Cardiovascular responses to hypoxia in the Adriatic sturgeon (Acipenser naccarii)

J. Appl. Ichthyol. 15 (1999), 67-72 0 1999 Blackwell Wissenschafts-Verlag, Berlin ISSN 0175-8659

Cardiovascular responses to hypoxia in the Adriatic sturgeon (Acipenser naccario By C. Agnisola’, D.J. McKenzie’, D. Pellegrino3, P. Bronzi4, B.Tota3 and E.W. Taylo? ’Dipartimento di Fisiologia Generale e Ambientale, Universit&di Napoli “Federico II”, Napoli, Italy; 2SchooI of Biological Sciences, University of Birmingham, Birmingham, UK; ’Dipartimento di Biologia Cellulare, Universit; della Calabria, Arcavacata di Rende (Cs). Italy; ‘ENEL, SRI-CRAM Cologno Monzese (Mr), Italy.

Summary The in vivo cardiovascular responses to hypoxia, and the intrinsic functional characteristics of the heart in vitro, were determined, and compared, in the Adriatic sturgeon (Acipenser nnccarii). During exposure to hypoxia in vivo, blood oxygen content (Cao,) declined as water 0, partial pressure (Pwo,) was reduced, despite an increase in haematocrit. The main cardiovascular response was a reduction in dorsal aortic blood pressure, with a slight bradycardia, while cardiac output remained constant. Reduced oxygen content of the perfusate had significant inhibitory effects on the intrinsic performance of the heart in vitro, causing a reduction in the heart rate; a reduction in the sensitivity of responses to increased preload (Frank-Starling response), and a more rapid decline in power output and stroke volume when afterload was increased. Overall, the in vitro results suggest that hypoxia depresses the contractility of the heart (i.e. its inotropic responses). The reduction in dorsal aortic pressure in vivo may, therefore, counteract the depressive effects of hypoxia on heart contractility, and thereby avoid a hypoxic depression of cardiac output. Introduction As sturgeon live in freshwater, marine and estuarine environments, and often migrate between them, they may experience a wide range of environmental variation, including low oxygen levels, or hypoxia. Teleost fish usually exhibit homeostatic regulation of oxygen uptake during exposure to hypoxia, maintaining uptake relatively independent of ambient oxygen levels. This occurs through reflex changes in ventilatory activity and/or cardiovascular function, which combine to increase the effectiveness of branchial gaseous exchange (Hughes and Shelton 1962). Little is known about how hypoxia influences the cardiovascular and respiratory physiology of sturgeon, which, as members of the Chondrostei, are an ancient and unique group of fish. In teleost fish the typical cardiovascular response to hypoxia includes a bradycardia and hypertension, with an increase of both ventral and dorsal aorta pressures, an increase in peripheral vascular resistance in the systemic circulation and a constancy or a reduction in resistance in the branchial circulation (Fritsche and Nilsson 1993). On the other hand, a small hypotension has been described in elasmobranchs (Piiper et al. 1970; Butler and Taylor 1971). In teleosts, cardiac output often remains constant due to compensatory changes in stroke volume, according to the FrankStarling response (Fritsche and Nilsson 1993). Similar responses have been described in an agnathan, the hagfish, Myxine glutinosa (Axelsson et al. 1990) and in an elasmobranch, the dogfish ScyIiorhinus canicula (Butler and Taylor 1971; Taylor et al. 1977; Short et al. 1979).

Apart from the direct effects of hypoxia on the vasculature and the heart, this response is mediated in teleosts and elasmobranchs by reflexes triggered by branchial and extrabranchial oxygen receptors, and involves both the autonomic nervous system and catecholamine release from chromaffin tissue (Randall and Taylor 1989). The reflex responses of the Adriatic sturgeon, Acipenser naccari, to stimulation of peripheral chemoreceptors were described by McKenzie et al. (1 995a). These reflexes were similar to those described in teleosts (Burleson et al. 1992), and included a bradycardia and hyperventilation. Acipenser naccarii was apparently unique in that it possessed no inhibitory cardiac vagal tone in normoxia, a fact that might influence cardio-circulatory responses and regulation of blood flow in hypoxia. A study by Burggren and Randall (1978) reported that the white sturgeon (Acipenser transrnontanus) exhibited an unusual metabolic and ventilatory response to hypoxia, behaving as an “oxygen conformer” and exhibiting a progressive reduction in rates of oxygen uptake and gill ventilation as water oxygen partial pressure (Pwo,) was reduced. A similar hypoxic depression of oxygen uptake has been described in the Adriatic sturgeon (McKenzie et al. 1995b). The potential role of the cardiovascular system in these responses to hypoxia is as yet unclear. The present paper describes the cardio-circulatory responses to hypoxia in the Adriatic sturgeon in vivo, and relates these to the intrinsic characteristics of the heart as determined in vitro. Materials and Methods Animals Immature Adriatic sturgeon (Acipenser naccarii Bonaparte) of 1.5 years of age, either sex, and with a mean live mass of 1.6 f 0.2 kg were maintained at the Experimental Thermal Aquaculture Plant “La Casella” (Sannato, PC, Italy) in indoor 4 mzfibreglass tanks with a continuous water supply (23 rt 1”C, pH 7.9, hardness 125 mg I-’ as CaCO,), and fed a diet of pelletted commercial sturgeon feed (Alma Storioni Prima Fase, Agros, Bolzano, Italy). An appropriate number of animals (specified below) were randomly selected as experimental subjects. All in vivo and in vitro experiments were performed at La Casella. Surgical procedures for the in vivo study Sturgeon were anaesthetised in a buffered solution of tricaine methane sulphonate (MS222, 1:lOOOO w/v in water) and transferred to a surgical table where they were artificially ventilated with a MS222 solution at 1:20000 w/v. Two different surgical procedures were used for the in vivo study, one for measuring arterial pressures, heart rate, blood 0, status and haematocrit, the other for measuring in vivo cardiac output. In the first case, a saline-filled cannula (PE 50 Intramedic) was

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implanted into the dorsal aorta, via the roof of the mouth and secured with a cuff and sutures (Randall et al. 1992). The cannula was flushed twice a day with heparinised (50 IU 1.') sturgeon saline (see below), and was connected to a pressure transducer for measurement of heart rate (beats m i d ) and dorsal aortic systolic and diastolic pressures (kPa). For measurement of cardiac output (plus associated heart rate), the ventral aorta was exposed from the side of the isthmus and a flow probe (Crystal Biotech VF-I Pulsed Doppler) was secured around the vessel. The probe lead was tunnelled caudally and firmly secured by skin sutures. Pressure and flow signals were displayed and recorded on a chart recorder (Gould Windograf). Following surgery, fish were transferred to individual darkened plexiglass chambers (volume 40 1) and allowed to recover for a minimum of 24 h in a continuous flow of water, aerated by passage through a gas-exchange column countercurrent to a stream of air.

records. Flow was determined volumetrically (Houlihan et al. 1988) and cardiac output expressed as ml m i d k g l . Power output (PO) was expressed as mW g'l of ventricle and was calculated as (afterload - preload) o cardiac output/60, where pressures were expressed as kPa and cardiac output transformed in ml min-' g-'. Afterload was defined as the mean output pressure and preload as the mean diastolic intra-atrial pressure. Protocols Hypoxic exposure in vivo.

Measurements of the effects of hypoxia on cardioventilatory and blood-gas variables were made with a protocol involving 30 min. stepwise exposures to 3 progressive levels of hypoxia. For the measurements of the effects on blood pressures, heart rate and blood variables, normoxic resting cardioventilatory variables were recorded and a 1 ml blood sample was collected for measurement of control, normoxic Pao,, Cao, and haematocrit (blood was replaced with an equal volume of saline). Water Po, was then reduced through two levels of hypoxia, 6.6 f 0.2 kPa Measurement of blood variables Under the conditions defined in the experimental protocols (see (moderate hypoxia), and 4.6 f 0.2 kPa (deep hypoxia). Hypoxia below), blood samples were withdrawn from the dorsal aortic was created by passing water counter-current to a flow of 100% catheter, and the collected blood was replaced with an equal N, through a gas-exchange column. At the end of the exposure volume of saline. Arterial blood oxygen partial pressure (Pao,) periods in moderate and deep hypoxia measurements of steadywas measured with a Radiometer oxygen electrode, thermostatted state heart rates and blood pressures were made and a blood to the same water temperature as the fish and attached to a sample was taken for measurement of the above mentioned blood Radiometer PHM73 acid-base analyser. Arterial blood total variables. Measurements of cardiac output (plus heart rate) were oxygen content (Cao,) was measured with the technique performed on a separate group of animals (N=5, mean weight described by Tucker (1967) using an Instrumentation 0.99 f 0.13 kg), following exactly the same protocol of stepwise Laboratories oxygen electrode and IL 1302 blood-gas analyser exposure to hypoxia. In vitro cardiacperformance. thermostatted to 37OC. Portions of whole blood were immediately centrifuged upon A group of 5 animals (mean weight 1.18*0.13 kg) was used to sampling and plasma decanted and weighed. The red cell pellet test the effect of hypoxia on the intrinsic properties of the heart, was also weighed and haematocrit was calculated from the employing an in vitro isolated working preparation. The weights of the liquid and cellular portions of the sample, using following criteria were used to set the basal perfusion conditions: previously determined measurements of density of each fraction the diastolic output pressure was set to 2.0 kPa, while preload was adjusted to obtain a stroke volume of about 0.2 ml kg". to calculate their relative volumes (McKenzie et al. 1997). Basal performance was set with reference to stroke volume rather Isolated heart preparation Each animal was anaesthetised with 0.2 g/l MS-222 and 1 ml/kg then cardiac output, because in vitro heart rate is lower than in heparin (80 IU/ml) injected into the caudal arteryhein, then vivo (Agnisola et al. 1996). After stabilisation under basal killed by a sharp blow to the head and pithed, and the heart was conditions, the isolated hearts were challenged with increasing dissected and cannulated according to Houlihan et al. (1988). A preloads to induce a maximal in vitro Frank-Starling response double cannula was set in the atrium to measure intra-atrial and power output. Preload was increased through three to four pressure. As there are valves at the end of the bulbus cordis (the steps to increase cardiac output and then PO, according to the most rostra1 contractile chamber of the heart, at the entrance to Frank-Starling relationship, up to a plateau for power output with the ventral aorta), the isolated preparation included a 7-10 mm. maximal volume loading (PO,,). Subsequently, output pressure length of the ventral aorta, and care was taken to ensure that was increased through the physiological range as far as possible cannulation of this vessel did not obstruct these valves. Oxygen without affecting cardiac output to induce a homeometric supply to the myocardium was sustained by perfusing the hearts response (defined as the heart's intrinsic ability to maintain with oxygenated saline (Davie et a]. 1992). A perfusion chamber resting stroke volume over a range for aortic output pressure, that allowed subambient extracardiac pressure to be developed Farrell and Jones 1992) under conditions of maximal volume during the cardiac cycle was used according to Acierno et al. loading. The maximum PO measured under these conditions was (1990). The perfusion temperature was 20°C. A modification of equivalent to the in vitro power output of the heart under Cortland's saline was prepared, with the following composition conditions of maximal volume and pressure loading (POvL,pL). The total perfusion time was 50 min. As the heart rate appeared (in mmol/l): 130 NaCI, 5.09 KCI, 0.1 NaH,PO,, 2.5 Na,HPO,, 1.14 MgCI,, and 2 CaCI,. Glucose was added (1 g 1"). Total to be independent of both preload and afterload (see below), the enabled evaluation of in vitro cardiac osmolarity of this saline was similar to that of sturgeon plasma values of PO,, and PO,,,,, (285 osM; Cataldi et al. 1995). pH was adjusted to 7.8 with scope (as given by the ratio between maximal and basal values) NaHCO, (about 1 g I-') to agree with direct measurements of in sturgeon under these specified conditions. This volume plus blood pH from chronically cannulated animals under normoxic pressure loading protocol was repeated twice on the same heart, conditions (Randall et al. 1992). The saline was gassed with using two different levels of oxygen content in the saline. As 99.5% 0,-0.5% CO, (0, tension, Po, = 65.1 kPa) or with 99.5% previously described by Agnisola et al. (1996) control perfusion air-0.5% CO, (Po, = 19.8 kPa). Intra-atrial pressure and aortic was with oxygenated saline (0, content = 23 ml r', n = 5), to pressure were measured with Elcomatic pressure transducers compensate for the reduced oxygen content of saline relative to content = 7 ml r', n = 5 ) was connected to a chart recorder [Unirecord, UGO BASILE, blood, while aerated saline (0, Comerio, Italy]. Heart rate was determined from the pressure considered to deliver a hypoxic stimulus to the heart.

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Data analysis Two-way ANOVA or repeated measure ANOVA, with StudentNewman-Keuls as post hoc test, or paired Student’s t-test were used to compare data as appropriate. Apparent differences between mean values were assigned significance at a confidence level of 95% (P< 0.05). 3.5

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The effects of hypoxia on cardiovascular function in sturgeon are described in Figure 1.Moderate hypoxia (6.6 kPa) induced a slight but significant bradycardia, associated with a significant reduction in dorsal aortic pressure, which was accounted for by a reduction of both unchanged by exposure to deep hypoxia. Measurement of blood flow revealed that the relatively slight hypoxic bradycardia was not accompanied by a reduction in cardiac output (Figure 2), systolic and diastolic pressures (Figure 1). These effects were indicating that a compensatory increase in stroke volume had occurred. Thus, the main circulatory response to hypoxia in sturgeon seems to be a reduction in blood pressure and vascular resistance, accompanied by a slight bradycardia.

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Fig. 1: Effect of hypoxia on in vivo cardiovascular functional parameters in sturgeon. fH (beats min -I) = heart rate; PDA(kPa) = dorsal aorta pressure (squares = systolic; circles = diastolic). fi, values: normoxia, 18.5 kPa; moderate hypoxia, 6.6 kPa; deep hypoxia, 4.6 kPa. Data are means (iSE) of 6 determinations. *significantdifference from the normoxic value (repeated measures ANOVA, P