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First published online October 19, 2007
Journal of Experimental Biology 210, 3749-3756 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008763
An examination of the metabolic processes underpinning critical swimming in Atlantic cod (Gadus morhua L.) using in vivo 31P-NMR spectroscopy
Alfred Wegener Institut fuer Polar und Meeresforschung, Am Handelshafen 12, 27570, Bremerhaven, Germany
* Author for correspondence (e-mail: Glenn.Lurman{at}awi.de)
Accepted 23 August 2007
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Key words: Atlantic cod, Gadus morhua, critical swimming speed, in vivo 31P-NMR spectroscopy, high-energy phosphates, Gibb's free energy, intracellular pH
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Brett defined the Ucrit as the swimming
velocity when the fish could no longer propel itself forward off the
downstream grid due to exhaustion. The Ucrit test provides
an easy way of directly assessing fitness in fish, and by proxy, the sum of
both the aerobic and anaerobic metabolic scopes. More specific tests, such as
the burst swimming test and the endurance swimming test, have been developed
to separately examine anaerobic and aerobic scope, respectively. Since its
inception, however, many experiments have used the critical swimming speed
test to assess the fitness of various fishes under different conditions, e.g.
after feeding, during hypoxia, in polluted waters (for a complete review, see
Hammer, 1995).
A great body of literature exists on the different swimming modes in fish
(Schultz and Webb, 2002;
Webb, 2002;
Jayne and Lauder, 1994;
Videler, 1993;
Videler, 1981;
Beamish, 1978;
Brett, 1964) and the metabolic
processes fuelling muscle contraction
(Jones, 1982). It has been
clearly demonstrated that red muscle is oxidative
(Johnston, 1977) and produces
the slow contractions during subcarangiform swimming
(Jayne and Lauder, 1994),
while white muscle is glycolytic and responsible for fast twitch contractions
(Johnston, 1977), which
produce tail kicks during burst swimming
(Jayne and Lauder, 1994). A
handful of studies have also found white muscle to be recruited during
subcarangiform swimming (Rome et al.,
1984; Jones, 1982;
Greer-Walker and Pull, 1973).
Yet to date, few studies have looked at the metabolic processes underpinning
the continuum, i.e. from subcarangiform swimming through the gait transition
to kick-and-glide bursts to exhaustion.
Although an increase in inorganic phosphate and an acidification of the
intracellular milieu is known to be involved in muscular fatigue, the exact
processes leading to muscular fatigue are ill-defined. Allen and Westerblad
(Allen and Westerblad, 2001)
contend that it stems from excess inorganic phosphate altering intracellular
concentration of Ca2+ and/or the Ca2+ sensitivity of the
myofilaments. Building on previous work
(Hibberd et al., 1985), Debold
et al. add that inorganic phosphate and H+ also reduce the force
generated by cross-bridge cycling (Debold
et al., 2004). Others have argued that a drop in the Gibbs free
energy of ATP hydrolysis (dG/d
ATP) below a certain
threshold results in fatigue (Hardewig et
al., 1998).
Nevertheless, the anaerobic products of kick-and-glide bursts leave a
`fingerprint' on the fish's metabolic state, and a small number of studies
have examined the relationship between swimming gait and metabolic processes.
For example, it was concluded that elevated post-exercise oxygen consumption
(EPOC) measured in salmon was a product of the anaerobiosis that fuels
swimming shortly before Ucrit
(Lee et al., 2003).
Furthermore, a clear relationship was demonstrated between anaerobic markers
(plasma and tissue lactate levels, tissue glycogen and EPOC), and
kick-and-glide swimming in smallmouth bass (Micropterus dolomieu
Lacepedé) (Peake and Farrell,
2004). Thus, Brett's traditional definition of
Ucrit is the speed that causes complete fatigue, i.e. when
energy demand has exceeded both the aerobic and anaerobic supply
(Brett, 1964).
Traditionally, metabolic critical points such as critical temperatures and
critical oxygen tensions, are points marked by a transition from steady state
aerobic metabolism to non-steady state anaerobic metabolism
(Pörtner, 2002;
Pörtner et al., 1985;
Claireaux and Dutil, 1992).
The traditional definition of Ucrit does not correspond
with these `critical' parameters. This was initially noted
(Brett, 1964) and further
discussed (Beamish, 1978) in
relation to swimming duration. Subsequent research has expanded upon this; for
example, the metabolic cost of swimming in common brief squid (Loliguncula
brevis Blainville) was investigated using the equivalent of a
Ucrit test, and it was found that anaerobiosis had
commenced long before the squid could no longer swim
(Finke et al., 1996). Pilot
studies in our laboratory have found similar results using Atlantic cod
(Pörtner et al., 2002).
While Brett's Ucrit test gives information about the
swimming performance, little is known about the underlying metabolic
processes, and in particular, what causes fatigue during exhaustive
exercise.
The use of in vivo 31P-NMR spectroscopy has proved to
be an exceptional method for non-invasively monitoring the metabolic status of
high-energy phosphates like PCr (phosphocreatine) and ATP, as well as the
formation of metabolic products such as intracellular phosphate (Pi), and
intracellular changes in pH in muscle
(Gadian, 1982). Furthermore,
it has been extensively used for mammalian muscle
(Cozzone and Bendaham, 1994).
A number of pioneering studies used 31P-MRS in freshwater fishes
(for reviews, see van den Thillart and van
Waarde, 1996; van der Linden
et al., 2004), and these methods have been further developed for
marine fishes (Bock et al.,
2001; Bock et al.,
2002b; Sartoris et al.,
2003). Early studies were carried out exclusively on restrained or
resting animals. However, a technique has recently been developed for
non-invasive studies of unrestrained fish under resting conditions (for
reviews, see Bock et al.,
2002b; Pörtner et al.,
2004). Furthermore, recent preliminary trials within our
laboratory have successfully investigated the use of in vivo
31P-NMR in Atlantic cod during swimming
(Bock et al., 2002a;
Pörtner et al.,
2002).
In this study we report on an online analysis of metabolic processes by in vivo 31P-NMR spectroscopy during Ucrit tests. The resulting data were used to examine three questions: (1) at what point in the Ucrit test do cod go from using steady state aerobic metabolism to time-limited, non-steady state anaerobic metabolism to fuel swimming and how does this tie in with the different swimming gaits; (2) how does the traditional definition of Ucrit compare with these underlying metabolic processes; (3) what are the metabolic processes that potentially cause fatigue in swimming Atlantic cod?
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Materials and methods |
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All fish were fed to satiation twice a week with a mixture of mussels (Mytilus edulis L.) and live common shrimp (Crangon crangon L.). Feeding was stopped a minimum of 5 days, maximum 7, before experimentation.
All experiments were carried out within German animal care legislature. Mortality during the entire holding period, i.e. from August 2004 to February 2006, was approximately 25%. No fish died during the course of the experiments; however, one died a week after experimentation.
Experimental setup
The swim tunnel consisted of three major parts
(Fig. 1). A PerspexTM pipe
was fed through a 40 cm inner diameter Bruker Biospec 47/40 (Ettlingen,
Germany) operating at 4.7 T. This pipe was attached to a circulation system.
When `closed,' a 256 l volume of seawater was hermetically sealed to measure
oxygen consumption. When `open,' a supplemental 444 l volume with a reservoir
of constantly aerated seawater was used to flush the system. Switching between
the two circulations was accomplished via three large taps. The
gas-tightness of the closed system was checked periodically by bubbling the
seawater with nitrogen gas overnight, then monitoring the seawater oxygen
content over a 12 h period. On no occasion during this test did the oxygen
content increase. A digital motion camera system connected to a computer was
used for observing the fish. Both circulations were temperature controlled to
within ±0.3°C. All swimming speeds were corrected for solid
blocking effects using the procedure outlined by Nelson et al.
(Nelson et al., 1994).
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),
measuring 3 cm 3 cm 0.2 cm, was sewn to the left side of the body, 15 cm
distal to the end of the caudal fin with two sutures on the leading edge. The
trailing edge was left free to move. This took less than 15 min. Fish were
then placed in a tubular shaped cage (15 cm diameter 60 cm length) within the
chamber. Here, the receive coil was positioned to allow optimal signal
transduction, while at the same time the fish was observed as it recovered
from anaesthesia. Complete recovery usually took approximately 15 min. The
cage was then pulled by means of a cord into the centre of the magnet and the
fish allowed to recover for at least another 4 h.
Exercise protocol
As part of a larger project looking at the effects of thermal acclimation
and acute temperature change on swimming performance (G.J.L., C.H.B. and
H.-O.P., manuscript in preparation), all fish were swum twice, once at the
acclimation temperature 10°C and once at the non-acclimation temperature
of 4°C; however, data for this study were only used from the 4°C swim.
Fish were allowed to recover from surgery for a minimum of 4 h before they
were cooled in a stepwise manner (2 3°C steps per 2 h) to 4°C and left
at minimal flow, i.e. 1 m3 h–1 (typically
0.15–0.19 BL s–1) overnight. The routine
metabolic rate was determined in fish swimming with minimal flow. The flow was
then increased in approximately 1 m3 h–1 (approx.
0.05 BL s–1) stepwise manner with each step lasting
30 min so that sufficient 31P-NMR spectra could be recorded (see
below). At sufficiently high water flows, fish would rest on the rear grid of
the cage. When two of these successive pauses lasted more than 20 s, a short 9
V electric current was manually applied to a grid downstream. The traditional
Ucrit was defined as the time when the fish was no longer
able to move from the grid (as per Nelson
et al., 1994), and calculated according to the formula
(Brett, 1964):
 | (1) |
Tail-beat frequency measurement
Tail-beat frequency was measured manually by counting the number of tail
beats in a 30 s period using the digital camera system. This was repeated
eight times at each of the 30 min swimming stages. The mean of these eight was
then taken as the tail-beat frequency. Eight 30 s sampling periods were not
always possible at Ucrit, so the mean was taken of as many
sampling periods as were permitted, minimum three. The time of the first kick
was also recorded.
Oxygen measurement
Oxygen was measured constantly at a sampling rate of 0.5 Hz using Fibox
optodes (Presens, Regensburg, Germany) with the temperature compensation
entered manually. Optodes were zeroed chemically with sodium dithionite in
seawater, and 100% was calibrated by placing the optode in the open
swim-tunnel circulation. This was checked periodically against a MultiLine P4
CellOx 325 oxymeter (WTW, Weilheim, Germany) calibrated to fully air saturated
seawater.
Oxygen consumption was calculated from the slope of the drop in water
oxygen content, which was monitored over a 20 min measurement period at each
speed. At no point did the seawater oxygen content drop below 80%. After the
initial 20 min measurement period the circulation was opened for a 10 min
flush/re-oxygenation. At the end of each experiment when the fish had been
removed, a `blank' oxygen consumption run was performed to quantify any
background microbial oxygen consumption. This was then subtracted from the
fish's respiration rate. Oxygen consumption rates were corrected for any
allometric size effects using the mass exponent of 0.8
(Saunders, 1963):
 | (2) |
O2 is the
standardised oxygen consumption rate in mg O2 kg–1
h–1, M is the mass of the fish in kg, and
O2m is the
measured oxygen consumption.
31P-NMR spectroscopy
In vivo 31P-NMR spectra included primarily white muscle
with a minor contribution from red muscle. Spectra were collected using a 200
s bp32 pulse with a flip angle of 45°, sweep width was 5000 Hz at 4 k, and
repetition time was 0.8 s. 256 scans were collected resulting in a total
acquisition time of around 3 min. In vivo 31P-NMR spectra
were recorded over the whole 30 min time period for each swimming speed.
Statistical analysis, data processing and modeling
31P-NMR spectra were acquired using Paravision 3.0 (Bruker,
Ettlingen, Germany). Spectra were processed in Topspin 1.5 (Bruker) first by
fast Fourier transformation, then filtered with line broadening in the range
of the half width of the PCr signal. Phase and baseline were corrected using a
specially adapted automatic correction routine (R.-M. Wittig, AWI, Germany).
Typically, 6–7 spectra were collected per swimming speed, i.e. per 30
min period. The best six spectra were then added for each swimming speed.
Metabolite concentrations were determined by operator defined integration
limits using the standard integration routine in Topspin 1.5 (Bruker).
The control PCr integral was converted into mol g–1 using
the intracellular concentration of 27.3 mol g–1 for resting
Atlantic cod (Sartoris et al.,
2003). All other concentrations, i.e. subsequent PCr measurements,
Pi and ATP, were then calculated relative to this. The intracellular pH was
calculated from the Pi chemical shift using the temperature compensated
formula given elsewhere (Bock et al.,
2001). Gibbs free energy change of ATP hydrolysis
(G/dATP) was estimated for NMR visible metabolites as
described earlier (Pörtner et al.,
1996; Satoris et al., 2003;
van Dijk et al., 1999), except
that creatine concentration was estimated using the following equation:
 | (3) |
).
All statistical analysis and modelling was performed using Graphpad Instat
3.0 and Prism 4.0 software (Graphpad Software, San Diego, CA, USA). For
comparative purposes, significant differences were tested using ANOVA with
Tukey's post-tests. Differences were considered significant when
P
0.05. Data are presented throughout as mean ± standard
deviation (s.d.).
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The oxygen consumption rate increased exponentially and significantly as a function of swimming speed up to the active metabolic rate (208±21.4 mg O2 h–1 kg–1; Fig. 2A). The standard metabolic rate (SMR) was estimated using non-linear regression of the oxygen consumption data at various speeds and extrapolating back to a swimming speed of 0 BL s–1. The mean SMR for all fish was 36.3±10.7 mg O2 h–1 kg–1.
The tail-beat frequency increased in a linear fashion up to the point when kicking was initiated and then levelled off (Fig. 2B). Any increase in swimming speed attained thereafter must have been accomplished by an increase in amplitude or the frequency of the kick-and-glide bursts. Kicking started before the traditional Ucrit was reached (at 89±2.5% Ucrit). Once initiated, the number of kicks increased in frequency until Ucrit, when the fish were kicking exclusively for several minutes before they stopped swimming altogether. This paralleled the initial decrease in the PCr signal, an increase in the Pi signal, and a resultant decrease in pHi (see below).
Difficulties were encountered in collecting sufficient 31P-NMR spectra from the moving animals. For optimal signal transduction, both the inductive and receive coils had to be parallel, with a minimal distance between them. Therefore, unless the fish was cooperative and swam near the coil, only a very weak signal could be detected. These difficulties were further exacerbated at higher speeds, in particular at Ucrit, when fish were kicking and gliding. Thus, it was possible to collect spectra from only three of the six fish at Ucrit. The loss of signal strength and the broadening of spectral bands due to swimming movements could have been mitigated by restricting the movements of the fish; however, this would have reduced the swimming performance.
Using in vivo 31P-NMR we were able to show a significant increase in the relative proportion of the inorganic phosphate signal above resting levels as the fish approached the critical swimming speed (Fig. 3A). Under minimal flow, the Pi signal was often below the detection limit. As the critical swimming speed was approached, the Pi signal began to intensify, until it was maximally and significantly elevated at the traditional Ucrit (9.5±3.4 mol g–1). Thereafter it decreased and was again basal at 2 h post exhaustive exercise.
The increase in Pi was coupled with a stoichiometric decrease in the relative proportion of the PCr from 27.3 mol g–1 at minimal flow, to 16.7±6.2 mol g–1 at Ucrit (Fig. 3B). A possible slight overshoot in the relative PCr proportion was apparent during recovery. The free ATP at control was 4.5±0.9 mol g–1 and did not change significantly throughout the course of the swimming bout, i.e. 3.8±1.4 mol g–1 at Ucrit, or recovery, although a slight dip to 2.2±3.0 mol g–1 was seen 90 min post Ucrit, at approximately the same time as the aforementioned PCr overshoot (Fig. 3C).
The pHi decreased significantly as the fish approached Ucrit (Fig. 4). At minimal flow pHi was 7.48±0.03. The pHi started to drop at approximately the same time as the Pi concentration began to increase (see below), i.e. between 72±2.6 and 91±3.0% Ucrit. At 91±3.0% Ucrit the increase became significant. The pHi was minimal, 6.81±0.05, at Ucrit and began to increase back to resting conditions during recovery.
As previously mentioned, under minimal flow conditions the Pi signal was
extremely small, sometimes undetectable, as also observed by other authors
(Bock et al., 2002b;
Sartoris et al., 2003), which
reflects the resting condition of the unrestrained fish. However, this made
determination of resting intracellular pH difficult. Some of the variation in
pHi at higher speeds, particularly near Ucrit,
where a very clear Pi signal could be discerned, may be a result of the
temporal resolution. As the acquisition of each spectrum took approximately
3.0 min, the pHi may have changed in this time. Furthermore, six of
these spectra were then summed for each swimming speed, potentially broadening
the Pi signal. In addition, the kicking at Ucrit would
have caused turbulences in the water that may have led to magnetic
inhomogeneities, resulting in broadening of all signals. As we predominantly
saw a broadening of the Pi signal alone, we believe the former explanation to
be correct.
At minimal flow the G/dATP was
–55.6±1.4 kJ mol–1 and had significantly
decreased to –49.8±0.7 kJ mol–1 at the
traditional Ucrit (Fig.
5). The drop in G/dATP was not a linear
function of the speed or time before Ucrit. The largest
drops in G/dATP of 2.96 kJ mol–1 and
4.04 kJ mol–1 were seen between 82±2.8% and
91±3.0% Ucrit and 91±3.0% and
Ucrit, respectively. Non-linear regression and
extrapolation to 0% Ucrit, i.e. resting conditions
(Fig. 5), gave a resting
G/dATP value of –57.3±1.5 kJ
mol–1.
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ATP below a threshold appears to be ultimately
responsible for fatigue.
It is worth noting that both the inductive coil and the receive coil
produced micro-turbulences. As a result, the flow in the swim tunnel behind
the receive coil was turbulent, and thus swimming conditions were not optimal.
When compared to literature values of similarly sized cod
(Table 1), an approximate
decrease of 30% in Ucrit was observed in the current
study, similar to that seen by Pörtner et al.
(2002), using a similar
set-up. Furthermore, fish in the present study were 10°C acclimated but
swum at 4°C. Parallel work found that an acute thermal change from
10°C to 4°C reduced swimming performance by approximately
10–15%, when compared to the performance at the acclimation temperature
of 10°C (G.J.L., C.H.B. and H.-O.P., manuscript in preparation).
Energetics of Ucrit
Several previous studies have used excessive post-exercise oxygen
consumption and blood parameters as measures of anaerobic metabolism
(Peake and Farrell, 2004;
Lee et al., 2003;
Nelson et al., 1994;
Brett, 1964), assuming that any
oxygen debt accumulated during the swimming trial was re-paid during recovery.
The online measurement of metabolic processes underpinning the entire swimming
trial, i.e. from slow swimming through the transition to kick-and-glide to
complete fatigue, confirm previous findings that Ucrit is
indeed the point of complete fatigue, when both the aerobic and
anaerobic resources have been fully expended.
In the current study, kicking started at 89±2.5%
Ucrit, just as the tail-beat frequency began to plateau
(Fig. 2B). At the same time Pi
began to increase and the pHi started to acidify. These kicks
avoided any increase in the tail-beat frequency required as speed increased.
Although it is possible that the fish were able to compensate by increasing
the tail-beat amplitude at the same frequency, we primarily observed that once
initiated, the number of kicks increased in frequency until close to
exhaustion (i.e. Ucrit), when the cod were kicking
exclusively. This strategy indicated the involvement of white muscle fibres
(Jones, 1982) and came at an
additional cost fuelled anaerobically on top of enhanced aerobic metabolic
rate.
Using electromyography, Rome et al.
(Rome et al., 1984) previously
found that as swimming velocity increased, mirror carp (Cyprinus
carpio L.) increasingly recruited more white muscle to increase the power
production before kicking was initiated. Similar findings have also been seen
with other fish species (Jayne and Lauder,
1994; Rome et al.,
1992). Although the precise time when kicking was first initiated
was not given, a significant increase in lactate and drop in PCr was seen at
70% Ucrit in rainbow trout (Onchorhynchus mykiss
Walbaum) using 31P-NMR (Burgetz
et al., 1998). This continued to rise at 80% and was maximal at
Ucrit. A more invasive study
(Nelson et al., 1994) looked
at, among other parameters, lactate production in Atlantic cod as a proxy for
anaerobic metabolism, and found that it had already significantly increased at
approximately 80% Ucrit. The reasons why this switch to
anaerobic metabolism occurred later in the present study, i.e. at 89%
Ucrit, are discussed below.
31P-NMR and energetic status
To our knowledge, only our previous study with Atlantic cod
(Pörtner et al., 2002)
and one other study with rainbow trout
(Burgetz et al., 1998) have
looked at exercised fish using 31P-NMR spectroscopy. In the latter
study, where the trout were swum to 70, 80 or 100% Ucrit
and then transferred to the NMR magnet, the exactitude of the measurements in
relation to swimming speeds was limited due to the potential stress incurred
during transfer. An artefactual decrease in PCr and an increase in Pi is
almost invariably seen in studies after the initial transfer of animals to the
NMR magnet (e.g. Sartoris et al.,
2003). However, because the trout were restrained, the spectra
were of a higher quality.
A previous study (Burgetz et al.,
1998) found that PCr had dropped to approximately one half of the
30 mol g–1 control values at 70% Ucrit.
This depletion continued and was maximal at Ucrit where
PCr was approximately 4 mol g–1. The severe reduction in the
PCr concentration, particularly at 70% Ucrit, is much
larger than in our study. This may be due to (i) a stress related artefact, as
discussed above, or (ii) species differences between athletic trout and
lethargic Atlantic cod.
In the same study (Burgetz et al.,
1998), biochemical analysis was used to determine lactate
concentrations in the same tissue. A strong correlation
(r2=0.83) was found between the tissue lactate
concentration and the pHi calculated from the Pi shift. At 70%
Ucrit, tissue lactate concentration had already increased
significantly (from 
13 mol g–1 tissue to 21 mol
g–1), and was maximal at Ucrit (47
mol g–1). In the current study pHi had dropped
significantly at 91±3.0% Ucrit. The disparity
between the time of onset and degree of intracellular acidification may be
attributable to the differences between the two species. For example, the
maximum plasma lactate concentration after exhaustive exercise was found to be
7 mmol l–1 in Scotian Shelf cod and 10 mmol
l–1 in Bras d'Or cod
(Nelson et al., 1994). In
comparison, other authors (Jain and
Farrell, 2003) found that plasma lactate levels could get as high
as 20 mmol l–1 in rainbow trout post exhaustive
exercise. We conclude from the cited evidence that Atlantic cod are
physiologically unable to exert themselves to the same degree as trout.
Additionally, the general level of fitness of our fish may have further
reduced their swimming capacity as they had been kept in our aquarium for more
than 1 year and there was only a slight current against which they could
exercise, possibly making them rather lethargic in comparison to freshly
caught, wild fish (Soofiani and Preide,
1985; Webb, 1971;
Bams, 1967;
Bainbridge, 1962;
Brett, 1958). The critical
swimming speed indicating onset of anaerobic metabolism may not be fixed but
be found at variable velocities in relation to Ucrit. In
cod it appears to be tied to the onset of kick-and-glide swimming.
Fatigue
One of the aims of the current study was to observe exactly when the
pHi decreased, i.e. at or before Ucrit, as this
would be an important step in determining the transition to non-steady state
anaerobic metabolism. It has previously been hypothesised that the
accumulation of physiological concentrations of Pi (e.g. 30 mol
g–1) is responsible for a decrease in the power generated in
the muscle as Pi moves into the sarcoplasmic reticulum and precipitates
Ca2+ (Allen and Westerblad,
2001), and reduces steady state tension by reducing/preventing
cross-bridge attachment (Hibberd et al.,
1985). The mean Pi concentration at Ucrit was
significantly elevated at 9.5±3.4 mol g–1, but we
expect the effect of Pi on fibre contraction would have been reduced, as
levels are 30% of those cited above. Two further points supporting the reduced
importance of Pi and pHi in exercise induced fatigue must be noted.
Firstly, increased intracellular Pi in fact increased force re-development
during rapid contraction–relaxation cycles
(Hibberd et al., 1985), which
are typical of kick-and-glide bursts seen here. Secondly, a drop in
pHi led to increased excitability of working skinned muscle fibres
from rats (Pedersen et al.,
2004).
More important were the changes observed in tissue energetics. The
stoichiometric relationship between the Pi increase and the PCr decrease
indicates that these cellular energetic stores were being depleted at
Ucrit to buffer cellular ATP concentrations. The
consequent drop in pHi indicated that anaerobic metabolism was also
being used to maintain ATP concentrations. Both Pörtner et al.
(Pörtner et al., 1996)
and Hardewig et al. (Hardewig et al.,
1998) have argued that as the cytosol becomes more acidic and,
more importantly, as the phosphagen (PCr in case of fish) is lysed to Pi and
Cr, there is a drop in G/dATP. For two species of
Zoarcid eelpouts, the free energy of ATP hydrolysis was observed to drop from
–60 kJ mol–1 to approximately –46 kJ
mol–1 and for rainbow trout, G/dATP
dropped from –60 to –47 kJ mol–1 after exhaustive
exercise (Hardewig et al.,
1998).
Various studies have looked at the effects of a drop in free energy values
and the detrimental effects on cellular ion transporters
(Jansen et al., 2003;
Kammermeier et al., 1982).
Hardewig et al. (Hardewig et al.,
1998) argued that below a threshold of approximately –52 kJ
mol–1, cellular processes such as Ca2+-ATPases,
essential to muscle function, can no longer derive enough energy to be
maintained. We suggest that the muscular fatigue observed in our fish was
predominantly due to a drop in G/dATP to
–49.8±0.7 kJ mol–1 at Ucrit,
which was below a certain threshold, potentially –52 kJ
mol–1 (Hardewig et al.,
1998), required by transporters to maintain ion gradients and fuel
the muscular machinery.
The control G/dATP values in the current study
(–55.6±1.4 kJ mol–1) lie below those reported
for resting eelpout (Hardewig et al.,
1998) and –61 kJ mol–1 for resting Atlantic
cod (Sartoris et al., 2003).
It seems reasonable to conclude that this is because resting values in the two
aforementioned studies were obtained from inactive fish, whereas our values
were obtained at minimal flow. A relatively linear decrease in
G/dATP with increasing swimming speed was previously
shown at moderate speeds in squid
(Pörtner et al., 1996).
Consequently, the slow swimming of our fish during control conditions would
have led to a shift in steady state energy status, thus reducing
G/dATP in comparison to values from inactive fish.
Extrapolation of our values back to resting
(Fig. 5) gave us a base value
of –57.3±1.5 kJ mol–1
(r2=0.90).
Conclusions
Through the use of in vivo 31P-NMR spectroscopy
combined with a Brett-type swim tunnel, we were able to show that as Atlantic
cod swimming speed increased, and gait was changed from subcarangiform
swimming to kick-and-glide swimming prior to traditional
Ucrit, i.e. at 89±2.5% Ucrit, a
graded decrease in intracellular pH was observed while the oxygen consumption
rate continued to increase exponentially. At the same time, phosphocreatine
levels fell and this was accompanied by a significant increase in inorganic
phosphate. All these changes were maximal at the traditional
Ucrit (i.e. exhaustion), when the fish were kicking
exclusively. These changes were subsequently restored during recovery. The
Gibbs free energy change of ATP hydrolysis was also minimal at the traditional
Ucrit (i.e. down from –55.6±1.4 at control to
–49.8±0.7 kJ mol–1), and this was argued to be
the leading cause for muscular fatigue leading to exhaustion of the cod. Thus,
a transition from steady state aerobic metabolism to non-steady state
anaerobic metabolism led to a complete exhaustion of aerobic and anaerobic
resources at the traditional critical swimming speed.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Allen, D. G. and Westerblad, H. (2001). Role of
phosphate and calcium stores in muscle fatigue. J. Physiol.
(Lond.) 536,657
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