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Environmental influences on the development of the cardiac system in fish and amphibians
Comparative Biochemistry and Physiology Part A 124 (1999) 407 – 412 www.elsevier.com/locate/cbpa
Review
Environmental influences on the development of the cardiac system in fish and amphibians Bernd Pelster * Department of Zoology, Uni6ersity of Innsbruck, Technikerstr. 25, A – 6020 Innsbruck, Austria Received 21 September 1998; received in revised form 10 December 1998; accepted 23 December 1998
Abstract In poikilothermic animals body temperature varies with environmental temperature, and this results in a change in metabolic activity (Q10 of enzymatic reactions typically is around 2 – 3). Temperature changes also modify gas transport in body fluids. While the diffusion coefficient increases with increasing temperatures, physical solubility and also hemoglobin oxygen affinity decrease. Therefore, an increase in temperature typically requires adjustments in cardiac activity because ventilatory and convectional transport of respiratory gases usually are tightly coupled in adults in order to meet the oxygen demand of body tissues. Hypoxic conditions also provoke adaptations in the central circulatory system, like the hypoxic bradycardia, which has been described for many adult lower vertebrates, combined with an increase in stroke volume and peripheral resistance. In embryos and larvae the situation is much more complicated, because nervous control of the heart is established only late during development, and because the site of gas exchange changes from mainly cutaneous gas exchange during early development to mainly pulmonary or branchial gas exchange in late stages. In addition, recent studies in amphibian and fish embryos and larvae reveal, that at least in very early stages convectional gas transport of the hemoglobin is not essential, which means that in these early stages ventilatory and convectional gas transport are not yet coupled. Accordingly, in early stages of fish and amphibians the central cardiac system often does not respond to hypoxia, although in some species behavioral adaptations indicate that oxygen sensors are functional. If a depression of cardiac activity is observed, it most likely is a direct effect of oxygen deficiency on the cardiac myocytes. Regulated cardiovascular responses to hypoxia appear only in late stages and are similar to those found in adult species. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Ontogeny; Development; Heart; Circulation; Oxygen consumption; Temperature; Hypoxia; Amphibian; Fish
1. Introduction While birds and mammals typically develop under more or less controlled and stable conditions under parental protection, embryos of lower vertebrates typically are free-living and thus develop in an environment that may be characterized by variable temperatures and changing oxygen availability. This means, that they usually are exposed to the same or a similar environment as the adults. Physiological changes and adaptations to reduced oxygen availability or a change in temperature in adult amphibians and fishes have been * Tel.: +43-512-5076180; fax: 43-512-5072930. E-mail address: [email protected] (B. Pelster)
analyzed in a large number of studies. These studies reveal a sophisticated coupling of metabolic demand of tissues, respiratory gas exchange and cardiac activity. Coupling is achieved by autonomic nervous control, and the combined action of nervous and humoral control mechanisms assure a coordinated response of these functions in the face of changing environmental conditions — at least if the environmental perturbations are not too severe [32,36]. Very little, however, is known about embryos and larvae in this respect. Several studies demonstrated, that, as a probably general trait in development, the nervous system controlling or modifying cardiac activity becomes functional only late during larval development [11,19,26,31]. This immediately raises the
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B. Pelster / Comparati6e Biochemistry and Physiology, Part A 124 (1999) 407–412
question, whether embryos and larvae are able to adjust to environmental changes, at least to a certain degree, or whether they show a more or less constant respiratory and cardiac activity, unable to compensate for environmental perturbations. If a coordinated response is not possible, this also implies that the, for adults well established, coupling between metabolic requirements, respiratory gas exchange and cardiac activity may not yet be established in early developmental stages. The analysis of cardiac and respiratory changes with changing environmental conditions during development must take into account that development per se may modify body functions. Development typically is characterized by an increase in body mass, and an increase in body mass will impose an additional demand on the convectional transport of nutrients and respiratory gases. Furthermore, a switch from cutaneous respiration to gill respiration with development, or from gill respiration to lung respiration, requires appropriate changes in the circulatory system. On the other hand, a comparison of the changes in cardiac activity with changes in body mass may already provide information about the coupling of metabolic demand and convectional transport.
2. Allometric changes Heart rate changes with development, and these changes are species dependent. There appears to be no specific difference between amphibians and fish [5]. In some species, heart rate initially increases and then slowly decreases or remains constant, in some species a continuous increase is observed, while in others heart rate continuously decreases with increasing body mass. In the bullfrog Rana catesbeiana and in the clawed frog Xenopus lae6is, resting heart rate scales with body mass to the exponent − 0.23 and − 0.06, respectively [4,16], while in rainbow trout Oncorhynchus mykiss heart rate increases with body mass to the exponent 0.26 [24]. In the little skate Raja erinacea, heart rate initially increases and then continuously decreases with increasing body mass [28]. Blood pressure increases with development, and in most species the increase in blood pressure appears to be related to a concomitant increase in body mass. Systolic blood pressure scales with body mass to the exponent 0.61 in fall and winter animals of Rana catesbeiana [30], lower values are reported for the little skate Raja erinacea and for Xenopus lae6is with mass exponent 0.45 and 0.20, respectively [16,28]. This suggests, that the increase in body mass causes a proportional increase in blood pressure (allometric change). There are, however, also changes that are not allometric. In Rana catesbeiana the mass exponent in summer animals is much lower than in winter animals [30], and in the
paradoxical frog, Pseudis paradoxus, for example, body mass decreases from larvae to adult, while blood pressure increases [1]. A crucial parameter to characterize cardiac activity is cardiac output. A mass specific comparison of cardiac output and oxygen consumption during early development reveals, that in Xenopus lae6is [25] as well as in rainbow trout Oncorhynchus mykiss [24] the mass exponent for oxygen uptake is significantly smaller than that for cardiac output. The increase in convectional blood transport with development therefore cannot simply be explained by the mass related increase in oxygen uptake. This suggests, that at least in early developmental stages of these species blood convection and metabolic demand are not yet tightly coupled.
3. Temperature effects Temperature is an important variable that modifies enzyme activity, as well as gas transport and solubility. An increase in temperature by 10°C typically increases metabolic activity by a factor of 2–3 (Q10 = 2 –3 for enzymatic reactions). An increase in temperature also increases Brownian motion and thus the diffusion coefficient, but the solubility of gases in aqueous media decreases at higher temperatures. As a consequence, Krogh’s constant of diffusion increases only by a factor of 1.1 for a temperature increase of 10°C [7], and at higher temperatures diffusive gas transport can hardly keep up with the increase in metabolic activity. A stimulation of convective gas transport (i.e. cardiac activity) in this situation thus could be interpreted as an attempt to reduce the necessity for diffusive gas transport. Thus, in poikilothermic organisms temperature changes do influence gas exchange. Not only fish, amphibians and reptiles are poikilothermic, but bird embryos also are poikilotherm during the first part of their development, as the ability to keep body temperature more or less constant is gained only just before or around hatching ([37,38]; see also Ref. [27]). Q10 for embryonic and larval metabolism in fish is about three, but only about two in juveniles and adults [33,35]. Even Q10 values of about 4–6 have been reported for larval Eleutherodactylus coqui [3]. The elevated Q10 values for oxygen uptake compared to adults may be a result of less efficient operation of the embryonic system. The activity and the efficiency of the Ca2 + pump of the sarcoplasmatic reticulum in vesicles from immature rabbit hearts, for example, is lower than in vesicles from adults hearts (see Ref. [21]). The effect of a temperature increase on heart rate appears to be less pronounced compared to the effect on energy metabolism. Q10 for heart rate for embryonic brook trout is 2.2–2.4 [9], and for Atlantic salmon embryos a value of 2.6 can be calculated based on the
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data of Klinkhardt et al. [20]. Similar results exist for larvae of the frog Eleutherodactylus coqui [3], where Q10 for heart rate is between 1.5 and 2 throughout development, while the Q10 for O2 uptake initially may even exceed a value of 5. We recorded cardiac activity between 14 and 20°C for the minnow (Phoxinus phoxinus) and obtained quite similar results. Q10 for heart rate decreases with development until about 5 days after hatching, typically to values around or even below 2, and this is also true for stroke volume (Fig. 1). Taken together these data indicate, that changes in heart rate and in stroke volume with temperature are much smaller than the changes in metabolism. This clearly suggests, that respiration and cardiac activity are not yet tightly linked at this stage of development. In contrast to our results, Mirkovic and Rombough [24] for rainbow trout larvae report a Q10 for cardiac output of 3.06, while the Q10 for oxygen uptake was 2.99. Although this coincidence at first glance suggests a tight supply and demand relationship for this species, the authors quite convincingly discuss, that this coincidence appears to be just incidental, and that the data do not support the idea of a tight coupling of convectional gas transport and metabolism. A very strong argument of Mirkovic and Rombough is, that the mass exponent for cardiac output is significantly larger than the mass exponent for oxygen consumption in larval rainbow trout. The data therefore collectively show, that the thermal sensitivity of cardiac activity is not parallel to that of metabolism. This suggests, that the linkage between metabolic demand and convective transport, which is well established for adults, is not tight in embryos and early larvae. An interesting hint towards the same conclusion comes from a study on exercising larvae. In
Fig. 1. Q10 values calculated for heart rate and stroke volume in the minnow, Phoxinus phoxinus. Heart rate and stroke volume were determined using a video system similar to the method of Hou and Burggren [17] in unrestrained larvae without anesthesia at a temperature of 14 and 20°C (T. Schwerte, L. Ivarsson and B. Pelster, unpublished).
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contrast to larger larvae or juveniles, small larvae of herring at the end of yolk-sac stage show no dependence of swimming speed on temperature [13].
4. The influence of hypoxia The response of adult amphibians and fishes to a decrease in oxygen availability has been analyzed in a large number of studies. A typical response would be a hypoxic bradycardia, sometimes offset by an increase in stroke volume, so that cardiac output remains more or less constant. Ventilation is enhanced, the ventilation/ perfusion ratio increases in water breathing lower vertebrates, the transfer factor of the gills for oxygen increases and peripheral resistance often shows an increase. Finally, venous PO2 decreases, resulting in a stable rate of oxygen uptake, at least if the hypoxia is not too severe [4,10,18,32]. Before discussing the impact of hypoxia on cardiac function during development, we need to briefly look at the gas exchange in embryos and point out some difficulties or complicating factors, that might influence cardiac development and function. First, the site of gas exchange varies with development, and this of course may require a more or less complicated reconstruction of capillary systems. Wells and Pinder [41] analyzed the gas exchange in Atlantic salmon and nicely showed that cutaneous gas exchange decreases with development, while branchial gas exchange increases. The yolk sac has been discussed as a possible site of gas exchange, but in rainbow trout larvae the blood vessel density of the yolk sac had no significant impact on gas exchange of this organ [35]. This appears to be a more general effect, because unstirred boundary layers in the range of several hundred microns limit gas exchange severely [34,35], except in organs like the fins that show quick and oscillatory movements. These movements largely reduce the thickness of the boundary layer. While adult fish and amphibians typically are socalled oxyregulators, i.e. they are able to maintain the rate of oxygen uptake at a more or less constant level during mild hypoxia, embryos and larvae quite often appear to be oxyconformers. Hastings and Burggren [14] analyzed oxygen uptake of Xenopus lae6is at various PO2 levels and found, that the critical PO2, i.e. the PO2 below which the rate of oxygen uptake significantly decreases, varies with development, and that these changes clearly were related to changes in gas exchange organs. A critical PO2 of 20 kPa is indicative for an oxyconformer. In early stages, Xenopus larvae appeared to be oxyconformers, except for a brief episode, when the external gills were present. During this time the critical PO2 significantly decreased, which suggests that the external gills can supply sufficient amounts of oxygen to support aerobic metabolism even at lower PO2
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Fig. 2. Oxygen consumption (A) and heart rate (B) during 8 h anoxia and subsequent recovery in embryonic Arctic char (Sal6elinus alpinus) at a temperature of 8°C. The initial bout in oxygen uptake during reoxygenation probably represents a restoration of oxygen stores in the perivitelline fluid and in body fluids. Oxygen consumption only slowly returns to pre-anoxic values and then remains elevated for several hours. Heart rate, in turn, returns to pre-anoxic values much earlier than oxygen uptake (Martin Ortner, unpublished).
values. The external gills most likely remove oxygen from the water for the benefit of other organs, which implies, that this oxygen has to be transported to other parts of the body by means of convection. This suggests that convectional gas transport might be of importance at this stage. Following the transition to internal gills as the respiratory organ until the point during development, when lung ventilation becomes most important for gas exchange, the larvae again were oxyconformers [14]. Embryos and larvae clearly have the capacity for aerobic and anaerobic metabolism, and trout heart has a considerable tolerance of hypoxia during early life. Thus, the critical PO2 for trout heart is in the range of 0.1 kPa or even below [35]. Unpublished experiments of Martin Ortner in our laboratory support this conclusion. Embryos of Arctic char, at about 80% of embryonic development, survive 8 h of anoxia. During anoxia heat production decreased to a few percent of control, indicating a severe metabolic depression down to a few percent of control. Anaerobic metabolism, however, was not restricted to the anoxic period, but made a significant contribution to total metabolism during the first 9 h of reoxygenation. Considering the influence of hypoxia or anoxia on cardiac function, the simultaneous changes in heart rate are quite interesting. Anoxia induced a rapid, severe decline in heart rate,
and during recovery heart rate returned to control values over about 10 h. Oxygen uptake at this time, however, was still much lower than the control level and required almost 20 h for recovery (Fig. 2). Clearly, the impact of hypoxia on heart rate differed from its impact on oxygen uptake. Orlando and Pinder [25] incubated larvae of Xenopus lae6is at various PO2 values and determined heart rate, stroke volume as well as cardiac output. In Nieuwkoop Faber stage 44–49.5 larvae and in stage 51–54 larvae there was initially no significant response to a decreasing PO2, but at the lowest PO2 of about 5.3 kPa a bradycardia developed. In contrast to the situation described for adults (see above), stroke volume also decreased at the lowest PO2, resulting in a significant drop in cardiac output. This was confirmed by Fritsche and Burggren [12], who looked at various stage groups and found a minor bradycardia combined with a decrease in stroke volume during hypoxia in early stages, while in later stages (beyond Nieuwkoop Faber stage 52–54) the bradycardia was more pronounced, and in part compensated by an increase in stroke volume. As pointed out by Orlando and Pinder [25] and by Fritsche and Burggren [12], the response of the early stages appears to be a direct effect of oxygen deficiency on cardiac muscle cells, and not a coordinated response to compensate for the reduced oxygen availability. The controlled adult response, characterized by a bradycardia combined with an increase in stroke volume, appears only late during development. The tachycardia observed in salmon larvae during hypoxia [15,22] does not fit into the picture developed with amphibian larvae. A tachycardia would require a coordinated response. The nerves controlling cardiac function appear to be functional only late during amphibian and bird development [19,26,31], and this most likely is also true for fish larvae. Experiments with chemical hypoxia provide useful information concerning the role of circulatory oxygen delivery and aerobic metabolism. Hypoxia or anoxia can experimentally be arranged by decreasing the oxygen content/oxygen partial pressure of the water. It can also be arranged as chemical hypoxia or anoxia, by using cyanide to block the respiratory chain, or by applying CO or phenylhydrazine, which blocks or chemically destroys the hemoglobin, to reduce the oxygen carrying capacity of the blood in order to reduce convectional oxygen transport in the blood stream. The zebrafish Danio rerio and the clawed frog Xenopus lae6is can be raised in an atmosphere containing 1–2% CO, which should be sufficient to essentially inhibit hemoglobin oxygen transport [29,39]. For both, zebrafish and Xenopus, the oxygen uptake of CO incubated animals does not differ from that of control animals during the first days of embryonic and larval development. Furthermore, the pattern of blood pres-
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sure changes, the absolute pressure and the time course of pressure generation, plotted as DP/DT, are the same in CO incubated and control zebrafish until about 2 days after hatching [29]. This clearly indicates, that hemoglobin oxygen transport is not important at this stage of development to support oxygen supply to the tissues. A third set of experiments needs consideration in this context, although at first glance, it has nothing to do with hypoxia. Increasing knowledge and activity in molecular techniques also resulted in the study of an increasing number of mutants. In Ambystoma mexicanum, the Mexican axolotl, a cardiac lethal mutant has been described, that develops until well after hatching. By microsurgery heartless axolotls were produced by removing the primordia. In mutants, as well as in these experimentally produced heartless axolotl, oxygen uptake was not significantly different from oxygen uptake of control animals [23]. Recently, it was observed that zebrafish embryos can survive without a functioning cardiovascular system for up to 5 days after fertilization [6], which clearly demonstrates that, at this stage, convective transport is not essential to support metabolism. And this clearly is not restricted to the convectional transport of oxygen, it also includes the convectional transport of nutrients. Collectively these results clearly indicate, that cardiac activity in early embryos and larvae is not linked to metabolic demands of the tissue as it is in adults, and hypoxia induced reductions in cardiac activity most likely represent a direct effect of oxygen shortage on cardiac muscle cells. In consequence, the oxygen supply to the embryonic tissue can be met by diffusion. According to the laws of diffusion, this is only possible up to a certain body size. After reaching this size — at latest — convective oxygen transport must take over, and this perhaps is the point where the supply and demand linkage between metabolic requirements and convective transport is established. This may also be the point where a regulated and coordinated response of the circulatory system becomes possible, so that the cardiac system may contribute to an adaptive response of the organism to changes in the environment. In spite of the fact that a coordinated response to environmental hypoxia is established only in late larval stages, sensors for oxygen are present and functional much earlier. In anuran larvae, for example, behavioral changes like an increase in surfacing activity are induced during hypoxic incubations in very early stages, even long before the lung is developed [2,8,40,42]. If the circulatory system initially is not linked to metabolic requirements, and if embryos even without functional circulatory system survive for several days, why then is the heart, in almost all embryos, the very first organ to operate? One possibility, which currently is under investigation, is that it is necessary for the proper development of tissue capillary networks.
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Acknowledgements This work was in part supported by the Austrian Fonds zur Fo¨rderung der wissenschaftlichen Forschung (P12571-BIO). References [1] Burggren WW, Bicudo JE, Glass ML, Abe AS. Development of blood pressure and cardiac reflexes in the frog Pseudis paradoxsus. Am J Physiol 1992;263:R602– 8. [2] Burggren WW, Doyle M. Ontogeny of heart rate regulation in the bullfrog, Rana catesbeiana. Am J Physiol 1986;251:R231–9. [3] Burggren WW, Infantino RL, Townsend DS. Developmental changes in cardiac and metabolic physiology of the direct-developing tropical frog Eleutherodactylus coqui. J Exp Biol 1990;152:129 – 47. [4] Burggren WW, Pinder AW. Ontogeny of cardiovascular and respiratory physiology in lower vertebrates. Annu Rev Physiol 1991;53:107 – 35. [5] Burggren WW, Warburton S. Patterns of form and function in developing hearts: contributions from non-mammalian vertebrates. Cardioscience 1994;5:183 – 91. [6] Chen J-N, Fishman MC. Genetic dissection of heart development. In: Burggren WW, Keller BB, editors. Development of Cardiovascular Systems: Molecules to Organisms. Cambridge: Cambridge University Press, 1997:7 – 17. [7] Dejours P. Principles of Comparative Respiratory Physiology. Amsterdam: Elsevier, 1981. [8] Feder ME. Responses to acute aquatic hypoxia in larvae of the frog Rana berlandieri. J Exp Biol 1983;104:79 – 95. [9] Fisher KC. The effect of temperature on the critical oxygen pressure for heart beat frequency in embryos of Atlantic salmon and speckled trout. Can J Res, Sec D 1942;20:1 – 12. [10] Fritsche R. Effects of hypoxia on blood pressure and heart rate in three marine teleosts. Fish Physiol Biochem 1990;8:85–92. [11] Fritsche R. Ontogeny of cardiovascular control in amphibians. Am Zool 1997;37:23 – 30. [12] Fritsche R, Burggren W. Development of cardiovascular responses to hypoxia in larvae of the frog Xenopus lae6is. Am J Physiol 1996;271:R912– 7. [13] Fuiman LA, Batty RS. What a drag it is getting cold: partitioning the physical and physiological effects of temperature on fish swimming. J Exp Biol 1997;200:1745 – 55. [14] Hastings D, Burggren WW. Developmental changes in oxygen consumption in larvae of the South African clawed frog Xenopus lae6is. J Exp Biol 1995;198:2465 – 75. [15] Holeton GF. Respiratory and circulatory responses of rainbow trout larvae to carbon monoxide and to hypoxia. J Exp Biol 1971;55:683 – 94. [16] Hou PCL, Burggren WW. Blood pressures and heart rate during larval development in the anuran amphibian Xenopus lae6is. Am J Physiol 1995;269:R1120– 5. [17] Hou PCL, Burggren WW. Cardiac output and peripheral resistance during larval development in the anuran amphibian Xenopus lae6is. Am J Physiol 1995;269:R1126– 32. [18] Jones DR, Milsom WK. Peripheral receptors affecting breathing and cardiovascular function in non-mammalian vertebrates. J Exp Biol 1982;100:59 – 91. [19] Kimmel PB. Adrenergic receptors and the regulation of vascular resistance in bullfrog tadpoles (Rana catesbeiana). J Comp Physiol B 1992;162:455 – 62. [20] Klinkhardt MB, Straganov AA, Pavlov DA. Motoricity of Atlantic salmon embryos (Salmo salar L.) at different temperatures. Aquaculture 1987;64:219 – 36.
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[21] Mahony L. Cardiac membrane structure and function. In: Burggren WW, Keller BB, editors. Development of Cardiovascular Systems: Molecules to Organisms. Cambridge: Cambridge University Press, 1997:18–26. [22] McDonald DG, McMahon BR. Respiratory development in Arctic char Sal6elinus alpinus under conditions of normoxia and chronic hypoxia. Can J Zool 1977;55:1461–7. [23] Mellish JA, Smith SC, Pinder AW. Oxygen transport in heartless vertebrates, Physiol Zool 1999; (in press). [24] Mirkovic T, Rombough PJ. The effect of body mass and temperature on the heart rate, stroke volume, and cardiac output of larvae of the rainbow trout, Oncorhynchus mykiss. Physiol Zool 1998;71:191 – 7. [25] Orlando K, Pinder AW. Larval cardiorespiratory ontogeny and allometry in Xenopus lae6is. Physiol Zool 1995;68:63–75. [26] Pappano AJ. Ontogenetic development of autonomic neuroeffector transmission and transmitter reactivity in embryonic and fetal hearts. Pharm Rev 1977;29:3–33. [27] Pelster B. Oxygen, temperature, and pH influences on the development of nonmammalian embryos and larvae. In: Burggren WW, Keller BB, editors. Development of Cardiovascular Systems: Molecules to Organisms. Cambridge: Cambridge University Press, 1997:227 –39. [28] Pelster B, Bemis WE. Ontogeny of heart function in the little skate Raja erinacea. J Exp Biol 1991;156:387–98. [29] Pelster B, Burggren WW. Disruption of hemoglobin oxygen transport does not impact oxygen-dependent physiological processes in developing embryos of zebra fish (Danio rerio). Circ Res 1996;79:358 – 62. [30] Pelster B, Burggren WW. Central arterial hemodynamics in larval bullfrogs (Rana catesbeiana): developmental and seasonal influences. Am J Physiol 1991;260:R240–6. [31] Protas LL, Leontieva GR. Ontogeny of cholinergic and adrenergic mechanisms in the frog (Rana temporaria) heart. Am J Physiol 1992;262:R150–61.
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[32] Randall D. The control of respiration and circulation in fish during exercise. J Exp Biol 1982;100:275 – 88. [33] Rombough PJ. Respiratory gas exchange, aerobic metabolism and effects of hypoxia during early life. In: Hoar WS, Randall DJ, editors. Fish Physiology, Vol. XI, The Physiology of Developing Fish. San Diego, NY: Academic Press, 1988:59 –161. [34] Rombough PJ. Intravascular oxygen tensions in cutaneously respiring rainbow trout (Oncorhynchus mykiss) larvae. Comp Biochem Physiol 1992;101A:23 – 7. [35] Rombough PJ. Piscine cardiovascular development. In: Burggren WW, Keller BB, editors. Development of Cardiovascular Systems: Molecules to Organisms. Cambridge: Cambridge University Press, 1997:145 – 65. [36] Taylor EW. Nervous control of the heart and cardiorespiratory interactions. In: Hoar WS, Randall DJ, Farrell AP, editors. Fish Physiology, Vol. XIIB, The Cardiovascular System. San Diego, NY: Academic Press, 1992:343 – 87. [37] Tazawa H, Wakayama H, Turner JS, Paganelli CV. Metabolic compensation for gradual cooling in developing chick embryos. Comp Biochem Physiol 1988;89A:125 – 9. [38] Tazawa H, Okuda A, Nakazawa S, Whittow GC. Metabolic responses of chicken embryos to graded, prolonged alterations in ambient temperature. Comp Biochem Physiol 1989;92A:613–7. [39] Territo PR, Burggren WW. Cardio-respiratory ontogeny during chronic carbon monoxide exposure in the clawed frog Xenopus lae6is. J Exp Biol 1998;201:1461 – 72. [40] Wassersug RJ, Seibert EA. Behavioral responses of amphibian larvae to variation in dissolved oxygen, Copeia 1975;86–103. [41] Wells PR, Pinder AW. The respiratory development of Atlantic salmon. II. Partitioning of oxygen uptake among gills, yolk sac and body surfaces. J Exp Biol 1996;199:2737 – 44. [42] West NH, Burggren WW. Reflex interactions between aerial and aquatic gas exchange organs in larval bullfrogs. Am J Physiol 1983;244:R770– 7.
Review
Environmental influences on the development of the cardiac system in fish and amphibians Bernd Pelster * Department of Zoology, Uni6ersity of Innsbruck, Technikerstr. 25, A – 6020 Innsbruck, Austria Received 21 September 1998; received in revised form 10 December 1998; accepted 23 December 1998
Abstract In poikilothermic animals body temperature varies with environmental temperature, and this results in a change in metabolic activity (Q10 of enzymatic reactions typically is around 2 – 3). Temperature changes also modify gas transport in body fluids. While the diffusion coefficient increases with increasing temperatures, physical solubility and also hemoglobin oxygen affinity decrease. Therefore, an increase in temperature typically requires adjustments in cardiac activity because ventilatory and convectional transport of respiratory gases usually are tightly coupled in adults in order to meet the oxygen demand of body tissues. Hypoxic conditions also provoke adaptations in the central circulatory system, like the hypoxic bradycardia, which has been described for many adult lower vertebrates, combined with an increase in stroke volume and peripheral resistance. In embryos and larvae the situation is much more complicated, because nervous control of the heart is established only late during development, and because the site of gas exchange changes from mainly cutaneous gas exchange during early development to mainly pulmonary or branchial gas exchange in late stages. In addition, recent studies in amphibian and fish embryos and larvae reveal, that at least in very early stages convectional gas transport of the hemoglobin is not essential, which means that in these early stages ventilatory and convectional gas transport are not yet coupled. Accordingly, in early stages of fish and amphibians the central cardiac system often does not respond to hypoxia, although in some species behavioral adaptations indicate that oxygen sensors are functional. If a depression of cardiac activity is observed, it most likely is a direct effect of oxygen deficiency on the cardiac myocytes. Regulated cardiovascular responses to hypoxia appear only in late stages and are similar to those found in adult species. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Ontogeny; Development; Heart; Circulation; Oxygen consumption; Temperature; Hypoxia; Amphibian; Fish
1. Introduction While birds and mammals typically develop under more or less controlled and stable conditions under parental protection, embryos of lower vertebrates typically are free-living and thus develop in an environment that may be characterized by variable temperatures and changing oxygen availability. This means, that they usually are exposed to the same or a similar environment as the adults. Physiological changes and adaptations to reduced oxygen availability or a change in temperature in adult amphibians and fishes have been * Tel.: +43-512-5076180; fax: 43-512-5072930. E-mail address: [email protected] (B. Pelster)
analyzed in a large number of studies. These studies reveal a sophisticated coupling of metabolic demand of tissues, respiratory gas exchange and cardiac activity. Coupling is achieved by autonomic nervous control, and the combined action of nervous and humoral control mechanisms assure a coordinated response of these functions in the face of changing environmental conditions — at least if the environmental perturbations are not too severe [32,36]. Very little, however, is known about embryos and larvae in this respect. Several studies demonstrated, that, as a probably general trait in development, the nervous system controlling or modifying cardiac activity becomes functional only late during larval development [11,19,26,31]. This immediately raises the
1095-6433/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 ( 9 9 ) 0 0 1 3 2 - 4
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B. Pelster / Comparati6e Biochemistry and Physiology, Part A 124 (1999) 407–412
question, whether embryos and larvae are able to adjust to environmental changes, at least to a certain degree, or whether they show a more or less constant respiratory and cardiac activity, unable to compensate for environmental perturbations. If a coordinated response is not possible, this also implies that the, for adults well established, coupling between metabolic requirements, respiratory gas exchange and cardiac activity may not yet be established in early developmental stages. The analysis of cardiac and respiratory changes with changing environmental conditions during development must take into account that development per se may modify body functions. Development typically is characterized by an increase in body mass, and an increase in body mass will impose an additional demand on the convectional transport of nutrients and respiratory gases. Furthermore, a switch from cutaneous respiration to gill respiration with development, or from gill respiration to lung respiration, requires appropriate changes in the circulatory system. On the other hand, a comparison of the changes in cardiac activity with changes in body mass may already provide information about the coupling of metabolic demand and convectional transport.
2. Allometric changes Heart rate changes with development, and these changes are species dependent. There appears to be no specific difference between amphibians and fish [5]. In some species, heart rate initially increases and then slowly decreases or remains constant, in some species a continuous increase is observed, while in others heart rate continuously decreases with increasing body mass. In the bullfrog Rana catesbeiana and in the clawed frog Xenopus lae6is, resting heart rate scales with body mass to the exponent − 0.23 and − 0.06, respectively [4,16], while in rainbow trout Oncorhynchus mykiss heart rate increases with body mass to the exponent 0.26 [24]. In the little skate Raja erinacea, heart rate initially increases and then continuously decreases with increasing body mass [28]. Blood pressure increases with development, and in most species the increase in blood pressure appears to be related to a concomitant increase in body mass. Systolic blood pressure scales with body mass to the exponent 0.61 in fall and winter animals of Rana catesbeiana [30], lower values are reported for the little skate Raja erinacea and for Xenopus lae6is with mass exponent 0.45 and 0.20, respectively [16,28]. This suggests, that the increase in body mass causes a proportional increase in blood pressure (allometric change). There are, however, also changes that are not allometric. In Rana catesbeiana the mass exponent in summer animals is much lower than in winter animals [30], and in the
paradoxical frog, Pseudis paradoxus, for example, body mass decreases from larvae to adult, while blood pressure increases [1]. A crucial parameter to characterize cardiac activity is cardiac output. A mass specific comparison of cardiac output and oxygen consumption during early development reveals, that in Xenopus lae6is [25] as well as in rainbow trout Oncorhynchus mykiss [24] the mass exponent for oxygen uptake is significantly smaller than that for cardiac output. The increase in convectional blood transport with development therefore cannot simply be explained by the mass related increase in oxygen uptake. This suggests, that at least in early developmental stages of these species blood convection and metabolic demand are not yet tightly coupled.
3. Temperature effects Temperature is an important variable that modifies enzyme activity, as well as gas transport and solubility. An increase in temperature by 10°C typically increases metabolic activity by a factor of 2–3 (Q10 = 2 –3 for enzymatic reactions). An increase in temperature also increases Brownian motion and thus the diffusion coefficient, but the solubility of gases in aqueous media decreases at higher temperatures. As a consequence, Krogh’s constant of diffusion increases only by a factor of 1.1 for a temperature increase of 10°C [7], and at higher temperatures diffusive gas transport can hardly keep up with the increase in metabolic activity. A stimulation of convective gas transport (i.e. cardiac activity) in this situation thus could be interpreted as an attempt to reduce the necessity for diffusive gas transport. Thus, in poikilothermic organisms temperature changes do influence gas exchange. Not only fish, amphibians and reptiles are poikilothermic, but bird embryos also are poikilotherm during the first part of their development, as the ability to keep body temperature more or less constant is gained only just before or around hatching ([37,38]; see also Ref. [27]). Q10 for embryonic and larval metabolism in fish is about three, but only about two in juveniles and adults [33,35]. Even Q10 values of about 4–6 have been reported for larval Eleutherodactylus coqui [3]. The elevated Q10 values for oxygen uptake compared to adults may be a result of less efficient operation of the embryonic system. The activity and the efficiency of the Ca2 + pump of the sarcoplasmatic reticulum in vesicles from immature rabbit hearts, for example, is lower than in vesicles from adults hearts (see Ref. [21]). The effect of a temperature increase on heart rate appears to be less pronounced compared to the effect on energy metabolism. Q10 for heart rate for embryonic brook trout is 2.2–2.4 [9], and for Atlantic salmon embryos a value of 2.6 can be calculated based on the
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data of Klinkhardt et al. [20]. Similar results exist for larvae of the frog Eleutherodactylus coqui [3], where Q10 for heart rate is between 1.5 and 2 throughout development, while the Q10 for O2 uptake initially may even exceed a value of 5. We recorded cardiac activity between 14 and 20°C for the minnow (Phoxinus phoxinus) and obtained quite similar results. Q10 for heart rate decreases with development until about 5 days after hatching, typically to values around or even below 2, and this is also true for stroke volume (Fig. 1). Taken together these data indicate, that changes in heart rate and in stroke volume with temperature are much smaller than the changes in metabolism. This clearly suggests, that respiration and cardiac activity are not yet tightly linked at this stage of development. In contrast to our results, Mirkovic and Rombough [24] for rainbow trout larvae report a Q10 for cardiac output of 3.06, while the Q10 for oxygen uptake was 2.99. Although this coincidence at first glance suggests a tight supply and demand relationship for this species, the authors quite convincingly discuss, that this coincidence appears to be just incidental, and that the data do not support the idea of a tight coupling of convectional gas transport and metabolism. A very strong argument of Mirkovic and Rombough is, that the mass exponent for cardiac output is significantly larger than the mass exponent for oxygen consumption in larval rainbow trout. The data therefore collectively show, that the thermal sensitivity of cardiac activity is not parallel to that of metabolism. This suggests, that the linkage between metabolic demand and convective transport, which is well established for adults, is not tight in embryos and early larvae. An interesting hint towards the same conclusion comes from a study on exercising larvae. In
Fig. 1. Q10 values calculated for heart rate and stroke volume in the minnow, Phoxinus phoxinus. Heart rate and stroke volume were determined using a video system similar to the method of Hou and Burggren [17] in unrestrained larvae without anesthesia at a temperature of 14 and 20°C (T. Schwerte, L. Ivarsson and B. Pelster, unpublished).
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contrast to larger larvae or juveniles, small larvae of herring at the end of yolk-sac stage show no dependence of swimming speed on temperature [13].
4. The influence of hypoxia The response of adult amphibians and fishes to a decrease in oxygen availability has been analyzed in a large number of studies. A typical response would be a hypoxic bradycardia, sometimes offset by an increase in stroke volume, so that cardiac output remains more or less constant. Ventilation is enhanced, the ventilation/ perfusion ratio increases in water breathing lower vertebrates, the transfer factor of the gills for oxygen increases and peripheral resistance often shows an increase. Finally, venous PO2 decreases, resulting in a stable rate of oxygen uptake, at least if the hypoxia is not too severe [4,10,18,32]. Before discussing the impact of hypoxia on cardiac function during development, we need to briefly look at the gas exchange in embryos and point out some difficulties or complicating factors, that might influence cardiac development and function. First, the site of gas exchange varies with development, and this of course may require a more or less complicated reconstruction of capillary systems. Wells and Pinder [41] analyzed the gas exchange in Atlantic salmon and nicely showed that cutaneous gas exchange decreases with development, while branchial gas exchange increases. The yolk sac has been discussed as a possible site of gas exchange, but in rainbow trout larvae the blood vessel density of the yolk sac had no significant impact on gas exchange of this organ [35]. This appears to be a more general effect, because unstirred boundary layers in the range of several hundred microns limit gas exchange severely [34,35], except in organs like the fins that show quick and oscillatory movements. These movements largely reduce the thickness of the boundary layer. While adult fish and amphibians typically are socalled oxyregulators, i.e. they are able to maintain the rate of oxygen uptake at a more or less constant level during mild hypoxia, embryos and larvae quite often appear to be oxyconformers. Hastings and Burggren [14] analyzed oxygen uptake of Xenopus lae6is at various PO2 levels and found, that the critical PO2, i.e. the PO2 below which the rate of oxygen uptake significantly decreases, varies with development, and that these changes clearly were related to changes in gas exchange organs. A critical PO2 of 20 kPa is indicative for an oxyconformer. In early stages, Xenopus larvae appeared to be oxyconformers, except for a brief episode, when the external gills were present. During this time the critical PO2 significantly decreased, which suggests that the external gills can supply sufficient amounts of oxygen to support aerobic metabolism even at lower PO2
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Fig. 2. Oxygen consumption (A) and heart rate (B) during 8 h anoxia and subsequent recovery in embryonic Arctic char (Sal6elinus alpinus) at a temperature of 8°C. The initial bout in oxygen uptake during reoxygenation probably represents a restoration of oxygen stores in the perivitelline fluid and in body fluids. Oxygen consumption only slowly returns to pre-anoxic values and then remains elevated for several hours. Heart rate, in turn, returns to pre-anoxic values much earlier than oxygen uptake (Martin Ortner, unpublished).
values. The external gills most likely remove oxygen from the water for the benefit of other organs, which implies, that this oxygen has to be transported to other parts of the body by means of convection. This suggests that convectional gas transport might be of importance at this stage. Following the transition to internal gills as the respiratory organ until the point during development, when lung ventilation becomes most important for gas exchange, the larvae again were oxyconformers [14]. Embryos and larvae clearly have the capacity for aerobic and anaerobic metabolism, and trout heart has a considerable tolerance of hypoxia during early life. Thus, the critical PO2 for trout heart is in the range of 0.1 kPa or even below [35]. Unpublished experiments of Martin Ortner in our laboratory support this conclusion. Embryos of Arctic char, at about 80% of embryonic development, survive 8 h of anoxia. During anoxia heat production decreased to a few percent of control, indicating a severe metabolic depression down to a few percent of control. Anaerobic metabolism, however, was not restricted to the anoxic period, but made a significant contribution to total metabolism during the first 9 h of reoxygenation. Considering the influence of hypoxia or anoxia on cardiac function, the simultaneous changes in heart rate are quite interesting. Anoxia induced a rapid, severe decline in heart rate,
and during recovery heart rate returned to control values over about 10 h. Oxygen uptake at this time, however, was still much lower than the control level and required almost 20 h for recovery (Fig. 2). Clearly, the impact of hypoxia on heart rate differed from its impact on oxygen uptake. Orlando and Pinder [25] incubated larvae of Xenopus lae6is at various PO2 values and determined heart rate, stroke volume as well as cardiac output. In Nieuwkoop Faber stage 44–49.5 larvae and in stage 51–54 larvae there was initially no significant response to a decreasing PO2, but at the lowest PO2 of about 5.3 kPa a bradycardia developed. In contrast to the situation described for adults (see above), stroke volume also decreased at the lowest PO2, resulting in a significant drop in cardiac output. This was confirmed by Fritsche and Burggren [12], who looked at various stage groups and found a minor bradycardia combined with a decrease in stroke volume during hypoxia in early stages, while in later stages (beyond Nieuwkoop Faber stage 52–54) the bradycardia was more pronounced, and in part compensated by an increase in stroke volume. As pointed out by Orlando and Pinder [25] and by Fritsche and Burggren [12], the response of the early stages appears to be a direct effect of oxygen deficiency on cardiac muscle cells, and not a coordinated response to compensate for the reduced oxygen availability. The controlled adult response, characterized by a bradycardia combined with an increase in stroke volume, appears only late during development. The tachycardia observed in salmon larvae during hypoxia [15,22] does not fit into the picture developed with amphibian larvae. A tachycardia would require a coordinated response. The nerves controlling cardiac function appear to be functional only late during amphibian and bird development [19,26,31], and this most likely is also true for fish larvae. Experiments with chemical hypoxia provide useful information concerning the role of circulatory oxygen delivery and aerobic metabolism. Hypoxia or anoxia can experimentally be arranged by decreasing the oxygen content/oxygen partial pressure of the water. It can also be arranged as chemical hypoxia or anoxia, by using cyanide to block the respiratory chain, or by applying CO or phenylhydrazine, which blocks or chemically destroys the hemoglobin, to reduce the oxygen carrying capacity of the blood in order to reduce convectional oxygen transport in the blood stream. The zebrafish Danio rerio and the clawed frog Xenopus lae6is can be raised in an atmosphere containing 1–2% CO, which should be sufficient to essentially inhibit hemoglobin oxygen transport [29,39]. For both, zebrafish and Xenopus, the oxygen uptake of CO incubated animals does not differ from that of control animals during the first days of embryonic and larval development. Furthermore, the pattern of blood pres-
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sure changes, the absolute pressure and the time course of pressure generation, plotted as DP/DT, are the same in CO incubated and control zebrafish until about 2 days after hatching [29]. This clearly indicates, that hemoglobin oxygen transport is not important at this stage of development to support oxygen supply to the tissues. A third set of experiments needs consideration in this context, although at first glance, it has nothing to do with hypoxia. Increasing knowledge and activity in molecular techniques also resulted in the study of an increasing number of mutants. In Ambystoma mexicanum, the Mexican axolotl, a cardiac lethal mutant has been described, that develops until well after hatching. By microsurgery heartless axolotls were produced by removing the primordia. In mutants, as well as in these experimentally produced heartless axolotl, oxygen uptake was not significantly different from oxygen uptake of control animals [23]. Recently, it was observed that zebrafish embryos can survive without a functioning cardiovascular system for up to 5 days after fertilization [6], which clearly demonstrates that, at this stage, convective transport is not essential to support metabolism. And this clearly is not restricted to the convectional transport of oxygen, it also includes the convectional transport of nutrients. Collectively these results clearly indicate, that cardiac activity in early embryos and larvae is not linked to metabolic demands of the tissue as it is in adults, and hypoxia induced reductions in cardiac activity most likely represent a direct effect of oxygen shortage on cardiac muscle cells. In consequence, the oxygen supply to the embryonic tissue can be met by diffusion. According to the laws of diffusion, this is only possible up to a certain body size. After reaching this size — at latest — convective oxygen transport must take over, and this perhaps is the point where the supply and demand linkage between metabolic requirements and convective transport is established. This may also be the point where a regulated and coordinated response of the circulatory system becomes possible, so that the cardiac system may contribute to an adaptive response of the organism to changes in the environment. In spite of the fact that a coordinated response to environmental hypoxia is established only in late larval stages, sensors for oxygen are present and functional much earlier. In anuran larvae, for example, behavioral changes like an increase in surfacing activity are induced during hypoxic incubations in very early stages, even long before the lung is developed [2,8,40,42]. If the circulatory system initially is not linked to metabolic requirements, and if embryos even without functional circulatory system survive for several days, why then is the heart, in almost all embryos, the very first organ to operate? One possibility, which currently is under investigation, is that it is necessary for the proper development of tissue capillary networks.
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