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First published online July 20, 2006
Journal of Experimental Biology 209, 2961-2970 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02319
Cardiovascular and haematological responses of Atlantic cod (Gadus morhua) to acute temperature increase
1 Ocean Sciences Centre, Memorial University of Newfoundland, St John's, NL,
A1C 5S7, Canada
2 Biology Department, Mount Allison University, 63B York Street, Sackville,
New Brunswick, E4L 1G, Canada
* Author for correspondence at present address: Department of Biology, University of Ottawa, 150 Louis Pasteur, Ottawa, ON, K1N 6N5, Canada (e-mail: mgollock{at}uottawa.ca)
Accepted 9 May 2006
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1.7°C h-1 to CTM) on the
cardiorespiratory physiology of Atlantic cod, we (1) carried out respirometry
on 10.0°C acclimated fish, while simultaneously measuring in vivo
cardiac parameters using Transonic® probes, and (2) constructed in
vitro oxygen binding curves on whole blood from 7.0°C acclimated cod
at a range of temperatures. Both cardiac output
(
) and heart rate
(fH) increased until near the fish's CTM
(22.2±0.2°C), and then declined rapidly. Q10 values for
and fH were 2.48
and 2.12, respectively, and increases in both parameters were tightly
correlated with O2 consumption. The haemoglobin (Hb)-oxygen binding
curve at 24.0°C showed pronounced downward and rightward shifts compared
to 20.0°C and 7.0°C, indicating that both binding capacity and
affinity decreased. Further, Hb levels were lower at 24.0°C than at
20.0°C and 7.0°C. This was likely to be due to cell swelling, as
electrophoresis of Hb samples did not suggest protein denaturation, and at
24.0°C Hb samples showed peak absorbance at the expected wavelength (540
nm). Our results show that cardiac function is unlikely to limit metabolic
rate in Atlantic cod from Newfoundland until close to their CTM, and we
suggest that decreased blood oxygen binding capacity may contribute to the
plateau in oxygen consumption.
Key words: Gadus morhua, temperature, cardiac output, heart rate, stroke volume, metabolic rate, haemoglobin, blood oxygen concentration
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Introduction |
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;
Jobling, 1981;
Beitinger et al., 2000). The
Atlantic cod Gadus morhua is a marine fish species whose thermal
biology has also received considerable attention
(Saunders, 1963;
Clark and Green, 1991;
Jobling, 1988;
Schurmann and Steffensen,
1997; Otterlei et al.,
1999; Claireaux et al.,
2000; Björnsson et al.,
2001; Despatie et al.,
2001; Peck et al.,
2003; Petersen and Steffensen,
2003). Recently, it was shown that North Sea cod thermal tolerance
is limited by the capacity of oxygen supply mechanisms
(Sartoris et al., 2003;
Lannig et al., 2004). Further,
Lannig et al. suggested, based on data collected by magnetic resonance imaging
(MRI), that there is a progressive mismatch between oxygen delivery and demand
above 5.0°C because temperature dependent increases in heart rate
(fH) do not result in similar increases in blood flow
(i.e. cardiac output) (Lannig et al.,
2004). The conclusion that oxygen delivery limits thermal
tolerance in this species is consistent with the current literature on marine
ectotherms (e.g. Pörtner,
2002). However, the reported insensitivity of blood flow to
temperature increases above 5.0°C is an interesting finding, which
suggests that the cardiorespiratory system of North Sea cod may respond
differently to an acute thermal challenge compared to other fish species. For
example, Overgaard et al. (Overgaard et
al., 2004) showed that as a result of increased
fH and the maintenance of stroke volume
(VS), maximum cardiac output
() increased with temperature
(Q10=1.8) in the in situ rainbow trout heart. Furthermore,
8.0°C acclimated winter flounder (Pleuronectes americanus)
exposed to acute elevations in temperature can increase
until approx. 1-2°C before their
critical thermal maximum (CTM) (25.0°C) (P. C. Mendonca and A.K.G.,
unpublished).
Although free swimming Atlantic cod held in thermally stratified water move
to preferred temperatures (Claireaux et
al., 1995), sea-caged fish are limited in their movement in the
water column. For example, in Newfoundland waters, cod can be exposed to
temperatures of up to 20.0°C (even at depths of 6 m) and short-term
(daily/weekly) temperature fluctuations of as much as 10.0°C during the
summer months (Fig. 1). Since
Atlantic cod are generally considered to have a preferred temperature of
8-15°C (Despatie et al.,
2001; Petersen and Steffensen,
2003), it is important to determine whether high temperatures
negatively impact oxygen delivery in Newfoundland Atlantic cod, as has been
suggested for the North Sea populations
(Lannig et al., 2004). This is
particularly pertinent, as there is evidence to suggest that physiological
responses to imposed challenges differ between cod populations
(Nelson et al., 1994).
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In this study, we fitted 10.0°C acclimated adult cod of Newfoundland
origin with Transonic® flow probes, and measured cardiac variables and
oxygen consumption
(
) as
water temperature was increased to the cod's CTM. Further, we constructed
in vitro haemoglobin-oxygen dissociation curves at 7.0, 20.0 and
24.0°C to investigate the effects of an acute increase in temperature on
blood oxygen-binding capacity.
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3000
litre tank in the ARDF supplied with aerated seawater at 10.0-11.0°C for
at least 2 months prior to experimentation. The fish were fed a commercial cod
diet daily, and photoperiod was maintained at 8 h:16 h light:dark. To perform the in vitro studies of haemoglobin-oxygen binding we used 7.0-8.0°C acclimated cod (0.383±0.016 kg; range=0.206-0.483 kg) reared at the ARDF. Prior to experimentation these animals were maintained in a 17 500 litre tank at the Ocean Science Centre. These fish were also fed commercial cod pellets daily, and maintained on an ambient photoperiod.
Surgical procedures
Fish were netted and placed in seawater containing tricaine
methanesulfonate (MS-222, Finquel; 0.15 g l-1) until ventilatory
movements ceased. The fish were then weighed and measured, before being
transferred to a surgery table, where oxygenated seawater containing MS-222
(0.055 g l-1) continuously irrigated their gills. The procedure
used to implant the flow probe was modified from that described
(Thorarensen et al., 1996) for
the rainbow trout. Briefly, the cod was placed on its right side on a wetted
sponge, and the operculum on the left side was lifted and secured in place to
allow access to the gill arches. Then, a surgical thread was placed around the
gill arches and tied to allow access to the ventral aorta. A small (approx.
7-10 mm) incision was made in the tissue just below the junction of the second
and third gill arches with a scalpel, and the ventral aorta was located by
carefully cutting away connective tissue. Without disrupting the pericardium,
the vessel was then freed from the surrounding tissue using blunt dissection,
and a 2.5S Transonic® blood flow probe (Transonic Systems, Ithaca, NY,
USA) was placed around the aorta. The cable of the flow probe was then secured
to the animal with 3 skin sutures (1-0 silk thread, American Cyanamid Company,
Pearl River, NY, USA); one close to the incision, one just ventral to the
pectoral fin, and finally, one close to the dorsal fin.
Respirometry and cardiac output measurements
Once surgery had been completed, fish were transferred to a 142-litre
custom-designed swim tunnel with the water speed set at 0.2 body lengths per
second (BL s-1). This current velocity allowed the fish to
hold position, without having to swim actively. All fish commenced ventilation
almost immediately after being transferred to the swim tunnel, and were given
at least 18 h to recover from surgery. Acute temperature challenges were
carried out the day after surgery, by increasing temperature from baseline
(10-11°C) by 1.7°C h-1 until the fish lost
equilibrium; the temperature at which the fish lost equilibrium was recorded
as the animal's CTM.
measurements were made during the last 12 min at each temperature, as we had
previously determined that temperature had reached a steady state by this
time. From preliminary experiments we estimated that the CTM was approx.
22.0°C, and therefore oxygen measurements were taken every 30 min,
starting 5.0°C before the fish's expected CTM.
measurements were not taken after the fish lost equilibrium, as water
temperature was rapidly reduced after brief (<1 min; see
Table 1) cardiovascular
measurements were made. This procedure was performed in an effort to recover
the animals, but only one fish survived.
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Oxygen concentration (mg O2 l-1) in the swim tunnel
was continuously measured by pumping water through an external circuit using a
peristaltic pump (Masterflex, Cole Palmer; Anjou, QC, Canada). The circuit was
constructed of tubing with an extremely low gas permeability (Tygon Food, ser.
6-419, Cole Parmer; Anjou, QC, Canada), and contained a D201 flow cell (WTW;
Weilheim, Germany) that housed a galvanic oxygen electrode (model CellOx 325,
WTW). This oxygen probe was connected to an oxygen meter (model 330, WTW) with
automatic temperature compensation so that water oxygen levels (mg
l-1) could be obtained. Dissolved oxygen in the water never dropped
below 6.5 mg l-1 (85% saturation at 22.0°C) during
the study, and
of the
fish (in mg O2 kg-1 h-1) was calculated as:
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where: 
O2 is the change in water oxygen content (mg
l-1), v is volume of the respirometer and external circuit
(142 l), Mb is mass of the fish (kg) and t is
time required to make the
measurement (h).
was directly measured by
connecting the flow probe lead to a blood flow meter (model T206 Transonic
Systems, Ithaca, NY, USA), which was interfaced with a MP100A-CE data
acquisition system (Biopac Systems Inc., Santa Barbara, CA, USA) and a laptop
PC running AcqKnowledge software (Biopac Systems Inc.). Data was recorded at a
frequency of 10 Hz, and records of
were obtained during each
measurement. Brief (<5 min) recordings of
were also made after the fish reached
its CTM. (ml min-1
kg-1) was calculated in AcqKnowledge by dividing the raw data (ml
min-1) by the mass of the fish (kg). fH (beats
min-1) was calculated by counting the systolic peaks during a 15-30
s measurement period, and VS (ml kg-1) was
calculated from
/fH. These
measurements were repeated three times per temperature, and the mean value
used for data and statistical analyses.
In vitro blood oxygen binding curves
Oxygen binding curves were constructed for Atlantic cod blood incubated at
7.0, 20.0 and 24.0°C. These temperatures were selected because they
represented baseline levels, the temperature at which fish in the in
vivo study started to show signs of sublethal stress (levelling off of
fH and an increase in VS), and a temperature
slightly above the highest CTM reached (23.2°C), respectively.
Fish were anaesthetized in seawater containing MS-222 (0.055 g
l-1) and 3 ml of blood was quickly withdrawn using caudal puncture
and heparinized syringes. Haematocrit (Hct) was then determined in duplicate
by centrifugation of blood in micro-haematocrit tubes at 10 000
g for 3-5 min, and the remaining blood sample adjusted to 20%
haematocrit using marine teleost saline
(Driedzic et al., 1985). After
adjusting Hct, blood samples were placed in heparinized round-bottom flasks in
a 7.0°C shaking water bath, and initially gassed with a humidified mix of
100% air/0.2% CO2 (blood PO2 16-20 kPa). For
experiments conducted at 7.0°C, these experimental conditions were
maintained for 1 h. In contrast, blood used in the 20.0 and 24.0°C
experiments was gradually warmed to the desired temperature for 1 h. After
this initial 1 h equilibration period, eight different O2 tensions
ranging from 20 to 1.2 kPa were achieved by adjusting the relative percentages
of N2 and air (CO2 remaining constant at 0.2%) using
flow meters and a Wösthoff gas-mixing pump (H. Wösthoff Co., Bochum,
Germany). Blood was allowed to equilibrate at each PO2
level for approx. 30 min prior to sampling using gas-tight Hamilton syringes.
Blood PO2 was determined by injecting blood into a small
thermostatted chamber containing a Clark-type oxygen electrode (Cameron
Instrument Co., Port Aransas, TX, USA) set to the experimental temperature
(7.0, 20.0 or 24.0°C), while blood oxygen content (Hb-O2) was
measured on 30 µl blood samples using Tucker's methods
(Tucker, 1967) and a custom
designed thermostatted Tucker chamber (volume=1.66 ml) maintained at
32.0°C. The oxygen electrodes were connected to an OM 200 oxygen meter
(Cameron Instrument Co.) and a desktop PC running AcqKnowledge software. At
each temperature blood from six individuals was used to generate the
haemoglobin-oxygen binding curve.
At each PO2 level, 50 µl blood samples were taken for the measurement of haemoglobin concentration ([Hb]), and immediately frozen in liquid nitrogen. [Hb] was subsequently measured in duplicate on 10 µl blood samples. These assays were performed using a commercially available haemoglobin assay kit (Sigma Chemical Co., St Louis, MO, USA) and a spectrophotometer (Beckman Coulter, Mississauga, ON, USA; model DU 640) set to a wavelength of 540 nm. Hct was determined at the end of the experiments (as above), and mean corpuscular haemoglobin content (MCHC) (g 100 ml-1) calculated as [Hb]/Hctx100.
To ensure that the lower values of [Hb] measured at 24.0°C were not due
to haemoglobin degradation, or alterations in the nature of the chemical
interaction between the Drabkin's reagent (used in the Hb assay) and the Hb
protein at this high temperature, we performed a brief experiment on two cod.
In this experiment, we placed cod blood with a Hct of 20% in heparinized round
bottom flasks, warmed the blood by approx. 2.0°C every 30 min, and
collected blood samples at 7.0, 16.0, 18.0, 22.0, 24.0 and 26.0°C. Then we
conducted two sets of subsequent analyses. First, we performed wavelength
scans (400-600 nm) on blood that was being analyzed for [Hb] to see if there
was a change in the optimum wavelength (usually 540 nm) or the shape of the
spectra. Secondly, we performed agar gel electrophoresis on 50 µl blood
samples using the protocol described by Petersen and Steffensen
(Petersen and Steffensen,
2003).
Data and statistical analyses
Haemoglobin oxygen binding curves were constructed for individual fish at
7.0, 20.0 and 24.0°C (Tucker,
1967) by plotting Hb-O2 as a function of
PO2, and fitting a 4-parameter sigmoidal curve to the data
using Sigmaplot 2001 (SPSS, Chicago, IL, USA). The P50
value and Hill Coefficient (n) for each fish were derived from Hill
plots (log[satHbO2/(1-sat HbO2] vs
logPO2).
Statistical analyses were carried out using SPSS (v.11.0; SPSS, Chicago,
IL, USA).
,
, fH and
VS measurements taken at each temperature were compared to
both baseline levels and maximum levels prior to CTM using analysis of
variance (ANOVA) and Dunnett's post-hoc tests. One-way ANOVAs
followed by Tukey's post-hoc tests were used to examine whether
resting, maximum and post-CTM cardiac parameters and
were
different, and to evaluate the effect of incubation temperature on in
vitro haematological parameters. Pearson's correlation analysis was
carried out to define the strength of the relationship between
and
both and fH.
Finally, a 2-way ANOVA with Tukey's post-hoc analysis was used to
determine the effect of temperature and PO2 on in
vitro haemoglobin levels. A result was considered significant when
P<0.05. All data presented in the text, figures and tables are
means ± standard error (s.e.m.).
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was
82.2±3.7 mgO2 kg-1 h-1, and values for
, fH and
VS were 21.5±0.8 ml min-1
kg-1, 36.3±1.7 beats min-1 and 0.6±0.04 ml
kg-1, respectively.
increased gradually as temperature rose, was significantly elevated above
basal (10.0-11.0°C) values after 14.0°C, and reached a maximum value
2.56 times baseline just prior to the cod's CTM (22.2±0.2°C)
(Table 1 and
Fig. 2). A similar pattern was
observed for , where values became
significantly elevated above basal values after 16.0°C
(=33.3±1.6 ml min-1
kg-1) and the maximum value reached 2.45x baseline
(Table 1,
Fig. 2D). In contrast, both
fH and VS showed different
relationships with temperature (Fig.
2B,C). Heart rate rose significantly above baseline after
14.0°C (fH=49.4±2.6 beats min-1),
peaked at a temperature of approx. 20.0°C (at 1.99x baseline), but
had declined slightly (by approx. 3 beats min-1) by the time cod
reached their CTM. Although VS appeared to be increasing
at temperatures above 19.0°C, no significant elevation in
VS was observed prior to the fish reaching their CTM
(Fig. 2C). Both
fH and declined
rapidly when the fish reached their CTM, returning to values not significantly
different from baseline. In contrast, VS showed a
significant increase (to 0.80±0.08 ml kg-1) after CTM was
reached (Fig. 2C).
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Fig. 4 shows the
relationship between
, and
fH (A) and (B),
for all individuals used in this study. When the data are displayed as a
scatter plot, it appears that the relationship between
and
the two cardiac parameters is similar; a conclusion supported by the
correlation coefficients for the two linear relationships
(fH, r=0.862;
, r=0.871). However, fitting third
order regressions to the mean values recorded at each temperature revealed a
subtle difference in the shape of the relationships. For example, the
relationship between
and
fH appears exponential in nature, whereas that between
and
is more sigmoidal, apparently because
cardiac output was still increasing after
had
plateaued.
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of
Atlantic cod from Newfoundland. We show that these cod are able to increase
in response to an acute temperature
increase until 2.0°C prior to the cod losing equilibrium, but that
heart function (based on the appearance of cardiac arrhythmias) is negatively
influenced 4.0°C before the fish's CTM. Our results show that
VS was not compromised even at the highest measured
fH, a result that contrasts with what would be expected
based on the negative force-frequency relationship exhibited by in
vitro heart muscle (Shiels et al.,
2002a) and in situ studies, which suggest that
limitations on cardiac filling may contribute to decreases in stroke volume at
high heart rates/temperatures (Graham and Farrell, 1990;
Farrell et al., 1996). This
latter finding suggests that in vivo homeostatic mechanisms
compensate for the negative effects of temperature and increased contraction
frequency on myocardial performance, and enable cod to increase routine
cardiac output to a level (53 ml min-1 kg-1) greater
than that measured at maximal exercise at 10°C [35 ml min-1
kg-1 (Webber et al.,
1998); 34 ml min-1 kg-1 (L.H.P. and A.K.G.,
unpublished)]. Finally, we provide in vitro data, which suggests that
decreased blood oxygen binding capacity may contribute to the plateau in
oxygen consumption prior to the fish's CTM.
As expected,
also
rose in response to the acute temperature increase, and there was a tight
relationship between
and
. However, it is unclear from our
results whether there was a mismatch between blood oxygen delivery
(xblood oxygen content) and the
cod's oxygen demand as temperatures approached CTM. First, we did not measure
demand, only oxygen consumption, and it is possible that tissue oxygen
utilization fell as the high temperatures began to impair cellular functions.
Second, from Fig. 4B, it
appears that was still rising when
was
beginning to plateau. Finally, the acute temperature increase negatively
affected in vitro [Hb], and both haemoglobin oxygen affinity and
binding capacity. A decrease in haemoglobin oxygen affinity and binding
capacity at temperatures approaching the cod's CTM could have limited oxygen
uptake at the gills, and consequently resulted in reduced arterial oxygen
content (Jensen et al., 1998)
and a plateau in
.
However, these in vitro results do not take into account the
potential benefits of catecholamine release on red blood cell oxygen-carrying
capacity and gill perfusion (Perry et al., 1995;
Reid et al., 1998), and thus
it is unknown whether blood oxygen-carrying capacity was diminished in
vivo.
In our study, the cod's CTM was 22.2±0.2°C, Q10
values for
,
and fH were 2.78,
2.48 and 2.12, respectively, and cardiac function and metabolic rate showed
similar patterns of increase until approx. 2.0°C below the fish's CTM. The
CTM of the Atlantic cod used in this study is in the range of that determined
for North Sea cod (19-22°C) (Sartoris
et al., 2003), and similar increases in metabolic rate and
fH have been recorded in salmonids exposed to an acute
temperature increase (Heath and Hughes,
1973; Brodeur et al.,
2001; Rodnick et al.,
2004). However, few studies have examined the effects of an acute
temperature increase to CTM (or near CTM) on
and VS in
vivo. In North Sea cod, it was suggested that above 7.0°C, although
fH increases,
does not, because VS is reduced
(Lannig et al., 2004). In
contrast, we found that VS was maintained in Atlantic cod
at high temperatures, and that this allowed
to increase until just prior to the
fish's CTM (22°C; Figs 2
and 4). These differing results
may not be surprising considering that cod from different habitats have
equivalent exercise performances, but achieve these by different physiological
mechanisms (Nelson et al.,
1994). Furthermore, Webber et al. highlighted differences between
the cardiac responses to exercise in cod from the Scotian Shelf, and those
from the North Sea (Webber et al.,
1998). For example, the percentage increase in
from 0 to 0.67 BL
s-1 was 100% in the Scotian Shelf cod
(Webber et al., 1998),
compared to 47% and 57% in North Sea cod
(Axelsson and Nilsson, 1986;
Axelsson, 1988).
It has been shown that the haemoglobin isotype expressed can have a
significant effect on the physiology of cod
(Brix et al., 2004;
Petersen and Steffensen,
2003). Atlantic cod caught off the coast of Newfoundland were
found to be 
90% HbI 2-2 - the `low temperature' isoform
(Sick, 1965) - whereas fish
caught from the German Bight are likely to be composed of >55% HbI 1-1 -
the `high temperature' isoform (Brix et
al., 2004). Data suggest that the occurrence of the `high
temperature' isoform (HbI 1-1) may have a beneficial effect on in
vivo oxygen transport when the fish are exposed to elevated temperatures
(Brix et al., 2004). Thus, it
is possible that differences in haemoglobin isotype and associated
physiological characteristics, as well as the prolonged exposure to differing
temperature profiles in the wild prior to being held in lab conditions,
allowed the German Bight cod used by Lannig et al.
(Lannig et al., 2004) to meet
the metabolic demands concomitant with elevated temperatures without having to
increase . It should also be noted,
however, that different techniques were used to measure blood flow (cardiac
output) in the two studies, and this may also account for some of the observed
differences. Magnetic resonance imaging, used by Lannig et al.
(Lannig et al., 2004),
provided a relative measure of blood flow in the caudal vein and dorsal aorta.
In contrast, the Transonic® flow probes used in the present study provided
a direct and accurate measure of .
Clearly, future research should focus on the degree of intra-specific
variation in cardiac function between cod populations, its relation to
haemoglobin isotype, and the influence of both these factors on the thermal
tolerance and biology of cod.
As indicated by the large decreases in
and fH, cardiac
function collapsed at the cod's CTM. There are several possible reasons why
this occurred. Bradycardia at high temperatures might occur as an adaptive
response to either internal or external hypoxia when fish are exposed to high
temperatures (Heath and Hughes,
1973). The concept that slowing of the heart is an adaptive
response to temperature extremes has also been promoted by Rantin et al.
(Rantin et al., 1998), who
suggest that a controlled decrease in fH may provide
protection by maintaining low intracellular Ca2+ levels. However,
we did not observe a decrease in fH until very close to,
or at, the cod's CTM, and the decrease in
would have resulted in a considerable
mismatch between the fish's metabolic demands and blood oxygen transport. This
strongly suggests that the decrease in fH was not
adaptive, but an indication that the fish was reaching its thermal limit, and
that homeostasis could no longer be maintained.
It is also possible that heart function was compromised just prior to CTM
due to a temperature-dependent increase in peripheral tissue oxygen demand,
and thus insufficient oxygen to supply the heart's needs. For example, the
oxygen gradient between the red muscle and the blood is maintained in rainbow
trout even during hypoxia (McKenzie et
al., 2004), and if a similar situation occurs during an acute
temperature increase, venous blood oxygen levels reaching the heart could be
limiting. Moreover, a right shift in the HBC, as we observed at the higher
temperatures, would allow oxygen to be unloaded more efficiently to the
tissues (Jensen et al., 1998).
For fish with a coronary blood supply, this right shift in the HBC could be
beneficial (Farrell and Clutterham,
2003). However, for fish such as gadids, that do not have a
coronary circulation and rely on returning venous blood for the heart's oxygen
supply, this would be detrimental. Venous PO2 values of
between 0.7 and 4 kPa, depending on water oxygen saturation, activity and
acclimation temperature, have been suggested as the minimum that will allow
O. mykiss to maintain oxygen supply to the myocardium
(Kiceniuk and Jones, 1977;
Steffensen and Farrell, 1998;
Farrell and Clutterham, 2003).
Although no values are given, we estimate from the work of Lannig et al.
(Lannig et al., 2004), that
PvO2 declines to 2.3 kPa at 19.0°C in North Sea
cod, and expect that levels would decline further as temperature approached
the fish's CTM (mean 22.2°C). More importantly, applying the 2.3 kPa
estimate of venous PO2 to our 20.0°C and 24.0°C
in vitro HBCs, suggests that Hb-O2 would be <0.25 ml
O2 100 ml-1 blood just prior to CTM, severely limiting
oxygen supply to the heart. The fact that
stabilized or declined during the
10 min prior to loss of equilibrium in most of the fish used in the study
would support this argument.
Finally, there are a number of other factors that could have independently,
or in concert, led to the observed loss of cardiac function. It is possible
that the decline in fH and subsequent
observed just prior to loss of
equilibrium, rendered the brain hypoxic, and subsequently resulted in neural
dysfunction. For example, it has been suggested that impaired neural circuit
function is a more likely factor in death caused by exposure to environmental
extremes than accumulating cell death in organs
(Robertson, 2004). High
temperatures may have disrupted signal production and/or transduction of the
heart's pacemaker. In the present study, arrhythmias initially occurred at
18.0°C, and increased in frequency and duration as temperature
increased. Further, Lennard and Huddart suggest that as a result of changes in
membrane fluidity, cessation of transmembrane ion transport causes the fish
heart to cease beating (Lennard and
Huddart, 1991). It has been shown that acclimation to different
temperatures, and acute temperature increases, affect the duration of the
action potential in both isolated plaice (Pleuronectes platessa)
pacemaker cells (Harper et al.,
1995) and O. mykiss ventricular myocardium strips
(Coyne et al., 2000). Changes
in action potential duration have been shown to negatively affect
intracellular Ca2+ flux in atrial myocytes during depolarisation
(Shiels et al., 2002b), and
this would potentially affect cod myocardial contractility, considering the
pronounced negative effect that temperature has on the myocardium's
force-frequency relationship [(Shiels et
al., 2002a), adapted from Shiels and Farrell
(Shiels and Farrell, 1997)].
However, in the present study, a decrease in VS was not
observed with increased temperature. Although the catecholamine sensitivity of
trout atrial myocytes is reduced when exposed to an acute increase in
temperature from 14.0 to 21.0°C, high levels of adrenaline (e.g. 1 µmol
l-1) still cause a 1.6-fold increase in L-type
Ca2+ channel current (Shiels et
al., 2003). Thus, it is possible that the cod released large
amounts of catecholamines during our CTM experiments, and that these hormones
had a positive inotropic effect on the heart that facilitated the maintenance
of VS.
In conclusion, our results show that the cardiorespiratory system of
Atlantic cod (Gadus morhua) from the waters surrounding Newfoundland
is able to cope with an acute temperature increase to near the fish's CTM by
increasing fH, and concomitantly
. Indeed both
and fH proved to
be tightly correlated with metabolic rate
(Fig. 4) during the acute
temperature exposure. Although the relationship between fH
and
has been shown to vary with a number of factors
(Lucas et al., 1993;
Lefrançois et al.,
1998), our data and that of Webber et al.
(Webber et al., 1998) show
tight relationships between both fH and
, and
, when
cod are exposed to acute temperature increases and exercise tests,
respectively. These data indicate that cardiac parameters may be valuable for
telemetered studies of metabolism in this species. Finally, based on our
in vitro studies, it appears that as temperature approaches/reaches
the cod's CTM, the blood's capacity to take up oxygen decreases due to
reductions in both haemoglobinoxygen affinity and binding capacity. This was
particularly evident at 24.0°C, where cell swelling occurred, and the
haemoglobin-oxygen binding curve was shifted considerably downward and to the
right. However, we are unsure whether blood oxygen-carrying capacity is
compromised at high temperatures in vivo, or whether
declined prior to CTM because of a decrease in blood oxygen transport or a
decline in tissue oxygen demand. Clearly, more in vivo experiments
must be performed before the inter-relationships between blood oxygen
transport, tissue oxygen demand and thermal tolerance in this species can be
understood.
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