Mutually exclusive muscle designs: the power output of the locomotory and sonic muscles of the oyster toadfish (Opsanus tau).

Animals perform a vast array of motor activities. Although it has generally been accepted that muscles are well suited to the function that they must perform, specialization for performing one function may compromise their ability for carrying out another. We examined this principle in the toadfish muscular system: slow-twitch red and fast-twitch white myotomal muscles are used for powering swimming at relatively low frequencies, while the superfast swimbladder muscle powers mating calls by contracting at 100 Hz. We measured muscle power output over a wide range of frequencies. The red and white locomotory muscles could not generate power over ca. 2.2 and 12 Hz, respectively and, hence, could not power sound production. In contrast, the swimbladder muscle has many specializations that permit it to generate power at frequencies in excess of 100 Hz. However, these specializations drastically reduce its power output at low frequencies: the swimbladder muscle generated only one-twentieth of the power of the red muscle and one-seventh of the power of the white muscle at the frequencies used during swimming. To generate the same total power needed for swimming would require unfeasibly large amounts of swimbladder muscle that could not fit into the fish. Hence, the designs of the swimbladder and locomotory muscles are mutually exclusive.

Quantitative electrophoretic analysis of myosin heavy chains in single muscle fibers.

To better understand the molecular basis of the large variation in mechanical properties of different fiber types, there has been an intense effort to relate the mechanical and energetic properties measured in skinned single fibers to those of their constituent cross bridges. There is a significant technical obstacle, however, in estimating the number of cross bridges in a single fiber. In this study, we have developed a procedure for extraction and quantification of myosin heavy chains (MHCs) that permits the routine and direct measurement of the myosin content in single muscle fibers. To validate this method, we also compared MHC concentration measured in single fibers with the MHC concentration in whole fast-twitch (psoas and gracilis) and slow-twitch (soleus) muscles of rabbit. We found that the MHC concentration in intact psoas (184 microM) was larger than that in soleus (144 microM), as would be expected from their differing mitochondrial content and volume of myofibrils. We obtained excellent agreement between MHC concentration measured at the single fiber level with that measured at the whole muscle level. This not only verifies the efficacy of our procedure but also shows that the difference in concentration at the whole muscle level simply reflects the concentration differences in the constituent fiber types. This new procedure should be of considerable help in future attempts to determine kinetic differences in cross bridges from different fiber types.

The influence of thermal acclimation on power production during swimming. I. In vivo stimulation and length change pattern of scup red muscle.

Ectothermal animals are able to locomote in a kinematically similar manner over a wide range of temperatures. It has long been recognized that there can be a significant reduction in the power output of muscle during swimming at low temperatures because of the reduced steady-state (i.e. constant activation and shortening velocity) power-generating capabilities of muscle. However, an additional reduction in power involves the interplay between the non-steady-state contractile properties of the muscles (i.e. the rates of activation and relaxation) and the in vivo stimulation and length change pattern the muscle undergoes during locomotion. In particular, it has been found that isolated scup (Stenotomus chrysops) red muscle working under in vivo stimulus and length change conditions (measured in warm-acclimated scup swimming at low temperatures) generates very little power for swimming. Even though the relaxation of the muscle has slowed greatly, warm-acclimated fish swim with the same tail-beat frequencies and the same stimulus duty cycles at cold temperatures, thereby not affording the slow-relaxing muscle any extra time to relax. We hypothesize that considerable improvement in the power output of the red muscle at low temperatures could be achieved if cold acclimation resulted in either a faster muscle relaxation rate or in the muscle being given more time to relax (e.g. by shortening the stimulus duration or reducing the tail-beat frequency). We test these hypotheses in this paper and the accompanying paper. Scup were acclimated to 10 degrees C (cold-acclimated) and 20 degrees C (warm-acclimated) for at least 6 weeks. Electromyograms (EMGs) and high-speed cine films were taken of fish swimming steadily at 10 degrees C and 20 degrees C. At 10 degrees C, we found that, although there were no differences in tail-beat frequency, muscle strain or stimulation phase between acclimation groups, cold-acclimated scup had EMG duty cycles approximately 20 % shorter than warm-acclimated scup. In contrast at 20 degrees C, there was no difference between acclimation groups in EMG duty cycle, nor in any other muscle length change or stimulation parameter. Thus, in response to cold acclimation, there appears to be a reduction in EMG duty cycle at low swimming temperatures that is probably due to an alteration in the operation of the pattern generator. This novel acclimation probably improves muscle power output at low temperatures compared with that of warm-acclimated fish, an expectation we test in the accompanying paper using the work-loop technique.

The influence of thermal acclimation on power production during swimming. II. Mechanics of scup red muscle under in vivo conditions.

We have previously shown that the power output of red muscle from warm-acclimated scup is greatly reduced when the fish swim at low temperatures. This reduction occurs primarily because, despite the slowing of muscle relaxation rate at cold temperatures, warm-acclimated scup swim with the same tail-beat frequency and the same stimulation durations, thereby not affording the slower-relaxing muscle any extra time to relax. We hypothesize that power output during swimming could be increased if the stimulus duration were reduced or if the relaxation rate of the red muscle were increased during cold acclimation. Scup were acclimated to 10 degrees C (cold-acclimated) and 20 degrees C (warm-acclimated) for at least 6 weeks. Cold acclimation dramatically increased the ability of scup red muscle to produce power at 10 degrees C. Power output measured from cold-acclimated muscle bundles driven through in vivo conditions measured from cold-acclimated scup swimming at 10 degrees C (i.e. work loops) was generally much greater than that from warm-acclimated muscle driven through its respective in vivo conditions at 10 degrees C. The magnitude of the increase depended both on the anatomical location of the muscle and on swimming speed. Integrated over the length of the fish, the red musculature from cold-acclimated fish generated 2.7, 8.9 and 5.8 times more power than the red musculature from warm-acclimated fish while swimming at 30 cm s(-)(1), 40 cm s(-)(1) and 50 cm s(-)(1), respectively. Our analysis suggests that the cold-acclimated fish should be able to swim in excess of 40 cm s(-)(1) with just their red muscle whereas the warm-acclimated fish must recruit their pink muscle well below this speed. Because the red muscle is more aerobic than the pink muscle, cold acclimation may increase the sustained swimming speed at which scup perform their long seasonal migrations at cool temperatures. We then explored the underlying mechanisms for the increase in muscle power output in cold-acclimated fish. Contrary to our expectations, cold-acclimated muscle did not have a faster relaxation rate; instead, it had an approximately 50 % faster activation rate. Our work-loop studies showed that this faster activation rate, alone, can increase the mechanical power production during cyclical contractions to a surprising extent. By driving cold-acclimated muscle through warm- and cold-acclimated in vivo conditions, we were able to partition the improvement in power production associated with increased activation rate and the approximately 20 % reduction in the duration of electromyographic activity found in the accompanying study. Depending on the position and swimming speed, approximately 60 % of the increase in power output was due to the change in the red muscle's contractile properties (i.e. faster activation); the remainder was due to the shorter stimulus duty cycle of cold-acclimated scup. Thus, by both shortening the in vivo stimulation duration and speeding up the rate of muscle activation as part of cold-acclimation, scup achieve a very large increase in the power output of their red muscle during swimming at low temperature. This increase in power output probably results in an increase in muscle efficiency and, hence, a reduction in the energetic cost of swimming. This increase in power output also reduces reliance on the less aerobic and less fatigue-resistant pink muscle. Both these abilities may increase the swimming speed at which prolonged aerobic muscle activity can occur and thus reduce the travel time for the long seasonal migrations in which scup engage.

Superfast contractions without superfast energetics: ATP usage by SR-Ca2+ pumps and crossbridges in toadfish swimbladder muscle.

1. The rate at which an isometrically contracting muscle uses energy is thought to be proportional to its twitch speed. In both slow and fast muscles, however, a constant proportion (25-40 %) of the total energy has been found to be used by SR-Ca2+ pumps and the remainder by crossbridges. We examined whether SR-Ca2+ pumps account for a larger proportion of the energy in the fastest vertebrate muscle known (the toadfish swimbladder), and whether the swimbladder muscle utilizes energy at the superfast rate one would predict from its mechanics. 2. The ATP utilization rates of the SR-Ca2+ pumps and crossbridges were measured using a coupled assay system on fibres skinned with saponin. Surprisingly, despite its superfast twitch speed, the ATP utilization rate of swimbladder was no higher than that of much slower fast-twitch amphibian muscles. 3. The swimbladder achieves tremendous twitch speeds with a modest steady-state ATP utilization rate by employing two mechanisms: having a small number of attached crossbridges and probably utilizing intracellular Ca2+ buffers (parvalbumin) to spread out the time over which Ca2+ pumping can occur. 4. Finally, although the total ATP utilization rate was not as rapid as expected, the relative proportions used by SR-Ca2+ pumps and the crossbridges were similar to other muscles.

The influence of temperature on power production during swimming. I. In vivo length change and stimulation pattern.

Ectothermal animals are able to locomote effectively over a wide range of temperatures despite low temperature reducing the power output of their muscles. It has been suggested that animals recruit more muscle fibres and faster fibre types to compensate for the reduced power output at low temperature, but it is not known how much low temperature actually reduces power output in vivo. 'Optimized' work-loop measurements, which are thought to approximate muscle function in vivo, give a Q(10) of approximately 2.3 for power output of scup (Stenotomus chrysops) red muscle between 10 degrees C and 20 degrees C. However, because of the slower muscle relaxation rate at low temperatures, 'optimizing' work loops requires stimulation duration to be reduced and oscillation frequency to be decreased to obtain maximal power output. Previous fish swimming experiments suggest that similar optimization may not occur in vivo, and this may have substantial consequences in terms of muscle power generation and swimming at low temperatures. To assess more precisely the effects of temperature on muscle performance and swimming, in the present study, we measured the length change, stimulation duration and stimulus phase of red muscle at various positions along scup swimming at several speeds at 10 degrees C and 20 degrees C. In a companion study, we determined the effects of temperature on in vivo power generation by driving muscle fibre bundles through these in vivo length changes and stimulation conditions, and measuring the resulting power output. The most significant finding from the present study is that, despite large differences in the in vivo parameters along the length of the fish (a decrease in stimulus duration, an increase in strain and a negative shift in phase) moving posteriorly, these parameters do not change with temperature. Thus, although the nervous system of fish could, in theory, compensate for slow muscle relaxation by greatly reducing muscle stimulation duration at low temperatures, it does not. This lack of compensation to low temperatures might reflect a potential limitation in neural control.

The influence of temperature on power production during swimming. II. Mechanics of red muscle fibres in vivo.

We found previously that scup (Stenotomus chrysops) reduce neither their stimulation duration nor their tail-beat frequency to compensate for the slow relaxation rates of their muscles at low swimming temperatures. To assess the impact of this 'lack of compensation' on power generation during swimming, we drove red muscle bundles under their in vivo conditions and measured the resulting power output. Although these in vivo conditions were near the optimal conditions for much of the muscle at 20 degrees C, they were far from optimal at 10 degrees C. Accordingly, in vivo power output was extremely low at 10 degrees C. Although at 30 cm s(-)(1), muscles from all regions of the fish generated positive work, at 40 and 50 cm s(-)(1), only the POST region (70 % total length) generated positive work, and that level was low. This led to a Q(10) of 4-14 in the POST region (depending on swimming speed), and extremely high or indeterminate Q(10) values (if power at 10 degrees C is zero or negative, Q(10) is indeterminate) for the other regions while swimming at 40 or 50 cm s(-)(1). To assess whether errors in measurement of the in vivo conditions could cause artificially reduced power measurements at 10 degrees C, we drove muscle bundles through a series of conditions in which the stimulation duration was shortened and other parameters were made closer to optimal. This sensitivity analysis revealed that the low power output could not be explained by realistic levels of systematic or random error. By integrating the muscle power output over the fish's mass and comparing it with power requirements for swimming, we conclude that, although the fish could swim at 30 cm s(-)(1) with the red muscle alone, it is very unlikely that it could do so at 40 and 50 cm s(-)(1), thus raising the question of how the fish powers swimming at these speeds. By integrating in vivo pink muscle power output along the length of the fish, we obtained the surprising finding that, at 50 cm s(-)(1), the pink muscle (despite having one-third the mass) contributes six times more power to swimming than does the red muscle. Thus, in scup, pink muscle is crucial for powering swimming at low temperatures. This overall analysis shows that Q(10) values determined in experiments on isolated tissue under arbitrarily selected conditions can be very different from Q(10) values in vivo, and therefore that predicting whole-animal performance from these isolated tissue experiments may lead to qualitatively incorrect conclusions. To make a meaningful assessment of the effects of temperature on muscle and locomotory performance, muscle performance must be studied under the conditions at which the muscle operates in vivo.

Trading force for speed: why superfast crossbridge kinetics leads to superlow forces.

Superfast muscles power high-frequency motions such as sound production and visual tracking. As a class, these muscles also generate low forces. Using the toadfish swimbladder muscle, the fastest known vertebrate muscle, we examined the crossbridge kinetic rates responsible for high contraction rates and how these might affect force generation. Swimbladder fibers have evolved a 10-fold faster crossbridge detachment rate than fast-twitch locomotory fibers, but surprisingly the crossbridge attachment rate has remained unchanged. These kinetics result in very few crossbridges being attached during contraction of superfast fibers (only approximately 1/6 of that in locomotory fibers) and thus low force. This imbalance between attachment and detachment rates is likely to be a general mechanism that imposes a tradeoff of force for speed in all superfast fibers.

Muscle activity in steady swimming scup, Stenotomus chrysops, varies with fiber type and body position.

The red and pink aerobic muscle fibers are used to power steady swimming in fishes. We examined red and pink muscle recruitment and function during swimming in scup, Stenotomus chrysops, through electromyography and high-speed ciné. Computer analysis of electromyograms (EMGs) allowed determination of initial speed of muscle recruitment and duty cycle and phase of muscle electromyographic activity for both fiber types. This analysis was carried out for three longitudinal positions over a range of swimming speeds. Fiber type and longitudinal position both affected swimming speed of initial recruitment. Posterior muscle is recruited at the lowest swimming speed, whereas more anterior muscle is not initially recruited until higher speeds. At more anterior positions, the initial recruitment of pink muscle occurs at a higher swimming speed than the recruitment of red muscle. The duty cycle of pink muscle EMG activity is significantly shorter than that of red muscle, reflecting a difference in the onset time of activation during each cycle of length change: pink muscle onset time follows that of red. The different patterns of usage of red and pink muscle reflect differences in their contraction kinetics. Because pink muscle generates force more rapidly than red muscle, it can be activated later in each tailbeat cycle. Pink muscle is used to augment red muscle power production at higher swimming speeds, allowing a higher aerobically based steady swimming speed than that possible by red muscle alone.

Quantitative analysis of muscle fibre type and myosin heavy chain distribution in the frog hindlimb: implications for locomotory design.

To investigate the design of the frog muscular system for jumping, fibre type distribution and myosin heavy chain (MHC) isoform composition were quantified in the hindlimb muscles of Rana pipiens. Muscles were divided into two groups: five large extensor muscles which were predicted to shorten and produce mechanical power during jumping (JP), and four much smaller muscles commonly used in muscle physiology studies, but that do not shorten or produce power during jumping (NJP). fibres were classified as one of four different types (type 1, 2, 3 or tonic) or an intermediate type (type 1-2) based on their relative myosin-ATPase reactivity and MHC immunoreactivity in muscle cross-sections according to previous nomenclature established for amphibian skeletal muscle. Type 1 fibres correspond to the fastest and most powerful of the twitch fibres, and type 3 fibres are the slowest and least powerful. Myosin-ATPase histochemistry revealed that the JP muscles were composed primarily of type 1 fibres (89%) with a small percentage of type 2 (7%) and intermediate type 1-2 fibres (4%). The fibre type composition of NJP muscles was more evenly distributed between type 1 (29%), type 2 (46%) and type 1-2 (24%) fibres. Tonic fibres comprised less than 2% of the muscle cross-section in both JP and NJP groups. Similarly, MHC composition determined by quantitative SDS-PAGE revealed that JP muscles were composed predominantly of type 1 MHC (86%), with a balance of type 2 MHC (14%). The opposite pattern was found for MHC composition in the NJP muscles: type 1 (28%), type 2 (66%) and type 3 (6%). These results demonstrate that the large extensor muscles that produce the power required for jumping have a fibre type distribution that enables them to generate high levels of mechanical power, with the type 1 isoform accounting for 85-90% of the total MHC content.

Some advances in integrative muscle physiology.

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) Ca(2+)-troponin kinetics, and (3) crossbridge kinetics. New technologies will soon enable us to better appreciate how different animal designs evolved.

Contraction kinetics of red muscle in scup: mechanism for variation in relaxation rate along the length of the fish.

We studied possible mechanisms for the twofold difference in red muscle relaxation times between the posterior (207.2 ms) and anterior (98.4 ms) musculature of scup Stenotomus chrysops, which has been shown to have a large effect on power generation during swimming. This difference was not due to contamination of the anterior bundles with faster fiber types, as histological examination showed that all bundles contained more than 98.9% red fibers. Further, maximum velocities of shortening (Vmax) at 20 degrees C were nearly identical, 5.37 MLs-1 (where ML is muscle length) for the anterior musculature and 5.47 MLs-1 for the posterior musculature, suggesting that the difference in relaxation times was not due to a difference in the crossbridge detachment rates associated with different myosin isoforms. The possibility of differences in the Ca2+ pumping rate influencing relaxation rate was explored using cyclopiazonic acid (CPA), a sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor. The concentration of CPA could be adjusted to slow the relaxation rate of an anterior muscle to that of a posterior muscle. However, SDS gels showed no difference in the intensity of SR Ca(2+)-ATPase protein bands between muscle positions. These results suggest that differences in the Ca2+ pumping could account for the observed difference in relaxation rate, but do not support the simplest hypothesis that the difference in relaxation rates is due to differences in numbers of Ca2+ pumps. Other possible mechanisms for this difference are explored.

Contraction dynamics and power production of pink muscle of the scup (Stenotomus chrysops).

Although the contribution of red muscle to sustained swimming in fish has been studied in detail in recent years, the role of pink myotomal muscle has not received attention. Pink myotomal muscle in the scup (Stenotomus chrysops) lies just medial to red muscle, has the same longitudinal fibre orientation and is recruited along with the red muscle during steady sustainable swimming. However, pink muscle has significantly faster rates of relaxation, and the maximum velocity of shortening of pink muscle (7.26 +/- 0.18 muscle lengths s-1, N = 9, at 20 degrees C, and 4.46 +/- 0.15 muscle lengths s-1, N = 6, at 10 degrees C; mean +/- S.E.M.) is significantly faster than that of red muscle. These properties facilitate higher mass-specific maximum oscillatory power production relative to that of red muscle at frequencies similar to the tailbeat frequency at maximum sustained swimming speeds in scup. Additionally, pink muscle is found in anatomical positions in which red muscle is produces very little power during swimming: the anterior region of the fish, which undergoes the lowest strain during swimming. Pink muscle produces more oscillatory power than red muscle under low-strain conditions (+/- 2-3%) and this may allow pink muscle to supplement the relatively low power generated by red muscle in the anterior regions of swimming scup.

Muscle function during jumping in frogs. I. Sarcomere length change, EMG pattern, and jumping performance.

We determined the influence of temperature on muscle function during jumping to better understand how the frog muscular system is designed to generate a high level of mechanical power. Maximal jumping performance and the in vivo operating conditions of the semimembranosus muscle (SM), a hip extensor, were measured and related to the mechanical properties of the isolated SM in the accompanying paper [Muscle function during jumping in frogs. II. Mechanical properties of muscle: implication for system design. Am. J. Physiol. 271 (Cell Physiol. 40): C571-C578, 1996]. Reducing temperature from 25 to 15 degrees C caused a 1.75-fold decline in peak mechanical power generation and a proportional decline in aerial jump distance. The hip and knee joint excursions were nearly the same at both temperatures. Accordingly, sarcomeres shortened over the same range (2.4 to 1.9 microns) at both temperatures, corresponding to myofilament overlap at least 90% of maximal. At the low temperature, however, movements were made more slowly. Angular velocities were 1.2- to 1.4-fold lower, and ground contact time was increased by 1.33-fold at 15 degrees C. Average shortening velocity of the SM was only 1.2-fold lower at 15 degrees C than at 25 degrees C. The low Q10 of velocity is in agreement with that predicted for muscles shortening against an inertial load.

Muscle function during jumping in frogs. II. Mechanical properties of muscle: implications for system design.

We characterized the design of the frog muscular system for jumping by comparing the properties of isolated muscle with the operating conditions of muscle measured during maximal jumps. During jumping, the semimembranosus muscle (SM) shortened with a V/Vmax (where V is shortening velocity and Vmax is maximal shortening velocity) where 90 and 100% of maximal power would be generated at 15 and 25 degrees C, respectively. To assess the level of activation during jumping, the SM was driven through the in vivo length change and stimulus conditions while the resulting force was measured. The force generated under the in vivo conditions at both temperatures was at least 90% of the force generated at that same V under maximally activated conditions. Thus the SM was nearly maximally activated, and shortening deactivation was minimal. The initial sarcomere length and duration of the stimulus before shortening were important factors that minimized shortening deactivation during jumping. Thus the frog muscular system appears to be designed to meet the three necessary conditions for maximal power generation during jumping: optimal myofilament overlap, optimal V/Vmax, and maximal activation.

The whistle and the rattle: the design of sound producing muscles.

Vertebrate sound producing muscles often operate at frequencies exceeding 100 Hz, making them the fastest vertebrate muscles. Like other vertebrate muscle, these sonic muscles are "synchronous," necessitating that calcium be released and resequestered by the sarcoplasmic reticulum during each contraction cycle. Thus to operate at such high frequencies, vertebrate sonic muscles require extreme adaptations. We have found that to generate the "boatwhistle" mating call (approximately 200 Hz), the swimbladder muscle fibers of toadfish have evolved (i) a large and very fast calcium transient, (ii) a fast crossbridge detachment rate, and (iii) probably a fast kinetic off-rate of Ca2+ from troponin. The fibers of the shaker muscle of rattlesnakes have independently evolved similar traits, permitting tail rattling at approximately 90 Hz.

Quantitative distribution of muscle fiber types in the scup Stenotomus chrysops.

Because the mass-specific power generated by myotomal muscle during swimming varies along the length of the fish, a realistic assessment of total power generation by the musculature requires integrating the product of mass-specific power and muscle mass at each position over the length of the fish. As a first step toward this goal, we examined the distribution of red, pink, and white muscle along the length of Stenotomus chrysops (scup) using histochemical and image analysis techniques. The largest cross-sectional area of red fibers occurs at 60% of total fish length and declines both anteriorly and posteriorly. By contrast, white fibers have the largest cross-sectional area in the anterior and decline dramatically moving posteriorly. The proportion of the fishes' cross-section occupied by red fibers increases from 1.37% to 8.42% moving posteriorly along the length of the fish. In contrast, the proportion of cross-sectional area occupied by pink fibers is constant (1.19%), while the proportional cross-sectional area of white fibers falls from 82.5% to 66.3%. The red, pink, and white fibers comprise 2.09, 0.73, and 51.1%, respectively, of total fish weight. We also compared the distribution of muscle in 10 degrees C- and 20 degrees C- acclimated animals. The value for red fiber volume, though slightly higher (13%) in cold-acclimated fish, is not statistically different. No difference was found in pink or white fibers. Finally, the finding that most of the red muscle is in the posterior half of the fish further supports the notion that most power for steady swimming at moderate speeds comes from posterior rather than anterior musculature.

Muscle length changes during swimming in scup: sonomicrometry verifies the anatomical high-speed cine technique.

Recent attempts to determine how fish muscles are used to power swimming have employed the work loop technique (driving isolated muscles using their in vivo strain and stimulation pattern). These muscle strains have in turn been determined from the anatomical high-speed cine technique. In this study, we used an independent technique, sonomicrometry, to attempt to verify these strain measurements and the conclusions based on them. We found that the strain records measured from sonomicrometry and the anatomical-cine techniques were very similar. The ratio of the strain measured from sonomicrometry to that from the anatomical-cine technique was remarkably close to unity (1.046 +/- 0.013, mean +/- S.E.M., N = 15, for transducers placed on the muscle surface and corrected for muscle depth, and 0.921 +/- 0.028, N = 8, in cases where the transducers were inserted to the average depth of the red muscle). These measurements also showed that red muscle shortening occurs simultaneously with local backbone curvature, unlike previous results which suggested that white muscle shortening during the escape response occurs prior to the change in local backbone curvature.