Download Developmental plasticity in the cardiovascular system of fish, with

Comparative Biochemistry and Physiology Part A 133 (2003) 547–553
Review
Developmental plasticity in the cardiovascular system of fish, with
special reference to the zebrafish夞
Bernd Pelster*
¨ Zoologie und Limnologie, Leopold-Franzens-Universitat
¨ Innsbruck, A-6020 Innsbruck, Austria
Institut fur
Received 2 February 2002; received in revised form 22 May 2002; accepted 28 June 2002
Abstract
During development the circulatory system of vertebrates typically starts operating earlier than any other organ. In
these early stages, however, blood flow is not yet linked to metabolic requirements of tissues, as is well established for
adults. While the autonomic nervous system becomes functional only quite late during development, in the early stages
control of blood flow appears to be possible by blood-borne andyor local hormones. This study presents methods based
on video-imaging techniques and fluorescence microscopy to visualize cardiac activity, as well as the vascular bed of
developing lower vertebrates, and tests the idea that environmental factors, such as hypoxia, may modify cardiac activity,
or even the early formation of blood vessels in embryos and larvae. In zebrafish larvae, adaptations of cardiovascular
activity to chronic hypoxia become visible shortly after hatching, and the formation of some blood vessels is enhanced
under chronic hypoxia. Exposure of early larval stages of zebrafish to a constant water current induces physiological
adaptations, resulting in enhanced swimming efficiency and increased tolerance towards hypoxia. Furthermore, application
of hormones such as NO can modify cardiac activity as well as peripheral resistance, and they can stimulate blood
vessel formation. In consequence, even during early development of fish or amphibian larvae, the performance of cardiac
muscle and of skeletal muscle can be modified by environmental influences and peripheral resistance can be adjusted.
Even blood vessel formation can be stimulated by hypoxia, for example, or by the presence of specific hormones. Thus,
at approximately the time of hatching the physiological performance of vertebrate larvae is already determined by the
combined action of environmental influences and of genetic information.
䊚 2002 Elsevier Science Inc. All rights reserved.
Keywords: Ontogeny; Circulatory system; Heart; Vascularization; Hypoxia; Hypoxemia; Zebrafish; Xenopus
1. Introduction
Form and functioning of the circulatory system
of animals has attracted attention for several hundred years. Only within recent years has the
夞 This paper was originally presented at ‘Chobe 2001’; The
Second International Conference of Comparative Physiology
and Biochemistry in Africa, Chobe National Park, Botswana
– August 18–24, 2001. Hosted by the Chobe Safari Lodge
and the Mowana Safari Lodge, Kasane; and organised by
Natural Events Congress Organizing ([email protected]).
*Tel.: q43-512-5076180; fax: q43-512-5072930.
E-mail address: [email protected] (B. Pelster).
development of new methods and technologies
opened up the possibility of including embryos
and larvae in this research. During development,
the circulatory system of vertebrates typically starts
operating earlier than any other organ. Observations of mutants that survive for a couple of days
without any heartbeat (Chen and Fishman, 1997),
and the fact that zebrafish (Danio rerio) or Xenopus larvae develop nicely for at least 1 or 2 weeks
in a CO-containing atmosphere (Pelster and Burggren, 1996; Territo and Altimiras, 1998; Territo
and Burggren, 1998) raised questions concerning
the main function of the circulatory system at this
1095-6433/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved.
PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 1 9 4 - 0
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B. Pelster / Comparative Biochemistry and Physiology Part A 133 (2003) 547–553
early state of development. Several studies raising
larvae at various temperatures revealed that the
temperature-induced increase in metabolic rate by
far exceeded the temperature-induced increase in
cardiac output (Mirkovic and Rombough, 1998;
¨
Schonweger
et al., 2000). The obvious explanation
for these observations was that in early developmental stages blood flow is not yet linked to
metabolic requirements of tissues, as is well established for adults.
The linkage between metabolism and cardiac
activity represents the main drive for adaptations
of the cardiovascular system to changing environmental conditions, for example. This raises the
question as to whether the cardiovascular system
during early development follows a genetic program and is not capable of a co-ordinated response
to environmental changes, or whether there is some
flexibility in the design and functioning of the
embryonic or larval cardiovascular system.
2. Visualization of cardiac performance and of
blood vessels
To analyze shape and performance of the developing cardiovascular system, the heart and blood
vessels must be visualized, but Doppler methods
are not suitable to measure blood flow in blood
vessels of capillary size (Burggren and Fritsche,
1995). Thus, video-imaging appears to be the
method of choice, but the contrast in a video
image of living tissues is rather poor and hardly
allows for morphometric analysis of the vascular
bed. A significant improvement can be achieved
by injecting a dye into the vascular system. Weinstein and co-workers (Weinstein et al., 1995; Isogai
et al., 2001) injected fluorescent microspheres into
the dorsal and ventral arteries of zebrafish larvae
and using confocal microangiography a threedimensional reconstruction of the vascular bed was
possible. The result is a remarkable atlas of the
vascular anatomy of zebrafish larvae at various
developmental stages.
A second approach to visualize the vascular bed
is a molecular approach. DNA constructs coding
for fluorescent protein can be inserted into the
genome. If introduced under the control of a tissuespecific promoter, expression is restricted to a
single tissue. Tie1 and Tie2 are the genes for
tyrosine kinase receptors, which are expressed only
in endothelial cells. Thus, a DNA construct was
generated with green fluorescent protein (GFP)
under the control of the endothelium specific
promoter Tie2 (Motoike et al., 2000). If this
construct is successfully introduced into Xenopus
sperm nuclei and these sperm nuclei are then used
for fertilization, transgenic Xenopus develop,
which, starting at approximately day 3 of development, express GFP exclusively in endothelial
cells. The vasculature of these animals is clearly
visible in video images, and similar experiments
have also been performed with zebrafish embryos
(Walsh and Stainier, 2001).
The third method to visualize the vascular bed
in transparent larvae was developed in our laboratory, and we named it digital motion analysis
(Schwerte and Pelster, 2000). Animals are videotaped using an inverted microscope and the vasculature is visualized by digital analysis of these
recordings. If the two fields of a video frame are
subtracted, any pixel in which no movement
occurred will have the grayscale value zero, while
in any pixel in which movement occurred the
grayscale value will be different from zero. Thus,
any movement that occurred within the 20 ms
necessary for the acquisition of one field can be
visualized. The length of the shifting vectors generated by this subtraction represents a direct measure for the velocity of a moving particle, i.e. an
erythrocyte in the vascular system. By accumulation, the differential images generated from several
subsequent video frames, a complete trace of the
routes on which erythrocytes moved can be
obtained. Thus, a cast of the vascular system,
except for those tiny vessels that are not entered
by erythrocytes, is obtained. Because the grayscale
value of any given pixel or any given group of
pixels increases with the number of erythrocytes
passing it, this method can also be used to visualize
the distribution of blood cells in transparent tissues.
Compared to the first two methods, this method is
much easier to use because it does not require
difficult dye injection and fixation of the animals,
and a developmental series can even be recorded
from a single animal without the necessity of
generating transgenic animals.
3. Stimulation of cardiac activity by lack of
oxygen
In order to test the possible flexibility of the
cardiovascular system during development, animals must be exposed to varied environmental
conditions, and one example is to raise animals
B. Pelster / Comparative Biochemistry and Physiology Part A 133 (2003) 547–553
under conditions of reduced oxygen availability
(Orlando and Pinder, 1995; Fritsche and Burggren,
1996). Soon after fertilization, fish eggs are surprisingly resistant towards a lack of oxygen.
Embryos of the Arctic charr (Salvelinus alpinus)
exposed to anoxia for 8 h show remarkable metabolic depression and a decrease in heart rate of
approximately 90% (Pelster, 1999), and zebrafish
embryos survive 24 h of anoxia in a state of
suspended animation, in which cardiac activity
ceases and mitotic activity of the blastomeres is
arrested (Padilla and Roth, 2001). The decrease in
heart rate and in cardiac output observed in very
early stages of fish and amphibians during periods
of oxygen deficiency has been attributed to a
direct effect of the lack of oxygen on the metabolism of cardiomyocytes (Orlando and Pinder,
1995; Fritsche and Burggren, 1996; Pelster, 1999).
In Xenopus larvae, co-ordinated stimulation of
cardiac activity in order to enhance convective
oxygen transport during periods of hypoxia was
only observed in later developmental stages.
While survival during episodes of severe hypoxia or even anoxia is only possible by marked
metabolic depression or by entering a state of
suspended animation, more or less normal developmental progress may be possible during chronic
exposure to mild hypoxia. We raised zebrafish
until complete opening of the swimbladder at
normoxia (i.e. PO2s20 kPa) and at a PO2 of 10
kPa at a temperature of 25, 28 and 31 8C. Heart
rate increased with development in normoxic as
well as in hypoxic animals at all three temperatures. At 25 and at 28 8C, heart rate was not
different in control and in hypoxic animals until
days 4 and 5 after fertilization, respectively. Then
heart rate was significantly elevated in hypoxic
animals, indicating that in hypoxic animals the
pacemaker was stimulated either by hormonal or
nervous activity.
Compared to heart rate, stroke volume appears
to be rather temperature-independent, with a Q10
value close to 1.0 in rainbow trout as well as in
zebrafish (Mirkovic and Rombough, 1998; Jacob
et al., 2002). A comparison of systolic and enddiastolic ventricular volume in zebrafish raised at
25 8C suggested that an increase in diastolic
volume in hypoxic animals caused an increase in
stroke volume at 4, 5 and 6 days post-fertilization
(dpf), which contributed to the increase in cardiac
output observed at this point of development.
Similar results were obtained at a temperature of
549
Fig. 1. Changes in cardiac output with development within the
first 4 (28 and 31 8C) to 5 days (25 8C) of development in
zebrafish larvae raised under normoxia and under hypoxia. 25
8C, ns10; 28 8C, ns16; 31 8C, ns12 (modified after Jacob
et al., 2002).
28 8C. In animals raised at 31 8C, however, the
hypoxia-induced stimulation of cardiac activity
was largely reduced compared to the effects
observed at lower temperatures. It could well be
that the high intrinsic activity of the ventricle at
this temperature significantly reduced the scope
for any further enhancement of cardiac activity
due to environmental conditions.
The influence of hypoxia on cardiac activity of
zebrafish larvae is nicely demonstrated by comparing the increase in cardiac output observed
between the first day of cardiac activity and the
time of complete opening of the swimbladder in
normoxic and in hypoxic animals (Fig. 1). In
normoxic animals, cardiac output increased in
animals raised at 31 8C, but there was only a
minor increase in animals raised at 25 8C and no
increase at all in animals raised at 28 8C. In
hypoxic animals, however, cardiac output significantly increased with development at all three
temperatures. Therefore, hypoxia clearly stimulates
cardiac activity in zebrafish larvae, and the first
response is observed at approximately 1 or maybe
even 0.5 days after hatching. Before this time,
severe hypoxia will only have a direct effect on
cell metabolism, resulting in inhibition of ventricular activity, but it cannot yet elicit a co-ordinated
response of the heart.
Not only cardiac activity, but also the distribution of blood to various tissues and even the
capillarization of tissues can be modified by
hypoxia in zebrafish larvae. After 7 days of hypox-
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Fig. 2. The influence of chronic hypoxia (7 days, PO2s10 kPa)
on gut perfusion and blood vessel formation in larval zebrafish.
At 7 dpf all larvae raised at normoxia have a perfused gut, but
in only 55% of hypoxic animals is blood flow to the gut
observed. The number of hypoxic animals showing the presence of intersegmental anastomosis connecting intersegmental
vessels in the tail region and the presence of the caudal vascular tree exceeds the number of normoxic animals showing
the presence of these blood vessels (S. Grillitsch, T. Schwerte,
B. Pelster, unpublished observation).
ia, we observed that in all control animals the gut
was perfused with blood. However, only in approximately 50% of the hypoxic animals was
blood flow to the gut observed (S. Grillitsch, T.
Schwerte, B. Pelster, unpublished observation). On
the other hand, blood vessel formation was
enhanced in hypoxic animals. Soon after hatching,
the intersegmental vessels become interconnected
by anastomosing vessels. In approximately 50%
of the normoxic animals the anastomosis is present
at 7 dpf, but approximately 80% of the hypoxic
animals show this pattern. In addition, a caudal
vascular tree is present in less than 10% of the
normoxic animals, but in approximately 15% of
the hypoxic animals (Fig. 2). Thus, hypoxia also
modifies organ perfusion and blood vessel
formation.
Shortage of oxygen supply can occur during
periods of reduced oxygen availability, but it can
also be induced by hypoxemia, i.e. by a reduction
in hemoglobin oxygen-carrying capacity. Hypoxemia can be experimentally induced by raising
zebrafish in the presence of CO, blocking the
hemoglobin oxygen-binding sites, or of phenylhydrazine, which destroys the hemoglobin. Zebrafish
raised until 4 or 5 dpf under these conditions have
a similar oxygen consumption compared to control
animals (Pelster and Burggren, 1996), and the
same is true for Xenopus larvae raised under
chronic CO exposure until Nieuwkoop Faber (NF)
stage 54 (Territo and Altimiras, 1998; Territo and
Burggren, 1998). Measurements of muscle lactate
concentration revealed no indication for a stimulation of anaerobic metabolism in zebrafish larvae
raised under hypoxemia until 14 dpf (Jacob et al.,
2002), and in Xenopus larvae raised under hypoxemia until NF stage 54 (Territo and Burggren,
1998). Cardiac performance of hypoxemic zebrafish is not different from control animals until 2
weeks after fertilization (Pelster and Burggren,
1996; Jacob et al., 2002). Territo and Altimiras
(1998) analyzed cardiac performance of Xenopus
raised under hypoxic, hypoxemic and hyperoxic
conditions and concluded that altering environmental PO2 does not modify global tissue perfusion
and cardiac output, but CO exposure enhanced
cardiac output in early developmental stages.
The obvious conclusion from these considerations is that convective oxygen transport is not
necessary in zebrafish larvae until approximately
2 weeks after fertilization if the PO2 gradient
through the skin is normal. If this gradient is
reduced due to a decrease in environmental PO2,
cardiac activity can be stimulated at approximately
0.5 or 1 day after hatching. This appears to be
even earlier than in the larger salmonid or Xenopus
larvae, where the hypoxic stimulation was not
observed before 1 day after hatching or even later.
Thus, the time in development at which a coordinated response of the ventricle becomes possible is not only governed by body mass.
4. The influence of chronic swimming activity
Hypoxic conditions can also result from
increased metabolic activity. This is possible, for
example, during periods of exercise, where the
increased activity of myosin ATPase significantly
stimulates oxygen consumption of the muscle cells.
Swim training leads to a modification of growth
rate and of food conversion efficiency in adult fish
(Wieser et al., 1988; Christiansen and Jobling,
1990). To assess possible influences of chronic
swimming activity on metabolism and growth,
zebrafish larvae were trained in a swim tunnel
system for approximately 15 h every day at a
water velocity of 5 bl sy1 (body length per second)
(Bagatto et al., 2001). Due to feeding require-
B. Pelster / Comparative Biochemistry and Physiology Part A 133 (2003) 547–553
551
(Johnston et al., 1977; De Graaf et al., 1990).
Adaptations are also possible on the level of
oxygen and nutrient supply to the muscle tissue.
This may include mitochondrial density, diffusion
distances in muscle tissue and an improvement in
convective oxygen transport (Hoppeler and Billeter, 1991).
Although thermal tolerance of larvae appears to
be lower in larval fish compared to adults and
sensitivity towards toxic chemicals appears to be
higher than in adult fish (Brett, 1964; Rombough,
1996), early larval stages do have some scope for
adaptations to changing environmental conditions.
Fig. 3. Active mass-specific oxygen consumption in free swimming zebrafish larvae as a function of water velocity. Larvae
were measured after the 11th day of the experimental period
of either no training (d) or training at 5 bl sy1 (m) (modified
after Bagatto et al., 2001).
ments, three different stage groups were trained:
yolk sac larvae, which cannot really be trained
because they cling to the swim tube and orient
towards the current; swim-up fry feeding on Paramecium; and free swimming larvae that feed on
Artemia. The training protocol had no influence
on growth of the larvae, although yolk absorption
was significantly faster in animals exposed to a
constant water current. An exciting observation
was that, compared to control animals, free swimming larvae after 11 days of training had a higher
MO2 at rest, and a significantly lower rate of
oxygen consumption in the swim tunnel at any
given water velocity (Fig. 3). The higher rate of
oxygen consumption at rest may be attributable to
an increased activity level at rest. The reduction
in MO2 during activity, however, clearly shows that
the efficiency of muscular contraction andyor the
efficiency of energy conversion have been
improved in trained animals. Furthermore, training
enhanced the survival of larvae during exposure
to low oxygen partial pressures (Bagatto et al.,
2001). Exposure of early larval stages of zebrafish
to a constant water current therefore induced physiological adaptations, resulting in enhanced swimming efficiency and in increased tolerance towards
hypoxia. The nature of these adaptations has not
yet been disclosed, but it could, for example, be
on the level of enzyme functioning in muscle cells.
Myosin ATPase activity or Ca2q pumping might
be more efficient, and the activity of enzymes
involved in energy metabolism may be involved
5. The influence of hormones
Several studies have shown that cholinergic and
adrenergic receptors are present and functional in
the vascular system of vertebrate larvae at the time
of hatching or even earlier (Kimmel, 1992; Pelster
et al., 1993; Fritsche, 1997; Fritsche and Jacobsson, 2000; Fritsche et al., 2000; Jacobsson and
Fritsche, 1999). In zebrafish larvae at 8 dpf, a
redistribution of blood can be achieved, for example by a-adrenergic stimulation (Schwerte and
Pelster, 2000). In addition, locally produced hormones such as NO (Fritsche et al., 2000) or
endothelin (Schwerte et al., 2001) contribute to
the regulation of peripheral resistance in zebrafish
and Xenopus larvae. In rainbow trout alevins
exposed for up to 4 weeks to the NO donor
isosorbide dinitrate (ISDN), significant dilation of
the vitelline vein was observed, combined with a
decrease in heart rate, but an increase in cardiac
output (Eddy et al., 1999). Thus, a redistribution
of blood can be achieved in early larval stages by
hormonal activity, and the responsiveness of the
vasculature to various hormones precedes the functional control of the cardiovascular system via the
autonomic nervous system (Fig. 4). Thus, control
mechanisms, an essential prerequisite for any adaptational response that may be elicited by environmental
perturbations,
are
available
at
approximately the time of hatching or even earlier.
The vasodilatory effect of NO contributes to
changes in local blood flow, and modifications of
sheer stress, for example, may in turn contribute
to angiogenesis. NO is an important modulator of
VEGF-induced vascular permeability and in
VEGF-induced angiogenesis (Conway et al., 2001;
Fukumura et al., 2001). Chronic exposure of
zebrafish to water containing SNP or ISDN, both
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B. Pelster / Comparative Biochemistry and Physiology Part A 133 (2003) 547–553
Fig. 4. Schematic drawing showing the timetable for the appearance of and or response to: CS, cholinergic stimulation; CT, cholinergic
tonus, CNF, cholinergic nerve fibers; AS, adrenergic stimulation; AT, adrenergic tonus; AEC, adrenergic endocrine cells; and ANF,
adrenergic nerve fibers, in three species of amphibians (modified after Fritsche and Jacobsson, 2000).
substances that release NO into the water, indeed
resulted in an earlier appearance of a small blood
vessel protruding ventrally from the caudal end of
the caudal vein, which was named the caudal
vascular tree. In control animals it is first observed
at 7 dpf, but only 10% of the animals show this
pattern at this time. At 12 dpf, 50% of the animals
show this caudal tree. In SNP-incubated animals,
this vessel first appears at 5 dpf, and by 7 dpf
almost 50% of the animals show it (S. Grillitsch,
T. Schwerte, B. Pelster, unpublished observation).
These results demonstrate that in early larval
stages peripheral resistance, as well as cardiac
activity, can be modified by various hormones.
NO also appears to exert an effect on blood vessel
formation. Although the autonomic nervous system
gains control over cardiac activity only late during
development (Kimmel, 1992; Protas and Leontieva, 1992; Fritsche, 1997; Jacobsson and Fritsche,
1999), hormonal control is apparently established
much earlier. This hormonal control, however,
should enable the larvae to respond to environmental changes.
6. Conclusion
and adjustments of cardiac activity to changing
environmental conditions in adult vertebrates, is
not established in embryonic and early larval
stages of fish or amphibians. Accordingly, at normoxic environmental PO2, convective oxygen
transport in the blood is not required until well
after hatching. Nevertheless, hormonal control of
peripheral resistance and of cardiac activity is
established prior to hatching. Using this control
system, larvae are capable of modifying cardiac
activity and peripheral resistance in response to
environmental changes, such as hypoxia or forced
exercise, even at a time when convective oxygen
transport is not yet essential to assure oxygen
supply to body tissues. Thus, there is some flexibility in the design and functioning of the embryonic or larval cardiovascular system, and it is not
operating simply on the basis of a given genetic
program.
Acknowledgments
Parts of the study were financially supported by
¨
the Fonds zur Forderung
der wissenschaftlichen
Forschung (FWF, P12571-BIO and P14976-BIO).
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