Stroke volume and cardiac output in juvenile elephant seals during forced dives
1 Department of Zoology, University of British Columbia, 6270 University
Blvd, Vancouver, British Columbia V6T 1Z4, Canada
2 Department of Biology, Sonoma State University, 1801 East Cotati Avenue,
Rohnert Park, CA 94928-3609, USA
3 Department of Biology, University of California, Santa Cruz, Santa Cruz,
CA 95064, USA
4 Department of Radiology, Stanford University, Lucas Center for MR Imaging
and Spectroscopy, Stanford, CA 94305, USA
* Author for correspondence (e-mail: thornton{at}zoology.ubc.ca)
Accepted 11 July 2005
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Summary |
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Key words: diving, elephant seal, Mirounga angustirostris, cardiac output, stroke volume, magnetic resonance imaging
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Introduction |
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In light of the reported variation in
measurements and
VS calculations in diving pinnipeds, as well as
differences in interpretation, our objective was to elucidate the effect of
diving on cardiac dynamics using MR Imaging and phase contrast flow analysis.
Using this approach,
was measured
before, during and after forced dives in restrained juvenile northern elephant
seals, Mirounga angustirostris (L).
Elephant seals are ideal subjects for a study of cardiac function because
they stand out among diving mammals for their deep, long-duration, continuous
diving (Le Boeuf et al., 1988,
1993
,
2000a
). The diving pattern of
juveniles, although reduced in scale, is similar to that of adults. By one
year of age, after having spent five months at sea foraging, they are
accomplished divers with oxygen stores equivalent to 84% of adult female
storage ability (Thorson and Le Boeuf,
1994
). The dives of yearlings average 15 min duration, with only 2
min at the surface between dives; 88% percent of the time at sea is spent
underwater (Le Boeuf et al.,
1996
). Heart rate of free-ranging juveniles decreases from a mean
107 beats min-1 at the surface (between dives) to a mean of 35
beats min-1 during dives, with maximum decreases observed as low as
3 beats min-1 (Andrews,
1997
; Webb et al.,
1998
; Andrews et al.,
2000
; Le Boeuf et al.,
2000b
). Juvenile elephant seals are also excellent subjects for MR
Imaging, as they are amenable to restraint, transport and handling.
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Materials and methods |
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Prior to imaging, animals were fitted with a diving helmet manufactured
from a 35 cm-diameter Plexiglas tube, an inner neoprene seal and a secondary
outer latex neck seal. The seal was allowed to acclimate for approximately 30
min, during which time a vacuum hose was attached to the helmet to ensure
sufficient airflow through the open valves. At the initiation of a diving
experiment, the vacuum hose was removed and the helmet was filled with cold
water. Timing of the forced apnea or `dive' commenced when the animal's
nostrils were completely submerged and continued until the helmet was drained
and the first inspiration occurred. Each animal was subjected to 2-5 forced
dives with a mean duration of 6.28±1.07 min (± S.D.).
These forced dives were approximately 63% of the mean duration of natural
dives of similar aged juveniles at sea (Le Boeuf, 1994) and slightly less
duration than sleep apneas of juveniles sleeping on the beach
(Blackwell and Le Boeuf, 1993).
Minimum time between dives was 13 min to allow for full recovery. Cardiac
measurements were collected during the pre-dive, dive and post-dive periods.
Values referred to as `resting' were obtained during the quiescent 5 min
period immediately preceding a dive.
Heart rate
At the MR unit, the seal was physically restrained while four ECG
electrodes were glued to the ventral surface of the animal using cyanoacrylate
adhesive. The seal was then placed in a custom-made conical nylon jacket and
strapped to a restraining board in a prone position. The board was placed in a
PVC cradle that served as a fluid containment unit to prevent water damage to
the magnet. Once in position on the magnet bed, the ECG leads were connected
and the strap of the respiratory bellows was threaded under the animal at the
level of the diaphragm. Heart rate data were collected throughout the
experimental protocol on a Macintosh IISI with an 8-channel PowerlabTM
(ADInstruments, Colorado Springs, CO, USA) interface and an ML 132 BioAmp.
Pre-dive heart rates were collected during the 5 min acclimation period
immediately preceding the first dive. Heart rate was acquired by recording
interbeat intervals using ChartTM (ADInstruments).
MR Imaging
All images were collected using a high-performance 1.5 T system (Signal
Horizon Echo Speed, GE Medical Systems, Milwaukee, WI, USA). Localizer images
were acquired in the axial plane at the base of the aortic bulb
(Fig. 1; Movie 1 in
supplementary material). Phase contrast (PC) MRI was used to acquire
through-plane velocity data using a conventional 2-D cine PC sequence and an
experimental cine-spiral PC sequence (Liao
et al., 1995). Scan parameters were TR=55, TE=6.2, NSA=1,
FOV=48x48 cm2 and slice thickness=7 mm. The spiral sequence
used 12-16 interleaves, allowing much faster data acquisition than the
conventional sequence.
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Statistical analysis
Statistical analysis of physiological parameters was conducted using JMP
3.2.1; reported values are means ± S.E.M. unless otherwise
indicated.
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Results |
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Cardiac output
Mean cardiac output () during
diving (4011±387 ml min-1) and resting (6530±1018 ml
min-1) was not significantly different (paired t-test;
P<0.055, N=4).
Stroke volume
Stroke volume (VS) increased significantly during
forced dives from a mean resting level of 104.9±4.1 ml to a mean value
of 126.1±3.9 ml (paired t-test; P<0.008,
N=4). fH ratio (mean diving
fH/pre-dive fH) represents the diving
bradycardic response (Fig. 2)
and is negatively correlated with stroke volume (r2=0.98,
P=0.01).
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Discussion |
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Experimental procedure
Velocity-encoded cine MR imaging, or MR flow velocity mapping, is
increasingly used clinically for the quantification of arterial and
transvalvular blood flow. Velocity and flow rate measurements with PC MRI have
been repeatedly validated (e.g. Evans et
al., 1993; Pelc et al.,
1992
). The principal source of error on the measured velocity is
eddy currents induced by switched gradients, which can introduce a non-zero
baseline. This is generally well controlled using corrections based on the
apparent velocity of tissues known to be static. Flow measurements require
integration over the vessel lumen, and errors can be introduced by imperfect
definition of this region. This is more serious for small vessels than for
channels as large as the aortic root. For more details on these effects, see
Pelc et al. (1994
).
Almost all cardiac MRI techniques employ a data acquisition protocol that is gated by ECG, which serves to eliminate motion artifacts due to the contractile motion of the heart. However, gated cardiac studies are reliant on the ability of the MR system to accurately assess each point in the cardiac cycle and require a significant amount of time to acquire a series of images. For the most part, the animals used in this study maintained a steady fH during the dive except during periods of struggling, where fH would increase during movement, then drop for a period of 5-10 s in the post-movement phase before leveling. We used a spiral sequence that employs 12-16 interleaves, allowing for much faster data acquisition than the conventional sequence. Application of this technique to 2-D slice data allows for computation of flow over the entire cardiac cycle by interleaving data from different beats. When the acquisition of a clear ECG signal was occasionally impaired by animal movement or lead displacement, the gating sequence would `stall' until a suitable ECG signal was obtained. The reconstructed cardiac cycle would then consist of images obtained over a period of 1-2 min. As the average fH for any given minute of the dive was not significantly different from the mean diving fH, flow data based on reconstructed cardiac cycles was accepted as an accurate representation of blood flow during the dive.
Diving ability
A key variable for explaining the results of forced dive studies on cardiac
function is the diving schedule imposed on the seal and the diving
capabilities of the subjects. Juvenile elephant seals have the capacity to
dive longer than adult California sea lions (Zalophus californianus),
grey seals (Haliochoerus grypus), harbor seals (Phoca
vitulina) and Weddell seals (Leptonychotes weddelli), the
subjects of other similar studies. The diving schedule we used, dives of 6 min
followed by a surface rest of at least 13 min for complete recovery, is
shorter in duration than dives performed in nature. On their first trip to
sea, juvenile elephant seals exhibit a continuous diving pattern similar to
adults and are submerged 85% of the time. The mean dive duration is about half
that of adults, with a maximum of 22.3 min. Dives at sea are normally
undertaken as part of a continuous diving cycle, or `bout', with brief surface
intervals between 1.4 and 1.8 min. This results in at-sea pre-dive heart rates
that are strongly influenced by the previous dive and are tachycardic when
compared with resting rates. The short surface interval could not be emulated
under our experimental conditions, as the animal was removed from the magnet
between dives to assess flipper temperature.
The selection of a 6 min experimental dive time was based on previous experience with juvenile elephant seals. In general, animal movement increases after 6 min of forced submergence, which would have negative effects on cardiac gating. Although fH has been observed to decrease slightly as a dive progresses in natural dives, no significant differences between Min 1 fH and Min 5 fH were observed in these experiments. This allowed us to use cardiac assessments gathered at different points within a dive.
Bradycardia
Assuming that the animals adapted to the experimental paradigm, as
evidenced by reduced signs of stress, the animals never had to `defend against
asphyxia' (Elsner and Gooden,
1983) by exhibiting extreme bradycardia. That is, diving
fH did not approach the 3 beats min-1 of which
they are capable. The response of juvenile seals in this study was much like
that observed in elephant seals sleeping on the beach in the hot sun with
their heads in a pool of water; a `diving response' is observed but the
decrease in heart rate from eupnea to apnea is less extreme than during dives
at sea, where the tachycardic interdive heart rate results in a greater degree
of bradycardia during the dive.
Juvenile elephant seals are tractable and appear to be tolerant of forced diving protocols. The animals used in this experiment were also subjects in a concurrent study and had been previously exposed to the diving protocol. The profound bradycardia that normally accompanies forced diving protocols was not observed in this study. Forced diving fHs are thought to be partly due to fear and the effects of restraint and handling; therefore, acclimation to the experimental protocol may have attenuated the bradycardic response.
Another possible contributing factor to the comparatively high forced
diving fHs (31.8±3.6 beats min-1) is
thermoregulation. Animals are only subject to facial immersion and not whole
body submergence, therefore heat retention may become a physiological
challenge over the course of an experiment. During such experimental
protocols, restraining devices may further restrict cutaneous heat loss from
the animal. Temperature regulation in phocids is accomplished primarily
through peripheral vasoregulation (McGinnis et al., 1971), therefore a
decrease in total peripheral resistance due to heat dissipation may have
resulted in a higher diving heart rate
(=mean arterial blood pressure/total
peripheral resistance). However, experiments on harbor seals
(Hammel et al., 1977
) showed
that diving-induced peripheral vasoconstriction overrides the increased
cutaneous blood flow normally elicited in the presence of a thermal load but
results in an increase in flipper vasodilation, as indicated by a 10°C
rise in flipper temperature. In our study, the animals' fore and rear flippers
were palpated before and after each dive (a commonly employed means of
assessing thermal stress in pinnipeds). Flippers remained cool to the touch,
indicating that animals were not dissipating excessive heat during the
experiments.
Stroke volume
In this study, elephant seal VS did not correlate with
fH during the pre-dive, dive or post-dive state. However,
there exists a negative correlation between VS and the
ratio of diving fH to pre-dive
fH (the ratio is indicative of the degree of bradycardia
expressed by the individual: mean diving fH/pre-dive
fH). Animals that exhibit a more profound bradycardia have
a higher VS than individuals who express a less profound
reduction in fH (Fig.
2). Although the degree of bradycardia correlated negatively with
VS, the absolute fH during the dive
did not show a correlation. This finding suggests that it is the magnitude of
the fH response to diving that is driving the observed
increase in VS, rather than the absolute rate of
contraction.
The extended diastolic filling time that accompanies bradycardia may
partially account for the increase in VS. An increased
strength of myocardial contraction is normally thought to accompany
bradycardia and compensate for the reduction in
caused by a decrease in cardiac
frequency. This positive inotropic effect is achieved through the increased
left ventricular filling time that is associated with extended diastole,
causing increased myocardial preload and ventricular performance (Starling
relationship). Right ventricular dilation is observed during diving,
indicating a significant increase in preload
(Hol et al., 1975
;
Blix and Hol, 1973
). An
increased ventricular volume also stimulates the ventricle to contract more
rapidly, further augmenting the interbeat filling time (Frank-Starling Law).
Contraction of the caval sphincter that occurs during diving in phocid seals
may serve as a mechanism for controlling the level of preload. Hepatic sinus
filling accounts for the increased volume of blood resulting from splenic
contraction (Thornton et al.,
2001
), but may not attenuate the increased preload resulting from
peripheral vasoconstriction. Thus, during diving, some increase in ventricular
pressure would be anticipated.
In one animal, the combination of a clear ECG signal and lack of movement
during the dive resulted in rapid acquisition of flow measurements
(Fig. 3). Aortic blood flow
during the dive peaked following ventricular contraction and aortic valve
opening. Peak flow was followed by a reduction of flow approaching zero, which
corresponds to closure of the aortic valve and phase lag effects
(Nichols and O'Rourke, 1990).
Flow was then maintained at a relatively constant rate throughout diastole, as
evidenced by the latter half of the flow trace. The maintenance of flow during
diastole is largely due to the windkessel effect of the aortic bulb, which
continues to deliver blood via the elastic recoil of the stretched
arterial walls (Jones,
1992
).
Cardiac output
Previous studies of pinniped s all
report significantly higher values than those predicted by allometric formulas
(Table 1;
Stahl, 1967
). Although it was
originally proposed that the metabolic rates of marine mammals scale
differently in relation to body mass than terrestrial mammals
(Platt and Silvert, 1981
),
this proposition was soundly disputed (Lavigne et al.,
1985
,
1986
). Eupneic
fHs in phocids are reportedly higher than the predicted
relationship (Mb-0.25, where
Mb is body mass;
Castellini and Zenteno-Savin,
1997
); however, the increase in fH is not
enough to account for the elevated
values. An increase in VS over predicted values is
required in order to produce the resting
values reported for pinnipeds. As
seal hearts scale isometrically with Mb, it is unlikely
that resting VSs would be 2-3 times higher than predicted
values. One explanation for the discrepancy in measured vs predicted
values is that the allometric equation is based on resting
measurements from a myriad of species. The definition of the phocid `resting'
condition is elusive, as many species spend a considerable amount of time in
the submerged, apneic and bradycardic condition
(Castellini and Zenteno-Savin,
1997
). In general, most studies (including this one) loosely
define a quiescent eupneic period as resting, introducing considerable
variability into this evaluation. It is plausible that the
measurements from previous pinniped
studies were obtained during a time when the animal was not in a resting
state, thus creating a disparity between the predicted
and the measured value.
|
In most systems, comparison of
between a control and experimental condition will illustrate differences in
systemic oxygen distribution and availability. These comparisons are based on
the reasonable assumption that the oxygen content
(CaO2) of arterial blood leaving the heart
remains relatively constant. However, when examining variations in phocid
cardiac output, one must take into account the effect of the increased
haematocrit and continued hemoglobin desaturation that accompanies diving. The
blood that is expelled from the heart in the pre-dive state does not carry as
much oxygen as that expelled in the first few minutes of diving. With respect
to oxygen distribution, VS and
are not directly comparable between
control and experimental conditions. This is further complicated by the fact
that, after an initial rise in CaO2 caused by
the increase in haematocrit, the blood oxygen content in the latter part of
the dive continuously decreases as hemoglobin desaturation occurs
(Qvist et al., 1986
). Although
the effect of decreased CaO2 on cardiac
contractile force in seals is not known, any chemoreceptor effect elicited by
a reduced oxygen content would likely be overridden by the vagal input.
Conclusions
During diving, a reduction in cardiac frequency occurs and is accompanied
by an increase in total peripheral resistance. In these experiments, the
degree of bradycardia is considerably less than that reported in the seven
previous studies reporting
measurements for diving pinnipeds and may be responsible for the observed
disparity in VS results. As the diving
fH values recorded in this study are close to those
observed in freely diving animals, the data presented suggest that
VS may increase during free dives when bradycardia is less
profound, rather than decrease as suggested by previous studies. These
findings indicate that the effect of diving on VS is
correlated to the degree of bradycardia, suggesting that
fH alone may not be an accurate indicator of oxygen
distribution in a diving animal.
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Footnotes |
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