Effect of heart rate and hypoxia on the performance of a perfused trout heart A. P. FARRELL, S. SMALL,A N D M . S. GRAHAM' Department of Biological Sciences, Simon Fraser University, Burnaby, B.C., Canada V5A IS6

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Received March 10, 1988 FARRELL, A. P., SMALL, S., and GRAHAM, M. S. 1989. Effect of heart rate and hypoxia on the performance of a perfused trout heart. Can. J. Zool. 67: 274-280. While adrenergic stimulation and increased filling pressure of the heart are recognized to increase cardiac stroke volume in the trout heart, the effects of factors such as heart rate and oxygen supply have not been examined. The present study used isolated, saline-perfused trout hearts to determine the maximum cardiac performance during hypoxic perfusion and during changes in pacing frequency similar to the range of heart rate observed in intact trout. The threshold oxygen tension of the perfusate was between 25 and 46 Torr (3.33 -6.13 kPa) for maintaining resting and maximum cardiac ouput, but was between 46 and 67 Torr (6.13 -8.93 kPa) for maintaining maximum power output. Increasing the pacing frequency from 30 to 58 beatslmin did not produce a proportionate increase in the maximum cardiac output because maximum stroke volume was reduced significantly. It is suggested that the reduction in maximum stroke volume occurs because atrial filling time is compromised at higher pacing frequencies in the isolated perfused heart. FARRELL, A. P., SMALL, S., et GRAHAM, M. S. 1989. Effect of heart rate and hypoxia on the performance of a perfused trout heart. Can. J. Zool. 67 : 274-280. Bien que la stimulation adrknergique et l'augmentation de la pression de remplissage du coeur soient reconnus comme des facteurs capables d'augmenter le volume systolique du coeur chez la truite, l'effet de facteurs tels que le rythme cardiaque ou le volume d'oxygkne n'a jamais CtC CtudiC. Des coeurs de truite isolCs et gardCs dans une solution saline ont servi B dCterminer la performance cardiaque maximale au cours d'une perfusion hypoxique et en rCponse B des changements de frkquence semblables aux changements de rythmes cardiaques qu'Cprouve une truite saine. La pression d'oxygkne seuil nkcessaire au maintien du dCbit cardiaque au repos ou du dCbit cardiaque maximal se situait entre 25 et 46 Torr (3,33 -6,13 kPa), mais cette pression se situait entre 46 et 67 Torr (6,13 -8,93 kPa) lorsqu'il s'agissait de maintenir la puissance cardiaque B son maximum. Une augmentation du rythme cardiaque de 30 B 58 pulsations cardiaquelmin n'entraine pas une augmentation proportionnelle du dCbit cardiaque maximum, car le volume systolique maximal diminue alors considCrablement. Nous croyons que cette reduction du volume systolique maximal se produit parce que la durCe du remplissage de l'oreillette est modifiCe i des rythmes cardiaques plus ClevCs dans un coeur isole en perfusion. [Traduit par la revue]

Introduction During exercise, vertebrates increase oxygen delivery to working tissues by increasing cardiac output (Q) and tissue oxygen extraction. While mammals primarily increase heart rate (fH) to increase Q, teleost fish rely more on changes in stroke volume of the heart (SVH) (Jones and Randall 1978). Rainbow trout (Salmo gairdneri) increased resting SVH 2.4 times and fH by 36% during sustained swimming (Kiceniuk and Jones 1977). These observations confirmed an earlier suggestion that swiming trout increase fH by 36-39% and resting SVH 2.1 -2.9 times (Priede 1974). Atlantic cod (Gadus morhua) also increased SVH slightly more than they increased fH during sustained swimming (Axelsson and Nilsson 1986). The mechanisms underlying the more pronounced changes in SVHhave not been examined to any degree in intact teleost fish, although the regulation of fH is reasonably well understood (for recent reviews, see Nilsson 1983; Laurent et al. 1983; Farrell 1984). Nonetheless, studies with perfused hearts have established that a modest increase in filling pressure of the heart (1 -2 cm H20; 1 cm H 2 0 = 98.1 Pa) can produce an increase in SVH comparable to that produced during exercise (Farrell et al. 1982, 1986; Farrell 1984). Furthermore, adrenergic stimulation of the trout heart increases the sensitivity of the heart to filling pressure (Farrell et al. 1986). Thus, it is likely that either small increases in atrial filling pressure and (or) adrenergic stimulation of the myocardium are major determinants of SVH during exercise in the trout. Even so, 'Present address: Vancouver Public Aquarium, P.O. Box 3232, Vancouver, B.C., Canada V6B 3x8. Printed in Canada I Imprimt au Canada

SVH will be influenced by factors other than filling pressure and inotropic state. The effects of oxygen supply and filling time of the heart are considered here. Teleost hearts are highly aerobic (Driedzic et al. 1983; Farre11 et al. 1985), yet they rely partially or wholly on venous blood for an oxygen supply (Farrell 1984; Santer 1985). The trout myocardium receives its O2 supply from arterial blood contained in the coronary artery and from venous blood being pumped through the heart chambers. The coronary circulation supplies the outer, compact myocardium, which represents approximately 40 % of the ventricle mass (McWilliam 1885, cited by Santer 1985; Hesse 1921; Grant and Regnier 1926; Cameron 1975; Poupa et al. 1974). During sustained exercise, arterial blood remains saturated (Kiceniuk and Jones 1977) and so the outer, compact myocardium has an adequate O2 supply provided coronary flow is not limiting. In contrast, the inner, spongy myocardium, which relies on venous blood, may become hypoxic as venous Po2 is reduced during exercise. As severe hypoxia debilitates cardiac function in teleosts (Turner and Driedzic 1980; Nielsen and Gesser 1984; Farrell et al. 1985), the present study investigated how a decrease in perfusate Po2 affects cardiac performance. As the isolated, saline-perfused trout hearts were electrically paced in the present study, we were particularly interested in how maximum SVH was affected by hypoxia. Increases in fH ultimately compromise cardiac filling time, but whether this occurs at physiological heart rates in fish is not known. As atrial filling is regarded as a relatively long event in fish (Chow and Chan 1975; Johansen and Gesser 1986), we examined whether maximum SVH of the perfused trout heart was compromised at higher pacing frequencies.

FARRELL ET AL.

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Materials and methods The experiments used an electrically paced, isolated heart preparation in which the coronary circulation was pump perfused and the atrium was filled from a constant pressure reservoir (Farrell 1987). The pacing frequency was altered to simulate changes in fH associated with exercise. The 0, tension of the perfusate was reduced to simulate the decrease in venous 0, tension also associated with exercise or environmental hypoxia. Maximum SVH, maximum Q, and maximum myocardial power output were compared for each perturbation. Coronary flow rate and the level of oxygenation of the coronary perfusate were unchanged during the experimental manipulations. Fish Rainbow trout, Salmo gairdneri, were obtained from local suppliers and held at Simon Fraser University in 2000-L tanks supplied with dechlorinated tap water. The fish were exposed to a photoperiod simulating that at 49"N, acclimated to a water temperature of 10°C for at least 2 weeks, and fed commercial trout chow a d libitum. Heart preparation Details of the heart preparation have been presented previously (Farrell 1987). In brief, the heart was removed from the animal and placed in a dish of ice-chilled oxygenated saline for the cannulation procedures. Stainless steel cannulae were secured in the ventral aorta and atrium (at the junction with the sinus venosus). A 0.8-cm long saline-filled, polyethylene cannula (PE 10, Clay Adams) was inserted into the coronary artery via a puncture hole made with either a 25or 23-gauge hypodermic needle. The tip of the cannula lay upstream of any branch point in the coronary artery. Coronary perfusion was started immediately and rapidly cleared the blood from the coronary circulation. Spontaneous beating of the heart cleared blood from the lumen of the heart by drawing saline from the operating dish into the atrium. Preparation time was 5- 15 min. Cardiac petjksion Once cannulated, the isolated heart was immersed in an organ bath filled with mineral oil. A water jacket around the organ bath maintained the heart at 10°C. The atrial input cannula was connected to a saline reservoir, also at 10°C, via a constant pressure head. The input pressure was adjusted to vary SVH. The heart was paced initially between 45 and 50 beatslmin (bpm) via electrical stimulation (Farrell 1987). Therefore, at a given fH, Q was set by adjusting SVH. Under control conditions, Q was set at about 17 mL min-' kg-I BM (BM, body mass) to simulate Q in resting trout (Kiceniuk and Jones 1977). The ventral aortic cannula was connected to a diastolic pressure head of about 45 cm H,O to simulate a physiological output pressure. The coronary circulation was perfused at a constant flow rate with a peristaltic pump (Haake Buchler MCP2500, Saddlebrook, NJ). The initial coronary flow rate of 0.33 mL min-I kg-' BM represented about 2 % of the resting Q in trout at 10°C. The coronary flow drained into the atrium via the coronary veins. The measured Q therefore represented the combined flows from the coronary circulation and the atrial input cannula. Protocols All hearts were perfused initially for 10-20 min under control conditions to allow thermal equilibration. Hearts were discarded if they showed either a poor response to elevated filling pressure, an irregular heart beat, or an unusually high coronary perfusion pressure because of poor cannula placement or air bubbles in the coronary circulation. Assessment of maximum cardiac pelformance The heart develops flow (Q) and pressure (output pressure minus input pressure); both can be increased either independently or simultaneously. Maximum cardiac performance was determined during each experimental perturbation with the following protocol (Farrell et al. 1988). First, filling pressure of the atrium was raised until there was no further increase in SVH. This was termed maximum SV,.

275

The corresponding Q was termed maximum Q as fH was constant. Then, the mean output pressure was raised from 50 to between 70 and 75 cm H,O: This further increased myocardial power output (the product of Q and pressure developed) because the decrease in maximum Q associated with the increase in output pressure was small relative to the increase in pressure development. The term maximum power output was used for the power output associated with elevated Q and output pressure. An assessment of maximum cardiac performance took about 15 min and was performed with each change in perfusate Po, or each change in heart rate. The terms maximum SVH, maximum Q, and maximum power output apply only to the isolated heart preparation as we recognize that it is probably impossible using in vitro preparations to replicate exactly the maximum mechanical performance of the heart in intact trout. Nonetheless, the almost twofold increase in SVH and the 50% increase in output pressure were comparable to the changes observed in swimming trout (Kiceniuk and Jones 1977). Hypoxia The effect of reducing the Po, of the atrial perfusate under conditions of constant coronary flow and Po, was examined in seven preparations. Preliminary experiments indicated that reducing the atrial perfusate Po, from 150 to 80 Torr (1 Torr = 133.322 Pa) had little effect on maximum Q. Therefore, the Po, of the atrial perfusate was reduced in a stepwise fashion from the control Po2 of 70 Torr. Po, was reduced to around 45 and 25 Torr in seven preparations, and to 5 Torr in two of these preparations. Maximum SVH, Q, and power output were established, as described above, after a 10-min equilibration period at each level of hypoxia. Stable levels of Q were obtained with all perturbations, except with extreme hypoxia when the heart could not maintain a stable Q as output pressure was elevated. At that point, further decreases in perfusate Po, were not attempted, but the effect of a two- to three-fold increase in coronary flow was examined in some preparations. Po, determinations were made on duplicate perfusate samples taken from the coronary perfusate, the atrial perfusate, and the perfusate leaving the ventral aorta with the intention of measuring myocardial oxygen consumption. However, as subsequent calculations of the mechanical efficiency of the heart were unusually high and flow dependent, we suspected that there was a significant, but small, problem with oxygen transfer across the flexible Silastic connections to the cannulae. Thus, the Po, measurements provided only an approximation of the oxygen gradient across the heart. Heart rate Whether physiological changes in fH affected maximum SVH was assessed at three pacing frequencies, 30,43, and 58 bpm, in six heart preparations. Petjksate The composition of the basic perfusate was as follows (in g L-'); NaCl, 7.25; KC1, 0.23; CaCl,, 0.22; M g S 0 , . 7 H 2 0 , 0.24; NaH2P04 . H,O, 0.014; Na2HP04, 0.35; NaHCO,, 0.95; dextrose, 1.O; polyvinylpyrrolidone (M, 40 OOO), 10. It had a pH of 7.9 at 10°C. The perfusate delivered to the atrium (atrial perfusate) was maintained at 10°C and gassed with either 0.5% CO; in air (pacing experiments) or 0.5% C 0 2 with a reduced 0, content using Wijstoff gas mixing pumps (hypoxia experiments). The coronary perfusate was contained in a separate water-jacketed reservoir (10°C) and was gassed with 0.25% C 0 , in air. The coronary perfusate was also filtered (8 pm, Nucleopore). Adrenaline was added to the perfusates to a final concentration of 5 nmol L-' to provide tonic cardiac stimulation (Farrell et al. 1988). Instrumentation Coronary input pressure, ventral aortic output pressure, and atrial input pressure were measured via saline-filled tubes connected to Micron pressure transducers (Narco Life Sciences, Houston, TX). The transducers were calibrated before each experiment and were regularly referenced to the fluid level in the organ bath during the

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TABLE 1. The effect of pacing frequency on the performance of isolated perfused trout hearts under resting conditions, increased filling pressure to increase stroke volume, and increased output pressure to further increase power output

Test condition Resting

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Maximum cardiac output Maximum power output

Stroke volume, mL kg-' BM

Heart rate, bpm

Filling pressure, cm H20

Output pressure, cm H20

Coronary pressure, cm H,O

Cardiac output, mL min-' kg-' BM

Power output, mW g-I VM

29.5 (0.2) 42.9 (0.1) 57.7 (0.5) 30.3 (0.9) 43.2 (0.4) 58.3 (0.6) 30.7 (1.1) 43.3 (0.3) 58.0 (0.7) --

- -

NOTE: Mean values (SEM) are presented for five or six fish with a mean body mass (BM) of 1.59 + 0.09 kg and a mean ventricle mass (VM) of 1.49 + 0.08 g. The ventricle was composed of 34.7 +_ 2.3% compact tissue. Values for a given variable within a test condition followed by different letters are significantly different (P < 0.05).

experiment. All pressure measurements were corrected for the cannula resistance. The resistance of the coronary cannula, with the securing thread in place, was measured after each experiment. The pressure signals were suitably amplified and displayed on a chart recorder (Gould, Cleveland, OH). Cardiac output (averaged over 10 consecutive heart beats) was measured gravimetrically with a toploading balance accurate to 0.01 g. Oxygen tension was measured at 10°C with a water-jacketed Radiometer oxygen electrode. Calculations

Pressures were measured in centimetres of H20 (1 cm H20 = 98.1 Pa). Cardiac output (mL min-') was determined from the product of fH and SVH.Myocardial power output (mW) was calculated from [cardiac output (mL min-')I601 x [output pressure filling pressure (cm H20)] x [980 (cm ~ - ~ ) / 1 0 0 0(mW 0 erg-')] (1 erg = J). The blotted wet mass of the ventricle was determined after each experiment. Myocardial power output and coronary flow were initially based on the body mass of the fish, as this value was known before the experiment, but they were subsequently normalized per gram ventricle mass (VM) for presentation of data. Vascular resistance of the coronary circulation was calculated from [coronary input pressure - atrial input pressure (cm H20)]/[coronary flow (mL min- ' g- ' VM)]. However, as coronary flow was constant, changes in coronary vascular resistance were evident from significant changes in the pressure differential for coronary perfusion. Values are presented as means with SEM in parentheses. Student's paired t-test and a repeated measures ANOVA with orthagonal contrasts were used, where appropriate, to determine statistically significant differences (P < 0.05). Each preparation served as its own control which permitted paired statistical analysis. Results Cardiac performance at the three pacing frequencies is presented in Table 1. Table 2 compares the effects of control and hypoxic levels of atrial infusate on-cardiac performance. Under well-aerated conditions, resting Q was set with a filling pressure of less than 1 cm H20. Maximum SVH was approximately twice the resting SVH and was achieved by increasing atrial filling pressure 1 -2 cm H 2 0 (Tables 1 and 2). When output pressure was increased, maximum SVH and maximum Q were reduced by approximately 15%. Nonetheless, the maximum power output was approximately 20 % greater than the power output associated with maximum Q (Table 1). - -

-

-

-

.

Effect of pacing frequency An increase in pacing frequency from 30 to 58 bpm pro-

duced significant increases in maximum Q and power output, but maximum SVH was decreased (Table 1, Fig. -1). However, maximum power output and maximum Q did not increase in a manner equivalent to fH because of the associated decrease in maximum SVH (Fig. 1). For example, when fH was increased by 93% (from 30 to 58 bpm), maximum power output increased only 38%. Similarly, when fH was increased by 43 % (from 30 to 43 bpm), maximum Q increased 34%, and a further increase in fH (from 43 to 58 bpm) had no significant effect on maximum Q. Attempts to increase pacing frequency to 75 bpm were unsuccessful because resting Q could not be maintained. The observation that maximum SVH was compromised by increased fH is consistent with maximum SVHbeing constrained by the associated decrease in the time available for atrial filling. When output pressure was increased to generate maximum output power, there was a significant (P < 0.05) increase in the coronary input pressure (35 -45 %) compared with the two other test conditions (resting and maximum Q; Table 1). This observation indicates that coronary vascular resistance increases when the heart is working against an elevated output pressure. Hypoxia The maximum power output of hearts receiving an atrial Po2 of 67 Torr (Table 2) was comparable to that found for the well-aerated hearts in the pacing experiments (Table 1). Compared with cardiac performance at 67 Torr, stepwise reductions of atrial perfusate Po2, with a constant Po2 of the coronary perfusate, decreased the mechanical performance of the heart (Table 2, Fig. 2). The resting Q was-maintained in all preparations at a Po2 of 46 Torr and resting Q was not significantly different at 25 Torr (Table 2). However, only three out of seven preparations were able to generate the resting Q at 25 Torr. Thus, the threshold for maintaining resting mechanical performance was probably an atrial Po2 near 25 Torr. Only two preparations performed at a Po2 less than 10 Torr; neither could generate the resting Q . Maxirnum Q was compromised at a higher atrial Po2 than resting Q . Maximum Q was not significantly reduced with a Po2 of 46 Torr (Table 2, Fig. 2). However, it was clearly reduced at 25 Tor! and was, in fact, not significantly different from the resting Q under control conditions (Fig. 2).

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TABLE 2. The effect of progressive hypoxia on the performance of isolated perfused trout hearts paced at 45 bprn under resting conditions, increased filling pressure to increase stroke volume, and increased output pressure to further increase power output

Test condition

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Resting Maximum cardiac output Maximum power output

Atrial perfusate Po,, Torr

Filling pressure, cm H,O

Output pressure, cm H,O

Coronary pressure, cm H,O

Cardiac output, Power output, mL min-' kg-' BM mW g-' VM

66.8 (1.4) 46.3 (1.4) 25.6 (2.2) 66.8 (1.4) 46.3 (1.4) 25.6 (2.2) 66.8 (1.4) 46.3 (1.4) 25.6 (2.2)

NOTE: Mean values (+ SEM) are presented for six or seven fish with a mean body mass (BM) of 1.39 0.07 kg and a mean ventricle mass (VM) of 1.03 0.05 g. The ventricle was composed of 46.4 2.4% compact tissue. Values for a given variable within a test condition followed by different letters are significantly different (P < 0.05).

+

+

Maximum power output was compromised at a higher atrial Po2 than maximum Q (Fig. 2). Maximum power output was reduced significantly and in all seven preparations at a Po2 of 46 Torr, unlike maximum Q (Table 2, Fig. 2). This was not surprising as the increase in output pressure that was used to obtain maximum power output increased myocardial oxygen demand without increasing oxygen supply (oxygen supply is set by inflow to the heart, which either was constant or decreased slightly in the present experiments). Thus, although the heart was receiving almost maximal atrial perfusion, oxygen demand outstripped oxygen supply. At a Po2 of 25 Torr, this problem was exacerbated as increasing output pressure from 50 to 75 cm H 2 0 completely stopped outflow in three preparations and reduced maximum Q by between 25 and 75 % in the other four preparations. These data therefore suggest that the threshold for maintaining maximum power output of saline-perfused trout heart was an atrial Po2 between 46 and 67 Torr. The importance of increasing coronary flow to the hypoxic heart was not examined in a thorough manner because the hearts were already severely debilitated by hypoxic perfusion before the procedure was attempted. However, increasing the flow of aerated coronary perfusate increased maximum Q by 2 - 13 mL rnin- kg- BM in three preparations and decreased maximum Q by 0.5 - 1.5 mL min-I kg-' BM in two preparations.

Discussion The present study used saline-perfused hearts to identify the hypoxic threshold of the trout heart and to establish that physiological increases in fH can compromise maximum SVH. Perfused preparations allow a study of cardiac function without evoking homeostatic mechanisms present in intact animals (Perry and Farrell 1988). However, it is important that in vitro conditions reflect the in vivo situation as closely as possible to permit meaningful interpretation of the data. Aftempts were made to do this in the present study. Resting Q and output pressure were similar to those found in resting trout. Pacing frequencies were similar to the range observed with sustained swimming in trout (46 - 54 bprn at 10 - 12"C, Stevens and Randall 1967; 32 -55 bprn at 6.5 "C, Priede 1974; 37.8 51.4 bprn at 10°C, Kiceniuk and Jones 1977). The range for

atrial perfusate Po2 spanned the decrease in venous Po2 from 33 to 16 Torr which was found in exercising trout (Kiceniuk and Jones 1977). Likewise, the assessment of maximum cardiac performance attempted to simulate the twofold increase in resting SVH and the 66% increase in mean output pressure encountered with maximum sustained swimming (Kiceniuk and Jones 1977), when it is presumed that the heart is working maximally (Farrell and Steffensen 1987). All the same, studies with perhsed hearts are not free of limitations (Farrell et al. 1985; Farrell 1987; Perry and Farrell 1988). The discussion focuses, therefore, on whether any of these limitations influenced the present results. The present study found that increasing fH from 30 to 58 bprn did not produce a proportionate increase in maximum Q because maximum SVH was reduced. In addition, attempts to pace the perfused heart faster than 60 bprn were usually unsuccessful because resting Q was reduced. This latter observation is consistent with an earlier finding that there was a maximum (and minimum) frequency for successfully pacing isolated trout hearts and that the range for pacing frequency was a function of temperature (Farrell 1987). A study with in situ perfused trout hearts found that the maximum SVHwas 0.92 mL kg-' at 5°C with fH at 39 bpm, whereas maximum SVH was significantly lower (0.71 mL kg-') at 15"C with a higher fH (68 bpm) (Graham and Farrell 1988). Whether this decrease in maximum SVHreflected an effect of fH on cardiac filling time, a temperature compensation, or both is not clear at this time. While the present study suggests that the minimum time for complete atrial filling of the isolated trout heart lies within the physiological range for fH in trout at 10°C, there are a number of considerations that must be addressed before we accept that this conclusion is also true for intact trout. First, it is important to consider whether the maximum SVH in vitro was comparable to in vivo values and, if it was not, whether the difference affects the extrapolation of the present study to intact trout. The maximum SVHin isolated trout heart was lower than that in either in situ heart preparations or intact trout. Maximum SVH in isolated hearts ranged from 0.63 to 0.82 mL kg- depending on the pacing frequency (present study; Farrell 1987). With in situ perfused hearts, maximum SVH was as high as 0.9 mL kg-' for fH around 50 bprn (Far-

CAN. J . ZOOL. VOL. 67, 1989

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Maximum Power Output, mW g-' VM

35Maximum Cardiac Output, mL min-' kg-' BM

1 1

Maximum Power output, mW g-' VM 2

B

* *

I

30-

I

I/I

Maximum Cardiac 20 Output, mL min-' kg-' BM

0Po2 o f Atrial Perfusate, Torr Relative Change inMaximum '' Stroke Volume, mL kg-' BM-0,2-

-

FIG. 2. The effect of a stepwise decrease in atrial perfusate Po2 on (A) maximum power output and (B) maximum cardiac output of isolated perfused trout hearts. Each point represents a mean value (vertical bar is SEM) for five to seven preparations. Asterisks denote statistically significant differences (P < 0.05) from the control value at a Po2 of 67 Torr; dagger denotes a significant difference between mean values at Po2 levels of 43 and 25 Torr. For reference, the SEM for the resting stippled area represents the mean value cardiac output and power output under control conditions.

+

Pacing Frequency, bpm

FIG. 1. The effect of pacing frequency on (A) maximum power output, (B) maximum cardiac output, and (C) maximum stroke volume of isolated perfused trout hearts. Maximum stroke volume is presented as a relative change from the maximum stroke volume at a pacing frequency of 30 bpm. Each point represents a mean value (vertical bar is SEM) for five or six preparations. Asterisks denote statistically significant differences (P < 0.05) from the mean value at a pacing frequency of 30 bpm.

re11 et al. 1986, 1988). The maximum SVH calculated for swimming trout was 1.0 mL kg-' at fH of 51 bpm (Kiceniuk and Jones 1977). Although maximum SVH for the isolated heart is lower than that in vivo, Farrell (1987) concluded that the absence of sinoatrial valving was the primary problem associated with the reduced maximum Q in isolated trout hearts. At this time there is no evidence to suppose that atrial backflow is dependent on fH. Furthermore, this concern is probably not important because the higher the SVH,the longer the atrial filling time, and the more likely pacing frequency will affect maximum SVH. A second concern relates to the level of adrenergic stimulation of the heart. Tonic levels of adrenaline were used in the present study. However, adrenergic stimulation of the heart probably increases during exercise presumably through adrenergic nerves because circulating catecholamines do not increase appreciably during sustained swimming (Butler 1986). While it is recognized that adrenergic stimulation improves maximum SVHthrough improving inotropy (Farrell et al. 1986), it is not known whether the increase in the speed of myocardial contraction and relaxation associated with adrenergic stimulation leads to an increase in the time available for atrial filling. If this proves to be so, adrenergic stimulation of the heart would be important in offsetting potential decreases in SVH at the same time as it increases fH.

A third concern relates to a possible influence of the pericardium and its important contribution to vis-a-fronte filling of the trout heart (Farrell et al. 1988). The working hypothesis for the present study was that insufficient atrial filling ultimately affects ventricular SVHas ventricle filling in teleosts is determined solely by atrial contraction (Randall 1970; Johansen and Gesser 1986). Measurements of atrial filling time indicate that atrial filling is apparently a relatively long event in teleosts, occupying about 50% of the cardiac cycle in Anguilla japonica (fH ranging from 53.8 to 77 bpm) and about 45 % of the cardiac cycle in an isolated trout heart (fH of 46 bpm; A. P. Farrell, unpublished observations). However, these observations were made either with the pericardium absent or with its integrity disrupted by hypodermic needles (Chow and Chan 1975; Farrell et al. 1988). Consequently, when the pericardium is functionally intact and cardiac filling is assisted by a vis-u-fronte effect in intact trout, it is possible that atrial filling is much faster because it is coupled to a rapid ventricular contraction which lasts only 2 1% of the cardiac cycle in the isolated trout heart (A. P . Farrell , unpublished observations). It follows then that vis-a-fronte filling of the atrium could occur about twice as fast as vis-a-tergo filling. The present experiments used vis-a-tergo filling and, therefore, it is possible that atrial filling is not compromised at a high fH in intact trout because atrial filling is faster with vis-a-fronte filling. However, the suggestion that atrial filling is faster with vis-a-fronte filling must be verified experimentally. In summary, a significant decrease in maximum SVHof the isolated perfused trout heart was associated with an increase in pacing frequency similar to the fH observed in intact trout. Whether this decrease in maximum SVH can be prevented in vivo by adrenergic stimulation and (or) vis-a-fronte filling has to be investigated. It is also important to note that there

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may be advantages to increasing fH, other than changes in Q, such as improved synchrony between gill perfusion and ventilation across the gill lamellae (Randall 1982). The effect of hypoxia on the resting mechanical performance of a perfused fish heart was examined previously in the sea raven (Hernitripterus arnericanus). The sea raven lacks a coronary circulation and therefore relies entirely on venous blood for its myocardial O2 supply (Farrell et al. 1985). The present study is therefore the first to examine the effect of hypoxia on a perfused teleost heart with a coronary circulation and at elevated levels of cardiac performance. The threshold for maintaining resting Q in the sea raven, and atrial Po2 between 60 and 80 Torr, was higher than the 25-46 Torr range found for the trout. This means that either the trout heart is more tolerant to hypoxia and (or), more likely, that the coronary circulation is effective in ensuring myocardial oxygen supply when venous Po2 is reduced through either exercise or environmental hypoxia. These in vitro observations are consistent with those made on intact fish exposed to environmental hypoxia. Intact trout maintain Q at an environmental Po2 of 40 Torr (Holeton and Randall 1967a, 1967b) whereas the lingcod (Ophiodon elongatus), a teleost fish that lacks a coronary circulation, reduces Q at a similar level of environmental hypoxia (Farrell 1982). There is limited information on cardiac performance in intact fish when oxygen delivery to the heart is compromised by reducing the blood Po2. At the maximum sustained swimming speed when venous Po2 is reduced from 33 to 16 Torr, trout can more than double SVH, increase fH by 36%, and increase ventral aortic blood pressure by 66% (Kiceniuk and Jones 1977). During environmental hypoxia when the venous Po2 is 20 Torr and the arterial Po2 is 40 Torr, trout maintain Q with a marked bradycardia (Holeton and Randall 1967a, 1967b). Thus, even with this limited information it is clear that the heart of an intact trout tolerates a greater reduction in Po2 than a saline-perfused heart. There are several reasons for this difference. First, a distinction must be made between the importance of oxygen tension versus oxygen content in terms of oxygen delivery to the heart. While oxygen tension determines the driving pressure for diffusion, oxygen tension can be reduced significantly if myocardial oxygen consumption is relatively high compared with oxygen content. This is true for hearts perfused with saline, but not for venous blood (Farrell et al. 1985). Thus, Farrell et al. (1985) suggested that the output rather than the input Po2 is probably a better index of the threshold Po2 for saline-perfused hearts. In the present study, the Po2 of the output perfusate was 5-20 Torr lower than input Po2. Second, facilitated diffusion by hemoglobin may be important in extending the hypoxic threshold of the heart. Third, the bradycardia associated with environmental hypoxia increases the blood residence time in the heart and lowers the diastolic pressure in the ventral aorta (Holeton and Randall 1967a, 1967b; Farrell 1982). It would seem that any reduction in the diastolic pressure in intact fish is advantageous in maintaining resting Q and consequently oxygen delivery to the heart, given the present observation that increasing output pressure could stop cardiac output in the hypoxic heart. Lastly, it seems likely that the major reason why the hypoxic threshold was higher in vitro than in vivo was that oxygen delivery by the coronary circulation was inadequate in the peffused heart. We used a coronary flow of 2% of the resting Q, which is within the range estimated for coronary flow in intact fish (0.5 -2.9 % ; Cameron 1975; Farrell 1987). This flow rate

would supply 5.7 pL O2 min-I to the compact tissue, assuming 40 % compact myocardium, an oxygen solubility coefficient of 0.0447 mL O2 L- Torr- (Graham 1986), and fully aerated perfusate. Given that the trout heart consumes 360 pL O2 min-l g-I VM (Farrell and Milligan 1986), the requirement of the 40% compact tissue (142 pL O2 min-l) greatly exceeds that delivered by the coronary perfusate. It is likely, therefore, that the Po2 gradient from atrial perfusate, being greater than that from venous blood in intact trout, enabled a compensatory oxygen delivery from the lumen to the compact myocardium under control conditions. Clearly, this could not be maintained during hypoxia when the Po2 of the atrial infusate was reduced. This conclusion is supported by the observation that when coronary flow rate was increased during hypoxic perfusion there was an improvement or stabilization of resting Q. Previously, it was demonstrated that perfused trout hearts generated a better power output with coronary flow than without coronary flow (Houlihan et al. 1988). The practical implication of this conclusion is that to simulate in vivo conditions as closely as possible, the coronary circulation should be perfused with blood because even oxygenated saline cannot adequately supply the O2 requirements of the compact myocardium. In conclusion, it was demonstrated that physiological increases in fH and a decrease in Po2 reduced maximum performance of the saline-perfused trout heart. By critically examining the limitations of the isolated heart preparation it was possible to elucidate the possible importance of these observations for cardiac control in intact trout.

Acknowledgements This study was supported by the Natural Sciences and Engineering Research Council of Canada through an operating grant to A. P. Farrell and a postdoctoral fellowship to M. S. Graham. We thank Jeff Johansen for his help with the data analysis. AXELSSON, M., and N~LSSON, S. 1986. Blood pressure control during exercise in the Atlantic cod, Gadus morhua. J. Exp. Biol. 126: 225 -236.

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