Reduction of blood oxygen levels enhances postprandial cardiac hypertrophy in Burmese python ( Python molurus )

Abstract

Physiological cardiac hypertrophy is characterized by reversible enlargement of cardiomyocytes and changes in chamber architecture, which increase stroke volume and VO2 max via augmented convective oxygen transport. Cardiac hypertrophy is known to occur in response to repeated elevations of O2 demand and/or reduced O2 supply in several species of vertebrate ectotherms, including postprandial Burmese pythons (Python molurus). Recent data suggest postprandial cardiac hypertrophy in P. molurus is a facultative rather than obligatory response to digestion, though the triggers of this response are unknown. Here we hypothesize that an O2 supply-demand mismatch stimulates postprandial cardiac enlargement in Burmese pythons. To test this hypothesis, we rendered animals anemic prior to feeding, essentially halving blood oxygen content during the postprandial period. Fed anemic animals had heart rates 126% higher than fasted controls, which, coupled with a 71% increase in mean arterial pressure suggests fed anemic animals were experiencing significantly elevated cardiac work. We found significant cardiac hypertrophy in fed anemic animals, which exhibited ventricles 39% larger than fasted controls and 28% larger than fed controls. These findings support our hypothesis that those animals with a greater magnitude of O2 supply-demand mismatch exhibit the largest hearts. The "low O2 signal" stimulating postprandial cardiac hypertrophy is likely mediated by elevated ventricular wall stress associated with postprandial hemodynamics.

Full text

The Journal of Experimental Biology
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© 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 1784-1789 doi:10.1242/jeb.092841
ABSTRACT
Physiological cardiac hypertrophy is characterized by reversible
enlargement of cardiomyocytes and changes in chamber
architecture, which increase stroke volume and V
·
O
2
,max via augmented
convective oxygen transport. Cardiac hypertrophy is known to occur
in response to repeated elevations of O2demand and/or reduced O2
supply in several species of vertebrate ectotherms, including
postprandial Burmese pythons (Python bivittatus). Recent data
suggest postprandial cardiac hypertrophy in P. bivittatus is a
facultative rather than obligatory response to digestion, though the
triggers of this response are unknown. Here, we hypothesized that
an O2supply–demand mismatch stimulates postprandial cardiac
enlargement in Burmese pythons. To test this hypothesis, we
rendered animals anemic prior to feeding, essentially halving blood
oxygen content during the postprandial period. Fed anemic animals
had heart rates 126% higher than those of fasted controls, which,
coupled with a 71% increase in mean arterial pressure, suggests fed
anemic animals were experiencing significantly elevated cardiac
work. We found significant cardiac hypertrophy in fed anemic
animals, which exhibited ventricles 39% larger than those of fasted
controls and 28% larger than in fed controls. These findings support
our hypothesis that those animals with a greater magnitude of O2
supply–demand mismatch exhibit the largest hearts. The ‘low O2
signal’ stimulating postprandial cardiac hypertrophy is likely mediated
by elevated ventricular wall stress associated with postprandial
hemodynamics.
KEY WORDS: Cardiac plasticity, Cardiovascular regulation, Python
molurus bivittatus, Reptile, Heart, Digestion, Postprandial, SDA,
Anemia
INTRODUCTION
Burmese pythons (Python bivittatus) (Kuhl, 1820), like many large
snakes, utilize an intermittent ‘sit-and-wait’ feeding strategy, where
prolonged fasts are punctuated by brief and voracious feeding bouts
when prey is available. Digestion of these large meals (up to 25–100%
of their body mass) is associated with pronounced upregulation of a
suite of digestive functions and a large postprandial increase in oxygen
uptake (V
·O
2
), termed specific dynamic action (SDA), where V
·O
2
may
exceed that during aerobic activity and last for several days (Benedict,
1932; Secor and Diamond, 1995; Secor and Diamond, 1997; Secor
and Diamond, 1998; Secor, 2008; Cox and Secor, 2008; Secor et al.,
2000b; Wang et al., 2001b). To support the high V
·O
2
during digestion,
RESEARCH ARTICLE
1Department of Ecology and Evolutionary Biology, University of California Irvine,
Irvine, CA 92697, USA. 2Zoophysiology, Institute for Biological Sciences, Aarhus
University, DK-8000 Aarhus, Denmark. 3Inderdisciplinary Nanoscience Center,
Aarhus University, DK-8000 Aarhus, Denmark.
*Author for correspondence (tobias.wang@biology.au.dk)
Received 20 June 2013; Accepted 13 November 2013
cardiac output increases drastically above resting values through a
combination of increased stroke volume and heart rate (fH) (Secor et
al., 2000a; Secor and White, 2010). This hemodynamic response is
mitigated largely by a reduction in cholinergic tone and positive
chronotropic effects of non-adrenergic, non-cholinergic (NANC)
factors, including an increased histaminergic tone (Wang et al., 2001a;
Skovgaard et al., 2009; Enok et al., 2012; Enok et al., 2013; Burggren
et al., 2014).
The rise in stroke volume has been linked with a 40% increase in
ventricular mass within 48 h of eating (Andersen et al., 2005) that
Riquelme et al. described as being ‘physiological’ in nature and
triggered by humoral factors, including increased levels of circulating
free fatty acids (Riquelme et al., 2011). The universality and stimulus
of the postprandial cardiac hypertrophy, however, remain unclear as
Jensen et al. found no postprandial cardiac hypertrophy in Burmese
or Ball pythons (Python regius) under a similar experimental protocol
(Jensen et al., 2011). They argued, therefore, that postprandial cardiac
hypertrophy should be considered a ‘facultative’ rather than
‘obligatory’ response to feeding (Jensen et al., 2011), and the lack of
postprandial cardiac hypertrophy was recently reported in two
additional studies (Hansen et al., 2013; Enok et al., 2013).
The correlation between the magnitudes of SDA and postprandial
cardiac hypertrophy is not well understood (Jensen et al., 2011).
Thus, while V
·O
2
consistently increases following feeding, the
postprandial cardiac hypertrophy is inconsistent. We hypothesize
that postprandial cardiac hypertrophy is triggered when systemic
metabolic demand outpaces systemic oxygen delivery. To
investigate this hypothesis, we established an oxygen supply–
demand mismatch in postprandial pythons by rendering specimens
anemic prior to feeding, with the prediction that anemic pythons
would exhibit greater postprandial cardiac hypertrophy than fasted
pythons with normal blood oxygen levels.
RESULTS
Hematological parameters and blood gases
Our experimental procedure for rendering animals anemic resulted
in significantly reduced blood oxygen carrying capacity (Table 1).
At the time of sampling, 72 h after surgery, anemic animals (both fed
and fasted) exhibited 61% lower hematocrit (Hct) than controls
(F1,28=103.0, P<0.0001) and 53% lower arterial blood oxygen
concentration (CO
2
) than control animals (F1,19=27.8, P<0.0001).
Among fed animals, CO
2
was significantly reduced in anemic
animals compared with control animals (F3,19=9.4, P<0.0001),
which was critical for testing the hypothesis. Arterial pH did not
differ between anemic and control snakes and was not affected by
digestion (F3,21=1.15, NS).
Cardiovascular parameters
While manipulation of Hct alone did not significantly elevate the fH
of fasting snakes, feeding elicited significant increases in fHamong
both anemic (50% increase) and control (78% increase) snakes.
Reduction of blood oxygen levels enhances postprandial cardiac
hypertrophy in Burmese python (Python bivittatus)
Christopher E. Slay1,2, Sanne Enok2,3, James W. Hicks1and Tobias Wang2,*

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RESEARCH ARTICLE The Journal of Experimental Biology (2014) doi:10.1242/jeb.092841
Coupling anemia with feeding, however, resulted in a 126%
difference between fasted controls and fed anemic snakes (Fig. 1A;
F3,20=9.2, P<0.001).
Mean arterial pressure (MAP) was 85% higher in fed controls
than in fasted controls and 71% higher in fed anemic animals than
in fasted controls (Fig. 1B; F3,20=5.9, P<0.05). As a consequence of
the markedly elevated fHand MAP, particularly in fed anemic
snakes, the rate–pressure product (RPP) was 2.9-fold higher in fed
anemic snakes than in fasted controls (Fig. 1C; F3,20=6.2, P<0.05).
The changes in fHwere attended by changes in autonomic tone on
the heart (Fig. 2). Feeding alone elicited a 42% reduction in
adrenergic tone among control animals, but the response was
blunted in anemic animals, resulting in a more modest 27%
reduction. The greatest reduction in adrenergic tone was the 47%
difference between fasted anemic snakes and fed control snakes
(Fig. 2A; F3,16=10.3, P=0.001).
There were significant effects of digestive status (F1,16=10.4,
P<0.05) and Hct (F1,16=5.0, P<0.05) on cholinergic tone (Fig. 2B),
with a modest difference existing between fasted controls and fed
controls (47%), and a greater difference between fasted controls and
fed anemic animals (73%) (Fig. 2B; F3,16=8.1, P<0.005).
The effect of feeding alone was significant in determining double-
blocked fH(F1,17=16.9, P<0.005), whereas Hct did not have a
significant effect (F1,17=2.0, NS). Fed anemic animals had a higher
double-blocked fHthan either group of fasted animals (F3,17=6.9,
P<0.005; Fig. 2C).
Cardiac hypertrophy
Heart mass of snakes with normal Hct did not increase during
digestion, and anemia did not elicit cardiac growth in fasting snakes
(Fig. 3A). However, the anemic snakes, 48 h into digestion, had a
ventricular mass of 1.8 g kg−1, which is 39% larger than the ventricle
of fasting snakes with normal Hct and 28% larger than the ventricle
of fed animals with normal Hct. Thus, there was a significant
difference (F3,30=3.0, P<0.05) in ventricular wet mass between
treatments. The effects of Hct on ventricular mass (F1,30=5.3,
P<0.05) were greater than the effects of digestion (F1,30=2.6, NS).
There was no significant difference in the dry mass:wet mass ratio
(Md:Mw) between treatments (Fig. 3B; F3,30=0.7, NS).
Total wet heart mass, i.e. combined ventricular and atrial wet
masses, also differed between treatments (Fig. 3C) (F3,30=3.9,
P<0.05), with fed anemic animals again having the largest hearts
(38% larger than hearts of fasted controls and 22% larger than hearts
of fed controls), but not significantly larger than hearts of fasted
anemic snakes. Hct exerted a greater effect on total heart mass
(F1,29=7.3, P<0.05) than digestion (F1,29=2.9, NS). Atrial wet mass
was also 36% greater in fed anemic animals than in fasted controls
(F3,30=4.3, P<0.05), again, with a significant effect of Hct (F1,29=8.8,
P<0.05) but not feeding status (F1,29=3.5, NS). There was no
significant difference in atrial Md:Mwbetween groups of animals
(F1,29=0.3, NS).
We correlated ventricular mass with RPP, where this value
estimates myocardial oxygen consumption and thus provides a
List of symbols and abbreviations
CO
2
blood oxygen concentration
fHheart rate
Hct hematocrit
MAP mean arterial blood pressure
NANC non-adrenergic, non-cholinergic
RPP rate pressure product
SDA specific dynamic action
SMR standard metabolic rate
Table 1. Blood parameters in fasting and fed pythons
Fasted Fed
Control Anemic Control Anemic
CO
2
(mmol l−1) 3.24±0.84a1.76±0.47b3.94±0.24a1.69±0.26b
Hct (%) 24.0±1.7a7.4±0.5b21.1±1.7a9.9±1.0b
pH 7.49±0.08a7.65±0.05a7.60±0.09a7.66±0.04a
CO
2
, arterial blood oxygen concentration.
Values with the same superscript letters are not significantly different from one another.
0
20
40
60
fH (beats min–1)
a
a,b
b,c
c
0
5
10
15
MAP (kPa)
aa,b
cb,c
Control
Anemic
Control
Anemic
0
200
400
600
RPP (beats kPa min–1)
Fasted Fed
a
a,b
b,c c
A
B
C
Fig. 1. Hemodynamic parameters in fasting and fed (48 h into digestion)
Burmese pythons (Python bivittatus). Heart rate (A; fH) was significantly
higher in digesting snakes than in fasted controls, with a greater difference
between fed anemic snakes and fasted controls. (B) Mean arterial blood
pressure (MAP) was significantly higher in digesting snakes than in fasting
control snakes, while anemia alone did not influence MAP. (C) The
rate–pressure product (RPP, a proxy for cardiac work) was also significantly
higher in digesting snakes than in fasted controls with a greater difference
between fed anemic snakes and fasted controls. Groups with the same
lowercase letter do not differ significantly. Data are presented as means ±
s.e.m. Fasted controls, N=4; fasted anemic, N=5; fed controls, N=6; fed
anemic, N=7.

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RESEARCH ARTICLE The Journal of Experimental Biology (2014) doi:10.1242/jeb.092841
proxy for cardiac work (Fig. 4). Ventricular mass was positively and
linearly correlated with RPP (P<0.05, R2=0.22).
Plasticity of the digestive organs
Stomach wet mass was significantly greater in fed control than in
fasted control animals (Table 2; F3,30=5.2, P<0.05), but there was no
difference in stomach dry mass between groups (F3,30=2.4, NS). Wet
mass of the small intestine was also significantly larger in digesting
snakes (F3,30=10.0, P<0.0001), with similar trends for dry mass (albeit
with no statistical difference between fasted anemic and fed control
intestines; F3,30=16.0, P<0.005). There were no significant differences
in large intestine mass (wet F3,30=1.5, NS; dry F3,29=2.3, NS) or liver
wet mass (F3,30=2.4, NS), whereas liver dry mass differed
significantly between groups (F3,30=16.5, P<0.005). Fed anemic
animals exhibited higher kidney wet mass than fasted controls (84%
enlargement; F3,30=5.3, P<0.05), but there were no significant changes
in kidney dry mass (F3,27=1.7, NS). While growth of the small
intestine was due only to digestion (F1,30=27.1, P<0.0001) and not Hct
(F1,30=1.7, NS), both digestion (F1,30=7.8, P<0.05) and Hct (F1,30=7.1,
P<0.05) had significant effects on kidney wet mass.
DISCUSSION
Our study confirms that feeding alone does not elicit postprandial
cardiac hypertrophy. Animals confronted with the simultaneous
challenges of increased O2demand (digestion) and reduced O2
supply (anemia) do, however, exhibit postprandial cardiac
hypertrophy when compared with fasted, un-manipulated controls.
This suggests that cardiac hypertrophy is triggered when oxygen
supply/delivery cannot meet the elevated metabolic demands of
digestion. Interestingly, cardiac mass of several other ectothermic
0
20
40
60
Adrenergic tone (%)
a,b a
c
b
0
20
40
60
Cholinergic tone (%)
a
bb,c
c
Control
Anemic
Control
Anemic
0
20
40
60
Double-blocked fH
(beats min–1)
Fasted Fed
aa,b
b,c c
A
B
C
0
0.5
1.0
1.5
2.0
2.5
Ventricular Mw (g kg–1)
aa,b a
b
0
0.1
0.2
0.3
Ventricular Md:Mw
Control
Anemic
Control
Anemic
0
0.5
1.0
1.5
2.0
2.5
Total heart mass (g kg–1)
Fasted Fed
aa,b a
b
A
B
C
aaa
a
Fig. 2. Adrenergic and cholinergic cardiac tone in fasting and
postprandial (48 h into digestion) Burmese pythons (P. bivittatus). Both
cholinergic (A) and adrenergic (B) tone were lower in digesting snakes, and
cholinergic tone was reduced during anemia in both fasting and digesting
snakes. Double blocked fH(C) was significantly higher in fed animals than in
fasted controls, with no significant effect of anemia alone. Groups with the
same lowercase letter do not differ significantly. Data are presented as
means ± s.e.m. Fasted controls, N=3; fed controls, N=4; fasted anemic, N=4;
fed anemic, N=7.
Fig. 3. Ventricular wet mass, dry to wet mass ratio and total heart mass
in fasting and postprandial (48 h into digestion) Burmese pythons (P.
bivittatus). Ventricular wet mass (A; Mw), was significantly higher in fed
anemic snakes than both fasting control and fed control snakes, while there
were no differences in dry to wet mass ratio (Md:Mw; B). Total heart mass (C)
was significantly higher in fed anemic snakes than in fasted and fed controls.
Groups with the same lowercase letter do not differ significantly. Data are
presented as means ± s.e.m. Fasted control, N=8; fasted anemic, N=6; fed
control, N=9; fed anemic, N=8.
0 200 400 600 800
0
0.5
1.0
1.5
2.0
2.5
RPP (beats kPa min–1)
Ventricular mass (g kg–1)
r2=0.22
P<0.05
Fasted controls
Fed controls
Fed anemic
Fasted anemic
Fig. 4. Correlation between ventricular wet mass and RPP across all
experimental groups (fasted control, fasted anemic, fed control and fed
anemic) of Burmese pythons (Python bivittatus). The RPP is equal to the
product of MAP and fH, and is a proxy for cardiac work.

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vertebrates also responds to oxygen supply and demand mismatches,
such as alligators reared in hypoxia (Warburton et al., 1995;
Crossley and Altimiras, 2005; Owerkowicz et al., 2009) and fish
rendered anemic (e.g. Sun et al., 2009; Simonot and Farrell, 2007).
Our findings conflict with the previous reports of an obligatory
postprandial cardiac hypertrophy (e.g. Andersen et al., 2005;
Riquelme et al., 2011), and support the proposal that postprandial
cardiac hypertrophy is a facultative response in pythons (Jensen et
al., 2011; Hansen et al., 2013; Enok et al., 2013). In contrast,
postprandial enlargement of the small intestine, liver and kidneys
seems consistent amongst studies (Secor and Diamond, 1995; Secor
and Diamond, 1998; Starck and Beese, 2001; Ott and Secor, 2007;
Cox and Secor, 2008; Jensen et al., 2011; Hansen et al., 2013; Enok
et al., 2013). Supporting the idea that expansion of the intestine is
stimulated by the presence of chyme (Secor et al., 2000b), there was
no effect of Hct reduction on the rise in intestinal mass during
digestion, though it is impressive that significant intestinal
hypertrophy occurs in animals with severe oxygen limitation.
Enlargement of the stomach seems to be another facultative
response to digestion, as it is noted in some studies (Secor and
Diamond, 1995; Jensen et al., 2011) but not others (Cox and Secor,
2008; Ott and Secor, 2007). As in other studies (Secor and Diamond,
1995; Jensen et al., 2011), kidney wet mass increased with digestion,
but we also note that snakes with reduced Hct had enlarged kidneys,
which may result from a stimulation of erythropoietic functions, but
dry kidney mass did not differ between groups.
As shown in earlier studies (Wang et al., 2001a; Skovgaard et al.,
2009; Enok et al., 2012; Enok et al., 2013), the postprandial
tachycardia is largely governed by a reduction of cholinergic tone on
the heart, whereas the adrenergic tone actually decreases during
digestion. In the double-blocked heart, there was also a rise in the
postprandial fHresulting from circulating NANC factors (Skovgaard
et al., 2009), although the specific nature of the stimulus remains to
be identified (Enok et al., 2012). Given that the NANC factor is likely
to be released in direct response to digestion, possibly as a peptide
from the digestive organs, it is not surprising that anemia did not affect
the double-blocked fH. The rise in fHof the anemic snakes was likely
a barostatic response to vasodilation and the attendant lowering of
total peripheral resistance in response to lowered blood CO
2
, but could
also result from the stimulation of chemoreceptors (Wang et al., 1994;
Wang et al., 1997; Andersen et al., 2003). In contrast to previous
studies on digesting snakes, the postprandial tachycardia in our study
was associated with a significant rise in MAP. However, because
MAP did not increase proportionally to the rise in fH, and because
stroke volume is likely to have been elevated, digestion was probably
attended by a reduced total peripheral resistance as blood flow to the
digestive organs increases during digestion (Secor et al., 2000a; Starck
and Wimmer, 2005; Secor and White, 2010). In addition, lowering of
Hct is likely to have reduced blood viscosity and hence could have
alleviated the workload on the heart. However, anemia did not
influence MAP, and the anemic snakes therefore did have a higher
RPP than animals with normal Hct.
The observation that the postprandial cardiac hypertrophy of
pythons is facultative rather than obligatory indicates that factors
other than circulating signal molecules are involved, and our results
suggest that increased cardiac work or myocardial oxygen
consumption stimulate the postprandial cardiac growth in pythons.
Compared with resting animals, postprandial cardiac growth was
elicited in anemic snakes with significantly higher RPP, suggesting
increased workload and greater mechanical stress on the ventricles.
In mammals, the molecular pathways stimulating physiologic
cardiac hypertrophy are stimulated by increased mechanical stress,
such that increased workload stimulates myocytes to synthesize and
release growth factors, including insulin-like growth factor I (IGF-
I) (Serneri et al., 1999; Hill and Olson, 2008). These growth factors
are then involved in paracrine and/or autocrine activation of the
phosphatidylinositol 3′-kinase (PI3K)–Akt-mTOR pathway, which
ultimately leads to synthesis of contractile elements (Dorn and
Force, 2005; Shiojima and Walsh, 2006; Dorn, 2007; Hill and
Olson, 2008). AMPK, Akt, GSK3β and mTOR, all signaling
molecules in mammalian physiologic hypertrophy pathways
mediated by mechanical stress, are known to be active in the python
model (Riquelme et al., 2011). This suggests that the cardiac
hypertrophy in pythons occurs in response to elevated mechanical
stress on ventricular myocytes. This obviously does not rule out the
possibility that circulating factors, such as free fatty acids (Riquelme
et al., 2011), may contribute to the postprandial hypertrophy.
Nevertheless, such humoral regulation does not appear adequate
without a sufficient elevation of cardiac work and mechanical stress.
General conclusions
Despite the universal presence of gastrointestinal hypertrophies in
fed pythons, our study supports the concept that postprandial cardiac
hypertrophy is not an obligatory response to elevated oxygen
demands associated with digestion in the python. We describe
postprandial cardiac hypertrophy in fed anemic animals, whose
hearts are operating at significantly elevated fH(as mediated by
reduced CO
2
, subsequently reduced cholinergic tone, and the
presence of a significant NANC tone), and elevated cardiac work
(as indicated by the RPP). We posit that regardless of the potential
for other humoral signals (Riquelme et al., 2011), significantly
elevated cardiac work is required to ‘trigger’ the postprandial
hypertrophy via common physiological hypertrophy signaling
pathways. However, the precise level of cardiac work needed to
induce cardiac hypertrophy is difficult to assess from the current
analysis, as the experimental paradigm depends on a group analysis.
Experiments measuring systemic flow, fH, MAP, V
·O
2
and heart
size/mass need to be correlated during fasting and digestion, within
Table 2. Visceral organ mass
Wet mass (g kg−1) Dry mass (g kg−1)
Fasted Fed Fasted Fed
Control Anemic Control Anemic Control Anemic Control Anemic
Stomach 12.9±0.8a15.2±1.1a,b 17.7±2.2b17.0±1.2a,b 2.7±0.2A2.7±0.1A3.6±0.4A3.6±0.3A
Small intestine 14.5±1.4a17.1±0.8a25.4±3.2b29.6±1.7b2.8±0.3A3.0±0.1A,B 4.6±0.9B,C 5.9±0.4C
Large intestine 8.7±0.7a10.7±1.0a10.2±0.8a13.2±2.6a2.2±0.5A1.45±0.1A1.4±0.2A1.7±0.2A
Liver 17.5±1.4a18.7±1.9a22.1±2.6a25.5±2.8a5.1±0.4A4.7±0.5 A6.1±0.9A,B 8.2±0.8B
Kidney 4.5±0.4a5.9±0.4a,b 6.0±0.6a,b 7.7±0.8b0.9±0.1A1.1±0.1A1.4±0.3A1.4±0.2A
Values with the same superscript letters (lowercase for wet mass and uppercase for dry mass) are not significantly different from one another.

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individual animals. Advanced imaging techniques, which are
becoming increasingly accessible to comparative physiologists (e.g.
Hansen et al., 2013), in combination with classical physiological
measurements would provide the information to determine the
trigger level needed to induce postprandial cardiac hypertrophy in
the Burmese python.
MATERIALS AND METHODS
Animal acquisition and husbandry
Burmese pythons (Python bivittatus; N=31) of both sexes were acquired
from commercial vendors and housed for several months prior to
experimentation at the vivarium facilities of Aarhus University or the
University of California, Irvine. Animals ranged from 0.24 kg to 11.5 kg
with a mean body mass of 1.83±0.52 kg. Snakes were kept in individual
vivaria at 27–30°C, and had access to heated surfaces that reached 32°C. A
12 h light:12 h dark photoperiod was maintained. All animals always had
access to water, vigorously consumed rodent meals every 1–2 weeks, and
gained mass during captivity. All snakes were fasted for a minimum of
28 days prior to experimentation. Animals were housed and treated
according to Danish Federal Regulations and UCI IACUC protocol 2009-
2821.
Surgical procedures
Snakes (27 of the 31) were instrumented with arterial catheters for
measurement of MAP and fH, as well as for withdrawal of arterial blood
samples to determine blood CO
2
and blood pH. To induce anesthesia,
individual snakes were placed in a sealed container containing gauze soaked
in isoflurane (Baxter, Allerød, Denmark) until they lost muscle tone and
could be intubated for artificial ventilation with 2% isoflurane at
5 breaths min−1and 50 ml kg−1tidal volume, using a vaporizer (EZ-155, EZ
Systems, Bethlehem, PA, USA) and an HI 665 Harvard Apparatus respirator
(Holliston, MA, USA). A 5 cm incision close to the cloaca enabled the
dorsal aorta to be accessed by blunt dissection, so a catheter (PE-50)
containing heparinized saline (50 IU ml−1) could be inserted and externalized
via a small cutaneous puncture and secured to the skin with 2-0 braided silk
suture. Approximately 0.15 ml of whole blood was then withdrawn from the
catheter to determine Hct by spinning the blood in glass capillaries for 3 min
at 12,000 rpm.
A subset of 14 randomly selected snakes was rendered ‘anemic’ (see
discussion of experimental groups, below) by withdrawing blood while the
snakes were still anesthetized. Aliquots of 10% of the estimated blood
volume (6–7% of body mass) (Lillywhite and Smits, 1984) were placed in
sterile 1.5 ml Eppendorf tubes and centrifuged at 6000 rpm for 5 min. The
supernatant plasma was returned via the arterial catheter. Hct was re-
measured 15 min after reinjection of plasma and the process was repeated
until Hct was reduced to ~10% (mean 10.1±0.3%).
The snakes were ventilated with room air until they regained muscle tone
and resumed spontaneous ventilation. They were then returned to their
enclosures, given access to water, and placed in a 30°C temperature-
controlled chamber. Animals were allowed to recover from surgery
undisturbed in their enclosures for 24 h to ensure low plasma catecholamine
levels (Olesen et al., 2008).
Experimental and feeding protocols
Following the 24 h recovery period, we measured MAP and fHfrom each
snake while they remained minimally disturbed in the climactic chamber.
The catheters were connected to pressure transducers (PX600, Baxter
Edwards, Irvine, CA, USA) calibrated with a vertical water column and
connected to an in-house built amplifier sampling at 200 Hz (MP100 BioPac
Systems, Inc., Goleta, CA, USA). MAP and fHwere analyzed over 5–10 min
intervals.
Each animal was randomly assigned to one of four treatments: fasted
control (N=8), fasted anemia (N=6), fed control (N=9) or fed anemia
(N=8). Following the measurements of MAP and fH, the ‘fasted’ animals
remained undisturbed at 30°C, whereas ‘fed’ animals consumed rodent
meals equivalent to 25±0% body mass. Contingent upon catheter patency,
48 h after recovery (72 h after surgery), MAP and fHwere obtained and the
RPP was calculated (fH× MAP) from each animal. Cardiac output and thus
work was not measured, but we used the RPP as a proxy for myocardial
work. From each animal, an arterial blood sample of ~0.5 ml was
withdrawn to determine CO
2
(Tucker, 1967), blood pH (glass electrode
maintained at 30°C and connected to a PHM 73; Radiometer, Copenhagen,
Denmark) and Hct. Immediately after blood sampling, adrenergic and
cholinergic tone were assessed by sequential infusion of atropine and
propranolol (see Enok et al., 2012) and calculated from the standard
equations, modified for use of fHrather than R–R interval (e.g. Altimiras
et al., 1997):
and
where fH,cont is the control heart rate, fH,atr is the heart rate following
administration of atropine, and fH,dbl is the double-blocked heart rate (i.e.
following administration of atropine and propranolol).
Tissue harvest
Immediately following assessment of autonomic tone, animals were killed
via intraperitoneal injection of sodium pentobarbital (>100 mg kg−1)
whereupon a long ventral incision allowed for the heart, liver, stomach,
small intestine, large intestine and kidneys to be removed. All organs were
rinsed with isotonic saline and blotted dry with gauze to remove blood and
chyme before determining wet mass. A small representative sample was
removed from each organ and weighed before and after it had been dried in
an oven at 60°C for 72 h to determine the Md:Mwratio.
Statistical analyses
Mass-specific organ mass (g tissue per kg body mass), CO
2
, fHand MAP data
were compared using two-way ANOVA and post hoc Tukey’s HSD in JMP
statistical software (Version 7, SAS Institute, Inc., Cary, NC, USA)
following assurance of homogeneity of variance and normal distribution of
data. Post hoc tests were performed only when the ANOVA yielded
significance (P≤0.05), and were considered significant when P≤0.05.
Hematocrit, adrenergic tone, cholinergic tone and Md:Mwwere arcsin
square-root transformed and compared using a two-way ANOVA in JMP.
Effects, where reported, are the results of the effect tests conducted as part
of the ANOVA model and are distinguished by the single degree of freedom.
Regression plots were generated using GraphPad Prism (Version 6,
GraphPad Software, La Jolla, CA, USA) and slopes were analyzed using the
software’s linear regression analysis. Slopes of the regression lines were
considered significantly different from 0 at the level of P≤0.05. All values
are reported as means ± s.e.m.
Acknowledgements
We owe many thanks to Rasmus Buchanan, Heidi Jensen, Kasper Hansen and
Alexander Jackson for assistance with equipment, animal husbandry and
dissections.
Competing interests
The authors declare no competing financial interests.
Author contributions
All authors contributed to experimental design. C.E.S. and S.E. conducted
experiments, performed statistical analyses, and generated figures. C.E.S. drafted
the manuscript, which was revised and approved in its final form by all authors.
Funding
This research was supported by the Danish Research Council (to T.W.), the
National Science Foundation [grant IOS-0922756 to J.W.H.; Graduate Research
Fellowship to C.E.S.] and the Society for Integrative and Comparative Biology
[Fellowship for Graduate Student Travel to C.E.S.].
=×Cholinergic (%)
–
100 (1)
ff
f
11
1
H,cont H,atr
H,dbl
=×Adrenergic (%)
–
100 , (2)
ff
f
11
1
H,dbl H,atr
H,dbl

The Journal of Experimental Biology
1789
RESEARCH ARTICLE The Journal of Experimental Biology (2014) doi:10.1242/jeb.092841
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