJTQ curve suggests that some "active" part of the cross-bridge can take up approximately 6 nm of shortening while maintaining a tension not much less than it exerts in an isometric contraction. An important point in the development of a new theory was the discovery that, at different sarcomere lengths, the size of the responses to a given size of step varied in direct proportion to the isometric tension, and hence to the amount of overlap. Thus the instantaneous elasticity, as well as the early recovery, is attributable to the cross-bridges. Another non-linearity in the early tension recovery is the fact that its time course varies with the direction and amplitude of the length step, being slowest in a large stretch, fastest in a large release, and varying continuously in between. A. F. Huxley and Simmons' 1971 theory. The general outline of the theory is that attached crossbridges can exist in a small number of stable positions and that the tension in the bridge influences the probability of it being in a particular position. The essentials of the scheme are illustrated in figure 10, which is framed in terms of the "tilting head" crossbridge model of H. E. Huxley (1969), although there are many other structural possibilities, some of which are outlined by A. F. Huxley (1974). In the model the stable positions are those where a pair of sites is combined. During shortening the head attaches, rotates clockwise into successive stable positions and detaches when it has reached the last position. Clockwise rotation into successive positions occurs because the stable positions have progressively less potential energy, whereas tension in the elastic element tends to cause backward movement. In response to a step reduction in fibre length, the tension in the elastic element is reduced and this increases the tendency

120 BRITISH JOURNAL OF ANAESTHESIA Thick filament LMM S-2 u > 11 TTTT z> 1 2 Thin filament FIG. 10. Huxley and Simmons' (1971) version of the "tilting head" cross-bridge model to incorporate the elastic and stepwise-shortening elements inferred from the experiments on tension transients. The strength of binding of the attached sites is greater in position 2 than position 1, and in position 3 than position 2. During isometric contraction the myosin head oscillates rapidly between its three stable positions. The myosin head can be detached from position 3 with the utilization of a molecule of ATP; this is the predominant process during shortening. During stretch, the myosin head can dissociate from position 1 without utilization of ATP. Huxley (1974), by permission of the Journal of Physiology. of the head to rotate clockwise. The larger the shortening step, the less the tension in each elastic element and the greater the tendency to rotate. This movement therefore occurs more rapidly, and shows itself in the experimental records as the fast time course of early recovery following a large release. This explanation of the early tension recovery is strengthened by the observations of Ford, Huxley and Simmons (1974) that the instantaneous stiffness is slightly decreased towards the end of the early tension recovery. Thus the early tension recovery takes place without an increase in the number of attached cross-bridges. Relaxation When stimulation ceases during an isometric tetanus the tension remains constant for a brief period, starts to decline slowly and then decreases exponentially to the baseline. At the end of the slow decline, 3 the tension "shoulder" is accompanied by a rapid lengthening of part of the fibre. The converse of this observation can be seen in figure 8 where the "spotfollower" device was being used to hold the middle part of the fibre at constant length: the onset of the rapid exponential phase has been considerably delayed. In the 1957 theory the value for the rate at which the attached side-pieces turn over during isometric contraction was derived from the rate constant of the late exponential decrease in tension, but there is no longer any basis for this because substantial internal length changes are occurring at the time tension is decreasing exponentially (Huxley and Simmons, 1973). The removal of free calcium from the sarcoplasm is believed to occur by means of the sarcopksmic reticulum calcium pump. However, the active accumulation of calcium by the sarcoplasmic reticulum in isolated vesicles and skinned fibres appears to be somewhat less than is required to account for relaxation (Endo, 1977). The functional roles of calsequestrin and high-affinity calciumbinding protein are not yet well established, and the role of parvalbumin, a calcium-binding protein in the sarcoplasm, is unknown (Endo, 1977; Curtin and Woledge, 1978; Tada, Yamamoto and Tonomura, 1978). The remaining stages in relaxation are switching on the inhibition of actin by the troponintropomyosin complex and the detachment of crossbridges, the latter being the direct cause, but not necessarily the rate-umiting step, of the decrease in isometric tension. ACKNOWLEDGEMENT I am indebted to Professor B. R. Jewell for many helpful suggestions on the manuscript. REFERENCES Adrian, R. H. (1978). Charge movement in the membrane of striated muscle. Ann. Rev. Biophys. Bioeng., 7, 85. Allen, D. G. (1978). Shortening of tetanized skeletal muscle causes a fall of intracellular calcium concentration. J. Physiol. (JUmd.), 275, 63P. Bastian, J., and Nakajima, S. (1974). Action potential in the transverse tubules and its role in the activation of skeletal muscle. J. Gen. Physiol., 63, 257. Blinks, J. R., Riidel, R., and Taylor, S. R. (1978). Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin. J. Physiol. (JLond.), 277, 291. Caputo, C. (1978). Excitation and contraction processes in muscle. Ann. Rev. Biophys. Bioeng., 7, 63. Costantin, L. L. (1970). The role of sodium current in the radial spread of contraction in frog muscle fibres. J. Gen. Physiol., 55, 703.

ACTIVATION AND CONTRACTION 121 Costantin, L. L. (1975). Contractile activation in skeletal muscle. Prog. Biopkys. Molec. Biol., 29, 199. Taylor, S, R. (1973). Graded activation in frog muscle fibres. J. Gen. Physiol., 61, 424. Curtin, N. A., and Woledge, R. C (1978). Energy changes and muscular contraction. Physiol. Rev., 58, 690. Ebashi, S. (1976). Excitation-contraction coupling. Ann. Rev. Physiol., 38, 293. Endo, M. (1968). Calcium ion and muscle contraction. Prog. Biophys. Molec. Biol., 18, 123. Ohtsuki, I. (1969). Control of muscle contraction. Q. Rev. Biophys., 2, 351. Edman, K. A. P. (1979). The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres. J. Physiol. (Lond.), 291, 143. Endo, M. (1977). Calcium release from the sarcoplasmic reticulum. Physiol. Rev., 57, 71. Ford, L. E., Huxley, A. F., and Simmons, R. M. (1974). Mechanism of early tension recovery after a quick release in tetanized fibres. J. Physiol. {Land.), 240, 42P. (1977). Tension responses to sudden length change in stimulated frog muscle fibres near slack length./. Physiol. (Lond.), 268, 441. Franzini-Armstrong, C (1970). Studies of the triad. I: Structure of the junction in frog twitchfibres.j. Cell. Biol., 47, 488. Fuchs, F. (1974). Stirated muscle. Ann. Rev. Physiol., 36, 461. Gordon, A. M., Huxley, A. F., and Julian, F. J. (1966a). Tension development in highly stretched vertebrate muscle fibres. J. Physiol. (.Lond.), 184, 143. (1966b). The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J. Physiol. (Lond.), 184, 170. Hill, A. V. (1938). The heat of shortening and the dynamic constants of muscle. Proc. R. Soc. B, 126, 136. Hodgkin, A. L., and Horowicz, P. (1960) Potassium contractures in single muscle fibres. J. Physiol. (Lond.), 153, 386. Huxley, A. F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. (Lond.), 117, 500. Homsher, E., and Kean, C. J. (1978). Skeletal muscle energetics and metabolism. Ann. Rev. Physiol., 40, 93. Huxley, A. F. (1957). Muscle structure and theories of contraction. Prog. Biophys. Biophys. Chan., 7, 255. (1971). The activation of striated muscle and its mechanical response. Proc. R. Soc. B, 178, 1. (1974). Muscular contraction (Review Lecture). J. Physiol. (Lond.), 243, 1. Niedergerke, R. (1954). Interference microscopy of living muscle fibres. Nature (Lond.), 173, 971. Peachey, L. D. (1961). The maximum length for contraction in vertebrate striated muscle. J. Physiol. (Lond.), 156, 150. Simmons, R. M. (1971). Proposed mechanism of force generation in striated muscle. Nature (Lond.), 233, 533. (1973). Mechanical transients and the origin of muscular force. Cold Spring Harb. Symp. Quant. Biol., 37, 669. Taylor, R. E. (1958). Local activation of striated muscle fibres. J. Physiol. (Lond.), 144, 426. Huxley, H. E. (1969). The mechanism of muscular contraction. Science, N.Y., 164, 1356. Julian, F. J., Sollins, M. R., and Moss, R. L. (1978). Sarcomere length non-uniformity in relation to tetanic responses of stretched skeletal muscle fibres. Proc. R. Soc. B, 200, 109. Peachey, L. D., and Schild, R. F. (1968). The distribution of the T-system along the sarcomeres of frog and toad sartorius muscles. J. Physiol. (Lond.), 194, 249. Sandow, A. (1970). Skeletal muscle. Ann. Rev. Physiol., 32, 87. Simmons, R. M., and Jewell, B. R. (1974). Mechanics and models of muscular contraction; in Recent Advances in Physiology (ed. R. J. Linden), p. 87. Edinburgh: Churchill Livingstone. Tada, M., Yamamoto, T., and Tonomura, Y. (1978). Molecular mechanism of active calcium transport by sarcoplasmic reticulum. Physiol. Rev., 58, 1. Ter Keurs, H. E. D. J., Iwazumi, T., and Pollack, G. H. (1978). The sarcomere length-tension relation in skeletal muscle. J. Gen. Physiol., 72, 565.