Cardiac performance in the zebrafish breakdance mutant
Institute of Zoology and Limnology, and Center for Molecular Biosciences, University of Innsbruck, Austria
* Author for correspondence: (e-mail: bernd.pelster{at}uibk.ac.at)
Accepted 29 March 2005
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Summary |
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Key words: Breakdance mutation, cardiac activity, temperature, development, cardiac arrhythmia, zebrafish, Danio rerio
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Introduction |
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One of these mutations identified in the Tübingen screen is the
breakdance-mutation (bretb218;
Chen et al., 1996), which has
been described as a mutation with an arrhythmic heart beat, i.e. the atrium
contracts twice while the ventricle beats only once
(Chen et al., 1996
;
Langheinrich et al., 2003
).
Similar arrhythmias have also been described for humans and linked to the
human ether-a-go-go-related gene (HERG). This gene encodes a channel
responsible for the rapidly activating delayed rectifier K+ current
IKr and is important for the repolarization phase of the cardiac
action potential (Vandenberg et al.,
2002
; Piippo et al.,
2000
; Sanguinetti et al.,
1995
). Langheinrich et al. cloned and sequenced the zebrafish
homolog of this gene (Zerg) and identified an amino acid replacement
in bre mutants (Ile59Ser;
Langheinrich et al., 2003
).
Furthermore, impairing the expression of this gene by using morpholinos
induced a 2:1 rhythm of the heart, an observation which supported the
conclusion that the bre mutation is a mutation in this K+
channel. This study, however, did not provide any evidence that the relaxation
period of the ventricle is indeed prolonged in the mutant.
Unfortunately, the only available physiological information about this mutant is that atrium and ventricle contract with a 2:1 rhythm. Particularly because the genetic basis of this mutation has been elucidated, it appears important to know what consequences a defect in this channel would have, for instance on cardiac output and blood pressure? Is there an indication for a prolonged relaxation period in the ventricle, as to be expected if this potassium channel (IKr) is affected? Is only the ventricle or the AV-node affected by this mutation or is there also a modification in the activity of the pacemaker cells? Furthermore, temperature has a significant influence on development and on cardiac activity in wild-type animals. Hence, it would be interesting to know if cardiac activity in these mutants is affected by temperature in a similar way. Answers to these questions will provide valuable insight into the pathophysiology of this cardiac arrhythmia and will provide further insight into the possible defects underlying this mutation.
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Materials and methods |
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Mortality recording
Larvae of breakdance and wild-type zebrafish were raised at
25°C, 28°C and 31°C. For both groups (bre and wdt) 10
independent batches with about 100 individuals in each batch were observed.
Larvae usually hatch at 3 d.p.f., and each subsequent day the dead larvae were
sorted out and the fraction of surviving larvae was calculated.
Experimental protocol and recording of cardiac activity
Cardiac activity was recorded between 3 d.p.f. and 14 d.p.f. Development
was faster at 31°C, so activity was recorded in this experimental group
until 9 d.p.f. in order to compare identical developmental stages. For
measurements larvae were removed from the incubation tank, anesthetized with
0.1 g l-1 tricaine (MS-222, pH 7.0) and embedded in 2.5% agarose
(dissolved in 0.1 g l-1 tricaine). To record cardiac activity the
embedded larvae were transferred to the incubation chamber in the
temperature-controlled microscope stage. The temperature was set to either
25°C, 28°C or 31°C. Increased mortality of the homozygous mutants
towards the end of the experiments meant that often only a few animals
survived until 14 d.p.f., so that the N-value had to be reduced for
older larvae. Cardiac activity was continuously recorded either for 1 min
(snapshot observation) or for 20 min. The 1 min period was chosen to measure
the incidence of the expression of the 2:1 rhythm in a large number of
animals. This value reflects the probability with which a mutant animal
selected out of a batch shows a 1:1 or a 2:1 rhythm during the first minute of
observation. After confirming that homozygous mutants do not show the 2:1
rhythm all the time, the 20 min period was chosen to measure heart rate and to
characterize individual variability in cardiac activity.
Video recording system
An inverted microscope (Leica, Vienna, Austria) was connected to a CCD
camera (Hamamatsu, Herrsching, Germany), which was plugged into the luminance
input of an SVHS video recorder (Sony, Vienna, Austria). The video cassette
recorder was remote-controlled via the RS232 serial communication
port. The recorded images were digitized by a monochrome frame grabber card
(Imagenation PX-610, Beaverton, OR, USA) with a personal computer (PIII 450
MHz).
Determination of heart rate, stroke volume and cardiac output
The rhythm of contraction (2:1 or 1:1) was classified during the beginning
of the observation period. Only animals showing a stable rhythm, either 2:1 or
1:1, were used for determination of heart rate, and animals that started
switching during the measurements were excluded from data analysis. Heart rate
(beats min-1) was determined by measuring the time interval
necessary for 30 heart beats. The average value obtained from triplicate
measurements was extrapolated to get the number of beats min-1 for
each individual fish. End-diastolic and end-systolic volumes of the ventricle
were surveyed by outlining the perimeter of the ventricle image using a mouse
or a graphic tablet. The perimeter was analyzed with a `fit-to-ellipse'
algorithm, which first calculated its center of mass and subsequently the
best-fitting ellipse (Jacob et al.,
2002). The major and minor axes were extracted and directly
transferred into a Microsoft Excel worksheet for calculation of stroke volume
using the formula for a prolate spheroid
(4/3*a*b2)
(Hou and Burggren, 1995
). For
analysis three diastoles and systoles were surveyed, and mean stroke volume
was calculated as the difference between diastolic and systolic ventricular
volumes. Cardiac output was calculated from heart rate and stroke volume.
Measurement of blood flow
Blood flow in the sinus venous was calculated from the velocity of the
erythrocytes, determined by digital analysis of the video recordings, and
blood vessel diameter. Details of this method have been described by Schwerte
and Pelster (2000).
Measurement of blood pressure
Blood pressure in intact slightly anesthetized larvae was measured
according to Pelster and Burggren
(1996) using a servo-null
micropressure system model 900A (World Precision Instruments, Berlin,
Germany). Larvae were anesthetized using 0.05 g l-1 MS222. A glass
electrode (tip diameter 5 µm) held in a micromanipulator was inserted
through the larval body wall into the lumen of either the ventricle or truncus
arteriosus to record central blood pressures. The output from the servo-null
system was recorded with a PC using the software package LabView. Mean
arterial pressure represents the arithmetic mean of the pressure curve
(LabView).
Statistical analysis
The acquired data were statistically analyzed by using a t-test
(software package STATISTICA). Significance was accepted when
P<0.05 and marked with an asterisk or listed in
Table 2. Data are presented as
means ± S.E.M.
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Results |
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Following the observation that the 2:1 rhythm was not expressed continuously in the larvae, we attempted to find out whether the expression of the 2:1 rhythm was related to development and/or temperature. A large number of larvae were screened for expression of the 2:1 rhythm at the onset of the observation period. The fraction of animals showing the 2:1 rhythm during the first minute at the beginning of the observation period changed with development and with incubation temperature (Fig. 2). At 3 d.p.f., only 35.0% of the animals raised at 25°C showed a 2:1 rhythm, while 52.5% of the larvae raised at 28°C showed the 2:1 rhythm at 3 d.p.f., and 75% of the animals raised at 31°C, respectively. Thus, at 3 d.p.f. the expression of the 2:1 rhythm in the mutants increased with increasing temperature, and the lowest fraction of animals with the 2:1 rhythm was observed at 25°C. With proceeding development, in the 28°C and 31°C groups the fraction of animals showing the arrhythmia significantly decreased. At 14 d.p.f. the fraction with a 2:1 rhythm amounted to 12.5% at 28°C, while at 31°C the fraction of animals with the 2:1 rhythm had decreased to 10% at 9 d.p.f. (Fig. 2). In animals raised at 25°C the number of animals showing the arrhythmia was very low at 4 d.p.f., and then increased to 21.67% at 7 d.p.f. Thereafter the fraction decreased again. In this series of experiments we attempted to reanalyze the same animals over several days. Given the high mortality at 31°C the same animals could not be observed throughout the experimental series, but it could be observed that some individuals expressing a more or less stable 2:1 rhythm had switched to the 1:1 rhythm on the next day and vice versa. Thus, the expression of the 2:1 rhythm decreased with development and was not sorted out by the dying of the animals showing the 2:1 rhythm.
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These experiments nevertheless revealed a higher mortality of mutant animals compared to wild-type larvae, especially at higher temperatures and at later developmental stages (Fig. 3). The highest survival rate of mutant animals was observed at 25°C, where bre larvae showed nearly the same survival rate as wdt animals until 11 d.p.f. Beyond 11 d.p.f. the survival rate of bre mutants was reduced compared to wild-type animals. In bre larvae raised at 28°C a steady decrease in the survival rate was observed from 9 d.p.f. on, reaching its minimum at 12-14 d.p.f. Survival of wild-type larvae also decreased from 11 d.p.f. on, but the mortality was significantly lower than in mutant animals. 31°C proved to be a very extreme situation. Deformations of the heart and impaired heart performance commonly occurred in both groups. Thus, survival had already started to decrease at 4 d.p.f., and the survival of bre larvae was significantly lower than that of wild-type larvae. At 5 d.p.f. only about 5% of the bre larvae survived, compared with 90% of wild-type animals. Afterwards survival remained almost constant, but at this temperature that of wild-type larvae was also remarkably low compared to lower temperatures. From 6 d.p.f. on it decreased continuously and very few larvae survived until the end of our observation period. In 6 of 10 batches none of the bre larvae survived until 9 d.p.f. Of the 1082 wild-type eggs at the onset of the experiment 809 larvae hatched, and on average only 30% of the wild-type larvae were still alive at 9 d.p.f.
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At 25°C most of the homozygous bre mutants showed a 1:1 rhythm. Heart rate started at about 110 beats min-1 at (3 d.p.f.), but then increased to 190 beats min-1 (8 d.p.f.), and dropped again to about 140 beats min-1 (13 d.p.f.; Fig. 4). These changes in cardiac activity with proceeding development paralleled the developmental changes in cardiac activity observed in wild-type animals, but the heart rate of bre mutants beating with the 1:1 rhythm was consistently lower than those of wild-type animals. Thus, throughout development, even with a 1:1 rhythm, bre mutants had a significantly lower heart rate (about 30-40 beats min-1) than wild-type animals (see Table 2 for significance). The bradycardia of bre mutants contracting in the 1:1 rhythm was even more pronounced at 28°C and 31°C. At both temperatures contraction rate in the 1:1 rhythm was 100-120 beats min-1, and there was no significant change with development (Fig. 4). In wild-type animals raised at these temperatures heart rate initially increased with development and than decreased, similar to the pattern observed in bre animals at 25°C.
Like heart rate, the stroke volume of larvae contracting with the 2:1 rhythm showed very little change with development at all three temperatures (Fig. 5). At 25°C the stroke volume of wild-type animals was significantly higher, but at 28°C lower, than in bre mutants (at 5 and 7 d.p.f. differences were statistically significant), except for the last few days of the observation period. After 11 d.p.f., stroke volume decreased in mutant animals, while that of wild-type animals increased, finally exceeding values of homozygous bre mutants. At 31°C, stroke volume of wild-type individuals was always lower than that of bre-mutants, and this difference was significant until 9 d.p.f.
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In an additional series of experiments blood pressure was measured in the ventricle and in the bulbus arteriosus of wild-type larvae and bre mutants at 2.5-3 d.p.f. (body mass 0.2-0.3 mg). At this early developmental stage, pressure could be measured by penetrating the body wall with the glass pipette, whereas at later stages the body wall became too thick, necessitating a preparation, which might have interfered with the pressure signal. While peak systolic pressure appeared to be similar in both experimental groups (Fig. 7), mean arterial blood pressure was slightly lower in breakdance larvae (0.31±0.02 mmHg; N=12), in contrast to 0.37±0.04 mmHg for wild-type larvae (N=17, Table 3; P<0.05). A comparison of pressure traces obtained from wild-type and bre animals showed that in mutants every second ventricular contraction was completely missing, and during this time ventricular and bulbus arteriosus pressure reached a plateau at about diastolic ventricular pressure. Compared to wild-type animals the final part of ventricular relaxation was extended. In wild-type animals the time between the increase of ventricular pressure at the onset of contraction and the return of the pressure trace to the diastolic level was 332.6±1.8 ms, while in bre mutants it was 464.7±3.8 ms (N=5; P<0.01). In the pressure traces this prolongation of ventricular contraction was sometimes clearly visible as a shoulder during the relaxation period (Fig. 7A). The delayed relaxation was not seen in either the pressure traces of the ventral artery (Fig. 7A), or in the bulbus arteriosus (Fig. 7C). Mostly the atrium contracted during this phase without emptying into the ventricle, inducing a visible back flow of erythrocytes away from the heart (Fig. 7D). The extended time period between subsequent ventricular contractions appeared to result in a slightly increased blood accumulation in the sinus venosus. Infrequently the ventricle took over additional blood from the second atrial contraction.
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Discussion |
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It is generally accepted that arrhythmias represent the end product of
abnormal ion-channel function, which can result from genetic mutations,
altered levels or spatial patterns of expression or from a modulation of
activity caused by ischaemia, for example
(Vandenberg et al., 2002).
Metabolic effects such as oxygen shortage can be excluded due to the small
size of the embryos, which do not need the circulatory system for oxygen
supply and are apparently well oxygenated just by bulk diffusion through the
skin (Territo and Altimiras,
2001
; Pelster,
1999
,
2002
). Thus, the slowed
repolarization of the ventricle can probably be attributed to a modification
of an ion channel. An unspecific cation channel, a Ca2+ channel and
a K+ channel (delayed rectifier channel) have been discussed in
context with cardiac arrhythmia (Baker et
al., 1997
). The phenotype of the bre-mutation appears to
phenocopy the human Long QT Syndrome
(Vandenberg et al., 2002
;
Shanbag et al., 2002
;
Curran et al., 1995
), which is
due to a mutation in a gene encoding a K+ channel
(Sanguinetti et al., 1995
). It
has been identified as a miss-sense mutation in the HERG-K+ channel
(HERG=human ether-a-go-go related gene)
(Huang et al., 2001
;
Piippo et al., 2000
), which
results in an abnormality in atrio-ventricular conductance and a prolonged QT
interval, and thus in an extended repolarization. Accordingly it appears
possible that a defective K+ channel is also underlying the
bre mutation in zebrafish, and Langheinrich et al.
(2003
) did indeed identify a
mutation in the Zerg gene that could be responsible for the
bre mutation. As a consequence of an extended QT repolarization, the
time course of the relaxation period following ventricular contraction may be
extended, and this was indeed observed in our pressure measurements. Compared
to wild-type animals the relaxation time of the ventricle was significantly
prolonged in bre mutants. This observation supports the conclusion of
Langheinrich et al. (2003
)
that the Zerg gene is responsible for this mutation, and that the 2:1
rhythm of bre animals is the consequence of an AV-block.
Not only was the relaxation time prolonged in bre mutants, but we
also observed a significant bradycardia in these animals in the 2:1 rhythm
(atrial frequency), and even more pronounced in the 1:1 rhythm, especially at
28°C and 31°C. This suggested that not only the QT interval of the
conductance system was affected, but that the pacemaker cells in the
sinoatrial node must be affected as well. Electrophysiological studies
revealed that the ERG channels (IKr) are expressed in mouse
pacemaker cells and pharmacological block of these channels results in a
bradycardia (Clark et al.,
2004). The bradycardia observed in bre animals thus
provides additional support for the conclusion that this mutation is due to a
defect in the delayed rectifier potassium channel. Slow mo has been
described as another zebrafish mutant with a chronically reduced heart rate,
and a defective pacemaker current (Ih) has been identified as
responsible for this mutation (Baker et
al., 1997
).
The influence of development
Our data clearly demonstrate that expression of the arrhythmia was
dependent on the developmental state of the larvae. With proceeding
development the expression of the arrhythmia was significantly reduced in
animals raised at 28°C and at 31°C. After the first beat, heart rate
typically increases with development, reaching a maximum after the first few
days of development and than decreasing slowly to an adult level
(Burggren and Warburton, 1994;
Pelster, 1997
). In zebrafish,
the highest heart rates are typically recorded until about 5 d.p.f. and 8
d.p.f., depending on temperature. During this time, the expression of the
arrhythmia had already dropped from about 55% at the onset of cardiac activity
to about 40% in 28°C animals, and at 31°C this decrease was even more
pronounced. Thus, in this phase of development the reduction in the expression
of the arrhythmia cannot be related to a concomitant change in heart rate. In
animals raised at 25°C the appearance of the 2:1 rhythm dropped until 5
d.p.f. and then increased again until 7 d.p.f. Several studies have discussed
possible reasons for development-related changes in membrane conductance in
pacemaker cells, but there appears to be no obvious explanation for this
change in the expression of the 2:1 rhythm with proceeding development. One
possibility might be a change in the expression pattern of ion channels
involved in the conductance of the electrical signal in cardiomyocytes. During
development different isoforms or even different ion channels may be
integrated into the cell membrane (Berthier
et al., 2002
; Schmidt et al.,
1999
) and so there may be an exchange of the defective channels by
functioning ones. Another possibility appears to be that the voltage
dependency of a channel changes with development, which has been shown for the
pacemaker current in rat ventricle, for example
(Robinson et al., 1997
). This
can be the consequence of a drifting of genes, a change in the structure of
the channel depending on the age of the larvae, or the presence or absence of
associated proteins. Growth of cells might also modify the electrical
properties and thus contribute to this phenomenon
(Verheijck et al., 2002
).
Apart from the prolonged relaxation period, comparison of the individual
contraction of the ventricle of a bre mutant and of wild-type animals
did not provide any indication for an impairment of ventricular performance in
mutant animals at the beginning of cardiac activity (3 d.p.f.). Peak systolic
blood pressure was similar in mutants and in wild-type animals, and normally
during the second atrial contraction ventricular filling did not increase,
obviously because ventricular pressure was still elevated and atrial pressure
could not open the atrioventricluar valve. Very rarely we observed that the
ventricle took over additional blood from the second atrial contraction.
Usually a retrograde movement of blood cells was observed in the sinus venosus
during the second contraction, and occasionally the shape change of the
chambers during the contraction without emptying caused a backwards movement
of red cells in the aorta dorsalis, but these cells did not return into the
ventricle. These observations clearly suggest a proper functioning of all
valves within the heart. Thus, the individual contraction of the ventricle
appeared to be normal except for the prolonged relaxation, and during
arrhythmia every second ventricular contraction was missing. This is in line
with previous descriptions of the mutant
(Chen et al., 1996;
Langheinrich et al., 2003
),
which stated that the arrhythmia was the only phenotype.
While the bradycardia was consistently observed in all mutants,
irrespective of the developmental stage and the temperature, the situation was
different for stroke volume. At 25°C, stroke volume was lower in
bre mutants than in wild-type animals, but at 28°C and at
31°C stroke volume was elevated compared to wild-type animals. In animals
raised at 25°C the bradycardia together with the reduced stroke volume
resulted in a significantly lower cardiac output in mutants, but even in
animals raised at higher temperatures the increase in stroke volume could only
partly compensate for the reduced heart rate, so that in all mutants cardiac
output was significantly lower than in wild-type animals. Until 8 d.p.f. at
28°C and 11 d.p.f. at 25°C the viability of mutants was not impaired
compared to wild-type animals, so that the reduced cardiac output did not
result in an increased mortality. This can probably be attributed to the fact
that in early developmental stages oxygen supply is achieved by bulk
diffusion, and convective oxygen transport is not required to ensure aerobic
metabolism of the tissues (Territo and
Altimiras, 2001; Pelster,
1999
,
2002
). Between 12 and 14
d.p.f., convective oxygen transport becomes essential to ensure adequate
oxygen supply to tissues (Schwerte et al.,
2003
; Jacob et al.,
2002
; Rombough,
2002
). The increased mortality of mutants observed at about 10 and
12 d.p.f. may therefore, at least in part, be related to the fact that at
about this stage convective oxygen transport becomes essential to meet the
oxygen requirements of the larvae, and the reduced cardiac output is not
sufficient to take on this task. At 31°C the situation was aggravated even
more. In our experiments at 31°C the viability of wild-type larvae was
actually significantly reduced compared to lower temperatures, despite
31°C being listed as a normal temperature for zebrafish development. The
mortality of mutants was extremely high from 5 d.p.f., and can probably be
attributed to the very high metabolic rate at this temperature, which requires
an earlier onset of convective oxygen transport in order to ensure aerobic
metabolism of all tissues.
The elevation of stroke volume observed at 28°C and at 31°C
obviously represented a partial compensation for the bradycardia caused by the
mutation in order to stabilize cardiac output. The pressure measurements
together with the data for cardiac output indicated that in mutants
compensatory changes also occurred in total peripheral resistance, which can
be calculated from the pressure difference P divided by blood
flow. While flow (cardiac output) was reduced by about 50% or more compared to
wild-type animals, systolic pressure was similar in mutants and in wild-type
animals, and mean arterial pressure was reduced by only 20% in bre
mutants, so that total peripheral resistance must be significantly elevated in
mutants. This suggests that a vasoconstriction may be present in order to
stabilize blood pressure in mutant animals.
Temperature dependence
A striking observation was an increasing expression of the arrhythmia at
higher temperatures. Screening of the larvae revealed that at 25°C only a
low percentage of the larvae expressed the 2:1 rhythm during the first days of
cardiac activity, while at 31°C the 2:1 rhythm was observed in more than
70% of the animals. It is well established that heart rate increases with
increasing temperature, and this is also true for vertebrate larvae during
early development (Pelster,
2002), which suggests that the expression of the 2:1 rhythm might
be especially pronounced at higher frequencies; but this conclusion was not
supported by the changes in the expression of the 2:1 rhythm observed with
development (see above). At 25°C, for example, expression of the 2:1
rhythm was higher at 3 d.p.f. than at 5 d.p.f., while heart rate usually
increases with development at this stage (see
Fig. 4, and also
Jacob et al., 2002
). At
28°C and at 31°C expression of the 2:1 rhythm was high in the earliest
stages, while the highest heart rate is observed about 2 days later (see
Fig. 4). Nevertheless, a
prolongation of the QT interval will most likely induce a 2:1 rhythm at high
frequencies. At low frequencies, low enough to permit completion of the
repolarization despite the fact that this repolarization is prolonged compared
to wild-type animals, the 1:1 rhythm should occur. If heart rate increases,
however, repolarization cannot be completed, and as a consequence the second
atrial depolarization reaches the ventricle in its refractory phase, and
atrio-ventricular conductance is interrupted (AV-block). The heart starts to
beat with a 2:1 rhythm. This may explain why expression of the 2:1 rhythm was
much lower at 25°C, but obviously cannot explain all of the
temperature-related changes in the expression of the 2:1 rhythm.
Studies on mutated ion channels of Drosophila revealed that
temperature may modulate the overall activity of ion channels. napts,
for example, is a recessive mutation affecting a sodium channel so that at
higher temperatures action potentials are lost
(Kernan et al., 1991).
Temperature has also been reported to regulate a potassium channel in
Drosophila selectively (Chopra
and Singh, 1994
). Therefore it may be that the effect of
temperature on the expression of the 2:1 rhythm is due to temperature related
changes in the activity of an ion channel.
Another striking observation was the temperature insensitivity of the
mutant heart, compared to wild-type animals. Ventricular frequency during the
2:1 rhythm remained at about 80-85 beats min-1, irrespective of
incubation temperature and development. In wild-type zebrafish larvae the
Q10 (i.e. the increase in activity encountered during a 10°C
increase in temperature) for heart rate varied between 1.2 and 2.5
(Jacob et al., 2002;
Barrionuevo and Burggren,
1999
). Accordingly, heart rate can be expected to increase with
rising temperature, but this was not observed after 5 d.p.f. at 28°C and
31°C and in mutants with the 2:1 rhythm. In the 2:1 rhythm mutants,
ventricular rate appeared to be independent of temperature, i.e. the
Q10 was about 1. An increase in incubation temperature, however,
caused a significant increase in the number of mutant animals expressing the
arrhythmia (see above).
A similar situation was observed when comparing heart rate in the 1:1 rhythm of animals raised at 28°C and 31°C. At both temperatures heart rate remained at about 110-120 beats min-1, demonstrating insensitivity to temperature. At 25°C the situation was different. In these animals heart rate showed some changes with development and reached values of about 160 beats min-1, which exceeded even that observed at higher temperatures, resulting in a reversed temperature effect between 25°C and 28°C. The results obtained with the 25°C group differed from the results of the higher temperature animals in several respects. In contrast to the 28°C and 31°C group, in this class developmental changes in heart rate were observed, and atrial contraction rate during the 2:1 rhythm and heart rate during the 1:1 rhythm were similar except for the time between 8 and 10 d.p.f. In the other two groups heart rate in the 1:1 rhythm was significantly lower than atrial rate during the 2:1 rhythm. Based on the general effect of temperature we would expect that heart rate in the 25°C group is lower than in the 28°C and 31°C class. Accordingly, this observation may again indicate that higher temperatures, and thus higher heart rates, facilitate the expression of the 2:1 rhythm, and that the negative aspects of this mutation can best be compensated at lower temperatures.
In summary, we can say that the bre mutation does not imply the
continuous expression of a 2:1 rhythm. An elevated ventricular pressure during
the relaxation phase and the bradycardia support the conclusion that an ion
channel involved in the repolarization of the ventricle is defective in mutant
animals. A possible candidate for this ion channel would be a potassium
channel, as proposed by Langheinrich et al.
(2003), but the mutation not
only affects the signal transduction to the ventricle, but also the pacemaker
itself. This was clearly demonstrated by the temperature sensitivity of mutant
hearts, which differed largely from the temperature sensitivity of wild-type
hearts. The bradycardia inevitably resulted in a significantly reduced cardiac
output, although at 28°C and at 31°C a compensating elevation of
stroke volume was observed. Measurements of blood pressure and of cardiac
output also indicated that in mutants peripheral resistance is elevated in
order to stabilize blood pressure. Nevertheless, at least in later stages, the
viability of mutants is significantly reduced compared to wild-type animals,
although bre mutants may survive until adulthood, especially at lower
temperatures.
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Acknowledgments |
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