Form and function of the bulbus arteriosus in yellowfin tuna (Thunnus albacares): dynamic properties
1 Department of Zoology, Cambridge University, Downing Street, Cambridge,
UK, CB2 3EJ,
2 Cooperative Marine Education and Research Program, Virginia Institute of
Marine Science, PO Box 1208, Greate Rd, Gloucester Point, Virginia 23062,
USA,
3 Department of Zoology, University of British Columbia, Vancouver, BC,
Canada, V6T 1Z4
4 Zoology Animal Care, 6199 South Campus Road, University of British
Columbia, Vancouver, BC, Canada, V6T 1W5
* Author for correspondence (e-mail: mhb31{at}cam.ac.uk)
Accepted 27 June 2003
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Summary |
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Key words: bulbus arteriosus, P-D loop, r-shaped curve, video dimensional analysis, tuna, Thunnus.
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Introduction |
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Like an artery, the bulbus is composed of elastin, collagen and smooth
muscle; however, it is highly modified, resulting in specialized inflation
properties (Braun et al.,
2003). Over the in vivo pressure range, an artery has a
J-shaped P-V (pressure-volume) loop, while the bulbus has an r-shaped P-V
loop. The bulbar curve can be broken into distinctive stages: (1) a sharp
initial rise in pressure for a relatively small volume change and (2) a
plateau stage where the bulbus is largely unaffected by even large changes in
volume. There is even some evidence to suggest that there is a third stage of
the bulbar inflation (Braun et al.,
2003
); when greatly distended, the bulbar material rapidly
increases in stiffness.
Stage 1 is due to the relationship between the wall tension, pressure and volume of the bulbus, as described by the Law of Laplace. The bulbar lumen is very small at low pressure and therefore bulbar expansion requires a large initial pressure increment. Stage 2 is a result of the specialized material properties of the bulbus. The bulbar wall has a very high elastin:collagen ratio and is almost entirely composed of novel elastin (low hydrophobicity, high solubility) aligned in a novel manner (loose fibrils, no lamellae). These modifications produce very low wall stiffness and the ability to undergo large strain changes and result in the compliance of the plateau. At large extensions, stiff adventitial collagen is recruited to resist the expansion of the bulbus.
Knowing the causes of the strange bulbar P-V loop is an important first step in understanding how the bulbus works. However, in order to make inferences based on the in vitro inflation curve, it is vital that the bulbus shows similar traits in vivo. To this end, in vivo changes in pressure and bulbar diameter during normal beating in anaesthetised yellowfin tuna were recorded using video dimensional analysis (VDA) and pressure recordings.
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Materials and methods |
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Yellowfin tuna were anaesthetized using 0.2 g l-1 ethyl p-amino benzoate and equimolar NaHCO3. Following anaesthesia, the fish were placed supine in a chamois leather cradle. A hose running aerated seawater was placed in the mouth of the anesthetized fish in order to simulate ram ventilation. A midline incision was made along the ventral surface to expose the pericardial cavity. The pericardium was opened and the heart exposed. During the experiment, anesthesia was maintained with Saffan (3 mg kg-1 intra-arterially; Glaxovet, Harefield, UK). To decrease heart rate, water flow over the gills was stopped for several seconds.
Arterial blood pressure was measured through a cannula inserted into the
bulbus and connected to a Unonics model P-106 pressure transducer (Wayland,
MA, USA) (Fig. 1). Pop tests
established the frequency response of the system to be 32 Hz, with damping
being 0.12 of critical damping (Jones,
1970). Changes in the diameter of the bulbus during systole and
diastole were measured using a video dimension analyzer (VDA; Instrumentation
for Physiology and Medicine, model 303). This system consists of a video
camera, a video processor and a monitor. The camera was focused on the bulbus,
and the signal fed through the processor. The VDA utilizes the video signal to
give a DC voltage that is proportional to the distance between two selected
contrast boundaries on the monitor. The VDA `window'
(Fig. 1) was used to track the
movement of the outside surface of the bulbus as it expanded and contracted
during systole and diastole. By calibrating the voltage generated, dynamic
dimensional changes were recorded. The VDA has a 15 Hz low-pass RC filter on
the output signal, which introduces 180° of phase delay at 15 Hz.
Appropriate corrections were applied to the diameter traces. For a more
in-depth explanation of the VDA, see Fung
(1981
). Voltages and pressures
were collected and stored using DASYLAB (Dasytec USA, Amherst, NH, USA).
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Following dynamic recordings of heart beats from tuna, static in vivo inflations of the bulbi were performed. The proximal ventral aorta and the bulbo-ventricular junction were ligated, and a T-junction was inserted into the pressure catheter to allow simultaneous bulbar inflation and pressure measurement. Measured volumes of saline (25°C) were injected into the bulbus, and the resultant pressure signal was amplified and recorded using DASYLAB software. Cycles of inflation and deflation were performed until consistent results were seen. Preconditioning usually required 5-10 cycles. These initial cycles were discarded. Each experiment consisted of 8-15 trials, and results from any trials in which a loss of more than 5% of the injected saline occurred were not used. After preconditioning, the data were recorded and plotted as pressure (kPa) versus volume (ml or µl). By simultaneously measuring the diameter changes due to each injection of fluid, it was also possible to create a plot of pressure versus diameter. Linear regressions of the curves yielded calibration curves describing the interactions between injected volume and diameter. Due to the differences in dimension along the bulbus, the dynamic and static measurements must be taken from the same locations. This was not the case for all observations, and, therefore, calibration curves could not be calculated for all recordings of pressure and diameter. These recordings of pressure and diameter were analyzed using Microsoft EXCEL.
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Results |
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When the heart was beating normally [i.e. heart rate approximately 1 Hz,
peak systolic pressure around 9.5-13.5 kPa and pulse pressure in the range of
5-6.5 kPa(Jones et al.,
1993)], the range of dynamic circumferential strains was
0.25-0.38. Ventricular movements moved the position of the bulbus within the
pericardial cavity so that finding the dynamic longitudinal range of strain
was not possible.
Static and dynamic P-V loops
The VDA followed the walls of the yellowfin tuna bulbus during both systole
and diastole and allowed mapping of dimensional changes associated with each
heartbeat (Fig. 1).
Fig. 2A compares the
dimensional and pressure changes occurring during a single heartbeat. The
rapid increase in pressure resulted in a sharp increase in diameter. Systolic
pressure of 9.3 kPa gradually declined to 4 kPa, while diameter initially fell
very rapidly, followed by a smoother decline that more closely followed the
fall in pressure. By plotting pressure against diameter for the heartbeat in
Fig. 2A, a pressure-diameter
(P-D) loop was generated (Fig.
2B),showing the inflation behaviour of the bulbus under in
vivo conditions. When this dynamic P-D loop was compared with a P-D loop
produced using the static inflation technique, the dynamic and static
behaviours matched well. In both cases, the slope initially rose sharply,
followed by a levelling off as the bulbus reached the plateau phase of the
inflation.
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The difference between the areas under the inflation and deflation curves is the amount of energy lost as heat. When this loss is normalized to the area under the inflation curve, the resulting percentage is known as hysteresis. There was significant hysteresis in both loops, indicative of a viscous element in the bulbar wall. In the dynamic loop, the larger hysteresis was due to the increased rate at which the bulbus was inflated. The faster the changes in the dimensions of a viscous element, the stiffer it becomes, and more energy is lost executing the changes.
During the initial rise of the r-curve, the inflation and deflation curves crossed over, indicating energy added. The bulbus lacks cardiac muscle and cannot contract beat-to-beat; therefore, the positive work loop was due to the changing length of the bulbus. The change in the length of the bulbus could be clearly seen at the end of the deflation (Fig. 2B). While pressure continued to drop, the diameter of the bulbus increased, indicating wider, upstream segments entering the field of view of the video camera.
The dynamic P-D loop in Fig.
2B demonstrates bulbar behaviour over the pressure range of 4-9.5
kPa. However, by looking at beats covering the pressure range of 2.5-21.5 kPa,
the features of the static bulbar inflation curve
(Fig. 3A) were recreated: the
initial steep rise, the plateau and the final steep rise at large inflations
and high pressures (Braun et al.,
2003). At the low end of the pressure range, the bulbus was
operating on the steep part of the inflation curve and small changes in volume
resulted in large, rapid changes in pressure
(Fig. 3B). A small,
low-pressure heart beat generated a very steep P-D loop. For a heart beat
covering the pressure range 11.3-12.6 kPa, the P-D loop showed that the bulbus
was inflating entirely on the plateau (Fig.
3C); the loop was horizontal, with very little vertical component.
The P-D loop from a heart beat over the range of 15.3-22.6 kPa showed that,
while the bulbus operated on the plateau for much of the beat, at very high
blood pressures bulbus stiffness rapidly increased
(Fig. 3D).
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In Fig. 4A, following a prolonged cardiac interval (marked with an asterisk), the smallest increase in diameter resulted in the generation of the largest pressure pulse. As peak pressure increased, subsequent pressure pulses became smaller while diameter changes increased. The first fluid injection after the long cardiac interval had a larger impact on the pressure than those that followed. The highlighted beat (marked with an asterisk) is equivalent to the sharp initial rise of the static inflation tests.
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Immediately after Saffan injection, the tuna hearts often deviated from
normal beating patterns by speeding up and/or increasing pressure. In a fish
recently injected with Saffan (Fig.
4B), the lowest diastolic pressure was 6.7 kPa, while the highest
pressure was 20.6 kPa. During a period of declining pressure, there was an
occasional small pressure `blip" (arrows in
Fig. 4B). The small increases
in pressure (0.13-0.67 kPa) were associated with large changes in the diameter
of the bulbus. These diameter changes were frequently as large as those
associated with pressure changes that were 20 times larger (diamond in
Fig. 4B). In the high-pressure
range (13.3-16 kPa), large volume changes (as evidenced by large changes in
bulbar diameter) result in relatively small changes in pressure, indicating
that the bulbus was on the plateau stage of static inflations.
At pressures greater than 17.3 kPa, however, small volume changes resulted in large changes in pressure. In Fig. 4C, the highlighted beats (triangles) had peak systolic pressures of 17.3 kPa, 22.7 kPa and 22.2 kPa. However, the resultant systolic diameters only varied slightly (1.077-1.095 cm) and the systolic-diastolic diameter changes were 0.18-0.23 cm. The insensitivity of the bulbar pressure to volume injections disappeared at very high pressures due to a rapid increase in the stiffness of the bulbar wall.
Dynamic P-D loops (Figs 2, 3) had the same features as the static P-V loops, and bulbar diameter seemed to be an accurate indication of bulbar volume. The validity of this assumption was checked by static inflations. Static r-shaped P-V loops were generated using the in situ VDA preparations after dynamic experiments (Fig. 5). The relationship between diameter and volume was linear for examples from both the anterior (Fig. 5C) and posterior (Fig. 5D) portions of the bulbus.
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Fig. 6 is an example of a typical beating pattern for an anaesthetized yellowfin tuna. The diameter and pressure were measured at a point near the middle of the bulbus. At a heart rate of 1 Hz, pulse pressure was approximately 6 kPa with a peak systolic pressure of 10 kPa. Diameter changes were about 0.1 cm, from 0.7 cm to 0.8 cm. Fig. 6B illustrates how these diameter changes translated into volume within the bulbus. The 0.1 cm-diameter change resulted in bulbar volume varying from 0.2 ml to 0.8 ml.
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Bulbar volume fell when blood pressure and heart rate decreased. In Fig. 7A,B, the heart was beating normally during the first 12 s, after which it began to slow from a rate of 1.2 Hz to 0.8 Hz. Bulbus diameter and internal volume began to fall, and, between 20 s and 25 s, the heart appeared to miss several beats, resulting in long diastolic periods. During these periods, bulbar volume fell close to zero. However, even at these low internal volumes, the pressure remained at 2.7 kPa (Fig. 7A,B).
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Fig. 7C shows a VDA recording of the ventricular end of the bulbus from a fish shortly after an inter-arterial injection of Saffan. The heart was beating extremely fast (4 Hz) and pulse pressure was approximately 2 kPa (10.6-12.6 kPa). Despite the small pulse pressure, the changes in diameter (0.08 cm) were nearly as large as in Figs 6B, 7B. This suggests that the bulbus was on the plateau phase of the r-shaped curve; large changes in volume generated small changes in pressure. Indeed, the volume changes (0.6-1 ml) seen in Fig. 7D were similar to those in Fig. 6.
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Discussion |
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Qualitatively, the linear relationship between diameter and volume allowed the inference that a change in diameter was due to an equivalent change in volume: if one heart beat resulted in a bulbus diameter change twice as large as another, then twice as much fluid entered the bulbus during that beat. Quantitatively, the fact that a linear regression closely described the interaction between diameter and volume (Fig. 5) allowed an analysis of the volume into and out of the bulbus with each beat.
The features of the static bulbar inflation curve
(Braun et al., 2003) occur
in vivo. Blood initially entering the bulbus caused a large jump in
pressure, followed by a stage in which large volume changes result in small
pressure differences. At very high pressures, bulbar stiffness rapidly rose,
and the ability to expand further was limited.
Both the sharp initial rise in pressure and the compliant plateau phase are
important in the bulbus' function as a pressure reservoir. Due to the Law of
Laplace (tension = pressure x radius), the relatively small internal
lumen of the bulbus results in a negligible tension in the bulbar wall at low
pressures (Braun et al., 2003).
This small internal radius necessitates a large pressure in order for
expansion to occur and results in the large initial jump in the bulbar P-V
loop (Fig. 8A). The larger
lumen radius of an artery allows much larger changes in volume at low
pressures due to the larger tension generated
(Fig. 8A). For the example in
Fig. 8B, the tension initially
created in the artery is over four times larger than in the bulbus. Arteries
generally expand 40-50% when pressurized to physiological ranges
(McDonald, 1974
). In the
yellowfin tuna bulbus, going from zero to physiological pressure requires a
strain of around 10%. The bulbus can reach the same pressure as an artery at a
fraction of the volume (Fig.
8A, broken arrow), which allows the bulbus to become `primed' to a
high pressure with a single heartbeat, regardless of cardiac output.
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When stroke volume is high, the compliance of the bulbus allows it to expand and `absorb' excess fluid while preserving a relatively constant pressure head. Even when stroke volume is low, the bulbus will maintain blood flow through the gills at a high pressure. Following a long diastolic period, the first heartbeat will have a larger effect on pressure than any following beats (Fig. 4A). The benefit of the bulbar design is that it allows the bulbus to behave similarly under both high and low cardiac outputs.
In rainbow trout (Oncorhynchus mykiss), the bulbus is most
compliant near the systolic pressure (Clark
and Rodnick, 1999), and the same phenomenon occurs in yellowfin
tuna. During systole, increasingly large changes in volume result in
relatively small pressure increases. Once systolic pressure has been reached,
the compliant plateau of the bulbus allows it to effectively `store' pressure,
despite large increases in volume. During diastole, the plateau allows the
bulbus to maintain a high pressure while internal volume is decreasing. In
fact, the bulbus can lose most of its volume, and pressure only falls by a
small amount (Fig. 8A, solid
arrows). In this manner, the bulbus extends the proportion of the cardiac
cycle during which blood flows into the gills
(Randall, 1968
;
Stevens et al., 1972
).
Over a physiological pressure range of 4 kPa, the bulbus can hold and
return 90% of its volume (Fig.
8A, thick, solid arrows), compared with only 15% in an artery.
Bulbar volume changes of 0.2-0.8 ml yield circumferential strain changes of
30-40% (Fig. 8B). This
behaviour is in stark contrast to arteries, which typically experience
circumferential strains of 2-7% during an inflation cycle
(McDonald, 1974). These large
differences between the behaviour of arteries and bulbi illustrate two
different means to the same end. Both bulbi and arteries are designed to
increase the capacitance in the circulatory system in order to depulsate and
attenuate flows and pressures. Capacitance in arteries is achieved through
length. Even a relatively inextensible tube can provide significant
capacitance if it is of sufficient length. Teleosts lack the luxury of a long
arterial tree separating the heart from the gills. Instead, capacitance is
increased by the bulbus and its r-shaped inflation. The tremendous compliance
of the bulbus on the plateau of its P-V loop results in a large volume change
(
V) over the physiological pressure range and allows a
relatively short bulbus to greatly increase the capacitance of the teleost
arterial system. Furthermore, an artery needs to be almost completely filled
in order to reach a high pressure, at which point a rapid increase in
stiffness occurs. Working against the very rigid walls of an artery-like
bulbus would increase the work of the heart. During diastole, a small amount
of fluid loss in an artery results in a rapid fall in pressure. The bulbus
ordinarily experiences large volume changes; in an artery-like bulbus, much of
the diastolic period would occur at low pressure, reducing the flow of blood
though the gills.
The bulbus is capable of both expanding to store cardiac output and
recoiling elastically to return the stored fluid to the circulation. When
contracted, bulbar volume is smaller than a single stroke volume. However, the
bulbus is capable of holding a very large blood volume: 200-300% of stroke
volume (Bushnell et al., 1992).
During diastole, it is important to have some way of maintaining blood flow
through the gills. Observation of the bulbus' in vivo functioning
shows that it maintains a reservoir and never completely empties during
diastole (Figs 6,
7). Following a bradycardia,
the bulbus `pumps' up until the reserves are replenished
(Fig. 7A,B). The bulbar
reservoir allows positive flow to occur during long diastolic periods. In ling
cod (Ophiodon elongatus), blood flow in the ventral aorta due to the
elastic rebound of the bulbus arteriosus represents about 29% of total cardiac
output (Randall, 1968
).
The central location of the bulbus has benefits to the teleost circulatory
system. A model study performed by Campbell et al.
(1981) showed that a large
compliance located far from the heart is equally good at raising diastolic
pressure as one located proximally, but only a compliance located directly
outside the heart effectively decreases peak systolic pressure. An elevated
diastolic pressure ensures continuing flow through peripheral vascular beds
during diastole, while decreasing the peak systolic pressure translates into
large cardiac energy savings by lowering the tension-time integral during
cardiac contraction. The majority of the heart's work is involved in
generating tension rather than in ejecting blood from the heart.
(Jones, 1991
). Therefore, the
position of the bulbus in the teleost circulation, just distal to the heart,
makes it of great importance for increasing the overall efficiency of the
piscine cardiovascular system.
At large volumes, the bulbus increases in stiffness
(Braun et al., 2003). As in
arteries, this feature may serve a similar strain-limitation function in the
bulbus. However, the pressures at which this rise in stiffness occurs are
extreme. In yellowfin tuna, pressures in excess of 20 kPa were required for
the final increase in stiffness to occur. Normal static inflations were never
taken to this level for two reasons: (1) the preparations would begin to leak
and (2) the very high pressures are far above the normal in vivo
pressure range (Jones et al.,
1993
). Clues suggesting that the bulbus did indeed possess a final
rise in stiffness came from several sources. Braun et al.
(2003
), after dissecting out
the bulbar media, demonstrated bulbar inflations with a rapid rise in
stiffness at large volumes. In the present study, the analysis of very high
(>20 kPa) blood pressure traces showed that, in contrast to what ordinarily
occurs on the plateau, large jumps in pressure were occurring for very small
changes in dimension (Figs 3,
4C), indicating an increased
stiffness. In reality, however, the bulbar location within the pericardium
means that the third phase may never be attained in an intact animal. An
extremely full and swollen bulbus could interfere with the functioning of the
atrium, reducing cardiac output and causing the bulbus to empty and shrink,
while the very rigid pericardium found in these fish would also limit the size
to which the bulbus could expand.
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Acknowledgments |
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