SUMMARY

Cardiovascular function and metabolic responses of the heart and other tissues during hypoxia exposure were compared between the hypoxia-tolerant epaulette shark (Hemiscyllium ocellatum) and the hypoxia-sensitive shovelnose ray (Aptychotrema rostrata). In both species, progressive hypoxia exposure caused increases in stroke volume and decreases in heart rate, cardiac output, cardiac power output (CPO, an assessment of cardiac energy demand) and dorsal aortic blood pressure, all of which occurred at or below each species' critical PO2 for whole-animal O2 consumption rate, (Pcrit). In epaulette sharks, which have a lower Pcrit than shovelnose rays, routine levels of cardiovascular function were maintained to lower water PO2 levels and the changes from routine levels during hypoxia exposure were smaller compared with those for the shovelnose ray. The maintenance rather than depression of cardiovascular function during hypoxia exposure may contribute to the superior hypoxia tolerance of the epaulette shark, presumably by improving O2 delivery and waste removal. Compared with shovelnose rays, epaulette sharks were also better able to maintain a stable cardiac high-energy phosphate pool and to minimize metabolic acidosis and lactate accumulation in the heart (despite higher CPO) and other tissues during a 4 h exposure to 40% of their respective Pcrit (referred to as a relative hypoxia exposure), which results in similar hypoxaemia in the two species (∼16% Hb–O2 saturation). These different metabolic responses to relative hypoxia exposure suggest that variation in hypoxia tolerance among species is not solely dictated by differences in O2 uptake and transport but also by tissue-specific metabolic responses. In particular, lower tissue [lactate] accumulation in epaulette sharks than in shovelnose rays during relative hypoxia exposure suggests that enhanced extra-cardiac metabolic depression occurs in the former species. This could facilitate strategic utilization of available O2 for vital organs such as the heart, potentially explaining the greater hypoxic cardiovascular function of epaulette sharks.

INTRODUCTION

The ability to tolerate environmental hypoxia varies greatly among fishes and the physiological attributes that underlie this variation in hypoxia tolerance remain incompletely understood. A growing body of evidence from comparative studies suggests that one important determinant of hypoxia tolerance in fishes is the ability to maintain O2 uptake at low water PO2 (PwO2), which is reflected by the PwO2 at which whole-animal O2 consumption rate () transitions from being independent to dependent on environmental O2; this inflection point is termed Pcrit (Mandic et al., 2009; Speers-Roesch et al., 2012). At PwO2 below Pcrit, hypoxic survival becomes dependent on maintenance of cellular energy balance in the face of decreases in aerobic energy supply (Richards, 2009). This may be achieved by an increased reliance on O2-independent energy supply (e.g. anaerobic glycolysis) as well as a profound, reversible metabolic rate depression in which large decreases in cellular energy demand occur (Richards, 2009).

The cardiovascular system is an essential component of the respiratory cascade and is driven by the heart, a hypoxia-sensitive organ with high energy demands. In fishes exposed to hypoxia the cardiovascular system is vital for transport of available O2 as well as in the distribution of fermentable fuel and the removal of waste. However, maintenance of cardiac function may be constrained by the limited energy supply during hypoxia exposure and in hypoxia-sensitive species catastrophic cardiac failure may occur as a result of a mismatch between energy supply and demand in the heart that leads to perturbed cardiac energy status (i.e. the levels of high-energy phosphate compounds) (Farrell and Stecyk, 2007). Hypoxia-tolerant fishes are able to avoid this fate, but the cardiovascular responses during hypoxia exposure (including PwO2 at and below Pcrit) that are associated with hypoxia tolerance remain unclear, in part because the responses reported are varied and because of the relatively few species studied. In most fishes exposed to hypoxia, bradycardia is observed and this generally occurs when PwO2 reaches Pcrit, suggesting that hypoxia-tolerant fishes with lower Pcrit may be able to maintain heart rate (fH) to lower PwO2 (Farrell, 2007; Speers-Roesch et al., 2010). However, responses of other cardiovascular parameters at and below Pcrit are more varied, confusing interpretation of their role in hypoxia tolerance. In some fishes, routine cardiac output () is defended to an extent by increases in stroke volume (VS), even as PwO2 falls below Pcrit (Butler and Taylor, 1975; Gamperl and Driedzic, 2009; Gamperl et al., 1994; Petersen and Gamperl, 2011). In other fishes, VS is unchanged and falls because of the decrease in fH below Pcrit (Iversen et al., 2010; Speers-Roesch et al., 2010; Stecyk and Farrell, 2006). Studies on fish species that allow to fall during hypoxia exposure, including tilapia (Oreochromis hybrid) and common carp (Cyprinus carpio), suggest that the reductions in fH and below Pcrit enable a decrease in cardiac power output (CPO), which represents a lowering of cardiac energy demand (Speers-Roesch et al., 2010; Stecyk and Farrell, 2006). Depression of CPO may be an important component of hypoxia tolerance because it allows cardiac energy demand to be matched to reduced energy supply at PwO2 below Pcrit where aerobic metabolism is limited, thus facilitating the maintenance of stable cardiac energy status and function (Farrell and Stecyk, 2007). The crucian carp (Carassius carassius), in contrast, has a low routine CPO that apparently can be sustained anaerobically, probably explaining how its remains relatively unchanged during hypoxia exposure (Stecyk et al., 2004). Overall, there is uncertainty about the degree to which depression or maintenance of cardiovascular function including CPO is associated with hypoxia tolerance and Pcrit in fishes.

Here, we carried out two series of experiments to assess cardiovascular and heart metabolic responses to progressive and relative hypoxia exposure in two species of elasmobranchs, the hypoxia-tolerant epaulette shark, Hemiscyllium ocellatum (Bonnaterre), and the comparatively hypoxia-sensitive Eastern shovelnose ray, Aptychotrema rostrata (Shaw). In one series of experiments, we report on cardiovascular function in epaulette sharks and shovelnose rays during progressive hypoxia exposure to examine how cardiovascular responses at and below Pcrit compare between a hypoxia-tolerant and a hypoxia-sensitive elasmobranch, given the lack of previous direct comparisons of the hypoxic responses of CPO between hypoxia-tolerant and -sensitive fishes. We hypothesized that routine cardiovascular function is maintained to a lower PwO2 in the epaulette shark because of its greater hypoxia tolerance and lower Pcrit compared with the shovelnose ray (Speers-Roesch et al., 2012). At progressively lower PwO2 below Pcrit, we hypothesized that there would be a greater depression of cardiovascular function including CPO in the epaulette shark compared with the shovelnose ray. In a second series of experiments, we assessed metabolic status (i.e. pH and levels of metabolites of energy metabolism, e.g. high-energy phosphates and lactate) in cardiac and other tissues of hypoxic epaulette sharks and shovelnose rays held for ≤4 h at a PwO2 representing the same percentage of each species' Pcrit. Our accompanying study revealed that exposure to the same percentage of Pcrit in these two species would yield a similar level of hypoxaemia (Speers-Roesch et al., 2012). Therefore, the present experiment allowed us to assess whether the greater hypoxia tolerance of epaulette sharks is associated with more stable hypoxic cardiac and extra-cardiac metabolic status compared with the shovelnose ray, even when variation in interspecific O2 supply is controlled for. In turn, this allowed us to test the hypothesis that the ability to take up and transport O2 at low PwO2, as indicated by Pcrit, dictates tissue-level hypoxia tolerance. In this case, we predicted that tissues of the two species during relative hypoxia exposure would have similar energy status, lactate accumulation and pH levels. Overall, these experiments and those in the accompanying study (Speers-Roesch et al., 2012) provide insight into the cardiorespiratory and metabolic responses that contribute to hypoxia tolerance in elasmobranchs and other fishes.

MATERIALS AND METHODS

Animals

Epaulette sharks and shovelnose rays of mixed sexes were collected and held in a recirculating seawater system (28°C) at Moreton Bay Research Station, North Stradbroke Island, QLD, Australia, as described in the accompanying paper (Speers-Roesch et al., 2012).

Experimental series I: cardiovascular responses to progressive hypoxia

Surgical protocol

Experimental series I is the same experiment as that described in our accompanying paper (Speers-Roesch et al., 2012) and the preparation of animals for surgery is fully described therein. In brief, cardiovascular responses to progressive hypoxia were monitored in epaulette sharks (1.29±0.04 kg, N=7) and shovelnose rays (1.54±0.06 kg, N=8) simultaneously with the measurements of and blood O2 transport properties. Measurement of dorsal aortic blood pressure (PDA) was via the caudal artery cannula that also allowed periodic blood sampling. To measure ventral aortic blood flow (i.e. cardiac output or ), an ultrasonic flow probe was fitted around the ventral aorta via a midline ventral incision made through the skin and overlying muscle anterior from the fifth gill slit. The connective tissue surrounding the ventral aorta was inspected for superficial vessels before being cut to expose the ventral aorta. Where present, vessels were ligatured with 4-0 silk to prevent bleeding. A 2.5 mm ultrasonic SB-type flow probe (Transonic Systems, Ithaca, NY, USA) filled with acoustic gel (Transonic Systems) was then fitted around the exposed ventral aorta distal to the third, fourth, and fifth afferent branchial arteries. This flow probe placement allows measurement of ∼37% of in elasmobranchs and this value does not change during hypoxia exposure [see Taylor et al. (Taylor et al., 1977) and Lai et al. (Lai et al., 1989), and references therein]. In vivo measurement of total is not possible in elasmobranchs because the afferent branchial arteries for the posterior gill arches arise as the conus arteriosus exits the pericardium and therefore no portion of the ventral aorta outside of the pericardium carries the entire cardiac output. Entering the pericardium is not an option in elasmobranchs because of its importance for cardiovascular function (Franklin and Davie, 1993; Stensløkken et al., 2004). After placement, the flow probe was secured with two 4-0 silk sutures tied to the surrounding muscle. The muscle incision was closed with interrupted 4-0 silk sutures and then the skin incision was closed with interrupted 1-0 silk sutures. The lead of the flow probe was secured to the skin and tied to the arterial cannula to prevent entanglement.

Experimental protocol

The experimental protocol and other details for the progressive hypoxia exposure are described in the ‘Experimental protocol’ section of the accompanying paper (Speers-Roesch et al., 2012). Routine cardiovascular variables (fH, , PDA) were continuously recorded at a normoxic PwO2 of approximately 16.0 kPa or 15.3 kPa for epaulette sharks and shovelnose rays, respectively (75–78% air saturation; 100% air saturation=20.4 kPa=153 Torr) for 1–2 h to ensure stable baseline conditions. After initial blood sampling and closing of the respirometer in which animals were exposed to progressive hypoxia, cardiovascular parameters were continuously monitored as PwO2 was depleted as a consequence of fish respiration. The effects on cardiovascular function of changes in water parameters (e.g. pH, PCO2) potentially associated with utilization of closed respirometry are considered to be negligible (Speers-Roesch et al., 2010). In fact, in both species increases in arterial PCO2 were minor and apparently had no effect on other measured parameters during progressive hypoxia exposure, as described in the accompanying study (Speers-Roesch et al., 2012). The PwO2 end points, rate of O2 depletion, duration of the progressive hypoxia exposures, and recovery in normoxic water for 60 min are described in the accompanying paper (Speers-Roesch et al., 2012). In some cases, flow probes became damaged by water exposure or by fish movements (typically during the overnight acclimation period) and therefore samples sizes of measured parameters vary slightly (see figure captions for final N values). At the end of the trials, fishes were terminally anaesthetized in seawater containing benzocaine and the ventricle was excised, emptied of blood, blotted dry and weighed.

Data acquisition and calculation of cardiovascular variables

The dorsal aortic cannula was connected to a pressure transducer (Capto SP844 model MLT844, MEMSCAP AS, Skoppum, Norway) calibrated against a static water column with the water surface in the experimental tank serving as the zero pressure reference. The transducer signal was amplified with a ML221 bridge amplifier (ADInstruments, Castle Hill, NSW, Australia). Dorsal aortic blood pressure recordings made in the respirometer were compensated for the small pressure change (∼0.5 kPa) that occurred depending on whether the respirometer was open or closed. The cannula was temporarily disconnected to allow for the periodic blood sampling described in the companion paper (Speers-Roesch et al., 2012). Cardiac output was recorded with a Transonic blood flow meter (Model T206, Transonic Systems, Ithaca, NY, USA). Flow probes were calibrated according to manufacturer guidelines at 28°C following the experiment to compensate for the effect of calibration temperature on flow readings. Water PO2 in the respirometer was monitored as described previously (Speers-Roesch et al., 2012). Signal integration and analysis were carried out using a Power Lab unit (ADInstruments) and LabChart Pro software (v. 6.0; ADInstruments), respectively.

Cardiovascular parameters were analysed over 5–10 min sampling periods bracketing PwO2 values at regular intervals that were similar to those used for calculation, from approximately 16.0 to 0.1 kPa in epaulette sharks and approximately 16.0 to 1.9 kPa in shovelnose rays. Because of periodic blood sampling and routine variability in individual traces, it was not possible to analyse data at exactly the same PwO2 values in each fish, so PwO2 values are provided with standard errors. Cardiovascular function during 60 min of recovery in normoxic water was analysed over 5–10 min intervals bracketing each time point (5, 15, 30, 45 and 60 min).

Cardiac output was calculated directly from the flow trace in LabChart Pro and corrected for the estimated loss of flow (63%) due to the location of the flow probes on the ventral aorta, as discussed previously (Lai et al., 1989; Taylor et al., 1977). PDA was calculated using the blood pressure analysis module in LabChart Pro. Because of limited animal numbers, we were unable to directly measure ventral aortic blood pressure (PVA). Therefore, we estimated PVA from our PDA measurements, using a percentage correction (PVA=1.3×PDA) based on previous simultaneous measurements of PVA and PDA in normoxia and hypoxia in epaulette sharks and spotted catsharks (Scyliorhinus canicula) (Short et al., 1979; Stensløkken et al., 2004; Taylor et al., 1977). CPO (mW g–1 wet ventricular mass) was calculated as the product of PVA (kPa) and (ml s–1) divided by the wet ventricular mass (g), where 1 J=1 kPa l. Heart rate (fH) was calculated from the pulsatile pressure or flow trace. Cardiac stroke volume (VS) was calculated as /fH and systemic peripheral resistance (RSYS) was calculated as PDA/, with the assumption that central venous blood pressure is zero. Cardiovascular parameters were plotted against PwO2 to identify the inflection points where each parameter ceased to be independent of PwO2 (i.e. the critical PwO2 of each cardiovascular parameter), as previously described for calculation of Pcrit of whole-animal (Speers-Roesch et al., 2012).

Experimental series II: tissue metabolic status during relative hypoxia exposure

Experimental protocol

Epaulette sharks (0.388±0.048 kg) and shovelnose rays (1.07±0.110 kg) were transferred from holding tanks to aquaria (∼300 l) supplied with aerated recirculating filtered seawater (28°C). Six to 10 epaulette sharks were distributed equally between two aquaria and the same was done for shovelnose rays in two separate aquaria. The fishes were allowed to acclimate for 12 h under well-aerated conditions. Then, one or two fish from the normoxic control group were gently removed from each aquarium and quickly immersed in a bucket of aerated seawater containing benzocaine (0.2 g l–1 benzocaine, initially dissolved in 95% ethanol). Animals struggled minimally during this procedure. Following anaesthesia (<1 min), mixed arterial–venous blood was sampled via caudal puncture and placed on ice until measurement of whole-blood pH as described before (Speers-Roesch et al., 2012). Following blood sampling, the fish was killed by severing the spinal cord posterior to the head. The heart was quickly removed, emptied of blood, blotted dry and frozen in liquid N2. White muscle from the caudal peduncle, liver and plasma obtained by centrifuging whole blood were also sampled. Frozen samples were transported to Canada in a dry shipper and kept at –80°C until analysis.

Following sampling of the normoxic fishes, hypoxia was induced by bubbling N2 into each aquarium, which was covered with plastic bubble wrap to prevent O2 ingress. Epaulette sharks were exposed to a PwO2 of 2.0 kPa (10% air saturation) and shovelnose rays were exposed to 3.1 kPa (15% air saturation), both of which represented 40% of each species' Pcrit and thus resulted in a similar level of physiological hypoxia (∼16% Hb–O2 saturation; ∼0.6 vol. % arterial O2 content) (Speers-Roesch et al., 2012). These exposures are referred to henceforth as relative hypoxia. The hypoxic levels were reached after ∼30 min of N2 bubbling. Levels of O2 were monitored using hand-held O2 meters and manually adjusted as needed by N2 bubbling. At 2 and 4 h of hypoxia exposure, fishes were sampled as previously described. This entire protocol was then repeated on subsequent days to achieve a total sample size of 4–7 fish per species and time point.

In a separate trial, the same protocol was repeated except epaulette sharks were exposed to a PwO2 of 1.0 kPa (5% air saturation) and shovelnose rays to 2.0 kPa (10% air saturation) in order to investigate the effects of deeper hypoxia in both species and to compare the effect of a similar level of environmental hypoxia (2.0 kPa) between species. At 2.0 kPa, however, shovelnose rays succumbed to hypoxia in <30 min, negating sampling, but epaulette sharks tolerated 1.0 kPa, allowing sampling at 2 and 4 h, as described previously.

Analytical protocols

Frozen tissue was broken into small pieces under liquid N2 using an insulated mortar and pestle. For extraction of metabolites, 1.0 ml of ice-cold 1 mol l–1 HClO4 was added to a microcentrifuge tube containing 50–100 mg of tissue and the mixture was immediately sonicated on ice for three bursts of 10 s using a Kontes sonicator on its highest setting. An aliquot was frozen at –80°C for measurement of [glycogen] (Bergmeyer, 1983). The remaining homogenate was centrifuged (10,000 g, 10 min, 4°C) and the supernatant neutralized with 3 mol l–1 K2CO3. Neutralized extracts were assayed spectrophotometrically for [adenosine triphosphate] ([ATP]), [CrP], [creatine] (heart only), [lactate] and [glucose] following methods described elsewhere (Bergmeyer, 1983). [Glycogen] was corrected for measured endogenous glucose levels. Intracellular pH (pHi) was measured in frozen heart tissue using previously published methods [(Pörtner et al., 1991), as validated by Baker et al. (Baker et al., 2009)] and a thermostatically controlled (28°C) BMS3 Mk2 capillary microelectrode with PHM84 pH meter (Radiometer, Copenhagen, Denmark). Levels of cardiac free adenosine diphosphate and adenosine monophosphate (ADPfree and AMPfree) were calculated as described previously (Speers-Roesch et al., 2010). Blood pH and plasma [lactate], [glucose] and [β-HB] were measured as described before (Speers-Roesch et al., 2012).

Statistics

The effects of species and O2 on cardiovascular measurements (experimental series I) were tested via a two-way ANOVA with Holm–Sidak (H–S) post hoc tests using data from 11 sampling points of overlapping PwO2 at approximately 16.0, 13.3, 11.7, 10.4, 8.3, 6.1, 5.1, 4.3, 3.1, 2.4 and 1.9 kPa and the points of statistical comparison are denoted by horizontal braces on the figures. Overlapping PwO2 values were not statistically different between species (Student's t-test, P>0.05). Data from other sampling points were omitted from these analyses. In order to fully assess the effect of O2 on cardiovascular parameters in each species, one-way ANOVA were run across all sampling points within each species, with H–S comparisons against the first normoxic resting value. The effect of O2 on measured parameters was found to be similar for both two-way and one-way ANOVA designs. The critical PwO2 values of cardiovascular variables were compared with each other and between species using a two-way ANOVA with H–S tests. The critical PwO2 of (Pcrit) measured in the accompanying study (Speers-Roesch et al., 2012) was included in this analysis to determine whether the critical PwO2 of cardiovascular variables coincided with Pcrit. Cardiovascular recovery values were compared between species and with the normoxic resting values measured at ∼16.0 kPa using a two-way ANOVA with H–S tests.

The effects of species and hypoxia exposure on physiological parameters in fishes exposed to relative hypoxia (experimental series II) were tested using a two-way ANOVA with H–S tests. The data for epaulette sharks exposed to a PwO2 of 1.0 kPa for 2 h were omitted from these analyses, but these were compared with the normoxic control using a Student's t-test.

Statistical significance was accepted when P<0.05. Analyses were carried out using SigmaStat 3.0. Data were log or square-root transformed prior to statistical analyses if assumptions of equal variance or normality were not met. Repeated measures ANOVA was not used because experimental constraints negated the use of data from the same animal at every sample period. In any case, the standard ANOVA procedures utilized here result in a conservative statistical assessment of our data.

RESULTS

Experimental series I: cardiovascular responses to progressive hypoxia

A hypoxia-induced bradycardia was observed in both species, with the onset (i.e. the critical PwO2 of heart rate) occurring at a significantly lower PwO2 in the epaulette sharks than in the shovelnose rays (Fig. 1A; Table 1). At PwO2 higher than ∼7.3 kPa, fH was similar in the two species, but at lower PwO2, fH was greater in epaulette sharks, even after bradycardia commenced in both species (Fig. 1A). fH showed a plateau below ∼3.3 kPa in shovelnose rays and both species showed a maximal fH depression of ∼65% compared with normoxic resting values (Fig. 1A).

Table 1.

Critical PwO2 (kPa) of cardiovascular parameters and whole-animal oxygen consumption rate in epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2

Critical PwO2 (kPa) of cardiovascular parameters and whole-animal oxygen consumption rate in epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2
Critical PwO2 (kPa) of cardiovascular parameters and whole-animal oxygen consumption rate in epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2

The responses of and CPO to progressive hypoxia exposure paralleled that of fH, with the decreases commencing at a lower PwO2 in the epaulette sharks than in the shovelnose rays (Table 1; Fig. 1B,C). Above ∼6.0 kPa, was similar in the two species, but it was significantly greater in epaulette sharks at lower PwO2 (Fig. 1B). A maximal depression of of approximately 50% and 60% was observed in shovelnose rays and epaulette sharks, respectively (Fig. 1B). CPO was significantly higher in epaulette sharks at all PwO2, and at similar PwO2 below the epaulette shark critical point for CPO, the percentage depression from the normoxic resting value was always greater in the shovelnose rays (e.g. at ∼3.1 kPa, CPO in epaulette sharks is 30% lower than the resting level compared with 55% lower in shovelnose rays; at ∼1.9 kPa, CPO is decreased 40% in epaulette sharks and 75% in shovelnose rays) (Fig. 1C).

In the two species, VS was similar under normoxic resting conditions and increased by 30–40% in response to progressive hypoxia exposure, with a plateau seen in shovelnose rays below ∼3.3 kPa (Fig. 2A). The increase occurred at a significantly lower PwO2 in epaulette sharks than in shovelnose rays (Table 1). A decrease in PDA (and calculated PVA, data not shown) of up to 45% was observed in both species during progressive hypoxia exposure (Fig. 2B). This decrease commenced at a significantly lower PwO2 in epaulette sharks than in shovelnose rays (Table 1). At PwO2 below ∼4.0 kPa, PDA was higher in epaulette sharks, whereas at higher PwO2, PDA was similar in the two species (Fig. 2B). Systemic peripheral resistance was similar for the two species and increased modestly during progressive hypoxia exposure in both species but reached statistical significance only for epaulette sharks (Fig. 2C). Critical PwO2 values for RSYS were not calculated because of the absence of major changes with decreasing PwO2.

The relationship between the critical PwO2 values of cardiovascular parameters and the critical PwO2 of (Pcrit) measured in the accompanying study (Speers-Roesch et al., 2012) were relatively similar between species. In each species the critical PwO2 values of , CPO and fH were statistically similar to one another and to Pcrit (Table 1). In each species the critical PwO2 of VS was significantly lower than that of only, except in epaulette sharks where it was also lower than the Pcrit (Table 1). In shovelnose rays the critical PwO2 of PDA was significantly lower than that of all other cardiovascular parameters as well as Pcrit whereas in epaulette sharks the same was true except that the critical PwO2 values for VS and fH were not significantly different from PDA (Table 1).

Fig. 1.

Heart rate (fH, A), cardiac output (, B), and cardiac power output (CPO, C) of epaulette sharks and shovelnose rays exposed to progressive decreases in water PO2 (PwO2) over 135±8 and 71±6 min, respectively, followed by up to 60 min of recovery in normoxic water. Data are means ± s.e.m. (N=5 for both species except epaulette sharks in recovery, where N=4 for fH and and N=3 for CPO). Recovery values are offset at each time point for clarity. *Significantly different from the first, normoxic resting value at ≥15.3 kPa within species. The two species values bracketed by horizontal braces (at statistically similar PwO2) are significantly different between species; the absence of a dagger indicates the two bracketed species values are not statistically different from each other. For recovery, a dagger denotes a significant difference between epaulette sharks and shovelnose rays at each time point.

Fig. 1.

Heart rate (fH, A), cardiac output (, B), and cardiac power output (CPO, C) of epaulette sharks and shovelnose rays exposed to progressive decreases in water PO2 (PwO2) over 135±8 and 71±6 min, respectively, followed by up to 60 min of recovery in normoxic water. Data are means ± s.e.m. (N=5 for both species except epaulette sharks in recovery, where N=4 for fH and and N=3 for CPO). Recovery values are offset at each time point for clarity. *Significantly different from the first, normoxic resting value at ≥15.3 kPa within species. The two species values bracketed by horizontal braces (at statistically similar PwO2) are significantly different between species; the absence of a dagger indicates the two bracketed species values are not statistically different from each other. For recovery, a dagger denotes a significant difference between epaulette sharks and shovelnose rays at each time point.

During the first 30 min of normoxic recovery from progressive hypoxia exposure, fH in shovelnose rays was significantly elevated above the normoxic resting value. In contrast, fH in epaulette sharks was significantly lower than the resting value after 5 min of recovery, returning to resting values by 15 min of recovery (Fig. 1A). returned to resting values within 5 min of recovery in both species and remained unchanged for the full 60 min recovery period (Fig. 1B). CPO returned to resting values after 5 min of recovery in both species, but as recovery progressed CPO continued to increase to above resting values in epaulette sharks but not in shovelnose rays (Fig. 1C). The same response was seen for PDA (and PVA, data not shown) during recovery resulting in significantly greater PDA during recovery in epaulette sharks than in shovelnose rays (Fig. 2B). During recovery in both species, VS and RSYS returned to resting values within 5 min and remained unchanged throughout the 60 min recovery period (Fig. 2A,C).

Experimental series II: tissue metabolic status during relative hypoxia exposure

Cardiac [ATP] was similar in the two species and was unaffected by 4 h of relative hypoxia exposure (Fig. 3A). The two species had similar normoxic levels of cardiac [CrP]. During the relative hypoxia exposure, heart [CrP] was unchanged in epaulette sharks but decreased significantly in the shovelnose rays (Fig. 3B). Cardiac [lactate] was similar in the two species under normoxic conditions and increased significantly during relative hypoxia exposure in shovelnose rays but not in epaulette sharks (Fig. 4A). Cardiac [glucose] was similar in the two species under normoxic conditions and was unaffected by relative hypoxia exposure (Table 2). Cardiac [glycogen] was significantly higher in epaulette sharks than in shovelnose rays in normoxia and relative hypoxia exposure had no effect on cardiac [glycogen] in either species (Table 2). Cardiac [ADPfree] and [AMPfree] were unchanged by relative hypoxia exposure in epaulette sharks whereas levels of both increased significantly in shovelnose rays (Table 2).

Table 2.

Levels of selected metabolites of energy metabolism in heart, liver, white muscle and plasma of epaulette sharks and shovelnose rays exposed either to normoxia or to 2 and 4 h of hypoxia representing 40% of each species' respective Pcrit (PwO2=2.0 and 3.1 kPa, respectively) and in epaulette sharks exposed to 20% of Pcrit (PwO2=1.0 kPa)

Levels of selected metabolites of energy metabolism in heart, liver, white muscle and plasma of epaulette sharks and shovelnose rays exposed either to normoxia or to 2 and 4 h of hypoxia representing 40% of each species' respective Pcrit (PwO2=2.0 and 3.1 kPa, respectively) and in epaulette sharks exposed to 20% of Pcrit (PwO2=1.0 kPa)
Levels of selected metabolites of energy metabolism in heart, liver, white muscle and plasma of epaulette sharks and shovelnose rays exposed either to normoxia or to 2 and 4 h of hypoxia representing 40% of each species' respective Pcrit (PwO2=2.0 and 3.1 kPa, respectively) and in epaulette sharks exposed to 20% of Pcrit (PwO2=1.0 kPa)
Fig. 2.

Stroke volume (VS, A), dorsal aortic blood pressure (PDA, B), and systemic resistance (RSYS, C) of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2 over 135±8 and 71±6 min, respectively, followed by up to 60 min of recovery in normoxic water. Data are means ± s.e.m. (N=5 for both species except epaulette sharks in recovery, where N=4 for VS and N=3 for PDA and RSYS). Recovery values are offset at each time point for clarity. See Fig. 1 legend for details on symbols denoting statistical comparisons.

Fig. 2.

Stroke volume (VS, A), dorsal aortic blood pressure (PDA, B), and systemic resistance (RSYS, C) of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2 over 135±8 and 71±6 min, respectively, followed by up to 60 min of recovery in normoxic water. Data are means ± s.e.m. (N=5 for both species except epaulette sharks in recovery, where N=4 for VS and N=3 for PDA and RSYS). Recovery values are offset at each time point for clarity. See Fig. 1 legend for details on symbols denoting statistical comparisons.

Cardiac pHi was similar between species in normoxia (Fig. 5A). Shovelnose rays showed a significant decrease in cardiac pHi throughout the relative hypoxia exposure, whereas a significant decrease was not observed until 4 h in epaulette sharks. At 2 h of exposure, epaulette sharks had significantly higher cardiac pHi compared with shovelnose rays, but not at 4 h (Fig. 5A). Blood pH remained unchanged during relative hypoxia exposure in epaulette sharks, but it decreased significantly in shovelnose rays (Fig. 5B).

White muscle [ATP] was unchanged in both species during relative hypoxia exposure but liver [ATP] decreased (Table 2), and did so more rapidly (within 2 h) in shovelnose rays than in epaulette sharks (within 4 h). White muscle [CrP] was similar in the two species in normoxia but was depleted more rapidly and by a greater amount in shovelnose rays during relative hypoxia exposure. Liver [CrP] was highly variable and unchanged in both species during relative hypoxia exposure (Table 2). Epaulette sharks had higher normoxic resting levels of liver [glycogen] compared with shovelnose rays (Table 2). Relative hypoxia exposure caused no change in white muscle [glycogen] whereas a significant decrease of liver [glycogen] occurred in shovelnose rays but not epaulette sharks (Table 2). White muscle [glucose] decreased after 4 h of relative hypoxia exposure in shovelnose rays but was unchanged in epaulette sharks. In liver, [glucose] was unaffected by relative hypoxia exposure (Table 2). [Lactate] in white muscle increased more rapidly and accumulated to a greater amount in shovelnose rays than in epaulette sharks exposed to relative hypoxia (Fig. 4B). Lactate accumulation in liver, however, was similar for the two species (Fig. 4C).

Similar to heart and white muscle, plasma [lactate] increased significantly during relative hypoxia exposure in both species, but the magnitude of accumulation was greater in shovelnose rays than in epaulette sharks (Fig. 4D). Plasma [glucose] was comparable between species and was unaffected by relative hypoxia exposure (Table 2). Plasma [β-HB] was higher in normoxia in epaulette sharks compared with shovelnose rays but levels were similar in the two species during relative hypoxia exposure because plasma [β-HB] in epaulette sharks decreased significantly whereas levels in shovelnose rays were unchanged (Table 2).

Compared with the normoxic group, the responses of metabolite levels in tissues of epaulette sharks exposed to 2 h of hypoxia at 1.0 kPa generally were similar to those exposed to 2.0 kPa (Table 2). However, the 2 h exposure at 1.0 kPa caused significant decreases in liver [ATP], white muscle [CrP] and cardiac pHi as well as significant increases in cardiac and white muscle [lactate], which at 2.0 kPa were only apparent after 4 h of exposure (Table 2; Fig. 4A,B; Fig. 5A). Qualitatively, a greater accumulation of plasma and liver [lactate] also was apparent in the epaulette sharks exposed to 1.0 kPa for 2 h compared with 2.0 kPa for 2 h (Fig. 4C,D). Finally, at 1.0 kPa, but not at 2.0 kPa, 2 h of hypoxia exposure caused a significant decrease in cardiac [glycogen] and a trend (P=0.06) of decreased liver [glycogen] (Table 2).

DISCUSSION

The impressive hypoxia tolerance of the epaulette shark is associated with enhanced cardiovascular function and more stable cardiac energy status during hypoxia exposure compared with the less hypoxia-tolerant shovelnose ray. Similar routine levels of cardiovascular function were maintained above Pcrit in the two species (5.10±0.37 kPa, epaulette shark; 7.23±0.40 kPa, shovelnose ray) (Speers-Roesch et al., 2012). Hypoxic cardiovascular responses occurred at or below Pcrit and always at a lower PwO2 in epaulette sharks. Thus, routine cardiovascular function was maintained to lower PwO2 in epaulette sharks compared with shovelnose rays. Also, epaulette sharks maintained greater levels of cardiovascular function, including higher CPO, during hypoxia exposure. Depression of cardiac energy demand may be of secondary importance for hypoxia tolerance compared with the ability to maintain greater cardiovascular function during hypoxia exposure.

Fig. 3.

Cardiac [ATP] (A) and [creatine phosphate] ([CrP], B) in epaulette sharks and shovelnose rays exposed either to normoxia or to 2 and 4 h of hypoxia representing 40% of each species' respective critical O2 tension of whole-animal O2 consumption rate, Pcrit (PwO2=2.0 and 3.1 kPa, respectively) and in epaulette sharks exposed to 20% of Pcrit (PwO2=1.0 kPa). Data are means ± s.e.m. (N=5–7 for epaulette sharks; N=4–7 for shovelnose rays). Values are offset at each time point for clarity. Values with different lowercase letters are significantly different from each other within species; between species differences at each time point are denoted by daggers. For epaulette sharks, a significant difference between the 1.0 kPa group and the normoxic group is denoted by different uppercase letters.

Fig. 3.

Cardiac [ATP] (A) and [creatine phosphate] ([CrP], B) in epaulette sharks and shovelnose rays exposed either to normoxia or to 2 and 4 h of hypoxia representing 40% of each species' respective critical O2 tension of whole-animal O2 consumption rate, Pcrit (PwO2=2.0 and 3.1 kPa, respectively) and in epaulette sharks exposed to 20% of Pcrit (PwO2=1.0 kPa). Data are means ± s.e.m. (N=5–7 for epaulette sharks; N=4–7 for shovelnose rays). Values are offset at each time point for clarity. Values with different lowercase letters are significantly different from each other within species; between species differences at each time point are denoted by daggers. For epaulette sharks, a significant difference between the 1.0 kPa group and the normoxic group is denoted by different uppercase letters.

The ability of epaulette sharks to maintain cardiac energy status and to minimize lactate accumulation and metabolic acidosis during hypoxia exposure is also superior to that of shovelnose rays. This is especially significant because the experimental exposure was carried out at a relative percentage of Pcrit, therefore equalizing the between-species variation in CaO2 (Speers-Roesch et al., 2012). Thus, while Pcrit accurately reflects O2 transport during hypoxia exposure (Speers-Roesch et al., 2012), it does not necessarily reflect tissue-level hypoxia tolerance, which may be affected by other factors such as tissue-specific metabolic rate depression. The more stable cardiac energy status of the epaulette shark is not apparently related to greater depression of cardiac energy demand, but perhaps is due to greater O2 delivery to the heart as a result of enhanced as well as extra-cardiac metabolic depression. Strategic O2 delivery to the heart, as well as greater blood O2 capacity (Speers-Roesch et al., 2012), may help explain the enhanced hypoxic cardiovascular function in the epaulette shark compared with the shovelnose ray.

Cardiovascular responses to progressive hypoxia and recovery

With the exception of CPO, which was higher in shovelnose rays, normoxic levels of cardiovascular function were similar between epaulette sharks and shovelnose rays (Figs 1 and 2). The resting fH and PDA of epaulette sharks match those measured previously in this species (Stensløkken et al., 2004). In epaulette sharks and shovelnose rays, resting fH, VS, , PDA and RSYS were similar to those in other elasmobranchs with similar activity levels and accounting for differences in experimental temperature (Butler and Taylor, 1975; Lai et al., 1989; Lai et al., 1990; Sandblom et al., 2006; Satchell et al., 1970). To our knowledge, the only other in vivo measurement of elasmobranch CPO is a mass-independent value for spotted catshark (Short et al., 1979), which is, in general, similar to our mass-specific values for epaulette sharks and shovelnose rays. Elasmobranchs have higher routine CPO values than typically seen in teleosts (0.5–3.0 mW g–1) (Speers-Roesch et al., 2010; Stecyk and Farrell, 2006).

Progressive hypoxia exposure elicited a similar cardiovascular response in epaulette sharks and shovelnose rays, including bradycardia, increased VS and decreased , CPO and PDA (Fig. 1; Fig. 2A,B). Bradycardia induced by hypoxia exposure is common in fishes and has been observed previously in elasmobranchs such as the epaulette shark, spiny dogfish (Squalus acanthias) and spotted catshark (Butler and Taylor, 1975; Sandblom et al., 2009; Stensløkken et al., 2004). Whereas spotted catsharks and spiny dogfish appear to achieve hypoxia-induced bradycardia via vagally mediated cholinergic inhibition, this is not the case in epaulette sharks where the mechanism is unclear (Stensløkken et al., 2004); nothing is known about mechanisms of hypoxic bradycardia in shovelnose rays. Acidosis has a negative chronotropic effect in fishes but does not appear to be a primary cause of bradycardia in epaulette sharks or shovelnose rays because, as shown in the accompanying study (Speers-Roesch et al., 2012), blood pH decreased at a lower PwO2 compared with the commencement of bradycardia [cf. fig. 5B in the accompanying study (Speers-Roesch et al., 2012) and Fig. 1A in the present study]. Bradycardia was initiated immediately below the PwO2 corresponding to a PaO2 just below the Hb–O2P50 in each species (Speers-Roesch et al., 2012) and direct hypoxaemic effects on fH probably contributed to the bradycardia. Further studies are needed to elucidate the mechanisms of hypoxic bradycardia in elasmobranchs and especially the epaulette shark, including the suggested involvement of α-adrenoceptors (Stensløkken et al., 2004).

Stroke volume increased in epaulette sharks and shovelnose rays during progressive hypoxia exposure but still decreased because of a large depression of fH (Fig. 1B; Fig. 2A). The large fall in with only a modest increase in RSYS resulted in the observed reduction in PDA in epaulette sharks and shovelnose rays, although the critical PwO2 for PDA was lower than that for (Table 1). Epaulette sharks show no change in gill resistance during hypoxia exposure so this is not likely to contribute to reduced PDA in this species (Stensløkken et al., 2004). The absence of a barostatic reflex to maintain arterial blood pressure is consistent with previous studies showing that the reflex is weak in response to hypotension in fishes and especially elasmobranchs (Olson and Farrell, 2006). Additionally, the barostatic reflex may be reset in the face of hypoxia-induced bradycardia in fishes (Stecyk and Farrell, 2006). Like the two species in the present study, an increase in VS and a decrease in PDA were seen in spotted catsharks exposed to progressive hypoxia, but no decrease in was observed, possibly because only moderate hypoxia was achieved (Butler and Taylor, 1975). In constrast, in spiny dogfish exposed to severe hypoxia, Sandblom and colleagues (Sandblom et al., 2009) observed decreases in and VS but PDA was unchanged. These data suggest that the pattern of cardiovascular responses to hypoxia, in particular VS and PDA, vary among elasmobranchs, as they do in teleosts.

Fig. 4.

[Lactate] in heart (A), white muscle (B), liver (C) and mixed arterial–venous plasma (D) in epaulette sharks and shovelnose rays exposed either to normoxia or to 2 h and 4 h of hypoxia representing 40% of each species' respective Pcrit (PwO2=2.0 and 3.1 kPa, respectively) and in epaulette sharks exposed to 20% of Pcrit (PwO2=1.0 kPa). Data are means ± s.e.m. (N=5–7 for epaulette sharks; N=4–7 for shovelnose rays). Values are offset at each time point for clarity. See Fig. 3 legend for details on symbols denoting statistical comparisons.

Fig. 4.

[Lactate] in heart (A), white muscle (B), liver (C) and mixed arterial–venous plasma (D) in epaulette sharks and shovelnose rays exposed either to normoxia or to 2 h and 4 h of hypoxia representing 40% of each species' respective Pcrit (PwO2=2.0 and 3.1 kPa, respectively) and in epaulette sharks exposed to 20% of Pcrit (PwO2=1.0 kPa). Data are means ± s.e.m. (N=5–7 for epaulette sharks; N=4–7 for shovelnose rays). Values are offset at each time point for clarity. See Fig. 3 legend for details on symbols denoting statistical comparisons.

Epaulette sharks and shovelnose rays showed a similar pattern of cardiovascular responses to progressive hypoxia and it remains unclear whether a specific suite of hypoxic cardiovascular responses correlates with hypoxia tolerance in fishes. There were, however, two key differences between the species: (1) all hypoxic cardiovascular responses in epaulette sharks occurred at a significantly lower PwO2 compared with shovelnose rays (Table 1); and (2) despite the presence of similar routine levels of cardiovascular function (i.e. above the critical PwO2 of each parameter; Table 1), the level of function of most parameters was greater at the same hypoxic PwO2 (below the critical PwO2) in the epaulette shark compared with the shovelnose ray (Fig. 1; Fig. 2A,B). In other words, compared with the shovelnose ray, the more hypoxia-tolerant epaulette shark can maintain cardiovascular function at routine levels to lower PwO2 and once hypoxic responses commence the change from routine levels is smaller. This may benefit hypoxia tolerance by improving O2 delivery and management of wastes.

The decreases in fH, and CPO in epaulette sharks and shovelnose rays coincided with the decreases in whole-animal seen during progressive hypoxia (Speers-Roesch et al., 2012) and the critical PwO2 of these parameters were statistically similar within species (Table 1). A correlation between Pcrit and the critical PwO2 value of heart rate appears to be a common phenomenon among fishes (Fig. 6), suggesting that Pcrit is a good predictor of the point at which hypoxic bradycardia is initiated. Likewise, co-occurrence of Pcrit and the critical PwO2 of and CPO also has been observed in fishes (Iversen et al., 2010; Speers-Roesch et al., 2010). These relationships are perhaps unsurprising considering the link between and convective O2 delivery (Webber et al., 1998). Other hypoxic cardiovascular responses (e.g. VS, PDA) were not necessarily correlated with Pcrit, although they always occurred at or below Pcrit (Table 1). Overall, these data suggest that in fishes exposed to hypoxia, Pcrit indicates the lowest PwO2 at which routine cardiovascular function is maintained and this may explain in part why epaulette sharks maintain routine cardiovascular function to lower PwO2. However, Pcrit may not fully predict the capacity for hypoxic cardiovascular function at PwO2 below Pcrit, because at the same relative percentages below Pcrit cardiovascular function remained closer to routine levels in epaulette sharks compared with shovelnose rays (e.g. Fig. 1; Fig. 2A,B). Because at relative percentages of Pcrit the and CaO2 are similar in the two species, this observation also suggests that the improved cardiovascular performance at PwO2 below Pcrit in epaulette sharks cannot be explained completely by its lower Pcrit and greater CaO2 (Speers-Roesch et al., 2012).

Cardiac energy demand as measured by CPO decreased as whole-animal fell in both epaulette sharks and shovelnose rays [cf. Fig. 1C in present study and fig. 1 in the accompanying paper (Speers-Roesch et al., 2012)], probably due to the depression of associated with bradycardia. A similar result has been observed in tilapia (Speers-Roesch et al., 2010). Unlike tilapia, however, reductions in PDA and PVA probably also contribute to depressed CPO during hypoxia exposure in elasmobranchs, including epaulette sharks (Fig. 2B) (Stensløkken et al., 2004). Our results support previous findings showing that the depression of CPO is an integral component of hypoxia-induced whole-animal metabolic rate depression in many fishes that may contribute to hypoxia tolerance by matching cardiac energy demand to lowered energy supply (Farrell and Stecyk, 2007; Speers-Roesch et al., 2010). There was no evidence, however, that depression of CPO was associated with the greater hypoxia tolerance of the epaulette shark, because epaulette sharks maintained a higher CPO at all similar PwO2, even when expressed as a percentage of the normoxic resting level (Fig. 1C). Thus, CPO in epaulette sharks was maintained to lower PwO2 and at higher levels during hypoxia compared with shovelnose rays, probably because of the similar maintenance of fH, and PDA. Only at ∼0.1 kPa did epaulette sharks reach the level of CPO depression seen at the lowest PwO2 (∼2.0 kPa) tolerated by shovelnose rays, and at or below 0.1 kPa epaulette sharks (and presumably their hearts) remain responsive for at least 45 min (Renshaw et al., 2002). While flow traces did not show marked cardiac arrhythmia in shovelnose rays or epaulette sharks, the plateau of fH and coupled with the continual decrease in CPO and PDA below a PwO2 of ∼2.7 kPa in shovelnose rays (Fig. 1; Fig. 2B) could be symptomatic of a failing heart, whereas no such pattern was evident for epaulette sharks.

Fig. 5.

Cardiac intracellular pH (pHi, A) and blood (mixed arterial–venous) pH (B) in epaulette sharks and shovelnose rays exposed either to normoxia or to 2 h and 4 h of hypoxia representing 40% of each species' respective Pcrit (PwO2=2.0 and 3.1 kPa, respectively) and in epaulette sharks exposed to 20% of Pcrit (PwO2=1.0 kPa). Data are means ± s.e.m. (pHi, N=4 for epaulette sharks and N=4–7 for shovelnose rays; blood pH, N=4–6 for epaulette sharks and N=3–6 for shovelnose rays). Values are offset at each time point for clarity. See Fig. 3 legend for details on symbols denoting statistical comparisons.

Fig. 5.

Cardiac intracellular pH (pHi, A) and blood (mixed arterial–venous) pH (B) in epaulette sharks and shovelnose rays exposed either to normoxia or to 2 h and 4 h of hypoxia representing 40% of each species' respective Pcrit (PwO2=2.0 and 3.1 kPa, respectively) and in epaulette sharks exposed to 20% of Pcrit (PwO2=1.0 kPa). Data are means ± s.e.m. (pHi, N=4 for epaulette sharks and N=4–7 for shovelnose rays; blood pH, N=4–6 for epaulette sharks and N=3–6 for shovelnose rays). Values are offset at each time point for clarity. See Fig. 3 legend for details on symbols denoting statistical comparisons.

Cardiovascular function returned to routine levels within 60 min of normoxic recovery following progressive hypoxia exposure in both epaulette sharks and shovelnose rays, with the exception of CPO in epaulette sharks, which steadily increased over time due to an increase in PDA (Fig. 1C; Fig. 2B). Shovelnose rays had elevated fH during the initial stage of recovery whereas epaulette sharks showed a non-elevated, gradual return to routine fH (Fig. 1A). Cardiac output showed qualitatively the same trends as fH (Fig. 1B). Further studies are needed to ascertain the role of these different cardiovascular responses for recovery from hypoxia exposure in epaulette sharks and shovelnose rays.

Fig. 6.

The relationship between Pcrit and critical PwO2 of heart rate (i.e. PwO2 at initiation of hypoxia-induced bradycardia) in fishes. (1) Hoplias malabaricus, 25°C (Rantin et al., 1993); (2) Oreochromis hybrid, 22°C (Speers-Roesch et al., 2010); (3) Piaractus mesopotamicus, 25°C (Rantin et al., 1998); (4) Leiopotherapon unicolor, 25°C (Gehrke and Fielder, 1988); (5) Hoplias lacerdae, 25°C (Rantin et al., 1993); (6) Hemiscyllium ocellatum, 28°C, present study and accompanying paper (Speers-Roesch et al., 2012); (7) Aptychotrema rostrata, 28°C, present study and accompanying paper (Speers-Roesch et al., 2012); (8) Gadus morhua, 10°C (McKenzie et al., 2009); (9) Gadus morhua, 10°C (Petersen and Gamperl, 2011); (10) Scyliorhinus canicula, 12°C (Butler and Taylor, 1975) (values estimated from graphical data); (11) Anguilla anguilla, 20°C (Iversen et al., 2010). The linear regression is significant (P=0.001, linear regression ANOVA).

Fig. 6.

The relationship between Pcrit and critical PwO2 of heart rate (i.e. PwO2 at initiation of hypoxia-induced bradycardia) in fishes. (1) Hoplias malabaricus, 25°C (Rantin et al., 1993); (2) Oreochromis hybrid, 22°C (Speers-Roesch et al., 2010); (3) Piaractus mesopotamicus, 25°C (Rantin et al., 1998); (4) Leiopotherapon unicolor, 25°C (Gehrke and Fielder, 1988); (5) Hoplias lacerdae, 25°C (Rantin et al., 1993); (6) Hemiscyllium ocellatum, 28°C, present study and accompanying paper (Speers-Roesch et al., 2012); (7) Aptychotrema rostrata, 28°C, present study and accompanying paper (Speers-Roesch et al., 2012); (8) Gadus morhua, 10°C (McKenzie et al., 2009); (9) Gadus morhua, 10°C (Petersen and Gamperl, 2011); (10) Scyliorhinus canicula, 12°C (Butler and Taylor, 1975) (values estimated from graphical data); (11) Anguilla anguilla, 20°C (Iversen et al., 2010). The linear regression is significant (P=0.001, linear regression ANOVA).

Metabolic status during relative hypoxia exposure

To investigate the effects of hypoxia on tissue metabolic status and to test the hypothesis that Pcrit dictates tissue-level hypoxia tolerance, we exposed epaulette sharks and shovelnose rays for up to 4 h to a PwO2 representing 40% of the respective Pcrit, which resulted in a similar level of arterial hypoxaemia in the two species [see fig. 2B in the companion paper (Speers-Roesch et al., 2012); interpolated CaO2 at 40% of respective Pcrit is ∼0.6 vol. % in both species]. Contrary to our prediction of similar interspecific metabolic status under these conditions, we found greater decreases in pH (Fig. 5), accumulation of lactate (Fig. 4A,B,D), and perturbation of tissue energy status (Fig. 3; Table 2) in shovelnose rays than in epaulette sharks. Perturbations of pH and energy status as well as high lactate load are thought to contribute to hypoxic death and hypoxia-tolerant animals are able to avoid or at least postpone such perturbation (Nilsson and Östlund-Nilsson, 2008). The more stable metabolic state of the epaulette shark compared with the shovelnose ray at a similar level of arterial hypoxaemia suggests that other factors work in concert with enhanced O2 supply to explain the epaulette shark's superior hypoxia tolerance, possibly including effective acid–base regulation, metabolic rate depression and strategic utilization of O2 (see below).

In the heart, [ATP] was stable in both species but whereas [CrP] was stable in epaulette sharks, it fell in shovelnose rays, leading to increases in [ADPfree] and [AMPfree] (Fig. 3; Table 2). The maintenance of cardiac energy status in epaulette sharks vs perturbation in shovelnose rays is consistent with previous results for hypoxia-tolerant vs -sensitive fishes exposed to hypoxia (Dunn and Hochachka, 1986; Jorgensen and Mustafa, 1980; Speers-Roesch et al., 2010). Although [CrP] stabilized at a lower level in hypoxia-exposed shovelnose rays, this may not represent a stable functional state considering that the increase of inorganic phosphate associated with CrP depletion, rather than ATP depletion, is thought to be a major contributor to hypoxic heart failure in mammals and fishes (Arthur et al., 1992; Neubauer, 2007). Notably, the PwO2 of the relative hypoxia exposure of shovelnose rays was only marginally higher than the PwO2 where possible signs of cardiac failure were seen during the progressive hypoxia exposure (see above). The energy status measurements suggest that matching of cardiac energy supply and demand during hypoxia exposure is much less perturbed in epaulette sharks than in shovelnose rays, even when hypoxaemia is similar. Under the more severe hypoxia exposure (PwO2=1.0 kPa), epaulette shark hearts still maintained stable energy status (Fig. 3), suggesting that energy supply and demand remain well matched even under severe hypoxic conditions where shovelnose rays cannot survive.

The ability of epaulette sharks to better match cardiac energy supply and demand is not due to a greater depression of energy demand (i.e. CPO) compared with shovelnose rays because epaulette sharks actually maintain higher levels of CPO during progressive hypoxia exposure, including when levels are compared at the species-specific PwO2 values used in the relative hypoxia exposure (Fig. 1C). Although CPO in epaulette sharks may have decreased more over the duration of the relative hypoxia exposure compared with the shorter progressive hypoxia exposure, we consider this unlikely because the first sample time point (2 h) of the relative hypoxia exposure is comparable in duration to the progressive hypoxia exposure. Also, in other fish, levels of CPO and other heart parameters measured at each PwO2 during progressive hypoxia exposure appear to be a good indicator of the levels that are seen during prolonged exposure at the same, stable PwO2 (Speers-Roesch et al., 2010).

Strategic delivery of available O2 to the heart during relative hypoxia exposure could explain, in part, how a stable cardiac energy status was maintained alongside greater cardiac function in epaulette sharks but not shovelnose rays, despite similar arterial hypoxaemia. Consistent with this hypothesis, there was minimal lactate accumulation in the heart of epaulette sharks and greater lactate accumulation in the heart of shovelnose rays during relative hypoxia exposure (Fig. 4A), even though cardiac energy demand was higher in the epaulette sharks (see above). Although not investigated in the present study, coronary perfusion of the myocardium could be more extensive in epaulette sharks, improving myocardial O2 supply compared with shovelnose rays. In epaulette sharks, hypoxia exposure causes changes in gill blood flow that may deliver blood directly to the heart (Stensløkken et al., 2004). Also, whole-animal metabolic rate depression, especially in non-essential tissues such as white muscle, could be greater in the epaulette sharks than in shovelnose rays, thus sparing O2 for the heart. The lesser accumulation of lactate in white muscle and plasma in epaulette sharks (Fig. 4B,D) supports this hypothesis because, at similar arterial hypoxaemia, lactate accumulation can be used as a rough proxy to compare energy demand between species. Liver [lactate] was similar between species (Fig. 4C), but in this case interpretation is complicated by the potential accumulation of lactate in liver for glycogen or glucose synthesis. A caveat is that the relative hypoxia exposure did not control for the higher seen during hypoxia exposure in epaulette sharks (Fig. 1B), which may facilitate tissue O2 delivery in this species despite CaO2 being the same in the two species. This may explain some of the species differences in lactate accumulation in non-cardiac tissues. Unfortunately, logistical constraints in the present study negated measurement of O2 content in blood returning to the heart and use of the Fick method produced unreliable estimates of venous O2 content, consistent with previous findings showing the inaccuracy of the Fick method during hypoxia exposure in elasmobranchs (Metcalfe and Butler, 1982). Further studies are needed to directly assess the idea that strategic cardiac utilization of O2 contributes to the superior hypoxia tolerance of epaulette sharks.

There was no increase in plasma [glucose] during relative hypoxia exposure in either species, similar to the results of the progressive hypoxia exposure (Speers-Roesch et al., 2012) and similar to previous studies on epaulette sharks and other elasmobranchs exposed to hypoxia (Routley et al., 2002; Speers-Roesch and Treberg, 2010). However, glycogen was mobilized in the liver (Table 2), suggesting that glucose flux increases. Plasma [β-HB] decreased during relative hypoxia exposure in the epaulette sharks (Table 2). The role of ketone bodies during hypoxia exposure in epaulette sharks warrants attention considering the protective effects of ketone bodies seen in mammalian ischaemia and because of the parallels with the hypoxic decreases of plasma free fatty acids seen in some hypoxia-tolerant teleosts, which could be related to metabolic rate depression (Speers-Roesch and Treberg, 2010; Speers-Roesch et al., 2010).

CONCLUSIONS

The results of the present study and the accompanying study (Speers-Roesch et al., 2012) show that in comparison with the relatively hypoxia-sensitive shovelnose ray, the hypoxia-tolerant epaulette shark possesses greater blood O2 transport, greater cardiovascular function and superior maintenance of pH and cardiac energy status during hypoxia exposure. These attributes probably contribute to the exceptional hypoxia tolerance of the epaulette shark and generally may be hallmarks of hypoxia tolerance in fishes. The enhanced hypoxic cardiorespiratory performance of the epaulette shark also highlights the importance for hypoxia tolerance of maintenance of a superior O2 supply including cardiovascular function in order to optimize aerobic energy production during hypoxia exposure.

A regulated depression of CPO in fishes is thought to be an important component of cardiac and consequently whole-animal hypoxia tolerance (Farrell and Stecyk, 2007; Speers-Roesch et al., 2010). We hypothesized that the hypoxia-tolerant epaulette shark would show greater hypoxia-induced depression of CPO compared with the hypoxia-sensitive shovelnose ray. In fact, the epaulette shark's greater hypoxic cardiovascular function was associated with a smaller reduction of CPO compared with the shovelnose ray (Fig. 1C). Although this finding does not refute the importance of depression of CPO for hypoxia tolerance, it does suggest that rather than outright cardiac depression, the maintenance of higher levels of cardiac function and therefore energy demand may be an equally important strategy for hypoxia tolerance in fishes because of the benefits for O2 supply and management of fuel and waste. Interestingly, unlike shovelnose rays, epaulette sharks appear to be able to avoid perturbation of cardiac energy status during hypoxia exposure (Fig. 3) despite maintaining a higher cardiac energy demand. Considering that this relative hypoxia exposure equalized CaO2 between species, these results and those for tissue [lactate] imply that during hypoxia exposure, O2 delivery to the heart in epaulette sharks is superior to that of shovelnose rays, possibly in part due to O2 sparing related to metabolic depression in non-essential tissues. Further studies are needed to test this hypothesis directly. Overall, epaulette sharks, unlike shovelnose rays, appear to be able to coordinate depression of cardiac energy demand (i.e. decreases in CPO) with improvements in cardiac energy supply (e.g. O2 supply) in order to achieve stable cardiac energy status, enhanced cardiac function and, consequently, improved hypoxia tolerance.

Finally, the present study provides insight into the use of Pcrit as a measure of hypoxia tolerance in fishes. Previous work has shown that Pcrit is an excellent indicator of the ability of a fish to take up and transport O2 at low PwO2 (Mandic et al., 2009; Speers-Roesch et al., 2012). Here, we also provide support for the idea that Pcrit provides an indication of the ability of a fish to maintain routine cardiovascular function to low PwO2. However, data from our relative hypoxia exposure show that Pcrit does not necessarily determine the metabolic status and hypoxia tolerance of tissues, where metabolic depression may also play a major role. Overall, our results suggest that Pcrit is an important measure of respiratory hypoxia tolerance and that improved hypoxic O2 supply associated with a low Pcrit is only one, albeit important, component of hypoxia tolerance in fishes.

LIST OF SYMBOLS AND ABBREVIATIONS

     
  • cardiac output

  •  
  • β-HB

    β-hydroxybutyrate

  •  
  • ADPfree

    free adenosine diphosphate

  •  
  • AMPfree

    free adenosine monophosphate

  •  
  • ATP

    adenosine triphosphate

  •  
  • CaO2

    arterial blood O2 content

  •  
  • CPO

    cardiac power output

  •  
  • CrP

    creatine phosphate

  •  
  • fH

    heart rate

  •  
  • Hb

    haemoglobin

  •  
  • Hb–O2P50

    haemoglobin–O2 binding affinity

  •  
  • whole-animal O2 consumption rate

  •  
  • PaO2

    arterial blood PO2

  •  
  • PCO2

    partial pressure of CO2

  •  
  • Pcrit

    critical O2 tension of whole-animal O2 consumption rate

  •  
  • PDA

    dorsal aortic blood pressure

  •  
  • pHi

    intracellular pH

  •  
  • PO2

    partial pressure of O2

  •  
  • PVA

    ventral aortic blood pressure

  •  
  • PwO2

    water PO2

  •  
  • RSYS

    systemic peripheral resistance

  •  
  • VS

    stroke volume

FOOTNOTES

FUNDING

Funding was provided by the Discovery Grant Program from the Natural Sciences and Engineering Research Council of Canada (NSERC) [to J.G.R., C.J.B., A.P.F. and Y.X.W.]. Funding was provided from Sea World Research and Rescue Foundation [to G.M.C.R.]. A.J.R.H. was supported by the University of Auckland Early Career Research Excellence award. C.J.B. was supported by a Killam Faculty Research Fellowship. B.S.-R. was supported by a Pacific Century Graduate Scholarship from the University of British Columbia and the Province of British Columbia, a War Memorial Scholarship from IODE Canada, a Journal of Experimental Biology Travelling Fellowship from the Company of Biologists, a Comparative Physiology and Biochemistry Student Research Grant from the Canadian Society of Zoologists, and a Graduate Travel Award from the Department of Zoology, University of British Columbia.

Acknowledgements

Kevin and Kathy Townsend and their staff at the Moreton Bay Research Station provided exemplary logistical and technical assistance. Craig Franklin kindly lent us equipment needed for a portion of this study. Miles Gray assisted with hypoxia exposures and sampling.

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