Am J Physiol Regul Integr Comp Physiol 294: R1024–R1032, 2008.
First published January 16, 2007; doi:10.1152/ajpregu.00755.2007.
Plasticity of muscle function in a thermoregulating ectotherm (Crocodylus
porosus): biomechanics and metabolism
Frank Seebacher1 and Rob S. James2
1
Integrative 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
may be constrained by
limited capacity to respond to environmental change, and
individuals that are most effective in compensating for environmental variability will gain a selective advantage (75).
Selection can be partitioned into a performance gradient and a
fitness gradient (4). The former relates variation in biochemical, physiological, or morphological traits to a measure of
ecologically relevant performance, and the latter quantifies the
effect of variance in performance on fitness (4, 56). Locomotion is closely linked to Darwinian fitness because of its
influence on foraging success and escape from predators (36,
40, 45, 63, 83, 84) as well as on social status (23). For example,
maximal sprint speed in male lizards (Crotaphytus collaris) is
a reliable predictor of territory size and defense (39, 68), and
faster lizards have significantly greater reproductive output (38).
Although locomotor performance is heritable (21, 46, 58,
88), there is considerable within-generation variability as a
result of environmental effects and gene-environment interac-
tions (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
Address for reprint requests and other correspondence: F. Seebacher, Integrative Physiology, School of Biological Sciences, Univ. of Sydney, NSW
2006, Australia (e-mail: [email protected]).
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.
body temperature; thermal acclimation; locomotion; work loop; enzymes
PHYSICAL PERFORMANCE OF VERTEBRATES
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Seebacher F, James RS. Plasticity of muscle function in a thermoregulating ectotherm (Crocodylus porosus): biomechanics and metabolism. Am J Physiol Regul Integr Comp Physiol 294: R1024–R1032, 2008.
First published January 16, 2007; doi:10.1152/ajpregu.00755.2007.—
Thermoregulation and thermal sensitivity of performance are thought
to have coevolved so that performance is optimized within the
selected body temperature range. However, locomotor performance in
thermoregulating crocodiles (Crocodylus porosus) is plastic and maxima shift to different selected body temperatures in different thermal
environments. Here we test the hypothesis that muscle metabolic and
biomechanical parameters are optimized at the body temperatures
selected in different thermal environments. Hence, we related indices
of anaerobic (lactate dehydrogenase) and aerobic (cytochrome c
oxidase) metabolic capacities and myofibrillar ATPase activity to the
biomechanics of isometric and work loop caudofemoralis muscle
function. Maximal isometric stress (force per muscle cross-sectional
area) did not change with thermal acclimation, but muscle work loop
power output increased with cold acclimation as a result of shorter
activation and relaxation times. The thermal sensitivity of myofibrillar
ATPase activity decreased with cold acclimation in caudofemoralis
muscle. Neither aerobic nor anaerobic metabolic capacities were
directly linked to changes in muscle performance during thermal
acclimation, although there was a negative relationship between
anaerobic capacity and isometric twitch stress in cold-acclimated
animals. We conclude that by combining thermoregulation with plasticity in biomechanical function, crocodiles maximize performance in
environments with highly variable thermal properties.
PLASTICITY OF MUSCLE FUNCTION
METHODS
Animal Maintenance and Acclimation
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 ⫻ 620 mm
height ⫻ 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
AJP-Regul Integr Comp Physiol • VOL
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
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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 warmacclimated 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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PLASTICITY OF MUSCLE FUNCTION
AJP-Regul Integr Comp Physiol • VOL
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.
RESULTS
Isometric Force
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).
Isometric rate of force development and relaxation times.
Caudofemoralis twitch force development time was significantly shorter in cold-acclimated animals at both test temperatures (F1,14 ⫽ 5.40, P ⬍ 0.04), and twitch force development
times decreased with increasing test temperature (F1,14 ⫽
106.50, P ⬍ 0.0001; Fig. 2A). Similarly, caudofemoralis twitch
relaxation time decreased in cold-acclimated animals at both
test temperatures (F1,14 ⫽ 4.94, P ⬍ 0.05), but there was no
effect of test temperature on twitch relaxation time (F1,14 ⫽
0.37, P ⫽ 0.55; Fig. 2B).
Acclimation treatment did not affect either tetanus force
development time (Fig. 2C) or tetanus relaxation time (Fig. 2D;
both F1,14 ⬍ 1.25, P ⬎ 0.25) in caudofemoralis muscle.
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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 crosssectional 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).
PLASTICITY OF MUSCLE FUNCTION
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There was a significant interaction in myofibrillar ATPase
activity between test temperature and acclimation treatment
(F1,14 ⫽ 7.41, P ⬍ 0.02; Fig. 4C). This interaction is reflected
in the Q10 values (cold-acclimated animals, Q10 ⫽ 2.16 ⫾
0.33; warm-acclimated animals, Q10 ⫽ 3.13 ⫾ 0.23), which
were significantly greater in warm-acclimated crocodiles
(t14 ⫽ ⫺2.39, P ⬍ 0.04).
Interindividual Comparisons
DISCUSSION
Fig. 1. Effect of acclimation temperature and test temperature on saltwater
crocodile caudofemoralis muscle isometric stress (twitch responses, A; tetanic
responses B). Black and white bars represent cold- and warm-acclimated
individuals, respectively. Acclimation treatment did not affect maximum
twitch or tetanus stress, but maximal twitch stress was significantly less at
30°C test temperature. Data represent means ⫾ SE; n ⫽ 8 for each acclimation
group.
However, both tetanic force development and relaxation times
decreased with increasing test temperature (both F1,14 ⬎ 5.20,
P ⬍ 0.04). There was no significant interaction between
acclimation treatment and test temperature in any of the twitch
or tetanus force development or relaxation times (all F1,14 ⬍
1.10, P ⬎ 0.32).
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).
Enzyme Activities
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).
AJP-Regul Integr Comp Physiol • VOL
Here we show that in crocodiles, changes in biomechanical
function of muscle occur following acclimation to simulated
winter and summer conditions, in addition to the previously
demonstrated compensation in sustained swimming performance (26).
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
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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).
In cold-acclimated animals, normalized work loop power
output at 20°C test temperature decreased with increasing
body mass (0.7 Hz: Y ⫽ 4.17x⫺2.39, r ⫽ 0.73; 1.0 Hz: Y ⫽
3.41x⫺0.34, r ⫽ 0.84; 2.0 Hz: Y ⫽ 1.86x⫺5.50; Fig. 3B). In the
same animals (at 20°C), mass corrected residuals of work loop
power output at 2.0 Hz decreased with increasing isometric
twitch force development and relaxation times (Fig. 3C; Table 2).
Additionally, in cold-acclimated animals at 20°C twitch stress
increased as force development and relaxation times decreased
(Table 2). LDH activity of cold-acclimated animals at 30°C
was negatively related to twitch stress (Table 1). Isometric
force development and relaxations times as well as work loop
power output at different cycle frequencies were related in all
animals (Tables 1 and 2).
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PLASTICITY OF MUSCLE FUNCTION
ation 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
AJP-Regul Integr Comp Physiol • VOL
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
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Fig. 2. Effect of acclimation temperature and test temperature on saltwater crocodile caudofemoralis muscle twitch and tetanus activation and relaxation times
(time to peak twitch, A; time from peak twitch to half relaxation B; time to half peak tetanus, C; time from last stimulus to half tetanus relaxation, D). *Significant
differences between acclimation treatments. Twitch activation times, tetanus activation times, and tetanus relaxation times significantly decreased with increasing
test temperature. Data represent means ⫾ SE; n ⫽ 8 for each acclimation group.
PLASTICITY OF MUSCLE FUNCTION
R1029
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
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Fig. 3. Effect of acclimation temperature and test temperature on saltwater
crocodile caudofemoralis muscle power output determined using the work loop
technique. A: power output data is normalized to muscle mass and plotted as
means ⫾ SE against the frequency of work loop cycles, n ⫽ 8 for each
acclimation group. Circles and diamonds represent data from 20°C and 30°C
test temperatures, respectively. Closed symbols and open symbols represent
data from cold- and warm-acclimated individuals, respectively. Power output
in cold-acclimated animals was significantly greater than in warm-acclimated
animals, and power output significantly increased with cycle frequency.
B: work loop power output decreased with body mass in cold-acclimated
crocodiles at 20°C test temperature at all cycle frequencies (0.7 Hz solid
inverted triangles, solid line; 1.0 Hz open circles, dotted line; 2.0 Hz open
triangles, dashed line). C: in the same animals at 2-Hz cycle frequency,
mass-corrected residuals of work loop power output significantly decreased
with increasing twitch activation time (open circles, dashed line) and increasing twitch relaxation time (closed circles, solid line). Regression lines are
shown for all relationships.
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
AJP-Regul Integr Comp Physiol • VOL
Fig. 4. Enzyme activities (means ⫾ SE) in caudofemoralis muscle of cold
(filled bars) and warm (open bars)-acclimated crocodiles measured at 20°C and
30°C test temperature. There was no effect of acclimation on lactate dehydrogenase (LDH; A) or cytochrome c oxidase (COX; B) activities. However, there
was a significant interaction between acclimation treatment and test temperature in myofibrillar ATPase activity (C).
294 • MARCH 2008 •
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R1030
PLASTICITY OF MUSCLE FUNCTION
Table 1. Correlation coefficients for enzyme activities and muscle biomechanical data of cold (c) and warm (w) acclimated
crocodiles at 30°C test temperature
ATP
LDH
COX
Twitch
Tw fd
Tw rel
Tet
Tet rel
0.7 Hz
1.0 Hz
2.0 Hz
0.21
⫺0.4
⫺0.58
⫺0.75*
⫺0.54
0.24
⫺0.52
0.52
0.09
⫺0.32
0.29
0.71*
⫺0.36
0.61
⫺0.18
0.74*
0.39
0.51
0.39
0.61
0.50
0.43
⫺0.021
0.28
⫺0.72*
⫺0.21
⫺0.20
⫺0.29
0.23
0.66
⫺0.32
⫺0.29
0.01
⫺0.41
0.28
⫺0.37
0.37
0.09
0.23
0.08
0.34
0.09
COX
Twitch
Tw fd
Tw rel
0.37
⫺0.41
0.14
⫺0.41
⫺0.37
0.13
0.14
⫺0.48
⫺0.13
⫺0.50
⫺0.46
⫺0.29
⫺0.47
⫺0.25
⫺0.56
⫺0.29
⫺0.33
⫺0.12
0.41
0.21
⫺0.35
⫺0.09
0.38
0.20
0.05
⫺0.15
⫺0.22
0.41
⫺0.46
0.30
⫺0.25
0.20
⫺0.42
0.04
0.60
⫺0.69
0.15
0.72*
0.88**
0.87**
0.63
0.22
0.26
0.49
0.19
0.33
⫺0.28
0.05
⫺0.18
⫺0.36
0.82*
⫺0.71*
0.75*
⫺0.27
0.50
⫺0.31
0.23
⫺0.25
⫺0.16
⫺0.20
Tet
Tet fd
Tet rel
0.7 Hz
1.0 Hz
⫺0.03
0.61
⫺0.39
0.32
0.33
0.72*
0.39
0.60
0.42
0.36
0.78*
0.17
0.50
0.27
0.31
0.22
⫺0.13
0.04
0.51
0.39
0.47
0.53
0.01
0.44
0.90**
0.94**
0.76*
0.81*
0.83*
0.93**
ATP, myofibrillar ATPase; LDH, lactate dehydrogenase; COX, cytochrome c oxidase; Twitch, twitch stress; Tw fd, twitch force development time; Tw rel,
twitch relaxation time; Tet, maximum tetanus stress; Tet fd, tetanus force development time; Tet rel, tetanus relaxation time; 0.7 Hz, 1.0 Hz, 2.0 Hz, work loop
power output at 0.7, 0.1, and 0.2 Hz cycle frequency, respectively. Truncated product method: cold, P ⫽ 0.0074, warm, P ⫽ 0.0011. *Significant at P ⬍ 0.05;
**significant at P ⬍ 0.01.
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.
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
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
Table 2. Correlation coefficients for enzyme activities and muscle biomechanical data of cold acclimated crocodiles at 20°C
test temperature
LDH
COX
Twitch
Tw fd
Tw rel
Tet
Tet fd
Tet rel
0.7 Hz
1.0 Hz
2.0 Hz
c
c
c
c
c
c
c
c
c
c
c
ATP
LDH
COX
Twitch
⫺0.23
⫺0.16
0.32
0.04
⫺0.01
⫺0.24
⫺0.40
⫺0.35
⫺0.36
⫺0.34
⫺0.21
⫺0.15
⫺0.02
⫺0.07
0.12
⫺0.31
⫺0.20
0.46
0.57
0.52
0.46
0.67
⫺0.51
⫺0.47
0.27
⫺0.32
⫺0.58
⫺0.26
⫺0.01
0.09
⫺0.85**
⫺0.84**
0.19
⫺0.83*
⫺0.85**
⫺0.10
0.38
0.55
Tw fd
0.93**
⫺0.26
0.79*
0.77*
⫺0.01
⫺0.61
⫺0.77*
Tw rel
Tet
Tet fd
Tet rel
0.7 Hz
1.0 Hz
⫺0.28
0.76*
0.78*
0.04
⫺0.56
⫺0.72*
0.20
⫺0.29
0.36
0.13
⫺0.01
0.61
0.23
⫺0.37
⫺0.61
0.30
⫺0.20
⫺0.36
0.69
0.45
0.95**
Truncated product method: P ⬍ 0.0001. *Significant at P ⬍ 0.05; **significant at P ⬍ 0.01.
AJP-Regul Integr Comp Physiol • VOL
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Tet fd
c
w
c
w
c
w
c
w
c
w
c
w
c
w
c
w
c
w
c
w
c
w
LDH
PLASTICITY OF MUSCLE FUNCTION
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?
GRANTS
R. S. James was supported by a Royal Society International Short Visit
grant, and this research was funded by an Australian Research Council
Discovery grant (to F. Seebacher).
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