Aortic blood flow and cardiac output in the hemoglobin-free fish Chaenocephalus aceratus

Cmtp. Bioch.

Physiot., 1972, Vol. 43A. pp. 1045 to 1051. Pwgaman Press. Printedin Great &it&a

AORTIC BLOOD FLOW AND CARDIAC OUTPUT IN THE HEMOGLOBIN-FREE FISH CliAENOCEPHALUS ACERATUS EDVARD A. HEMMINGSENr, EVERETT L. DOUGLAS2, KJELL JOHANSENa and RONALD W. MILLARD3 lPhysiologica1 Research Laboratory, Scripps Institution of Oceanography, La Jolla, California 92037; *Division of Biological Sciences, University of Missouri, Columbia, Missouri 65201; and 8Department of Zoology and Center for Bioengineering, University of Washington, Seattle, Washington 98105 (Received 12 Febrtmy

1972)

Abstract-l. The cardiac output of C%ae~~ocephalus aceratus, determined by the Fick principle, ranged from 99 to 153 ml/kg per min in unrestrained specimens at rest. These values are several-fold higher than those of other fishes. 2. The combined energy cost for cardiac and respiratory work at rest was estimated to be nearly half, or more, of the total oxygen consumption. 3. The ventral aortic blood flow, measured by electromagnetic flowmeter, increased during hypoxia and decreased during hyperoxia. The cardiac output was regulated by changes in the stroke volume and not heart rate. 4. The ventral and/or dorsal aortic blood pressures were monitored simultaneously with the blood flow. 5. Infusions of cardio- and vasoactive drugs revealed typical responses. INTRODUCTION THE ANTARCTIC

“icefish” of the family Chaenichthyidae have a surprisingly effective oxygen uptake and transport system in spite of the lack of hemoglobin in their blood. The main compensations for the absence of hemoglobin are increases in blood volume and in the rate of blood circulation, coupled with a lowered oxygen demand (Hemmingsen & Douglas, 1970, 1972; Holeton, 1970). Recently we had an opportunity (Hemmingsen, 1971) to examine further some cardiovascular functions in these specialized fish. Working with unrestrained specimens of C~~~~aZ~ aceratuf, we were able to obtain direct measurements of ventral aortic blood flow with simultaneous measurements of ventral and/or dorsal aortic blood pressures during normal resting conditions, during hypoxia and hyperoxia and during infusion of cardio- and vasoactive drugs, Also, the cardiac output was obtained by the Fick principle. MATERIALS

AND METHODS

Specimens of C. ucerutus were caught on hand lines at Anvers Island, Antarctic Peninsula. Storage and handling of the fish and some of the experimental procedures undertaken have been described in the preceding article (Hemmingsen & Douglas, 1972). Animals used in the present experiments ranged in weight from 1457 to 2027 g. Polyethylene catheters (PE 60 or 90) were inserted into the dorsal and/or ventral aorta and into

1045

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EDVARDA. HEMMING~ENet al.

the caudal vein through hypodermic needles. The fish were tranquilized or anaesthetized with MS 222 during the cannulating procedures. The catheters allowed continuous blood pressure recording and blood sampling for the determination of PO,. In two specimens, electromagnetic flow probes (Micron Probe size 25-34 mm) were placed around the ventral aorta between the second and third pair of afferent branchiaf arteries. The ventral aorta was reached for probe placement through a small incision in the buccal floor. It was not possible to place the probe closer to the heart without damaging the pericardium. The probe was secured in a position which did not impede or obstruct the blood flow; the leads were passed out through the incision and they did not interfere with normal breathing movements. In these specimens, the ventral aortic catheter was placed in an afferent bran&al artery adjacent to the flow probe. During the recordings and blood samplings, the fish, with their implanted catheters and flow probes, were unrestrained and at rest in water between 1 and 2°C. UsualIy no recordings were used until at least 6 hr had been allowed for recovery of the fish from anaesthesia. In some cases, a specimen was used over a period of 2-3 days before it was terminated. The blood pressures were measured with Statham pressure transducers (Type P23BB) and Brush Mark 220 recorders and smplifiers. The blood flow was monitored with a Micron RC 1000 electromagnetic flowmeter (Micron Instruments, Inc., Los Angeles, California) and Brush recorders. Hypoxic and hyperoxic conditions were established by bubbling nitrogen or oxygen through the water. Normal conditions were obtained with the fish resting in a tank with running sea water. Two sequential injections through the catheter in the caudal vein were made for each drug. The heart rate, blood flow and blood pressures were allowed to return to pre-injection values before administration of a diierent drug.

RESULTS

Figure l(a) shows an example of the simultaneously recorded ventral aortic blood flow and the ventral and dorsal aortic blood pressures. Figure (lb) shows the changes in blood flow in one fish which was sequenti~y exposed to normal, hyperoxic and hypoxic water. In all of these situations, a positive blood flow prevailed during the entire cardiac cycle and it never dropped to zero. One specimen (1457 g), kept in normally aerated water, had a mean resting flow, measured at intervals during a 30-hr period, which ranged from around 11 to 15 ml/kg per min; the pulse flow was 10 to 14 ml/kg per min. The flow rates obtained in a second specimen (1750 g) were somewhat lower. The m~mum values for mean flow and pulse flow were 9 and 10 ml/kg per min, respectively. However, in this latter case, the experiment had to be terminated before complete recovery from anaesthesia had occurred. It was noted that the MS 222 greatly reduced the flow rates. On one occasion a brief period of activity in one specimen in normally aerated water resulted in a flow increase of 45 per cent above the resting level while the heart rate remained unchanged. During exposure to hypoxic water [Fig. (lb)] (pOa, 50-70 torr), both the mean flow and the pulse flow increased gradually by as much as 50 per cent above the values obtained under normal conditions, The heart rate, which remained at 17-18 beats/min, did not change significantly. Overall there was little change in the blood pressures. The most pronounced pressure change occurred in the dorsal

AORTIC BLOOD FLOW AND CARDIAC OUTPUT

IN HEMOGLOBIN-FREE

FISH

1047

FIG. l(a). Simultaneously recorded ventral and dorsal aortic blood pressures and ventral aortic blood flow in C. acerutus. (b). Ventral aortic flow during hyperoxic and hypoxic conditions.

aorta, where the pressure dropped from the normal value of about 17/12 mm Hg (systole/diastole) to 14/10 mm Hg. A 30 per cent decrease in the blood flow and a slight increase in heart rate were observed in specimens exposed to hyperoxic water (PO,, 300-400 torr). In two experiments where only the dorsal aortic blood pressures were monitored during the hyperoxia, the dorsal aortic pressure increased from 15/10 to 18/10 mm Hg, and from 11/7 to 17/9 mm Hg, respectively. In both the hyperoxic and hypoxic situations, the caudal venous pressure remained unchanged at about 1.5 mm Hg or less systolic value. Injection of 1 pg acetylcholine promptly increased the peak flow by 38 per cent and the stroke volume (i.e. pulse flow) by 24 per cent. The heart rate decreased about 20 per cent. The dorsal aortic pressure dropped to half the pre-injection level. A second injection of O-2 pg acetylcholine (Fig. 2) resulted in a more pronounced bradycardia, but had little further effect on the dorsal aortic pressure. The acetylcholine caused the diastole flow level to reach zero. This was the only case where the blood flow was not continuous during the cardiac cycle. Injection of 1 pg epinephrine restored the heart rate and aortic pressure to essentially preinjection levels. Injection of 500 pg atropine caused a slight increase in the heart rate, from 16 to 18 beats/min, with no clear change in blood flow. The dorsal aortic pressure dropped by 12 per cent. A second injection of 500 pg atropine gave a barely perceptible increase in flow and a slight further decrease in dorsal aortic pressure. Several control injections of saline gave no detectable changes in flow, pressure or heart rate.

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2. Responses in ventral aortic blood flow and dorsal aortic pressures of C. aceratur to intravenous infusions of acetylcholine and atropine.

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Representative values of blood oxygen tensions, blood pressures and cardiac output are presented in Table 1. Cardiac output was estimated from ventral and dorsal aortic oxygen tensions and oxygen consumption data according to the Fick TARLIZ &-BLOOD (~,a.)

PRIBSURR AND OXYGEN TENSION ($03

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IN FOUR SPECMRNS OF

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Blood pressure* (mm Hg) Fish

IN THR VRNTRAL AORTA

(d.a.) MXI THII CARDIACOUTPUT

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D.a.

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D.a. 79 10.5 106 80

Cardiac output (ml OS/kg per min) 153 loo 134

* Syatolejdiastole. $ Blood flow data from this specimen are shown in Figs. 1 and 2.

AORTIC

BLOOD

FLOW

AND

CARDIAC

OUTPUT

IN

HEMOGLOBIN-FRRE

FISH

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principle. Resting 0, uptake was set at a value of 0.020 ml 0,/g per hr (Hemmingsen & Douglas, 1970) and a coefficient of 0.032 ml O,/ml blood per atm was used for the oxygen solubility in the blood (Ruud, 1954). The mean value obtained for the cardiac output was 119 ml/kg per min at the prevailing heart rates of 18 beats/min, corresponding to a mean stroke volume of 6.6 ml/kg. DISCUSSION

Although parts of the present study must be regarded as preliminary, some aspects of the cardiovascular functions in C. aceratus differ to a very striking degree from those in other fish. Thus, the present ventral aortic flow rates are very high for fish, particularly since blood flow through the last two branchial pairs is not inBecause of the anterior placement of the flow transcluded in the measurements. ducer on the ventral aorta, the aortic flow measurements do not allow a direct assessment of total cardiac output; however, if we assume that the relative distribution of flow in the branchial vessels remains constant, the observed changes in aortic flow may be used as an indication of relative changes in cardiac output. The values obtained for cardiac output in C. acerattls using the Fick principle ranged from 99 to 152 ml/kg per min. The mean value of 119 ml/kg per min is significantly higher than that obtained by Holeton (1970) even though the two ranges of values overlap. Although some uncertainties are inherent in these evaluations due to cutaneous respiration (Hemmingsen & Douglas, 1970) and to possible variations in oxygen consumption, there is a striking several-fold increase in the cardiac output as compared to the common values for hemoglobin-containing fish which have been compiled by Garey (1970) and Satchel1 (1971). The stroke volume in C. ace~atus is elevated to the same proportion as is the total cardiac output relative to hemoglobin-containing fish. In view of the very low oxygen capacity of the blood and the compensatory high cardiac output, the continuous blood flow during the entire cardiac cycle may be of specific importance for keeping a maximum volume of blood in contact with the branchial gas exchange surfaces for the minimum time necessary for oxygen saturation. A large compliance of the prebranchial vessels would dampen the effect of the beats. Based on visual inspection during implantation of the flow probes, the compliance of the cardiac outflow tract including the bulbus arteriosus as well as the ventral aorta and afferent branchial arteries appeared to be unusually large. Pressure-volume studies and assessment of e&&r/collagen ratios in the central arteries would prove most interesting in comparison with similar data recently published for other fish (Satchell, 1971). The energy cost of the cardiac work performed is modest in fish in general. In carp, for instance, it has been estimated to be about 5 per cent of the resting metabolic rate (Garey, 1970). The cardiac work in C. ucerutzls can be estimated from the ventral aortic blood pressures and the cardiac output by using assumptions similar to those employed by Garey (1970). The work is equal to (26 cm) X (124 g/kg per min) x l/0*2, using a blood density of 1.04 (Hemmingsen & Douglas, 1970) and a cardiac muscle efficiency of 20 per cent. This work equals 16,120

1050

EDVAFID A. HEMMINGSEN et al.

gem/kg per min, or about 27 per cent of the total energy production of the animal, which would be about 60,000 gem/kg per min for an oxygen consumption of 0.020 ml/g per hr (or 0.33 ml/kg per min) and an all fat metabolism. Thus, in spite of the low blood viscosity (Hemmingsen & Douglas, 1972) and the low blood pressure in C. aceratus, the energy cost of the high cardiac output is very substantial and much higher than in other fish. If the energy cost of normal gill ventilation which has been estimated to be up to 15 per cent (Cameron & Cech, 1970; Edwards, 1971) or even more than 30 per cent (Schumann & Piiper, 1966) of the total oxygen consumption, is added to the cardiac work, the energy cost of gas exchange and transport could easily be 50 per cent in resting C. aceratus. The value could be considerably higher during activity and hypoxic stress, when both the ventilation and the cardiac output increase. It appears that the regulation of cardiac output in C. aceratus takes place by changes in stroke aohme rather than heart rate as indicated by the substantial changes in aortic flow rates and negligible changes in heart rates during both the hyperoxia and hypoxia. This conforms to the compensatory mechanism observed or suggested for fish and many other vertebrates (Shannon & Wiggers, 1939; Johansen, 1962, 1971; Stevens & Randall, 1967a, b; Randall, 1970), but contrasts to the case in mammals where the cardiac output normally is regulated by changes in the heart rate. The results of the pharmacological experiments lend further support to the view that the cardiac output in the chaenichthyids cannot be raised very much by an increase in heart rate. Thus, the rate observed in resting specimens may be the Release of vagal inhibition to the maximum one possible at a given temperature. heart with atropine produced only a slight increase in heart rate, a strong indication that vagal tone is very low in C. aceratus, even at rest. In other fish, acceleration of heart rate by atropine is common (Johansen, 1971). The responses to epinephrine and acetylcholine are more complex and more Among other effects, these agents are reported to have difficult to interpret. branchial dilatory and constrictive effects, respectively (ostlund & FPnge, 1962; Johansen, 1971). The sudden drop in dorsal aortic pressure after injection of acetylcholine into C. aceratus is in accordance with such a response, as also seen in other fish. The changes in dorsal aortic pressures in C. aceratus during hypoxia and hyperoxia are similar to those which have been observed in some elasmobranchs (Satchell, 1962, 1971; Butler & Taylor, 1971). However, the changes differ somewhat from those which prevail, for instance, in trout where aortic pressure has been found to increase during hypoxia (Holeton & Randall, 1967). It is possible that these differences in responses are dependent on the rate of decrease of the oxygen tension in the water during hypoxic stress. When further data on transbranchial pressure gradients and selective branchial and vascular bed resistance become available, the comparative aspects can be discussed in more detail. Acknowledgements-The research grants GV-25401

main support for the present investigation was provided by and GB-24816

from the National

Science Foundation.

The

AORTICBLOODFLOW ANDCARDIAC OUTPUTIN HEMOGLOBIN-FREE FISH

1051

work was carried out at Palmer Station, Antarctica, and on board the R/V Alpha Helix during February 1971. The excellent support of the vessel’s crew and the station’s personnel is greatly appreciated. REFERENCES BUTLER P. J. & TAYLOR E. W. (1971) Response of the dogfish (Scyliorhinus canicula L.) to slowly induced and rapidly induced hypoxia. Camp. Biochem. Physiol. 39A, 307-323. CAMERONJ. N. & CECH J. J. (1970) Notes on the energy cost of gill ventilation in teleosts. Comp. Biochem. Physiol. 34, 447-455. EDWARDSR. R. C. (1971) An assessment of the energy cost of gill ventilation in the plaice (Pleuronectes platessa L.). COT@. Biochem. Physiol. MA, 391-398. GAREYW. (1970) Cardiac output of the carp (Cyprinus carpio). Comp. Biochem. Physiol. 33, 181-189. HEMMINGSENE. A. (1971) The R/V Alpha Helix Antarctic expedition, 1971. Bioscience 21, 869-870. HEMMINGSENE. A. & DOUGLASE. L. (1970) Respiratory characteristics of the hemoglobinfree fish Chaenocephalus aceratus. Camp. Biochem. Physiol. 33, 733-744. HEMMINGSENE. A. & DOUGLASE. L. (1972) Respiratory and circulatory responses in a hemoglobin-free fish, Chaenocephalus aceratus, to changes in temperature and oxygen tension. Comp. Biochem. Physiol. 43A, 1031-1043. HOLETONG. F. (1970) Oxygen uptake and circulation by a hemoglobinless antarctic fish (Chaenocephalus aceratus Lonnberg) compared with three red-blooded antarctic fish. Comp. Biochem. Physiol. 34, 457-471. HOLETONG. F. & RANDALLD. J. (1967) The effect of hypoxia upon the partial pressure of gases in the blood and water afferent and efferent to the gills of rainbow trout. J. exp. Biol. 46, 317-327. JOHANSENK. (1962) Cardiac output and pulsatile aortic flow in the teleost, Gadus morhua. Comp. Biochem. Physiol. 7, 169-174. JOHANSENK. (1971) Comparative physiology: gas exchange and circulation in fishes. Ann. Rev. Physiol. 33, 569-612. JOHANSFNK., FRANKLIN D. L. & VAN CITTERS R. L. (1966) Aortic blood flow in freeswimming elasmobranchs. Camp. Biochem. Physiol. 19, 151-160. &TLUND E. & F-GE R. (1962) Vasodilation by adrenaline and noradrenaline, and the effect of some other substances on perfused fish gills. Camp. Biochem. Physiol. 5, 307-309. RANDALLD. J. (1970) Gas exchange in fish. In Fish Physiology (Edited by HOARW. S. & RANDALLD. J.), Vol. IV, pp. 253-292. Academic Press, New York. RUUD J. T. (1954) Vertebrates without erythrocytes and blood pigment. Nature, Lond. 173, 848-850. SATCHELLG. H. (1962) Intrinsic vasomotion in the dogfish gill. J. exp. Biol. 39, 503-512. SATCHELLG. H. (1971) Circulation in Fishes. Cambridge University Press, London. SCHUMANND. & PIIPBB J. (1966) Der Sauerstoffbedarf der Atmung bei Fischen nach Messungen an der narkotisierten Schleie (Tinca tinca). PjEigem Arch. Ges. Physiol. 288, 15-26. SHANNONE. W. & WIGGERSC. J. (1939) The dynamics of the frog and turtle hearts-The non-refractory phase of systole. Am. J. Physiol. 128, 709-715. STEVENSE. D. & RANDALLD. J. (1967a) Changes in blood pressure, heart rate and breathing rate during moderate swimming activity in rainbow trout. y. exp. Biol. 46, 307-315. STEVENSE. D. & RANDALLD. J. (1967b) Changes in gas concentrations in blood and water during moderate swimming activity in rainbow trout. r. exp. Biol. 46, 329-337. Key Word Index-Antarctic fish; Chaenocephalus aceratus; hemoglobin absence; cardiac output; cardiac work; blood flow; blood gases; blood pressures.