Approximately half of all fishes have, in addition to the luminal venous O2 supply, a coronary circulation supplying the heart with fully oxygenated blood. Yet, it is not fully understood how coronary O2 delivery affects tolerance to environmental extremes such as warming and hypoxia. Hypoxia reduces arterial oxygenation, while warming increases overall tissue O2 demand. Thus, as both stressors are associated with reduced venous O2 supply to the heart, we hypothesised that coronary flow benefits hypoxia and warming tolerance. To test this hypothesis, we blocked coronary blood flow (via surgical coronary ligation) in rainbow trout (Oncorhynchus mykiss) and assessed how in vivo cardiorespiratory performance and whole-animal tolerance to acute hypoxia and warming was affected. While coronary ligation reduced routine stroke volume relative to trout with intact coronaries, cardiac output was maintained by an increase in heart rate. However, in hypoxia, coronary-ligated trout were unable to increase stroke volume to maintain cardiac output when bradycardia developed, which was associated with a slightly reduced hypoxia tolerance. Moreover, during acute warming, coronary ligation caused cardiac function to collapse at lower temperatures and reduced overall heat tolerance relative to trout with intact coronary arteries. We also found a positive relationship between individual hypoxia and heat tolerance across treatment groups, and tolerance to both environmental stressors was positively correlated with cardiac performance. Collectively, our findings show that coronary perfusion improves cardiac O2 supply and therefore cardiovascular function at environmental extremes, which benefits tolerance to natural and anthropogenically induced environmental perturbations.
Rapid changes in water temperature and O2 levels are normal phenomena in aquatic ecosystems, but their incidence and magnitude are exacerbated from ongoing anthropogenic activities such as eutrophication, habitat degradation and climate change; trends that are projected to continue and worsen in the future (Breitburg et al., 2018; Diaz, 2001; Ummenhofer and Meehl, 2017; Woodward et al., 2010). To predict how fish and other aquatic organisms will be impacted, there is a need to understand the physiological mechanisms underlying tolerance to these environmental perturbations. Heat and hypoxia tolerance are often correlated in fish, indicating a functional relationship between the physiological processes that dictate these environmental boundaries (Anttila et al., 2013; Zhang et al., 2018).
The overall metabolism and thus O2 demand increases in fish during warming, which is met by elevations in cardiovascular O2 delivery and tissue O2 extraction. This coincides with elevations in cardiac O2 demand as cardiac output and cardiac workload (a product of cardiac output and blood pressure) increases along with a direct stimulatory effect of warming on cardiac cellular metabolism (Ekström et al., 2016; Eliason and Anttila, 2017). Similarly, cardiac work appears to increase within a certain hypoxia range in some fish species, e.g. rainbow trout (Oncorhynchus mykiss), as cardiac output is either maintained or elevated while ventral aortic blood pressure increases (Holeton and Randall, 1967; Perry and Desforges, 2006; Steffensen and Farrell, 1998; Wood and Shelton, 1980), whereas other species reduce cardiac work (see table in Stecyk, 2017). Even so, both warming and hypoxia may constrain the supply of O2 to the heart as arterial blood oxygenation at the gills is impaired, tissue O2 extraction increases and the partial pressure of O2 (PO2) in the venous blood returning to the cardiac lumen decreases (Clark et al., 2008b; Ekström et al., 2016; Steffensen and Farrell, 1998; Thomas et al., 1994). Reductions in PO2 are well known to impair the contractility of cardiac muscle strips in vitro (Roberts and Syme, 2018), as well as perfused heart preparations (Davie and Farrell, 1991; Davie et al., 1992; Petersen and Gamperl, 2010), suggesting a linkage between cardiac O2 supply, cardiac performance and environmental tolerance limits.
The hearts of many fishes are composed exclusively of spongy myocardium, which generally relies entirely on the luminal venous blood for O2 supply. Other fishes (e.g. salmonids) have a coronary circulation, which delivers oxygenated arterial blood and is generally, although not exclusively, associated with myocardial compaction (Axelsson, 1995; Farrell et al., 2012; Farrell and Smith, 2017). The primordial fish heart was hypothesized to be fully vascularized and composed of a mix of spongy and compact myocardium. Yet, the adaptive trade-offs of this cardiac arrangement remain unclear because more than half of all extant fish species lack coronary arteries (Durán et al., 2015; Farrell et al., 2012). Still, hypoxia and cardiac work are thought to be the main evolutionary drivers for coronary circulations (Farrell et al., 2012), highlighting the possibility that exposure to physiological and environmental extremes that induce increased myocardial work and reduced cardiac luminal O2 supply, have been important selection pressures in the evolutionary history of the coronary circulation. Consequently, coronary perfusion capacity can be hypothesized to play an important role in determining the ability of fishes to withstand environmental challenges.
Salmonid fishes have a coronary circulation that perfuses the compact myocardium of the ventricle, which comprises ∼20–50% of the total ventricular mass (Brijs et al., 2017; Ekström et al., 2017, 2019; Farrell et al., 2009; Farrell and Smith, 2017). Yet, chronic surgical ligation of the coronary artery has been shown to either have little influence on routine cardiac function (heart rate, stroke volume and cardiac output; Gamperl et al., 1994a) or slightly elevate heart rate. This latter response is likely a compensation for compromised cardiac contractility and stroke volume to maintain cardiac output and arterial blood pressure, but there is currently no information available on absolute cardiac output after coronary ligation to substantiate this (Ekström et al., 2018, 2017, 2019; Steffensen and Farrell, 1998). However, consistent with the idea that the adaptive benefits of the coronary circulation are predominantly evident when fish are exposed to environmental and physiological challenges, coronary blood flow in salmonids increases markedly during acute hypoxia (Axelsson and Farrell, 1993; Gamperl et al., 1994b, 1995) and warming (Ekström et al., 2017), as well as during exercise (Gamperl et al., 1995). The functional significance of this response is evident after coronary artery ligation, which reduces relative stroke volume and cardiac output in rainbow trout exposed to acute warming (Ekström et al., 2017; Ekström et al., 2019), and impairs ventral aortic blood pressure generation in rainbow trout swimming in hypoxia (Steffensen and Farrell, 1998). Moreover, coronary ligation reduced the critical thermal maximum (CTmax; Ekström et al., 2017; Ekström et al., 2019) and compromised cardiac and aerobic metabolic capacity in rainbow trout in normoxia (Ekström et al., 2018), as well as the maximum sustained swimming speed in chinook salmon (Farrell and Steffensen, 1987). However, there is currently no information detailing the influence of coronary flow on absolute cardiac output responses during hypoxia or warming in salmonids, or in governing tolerance to hypoxia in fish in general.
The specific mechanisms dictating hypoxia tolerance are multi-faceted and can be broadly related to various combinations of a fish's capacity to: (i) maintain aerobic metabolism during hypoxia, (ii) depress overall metabolism and/or (iii) sustain anaerobic metabolism (for review, see Mandic and Regan, 2018; Richards, 2011). Hypoxia tolerance is often quantified by the critical PwO2 (Pcrit), defined as the PwO2 below which the fish cannot sustain the O2 consumption rate (ṀO2; a proxy for aerobic metabolic rate) needed for aerobic standard metabolic rate (SMR) in hypoxia (Negrete and Esbaugh, 2019; Ultsch and Regan, 2019). When the animal cannot sustain SMR below Pcrit, a mismatch between tissue ATP supply and demand develops and ultimately leads to loss of equilibrium at a certain PwO2 (i.e. PLOE; Claireaux and Chabot, 2016; Negrete and Esbaugh, 2019; Rogers et al., 2016). There are several cardiovascular adjustments thought to promote O2 extraction, transport and delivery to tissues in hypoxia (Gamperl and Driedzic, 2009; Sandblom and Axelsson, 2011; Stecyk, 2017). Below a certain PwO2, obligate water-breathing fishes typically develop a vagal reflex inhibition of heart rate (i.e. hypoxic bradycardia; Farrell, 2007; Sandblom and Axelsson, 2005; Sandblom et al., 2009; Stecyk, 2017), which is initiated by stimulation of O2-sensitive chemoreceptors in the gills (see Milsom, 2012). This bradycardia has been suggested to benefit cardiac performance in hypoxia by, for example, decreasing cardiac work and O2 demand (Speers-Roesch et al., 2010), as well as improving the O2 delivery to the myocardium by increasing end-diastolic volume and the residence time of blood in the heart lumen, which promotes O2 diffusion into the spongy myocardium (for review, see Farrell, 2007; Stecyk, 2017). The prolongation of diastole with bradycardia also elevates stroke volume through increased diastolic filling and contractile force (i.e. Frank–Starling mechanism and the negative force–frequency relationship; for reviews, see Sandblom and Axelsson, 2007; Shiels et al., 2002). This allows cardiac output to be maintained, or even increase in hypoxia (Stecyk, 2017; Wood and Shelton, 1980). Interestingly, coronary blood flow may play an important role in this response, as coronary perfusion improves the contractility of isolated heart preparations during hypoxic conditions (Davie and Farrell, 1991; Davie et al., 1992). Furthermore, the hypoxic bradycardia likely enhances coronary blood flow by extending the diastolic period when coronary blood flow peaks (Axelsson and Farrell, 1993; Farrell, 2007). Taken together, these observations strongly indicate an important role of the coronary arteries for promoting cardiac performance in hypoxia, which likely extends to governing hypoxia tolerance in fish.
The aim of this study was to determine the role of coronary O2 supply to the heart during exposure to acute hypoxia and warming, and to study the mechanisms dictating cardiac and whole-animal tolerance to these environmental extremes. We determined the effects of coronary ligation on cardiac (absolute cardiac output, heart rate and stroke volume) and respiratory performance (ṀO2) in rainbow trout subjected to progressive acute hypoxia to determine Pcrit and PLOE, as well as a standardized thermal challenge protocol to determine CTmax. Specifically, we hypothesized that coronary ligation would impair cardiac performance and the ability to sustain aerobic metabolism in hypoxia and during warming, which would compromise tolerance to hypoxia (i.e. elevate Pcrit and/or PLOE) and heat (i.e. reduce CTmax). We also analysed the relative contribution of key cardiorespiratory traits underlying intra-specific variation in environmental tolerance limits, as well as the relationship between hypoxia and heat tolerance across experimental treatment groups to unravel potential common underlying cardiorespiratory mechanisms.
MATERIALS AND METHODS
Fish and holding conditions
Rainbow trout (Oncorhynchus mykiss Walbaum 1792; see Table 1 for morphological details) of mixed sex where obtained from a commercial fish farm (Vänneåns Fiskodling AB, Halland, Sweden). The fish were acclimated to laboratory conditions for a minimum of 4 weeks and were held in 1000 litre tanks continuously supplied with aerated recirculating freshwater at 10±0.5°C under a 12 h:12 h light:dark photoperiod. The fish were fed pellets (7 mm, Protec Trout pellets, Skretting, Norway) twice a week until the start of the experiments, and were fasted for 3 days before the day of the surgical procedure to ensure they were in a post-absorptive state throughout the protocol. All experimental procedures were covered by ethical permit 165-2015, approved by the regional ethical committee in Gothenburg.
Surgery and instrumentation
Individual fish were anesthetized prior to surgery in freshwater containing 150 mg l−1 MS-222 (Tricaine methanesulfonate, Scan Aqua AS, Årnes, Norway) buffered with 300 mg l−1 NaHCO3. Once ventilatory movements ceased, indicating a surgical plane of anaesthesia, a 300 μl blood sample was drawn from the caudal vessel for analysis of haematological variables (haematocrit and [haemoglobin]). Fork length and body mass (Mb) were recorded, and the fish was then placed on its left side on a surgery table covered with wet foam. During the surgery, the gills were perfused with a continuous flow of aerated freshwater (10°C) containing 75 mg l−1 MS-222 and 150 mg l−1 NaHCO3. A small incision was made in the isthmus and blunt dissection was used to expose the ventral aorta and the coronary artery. Care was taken not to damage surrounding vessels and nerves and to ensure that the pericardium remained intact. One group of fish had their coronary artery ligated with a 6-0 silk suture (coronary-ligated group), downstream from where the coronary artery branches from the hypobranchial artery. Another group of fish underwent an identical surgical protocol with the exception that the coronary artery was not ligated and left intact (sham-operated group). A 2.5 mm Transonic transit-time blood flow probe (L type; Transonic Systems, Ithaca, NY,USA) was then placed around the ventral aorta to allow recordings of ventral aortic blood flow (cardiac output). The probe was secured with 3-0 silk sutures inside the opercular cavity and in the skin just outside of the opercular cavity and a final (2-0) suture in front of the dorsal fin. Following instrumentation, the fish were placed individually in 10 litres Perspex respirometers submerged in a ∼120 liters experimental tank filled with aerated recirculating freshwater at 10.5±0.05°C. The fish were allowed to recover from surgery for ∼20 h, throughout which ṀO2 was continuously recorded (see below for details). The experiments were run in pairs daily with one sham-operated and one coronary-ligated fish in each run. All fish were then subjected to a hypoxia challenge followed by a temperature challenge as described below.
Measurements of cardiorespiratory variables in normoxia commenced in the morning and were recorded for a minimum of 2 h before the start of the hypoxia challenge. Once steady state heart rate values had been obtained during the initial recording period, the hypoxia challenge commenced and was performed using closed circuit respirometry following previously described protocols (Negrete and Esbaugh, 2019; Reemeyer and Rees, 2019). Briefly, by turning off the inflow of normoxic water (PwO2 ∼21 kPa at 100% air saturation) into the respirometers, the O2 consumption of the fish caused a gradual decrease in PwO2 to hypoxic levels until the fish reached PLOE (Nilsson and Östlund-Nilsson, 2004), which was defined as the PwO2 where the fish lost equilibrium for longer than 10 s. Cardiac measurements were recorded at PLOE for one more minute, after which the individual respirometer was reperfused with aerated water to bring the PwO2 inside the respirometer back to normoxic conditions. The duration of the hypoxia challenge averaged 61 min and ranged between 25 to 109 min. All fish recovered once normoxic conditions were restored.
Following >20 h of recovery from the hypoxia challenge, which was sufficient for all cardiorespiratory variables to return to routine values, baseline values were again recorded for a minimum of 2 h before the start of an acute heating challenge. The temperature was then increased from 10.5°C to 20°C in 5°C h−1 increments, with the temperature ramp lasting approximately 30 min and the target temperature being maintained for another 30 min. When 20°C was reached, the temperature was increased further by 1°C steps every 30 min (2°C h−1), where the ramping lasted approximately 10 min and the target temperature was maintained stable for 20 min. CTmax was determined as the temperature at which the fish lost the ability to maintain an upright body position for more than 10 s. Before each temperature increment, two ṀO2 slopes were recorded by turning off the flush pump and letting the % air saturation decrease by ∼10% before reinitiating flushing. Once CTmax was reached, the fish were removed from the respirometer and euthanized via blunt trauma to the head, immediately followed by drawing a blood sample from the caudal vessels for analyses of haematological variables. To ensure that the coronary artery had been successfully ligated, the position and integrity of the suture was verified post mortem. Finally, the wet mass of the spleen and heart ventricle (after careful removal of atrium, bulbus and any remaining blood in the lumen) were determined. The ventricle was preserved in 70% ethanol for further analyses (see below).
Data acquisition and calculations
The % air saturation inside the respirometer was continuously measured using an O2 optode connected to a Firesting O2 system (PyroScience, Aachen, Germany). ṀO2 was determined using automated intermittent closed respirometry and calculated from the decline in % air saturation (i.e. slope) in the respirometer between flush cycles. ṀO2 measurements at rest were typically performed in continuous 20 min cycles with the flush cycle set to 15 min. However, to ensure that water air saturation remained >85%, the cycles were reduced to 15 min cycles with 12 min flushing periods for some fish.
where Vr is the volume of the respirometer, Vf is the volume of the fish assuming that 1 g of fish equals 1 ml of water, Δ%Sat/t is the change in % O2 saturation per unit time, α is the solubility coefficient of O2 in freshwater and adjusted for the different experimental temperatures and Mb is the body mass of the fish (Clark et al., 2013). SMR was calculated as the lowest 20th percentile of the ṀO2 values (Chabot et al., 2016) obtained throughout the ∼20 h overnight post-surgery recovery period up until the start of the hypoxia challenge. Routine metabolic rate (RMR) throughout the hypoxia protocol was calculated from the average slope at 10% air saturation intervals until the air saturation reached 50%, and then at 5% intervals until PLOE (i.e. starting at 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 and 10% air saturation). SMR values were then plotted against PwO2, and Pcrit was determined by fitting a linear regression to RMR values during the hypoxia exposure that were below the normoxic SMR to identify the PwO2 where the regression line intercepted the SMR (Pcrit) obtained in normoxia (see supplementary material, Fig. S1; McBryan et al., 2016). Prior to the temperature challenge, RMR at 10.5°C was obtained by calculating the average from four ṀO2 slopes when the fish was in a steady state, as determined by a low and steady heart rate. During the temperature challenge, mean values were calculated from the two ṀO2 slopes at each temperature step when the temperature had stabilized. Measurements of bacterial background respiration were performed following each trial after the removal of the animals from the respirometers and the acquired slopes were subtracted from the ṀO2 slopes of the fish.
The Transonic flow probe was connected to a Transonic blood flow meter (model T206; Transonic Systems, Ithaca, NY) and the signals where recorded using a PowerLab system (ADInstruments, Castle Hill, Australia) at a sampling rate of 10 Hz using LabChart pro data acquisition software (v.7.3.2, AD Instruments, Castle Hill, Australia). To correct for any temperature effects on the Transonic flow probe readings, all probes were individually bench calibrated as specified by the user manual at 10.5, 15, 20, 22, 24, 26, 28 and 30.0°C. Briefly, the transonic flow probe was mounted on a calibration tubing submerged in a temperature controlled water bath. A flow of temperature-regulated water was pumped through the tubing using a peristaltic pump (Gilson 312 Minipuls 3, Villiers-Le-Bel, France) for 1 min. The outflowing water was collected and weighed to determine the flow gravimetrically. This process was repeated at increasing flow rates covering the flow range of the probe (1 to 100 ml min−1). The values obtained from the transonic flow probe were plotted against the recorded gravimetric flow and the resulting regression equation was used to correct the obtained in vivo cardiac output values. The heart rate was calculated from the pulsating blood flow traces using the cyclic measurements module in LabChart Pro. Stroke volume was calculated as the quotient of cardiac output divided by heart rate.
The PwO2 at the onset of hypoxic bradycardia was determined via segmental linear regression analyses using GraphPad prism 8.3.0, according to established methods for identifying critical break points (Yeager and Ultsch, 1989). Briefly, this method fits two lines that intersect at the PwO2 where heart rate starts to decrease. The PwO2 and average value for peak stroke volume in hypoxia (peak SVhypoxia), and cardiac output at peak SVhypoxia (CO at peak SVhypoxia) were determined for each fish. We also determined the temperatures and average peak values for cardiac output, heart rate and MO2 during warming (peak COwarming, peak heart rate and peak ṀO2, respectively), as well as the average stroke volume at peak COwarming (SV at peak COwarming).
Statistical analyses were performed using SPSS statistics 24 for Windows (IBM Corp., Armonk, NY, USA). Differences between groups (i.e. sham-operated versus coronary-ligated) in morphological variables, environmental tolerance indices (i.e. CTmax, Pcrit and PLOE), specific cardiorespiratory indices (e.g. peak responses, bradycardia) and mean routine cardiorespiratory values (at ≥18.9 kPa and 10.5°C) were analysed using independent-sample t-tests, Welch t-test (if variances were unequal) or Mann–Whitney U-tests (if data were not normally distributed). Differences in haematological variables where analysed using a linear mixed model with an unstructured repeated covariance structure, with fish ID as subject variable and sampling time (initial versus final), treatment (sham-operated versus coronary-ligated) and their interaction as fixed factors. Differences in cardiorespiratory variables during the hypoxia and temperature challenges were analysed using a linear mixed model with a within-subjects factor (PwO2 or temperature), treatment and the interaction between the within-subject factor and treatment as fixed factors, and fish ID as subject variable. Either a first-order autoregressive or a heterogeneous first-order autoregressive repeated covariance structure was used, which provided best fit to the models (as indicated by the lowest Akaike's information criterion, AIC). PwO2 values ranging from ≥18.9 to 2.1 kPa were included in the models assessing the effects of hypoxia and values at PLOE were excluded. Models assessing the effects of temperature included temperatures ranging from 10.5 to 22°C, as this was the lowest temperature at which individual fish peaked across all variables. If significant interactions were found, these were further explored with between- and within-treatments pair-wise comparisons, where confidence intervals were adjusted for multiple testing using Bonferroni correction. For the statistical analysis of the hypoxia challenge, cardiac output, heart rate and ṀO2, as well as ṀO2 during the temperature challenge, were transformed to their natural logarithm to comply with the assumption of homoscedasticity of the residuals. Pcrit was transformed to its natural logarithm to comply with the assumption of normality.
Multiple regression models were used to determine the relative contribution of key variables on the variances in hypoxia (PLOE) and temperature tolerance (CTmax). In these analyses, values for sham-operated and coronary-ligated treatments were pooled. Moreover, to determine potential relationships between temperature and hypoxia tolerance, additional regression analyses were carried out with CTmax as the dependent variable and PLOE, Pcrit and treatment (sham-operated versus coronary-ligated) as independent variables. The variables used in the multiple regression models were selected as they lacked multicollinearity and appeared to follow a linear relationship with the dependent variables (PLOE and CTmax) when assessed by visual inspection of partial regression plots and fitted the assumptions of the model. Variables were subsequently gradually eliminated from the model using stepwise backward regression until only the variables that best explained the variability in the dependent variable remained. Only adjusted R2 values are reported. Regression coefficients, standard errors and confidence intervals are included in Table S1. In addition, correlations between peak cardiac variables and cardiac morphological variables were analysed for each treatment using Pearson's correlations (Pcorr). Statistical significance was accepted at P<0.05. All data are presented as means±s.e.m.
Morphological and haematological characteristics
There were no differences in overall body characteristics (Mb, fork length and condition factor), cardiac morphology (relative ventricular mass and % compact myocardium), haematological variables or relative spleen mass between treatment groups (Table 1).
Impacts of coronary blood flow on hypoxia tolerance and cardiorespiratory performance
In normoxia, there were no differences in SMR between sham-operated control and coronary-ligated trout (58.6±4.5 versus 60.8±2.8 mg O2 kg−1 h−1, respectively; Fig. S1). Similarly, there were no differences between control and coronary-ligated fish in routine cardiac output (13.4±1.2 versus 11.4±1.3 ml min−1 kg−1, respectively; Fig. 1B) in normoxia. Even so, heart rate was significantly elevated after ligation (57.1±1.9 versus 47.6±3.9 beats min−1; t11.888=−2.213, P=0.047; Fig. 1C), which coincided with a significantly reduced stroke volume relative to control trout (0.20±0.03 versus 0.29±0.02 ml kg−1; t21=2.461, P=0.012; Fig. 1D).
Given the methodology used, body mass affected the rate of decline in PwO2 during the gradual hypoxia exposure. However, there was no linear relationship between the duration of the hypoxia protocol or body mass with Pcrit or PLOE (as indicated by visual inspection of scatterplots; data not shown). Throughout the hypoxia challenge, there were no significant differences in ṀO2 between treatment groups (Fig. 1A). Moreover, Pcrit occurred at similar PwO2 levels in control and coronary-ligated trout (Table 2). However, PLOE was significantly higher in coronary-ligated trout (t21=−2.284, P=0.033; Table 2). As hypoxia progressed, an increasingly pronounced bradycardia developed in both groups (Fig. 1C), but the onset of bradycardia occurred at a significantly higher PwO2 by 2.6 kPa in coronary-ligated fish (t17=−2.384, P=0.029; Table 2). Moreover, while cardiac output was maintained in hypoxia via a significant increase in stroke volume in the sham-operated control fish (Fig. 2B and D), the stroke volume in coronary-ligated trout was significantly lower during the hypoxia exposure (Fig. 1D). Consequently, the peak SVhypoxia was significantly lower (t21=4.747, P<0.001) and occurred at a higher PwO2 (U=102, P=0.021; Table 2), which coincided with a significantly lower cardiac output in coronary-ligated fish during the hypoxia exposure (Fig. 1B). Multiple regression analyses revealed that relative ventricular mass, percentage compact myocardium and peak SVhypoxia combined, significantly explained part of the variation in PLOE (F3,19=4.896, P=0.011, R2=0.35).
Impacts of coronary blood flow on temperature tolerance and cardiorespiratory performance
There were no differences between control and coronary-ligated trout in normoxia at 10.5°C, following the recovery from the hypoxia exposure, with regards to routine ṀO2 (53.0±4.6 versus 59.5±4.0 mg O2 kg−1 h−1, respectively; Fig. 2A) and cardiac output (11.2±1.2 versus 10.0±1.0 ml min−1 kg−1, respectively; Fig. 2B). Moreover, although routine heart rate was not significantly different between groups (41.3±3.5 versus 50.8±3.0 beats min−1, respectively), a trend towards a higher heart rate was observed in coronary-ligated trout (t21=−2.003, P=0.058; Fig. 2C), which coincided with a significantly lower stroke volume (0.29±0.03 versus 0.20±0.02 ml kg−1; t21=2.248, P=0.035; Fig. 2D).
ṀO2 did not differ between treatment groups and increased with warming at a similar rate across treatment groups (Fig. 2A). However, the overall temperature tolerance was significantly reduced in coronary-ligated trout as indicated by a 1.3°C reduction in CTmax (U=20, P=0.004; Table 3). Consistent with the increase in ṀO2, cardiac output also increased with temperature in both groups, although the increase was greater in control fish from 21°C onwards (temperature effectsham: F4,51=12.528, P<0.001; temperature effectligated: F4,51=3.947, P=0.007; Fig. 2C). The increased cardiac output was governed by elevations in heart rate in both groups (Fig. 2B,C). Stroke volume remained significantly lower in coronary-ligated trout throughout the thermal challenge relative to the control group (Fig. 2D). Consequently, the peak COwarming was lower in coronary-ligated fish (t12.275=3.696, P=0.003; Table 3), which was due to a decreased SV at peak COwarming (t11.902=3.476, P=0.005; Table 3). Moreover, the temperatures at peak heart rate, peak ṀO2 and peak COwarming were lower in the ligated compared with the sham-operated trout (U=16, P=0.002; U=23, P=0.047 and U=22, P=0.006, respectively; Table 3). Multiple regression analyses revealed that the peak COwarming significantly explained part of the variation in CTmax (F1,21=9.685, P=0.005, R2=0.28), such that CTmax was positively correlated with COwarming (Pcorr=0.548).
Haematocrit was significantly increased following the temperature challenge in both control and coronary-ligated fish (Table 1), whereas [Haemoglobin] only increased significantly in the control group (Table 1). There were no significant differences in MCHC between treatment groups at the end of the temperature challenge (treatment effectafterCTmax: F1,20=2.964, P=0.101; Table 1). However, MCHC was significantly reduced compared with pre-surgery values in coronary-ligated trout indicating pronounced red blood cell swelling, but this was not observed in sham-operated fish (Table 1).
Linkages between hypoxia and temperature tolerance
A multiple regression was run to predict CTmax from Pcrit, PLOE and treatment. The model revealed that treatment (F1,21=12.352, P=0.002, R2=0.34), PLOE (F1,21=8.485, P=0.008, R2=0.25) and treatment in combination with PLOE (Fig. 3) significantly predicted CTmax. This indicates that individuals with lower PLOE (and thus greater hypoxia tolerance) were also the most temperature tolerant, which strengthens the above findings that coronary ligation reduces tolerance to both environmental extremes.
A significant negative correlation was found in the coronary-ligated fish between the percentage compact myocardium and peak SVhypoxia (Pcorr=−0.698; P=0.008; Fig. 4A), and consequently CO at peak SVhypoxia (Pcorr=−0.609; P=0.027). Similarly, peak COwarming (Pcorr=−0.655; P=0.015; Fig. 4B) and SV at peak COwarming (Pcorr=−0.629; P=0.021) were also negatively correlated with percentage compact myocardium. Thus, the acute coronary ligation in individuals with a large proportion of compact myocardium (which was presumably devoid of coronary blood supply) resulted in lower peak stroke volume and cardiac output during exposure to both hypoxia and warming. These significant correlations were not observed in the sham-operated control group (Fig. 2A,B).
Coronary blood flow promotes cardiac stroke volume in trout at rest
Similarly to previous studies, coronary ligation resulted in either a trend towards an elevated routine heart rate (prior to the heating protocol) or a significantly elevated routine heart rate (prior to hypoxia protocol) in rainbow trout (Ekström et al., 2018, 2017, 2019; Steffensen and Farrell, 1998). Here, we can show for the first time that this response serves to maintain O2 consumption rate and cardiac output by compensating for a ∼44% decrease in stroke volume following the coronary ligation. The elevations in heart rate are most likely mediated via a reduced cholinergic (i.e. vagal) tone on the heart, presumably by a reduction in ventral aortic blood pressure inducing a barostatic reflex (see Sandblom and Axelsson, 2011). Indeed, Ekström et al. (2019) showed that treatment with the muscarinic antagonist atropine, which normally would abolish the cholinergic tone on the heart thus leading to an elevated heart rate, had no effect on heart rate in coronary-ligated rainbow trout suggesting that the ligation per se had already caused a release of cholinergic tone.
Restrictions of coronary blood flow impairs cardiac performance in hypoxia and affect whole-animal hypoxia tolerance
In both treatment groups, the bradycardic responses to hypoxia occurred at a PwO2 (7.5–10.1 kPa), which is within the range of previously reported values for this species (see table in Stecyk, 2017). The onset of bradycardia occurred at a PwO2 that was 2.6 kPa higher in the ligated group compared with the control group. It is possible that this was because the input from the branchial O2-sensitive chemoreceptors in hypoxia overrode the barostatic response that may initially have kept heart rate elevated in the coronary-ligated fish. Even so, below a PwO2 of ∼8 kPa the heart rate was similar between treatment groups, suggesting that the hypoxic bradycardia response dominated. However, in contrast to the control trout where stroke volume increased substantially as the hypoxic bradycardia developed, the stroke volume was only marginally elevated in trout with ligated coronary arteries, which meant that cardiac output collapsed and was halved immediately prior to PLOE relative to the cardiac output recorded in normoxia. Thus, the abolished O2 delivery to the compact myocardium hampered the capacity to elevate stroke volume and maintain cardiac output at lower PwO2 levels, in contrast to trout with an intact coronary flow, which were far better able to maintain cardiac output at reduced ambient PwO2. Another possible contributing factor that requires further testing is that along with an insufficient myocardial oxygenation, ligation of the coronary circulation also prevents catecholamines from reaching and effecting the compact myocardium. These are normally released during acute hypoxia (Perry and Reid, 1994, 1992) and protect myocardial function from hypoxia, acidosis and hyperkalaemia (see Driedzic and Gesser, 1994; Hanson et al., 2006; Roberts and Syme, 2018).
Despite a cardiac output that was twice as high in the control relative to coronary-ligated trout immediately prior to PLOE, coronary-ligated trout were only marginally less tolerant to acute hypoxia as their PLOE was only elevated by 0.6 kPa (∼3% air saturation). Moreover, the Pcrit for both treatment groups were within the range of previously reported values for rainbow trout (Williams et al., 2019; Wood, 2018), and while Pcrit was numerically slightly higher in the ligated fish (by 0.8 kPa), this was not statistically significant. Thus, it seems likely that the coronary-ligated fish initiated some response in hypoxia that compensated for the severely reduced capacity for circulatory O2 delivery to the tissues, which in turn minimized the negative impacts of the coronary obstruction on overall hypoxia tolerance. Indeed, both treatment groups maintained a similar ṀO2 throughout most of the hypoxia protocol, suggesting a significantly increased tissue O2 extraction in the coronary-ligated trout when cardiac output collapsed in hypoxia. Given that Steffensen and Farrell (1998) found no difference in venous PO2 between sham-operated and coronary-ligated rainbow trout, and since blood O2 carrying capacity (as indicated by a similar haematocrit) did not differ between treatments, one possibility is that there was a right (Bohr effect) and possibly downward shift (Root effect) in the haemoglobin O2 dissociation curve in the coronary-ligated trout, thus augmenting O2 unloading at the tissues (Harter and Brauner, 2017; Rummer and Brauner, 2015; Rummer et al., 2013). This shift could have been caused by an exacerbated acidosis in coronary-ligated fish in hypoxia, but unfortunately neither blood pH nor blood O2 content was measured in the current or earlier studies to confirm this. Although the overall effects of acute coronary ligation on hypoxia tolerance where relatively mild, the effects of a sustained impaired coronary O2 delivery over the long-term on organismal hypoxia tolerance remains to be explored.
Coronary blood flow restrictions during warming are linked to a compromised stroke volume, early onset of heart rate collapse and reduction in upper thermal tolerance
Our study is the first to demonstrate the role of the coronary circulation in maintaining cardiac output and stroke volume along with respiratory performance during warming. Cardiac output is mainly elevated via heart rate increases in response to warming (Eliason and Anttila, 2017). Similarly to previous observations (Ekström et al., 2017; Ekström et al., 2019), the heart rate peaked at a ∼1.6°C lower temperature in coronary-ligated trout, although the peak heart rate was similar for both treatment groups. It is possible that the lower temperature for peak heart rate during warming was simply an effect of the slightly higher routine heart rate often observed in coronary-ligated trout (Ekström et al., 2018, 2017, 2019; Steffensen and Farrell, 1998). The peak COwarming was driven by the peak heart rate in both control and ligated fish, which meant that the peak COwarming occurred at 1.8°C lower temperature in trout with ligated coronary arteries. However, despite the similar peak heart rate response, the peak COwarming was reduced by 45% in ligated fish. Similarly to the situation in hypoxia, this impaired capacity to elevate cardiac output with warming was explained by a severely constrained stroke volume (reduced by ∼44%) at peak COwarming in coronary-ligated trout. This is consistent with the ∼50% reduction in peak relative cardiac output (recorded using Doppler flow probes) in coronary-ligated trout observed by Ekström et al. (2019). Despite the reduced cardiac output during warming, peak ṀO2 was not statistically different between groups; again, however, the peak occurred at a lower temperature in ligated fish. Nevertheless, the earlier collapse of cardiorespiratory function in the coronary-ligated trout was associated with a lower CTmax, indicating a loss of heat tolerance (Ekström et al., 2017; Ekström et al., 2019).
Following thermal ramping, haematocrit and [haemoglobin] increased in the control fish, indicating splenic contraction (Perry and Kinkead, 1989), whereas haematocrit increased and MCHC was reduced in the coronary-ligated group, indicating erythrocyte swelling (Nikinmaa, 1982; Templeman et al., 2014). Both responses are consistent with an increase in circulating catecholamines, which commonly occurs following acute warming and increases blood O2 carrying capacity (Templeman et al., 2014) and haemoglobin O2 affinity by elevating erythrocytic intracellular pH (Nikinmaa, 1982). However, as the reduction in MCHC was more pronounced in the coronary-ligated trout, it is possible that this reflects a more pronounced increase of circulating catecholamines in this group in an attempt to mitigate the effect of blood acidosis on haemoglobin O2 affinity and to increase blood O2 carrying capacity in face of hypoxemia at elevated temperatures (Boutilier et al., 1986; Perry et al., 1989).
Myocardial oxygenation and cardiac performance constitute a common underlying mechanism influencing hypoxia and heat tolerance
Although acute tolerance limits to both hypoxia and warming have been previously linked to cardiac function, it is less clear if there are common underlying mechanisms that confer tolerance to both environmental extremes. For example, intra-specific differences in whole-animal hypoxia tolerance in European seabass (Dicentrarchus labrax) were positively correlated with contractile force production of ventricular strip preparations under both normoxia and hypoxia (Joyce et al., 2016). Moreover, studies in various teleost species indicate that intra-specific warming tolerance is positively correlated with cardiac morphological traits such as ventricle size (Anttila et al., 2013; Ozolina et al., 2016) and cardiac myoglobin levels (Anttila et al., 2013). Anttila et al. (2013) also found a positive association between hypoxia and thermal tolerance, indicating that a better O2 supply to the heart (i.e. due to increased myoglobin levels) and a capacity for maintaining a higher cardiac output (given the positive association between cardiac output and ventricle size; Franklin and Davie, 1992) could be a common trait associated with tolerance to both environmental drivers. In the current study, 33% of the variation in PLOE could be explained by the combined variation in relative ventricular mass, percentage compact myocardium and peak SVhypoxia, whereas peak COwarming had the most pronounced influence on temperature tolerance and accounted for 26% of the individual variability in CTmax. This highlights not only the importance of cardiac function in determining overall hypoxia and temperature tolerance but also some of the underlying mechanisms that allow for such enhanced performance. Indeed, a relatively larger ventricle can generate a larger stroke volume for a given size fish (Franklin and Davie, 1992) and a larger percentage compact myocardium may allow for a greater pressure generating capacity, especially as hearts become larger and require a greater ventricular wall tension production (Laplace's law; Brijs et al., 2017; Farrell, 1991; Farrell et al., 2009). Similarly, ventricle size and percentage compact myocardium has been shown to positively correlate with other fitness traits such as migratory capacity in sockeye salmon (Oncorhynchus nerka), highlighting the relationship between these cardiac morphological traits and the ability of the fish to overcome the challenges they face in their upstream spawning migrations (Eliason et al., 2011). Our data also indicate that the impaired cardiac performance in hypoxia and during warming after coronary ligation was specifically related to an ischaemic compact myocardium, because there was a negative relationship between percentage compact myocardium and the peak SVhypoxia and CO at peak SVhypoxia, as well as between percentage compact myocardium and peak COwarming and SV at peak COwarming. We interpret this finding as individuals with a larger proportion of O2-deprived compact myocardium suffered greater reductions in ventricular contraction force as luminal O2 became increasingly limiting in hypoxic and warming conditions.
Although coronary arteries appear to have been present in the hearts of evolutionarily ancient fishes, they have been lost multiple times throughout their evolutionary history (Durán et al., 2015; Farrell et al., 2012), raising questions regarding the adaptive significance of their presence or absence (Farrell et al., 2012). Our results highlight that while coronary blood flow is not essential for the maintenance of routine cardiac output, its importance increases when fish are exposed to environmental extremes such as severe hypoxia and high temperatures. This allows fishes with high aerobic capacity like salmonids to increase cardiac work even during conditions and physiological states when cardiac luminal O2 supply is severely constrained. As a corollary, our findings also highlight that pathological restrictions in coronary blood flow (e.g. coronary arteriosclerosis), which are known to be highly prevalent in both farmed and wild salmonids (Brijs et al., 2020; Farrell, 2002), may severely impact on cardiac capacity and animal performance traits. The coronary O2 delivery is likely of particular adaptive benefit during episodes of elevated physical activity and high cardiac workloads (e.g. swimming during challenging spawning migrations), specifically when combined with environmental conditions that constrain luminal O2 supply such as extreme temperatures and hypoxia as examined here.
We gratefully acknowledge Jeroen Brijs for the advice regarding the data analyses. The authors also acknowledge Julija Česnulaitytė, Helena Delgado, Laura Glasner, Frida Pallapies, Staffan Persson, Teemu Piippolainen, Álvaro Pradal, Saga Samuelsson, Frida Seger, Jaime Villaverde and Yannic Wocken for the practical assistance during some of the experiments.
Conceptualization: E.S., A.E.; Validation: D.M., A.E.; Formal analysis: D.M., T.M., A.G.; Investigation: D.M., T.M., A.E.; Resources: M.A., E.S.; Data curation: D.M.; Writing - original draft: D.M.; Writing - review & editing: D.M., T.M., A.G., M.A., E.S., A.E.; Visualization: D.M.; Supervision: E.S., A.E.; Project administration: E.S., A.E.; Funding acquisition: E.S., A.E.
This work was supported by grants from the Swedish Research Council FORMAS (E.Sa.) [2016-00729 and 2019-00299], the Helge Ax:son Johnson Foundation to (A.E.) and the Wilhelm and Martina Lundgren Research Foundation (D.M.).
Data are available from the Dryad digital repository (Morgenroth, 2021): dryad.kprr4xh4g
The authors declare no competing or financial interests.