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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY
1Integrative Physiology, School of Biological Sciences, University of Sydney, New South Wales, Australia; and 2Department of Biomolecular and Sport Sciences, Faculty of Health and Life Sciences, Coventry University, Coventry, United Kingdom
Submitted 17 October 2007 ; accepted in final form 11 January 2008
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ABSTRACT |
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body temperature; thermal acclimation; locomotion; work loop; enzymes
Although locomotor performance is heritable (21, 46, 58, 88), there is considerable within-generation variability as a result of environmental effects and gene-environment interactions (14, 58, 73). Locomotor performance is determined by underlying intrinsic muscle properties, such as muscle activation and relaxation rates, maximal shortening velocity, and maximal force production (8, 35, 44). The magnitude of these biomechanical functions may depend on metabolic capacities to produce ATP via oxidative and glycolytic pathways (1, 17, 20, 22, 30) and to hydrolyze ATP by myofibrillar ATPase activity (24, 67). However, there is not always a direct link between biochemical indices and whole animal locomotor performance (25). Additionally, there may be a trade-off between aerobic and anaerobic metabolic capacities and associated endurance and burst locomotor performance as a result of differential expression of fast and slow twitch fibers in muscle (27, 29, 66, 96, 97).
Temperature influences locomotor performance via its effect on metabolism (30, 49) and muscle physiology (51, 69, 72, 87). At lower temperatures, muscles take longer to activate and relax and maximum shortening velocity is reduced, thereby causing a decrease in power output (42, 52). In addition to an acute effect of temperature, many ectotherms reversibly change their muscle and metabolic phenotypes in response to long-term (e.g., seasonal) changes in their thermal environment (30, 52, 77). The best examples of acclimation or acclimatization to changes in the thermal environment stem from fish. Thermoregulation in fish differs from terrestrial vertebrates because aquatic environments, particularly marine environments, are thermally more homogenous than terrestrial environments, and the great heat capacity of water means that body temperatures are closely tied to water temperatures. With the exception of endothermic sharks and tuna (17), most fish experience long-term (e.g., seasonal) fluctuations in body temperature, which parallel changes in water temperature. Acclimation responses require a relatively stable environmental cue (75), which is more common in an aquatic environment than in a terrestrial environment. Hence, many species of fish possess plastic phenotypes that compensate for variation in water temperature so that muscle function and metabolic capacities are maintained constant or near constant across a wide range of temperatures (5, 85, 92, 93).
In contrast, most terrestrial vertebrates regulate their body temperature to within a narrow set point range relative to operative temperature fluctuations by behaviorally exploiting different thermal microhabitats and by regulating metabolic heat production (10, 32, 64). It has been suggested that the thermal sensitivities of biochemical and physiological functions have coevolved with the set point body temperature range so that rate functions are optimized at the regulated body temperature (3, 19). In consequence, terrestrial ectotherms are thought to have limited ability to acclimate metabolic capacities and muscle function (2, 6, 7). Nonetheless, it may be advantageous for terrestrial animals to respond to environmental temperature change by concomitant adjustments of set point body temperature ranges and thermal sensitivities of cellular functions (77, 78). For example, regulated body temperatures of American alligators (Alligator mississippiensis) in the wild change with season (80), while the thermal sensitivities of muscle metabolic capacities change in parallel with seasonal body temperature changes (81). Additionally, locomotor performance acclimates perfectly in the saltwater crocodile (Crocodylus porosus), so that sustained swimming speed is the same in cold-acclimated animals at 20°C as it is in warm-acclimated animals at 30°C (26). In parallel with acclimation of locomotor performance, state III mitochondrial respiration and the activities of metabolic enzymes change, counteracting the depressing effects of lowered body temperatures (26).
Our aim in this study was to investigate plasticity of muscle function in a thermoregulating ectotherm (C. porosus). In particular, we aimed to relate metabolic indices to muscle biomechanics to provide a greater resolution of the performance gradient during thermal acclimation, and to test the hypothesis that metabolic and biomechanical parameters are optimized at the body temperatures selected in different thermal environments. Hence, we related indices of anaerobic [lactate dehydrogenase (LDH)], aerobic [cytochrome c oxidase (COX)] metabolic capacities, and myofibrillar ATPase activity to the biomechanics of isometric and work loop performance in isolated muscle. Specifically, we tested the hypotheses that 1) muscle performance acclimates to different temperatures and therefore can explain differences in locomotor performance, 2) myosin ATPase activity is a rate-limiting reaction (47, 49) that correlates positively with force production and isometric activation times, and 3) metabolic capacities are positively related to force production.
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METHODS |
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Juvenile estuarine crocodiles (C. porosus, n = 16), were obtained from a crocodile farm (Wildlife International, NT, Australia). Crocodiles were housed in plastic tanks (830 mm width x 620 mm height x 1,250 mm length; 2 or 3 animals/tank), which were designed so that animals could thermoregulate behaviorally. Shelter and water (up to 200 mm depth) were provided at one end, and a dry basking space at the other end. A full-spectrum light source (ReptiGlo) was suspended above each tank, and an infrared heat lamp delivered 400 W/m–2 to the dry basking area in the tank, which is comparable to solar radiation received while basking in spring (76). A constant 12:12-h light-dark cycle was maintained throughout the experiments. Crocodiles were fed live crayfish (farmed Cherax destructor) and insects 3 times per week, which closely resembles their natural diet (94).
Crocodiles were acclimated using the same experimental conditions as in Ref. 26. Thermal conditions for two acclimation treatments were chosen to resemble winter (cold) and summer (warm) in C. porosus' natural habitat in North Queensland, Australia (79). Treatments were conducted in controlled environment rooms (1 tank/room); in the cold treatment (n = 8 animals, mass = 719.4 ± 26.9 g; mean ± SE) mean air temperature was 20.2 ± 0.03°C, resulting in slightly lower water temperatures (19.5 ± 0.03°C), and basking opportunity was provided for 6 h/day. In the warm treatment (n = 8, mass = 726.9 ± 17.0 g) air temperature was 29.5 ± 0.01°C, water temperature was 29.2 ± 0.04°C, and basking opportunity was provided for 9 h/day. Crocodiles were acclimated for 30 days (9) before experimentation; after this we processed two animals per day, alternating between warm- and cold-acclimated animals. Crocodiles were euthanized by an injection of dilute sodium pentabarbitone (200 mg/kg) immediately before experimentation. All experimental and animal handling procedures were approved by the University of Sydney Animal Ethics Committee (approval no. L04/1-2007/3/4513).
Muscle Mechanics
Tissue samples. Caudofemoralis muscle, the main swimming muscle in the tail (70), was dissected out for subsequent muscle mechanics analysis in crocodile Ringer solution [composition in mmol/l: 110 NaCl, 4.0 KCl, 2.6 NaH2PO4, 1.4 MgSO4, 23.8 NaHCO3, 5.6 glucose, 2.8 CaCl2, pH 7.3 at 22°C (55)] leaving part of the most caudal vertebra that the muscle attached to at one end and part of the femur attached at the other end.
Isometric Studies
Isometric studies were conducted to determine the twitch and tetanus kinetics of isolated caudofemoralis muscle. The vertebra at the caudal end of the muscle preparation was clamped via a crocodile clip to a strain gauge (model UF1; Pioden Controls), and the tendon at the other end was clamped via a crocodile clip to a motor arm (model V201; Ling Dynamics Systems) attached to a linear variable displacement transformer (model DFG 5.0; Solartron Metrology). The linear variable displacement transformer was used to monitor the length changes delivered to the muscle preparation. The whole of the muscle, tendon, and bone preparation was then allowed to equilibrate to the test temperature (10 min at 20°C or 30°C) in an organ bath with circulating, aerated crocodile Ringer solution. The preparation was then held at constant length and stimulated via parallel platinum electrodes to generate a series of twitches. Stimulus amplitude and muscle length were adjusted to determine the stimulation parameters and muscle length corresponding to maximal isometric twitch force. Time to peak twitch (force development time) and time from peak twitch to half relaxation were measured using a Powerlab (AD Instruments, Sydney, Australia) and viewed with Chart 5.0 software (AD Instruments). An isometric tetanic force response was elicited by subjecting the muscle to a 600 ms train of stimulation. Stimulation frequency (80 to 100 Hz) was altered to determine maximal tetanic force. Time from first stimulus to half peak tetanic force (force development time) and time from last stimulus to half tetanic force relaxation were measured. A rest period of 5 min was allowed between each tetanic response.
Work loop analysis. The work loop technique was used to determine the power output of muscles during cyclical length changes (53). Unlike fixed-length isometric studies and fixed-load isotonic studies of muscle performance, the work loop technique allows measures of muscle power output under length and activation changes that are more indicative of in vivo contractile performance (12, 42). Each muscle preparation was subjected to a set of four sinusoidal length changes symmetrical around the length found to generate maximal twitch force. The muscle was stimulated using the stimulation amplitude, and frequency was found to yield maximal isometric force. Electrical stimulation and length changes were controlled via a data acquisition board and a custom-designed program developed with TestPoint software (Measurement Computing, Norton, MA). Muscle force was plotted against muscle length for each cycle to generate a work loop, the area of which equated to the net work produced by the muscle during the cycle of length change (53). The net work produced was multiplied by the frequency of length change cycles to calculate net power output. The cycle frequency of length change was altered from 1 Hz, to 2 Hz, to 0.7 Hz to generate power output-cycle frequency curves across the range of tail-beat frequencies previously found during swimming in saltwater crocodiles of similar size to those used in the present study (82). Muscle strain was not altered between each cycle frequency, as previous work (82) found that tail-beat amplitude did not alter across the range of tail-beat frequencies we have simulated. As the muscle strain waveform in caudofemoralis is not known for swimming crocodiles, we assumed that a sine wave would be a reasonable approximation in such cyclic undulations of the tail that appear similar to those seen in some swimming fish. Preliminary experiments determined that a total strain of length change cycles of 0.12 (which equated to ±6% of resting muscle length) yielded near maximal power output at each cycle frequency; therefore, this strain was maintained throughout all work loop experiments. Every 5 min the muscle was subjected to a further set of four work loop cycles with stimulation duration and stimulation phase parameters being altered in between each set until maximum net work was achieved at each cycle frequency. A set of control sinusoidal length change and stimulation parameters were imposed at 1-Hz cycle frequency on the muscle every three to four sets of work loops to monitor variation in the muscles’ ability to produce power/force over the time course of the experiment. Any variation in power was found to be due to a matching change in ability to produce force. Therefore, the ability of each preparation to produce power was corrected to the control run that yielded the highest power output, assuming that alterations in power generating ability were linear over time. On completion of the power-output cycle frequency curve at 20°C the temperature of the Ringer solution bathing the muscle was increased to 30°C over at least 20 min, allowing at least a further 10 min for the muscle to equilibrate at the new test temperature. The above isometric and work loop studies were then repeated at 30°C. The temperature of the Ringer solution bathing the muscle was decreased to 20°C over at least 20 min, allowing at least a further 10 min for the muscle to equilibrate at this final test temperature. The muscle was then subjected to a further twitch, tetanus, and control set of work loops at 20°C.
At the end of the isometric and work loop experiments, the bones and tendons were removed from each preparation and each muscle was blotted on absorbent paper to remove excess Ringer solution. Wet muscle mass was determined to the nearest 0.001 g using an electronic balance. Mean muscle cross-sectional area was calculated from muscle length and mass assuming a density of 1,060 kg/m–3 (62). Maximum isometric muscle stress at each test temperature was then calculated as maximum tetanic force divided by mean muscle cross-sectional area (kN/m–2). Normalized muscle power output at each test temperature was calculated as power output divided by wet muscle mass (W/kg).
Enzyme assays. Tissue samples for enzyme assays were collected from the caudofemoralis muscle that was not used for the biomechanical experiments. Fresh muscle samples (0.05–0.1g) were homogenized immediately in 9 volumes of ice-cold extraction buffer (in mmol/l: 50 imidazole, 2 MgCl2, 5 EDTA, 0.1% Triton, and 1 glutathione, pH 7.5 at 0°C) using a rotor-stator homogenizer (model Pro200; Pro Scientific). LDH and COX activities were determined according to 81. LDH and COX activities were expressed as micromoles of substrate converted per minute per gram of wet tissue mass.
Myofibrils were isolated from muscle tissue samples as described previously (5, 48), and ATPase activity was assayed in a linked enzyme assay that measured the disappearance of NADH at 340 nm (28, 31). The assay medium contained (in mmol/l) 50 KCl, 40 imidazole, 7 MgCl2, 5 CaCl2, 5 ATP, 1 phosphoenolpyruvate, 0.7 NADH, and 6 U/ml each of LDH and pyruvate kinase (all chemicals were obtained from Sigma, Sydney, Australia); saturating concentrations were ascertained before experimentation. The protein concentration of the myofibrillar solution was determined by the bicinchoninic acid method (Sigma,) according to the manufacturer's instructions. All enzyme assays were conducted in duplicate at 20°C and 30°C in a spectrophotometer with a temperature-controlled cuvette holder (Ultrospec 2100 pro UV; Amersham Pharmacia, Sydney, Australia).
Statistical Analysis
We used univariate analysis of variance with acclimation treatment as a fixed factor, test temperature as a repeated measure to analyze isometric stress, and isometric rate of force development and relaxation times, as well as enzyme activities. Cycle frequency was also included as a factor in the analysis of work loop power output. We log transformed enzyme activity data to meet assumptions of normality.
Additionally, we tested whether any of the variables (enzyme activities and muscle biomechanics) were dependent on body mass by fitting a nonlinear power function to the data (in CurveExpert version 1.3 software), and using the correlation coefficient r as an indicator of significant relationships; there were no significant relationships between mass and any of the variables except for work loop power output in cold-acclimated animals at 20°C test temperature. We therefore used mass as a covariate in the analysis of work loop data.
Q10 values were calculated according to Van't Hoff's equation: Q10 = (k1/k2)10/(t2-t1) where k1 and k2 are reaction rates at temperatures t1 (20°C) and t2 (30°C); we used t-tests to analyze Q10 values between acclimation treatments within muscles.
We performed Pearson product moment correlation analysis to determine interindividual relationships between biochemical indicators and muscle performance (8, 20, 88). We performed separate analyses for cold- and warm-acclimated animals, and for data collected at 20°C and 30°C test temperatures. We used the truncated product method (65, 98) to assess the effect of multiple comparisons on the validity of P values in the correlation matrices. Briefly, the truncated product method considers the distribution of P values from multiple hypothesis tests to provide a table-wide P value for the overall hypothesis that significant results in the set were truly significant rather than being due to chance (98). Significance levels in the analysis of warm-acclimated animals at 20°C are likely to be an artifact of multiple comparisons (truncated product method P = 0.14), and we did not consider these data further. The P values in the remainder of the correlation analyses represent truly significant differences (truncated product method, all P < 0.01). For work loop power output measured in cold-acclimated animals at 20°C, we used residuals of the power functions that relate mass to work loop power output. All means are presented ± SE.
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RESULTS |
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Acclimation treatment did not affect either maximal twitch or tetanic stress (force per cross-sectional area) in caudofemoralis muscle, and there was no significant interaction between test temperature and acclimation treatment (all F1,14 < 2.9, P > 0.1; Fig. 1). However, maximal twitch stress was significantly less at 30°C test temperature compared with 20°C (F1,14 = 68.25, P < 0.0001; Fig. 1A), whereas maximal tetanic stress increased with increasing test temperature (F1,14 > 26.30, P < 0.0001; Fig. 1B).
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Work loop power output. Caudofemoralis work loop power output was significantly greater in cold-acclimated compared with warm-acclimated animals at both test temperatures (F1,41 = 9.25, P < 0.005; Fig. 3A). Additionally, work loop power output increased with increasing cycle frequency (F2,41 = 19.84, P < 0.0001), and there was a significant interaction between test temperature and cycle frequency (F2,41 = 37.42, P < 0.0001; Fig. 3A).
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Activities of LDH and COX increased with increasing test temperature in caudofemoralis (tail) muscle (both F1,14 > 85.66, P < 0.0001; Fig. 4, A and B). However, acclimation treatment did not affect either LDH or COX activities (both F1,42 < 0.50, P > 0.50; Fig. 4, A and B), and there were no significant interactions between test temperature and acclimation treatment (F1,14 < 1.30, P > 0.27).
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Interindividual Comparisons
In warm-acclimated animals at 30°C, maximum tetanus stress and tetanus relaxation time were positively related to myofibrillar ATPase activity, and work loop power output at a cycle frequency of 0.7 Hz was positively related to maximum tetanus stress. (Table 1).
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DISCUSSION |
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Neither the twitch stress (force per muscle cross-sectional area) nor tetanic stress produced by the main swimming muscle (caudofemoralis) of C. porosus change with acclimation. However, isometric twitch force development and relaxation times are significantly shorter in cold-acclimated C. porosus. Both cold-acclimated fish (47, 86) and frogs (Xenopus laevis; 95) show similarly lower twitch relaxation times compared with warm-acclimated animals, although in each case force development times did not differ between acclimation groups. Additionally, isometric stress increases with cold acclimation in some fish and frogs (50, 95).
Work loop measurements are a biologically more realistic measure of dynamic muscle function than isometric measurements (41, 53, 86), and in C. porosus muscle, power output is significantly greater at both test temperatures in cold-acclimated animals compared with warm-acclimated animals. Muscle power output should increase with increasing rates of muscle force development and relaxation (13, 42), and, hence, the decreased twitch force development and twitch relaxation times in C. porosus can at least partially explain the significant increases in work loop power output with cold acclimation. Also in the present study, we found that normalized muscle power output (W/kg muscle mass) decreased with increasing body mass and that this decrease in power output was related to both increased twitch force development and twitch relaxation times, i.e., that as body mass increased, contractile rates slowed down, as has been found in previous scaling studies looking at larger body size ranges (43, 61). During fast but sustained swimming, crocodile muscle contracts at a frequency of around 2 Hz (82), and the negative relationship between muscle power and force development and relaxation rates implies these rates modulate power output during acclimation. Similarly, muscle relaxation rates are also correlated with stride frequency at lower test temperatures in lizards, leading to the conclusion that the relaxation rate is restricting locomotor performance at lower temperatures (60).
A likely mechanism underlying rate of muscle contraction is Ca2+ flux within the myocyte. Muscle excitation-contraction coupling is modulated by Ca2+ flux, where Ca2+ binding with troponin controls the interaction between actin and myosin, and thereby muscle contraction (18). Muscle relaxation times in carp are associated with Ca2+-ATPase activity in the sarcoplasmic reticulum (50), and it will be interesting to determine whether acclimation of locomotor function in the crocodile involves modification of Ca2+ transport to and from the myofibril.
Hydrolysis of ATP by myosin ATPase activity drives the cycle of interaction between actin and myosin, which moves the two filaments past each other. Hence, ATPase activity is functionally related to force production, and its activity can also influence muscle force development and relaxation times (11, 24, 33). Faster contraction times at low temperatures or during cold acclimation may be achieved by expression of different fiber types (57) and altered expression of myosin light chains (5). The myofibrillar ATPase activity of caudofemoralis muscle in C. porosus is similar to that of fish, e.g., sculpin, goldfish, and killifish (5, 47). Unlike many fish species (34, 50), however, myofibrillar ATPase activity does not increase with cold acclimation in crocodiles. However, the thermal sensitivity of ATPase activity decreases with cold acclimation, even though there is no complete thermal compensation for ATPase activity in caudofemoralis muscle of cold-acclimated crocodiles. This change in thermal sensitivity of ATPase activity may reflect that enzyme activity of cold-acclimated animals is impaired at higher temperatures as a result of denaturation of the protein (34). Hence, changes in ATPase activity resulting from cold acclimation are not beneficial if cold-acclimated animals were exposed to the warmer temperature. Similar to previous findings in other ectotherms (52), we found that in warm-acclimated crocodiles, myofibrillar ATPase activity is positively related to maximum tetanus stress. However, tetanus relaxation times increased with increasing ATPase activity in crocodiles; this result may simply reflect that relaxation times increase with an increase in force production as the rate of relaxation stays the same. It should be noted that the relatively low sample size for our correlative analyses (n = 8) poses the risk for a type 2 error; that is, we did not detect a significant relationship when, in fact, it exists. This means that we cannot interpret the absence of significant relationships.
Acclimation does not affect activities of LDH or COX. In contrast, LDH activity in longissimus caudalis muscle increased with cold acclimation in C. porosus (26), which emphasizes that different tissues within the same animal may respond differently to thermal change. Within-individual differences in thermal acclimation of muscle metabolic enzyme activities also occur in frogs, but as a function of sex-specific reproductive behavior (71). In cold-acclimated carp, increases in isometric force production are accompanied by increases in COX and citrate synthase activities, but there are no changes in LDH activity. In this case, increases in aerobic ATP production appear to be necessary to support acclimation of muscle contractile properties and, in particular, to recruit fibers for sustained locomotion (49). The lack of acclimation of COX and LDH in C. porosus indicates that aerobic or anaerobic ATP production in caudofemoralis muscle is not limiting cold acclimation of muscle contractile properties or locomotor performance.
In cold-acclimated C. porosus at 30°C there is a negative relationship between LDH activity and isometric twitch stress. Lactate accumulation from glycolysis can inhibit contraction and Ca2+ cycling causing inhibition of contractile force (74) so that twitch stress of more anaerobically poised animals may be lower, especially when LDH activity is high at higher temperatures. In cold-acclimated C. porosus, LDH activity is significantly upregulated in liver compared with warm-acclimated animals (26), and at 30°C, the accumulation of lactate in cold-acclimated animals could compromise muscle function.
Perspectives and Significance
Unlike other terrestrial ectotherms, most ecologically important behaviors of crocodilians occur in water (78, 91, 94), including long distance migrations and dispersal (89, 90). Unlike fish, however, crocodiles regulate their body temperature by basking on land (76, 79). These ecological correlates pose an interesting set of constraints. According to the evolutionary theory of coadaptation between selected body temperatures and performance optima (3, 19), it would be expected that rate functions are optimized around narrow mean body temperature ranges. On the other hand, prolonged time spent in the water, or even short periods in smaller animals, preclude thermoregulation and would favor more generalist phenotypes. By combining thermoregulation with plasticity in biochemical and biomechanical function, crocodiles transcend the "generalist-specialist trade-off" (37, 59) and maximize fitness in environments with highly variable thermal properties. Nonetheless, the theory of coadaptation between body temperature and thermal sensitivity implies that thermoregulation and within-individual plasticity should not occur together. It seems likely, however, that the regulatory networks underlying metabolism and muscle function are inherently plastic. That is, the integrated response of regulatory mechanisms, such as metabolic, transcriptional, or translational control, can produce different phenotypes in response to persistent differences in environmental inputs (15). The regulatory and sensory machinery underlying metabolic and muscle phenotypes is present at all times so that capacity for reversible plasticity or acclimation has little associated cost (16). Hence, it is likely that the potential for plasticity is present in all species, and the ecologically relevant question is, what elicits or prevents the realization of that potential?
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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