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First published online April 17, 2009
Journal of Experimental Biology 212, 1270-1276 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.022764

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Intrinsic mechanical properties of the perfused armoured catfish heart with special reference to the effects of hypercapnic acidosis on maximum cardiac performance

Linda M. Hanson^1,*, Daniel W. Baker¹, Louise J. Kuchel^1,, Anthony P. Farrell², Adalberto L. Val³ and Colin J. Brauner¹

¹ Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC, V6T 1Z4 Canada
² Faculty of Land and Food Systems and Department of Zoology, University of British Columbia, Vancouver, V6T 1Z4 Canada
³ Laboratory of Ecophysiology and Molecular Evolution, Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil

^* Author for correspondence (e-mail: hanson{at}zoology.ubc.ca)

Accepted 9 February 2009

	Summary

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
The armoured catfish, Pterygoplichthys pardalis, is known to be extremely tolerant of environmental hypercarbia (elevated water CO₂ tensions), which occurs in their natural environment. In addition, previous studies have demonstrated that during exposure to hypercarbia, P. pardalis does not exhibit extracellular pH compensation and thus the heart and other organs must continue to function despite a severe extracellular acidosis. We used an in situ perfused heart preparation to determine the effects of an extracellular hypercapnic (elevated CO₂ in the animal) acidosis (1–7.5% CO₂) on heart function, specifically cardiac output, power output, heart rate and stroke volume. The present study is the first to comprehensively examine cardiac function in an acidosis-tolerant teleost. When compared with control conditions, maximum cardiac performance was unaffected at levels of CO₂ as high as 5%, far exceeding the hypercapnic tolerance of other teleosts. Moreover, P. pardalis exhibited only a moderate decrease (35%) in cardiac performance when exposed to 7.5% CO₂, and full cardiac performance was restored in six out of seven hearts upon return to control conditions. Myocardial intracellular pH (pH_i) was protected in situ, as has been found in vivo, and this protection extended to the highest level of CO₂ (7.5%) investigated. Thus, maintained heart function during a hypercapnic acidosis in P. pardalis is probably associated with preferential pH_i regulation of the heart, but ultimately is not sufficient to prevent loss of cardiac function. Our findings suggest the need for further study to elucidate the mechanisms behind this remarkable cardiac hypercapnic tolerance.

Key words: hypercapnia, heart, carbon dioxide, intracellular pH, Pterygoplichthys pardalis, acid–base physiology

	INTRODUCTION

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
The waters of the Amazon are prone to episodes of both hypoxia (low dissolved oxygen) (Val et al., 1995) and hypercarbia (high dissolved carbon dioxide). These events are the result of high biomass and water stratification, and occur most frequently at night; CO₂ tensions as high as 8 kPa have been measured under dense vegetative mats (Heisler, 1982; Ultsch, 1996), suggesting that severe hypercarbia may be a relatively common occurrence in tropical environments. CO₂ levels of this magnitude will induce a severe extracellular acidosis in fishes, which no fish studied to date is able to compensate for. Consequently, organs which are bathed in, and rely on, venous blood, such as the heart, must continue to function in spite of the potentially debilitating effects of an extracellular acidosis if the animal is to survive. As a result of considerable study on the inotropic (force of contraction) and chronotropic (frequency of contraction) effects of hypercapnic acidosis in both isolated cardiac muscle strips (Poupa et al., 1978; Gesser and Jorgensen, 1982; Gesser et al., 1982; Kalinin and Gesser, 2002) and working perfused heart preparations (Farrell et al., 1986; Farrell and Milligan, 1986; Farrell et al., 1988; Hanson et al., 2006), it is believed that the origin of the negative effects on both myocardial contractility and pacemaker cells is in fact intracellular acidification (Satoh and Hashimoto, 1983).

Despite periodic hypercarbic challenges, a great number of teleost species thrive in the Amazon. One such species, the armoured catfish, Pterygoplichthys pardalis (formerly known as Liposarcus pardalis), is a facultative air breather and possesses a great capacity to tolerate aquatic hypercarbia (Brauner et al., 2004). This tolerance is not related to extracellular pH (pH_e) compensatory capacity as, during exposure to hypercarbia, P. pardalis does not compensate for the extracellular acidosis, and thus pH_e can decrease from pH 7.9 to below pH 7.0 for hours or days without apparent adverse effects (Brauner et al., 2004). In most fishes studied to date, decreases in pH_e are qualitatively matched by decreases in intracellular pH (pH_i) in tissues such as the heart, white muscle and liver (Milligan and Farrell, 1986; Milligan and Wood, 1986; Wood and LeMoigne, 1991; Wood et al., 1990; McKenzie et al., 2002). Intriguingly, P. pardalis has been shown to regulate heart, liver and white muscle intracellular pH at normocarbic levels during the severe extracellular acidosis induced by hypercarbia (Brauner et al., 2004). This unusual pattern of pH_i protection during severe pH_e depression, which has only been observed in P. pardalis, Acipenser transmontanus (Brauner and Baker, 2009) and Synbranchus marmoratus (Heisler, 1982), may be the basis for CO₂ tolerance in fishes (Brauner and Baker, 2009) and we hypothesize here, protection of heart function in P. pardalis. The objective of this study was to determine the effects of a severe extracellular acidosis on maximum cardiac performance of P. pardalis, using an in situ perfused heart preparation subjected to different levels of hypercapnia. In order to appropriately examine these effects, characterization of baseline cardiac parameters and cardiac function was necessary, as these variables have not been previously reported in this species. Of special interest was the effect of hypercapnia on heart pH_i in this in situ preparation, where hearts were forced to work maximally, representing a very different condition to that examined previously in vivo (Brauner et al., 2004). This study provides insight into the physiological basis for CO₂ tolerance in fish that has recently been considered to be associated with the evolution of air-breathing (Brauner and Baker, 2009).

	MATERIALS AND METHODS

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
Experimental animals
Armoured catfish (Pterygoplichthys pardalis Castelnau 1855) of both sexes (mass, 473±39 g; relative ventricular mass, 0.028±0.002%) were obtained from a local commercial fish supplier and held in aerated well water at Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, Brazil prior to and during experimentation. Fish were maintained under a natural photoperiod in aerated outdoor tanks containing well water at 28°C.

Surgical procedures
Fish were anaesthetized in an oxygenated solution of buffered tricaine methane sulfonate (MS-222, 0.15 g l^–1, with 0.30 g l^–1 NaHCO₃), weighed and placed on an operating table where their gills were continuously irrigated with chilled, oxygenated anaesthetic (0.05 g l^–1 MS-222 buffered with 0.1 g l^–1 NaHCO₃). Fish were then injected with 1 ml kg^–1 of heparinized saline (150 i.u. ml^–1) into the caudal vessels. An in situ perfused heart preparation was prepared as previously described (Farrell et al., 1986) but modified (Farrell et al., 1989); however, further modifications were necessary because of the anatomical differences between rainbow trout and armoured catfish. A shallow lengthwise incision was made from the anal opening to an area just posterior to the pectoral girdle to expose the viscera and allow a stainless steel input cannula to be introduced into the sinus venosus via a hepatic vein. In armoured catfish there are two major hepatic veins, one from each lobe of the liver. These two vessels are highly visible as they exit the liver and merge to form a single vessel. This single vessel is only visible for a very short distance before it becomes enveloped by the layer of connective tissue that encloses the entrance to the pericardium. Owing to these anatomical limitations, the input cannula was always inserted into the left hepatic vein at a point just upstream (posterior) from the junction. The input cannula was advanced along the vessel to the point at which the hepatic vein joined the sinus venosus. In the ideal preparation, the tip of the input cannula was introduced fully into the sinus venosus; however, the anatomy of the junction between the two structures was such that this was not always possible. Consequently, even with careful placement, partial occlusion of the tip of the cannula by the walls of the hepatic vessel was often unavoidable, thus high input pressures (0.11–0.25 kPa) were needed to supply adequate perfusate. The use of high perfusion pressures is not ideal; nevertheless, it provides far superior results than the only alternative, a preparation in which the integrity of the pericardium is not maintained. In addition, previous studies have used even higher perfusion pressure (Stuart et al., 1983) with no apparent ill effects.

Following insertion of the input cannula, the heart was immediately perfused with saline (composition below) containing 500 nmol l^–1 adrenaline (adrenaline bitartrate salt; AD) and 10 i.u. sodium heparin per millilitre. A stainless steel output cannula was then secured into the ventral aorta at a point confluent with the bulbus arteriosus. This was facilitated by removal of the lower jaw and gills. The total time to prepare the perfused heart preparation was 15–20 min. All experimental procedures complied with the policies of INPA, the University Animal Care Committee of the University of British Columbia, and the Canadian Council on Animal Care.

Following surgery, the fish was transferred to a physiological saline bath (7 NaCl). The input cannula was immediately connected to an adjustable, constant-pressure reservoir, and the output cannula was connected to a separate constant pressure head set at 3.0 kPa. The height of the input pressure reservoir was adjusted to set routine cardiac output () at approximately 35 ml min^–1 kg^–1. Input (P_in) and output (P_out) pressure were measured through saline-filled side arms (PE50 tubing) connected to disposable pressure transducers (DPT 6100, Smiths Medical, Kirchseeon, Germany). Cardiac outflow was continuously measured through the output line with an in-line electromagnetic flow probe (SWF-4, Zepeda Instruments, Seattle, WA, USA) that had been previously calibrated with known flow rates of perfusate. Hearts were allowed to equilibrate for 5-10 min under control conditions (see below) before the experiment commenced. Previous examination of P. pardalis ventricles revealed no evidence of a coronary circulation.

Perfusate composition
For the control perfusate, freshwater fish saline (125.0 mmol l^–1 NaCl, 3.0 mmol l^–1 KCl, 1.0 mmol l^–1 MgSO₄·7H₂O, 2.5 mmol l^–1 CaCl₂·2H₂O, 5.6 mmol l^–1 D-glucose, 11.9 mmol l^–1 NaHCO₃; all chemicals from Sigma-Aldrich, Oakville, ON, Canada) was aerated with 1.0% CO₂ (balance air), supplied by a Wösthoff gas mixing pump (Bochum, Germany), to achieve a pH of 7.8 and an oxygen level of 20 kPa. Previous experiments in our laboratory (Hanson et al., 2006) have shown no significant difference in maximum cardiac performance between hyperoxic hearts (95.5% O₂, 0.5% CO₂) and hearts perfused with air-saturated saline, which was the control level of oxygen for all experiments. For the hypercapnic test conditions the perfusate was aerated with varying levels of CO₂ (2.5, 5.0 and 7.5% CO₂, balance air), however, the ionic composition of the perfusate remained the same. Regardless of the test conditions, the perfusate contained 500 nmol l^–1 AD. Preliminary studies suggested that this high level of adrenergic stimulation was necessary for the heart to maintain consistent performance. Furthermore, previous studies on cardiac strips (trout and eel) demonstrated that adrenaline increases the force of contraction during hypercapnia without altering the relative changes in force seen under different exposure conditions [anoxia, hypercapnia, recovery (Gesser et al., 1982)], thus minimizing any concerns that potentially excess adrenergic stimulation affected the results of the present study. Additionally, 500 nmol l^–1 AD was shown not to affect pH_i during extracellular acidosis in perfused rainbow trout hearts, although contractility was restored in failing hearts (Farrell and Milligan, 1986).

Experimental protocols
Maximum cardiac performance was assessed in each heart preparation under every test condition. By initially measuring both maximum cardiac output (_max) and maximum cardiac power output (PO_max) under control conditions, each heart acted as its own control. To determine _max, P_in was gradually increased in increments of approximately 0.05 kPa until cardiac output reached a plateau (usually around 0.4 kPa). To assess PO_max, P_in was left at its maximum and P_out was increased in a stepwise fashion in 0.1 kPa increments until PO reached a plateau. After PO_max was determined, P_out and P_in were returned to resting levels and the heart was allowed to recover (5 min) before being exposed to the next perfusate. To mimic natural conditions all experiments were performed at 27.0±0.2°C.

Series 1: characterization of cardiac performance
The purpose of this series was to characterize the Starling response (the change in cardiac performance versus preload) and the pressure development of armoured catfish hearts. Following 5–10 min acclimation period to control conditions, the Starling response was determined by increasing P_in in 0.05 kPa increments, with the heart being allowed to equilibrate for approximately 1 min at each step. P_in was increased until such time as reached a maximum. Immediately following this procedure maximum pressure generation was determined by increasing P_out in a similar stepwise fashion (with P_in held constant at its maximum) until PO reached a maximum. Hearts were then returned to resting levels of P_in and P_out so that their routine, post-test performance could be compared with their initial performance to assess preparation viability and ensure that the heart had not been damaged by the test.

Series 2: hypercapnia
The purpose of Series 2 was to quantify the effect of hypercapnia and associated acidosis on mechanical characteristics of the perfused heart in maximally stimulated hearts (i.e. presence of 500 nmol l^–1 AD). Recovery from severe hypercapnia was assessed by re-testing hearts with control perfusate following the hypercapnic exposures. After assessing _max as described above (i.e. control, 1% CO₂, resulting in a pH of 7.83), hearts were then sequentially subjected to: (1) 2.5% CO₂ (resulting in a pH of 7.56), (2) 5.0% CO₂ (resulting in a pH of 7.26), (3) 7.5% CO₂ (resulting in a pH of 7.10), (4) control with a pH of 7.83 and (5) 7.5% CO₂ (resulting in a pH of 7.10). Hearts were re-exposed to 7.5% CO₂ during this final step so that intracellular pH (pH_i) could be measured under these conditions. Hearts were exposed to each perfusate for a total of 15 min during which time _max, PO_max, heart rate and stroke volume were measured; this time period also ensured continued viability of the photosensitive AD. Following the sixth and final 15 min exposure the heart was rapidly excised, the ventricle was dissected out, weighed and frozen in liquid nitrogen for later measurement of pH_i as described below.

Series 3: hypercapnic preconditioning
The purpose of this series was to determine if the maximum cardiac performance observed during the highest level of hypercapnia (7.5% CO₂ resulting in a pH of 7.10) in Series 2 was affected by the previous exposure to an intermediate level of hypercapnia. Individual hearts were subjected to the following protocol: (1) control (1% CO₂) resulting in a pH of 7.83, (2) 7.5% CO₂ resulting in a pH of 7.10, (3) control with a pH of 7.83. As in the previous experiments, maximum cardiac performance was assessed under each test condition. Following the final (15 min) step hearts were excised, ventricle removed, weighed and frozen in liquid nitrogen for later measurement of pH_i (termed recovery pH_i) as described below.

Intracellular pH determination
Intracellular pH was determined on freeze clamped ventricles using the tissue homogenate technique (Pörtner et al., 1990). In brief, the method involved homogenization of ventricular tissue using a liquid nitrogen cooled mortar and pestle. Pulverized tissue was then transferred using a pre-cooled metal scoop to a pre-cooled 1.5 ml centrifuge tube. An 800 µl aliquot of an isotonic metabolic inhibitor solution (150 mmol l^–1 KCl and 5 mmol l^–1 nitrilotriacetic acid disodium salt) was then added to the tissue, and pH of the resultant mixture was measured using a thermostated capillary pH electrode (Radiometer, BMS 2, London, Ont., Canada). Although this technique has been validated for both water breathing (blood 0.5% CO₂) and air breathing (blood 3–4% CO₂) animals, further validation (Baker et al., 2009) was provided by comparing measurements of pH_i of red blood cells separated from blood exposed to high CO₂ in tonometers (between 0.5% and 10% CO₂) using the freeze–thaw technique (Zeidler and Kim, 1977) and the metabolic inhibitor homogenate method (Pörtner et al., 1990). The high correlation between results (R²=0.95) indicated that the latter can be used to measure pH_i at very high CO₂ tensions despite the potential for CO₂ loss during tissue processing. For comparison purposes pH_i was also measured on hearts taken immediately from euthanized, uncannulated, resting control fish (N=6), hereafter referred to as control pH_i.

Calculations and statistical analysis
All experimental data were collected using data acquisition software (Labview version 5.1, National Instruments, Austin, TX, USA), which allowed for real-time measurements of f_H, P_in, P_out, and PO. Statistical differences within test groups were determined by one-way repeated measures analysis of variance (ANOVA). When warranted, the Holm–Sidak procedure was used for post-hoc multiple comparisons. Comparisons of pH_i between test groups were made using a Students' t-test. Sigma Stat (3.0, SPSS, San Rafael, CA, USA) was used for all statistical analysis. For statistical comparisons, =0.05 was used for determining statistical differences.

Owing to the anatomical limitations discussed above, control preloads ranged from 0.11 kPa to 0.25 kPa. Consequently, results in Fig. 1 are presented as change from routine P_in, where routine P_in is defined as the preload necessary to achieve a of 35 ml min^–1 kg^–1. Preloads were normalized by fitting (r²>0.98) the raw data for each individual fish (N=6) to a first order sigmoidal equation of the form =a/{1+e^{–[(Pin–routine Pin)/b]}} where a and b are coefficients derived from the fit for each individual fish. These equations were used to calculate for individual fish at specific, relative preload values. Results are presented as the mean for each preload value ± s.e.m.

View larger version (9K): [in this window] [in a new window]   Fig. 1. The effect of changes in input pressure on cardiac output (A) and cardiac stroke volume (B) in in situ perfused Pterygoplichthys pardalis hearts under control, normocapnic conditions at 27.0±0.2°C (N=6). Results are presented as change from routine preload pressure and were normalized to a first order sigmoidal equation (r2>0.98) as discussed in the Results. Data are presented as the means for each preload value ± s.e.m.

	RESULTS

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

View larger version (5K): [in this window] [in a new window]   Fig. 2. The effect of changes in after-load on cardiac power output in in situ perfused Pterygoplichthys pardalis hearts under control, normocapnic conditions at 27.0±0.2°C (N=6). Data are presented as means ± s.e.m.

View larger version (15K): [in this window] [in a new window]   Fig. 3. The effect of hypercapnia on (A) maximum cardiac power generation, POmax, (B) maximum cardiac output, max, (C) cardiac stroke volume and (D) heart rate of in situ perfused Pterygoplichthys pardalis hearts at 27.0±0.2°C (N=5). Hearts were sequentially exposed to CO2 tensions of 1.0% (control), 2.5%, 5.0% and 7.5% before being returned to 1.0% CO2 to assess recovery (open circles). Hearts were then re-exposed to 7.5% CO2 to permit sampling for intracellular pH determinations (marked circles). One heart failed to recover from exposure to extreme hypercapnia and thus has been presented separately (squares).

The effects of hypercapnia on heart function were usually not permanent as performance was restored following recovery to control conditions in all but one preparation (Fig. 3). Note that although results are only shown for five fish an additional two fish were tested under this protocol and also showed a complete recovery upon return to control conditions. Unfortunately, recordings from these two fish were lost due to equipment malfunction.

Series 3: hypercapnic preconditioning
Hearts exposed directly to 7.5% CO₂ following control perfusate showed a trend toward an additional 17–20% decrease in _max and PO_max when compared with the cardiac performance of hearts exposed to 7.5% CO₂ following a stepwise increase (Series 2). However, this difference between the two exposure methods was not statistically significant (possibly because of the small sample size, N=3). Nevertheless, these preliminary results suggest the potential for hypercapnic preconditioning, such that a gradual (stepwise) exposure to extreme hypercapnia reduces the impact of the final hypercapnic exposure. However, confirmation of this will need further study. Similarly to Series 2, the reduction in cardiac performance seen under hypercapnia appeared to result from decreases in both f_H and V_S.

Intracellular pH (pH_i)
Intracellular pH of hearts exposed to 7.5% CO₂ (Series 2) was found to be significantly (P<0.05) higher than that of non-perfused control hearts (7.02±0.05, N=6 versus 6.92±0.04, N=6). However, there was no significant difference in pH_i between hearts exposed to 7.5% CO₂ compared with those sampled under normocapnic conditions (1.0% CO₂) during hypercapnic recovery (7.06±0.01, N=3, Series 3). In addition, regression analysis showed that myocardial pH_i under hypercapnia was negatively correlated with cardiac performance (_max) under hypercapnia (r²=0.64; P<0.01) such that the ventricle with the lowest pH_i (i.e. closest to control values) showed the least decline in performance.

	DISCUSSION

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
This study demonstrates the great capacity of the heart of P. pardalis to both tolerate and perform maximally under a severe hypercapnic acidosis. Maximum cardiac performance was not significantly different from that seen under normocapnic conditions at levels of hypercapnia as high as 5% CO₂ (5.1 kPa). Moreover, P. pardalis exhibited only moderate decreases (35%) in cardiac performance when exposed to 7.5% CO₂. Equally as remarkable, full cardiac performance was restored upon return to normocapnic conditions in six out of seven preparations.

The present study is the first to comprehensively examine cardiac function in an acidosis-tolerant teleost. Previous studies looking at cardiac function in intact hearts during hypercapnic acidosis have all been conducted on species considered to be intolerant of acidosis (or sensitive to pH perturbation), i.e. rainbow trout (Farrell and Milligan, 1986; Farrell et al., 1986; Farrell et al., 1988), ocean pout (Turner and Dredzic, 1980; Farrell et al., 1983) and sea raven (Turner and Dredzic, 1980; Farrell et al., 1983). When compared with P. pardalis (present study) these species show significant decreases in cardiac performance (14–58%) during exposure to a far less severe hypercapnia (1–2% CO₂; Table 1). The degree of acidosis associated with a particular CO₂ tension in previous studies is not as great as in the present study (i.e. for equivalent CO₂ tensions hearts in the present study are exposed to a lower pH), but this is largely due to the effect of temperature on pH (0.016 pH°C^–1). When ocean pout hearts at 10°C were perfused with saline containing 2% CO₂, the pH of the perfusate was 7.4 and they exhibited a 20% decline in cardiac performance (Farrell et al., 1983). Conversely, performance of P. pardalis hearts at 27°C perfused with an equivalent CO₂ tension but at a lower pH (pH 7.2) did not differ from normocapnic values (present study). Thus, cardiac function is clearly maintained in P. pardalis at CO₂ tensions that greatly reduce cardiac function in seemingly acidosis-sensitive fishes.

Eels and sturgeons are known to tolerate severely hypercarbic water (10% and 3% CO₂, respectively) for short durations (several hours) with no decrease in cardiac output in vivo (Crocker et al., 2000; McKenzie et al., 2002). However, equivalent in situ studies of heart function have not been conducted for direct comparison with the present work. In addition, it is difficult to extrapolate the results of previous in vitro findings on isolated cardiac muscles to the present work. Nevertheless, if one assumes that isometric cardiac force generation is analogous to maximum power generation then P. pardalis would be slightly less acidosis tolerant than the turtle (Trachemys scripta, formerly Pseudemys scripta). In vitro turtle myocardium preparations showed no significant decrease in contractile force after 15–30 min at a similar level of respiratory acidosis (Poupa et al., 1978; Gesser and Jorgensen, 1982) as that which resulted in a 35% decrease in performance in the present study. Thus, although it appears that P. pardalis is remarkably acidosis tolerant for a teleost, and falls within the range of the few other acidosis-tolerant vertebrates, caution should be used when comparing air and water breathers since the former have higher in vivo resting blood CO₂ tensions and lower blood pH.

In the absence of published values for in vivo cardiovascular performance of P. pardalis, we compare our values with those reported for other teleost species and consider the effect of temperature on cardiac output ( increases with temperature). Given the rather sedentary nature of P. pardalis, maximum cardiac performance under routine (normocapnic) conditions (_max=62.8±4.1 ml min^–1 kg^–1; PO_max=10.5±0.3 mW g^–1 ventricle) appears impressive compared with rainbow trout at 18°C, where _max ranges from 54–78 ml min^–1 kg^–1 and PO_max ranges from 5.9–9.3 mW g^–1 ventricle (Keen and Farrell, 1994; Farrell et al., 1996; Hanson and Farrell, 2007). Although maximum cardiac power output of P. pardalis under normocapnic conditions was comparable to that seen in rainbow trout, the maximum pressure generation was not (4 kPa versus 6–8 kPa). This suggests that P. pardalis probably possesses a much lower arterial blood pressure. Maximum V_S of P. pardalis (0.55±0.05 ml kg^–1) is also lower than that of other teleosts where V_S ranges from 0.8–1.1 ml kg^–1 (Farrell et al., 1986; Mendonça et al., 2007), as is its relative ventricular mass (0.03% versus 0.07% in salmonids). If V_S is expressed per gram of ventricle (2.09±0.10 ml g^–1 ventricle), it is similar to that found in another benthic species, winter flounder (2.3 ml g^–1 ventricle) and greater than that of more active species [rainbow trout 1.2 ml g^–1 ventricle (Farrell et al., 1986); Atlantic salmon, 1.4 ml g^–1 ventricle, Atlantic cod, 1.7 ml g^–1 ventricle (Mendonça et al., 2007)]. In addition to the present results, a previous study (Mendonça et al., 2007) suggests that winter flounder also has a comparatively low arterial blood pressure. These sorts of comparisons lend some support to the possibility that higher relative ventricular mass in active fishes is more important for pressure generation than for control of stroke volume (Gamperl and Farrell, 2004). Our heart rates (110 min^–1) are higher than those recorded previously (76 min^–1) in resting individuals of this species (MacCormack et al., 2003a). We attribute the majority of this discrepancy to the removal of vagal cholinergic tone in the present experiment. Previous studies have found cholinergic tone to be exceptionally high in air breathing fishes (Sundin et al., 1999; McKenzie et al., 2007) and thus the removal of cholinergic control can have dramatic effects on heart rate. For example, administration of a cholinergic antagonist caused heart rate to nearly triple in the facultative air breathing jeju (Hoplerythrinus unitaeniatus) (McKenzie et al., 2007). An additional explanation is that some of the increase in heart rate may be due to adrenergic stimulation. Tonic catecholamine concentrations are currently unknown for P. pardalis and thus preliminary experiments were conducted to determine the appropriate level of adrenergic stimulation. These experiments revealed that high levels of adrenaline (500 nmol l^–1) were necessary to ensure a stable preparation.

When perfused hearts were exposed to the most severe hypercapnia (7.6 kPa; 7.5% CO₂) both _max and PO_max decreased by 35%. The reduction in both measures of cardiac performance appears to be mainly due to hypercapnic bradycardia (23 beats min^–1 at _max and 19 beats min^–1 at PO_max), although in the case of PO_max the 20% reduction in f_H (19 beats min^–1) was not statistically significant. A similar degree of hypercapnic bradycardia has been well documented in other species (Farrell et al., 1983; Farrell et al., 1986; McKendry and Perry, 2001). The current study used CO₂ aeration to induce an extracellular acidosis, however, the acidosis did not manifest intracellularly. This suggests that preferential regulation of myocardial pH_i is occurring in this in situ preparation, as CO₂ tensions within the myocardium are expected to equilibrate with the perfusate quite rapidly (Gesser and Jorgensen, 1982). Previous studies on the CO₂-sensitive rainbow trout demonstrated that during a hypercapnic acidosis both in vivo and in situ myocardial pH_i fell within 3 h (Farrell and Milligan, 1986; Wood and LeMoigne, 1991).

Interestingly, in P. pardalis myocardial pH_i is regulated both in situ (present study) and in vivo (Brauner et al., 2004). Robust tissue pH regulation has been identified in only a few other fishes, for example, the white sturgeon, Acipenser transmontanus (Brauner and Baker, 2009), and the facultative air breather, the marbled swamp eel, Synbranchus marmoratus (Heisler, 1982). In both of these species, intrinsic buffering was not great enough to account for pH_i protection, and therefore, active cellular pH regulation was hypothesized to be driving pH_i compensation. Trans-membrane cellular pH_i regulation capacity could explain the incongruity between the findings in rainbow trout and the armoured catfish. Elucidation of these mechanisms will have to await further study.

This study is the first to comprehensively examine cardiac function in a CO₂-tolerant teleost. Our results reveal that the heart of P. pardalis possesses a remarkable ability to both tolerate and perform maximally in the face of a severe hypercapnic acidosis; a situation that freshwater tropical fish may experience in their natural environment (Ultsch, 1996), and a condition that also occurs when a facultative air-breathing fish breathes air during exposure to hypoxia (Heisler, 1982). Our results also imply a role for pH_i protection in maintaining heart function at high CO₂ tensions, consistent with our hypothesis that CO₂ tolerance in fishes is associated with preferential pH_i regulation; although this regulation is not sufficient to ultimately prevent loss of cardiac function. Recent studies have suggested that P. pardalis is better able to regulate myocardial intracellular calcium (MacCormack et al., 2003b) than more acidosis-sensitive species. Intracellular calcium levels and myofilament calcium sensitivity play a vital role in determining myocardial contractility. In addition, the positive inotropic effects of intracellular calcium are thought to counteract the negative inotropic effects of hypercapnic acidosis. Consequently, intracellular calcium handling has been implicated in the restoration of force and contractility during hypercapnic acidosis, however, little is currently known about the mechanistic basis for this. If improved intracellular Ca²⁺ handling is involved in alleviating the effects of hypercapnia, future exploration of this topic may reveal the mechanisms responsible for the remarkable hypercapnic tolerance of P. pardalis.

List of abbreviations

AD

adrenaline

f_H

heart rate

pH_e

extracellular pH

pH_i

intracellular pH

P_in

input pressure

P_out

output pressure

PO

cardiac power output

PO_max

maximum cardiac power output

cardiac output

max

maximum cardiac output

V_S

stroke volume

	Footnotes

 
Present address: School of Integrated Biology, University of Queensland, Brisbane, QLD 4072 Australia

This research was supported by Natural Sciences and Engineering Research Council (NSERC) of Canada Discovery grants to C.J.B. and A.P.F., a CNPq Brazil research grant to A.L.V. and NSERC CGS, CSZ Research Travel Award and SEB COB Research Travel Grant to D.B. We thank Drs M. Axelsson and J. Altimiras for writing and providing the perfused heart data acquisition and analysis program for Labview, D. Jackson for valuable technical assistance, and Nazaré Paula da Silva for tremendous logistical support during our stay.

	References

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 

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