Some advances in integrative muscle physiology

Comparative Biochemistry and Physiology Part B 120 (1998) 51 – 72

Review

Some advances in integrative muscle physiology Lawrence C. Rome * Department of Biology, Leidy Labs, Uni6ersity of Pennsyl6ania, Philadelphia, PA 19104, USA Received 3 June 1997; received in revised form 13 August 1997; accepted 22 August 1997

Abstract Integrative muscle physiology has evolved from black box correlations to an understanding of how muscular systems are designed at the molecular level. This paper traces some of the obstacles facing integrative muscle physiology and some of the intellectual and technological breakthroughs which led to the field’s development. The ability to determine (1) which fiber types are active, (2) over what sarcomere lengths and velocities they shorten during locomotion and (3) their respective force –velocity relationships, enabled us to show that many muscular systems are designed so that muscles operate at optimal myofilament overlap and at optimal V/Vmax (where maximum power is generated). The ability to impose the in vivo length change and stimulation pattern on isolated muscle has further showed that fish muscle has a relatively slow relaxation rate, and thus rather than generating maximum power during swimming, the muscle appears designed to generate power efficiently. By contrast, during the single shot jump, frog muscle remains maximally activated during shortening and generates maximum power. Recently biophysical techniques have shown that relaxation rate can be altered during evolution by changing (1) Ca2 + transient duration; (2) Ca2 + -troponin kinetics, and (3) crossbridge kinetics. New technologies will soon enable us to better appreciate how different animal designs evolved. © 1998 Published by Elsevier Science Inc. All rights reserved. Keywords: Integrative muscle physiology; Fibre types; Force – velocity relationships; Frog muscle

1. Introduction The field of integrative muscle physiology has undergone tremendous changes in the last 20 years. The field has evolved from simple unrelated observations of muscle function or locomotion, to ‘black box’ correlations, to an understanding of how muscular systems are designed from a phenomenological viewpoint, and to the beginning of understanding how muscular systems are designed at the molecular level. In the near future, new technologies will enable us to accurately predict and to empirically test how single molecular changes alter animal’s motor abilities, and this will enable us to better appreciate the evolution of different animal designs. * Corresponding author. [email protected]

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Rather than just summarizing results, in this paper I will also trace some of the obstacles facing integrative muscle physiology and the intellectual and technological breakthroughs which enabled the field to develop. In addition, I will also try to provide a vision of the future. For the sake of coherence and space, this paper will primarily focus on the research efforts of my laboratory. The research foci of other prominent members of the field are highlighted in the other papers in this series. The ability to pose and answer new questions sometimes reflects the influence of others. Dick Taylor is, in large part, responsible for the emergence of this field— through his own work, through organizing meetings which brought people together from different fields, and through inspiring a generation of young scientists. He has help to transform the field of integrative physiology into a rigorous scientific endeavor.

0305-0491/98/$19.00 © 1998 Published by Elsevier Science Inc. All rights reserved. PII S0305-0491(98)00023-6

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Fig. 1. Experimental design for comparing the effect of temperature on isolated muscle and whole animal locomotory energetics. Although this set of experiments was designed to assess the relative importance of performing work and generating force during running, there are a number of problems in making this comparison (see text).

I vividly remember my first meeting with Dick Taylor. As I was finishing my graduate school interview we were standing in the parking lot of the Concord Field Station where Dick was talking about integrating cellular muscle physiology and whole animal locomotion studies. He said that I could work at Harvard Medical School with Marty Kushmerick and Elwood Henneman and at the Concord Field Station with him and then integrate all the information. Although I hadn’t had any prior knowledge of fields of muscle physiology or locomotion—I was hooked by the beauty of the system, the ability to integrate, and by Dick’s enthusiasm. Of course it has taken me over 20 years to accomplish some of the items that Dick was expecting for my thesis—but optimism was Dick’s strong suit.

2. Using temperature to relate the mechanics and energetics of isolated muscle to the mechanics and energetics of animal locomotion When I was a graduate student, Dick and Norm Heglund were in the process of relating whole animal energetics to whole animal mechanics and developing their ‘Force Hypothesis’. The failure of differences of mechanical work production explaining the difference in energetic cost of locomotion with size [16] combined with the results of the energetics of running animals carrying different loads [57] led Dick and Norm and others to propose the ‘Force Hypothesis’. This hypothesis suggested that the energetic cost of terrestrial locomotion is set by the energetic cost of generating muscle force. A corollary was that the energetic cost of gener-

ating force was higher in small animals than in large ones, and this causes the energetic cost of locomotion to be higher in small animals. At this time I had developed considerable expertise in isolated muscle energetics and whole animal energetics. I had also developed a strong interest in temperature effects on muscle and locomotion from time spent in the laboratory of the late Frank Carey who studied ‘warm blooded’ tuna. I developed a thesis project using temperature as a tool to compare the energetics of isolated muscle and running animals to provide a simple test of the force hypothesis. Temperature was known to have a large effect on the economy of force production (Q10 = 2–3) but little effect on maximum efficiency (mechanical power output/metabolic energy input) [17]. Hence, because lizards running at a given speed at different temperatures should generate the same forces, if force generation was in fact responsible, there should be a large Q10 in the energetic cost of running. In the first set of experiments, I measured the cost of generating force in the isolated frog sartorius muscle between 10 and 30°C finding that indeed that the cost of generating force has a Q10 of 1.8–3.0 across the whole range of temperatures [43] (Fig. 1). I then ran savannah monitor lizards at the same speed at 28 and 38°C, and found that stride frequency and the net cost of running (active–resting metabolism) was exactly the same at both temperatures (i.e. the Q10 = 1) [35]. What I thought might be a likely explanation for this apparent paradox is that at low temperatures, the lizard might have to recruit faster fiber types than at high temperatures and this would reduce the Q10 of the energetic cost of running (i.e. faster fibers at low tem-

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peratures use ATP as rapidly as slower fibers at high temperatures) [35,38]. In addition to the seemingly paradoxical results which led to a new theory of muscle fiber recruitment (see below), this study was important as it represents one of the first direct comparison of isolated muscle properties and whole animal movements — a comparison that had been rarely done to that point. Although this study was cutting edge for 1980, there were several intellectual problems with this approach which became evident when trying to reconcile these seemingly paradoxical findings. The recognition of these problems and trying to solve them has helped to drive my research program over the past 15 years.

3. Problems

3.1. Problem 1 In these experiments we were comparing frog muscle to lizard muscle. At the time I was constrained to work only on whole muscles which had to be thin to avoid O2 diffusion limitations. This muscle geometry was exemplified by frog sartorius. Further, because of being constrained to work on frog muscle and because frog muscle could not be run at the high temperatures at which lizards locomote, the muscles could not be compared over the same range of temperatures [43]. What was necessary was developing expertise in making mechanics and energetic measurements on small bundles or single fibers from the animal in question.

3.2. Problem 2 Even if we were able to work with lizard muscle at the same temperatures, the problem still remains that whereas in the isolated experiment all the muscle fibers are being activated by direct electrical stimulation, in the locomoting animal the recruitment is controlled by the nervous system and generally only a fraction of the muscle fibers are recruited [35,38,43]. Thus the comparison might be invalid because we may be examining the properties of different fiber types in the isolated and whole animal experiments. Thus what was needed was an experiment model where it could be determined which muscle fiber types are being recruited at particular locomotory speeds.

3.3. Problem 3 According to previous energetic and mechanics measurements, the force, power output, efficiency, and cost of generating force are all dependent on the velocity at which the muscle is shortening (as well as its Vmax) [17,28]. In this study we had no idea at what velocity

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the lizard muscles were shortening and thus could not make an accurate prediction of the effect of temperature on energetic cost of locomotion. What was needed was a way to measure the length changes of the active muscles. It has become clearer through the years that the concept of comparing Q10s to try to deduce how muscles were operating is problematic. Q10s vary greatly depending on the length change and stimulation pattern of the muscle in the locomoting animal and there is not a unique Q10 for different classes of contractions [50]. Thus to understand what types of contractions the muscles are performing, it is necessary to measure them directly. Interestingly, some of these issues were addressed, albeit in an indirect way, in a parallel set of experiments during my thesis comparing the mechanics of isolated frog muscle to jumping performance of frogs at different temperatures. Because the whole frog generates approximately the same power during jumping as the product of the mass of the extensor musculature and the maximum power output of the isolated sartorius, I concluded that (1) all the extensor muscle fibers must be recruited and maximally activated and (2) they must be all shortening at the appropriate velocity for power generation (although this precise speed was not known) [18,36]. These conclusions have held, in general, through the intervening years, but they have become more convincing through direct measurements [29]

4. Solving the Problems Over the next 5 years I sought to solve these experimental problems by developing (or learning) appropriate techniques. The general experimental goal was to determine where muscle shortens over its sarcomere length-tension and force-velocity curves during locomotion

4.1. Problem 2 To determine what fiber types are recruited in a locomoting animal required a comparative approach, that is switching to fish where the fiber types are anatomically separated and can be monitored by EMG [45,46] (Fig. 2). Although we had tried a glycogen loss technique in savannah monitor lizards to determine which fibers were recruited during running, it did not work. Further, although recruitment of different fiber types had been measured in cats, this required impaling motor neurons in the ventral root—a technique which may be restricted to this species because of practical reasons. At the time I was finishing my PhD, Geoff Goldspink, a pioneer in fish muscle function, was on sabbatical at the Field Station, and invited me back to England to do a postdoc. At that time there was

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Fig. 2. Longitudinal view (a), dorsal view (b) and cross-section (c) of carp. The red muscle represents a thin sheet of muscle just under the skin which extends to a depth of only 10% of the distance to the backbone (the cross-section of the red muscle is exaggerated for illustrative purposes). Because the red fibers run parallel to the body axis, SL excursion depends on both curvature of the spine and distance from the spine. The trajectories of the white muscle fibers shown in a and b are based on Alexander’s [33] description. The white fibers lie closer to the median plane than the red ones, and they run helically rather than parallel to the long axis of the body. Consequently, they shorten by only one quarter as much as the red ones for a given curvature change of the body. Placement of electromyography (EMG) electrodes used to determine the activity of the red and white muscles are shown in c. Reproduced from [25].

considerable confusion about the recruitment of muscle fibers in a swimming fish — with claims that white muscle are used at all speeds [7,20]. We developed a method to monitor the fish movements and EMGs simultaneously which clearly showed that when fish swim steadily at a given speed, they recruit only their slow twitch red fibers [45,46]. At higher speeds, and during non-steady swimming (i.e. burst and coast), the fast twitch white muscle is also used. Fish recruited muscle fiber types in the same order at low and high temperatures; however the recruitment order is compressed at low temperatures. That is to compensate for the lower power output of muscle at low temperatures, the animal recruits more muscle fibers and faster fiber types [45,46]. This theory was later formalized and generalized and called the ‘‘Compression of the recruitment order theory’’ and was extended to cold blooded animals over a wide range of locomotory activity [37,38].

4.2. Problem 1 My switch to fish was made because the anatomical separation of their different fiber types permitted the use of EMG techniques to tell which fiber types are active. This same separation of the fiber types opened the possibility of using fine dissection techniques to obtain small bundles pure in fiber type with which the mechan-

ical properties of different fibers types can be measured. Much of the techniques for fine dissection had already been developed during the mechanical studies of single frog fibers in the 1960s [15]; however these techniques were applied only to a very narrow group of muscle fiber types. To solve problem 1, I did postdoctoral research with Fred Julian, where I learned to dissect single live and skinned fibers, and to make force and length measurements on single fiber preparations [26,27]. As soon as I obtained my first faculty position at the University of Tennessee, I applied these techniques to different fiber types in fish. Other groups, too, recognizing the advantage of this anatomical separation, used this fish model to explore muscle mechanics and energetics (e.g. [2,3,11,12,21]).

4.3. Problem 3 Finally, I needed a way to measure muscle length changes during locomotion. Again, as soon as I had my first position, I contacted Neill Alexander concerning how this might be accomplished. Alexander’s lab worked out a method of determining backbone curvature from cine’ films, and then relating that curvature to sarcomere length measured in fixed fish. We were able to improve the accuracy and speed using computer techniques to analyze the film, and were able to im-

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prove the accuracy of the sarcomere length measurements by incorporating frozen sections which did not shrink [42,48].

5. Myofilament overlap and V/Vmax — two design ‘goals’ of the muscular system With the ability to measure in a single species (1) which fibers types are active; (2) how they shorten during locomotion and (3) their respective Vmax values, we were in a position to test some specific hypotheses of muscular system design. Making all these measurements in one species was a major improvement over the more common procedure of combining data from disparate species (and sources), as this can lead to qualitatively incorrect conclusions. Rather than examining how the muscles of animals function, and devising a post hoc explanation, I believe it is necessary to develop a priori hypotheses so we know what to measure. Given that there are different types of muscular systems designed to perform different types of activities, rather than making narrow hypotheses about how a particular animal’s muscular system functions, we have made a series of more global hypotheses and tried to generalize the results to most vertebrates. Although our conclusions will not necessarily be true for every muscle in every animal, the emergent ‘design rules’ provides a benchmark from which divergence can be recognized and further explored. From cell physiology, we may anticipate that there are some rules [39,44] that are followed when an animal muscular system evolves. During steady activation, the force muscle generates depends on the amount of filament overlap between myosin and actin, or more precisely, the number of myosin crossbridges which can interact with actin sites [15]. It would seem sensible for animals to vary the gear ratio of their muscle fibers and their myofilament lengths, so that no matter what movements the animal makes, the muscle would operate at optimal myofilament overlap (i.e. where the muscle generates near maximal force). As such, gear ratio and myofilament lengths can be viewed as the design parameters (those components that can be varied during evolution). Myofilament overlap can be viewed as a design constraint or design goal (i.e. the rule by which the variation in parameters is adjusted). As both design parameters are anatomical features of the muscle, one being at the organ level and the other at the molecular level, this can be viewed as a structural design consideration. There is also a dynamic design consideration which takes into account that muscle shortens during locomotion. The force muscle generates is a function of V/ Vmax, where V is the velocity of shortening. More importantly, the mechanical power that a muscle gener-

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ates and the efficiency with which it generates the mechanical power are functions of V/Vmax as well. Again we might anticipate that the muscular system of animals who need to generate power (swimming fish, jumping frogs, flying birds) would be designed in a manner so that no matter what movement the animal makes, the muscle fibers operate over a range of V/ Vmax values (0.15–0.40) where the fibers generate maximal power with near maximal efficiency. Thus, the design parameters V and fiber gear ratio are varied in such a way that they operate under the design constraint of V/Vmax. (Note that during terrestrial locomotion the primary function of muscle is to generate force (as opposed to work), and hence these muscles may be designed to operate at a low V/Vmax, where the economy of force generation is highest [35,56]. It is important to emphasize that myofilament overlap and V/Vmax are potential constraints, which are derived exclusively from experiments on isolated muscle. We needed to determine whether animals actually use their muscles over this narrow range of values during their full range of locomotion, which had never been previously determined.

6. Fish Studies

6.1. Myofilament o6erlap Experiments on fish have provided considerable insight into myofilament overlap in vertebrates. During caudal fin propulsion, most fish bend their backbone. By a combination of high speed motion pictures, and anatomical and mathematical approaches which relate SL to backbone curvature, we [41,42,48] found that at low swimming speeds in carp, the red muscle (Fig. 3), which powers this movement, undergoes cyclical SL excursions between 1.89 and 2.25 mm centered around a sarcomere length of 2.07mm (Fig. 4A). Further, the thick and thin filament lengths of the red (1.52 and 0.96 mm) and white (1.56 and 0.99 mm) muscle in carp are similar to that in frog [53]. Using the frog SL-tension relationship to approximate that of the red and white muscle, shows that the red muscle operates over a range of SLs where no less than 96% maximal tension is generated (Fig. 4A) [41,42,48]. The most extreme movement carp make, the escape response (pictured in Fig. 5B) involves a far greater curvature of the backbone than steady swimming. If the red muscle were powering this movement, it would have to shorten to a SL of 1.4 mm where low forces and even irreversible damage can occur (Fig. 4B) [42,48]. Rather, it is the white muscle which performs the movement because the white muscle has a different fiber orientation than the red. The red muscle fibers run parallel to the long axis of the fish (Fig. 2) just beneath the skin.

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The white muscle fibers, by contrast, run in a helical orientation with respect to the long axis of the fish. As predicted by Alexander [1] and experimentally verified by Rome and Sosnicki [48], the helical orientation endowed the white fibers with a 4-fold higher gear ratio than the red fibers (i.e. the white muscle can produce a given backbone curvature while undergoing only 1/4 of the SL excursion). Thus to power this most extreme movement of fish, on average the white muscle must shorten to a SL of 1.82 mm and at this SL the muscle generates about 92% maximal force [48]. As shown above, the myofilament overlap is never far from its optimal level even in the most extreme movements. It appears, therefore, that animals are designed in such a way that no matter what the movement, the muscles used generate nearly optimal forces.

Fig. 4. Design constraint 1-Myofilament overlap. During all movements, muscle is used at nearly optimal myofilament overlap. During steady swimming (A), carp use red muscle over a SL of 1.91–2.23 mm, where no less than 96% maximal tension is generated. If the red muscle had to power the more extreme escape response (B), it would have to shorten to 1.4 mm where it generates little tension and can be damaged. Instead the white muscle which has a 4 ×greater gear ratio is used. In the posterior region of the fish, the white muscle shortens to only 1.75 mm, where at least 85% maximal tension is generated. In the rest of the fish the excursion is smaller (average 1.82 mm) and the force higher (average 92%). Reproduced from [26].

As such, myofilament overlap can be considered a design constraint (i.e. a part of the system that is kept constant). Given the movements that fish need to make, two design parameters (fiber gear ratio and myofilament lengths) are adjusted during evolution such that the muscle fibers being used always operate at near maximal myofilament overlap and force generation [44,48].

6.2. V/Vmax

Fig. 3. Electromyograms from red (R) and white (W) muscle of carp swimming at 10 and 20°C. From [12].

The two main fiber types in fish, red muscle and white muscle, have different Vmax values in addition to the aforementioned different gear ratios (N.B., pink muscle is abundant in some species, has intermediate properties and appears to follow the same set of design rules [10]). The first question which was asked is why

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Fig. 5. Carp swimming steadily at 25 cm s − 1. Five film frames separated by 0.1 s are shown (A).The escape response of a carp. Six consecutive frames at 5ms intervals (B). A resting carp received a 100 ms, 150 Hz sound pulse through an underwater speaker in the aquarium about 30 cm from the fish. In all cases fish were filmed at 200 frames per s. Note the difference in curvature and speed of these movements. The steady swimming is powered by slow red muscle which runs parallel to the long axis and the escape response is powered by fast white muscle which run in a helical pattern.

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do animals have different fiber types and are the faster fibers used to power faster movement (higher V) while operating at the same V/Vmax? As illustrated in Fig. 6, Rome et al. [42] found that the Vmax of carp red muscle was 4.65 muscle lengths s − 1 (ML s − 1) and the Vmax of carp white muscle was 2.5 times higher, 12.8 ML s − 1. During steady swimming the red muscle is used over ranges of velocities of about 0.7–1.5 ML s − 1 (Fig. 6A, shaded part of the curve; [41,42,47]). This corresponds to a V/Vmax of 0.17–0.36, which is where maximum power is generated. At higher swimming speeds (higher V) the fish recruited their white muscle because the mechanical power output of the red muscle actually declines. It is clear from Fig. 6A, that the red muscle cannot possibly power the escape response. To power the escape response, the red muscle would have to shorten at 20 ML s − 1, which it clearly cannot do, as this is four times its Vmax. Even if white muscle were placed in the same orientation occupied by the red (i.e. same gear ratio), it could not power the escape response either, because its Vmax is only about 13 ML s − 1. However, because of its 4-fold higher gear ratio, the white muscle need shorten at only 5 ML s − 1 to power the escape response (Fig. 6B). This corresponds to a V/Vmax of about 0.38, which is where white muscle generates maximum power [42,48]. If the white muscle does so well at producing fast movements, then why don’t fish have only one fiber type and let the white muscle power the slow swimming movements as well? The white muscle could certainly power slow swimming, but it is not used because its high Vmax and 4-fold higher gear ratio would make its V/Vmax at slow swimming speeds, so low (i.e. 0.01– 0.03), that the muscle is nearly isometric and efficiency is nearly 0 (shaded portion of Fig. 6B). Thus the red and white muscle forms a 2 gear system. To achieve a wide repertoire of movements fish must use different fiber types with different Vmaxs and different gear ratios to power different movements. The red muscle powers slow movements, while the white muscle powers very fast movements, both while working at the appropriate V/Vmax (0.17–0.38). The effectiveness of white muscle to power fast movements depends on the product of its gear ratio and Vmax. In terms of backbone curvature, the white muscle can produce 10-fold faster movements (2.5-fold higher Vmax × 4-fold higher gear ratio) than the red muscle. If the red muscle had the same gear ratio as the white, it could not produce the escape response, nor could the white muscle if it had the gear ratio of the red. What is needed is both the correct Vmax and the correct gear ratio to produce very rapid movements [42,48]. Additional studies have revealed that fast and slow swimming species of fish [40,49], fish swimming at low

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Fig. 6. Design constraint 2-V/Vmax in carp. During slow movements and fast ones the active 6fibers always shorten at a V/Vmax of 0.17 – 0.38 where maximum power and efficiency are generated. During steady swimming (red muscle), the fibers are used at a V/Vmax of 0.17 – 0.36 (A). The red fibers cannot power the escape response because they would have to shorten at 20 ML s − 1 or 4 × their Vmax. The escape response is powered by the white muscle which need shorten at only 5 ML s − 1 (V/Vmax =0.38) because of its 4×higher gear ratio (B). The white muscle would not be well suited to power slow swimming movements, as it would have to shorten at a V/Vmaxof 0.01 – 0.03, where power and efficiency are low. Thus fast movements are obtained with fibers with a high Vmax and a large gear ratio.

and high temperatures [40,41,47,49], and even jumping frogs [29] also use their muscle at optimal V/Vmax (0.17 –0.36). This provides additional evidence that in animals which need to produce high mechanical power, V/Vmax is an important design constraint. It appears from these examples that many animals use their muscles over a narrow range of myofilament overlap and over a narrow range of V/Vmax where muscle generates maximum force and maximum power with optimal efficiency. Therefore during evolution, three design parameters (gear ratio, Vmax and myofilament lengths), appear to have been adjusted so as to obey these design constraints no matter what movement is made. Hence, these design constraints appear to constitute two of the rules by which muscular systems have been put together.

7. The importance of activation and relaxation processes in muscular system design: what is the design constraint? It is important to realize that the SL-tension curve and force–velocity curves are steady-state properties of maximally activated crossbridges and do not account for the fact that muscle must be turned on and off during locomotion. To fully understand the design of the muscular system, it is important to examine the kinetics of activation and relaxation. Bob Josephson led the way in expressing the importance of activation and relaxation processes in muscle design and pioneered the use of the so-called work loop technique on synchronous muscle [22–24]. In this type of experiment the muscle is driven under oscillatory

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length changes, stimulated under various conditions and the resulting force and work measured. This examination demonstrated that mechanical work production was influenced dramatically by the stimulation pattern and that the net power during cyclical contractions was lower than that predicted from one-half of the instantaneous power during steady shortening. Josephson also showed that the power output of the muscle could be optimized by adjusting the length changes and stimulation pattern. This procedure which we will call the ‘optimized workloop technique’ was seized upon by a number of groups because it was claimed that the workloop was a more realistic representation of muscle function during locomotion than had been the force – velocity curve, e.g. [3,4]. My laboratory was slow to incorporate the technique as we did not believe that driving a muscle through arbitrary length changes, with an arbitrary stimulation duration and phase would represent muscle function in vivo either. In our eyes, a more robust use of the ‘work loop’ approach would be to measure the length changes and stimulation pattern the muscle actually undergoes in vivo, and then drive isolated muscle through these same length change and stimulation conditions and measure the resulting force and power. We thus moved into this area when we had developed the means to make in vivo length changes and stimulation pattern measurements. We believed that the in vivo workloop approach could be used to determine the design constraints for setting the kinetics of activation and relaxation. Fig. 7 shows two possible design constraints relevant to cyclical locomotion—one for maximum power output and the other for maximum efficiency. Intuitively it seems beneficial that fibers be fully ‘activated’ during shortening and to fully ‘relax’ prior to relengthening. If the muscle were able to instantaneously activate and instantaneously relax, then the muscle could be maximally activated throughout shortening and generate maximum power (Fig. 7, left panel). However, there is a problem at the molecular level. To understand how muscles are designed to activate and relax at different speeds, it is necessary to examine the molecular processes of activation and relaxation. Muscle is activated by the release of calcium from the sarcoplasmic reticulum (SR, which stores Ca2 + ) into the myoplasm, Ca2 + in turn then binds to troponin removing the inhibition from the thin filament, and thereby allowing the myosin crossbridges to attach and generate force (Fig. 8). To get the muscle to relax, the process must be reversed; Ca2 + must unbind from troponin, so that inhibition can be returned to the thin filament. Thus the myoplasmic calcium must be lowered and this is done by Ca2 + being pumped into the SR, which requires ATP. Ca2 + must then unbind from troponin, and finally the crossbridges must detach (Fig. 8).

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If the muscle is maximally activated throughout shortening, then it must relax (i.e. Ca2 + must be sequestered, Ca2 + must come off troponin and crossbridges must detach see Fig. 8) instantly at the end of shortening so that the muscle would generate little resistance when it is relengthened. However, a muscle built so that is can relax ‘instantly’ is very expensive to run (see Sound Producing muscles). There are two kinetic reasons. First, to relax quickly, muscles must pump Ca2 + very rapidly back into the SR. If the pumps were dormant until the end of shortening and then turned on, fast pumping would not be more costly than slow pumping because a fixed amount of Ca2 + would have to be pumped and it does not matter how fast this is done. However, Ca2 + pumps continue pumping throughout contraction, hence additional Ca2 + must be released during the contraction to keep the muscle activated. Thus, if Ca2 + is pumped at a high rate, Ca2 + must be released at a high rate to keep the muscle activated, and the total amount of Ca2 + pumped, and thus the amount of ATP associated with Ca2 + pumping, will be high. Second, ‘instantaneous’ relaxation requires crossbridges to detach very rapidly. To maintain force, crossbridges would also attach very rapidly, which leads to fast cycling. Fast cycling crossbridges also use ATP much faster than slower cycling crossbridges [19]. By contrast, a muscle with more moderate calcium pumping and crossbridge cycling rates (Fig. 9 right panels) will be energetically less expensive, and may perform mechanical work more efficiently (mechanical power output/metabolic power input). This is of special concern for a muscle such as the red swimming muscle of a scup which is used continuously during seasonal migrations of several hundred miles. To guarantee that in a slow relaxing muscle, the muscle is nearly relaxed prior to being relengthened, the stimulation duty cycle must be shortened, and shifted to start during lengthening and actually end right after the beginning of shortening. Ironically, (and unavoidably) this means that during the power stroke (shortening phase) the muscle is actually in the process of relaxing. Hence the net work level is far less than in the fast relaxing muscle. Thus the question is whether fish are designed with fast relaxing but energetically costly muscle, or with a slow relaxing, but less energetically expensive muscle. The only way to answer this question is to determine the nature of the workloops in the locomoting animal. To determine the nature of the workloop in swimming fish, we first swam fish at 80 cm s − 1 and measured EMGs and muscle length changes of the red muscle at 4 places along the length of the fish (ANT1, ANT2, MID, POST) (Fig. 9) [51]. It was found that moving caudally along the length of the fish, the length change of the muscle becomes larger. Strains were

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Fig. 7. Hypothetical workloops for muscles driven under oscillatory length changes. The left panel shows a muscle with sufficiently fast activation and relaxation rates that the processes are completed in a small portion of the cycle, and hence appear instantaneous. The right panel shows a slow relaxing muscle driven under the same length charges. To permit the muscle to relax prior to relengthening requires shortening the duration and shifting the stimulus to precede shortening and to cease just after the beginning of shortening.The amount of net work the muscle generated during a length cycle, is graphically equivalent to the area contained within the force – length loop bottom panel. A muscle generates positive work only when it is shortening (FxDL), and the quantity is equal to the area under the force – length curve during the shortening phase. A muscle generates negative work when it is forcibly lengthened by a servomotor (or contralateral muscle) and the quantity (FxDL) is equal to the area under the force – length curve during lengthening phase. Hence, the net work is the area of the curve. The timing of the stimulus is signified by at thick endline.

9 1.5% of resting muscle length at ANT-1 increasing to 95.7% at the POST; (Fig. 9B). In addition, the EMGs had a longer duty cycle in the anterior of the fish than in the posterior (Fig. 9A). This is due primarily to a large difference in the onset time of the EMG in the anterior compared to the posterior, but nearly simultaneous off times. Finally EMGs precede the length changes by increasing amounts along the length of the fish towards the tail (Fig. 9A, B; Fig. 10A, B). To determine the mechanical performance of the muscle during this swimming behavior, we [51] drove red muscle bundles isolated from the four positions

on the fish through the length changes and stimulation conditions that they undergo during swimming and measured the resulting force and power the muscle generated. We found that despite the fact that the POST muscle is stimulated primarily during lengthening (Fig. 10), it generated large amounts of work. This finding demonstrates that the time course of muscle activation differs dramatically from the time course of EMG activity—assuming that they are the same (which is typically done) can lead to qualitatively incorrect conclusions. The anterior muscle, on the other hand, generated significantly less work under its in vivo conditions (Fig. 9, Fig. 10A, B).

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The cause of the low power generated in the anterior region of the fish is not due to an intrinsic deficiency of the anterior musculature, rather it is due to the specific length change and stimulation pattern that the anterior muscle undergoes. For instance when the ANT-1 muscle is driven through the length change and stimulation pattern that the POST muscle normally undergoes in vivo, it generates just as much power as the POST muscle (Fig. 10). The main reason for the low power output in the anterior region is the small strain (dl). A small dl reduces work output (Fxdl) per tailbeat. In addition, muscles undergoing small strains do not relax sufficiently fast. Relaxation is sped up greatly by muscle shortening (shortening deactivation), but the shorter the strain, the smaller is this enhancement of relaxation [3,25,50]. The fact that the ANT-1 muscle can produce any work at all during swimming has required a significant modification of the muscle. For instance, when the POST muscle is driven through the length change and stimulation pattern the ANT-1 muscle normally undergoes in vivo, it performs even worse than the ANT-1 muscle and generates no net work (compare Fig. 10II and III). The primary reason that the ANT1 muscle can generate significant power despite undergoing only a small strain (Fig. 10II) is that it has the

Fig. 8. Major kinetic steps in muscle activation and relaxation. Activation: (1) Ca2 + is released from the SR into the myoplasm. (2) Ca2 + binds to troponin, releasing inhibition of the thin filament. (3) Crossbridges then attach. Relaxation: (4) Ca2 + is resequestered from the myoplasm by the Ca2 + pumps. (5) Ca2 + comes off troponin, thereby preventing further crossbridge attachment. (6). Cross-bridges then detach. For a muscle to relax rapidly, steps 4–6 must all be very fast (see sound producing muscles).

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intrinsic ability to relax much more rapidly than the muscle in the posterior (Fig. 10A). Because of its slow intrinsic relaxation rate, POST muscle driven through the ANT-1 conditions does not come close to relaxing between stimulus trains, whereas the ANT-1 muscle relaxes almost completely (Fig. 10IID,IIID). Why does the ANT-1 muscle relax faster? We have eliminated differences in Vmax (i.e. the ANT-1 and POST have the same Vmax), and we believe it is due to prolonged troponin occupancy in the POST [55]. For instance, pharmacologically slowing of the Ca2 + pumps (with cyclopiazonic acid) leads to a slower relaxation rate and reduced power production [54,55]. Although we have not yet demonstrated the exact mechanism, these results show that relaxation rate is malleable and that it can greatly effect power production by the muscle. How is the muscle designed? Fish muscle is designed with a relatively slow activation-relaxation rate. Accordingly, during swimming, that stimulation duty cycle of the POST muscle (which generates most of the power) was short (27%) and in fact started during the last half of lengthening and ceased just after the beginning of shortening (compare Fig. 10, left panel to Fig. 7). Thus, the muscle is in the process of relaxing during the power stroke enabling the muscle to be nearly relaxed prior to being relengthened. The net power in the posterior muscles during swimming is close to the maximum power the muscle can generate during cyclical length change. However, the power was far less than the muscle could generate if it remained maximally activated during shortening, and then instantly relaxed at the end of shortening [49]. Apparently, to avoid the high cost of fast Ca2 + pumping, there is a compromise made that reduces power production. Hence, rather than being designed to generate maximum power, the muscle appears designed so as to generate power efficiently [51]. This hypothesis needs to be verified empirically by measuring efficiency of power production while the muscle is undergoing its in vivo length change and stimulation pattern. Prior to describing the use of this general technique to examine another form of locomotion (frog jumping), it is useful to reflect upon the importance of the ‘in vivo workloop’ technique (also used by Marsh and colleagues on scallops [33]). These experiments demonstrate several crucial features of this technique for integrative physiology. First, one cannot tell how a muscle has been designed by the properties of the isolated muscle, in the absence of knowing how the muscle is used in vivo. For instance, without knowing the length changes and stimulation pattern the ANT1 muscle undergoes during swimming, the performance of ‘optimized workloops’ would erroneously

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Fig. 9. Length changes, stimulation pattern, force production and work output of red muscle of scup during swimming. Step1 was to measure in a swimming (80 cm s − 1) fish the EMGs (A) and length changes (B) for the red muscle at four places along the length of the fish pictured in G. Step 2 was to impose on muscle bundles isolated from these four positions the length changes (D) and stimulation pattern (C) that were observed during swimming. Step 3 was to measure in the isolated muscle the resulting force production (E) and work production (F). Note that the reason for the apparent discrepancy between traces A–B and C–D is that A – B represent records from one of the fish (tail beat frequency =6Hz) while C–D represent the record for a muscle driven through the average swimming values (i.e. tail beat frequency = 6.4Hz). Traces A – E are all functions of time. Trace F is a plot of force produced against length changes where the area of the enclosed loop is the work produced during a tailbeat cycle. This value is much larger in the POST than in the ANT-1 position. Adapted from [43].

suggest that the ANT-1 muscle is capable of generating high mechanical power during swimming. Its anatomical position in the fish precludes this. Second, in addition to simply measuring power output, this technique permits one to partition the effects of muscle properties versus length change and stimulation conditions on power production. For instance, by being able to drive the ANT-1 and POST muscle types through the ANT-1 in vivo conditions (conditions that the POST muscle would ordinarily never see), we were able to answer a ‘what if?’ question. The answer conclusively showed that the low power output of the ANT-1 muscle in vivo is due to non-optimal length change and stimulation conditions, not to the contractile properties itself. This general paradigm can be used in a variety of ways to better appreciate muscular system design by asking other ‘what if?’ questions. For instance, if during thermal acclimation the output of the nervous system changes, how might power production change?

8. One shot locomotion—frog jumping The fundamental difference between frog jumping and fish swimming is that muscles used for jumping need only be designed to perform single shortening strokes, whereas those used for swimming must perform cyclical contractions [11,43]. This results in important differences in the respective designs of the muscular systems. During a maximal jump, a frog accelerates from a stationary crouched position to high vertical and forward velocities in under 100 ms [8,18]. To produce these rapid increases in potential energy and kinetic energy, the muscles of the frog must generate high mechanical powers. Ultimately, the distance frogs travel is directly proportional to power production [18]. For the muscles of the frog to generate their maximum mechanical power, they must (1) operate over the plateau of the sarcomere length (SL)-tension curve where maximum force is generated; (2) operate at the optimal V/Vmax where maximum power is generated,

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Fig. 10. Mechanical properties of ANT-1 and POST muscles. Columns I and II show a POST and a ANT-1 muscle bundle undergoing their respective length changes and stimulation pattern that the muscle undergoes during swimming. By contrast column III shows a POST muscle undergoing the stimulation pattern and length changes that is encountered by the ANT-1 muscle during swimming. Trace A shows the isometric twitch of the muscles in question. Traces B and C show the imposed stimulation pattern and length changes determined during swimming experiments. The resulting force is shown in trace D and the resulting work (area enclosed by loop) in E. A – D are functions of time, whereas E is force as a function of length. Note the large strain in the POST compared to the ANT-1 and the much larger work produced in that muscle. Note also that relaxation is much faster in muscle undergoing shortening (D, caused by shortening deactivation) than that being held isometrically (A). Finally note that relaxation is much faster in ANT-1 muscle than POST (A). This permits the ANT-1 muscle (IIE) to perform work under conditions where the POST muscle cannot (IIIE). Adapted from [43].

and (3) be maximally activated. Lutz and Rome [29], using similar techniques as used in fish, examined the function of the semimembranosus muscle (SM) in Rana pipiens by reproducing the in vivo length change and stimulation pattern in isolated muscle (Fig. 11).

8.1. Myofilament o6erlap Because measurements of tension and active SL showed that the standard frog SL-tension curve [15] fit the SM data, it was used to represent the SL-tension curve of the SM (Fig. 12). During jumping, the SM

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Fig. 11. Muscle function during jumping in frogs. Figure A, B, show the length change and stimulation pattern the SM undergoes during a maximal jump. C, D, show an isolated muscle bundle driven through the in vivo length change and stimulation pattern, and E shows the resulting force production of the muscle.The stimulation duration was determined from the EMG.The phase of the stimulus with respect to the length change was determined in the following fashion.The initiation of shortening was determined by extrapolating the constant velocity portion of the length record back to zero length (B). The lag between the stimulus and shortening was defined as the time between the onset of the EMG and the initiation of shortening. Because during jumping, the early portion of the length record was curved, digital smoothing was used to obtain the correct shape of the computer generated length change (D). The dashed line (E) is isometric force and the dotted line is the steady-state force generated by the same muscle at the same V during a force-velocity experiment. The jump shown in A,B is the longest measured (distance = 0.8 m), and this was reproduced in C–E. Reproduced from [11].

operates largely over the plateau of the SL-tension curve where maximum force is generated. The fact that the SM shortened somewhat beyond the plateau of the SL-tension curve deserves some attention. We might have expected that the muscle is used only over the plateau of the relationship. However, because the width of the plateau is only 10%, the 25% SL change which occurs during the jump obviously cannot fit entirely on the plateau. Nonetheless, even though the muscle operates on either side of the plateau, it generates at least 90% of the maximum tension of which it was capable throughout shortening, which is close to maximal (Fig. 12). Why isn’t the length change reduced so it fits on the plateau? Jump distance is set by the total amount of work done

at takeoff, and work is the product of force and length change (i.e. work= FxD length). Thus the long sarcomere length changes enabled the muscle to do far more work, and hence the frog jumps further while incurring only a small decrement in force. Further, additional measurements showed that given the large length change that the muscle does undergo, the placement on the SL-tension relationship is almost perfect [30,31]. Because of more subtle properties of the muscle, setting the initial SL to either a longer SL or a shorter SL would both result in lower work production. Thus it appears that the sarcomeres function in a very effective manner. How is this effective sarcomere length change pattern achieved? There must be matching between the frog’s

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Fig. 12. Where does the SM muscle operate on its SL-tension and force-velocity curves? (A) The muscle shortens from a SL of 2.34 to about 1.83 mm at take off. Even at 1.83 mm, however, the muscle still generated over 90% tension. (B) Shows typical force – velocity and power–velocity curves. The power curve was simply calculated from the force – velocity fit. At the V used during jumping, the muscle operates over the portion of the power curve where at least 99% of maximum power is generated. Reproduced from [11].

skeleton, the joint angle changes during jumping, the position of muscle attachments (moment arm or gear ratio), the myofilament lengths (i.e. different muscles have different myofilament length and thus different SL-tension curves), and the number of sarcomeres in series. To put it more simply, given the joint angle changes, the moment arm of the muscle, the myofilament lengths, and the sarcomere number must be appropriately set so that the initial sarcomere length in the ‘crouched’ position represents a nearly optimal solution for shortening during jumping.

8.2. V/Vmax Vmax of the SM was 10.35 ML s − 1 and the V at which maximum power was generated was 3.44 ML

s − 1 (Fig. 12B) which matches closely the average V during jumping (3.43 ML s − 1). This occurs at a V/ Vmax = 0.33 (Fig. 12). These results suggest that the Vmax has been adjusted during evolution such that during jumping, the muscle operates at the appropriate V/Vmax for maximal power generation.

8.3. Kinetics of acti6ation, kinetics of relaxation Although the SM shortens at the correct SLs and V/Vmax for maximum power generation, the question arises whether the muscle is stimulated for a sufficient time before shortening to be maximally activated during jumping. In terms of frog jumping, ‘activation’ was considered to be maximal if the muscle generated the same force (and power) under in vivo conditions as it

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does at the corresponding V during force-velocity experiments (where maximal activation is achieved by stimulating for 60 ms before shortening). Despite the short lag between the EMG and muscle shortening (18 ms), the force the muscle generates under the in vivo conditions (Fig. 11, C to E), is greater than or equal to the force generated at that V on the force-velocity curve (Fig. 12), suggesting that the muscle is maximally activated. Further mechanical studies showed that the fibers were maximally activated by the beginning of shortening and hence throughout the shortening phase. Thus the rate of activation is sufficiently rapid to ensure maximal activation during jumping. Comparison of isolated muscle and whole animal power production further support the conclusion that fibers were maximally activated and show that most of the extensor muscles in the frog hindlimb probably behaved similarly to the SM during jumping. The peak power required to accelerate the frog during jumping (determined from films of jumping frogs) was 67.2 W kg − 1 of body mass. Because the maximum power generated by the SM was 371 W kg − 1 of muscle and only about 17% of the body mass can be involved in powering jumping, the maximum possible whole animal power =371 W/(kg muscle)×0.17 (kg muscle/kg of body mass) =63 W/(kg of body mass). The close match between the power required to accelerate the frog (from films) and the maximum available from the muscle suggest that all the muscle fibers from each extensor muscle are probably recruited, and are stimulated at a sufficiently high frequency, and shorten over the appropriate SLs and V/Vmax for maximum power generation during jumping. This conclusion is further supported by our recent finding that unlike smaller muscles typically used in muscle physiology experiments (e.g. sartorius muscle which contains a significant amount of slower fiber types), the large extensor muscles of R. pipiens are composed almost entirely of the fastest fiber type (amphibian type 1) [32]. Thus the rates of activation (which includes calcium release, binding to troponin, crossbridge attachment and force generation) are all sufficiently rapid so that the muscle is maximally activated prior to the beginning of shortening. This is crucial point, because if the muscle starts to shorten before the muscle has been stimulated for sufficient time, then the muscle never becomes maximally activated during the jump. The time taken between the initiation of stimulation and the start of shortening involves the biomechanical arrangement of the muscles and the limbs, the inertia of the frog, and the kinetics of force generation. These parameters are set so that the muscle does not start to shorten appreciably prior to achieving maximal activation.

In short, the muscular system of jumping frogs appears to have been designed to generate maximum power. Not only do the muscles shorten at the appropriate SL and at the appropriate V/Vmax, but (1) the rate of activation is coordinated with the frog biomechanics, so that the muscle can maximally activate prior to shortening and (2) the muscle shows relatively little shortening deactivation and hence generates maximum power throughout the jump [30,31] Thus, the muscles of frog and those of fish are used in fundamentally different manners and have different properties. Frog muscle is designed to generate maximum power during a single shortening stroke, thus force stays high throughout shortening (Fig. 13 left). By contrast, the force the fish muscle generates during shortening declines dramatically (Fig. 13 right) due both to the early cessation of the stimulus and to intrinsic shortening deactivation which is far more pronounced in fish than frog muscle. This is necessary, however, if the muscle is to be nearly relaxed prior to being relengthened—a requirement for power generation during cyclical activity. One common factor between the locomotory muscles of frog and fish is that they have relatively slow relaxation rates.

Fig. 13. Different muscle designs for different types of locomotion. Force-length records for frog muscle during jumping and fish muscle during swimming.Thickened lines signify when in the contraction the muscle is being stimulated. Note that the force falls dramatically during shortening in the fish to ensure relaxation prior to relengthening. By contrast in the single shot jump of the frog where the muscles are not so constrained force remains high throughout shortening, thereby ensuring maximum power generation.

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Fig. 14. Twitch tension (upper) and calcium-transients (lower) of three fiber types from toadfish at 16°C (A) and of sonic fibers at 16 – 35°C (B). In each case, the force and the calcium transient have been normalized to their maximum value. (A) The twitch and calcium transient become briefer going from a the slow-twitch red fiber (r), to the fast-twitch white fiber (w) to the superfast-twitch swimbladder fiber(s). (B) Records from rattlesnake shaker fibers (RS; dotted) at 16 and 35°C and swimbladder fibers (s, solid) at 16 and 25°C.Note that the time scale is expanded about 30 ×in B. The same traces from the swimbladder at 16°C are shown in both A and B. Adapted from [52].

9. Sound producing muscles Up to this point our measurements of activation and relaxation have been phenomenological in nature. The question still remains what adjustments are made at the molecular level to set the appropriate activation and relaxation rate? Or, in the case of the ANT-1 muscle in scup, what molecular modifications endows it with the faster relaxation rate which has such a dramatic impact on function? To explore the activation and relaxation from a more mechanistic viewpoint, we examined one of the most spectacular examples of adaptation— sound producing muscles. The male toadfish (Opsanus tau) produces a ‘boatwhistle’ mating call 10 – 12 times min − 1 for many hours to attract females to its nest. This tone is generated by oscillatory contractions of the muscles encircling the fish’s gas-filled swimbladder at 200 times s − 1, making it the fastest vertebrate muscle known. If these sonic muscles were replaced with typical slow-relaxing locomotory muscles and stimulated at 200 Hz, they would be unable to relax between stimuli and consequently produce completely fused (i.e. constant force) tetani. Such maintained tension would simply compress the bladder and prevent it from vibrating. Thus, sonic muscles must be specifically adapted to turn on and off rapidly. To better understand the modification of the muscles which permit them to turn on and off so rapidly, we

[52] examined the changes in physiological properties of different fiber types from toadfish as the rate of muscle activation-relaxation varies by  50-fold between locomotory and sonic fiber types. Toadfish red muscle (used for slow steady swimming at  2Hz) has a twitch half-width (the time duration at the 50% force level) of about 500 ms, compared to approximately 200 ms for white muscle (used for burst swimming at  5Hz), and about 10 ms for the swimbladder muscle (upper panel of Fig. 14A). For a muscle to activate and relax rapidly, two conditions must be met. First, calcium, the trigger for muscle contraction, must enter the myoplasm rapidly and be removed rapidly (Fig. 8, step 1,4). Second, myosin cross bridges must attach to actin and generate force soon after the calcium level rises and then detach and stop generating force soon after the calcium level falls (Fig. 8, Steps 2,3,5,6). To test for the first condition, we measured the change in myoplasmic free (Ca2 + ) during contractions [52]. This was accomplished by injecting muscle cells with Ca2 + sensitive dyes, which change their fluorescence depending on whether Ca2 + is bound to the dye or not (Fig. 14). Thus by tracking fluorescence with time, we can determine the time course of Ca2 + release and reuptake. We found that the calcium transient in the sonic muscles is the fastest ever measured for any fiber type (a half-width of  3.4 ms at 16°C and 1.5 ms at 25°C). The importance of the calcium transient

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Fig. 15. Calcium transients and force production during repetitive stimuli. (A) Slow twitch red fiber stimulated at 3.5 Hz. The threshold [Ca + 2] for force generation was derived from Fig. 16 and is shown with a dotted line. (B) Swimbladder stimulated at 67 Hz. Note that the threshold is much higher for the swimbladder than for the red fiber. Note also the large magnitude of the first swimbladder Ca2 + transient compared to subsequent ones, and the different calibration for the ordinate. Both toadfish fiber experiments were performed at 16°C. (C) Rattlesnake shaker fibers at 16°C stimulated at 30 Hz. (D) Shaker fibers at 35°C stimulated at 100 Hz. Calcium thresholds are not shown for shaker fibers. Reproduced from [52].

duration in setting the twitch duration can be seen in Fig. 14, which shows that in going from the slow red fibers to the super-fast swimbladder fibers, both durations sped up in parallel by 50 fold. The significance of a fast Ca2 + transient is best illustrated during repetitive stimulation. During stimulation of slow red muscle at a modest 3.5 Hz (Fig. 15), the decay of the Ca2 + level is so slow that [Ca2 + ] does not have time to return to baseline between stimuli. Even the lowest [Ca2 + ] during the contraction is above the threshold required for force generation in this fiber type, and therefore the relaxation of force between stimuli is incomplete, resulting in a partially fused tetanus. By contrast, the swimbladder’s calcium transient is so rapid that even with 67 Hz stimulation, [Ca2 + ] returns easily to baseline between stimuli (Fig. 15; note the 50 × faster time base). In addition, except for the first stimuli, [Ca2 + ] is below the threshold for force generation more than half of the time. Hence, the Ca2 + transient is sufficiently rapid to permit the oscillation in force required for sound production. Even though [Ca2 + ] returns rapidly to baseline, the swimbladder fiber could not relax quickly unless its troponin rapidly released bound Ca2 + (Fig. 8, Step 5). Indeed, kinetic modelling [52] indicates that if the swimbladder troponin had the off-rate for calcium (koff)

estimated to apply to fast twitch fibers of frog (115 s − 1) then occupancy of its troponin sites with Ca2 + would not decline sufficiently rapidly to permit the observed rapid and nearly complete fall in force. To assess possible adaptations in the Ca2 + -troponin control sys-

Fig. 16. Force-pCa curve for fast twitch fibers of the frog Rana temporaria was also measured for comparison. For each fiber type an individual force-pCa data set is shown along with a curve fitted using the equation: % force =(100 ×(10 − pCa)N/(10 − pCa)N +(10−pCa50)N). (2) Note that the force from swim bladder fibers rose much more sharply than the fitted curve at forces below 50% and more gradually than the fitted curve at forces above 80%. Reproduced from [52].

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Fig. 17. Force-velocity curves of toadfish and rattlesnake fibers. For each fiber type an individual force-velocity data set is shown. The corresponding unconstrained Hill equation is fitted up to 80% isometric force (solid), beyond which the curve is approximated with a spline fit (dotted).The force-velocity curves are shown for the red (r), white (w), and swimbladder (s) of toadfish, and the rattlesnake shaker fibers (rs; open symbols, dashed curves). All toadfish fibers were measured at 16°C. Adapted from [52].

tem, the force-pCa relationship was measured (Fig. 16). As expected, a right shift (decreased Ca2 + sensitivity) was found in the curves for the white fibers with respect to the red fibers. In addition an even lower Ca2 + sensitivity was found for the super-fast fibers. Based on the 3- fold right shift of the force – pCa curve of swimbladder fibers with respect to frog fibers (Fig. 16), these results suggest that koff of swimbladder troponin is three times faster than that of frog. With this higher koff, the modeled rate of troponin deactivation no longer appears limiting. Thus the underlying benefit of the less sensitive troponin may be the possibility of faster relaxation through a more rapid koff [52]. Finally, for force to drop quickly following the dissociation of Ca2 + from troponin, a fast crossbridge detachment rate is required (Fig. 8, Step 6). Indeed, the Vmax (which is thought to be affected by crossbridge detachment rate) of swimbladder muscle ( 12 ML s − 1) is exceptionally fast, 5- and 2.5-fold faster than toadfish red and white muscle (Fig. 17). In recent direct measurements made in collaboration with Yale Gold-

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man’s laboratory, we measured the crossbridge detachment rate directly and found that it is exceptionally fast ( 200 s − 1 or about 100 times faster than rabbit fast fibers). This fast detachment rate would permit the rapid relaxation rate which is observed [9]. The toadfish experiments have thus identified a number of kinetic variables that change progressively as twitch speed increases from the slow twitch of red fibers to the superfast twitch of swimbladder fibers: (1) The duration of the calcium transient becomes shorter, which in turn requires more rapid calcium release and reuptake. Ultrastructural and biochemical studies suggest that this is achieved principally by an increased density of SR calcium release sites and SR calcium pumps [5]. (2) Troponin needs a faster off-rate for Ca2 + , which requires molecular modification of troponin to a lower affinity type. (3) Crossbridges detach more rapidly, which involves molecular modification of myosin. Most importantly, changes in all of these parameters in concert enable swimbladder fibers to perform the mechanical work at high operating frequencies. To emit continuous sound, sonic muscle must generate work to produce sound energy and to overcome frictional losses in the sound producing system. Workloop experiments, similar to those performed on swimming fish, show that swimbladder fibers can perform work at frequencies in excess of 200 Hz at 25°C, representing the highest frequency for work production ever recorded in vertebrate muscle. By contrast, locomotory muscles lack the combination of fast Ca2 + pumping and fast crossbridge cycling necessary to generate work at these high frequencies. The highest frequency for vertebrate locomotory muscles is an order of magnitude lower; 25– 30 Hz for mouse and lizard fast-twitch muscles at 35°C, and the red and white muscle of toadfish can not generate work at higher than 15 Hz. Further evidence that all three of these modifications are in fact necessary for rapid sound production was obtained in experiments on the shaker muscle of rattlesnakes. Rattlesnakes in the genus Crotalus also need very fast muscles. They use the shaker muscle at the base of their tail to shake their rattle as a warning for other species. Rattling is a loud and effective warning device that renders these animals, like many venomous animals, very conspicuous. At 15°C, the shaker fibers have a very rapid calcium transient (halfwidth= 4–5 ms), which is only 1–2 ms slower than that of the swimbladder (Fig. 14 B). However, the twitch duration of the shaker muscle twitch is far longer (half-width = 25 vs. 10 ms) than that of swimbladder (Fig. 14 upper). This slower twitch likely reflects the effects of slower crossbridge detachment (the shaker’s Vmax of  7 ML s − 1 is only about half that of the swimbladder; Fig. 17) and probably a slower troponin koff. This analysis suggests that in fact all three

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systems, not just the Ca2 + transient, must be very rapid to produce the fastest contractions. Thus at 15°C, shaker fibers can be stimulated up to only  20 Hz before force begins to summate. Accordingly, rattling frequency at 15°C is only about 30 Hz. To provide faster contractions and rattling frequencies, these reactions must be sped up. This is accomplished by increasing the temperature. At 35°C, where snakes rattle at 90 Hz, the calcium transient (Fig. 14) and the Vmax (Fig. 17) are even faster than for the swimbladder muscle at 15°C. In addition, koff is likely very rapid as well. The resulting briefer twitch (halfwidth = 8–9 ms; Fig. 14B) enables the shaker fibers to be stimulated at high frequencies without complete fusion of force, and to perform the requisite mechanical work at 90 Hz. The similarity of the modifications in this sound producing muscle to those in toadfish is suggestive of convergent evolution, which supports the hypothesis that these modifications are necessary for high frequency direct-coupled sound production by synchronous muscle.

10. Future We are beginning to understand how muscles are designed for different functions at the molecular level. Although a tremendous amount has been learned about muscle design over the last decade, as we move into the next century the field of integrative muscle physiology will undergo a revolution fueled by technological improvements in four main areas.

10.1. Better in 6i6o measurements of length change, stimulus pattern, and force production As described above, the ‘in vivo’ workloop technique permits one to observe how muscles are designed and to pose and answer crucial questions concerning how the output of the muscle might change if the in vivo conditions changed in a specific manner. What is needed are better ways of monitoring length change, stimulation pattern, and force production in vivo. Ingenious use of sonomicometry [33,34], strain gauge techniques [6,13,34] and anatomical and modeling techniques [14] are providing new and more accurate information.

10.2. Better mechanistic understanding of muscle function Our ability to integrate from molecular components to whole animal locomotion is presently limited by our lack of complete understanding of the detailed mechanisms of muscle contraction. This is now being solved by using new biophysics techniques to better

understand muscle design at the molecular level. Over the last 10 years there has been great advancement in the field of muscle biophysics. The use of fast Ca2 + sensitive dyes have permitted the measurement (or modeling) of the time course of calcium release from and re-uptake by the SR, and Ca2 + binding to and unbinding from troponin. Further techniques involving caged compounds (which release ATP or Ca2 + from photo-active compounds) have permitted the measurement of crossbridge kinetic rate constants. Finally, stop flow kinetics measurements have permitted measurement of on and off rate kinetics of Ca2 + for troponin. Judicious use of these and other new approaches will provide new insights into how, at a mechanistic level, the muscle is matched to its function.

10.3. Modelling of muscular systems Software is now available that permits the building of virtual animals with the appropriate anatomy (limb and muscle dimensions) and mass distribution [14]. This software is a powerful tool which enables us to predict whole animal movements from mechanical properties of muscle. Thus if one orders the muscles to contract in their normal sequence, a virtual frog should jump like the real one. By incorporating a molecular model of muscle we could answer ‘What if?’ questions. For instance, how would frog jumping performance be altered if myosin kinetics were changed in a specific manner? This approach will give us greater insight about the sensitivity of whole animal performance to changes in single molecular components, and a better appreciation for why there is so much variation in the molecular components of muscle.

10.4. Genetic approaches to studying muscle design Finally, although the above modelling approach will provide insights into how a change in molecular properties might affect whole animal locomotor performance, it is desirable to test these predictions empirically. This can be done by taking advantage of sometimes fortunate variation in nature, and eventually by using genetic engineering to alter component of the muscular system in a desired manner, and then test the prediction. The use of all these techniques, coupled with phylogenetic approaches and the comparative approach to pick the right animal, will not only give us a better understanding of how muscles are designed, but also how these different designs evolved. Only then we will be able to answer many of the questions Dick Taylor raised in the parking lot so many years ago.

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Acknowledgements This work was supported by NIH AR38404 and NSF IBN-9514383.

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