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.

Alternative exon-encoded regions of Drosophila myosin heavy chain modulate ATPase rates and actin sliding velocity.

To investigate the molecular functions of the regions encoded by alternative exons from the single Drosophila myosin heavy chain gene, we made the first kinetic measurements of two muscle myosin isoforms that differ in all alternative regions. Myosin was purified from the indirect flight muscles of wild-type and transgenic flies expressing a major embryonic isoform. The in vitro actin sliding velocity on the flight muscle isoform (6.4 microm x s(-1) at 22 degrees C) is among the fastest reported for a type II myosin and was 9-fold faster than with the embryonic isoform. With smooth muscle tropomyosin bound to actin, the actin sliding velocity on the embryonic isoform increased 6-fold, whereas that on the flight muscle myosin slightly decreased. No difference in the step sizes of Drosophila and rabbit skeletal myosins were found using optical tweezers, suggesting that the slower in vitro velocity with the embryonic isoform is due to altered kinetics. Basal ATPase rates for flight muscle myosin are higher than those of embryonic and rabbit myosin. These differences explain why the embryonic myosin cannot functionally substitute in vivo for the native flight muscle isoform, and demonstrate that one or more of the five myosin heavy chain alternative exons must influence Drosophila myosin kinetics.

Determining structure/function relationships for sarcomeric myosin heavy chain by genetic and transgenic manipulation of Drosophila.

Drosophila melanogaster is an excellent system for examining the structure/function relationships of myosin. It yields insights into the roles of myosin in assembly and stability of myofibrils, in defining the mechanical properties of muscle fibers, and in dictating locomotory abilities. Drosophila has a single gene encoding muscle myosin heavy chain (MHC), with alternative RNA splicing resulting in stage- and tissue-specific isoform production. Localization of the alternative domains of Drosophila MHC on a three-dimensional molecular model suggests how they may determine functional differences between isoforms. We are testing these predictions directly by using biophysical and biochemical techniques to characterize myosin isolated from transgenic organisms. Null and missense mutations help define specific amino acid residues important in actin binding and ATP hydrolysis and the function of MHC in thick filament and myofibril assembly. Insights into the interaction of thick and thin filaments result from studying mutations in MHC that suppress ultrastructural defects induced by a troponin I mutation. Analysis of transgenic organisms expressing engineered versions of MHC shows that the native isoform of myosin is not critical for myofibril assembly but is essential for muscle function and maintenance of muscle integrity. We show that the C-terminus of MHC plays a pivotal role in the maintenance of muscle integrity. Transgenic studies using headless myosin reveal that the head is important for some, but not all, aspects of myofibril assembly. The integrative approach described here provides a multi-level understanding of the function of the myosin molecular motor.

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.

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.

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.