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First published online October 31, 2008
Journal of Experimental Biology 211, 3588-3593 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.024489
Physiological importance of the coronary arterial blood supply to the rattlesnake heart
1 Department of Zoophysiology, Institute of Biological Sciences, University of
Aarhus, Denmark
2 Departamento de Zoologia, Centro de Aquicultura, UNESP, Rio Claro, São
Paulo, Brazil
3 Department of Cardiology, Research Unit, Aarhus University Hospital (Skejby),
Denmark
* Author for correspondence (e-mail: mette.hagensen{at}ki.au.dk)
Accepted 25 September 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: reptile, coronary artery occlusion, blood pressure, heart rate, Microfil
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Helle, 1983;
Tota, 1983;
Farrell et al., 1998;
Ostadal et al., 1999). The
spongious part of the myocardium is lost during the embryological development
in mammals and birds, and the adult heart of the endothermic vertebrates,
therefore, consists entirely of compact muscle. This morphological transition
is probably correlated with increase in heart rate and blood pressure that
attended the rise in metabolism with the evolution of endothermy
(Tota, 1983;
Burggren et al., 1997). The
compact myocardium is often perfused with coronary arteries, whereas the
spongy layer is avascular and depends entirely on oxygen diffusing from the
lumen (Ostadal et al., 1999).
Thus, the inner spongy myocardium consists of a network of intramyocardial
channels and sinusoids providing a large endocardial surface area for gas and
nutrient exchange, and the thin spongy network only represents a small
diffusion distance between the ventricular chamber and the deepest muscle
cells (Roethy et al.,
1999).
The general form and structure of the coronary arteries have been described
in representatives of most of the major taxonomic groups of reptiles
(Grant and Regnier, 1926;
Mackinnon and Heatwole, 1981;
Farrell et al., 1998). In most
species, the coronary artery arises at the root of the right aortic arch and
bifurcates to ventral and dorsal branches that perfuse most of the ventricle
(Grant and Regnier, 1926;
Erhart, 1935;
Mackinnon and Heatwole, 1981;
Farrell et al., 1998). Very
little, however, is known about the functional significance of the coronary
blood supply to the ventricle in reptiles and its physiological role has not
been addressed experimentally in any species of reptiles. The hearts of many
reptiles is generally very hypoxia tolerant (e.g.
Bing et al., 1972;
Poupa et al., 1978;
Overgaard et al., 2007), but
force production of isolated ventricular strips of some snakes is markedly
sensitive to hypoxia (Zaar et al.,
2007). In turtles, hypoxia can affect the electrocardiogram,
particularly during exercise when cardiac output is elevated and when the
oxygen concentration of the venous blood is reduced
(Farmer and Hicks, 2002).
Here we describe the coronary circulation of the South American
rattlesnakes (Crotalus durissus). The cardiovascular physiology of
this species of snake has been studied extensively (e.g.
Galli et al., 2005;
Skals et al., 2005;
Skovgaard et al., 2005). To
evaluate the importance of the coronary circulation, we compare the heart
rate, blood pressure and electrocardiograms (ECGs) of snakes with normal, and
snakes with occluded coronary circulation at rest and during enforced activity
under the hypothesis that lack of coronary supply should impair the ability to
maintain cardiac work.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Surgery and instrumentation
Snakes were anaesthetised by halothane (5%) inhalation. When the animals no
longer responded to tactile stimuli, local anaesthesia (Lidocaine, 1%) was
injected and a 5 cm ventral incision was made cranial to the heart, so the
pericardium could be opened to expose the heart. In six snakes, the coronary
arteries were occluded by burning close to the outflow from the right aortic
arch. Then, the pericardium was closed with two or three sutures (4–0
silk), and the cranial branch of the vertebral artery occlusively cannulated
by insertion of a PE-50 catheter containing heparinized saline (50 i.u.
ml–1). This catheter was used for measurements of systemic
arterial blood pressure (Psys). The catheter was
externalised and secured to the skin by several sutures. Finally, three ECG
leads, constructed from the ends of 25 gauge hypodermic needles (Terumo,
Europe NV Leuven, Belgium), were placed subcutaneously on the back to
triangulate the heart. The leads were fastened with sutures and the snakes
were given an intraperitoneal injection of the antibiotic enrofloxacin
(Baytril®; 2–3 mg kg–1) to prevent infection. The
surgery normally lasted 30 min and the snakes spontaneously resumed breathing
within 15 min and appeared to exhibit normal behaviour on the following day.
In four other snakes, the heart was burned in several places avoiding any
occlusion of coronary arteries, as a sham operation.
Measurement of blood pressure, heart rate and electrocardiograms
Arterial blood pressures were measured by connecting the catheters to
disposable pressure transducers (Baxter Edward model PX600, Irvine, CA, USA)
positioned at heart level, and heart rate (fH) was derived
from the pulsatile arterial pressure. The signal from the pressure transducers
was amplified by an in-house-built preamplifier and calibrated daily against a
static water column. The ECG signal was amplified 200 times using an
in-house-build preamplifier and filtered with a low band/high band pass filter
(0.4 Hz and 40 Hz, respectively). All signals were collected at 100 Hz using a
BioPac MP100 data acquisition system (BioPac, Goleta, CA, USA) connected to a
personal computer.
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Visualisation of the coronary vessels and verification of the coronary ablation
Upon termination of the physiological measurements, all snakes were
sacrificed by injection of the barbiturate mebumal (50 mg
ml–1) and the hearts were removed and arrested in diastole by
transfer to phosphate buffer for 15 min. The coronary network was cleared of
blood during this period. To evaluate whether the coronary arteries were
indeed ablated, the right systemic arch was cannulated with a metal cannula
and coloured silicone rubber (Microfil MV-122; Flow Tech, Carver, MA, USA) was
infused at a pressure of 6 kPa over 12 min. Infusion of Microfil in
non-occluded control hearts served to visualise the coronary vasculature. The
hearts were then immersed in a 4% formaldehyde solution for 48 h and stored in
phosphate buffer at 4°C.
Data analysis and statistics
The data were analysed using Acknowledge (3.0) data analysis software
(Biopac Systems, Inc., CA, USA). All values are means ± s.e.m.
Differences considered statistically significant from control values at a 95%
level of confidence (P<0.05) were evaluated using paired and
unpaired t-tests.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Injection of Microfil in the occluded hearts confirmed that occlusion of the coronary arteries was successful in all snakes, as shown in the example from one snake in Fig. 1E.
Haemodynamic variables
Mean systemic blood pressures (Psys) and heart rates
(fH) at rest and during enforced activity at 1 and 4 days
after surgery are presented in Fig.
2. Most snakes exhibited minor periodic increases in heart rate,
probably associated with ventilation, and mean values were taken for
non-ventilatory periods. Both at days 1 and 4, enforced activity caused an
immediate rise in Psys of both sham-operated snakes and
snakes with coronary occlusion. Occlusion of the vessels had no significant
effect on Psys during enforced activity, although
Psys tended to be higher in the snakes with coronary
occlusion. Both at rest and during enforced activity at day 4, there was a
significant reduction in Psys compared with day 1.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Erhart, 1935;
Mackinnon and Heatwole, 1981;
Farrell et al., 1998). The
Microfil injections revealed an extensive epicardial coronary arterial network
with multiple anastomoses connecting epicardial coronary arteries from
adjacent perfusion beds. It is, however, difficult to establish whether the
coronary circulation of Crotalus is more extensive than other
reptiles because previous studies on reptiles only described the major vessels
(see Farrell et al., 1998).
Clearly it would be of interest to use Microfil for detailed descriptions of
the coronary arteries in other reptiles. In spite of the extensive coronary
circulation to the outer myocardium, our study shows that ablation of the
coronary blood supply does not affect the ability to maintain blood pressure
and heart rate at rest and during short-term activity or cause abnormalities
in the electrocardiogram. We also did not observe any indication of necrosis
in the hearts with ablated coronary blood supply (data not shown).
Heart rate and blood pressures were similar to previous reports on
cannulated awake rattlesnakes (Skals et
al., 2005), but fH was higher than in
rattlesnakes that had recovered longer from a less invasive procedure
(Campbell et al., 2006). The
tachycardia 4 days after surgery in snakes with occluded coronaries is
probably due to the more invasive treatment, but shows that Crotalus
can maintain high blood pressure and heart rate without a coronary blood
supply. As in other snakes, Psys and
fH increased immediately during activity, which is likely
to stem from increased sympathetic tone
(Stinner and Ely, 1993;
Wang et al., 2001). The
cardiovascular response to enforced activity was similar to previous
measurements on rattlesnakes, and heart rate attained maximal values for that
temperature (T.W. and A.S.A., unpublished observations). Occlusion of the
coronary arteries did not influence the rise in Psys and
fH during enforced activity and shows that rattlesnakes
can increase cardiac work substantially during short bouts of intensive
exercise without coronary perfusion. This may imply that the cardiomyocytes in
the outer compact myocardium are either very hypoxia-tolerant or that they
derive oxygen from the lumen. Given that the compact myocardium seems
surprisingly thin, it is possible that diffusion distance is sufficiently
short to maintain oxygen delivery. However, venous
PO2 is probably very low during exercise, which
would reduce the PO2 gradient driving diffusion
of oxygen from the lumen to the outer myocardium. It is not known how much the
compact layer contributes to force development during each heart beat, so it
is also possible that its major function is to provide a surrounding shell to
prevent cardiac distension. Nevertheless, given the extensive network of the
coronary arteries, it seems that the compact layer receives a large supply of
oxygen, which would indicate a high oxygen requirement of that tissue.
Electrically paced ventricular strips from the European viper, which
hibernates at low temperature, shows a pronounced anoxia tolerance
(Poupa et al., 1978), whereas
contractility of heart strips from pythons is markedly reduced in hypoxia
(Zaar et al., 2007). The
hypoxia sensitivity of the cardiac muscle of rattlesnakes remains to be
studied, but given the high temperature in our studies, it seems very unlikely
that these hearts would be able to continue force production for 4 days
without oxygen. The surprising thing is how much blood supply it appears to
receive.
Although our study shows that rattlesnakes with ablated coronary arteries
can sustain normal cardiovascular response to short bouts of exercise, our
study does not rule out the possibility that the coronary arteries may be
important for cardiac performance during longer periods with elevated
metabolism. We studied the snakes at 30°C to increase metabolism, but it
would be interesting to assess importance of the coronary circulation on
myocardial oxygen supply during digestion where myocardial oxygen demand is
high and when venous blood returning to the heart is low in oxygen
(Andrade et al., 1997;
Overgaard and Wang, 2002;
Arvedsen et al., 2005). As in
rattlesnakes, the fish heart is not solely dependent on coronary blood flow
(Daxboeck, 1982;
Farrell and Steffensen, 1987;
Farrell, 2002), but the
coronary circulation becomes essential during hypoxia and intense swimming in
rainbow trout (Steffensen and Farrell,
1998). As a notable exception, skipjack tuna, where 60% of the
heart consists of compact muscle seems to depend on coronary circulation
(Farrell et al., 1992).
In turtles and alligators, the compact myocardium also receives some blood
supply directly from the ventricular lumen and this transmyocardial blood
supply may account for as much as 30% of the coronary perfusion
(Brady and Dubkin, 1964;
Voboril and Schiebler, 1970;
Kohmoto et al., 1997). Whether
a similar contribution is present in the rattlesnake remains uncertain. It is
possible that there are channels between the spongy and compact myocardium, as
in alligators (Kohmoto et al.,
1997) that delivers sufficient oxygen to the heart. Turtles have a
large left–right shunt during exercise
(Shelton and Burggren, 1976;
West et al., 1992;
Krosniunas and Hicks, 2003)
and it was suggested that the shunting of oxygen-rich blood throughout the
ventricle serves to oxygenate the myocardium
(Farmer, 1999;
Farmer and Hicks, 2002). It is
not known whether Crotalus exhibit a similar shunt pattern during
exercise, but adrenergic stimulation can certainly cause a large
left–right shunt (Galli et al.,
2007).
The unaffected fH and Psys were
consistent with a lack effects on the ECG. Electrical anomalies and arrhythmia
have been reported during myocardial hypoxia in the turtle Trachemys
scripta (Farmer and Hicks,
2002), where hypoxia increased the Q wave, elevated the ST segment
and lengthening the P–R interval
(Farmer and Hicks, 2002).
These changes are similar to those observed in mammals, in which an inverted
T-wave is a reliable indicator for cardiac ischemia
(Kern, 2005). Although a
similar interpretation was proposed for turtles
(Farmer and Hicks, 2002), this
does not seem to be the case for the rattlesnakes because inverted T waves
were observed equally often in sham-operated snakes
(Fig. 3). Similarly, the T-wave
is often inverted in toads and lizards
(Zaar et al., 2004;
Liu and Li, 2005), and may be
explained by anatomical differences affection the direction of the ventricular
repolarisation. In humans, transmural infarct of the left ventricle elevates
the ST interval, whereas a depression of the ST interval results from
subendocardial infarction. Occlusion of the coronary circulation did not
affect the ST interval in rattlesnakes.
In conclusion, Microfil injection revealed an extensive epicardial coronary arterial network in Crotalus, and the snakes were able to maintain normal blood pressures and heart rate at rest as well as the ability to increase blood pressure and heart rate during short-term activity for at least 4 days after occlusion of the coronary arteries.
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Acknowledgments |
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significant
difference from rest; 
significant difference from
sham-operated snakes.
