Aortic blood flow in free-swimming elasmobranchs

Comp. Biochem. Physiol., 1966, Vol. 19, pp. 151 to 160, Pergamon Press Ltd. PHnted in Great Britain

AORTIC BLOOD FLOW IN FREE-SWIMMING ELASMOBRANCHS* K. J O H A N S E N t , D. L. F R A N K L I N and R. L. V A N C I T T E R S Departments of Zoology and Physiology and Friday Harbor Laboratories, University of Washington, Seattle, Washington; and Department of Marine Biology, University of California, La Jolla, California ( R e c e i v e d 15 iVlarch 1966)

A b s t r a c t - - 1 . Ventral aortic blood velocity and pressure in free-swimming

elasmobranchs were recorded with the Doppler ultrasonic telemetry blood flowmeter. 2. Ejection time from the heart varied with heart rate. Peak ejection velocities ranged from 12-14 cm/sec in Horn sharks, 12-20 cm/sec in skates and 8-15 cm/sec in dogfish. Acceleration of the blood lasted from 120 msec in the Horn shark (T = 24°C) to 450 msec m the skates (T = 9°C). Deceleration of flow was slow in all species. Oscillations from bulbus cordis contraction and respiratory movements were superimposed on the basic waveform. Participation of the bulbar contraction in the cardiac sequence varied widely. 3. Heart rates increased mildly during short-lasting exercise. Peak velocity and flow were largely unchanged. Peak velocity and flow frequently increased in the post-exercise period. 4. The hemodynamic effects of atropine, acetylcholine and adrenaline are discussed in relation to the fact that the fish heart receives parasympathetic but not, apparently, sympathetic innervation. INTRODUCTION THE circulation of fishes offers special features not met with in other vertebrates. T h e fish heart consists of four chambers in series: the sinus venosus, the atrium, the ventricle and the bulbus cordis (in teleosts, bulbus arteriosus). T h e respiratory exchange vessels and the systemic vascular beds are in series. Blood volume is less than half of that in other vertebrates. T h e heart is known to receive inhibitory innervation through vagal fibers, but sympathetic innervation seems to be lacking. T h e heart rate and blood pressure of fishes are subject to diffuse and widespread reflexogenic influences (McWilliam, 1885). Tactile stimuli, such as gentle * This work was supported by Grant GB-358 from the National Science Foundation, and grants from the Washington State Heart Association and its Northeastern Chapter. Established Investigator, American Heart Association. 151

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K. JOHANSEN,D. L. FRANKLINANDR. L. VAN CITTERS

touch of the gills or skin, lead to reflex inhibition of the heart. Thus cardiac function in a physiological preparation is almost inevitably distorted. Ideally, meaningful data on cardiovascular function in fishes should be acquired from freeswimming undisturbed animals. Such data are virtually non-existent. Recent developments in cardiovascular instrumentation enable direct measurement and radio-telemetry of pulsatile blood velocity in totally unrestrained animals; when adapted for use in fishes this approach permits reconsideration of problems in fish circulation. In the present study, simultaneous measurements of ventral aortic blood velocity and pressure recorded from free-swimming sharks and skates are described. MATERIALS AND METHODS These experiments were performed on two specimens of the California Horn shark (HeterodontusfranciscO, eight Pacific dogfish (Squalus suckley0 and four large skates (Raja binoculata). Blood pressure was sampled via catheters chronically implanted in one afferent branchial artery (ventral aortic pressure) or in the coeliac or mesenteric arteries (dorsal aortic blood pressure). Blood pressure was measured with Statham P 23dB strain gage manometers. Blood velocity was measured with the Doppler ultrasonic telemetrybloodflowmeter (Franklin et al., 1964, 1966). In this technique, a transducer, consisting of a hinged cylindrical plastic cuff, is clamped snugly around the blood vessel. Two piezo-electric crystals are mounted diametrically inside the cuff with their acoustical axes oriented at a 60 ° angle with the axis of the enclosed vessel. One crystal emits 5 Mc/s sound into the blood stream; part of the sound is backscattered to the opposite crystal by the blood cells. According to the Doppler equation, the change in frequency of the backscattered sound is a linear function of the blood velocity. Velocity can be converted to volume flow provided that vessel diameter is fixed, i.e. the transducer fits snugly. For telemetry applications, the frequency shift modulates a 100 Mc/s FM transmitter, and an F M / F M signal is radiated. This signal is demodulated remotely by a standard FM receiver, and converted to an analog voltage for chart recording by a frequency-to-voltage converter. The two Horn sharks were caught and studied during an expedition to Guadalupe Island, Mexico, aboard a research vessel of the Scripps Institute of Oceanography. After they had recovered from surgery, the sharks were tethered to a float containing the flowmeter, transmitter and antennae. They could swim freely, and towed the float several hundred meters from the research vessel. Throughout this time their telemetered blood velocity was received and tape recorded on board the vessel. Experiments on dogfish and skates were carried out while they swam about in large aquaria at the University of Washington's Friday Harbor laboratories. Fine wires led from transducers implanted in these fish to flowmeters suspended above or alongside the aquaria, and flow information was telemetered to a remotely located receiver and recorded.

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Blood velocity and arterial pressure were recorded during rest and voluntary exercise, and in conjunction with drug injections and partial asphyxiation. RESULTS Horn shark A record of blood velocity in the ventral aorta of a California Horn shark during moderately active swimming is shown in Fig. 1. In general the outflow pattern through the ventral aorta was dependent on heart rate. At the prevailing rate of 36 beats/min, flow in the ventral aorta was continuous through most of the cardiac cycle. At very low heart rates the ejection time was less, at times down to one-fifth cm/sec

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FIG. 1. Ventral aortic (VA) blood velocity telemetered from a free-swimming California Horn shark (Heterodontus francisci).

of the cardiac cycle. Flow in the ventral aorta is primarily due to ventricular contraction, but is also augmented by contraction of the bulbus cordis and by the respiratory movements. Peak ejection velocity of 12-14 cm/sec is reached about 120 msec after the aortic valve opens; acceleration of the blood reaches about 120 cm/sec2. Deceleration of flow is much slower and small oscillations resulting from bulbar contraction and respiratory movements may be superimposed on the basic waveform (Fig. 1). No significant increase in heart rate, peak velocity or flow was detectable during short periods of exercise; however, peak velocity frequently increased during the post exercise period. Skate

The skates were ordinarily sluggish and remained on the bottom. Figure 2 shows simultaneous recordings of ventral aortic pressure and blood velocity from a large skate (20 kg). Heart rate is 5-6 beats/min at water temperature of 9°C, and ejection requires approximately 40-50 per cent of the cardiac cycle length. At higher heart rates ejection was prolonged, sometimes occupying the entire cycle. Distinct pulsations visible in the outflow pattern were correlated successively with ventricular contraction, bulbar contraction and respiratory movement. Participation of the bulbar contractions in the cardiac sequence varied widely; at times it was prominent, obviously adding propulsive energy as well as prolonging cardiac systole. For example, Fig. 3 shows a case in which bulbar contraction has prolonged cardiac systole to more than twice the duration of ventricular contraction, and almost eliminated the period of diastasis. Peak velocities in the ventral aorta varied between

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12-20 cm/sec. Acceleration lasted about 450 msec or through about one-fifth of ventricular ejection. Bulbar contraction prolonged ejection by about one-quarter; in other experiments this contribution ranged between one-fifth and two-thirds. Spontaneous abrupt changes in heart rate were seldom observed. When they did occur, the transition to higher rates was usually transient and showed no obvious correlation to change in activity or conditions in the environment. Such

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a change in heart rate in a skate is shown in Fig. 4. The tachycardia was accompanied by a considerable reduction in peak ejection velocity and stroke volume, so that cardiac output was scarcely changed. T h e decrease in the ejection velocity implied that the peripheral bed was constricted during the tachycardia, i.e. that the mechanism of tachycardia may be an integrated response. cmH20

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FiG. 4. Ventral aortic blood pressure and velocity in a skate during a spontaneous transient increase in heart rate. Injecting 10/~g acetylcholine into the ventral aorta of a large skate produced marked bradycardia and hypotension which were followed by tachycardia and increased blood pressure. Ejection velocity fell as pressure increased (Fig. 5). lOpg Acelylcholine

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Dogfish Hemodynamics in the Pacific dogfish Squalus suckleyi resembled those for the Horn sharks and skates. Peak ejection velocity in the ventral aorta normally ranged between 8-15 cm/sec. During short exercise (swimming) no significant changes in heart rate, blood pressure or ventral aortic blood flow were evoked; however, blood flow increased temporarily in the immediate post-exercise period (Fig. 7). During prolonged swimming (20 min or more), the heart rate increased mildly, but never more than 20-30 per cent of the initial rate. Resting

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FIG. 7. Ventral aortic blood pressure and velocity in a dogfish (,_qqualussucklori) before, during and after a short period of swimming. The effects of atropine on heart rate in a resting dogfish are shown in Fig. 8 (A). Heart rate was doubled. Atropinization also elevated heart rate in continuously swimming animals (Fig. 8B). The effect of atropine on ventral aortic blood flow is shown in Fig. 9. Heart rate increased from 14 to 24 beats/min while

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DISCUSSION Direct measurement of blood flow in free-swimming fishes has hitherto not been reported. Cardiac output has been estimated indirectly from information extrapolated from the literature, or assumed values (Mott, 1957). Equally simple methods have been reported in which the ventricle was tied off in a systole and diastole, with cardiac output values being computed from weight difference (Hart, 1943). Johansen (1962), using the electromagnetic flowmeter, recorded pulsatile blood flow in the ventral aorta of the teleost, Gadus morhua. However, the fishes were restrained and artificially ventilated during the experiments. One specimen (weight 2.91 kg) yielded a stroke volume of 0.9 ml, giving a cardiac output of 27-0 ml./min. More recently, Murdaugh et al. (1965) have estimated cardiac output in unrestrained dogfish, Squalus acanthias, with the dye dilution method. They arrived at an average cardiac output value of 26.6 ml/kg/min with a standard deviation as high as 16"6 ml/kg/min. The values obtained here for ventral aortic blood velocity do not permit total cardiac output to be evaluated directly since the flow transducers were placed distal to the first set of branchial arteries. It seems reasonable to assume that outfow from the heart is distributed fairly evenly between afferent branchial arteries and that changes in total outflow are reflected in the flow past the distal segment of the ventral aorta. Evidence from pressure measurements (Johansen, 1965; Haberich, 1965) have indicated that the bulbus cordis, representing a discrete chamber of the elasmobranch and amphibian heart, contributes propulsive energy to the blood leaving the heart. The present study substantiates this by showing that the bulbus contraction extends the ejection time from the heart, and also increases the volume ejected. The bulbus contribution varied greatly, minimally prolonging cardiac ejection by at least one-fifth. Respiratory movement was reflected in the ventral aortic pressure tracings as well as in the blood velocity records. This observation supports the concept that the respiratory movements provide modest but definite accessory blood propulsion. This phenomenon has been documented earlier in the specialized muscular gills of myxinoid fishes (Hol & Johansen, 1960). The possibility remains that pulses synchronous with the respiratory movements are artefacts due to mechanical displacement of the transducer with each respiratory movement. This possibility is largely minimized, however, by the extremely light weight of the transducers and their snug fit around the blood vessel. The data do not document any immediate change in blood flow during moderately long exercise (3-20 min), but do demonstrate an elevated peak blood velocity in the post-exercise period. Similarly the heart rate remained largely constant during swimming. Available evidence suggests large differences between species of fish in their heart rate response to exercise. Current concepts suggest that the fish heart (hagfish excepted) is only supplied by inhibitory fibers via the vagus with accelerator fibers lacking (von Skramlik, 1935). Sporadic pharmacological investigations have indicated that accelerating the fish heart is possible without lessening the vagal tone (Izquierdo, 1930). Atropinizing a dogfish which had

B L O O D F L O W I N SHARKS

159

rested quietly at the bottom for more than 40 min doubled the heart rate (Fig. 8A); atropinizing a fish which had been swimming for 30 min increased heart rate about 30 per cent (Fig. 8B). These findings suggest that in spite of prolonged swimming the heart's vagal tone did not appear to be fully released. In Fig. 9 ventral aortic blood pressure and blood velocity were simultaneously recorded during atropinization in a fish which was swimming quietly. When vagal tone was released, heart rate increased from 14 to 24 beats/rain, but in spite of minor changes in blood pressure, the ventral aortic blood flow was reduced. Based on the evidence above, one may wonder to what extent an increased heart rate represents a normal adjustment for increased cardiac output in the fishes investigated. Increasing heart rate pharmacologically admittedly does not mimic the increased heart rate attending spontaneous exercise, as the latter may be correlated with other measures of adjustment, such as increased venous return and changes in peripheral resistance. The concept that an optimal heart rate prevails at resting conditions in turtles and frogs was put forth by Shannon & Wiggers (1939). They concluded that turtles and frogs, unlike mammals, do not increase the minute output by an increase in heart rate. Johansen (1963) reached a similar conclusion from work on another amphibian. The current view in mammalian physiology is that adjustment to an increased cardiac output during exercise is effected by heart rate changes with small or no changes in stroke volume. Further experimentation on a wider selection of species is necessary before any generalizations can be made on this subject. The effects of adrenaline and acetylcholine are complex and difficult to interpret. The drugs have a dichotomous effect in fishes in that adrenaline dilates the branchial vessels and constricts the systemic vessels, whereas acetylcholine is reported to have the opposite effect (Ostlund & F~inge, 1962; Steen & Kruysse, 1964). It is likely that the bradycardia and general fall in blood pressure initially following acetylcholine injection is promptly compensated for via the widespread and diffusely located stretch receptors in the branchial region of fish (Lutz & Wyman, 1932; Irving et al., 1935). Stimulating these receptors tends to increase the sympathetic discharge to peripheral vessels while lessening the vagal tone to the heart. As acetylcholine selectively constricts the gill vessels, the blood pressure rises above the preinjection level. The resulting reduction in outflow from the heart perhaps may be attributed to the lack of any compensatory inotrophic effect on the heart due to its apparent lack of a sympathetic nerve supply. On this background it is noteworthy that adrenaline injection, although causing a similar increase in peripheral resistance, is accompanied by an increased outflow from the heart (Fig. 6). The difference can suggestively be linked to the direct inotrophic effect of adrenaline on the heart. REFERENCES FRANKLIND. L., WATSONN. W., PIERSONK. E. & VAN CITTERSR. L. (1966) A technique for radio-telemetry of blood flow velocity from unrestrained animals. Am. J. reed. Electron. 5, 24-28.

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FRANKLIN D. L., WATSONN. W. & VAN CITTI~RSR. L. (1964) Blood velocity telemetered from untethered animals. Nature, Lond. 203, 528-530. FRANKLIN D. L., WATSON N. W., VA.r~ CtTTERS R. L. & SMITrI O. A., JR. (1964) Blood flow telemetered from dogs and baboons. Fed. Proc. 23, 303. H~ERICH F. T. (1965) The functional separation of venous and arterial blood in the univentricular frog heart. Ann. N . Y . Acad. Sci. 127, 459-480. HART J. S. (1943) The cardiac output of four freshwater fish. Can. ft. Res. D 21, 77-84. I-Ior. R. & JOHANSEN K. (1960) A cineradiographic study of the central circulation in the hagfish, Myxine glutinosa L. ft. exp. Biol. 37, 469-473. IRVINGL., SOLANDTD. T. & SOLANDTO. M. (1935) Nerve impulses from branchial pressure receptors in the dogfish, ft. Physiol. 84, 187-190. IZQUmROo, J. J. (1930) On the influence of the extra cardiac nerves upon sinoauricular conduction in the heart of Scyllium. ft. Physiol. 69, 29-47. JOrtaNSEN K. (1962) Cardiac output and pulsatile aortic flow in the teleost, Gadus morhua. Comp. Biochem. Physiol. 7, 169-174. JOHANS~.N K. (1963) Cardiovascular dynamics in the amphibian, Amphiuma tridactylum. Acta Physiol. scand. 217, 82. JOHANSEN K. (1965) Cardiovascular dynamics in fishes, amphibians and reptiles. Ann. N . Y . Acad. Sci. 127, 414--442. LUTZ B. R. & W ~ v i ~ L. C. (1932) Reflex cardiac inhibition of branchiovascular origin in the elasmobranch, Squalus acanthias. Biol. Bull., Woods Hole 62, 17-22. McWILLIa_~ J. A. (1885) On the structure and rhythm of the heart in fishes, with especial reference to the heart of the eel. ft. Physiol. 6, 192-245. MOTT J. C. (1957) The cardiovascular system. In The Physiology of Fishes, Vol. 1 (Edited by BROWN M.E.). Academic Press, New York. MURDAUGH H. V., ROBIN E. D., MILLEN J. E. & D~WRY W. F. (1965) Cardiac output determinations by the dye dilution method in Squalus acanthias. Am. ft. PhysioL 209, 723-726. OSTLUND E. & FXNCE R. (1962) Vasodilation by adrenaline and noradrenaline, and the effects of some other substances on perfused fish gills. Comp. Biochem. Physiol. S, 307309. SHANNON E. W. & WIGG~.BSC. J. (1939) The dynamics of the frog and turtle hearts.--The non-refractory phase of systole. Am.ft. Physiol. 128, 709-715. YON S ~ L I K E. (1935) ~ b e r den Kreislauf bei den Fischen. Ergebn. Biol. 11, 1-130. STEEN J. B. & KRUYSSE A. (1964) The respiratory function of teleostean gills. Comp. Biochem. Physiol. 12, 127-142.