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tapraid4/zh6-areg/zh6-areg/zh600909/zh66913d09z xppws S⫽1 7/16/09 20:26 MS: R-00102-2009 Ini: 07/lak/dlh
Am J Physiol Regul Integr Comp Physiol 297: R000 –R000, 2009.
First published July 9, 2009; doi:10.1152/ajpregu.00102.2009.
AQ: 1
Correlation of cardiac performance with cellular energetic components in the
oxygen-deprived turtle heart
Jonathan A. W. Stecyk,1 Christian Bock,2 Johannes Overgaard,3 Tobias Wang,3 Anthony P. Farrell,4
and Hans-O. Pörtner2
1
Department of Zoology, University of British Columbia, Vancouver, BC, Canada; 2Alfred-Wegener-Institute for Marine and
Polar Research, Bremerhaven, Germany; 3Zoophysiology, Department of Biological Sciences, University of Aarhus, Aarhus,
Denmark; and 4Department of Zoology and Faculty of Food and Land Systems, University of British Columbia, Vancouver,
BC, Canada
Submitted 13 February 2009; accepted in final form 6 July 2009
AQ:2
Stecyk JA, Bock C, Overgaard J, Wang T, Farrell AP, Pörtner
H. Correlation of cardiac performance with cellular energetic components in the oxygen-deprived turtle heart. Am J Physiol Regul Integr
Comp Physiol 297: R000 –R000, 2009. First published July 9, 2009;
doi:10.1152/ajpregu.00102.2009.—The relationship between cardiac
energy metabolism and the depression of myocardial performance
during oxygen deprivation has remained enigmatic. Here, we combine
in vivo 31P-NMR spectroscopy and MRI to provide the first temporal
profile of in vivo cardiac energetics and cardiac performance of an
anoxia-tolerant vertebrate, the freshwater turtle (Trachemys scripta)
during long-term anoxia exposure (⬃3 h at 21°C and 11 days at 5°C).
During anoxia, phosphocreatine (PCr), unbound levels of inorganic
phosphate (effective P2⫺
i ), intracellular pH (pHi) and free energy of
ATP hydrolysis (dG/d␰) exhibited asymptotic patterns of change,
indicating that turtle myocardial high-energy phosphate metabolism
and energetic state are reset to new, reduced steady states during
long-term anoxia exposure. At 21°C, anoxia caused a reduction in pHi
from 7.40 to 7.01, a 69% decrease in PCr and a doubling of effective
P2⫺
i . ATP content remained unchanged, but the free energy of ATP
hydrolysis (dG/d␰) decreased from ⫺59.6 to ⫺52.5 kJ/mol. Even so,
none of these cellular changes correlated with the anoxic depression
of cardiac performance, suggesting that autonomic cardiac regulation
may override putative cellular feedback mechanisms. In contrast,
during anoxia at 5°C, when autonomic cardiac control is severely
blunted, the decrease of pHi from 7.66 to 7.12, 1.9-fold increase of
effective P2⫺
i , and 6.4 kJ/mol decrease of dG/d␰ from ⫺53.8 to ⫺47.4
kJ/mol were significantly correlated to the anoxic depression of
cardiac performance. Our results provide the first evidence for a close,
long-term coordination of functional cardiac changes with cellular
energy status in a vertebrate, with a potential for autonomic control to
override these immediate relationships.
high-energy phosphate metabolism; anoxic turtle cardiac performance; in vivo magnetic resonance spectroscopy
minutes when deprived of oxygen
(anoxia). This intolerance to anoxia is, at least in part, due to
cardiac failure caused by the inability of anaerobic metabolism
to match ATP supply to demand, leading to a decline in cardiac
energy state (11). Nonetheless, while it seems obvious that
perturbation of cardiac energetics contributes to the failure of
an oxygen-starved heart, the exact mechanisms underlying the
decline in cardiac contractile function during oxygen deprivation remain equivocal despite decades of research (1, 81).
Some studies propose that depletion of high-energy phosphates
MOST VERTEBRATES DIE WITHIN
Address for reprint requests and other correspondence: J. A. W. Stecyk,
Physiology Programme, Dept. of Molecular Biosciences, Univ. of Oslo, P.O.
Box 1041, NO-0316 Oslo Norway (e-mail: [email protected]).
http://www.ajpregu.org
[ATP and phosphocreatine (PCr)] and/or accumulation of metabolic by-products such as H⫹, ADP, and inorganic phosphate
(Pi) causes cardiac function to deteriorate (2, 15, 16, 22, 26, 27,
50, 63, 81). Others argue against such mechanisms (3, 14, 44,
50, 62). Moreover, the potential roles of decreased turnover of
high-energy phosphate compounds (7), reduced phosphorylation potential (i.e., [ATP]/[ADP] ⫻ [Pi]) (14), and the decrease
in free energy released from ATP hydrolysis (dG/d␰) (41, 42,
49) are equally ambiguous.
These contradictory conclusions have all arisen from studies
using Langendorff preparations or in situ heart preparations
with various mammalian species. Because cardiac energy status and performance decline precipitously within seconds to
minutes in oxygen-deprived mammalian hearts, the discrepancies among studies could easily arise from the experimental
difficulty associated with their short-term nature. Accordingly,
to compensate for the limitations of modern measurement
techniques, complex computational models have recently been
developed to explain the relationship between cardiac highenergy phosphate metabolism and performance (e.g., 6, 12, 81,
84). This theoretical approach, nevertheless, only provides hypotheses and predictions for future experimental testing (81).
An alternative tactic is to take advantage of a comparative
approach and study species that have evolved to tolerate
prolonged anoxia. The anoxia-tolerant freshwater turtles (genera Chrysemys, Chyledra, and Trachemys) present an interesting animal model in this context for a number of reasons.
Foremost, these reptiles are eminently amenable for long-term
correlation studies of cardiac cellular energy state and function
because they can survive anoxia for up to 24 h at high
temperatures (20 –25°C) and several months at low temperatures (3–5°C) (73). Thus, the anoxic turtle heart continues to
beat and generate work, albeit at a reduced level (see below),
for hours to months depending on ambient temperature. This
much longer time course of anoxia opens up new possibilities
to correlate cardiac function and energy state.
Secondly, turtles are well suited for noninvasive in vivo
NMR experiments because their anoxic cardiac performance at
both warm and cold temperatures is well documented and
consistent among studies (28, 29, 30, 31, 67, 69). Briefly, a
progressive, profound anoxic bradycardia reduces systemic
cardiac output (Qsys) and power output (POsys) by 4.5- to
20-fold to a new steady state within 1 h and ⬃3 days of anoxia
at warm and cold temperature, respectively. Thus, anoxic turtle
heart rate (fH), Qsys, and POsys are closely coordinated (29, 31,
67, 69) (see Supplemental Fig. 1 in the online version of this
article). Consequently, measures of turtle fH and Qsys, which
0363-6119/09 $8.00 Copyright © 2009 the American Physiological Society
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AQ: 3
AQ: 4
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R2
IN VIVO ENERGETICS OF THE ANOXIC TURTLE HEART
can be obtained with established in vivo MRI techniques, can
be regarded as suitable proxies for other measures of cardiac
performance, such as force development, contractility, and
work output that cannot be measured noninvasively by MRI.
Finally, a dichotomy in autonomic cardiovascular control
between warm- and cold-acclimated turtles (30, 67) renders
anoxic turtles unique for an in vivo examination of a temporal
relation between intrinsic cardiac performance and high-energy
phosphate metabolism. At high temperatures, autonomic control of the heart is retained during anoxia exposure, and
cholinergic cardiac inhibition contributes to ⬃36 – 48% of the
anoxic bradycardia (30, 31). In contrast, autonomic cardiac
control is severely blunted at low temperature and does not
contribute to the cardiac downregulation (30). Moreover, at
both high and low temperatures, other mechanisms such as
␣-adrenergic (69) and adenosinergic (67) inhibition are not
involved in the unaccounted cardiac inhibitory mechanisms.
Instead, modifications intrinsic to the heart have been implicated to contribute to the cardiac downregulation (66). Intrinsic
cardiac modifications could arise easily from alteration of
cardiac high-energy phosphate concentrations and, consequently, cellular energetic status. Therefore, the roles of cardiac metabolism and autonomic control in anoxic cardiac
depression can be separated in vivo with investigations at both
warm and cold temperatures.
High-energy phosphate metabolism of the anoxic turtle heart
has been investigated previously by 31P-NMR measurements
on isolated, working in vitro heart preparations exposed to
anoxia, acidosis, or combined anoxia and acidosis at warm
temperature (37, 76, 79) and on tissues terminally sampled
from 3°C turtles after 12 wk of anoxia exposure (38). Results
from the in vitro studies argue against any direct causal
relationship between cardiac function and high-energy phosphate compounds (ATP, PCr, and Pi) during anoxia or acidosis
exposure but revealed that anoxia and acidosis act synergistically to depress cardiac function. However, in vitro studies
depend greatly on the relevance of the in vitro extracellular
conditions and cardiac performance to those in vivo. Therefore,
the isolated heart preparations that were electrically paced (37)
and performing at subphysiological levels (76, 79) may not
create similar energetic conditions as in vivo. The terminal
sampling revealed a ⬃40% decrease of cardiac ATP and PCr,
a fall of intracellular pH (pHi) of 0.2 units, and unchanged Pi
levels (38). However, with terminal sampling, the rapidity with
which tissues can be sampled from a hard-shelled animal is
always a concern, and the temporal changes are undetermined.
Lacking, therefore, is a clear understanding of the temporal
changes in cardiac energy state that occur in vivo in warm and
especially in cold turtles during anoxia.
In the present study, we provide the first continuous measurements of in vivo cardiac energetic state of an anoxiatolerant vertebrate during prolonged anoxia. We used in vivo
31
P- NMR spectroscopy for direct and repeated measurements
of cardiac high-energy phosphates and pHi of unanesthetized
turtles (Trachemys scripta) during prolonged anoxia at 21°C
and 5°C. In addition, by using flow-weighted MRI techniques
to monitor fH, as well as aortic and pulmonary blood flows, we
could establish a time course for changes in cardiac activity
that could be directly compared with the changes in highenergy phosphates, pHi, and energetic state of the heart.
AJP-Regul Integr Comp Physiol • VOL
MATERIALS AND METHODS
Experimental animals and ethical approval. Fourteen red-eared
slider turtles (T. scripta, gray) with body masses ranging between 546
and 748 g (630 ⫾ 76 g, means ⫾ SD) were obtained from Lemberger
(Oshkosh, WI). The seven turtles studied at 21°C were held at room
temperature and a 12:12-h light-dark photoperiod for several weeks in
aquaria with free access to basking platforms and water. They were
fed several times a week with commercial turtle food pellets, but food
was withheld for 4 days before experimentation. The other seven
turtles were acclimated and studied at 5°C. These turtles had been kept
in aquaria within a temperature-controlled room (5°C) for 5 wk before
experimentation and had been fasted during the entire acclimation
period. All experimental procedures were carried out at the AlfredWegener-Institute for Marine and Polar Research in accordance with
German legislation.
Experimental protocol. Twenty-four hours before the in vivo MR
measurements, turtles were placed individually in an enclosed, watercontaining plastic chamber with access to air and were restrained by
two Velcro straps glued to the bottom of the chamber to prevent large
body movements and associated motion artefacts. The turtles could
freely move the appendages and the head. MR measurements were
carried out first under normoxic conditions and then at regular intervals during a prolonged anoxia exposure, so each animal could serve
as its own control. For MR measurements during normoxia, the
temperature within the magnet was set to the acclimation temperature
of the animal, and the chamber containing the turtle was placed within
the magnet [Bruker 47/40 Biospec DBX system with a 40-cm-wide
bore and actively shielded gradient coils (50 mT/m)], and the heart
was centered over a triple tuneable surface coil (31P, 13C, 1H; 5 cm
diameter) that was used for 31P-NMR spectroscopy. An actively
decoupled 1H cylindrical birdcage resonator (20-cm diameter) was
used for the MRI experiments. The coil circuit and field homogeneity
were optimized to the experimental setup, and the location of the heart
within the magnet was confirmed via coronal, sagittal, and transverse
scout images collected using a gradient echo sequence [excitation
pulse shape, hermite; pulse length, 2,000 ␮s, ␣ ⫽ 22.5°; matrix size,
128 ⫻ 128; field-of-view (FOV), 12 cm2; slick thickness, 3 mm;
repetition time (TR), 100 ms; echo time (TE), 5 ms; resulting scan
time, 25 s] (Supplemental Fig. 2). Data presented for control normoxic
31
P-NMR spectra and MR images were averaged from results of 4 or
5 sets of measurements that were obtained over a period of 45– 60 min
(see below for MR spectroscopy and imaging measurement parameters).
To create prolonged anoxia after the normoxic measurements, the
chamber was filled with water of the appropriate temperature and
continuously bubbled with N2 (water PO2 ⬍ 0.3 kPa). For MR
measurements during anoxia, the chamber was recentered in relation
to the magnet, surface coil, and resonator. For the 2.85 h anoxia
exposure at 21°C, 31P-NMR spectra and MR images were acquired
every 10 –15 min. For the 11-day anoxia exposure at 5°C, 31P-NMR
spectra and MR images were acquired every ⬃15 min for the first
⬃18 h and then on days 3, 7, and 11 (as averages from 4 or 5 sets of
31
P-NMR spectra and MR images obtained over a period of 45– 60
min). The duration of the anoxia exposures were similar to previous
studies of cardiac control during prolonged anoxia (29, 30, 31, 66, 67,
69, 70). When not in the magnet, the 5°C anoxic turtles were returned
to the cold room, and the housing chambers continuously bubbled
with N2.
31
P-NMR spectroscopy. In vivo 31P-NMR spectroscopy parameters
were as follows: sweep width, 4,000 Hz; flip angle, 60° (pulse shape,
bp32; pulse length, 200 ␮s); TR, 1 s; scans, 512; total acquisition
time, 8 min 32 s. 31P-NMR spectra were processed using TopSpin
v1.0 software (BrukerBioSpin MRI, Ettlingen, Germany) and an
automatic analyzing routine (written by R.-M. Wittig, Alfred-WegenerInstitute for Marine and Polar Research) to yield integrals of all major
peaks within the spectrum (Fig. 1), as these correlate with the amount
297 • SEPTEMBER 2009 •
www.ajpregu.org
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IN VIVO ENERGETICS OF THE ANOXIC TURTLE HEART
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Fig. 1. Representative in vivo 31P-NMR spectra of a 21°C normoxic turtle (A), a 21°C turtle at 2.85 h of anoxia (B), a 5°C normoxic turtle (C), and a 5°C turtle
on day 11 of anoxia exposure (D). PME, phosphomonoester; Pi, inorganic phosphate; PDE, phosphodiester; PCr, phosphocreatine. ␣-, ␤- and ␥-ATP correspond
to the three phosphates of ATP; ␣-ADP and ␤-ADP correspond to the two phosphates of ADP.
of substance within the detection volume of the 31P-NMR coil (9).
Briefly, a fit function consisting of a combination of Gaussian and
Lorentz line shapes (BrukerBioSpin, Ettlingen, Germany) was adjusted semiautomatically to all signals, resulting in signal integrals.
This procedure allows the quantification of overlapping signals.
Chemical shifts of the signals were determined using an automatic
peak picking routine within the software package TopSpin v1.0
(BrukerBioSpin GmbH, Ettlingen, Germany).
MR imaging. Alternating with spectroscopy, flow-weighted MR
imaging methods, previously used successfully for crustaceans and
fish (8, 10, 47), were applied to measure fH, as well as aortic and
pulmonary blood flows. fH was measured using a single slice fast
gradient echo MRI sequence [Snapshot Flash (25)] with the parameters: excitation pulse shape, hermite; pulse length, 2,000 ␮s; flip
angle, 80°; FOV, 6 cm; one axial slice, slice thickness, 2 mm; matrix,
128 ⫻ 64; TR, 8.53 ms; TE, 3.1 ms; resulting scan time, 545 ms;
dummy scans, 117; repetitions, 32 for 21°C experiments, 64 for 5°C
experiments; receiver gain (rg), 500. Blood flows in the left aorta and
pulmonary artery were determined using the same axial view from a
flow-weighted gradient echo MRI sequence similar to Bock et al. (8)
with the parameters: excitation pulse shape, hermite; pulse length,
2,000 ␮s; flip angle, 80°; FOV, 6 cm; slice thickness, 2 mm; matrix
128 ⫻ 128 averages, 4; dummy scans, 59; rg, 1,500.
Data analysis and statistics. Concentrations of ATP, PCr, and Pi
were expressed as a percentage of the total 31P-NMR signal [i.e., the
sum of the 7 major peaks: phosphomonoester (PME), Pi, phosphodiAJP-Regul Integr Comp Physiol • VOL
ester (PDE), PCr, ␥-ATP, ␣-ATP, and ␤-ATP; see Fig. 1] to control
for 1) possible differences in 31P-NMR signal intensities that can
occur from slight movement of the animal, and 2) minor differences
in the position of the turtle in the magnet before and following
commencement of anoxia, as well as between measurement days. This
approach assumes that no major phosphate export from turtle cardiac
muscle occurs with anoxia exposure. Although no previous study has
reported on the effect of anoxia on all phosphate compounds in the
turtle heart, data from Jackson et al. (38) show that the sum of Pi. PCr,
␤-ATP, PDE, and PME does not change in anoxic 20°C-acclimated
turtle hearts but is reduced slightly (⬃13%) in anoxic 3°C-acclimated
hearts. Similarly, our approach was validated by observing no statistically significant changes in total 31P signal during anoxia at 21°C,
and a small, but insignificant 13% decrease with anoxia exposure at
5°C (see Supplemental Fig. 3 in the online version of this article).
Relative metabolite concentrations were transformed to micromoles
per gram quantities by setting the mean 21°C control normoxic
␤-ATP value to 2.9 ␮mol/g wet weight (38) and using this value as a
conversion factor to calculate the other metabolite concentrations for
both warm and cold turtles. pHi was calculated from the chemical shift
of Pi relative to PCr using previously published formulas, describing
the relationship between pH and chemical shift difference for warm
(76) and cold (38) turtles.
The free energy of ATP hydrolysis (dG/d␰) was estimated from pHi
and ␤-ATP, PCr, and Pi concentrations as described earlier (58) and
is expressed as kilojoules per mole, where more negative values
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IN VIVO ENERGETICS OF THE ANOXIC TURTLE HEART
indicate greater free energy available from the hydrolysis of ATP to
drive ATP-requiring reactions. For the calculation of dG/d␰, molar
concentrations of metabolites were calculated assuming water content
of turtle cardiac tissue to be 80% (21), and levels of unbound effective
P2⫺
i , free ADP (ADPf), and free AMP (AMPf) were calculated on the
basis of the apparent equilibrium constants of creatine kinase and
adenylate kinase, which were corrected for experimental temperature
and pH (48). Cytostolic [Mg2⫹] was assumed to be 1 mM under all
experimental conditions as ␤-ATP peak position was found not to
vary significantly between acclimation temperatures or with anoxia
exposure at 5°C or 21°C (data not shown). Creatine content (Cr) was
estimated as the difference between the total Cr content in the turtle
heart and 31P-NMR measured PCr content. For 21°C turtles, total Cr
is 8.14 ␮M/g (53). Because no previous study has reported total Cr
content of 5°C turtle hearts and because PCr concentration of 5°C
hearts (8.4 ⫾ 0.5 ␮mol/g; Table 1) was greater than 8.14 ␮M/g, total
creatine content was estimated to be 9.95 ␮mol/g at 5°C using the
same ratio of Cr:PCr as for 21°C.
fH was calculated from the time interval between Snapshot Flash
MR images that depicted blood flow through the central blood vessels.
Relative changes in aortic and pulmonary blood flow were determined
by manually selecting regions of interest (ROIs) in the flow-weighted
gradient echo MR images and comparing changes in the mean signal
intensity of the ROIs. Depending on the location of the heart and
quality of image, blood flow was measured from either the left or right
aortic arch, as a previous study has shown blood flow through these
vessels to be equivalent independent of acclimation temperature or
anoxia exposure (67). Likewise, blood flow was measured from either
the right or left pulmonary artery under the assumption that blood flow
is equivalent in both vessels. To better compare data among turtles
and compensate for potential contrast changes between images, baseline corrections were applied to individual ROIs by subtracting signal
intensity of a ROI placed in a region of the image, in which flow
effects could be excluded (similar to Ref. 8; see Fig. 2B). The latter
ROIs are considered as noise. In some instances, especially with
prolonged anoxia at 5°C when fH is less than 1 min⫺1, pulmonary ROI
mean signal intensity was less than the noise ROI mean signal
intensity. Consequently, modest, negative values of pulmonary flow
could be obtained (see Fig. 3A). However, because our MR imagingdetermined changes in aortic and pulmonary flow closely match
previous invasive measurements (see RESULTS), we interpreted the
negative values as a complete cessation of pulmonary blood flow.
Statistically significant changes in measured or calculated parameters between acclimation temperatures were determined using t-tests.
Statistically significant changes in measured or calculated parameters
over time with prolonged anoxia exposure were determined using a
one-way repeated-measures analysis of variance. Where appropriate,
multiple comparisons were performed using Student-Newman-Keuls
tests, and in all instances, significance was accepted when P ⬍ 0.05.
All results are expressed as means ⫾ SE.
RESULTS
Normoxia: phosphorous metabolites, pHi and dG/d␰. Our in
vivo 31P-NMR spectroscopy distinguished the resonance peaks
previously reported for isolated perfused turtle hearts at warm
temperature (38, 76, 77) (Fig. 1). Further, our measurements of
cardiac high-energy phosphate content in normoxia at 5°C and
21°C were similar to previous 31P-NMR studies of isolated
perfused hearts or cardiac strips from Chrysemys picta bellii
(37, 38, 75–77, 79) as well as traditional biochemical measurements (24, 53).
We found a temperature dependence of cardiac high-energy
phosphate metabolism (Table 1). Cardiac PCr, PME, PDE,
ADPf, and AMPf content at 5°C were 1.4⫻, 2.7⫻, 2.6⫻, 3⫻,
and 70⫻ greater, respectively, than at 21°C. Similarly, normoxic pHi, was 0.16 units greater at 5°C. In contrast, estimated
dG/d␰ values revealed that 5.8 kJ/mol less energy was available from the hydrolysis of ATP at 5°C. Likewise, cardiac ATP
content was 48% lower at 5°C than at 21°C.
Prolonged anoxia: fH and blood flow responses. Our MRI
techniques accurately resolved fH and central vascular blood
flows, yielding similar values to those measured directly using
implanted blood flow probes (29, 30, 31, 67, 69) (Figs. 3–5).
Prolonged anoxia at 5°C reduced aortic blood flow by 80 –90%
(Figs. 2A and 3A), and fH decreased from 4.2 ⫾ 0.6 min⫺1 to
⬃1 min⫺1 (Fig. 4A). A steady state in fH was reached after 3
days. A novel finding was the cessation of pulmonary blood
flow with prolonged anoxia exposure at 5°C (Figs. 2B and 3A).
At 21°C, anoxia reduced aortic and pulmonary blood flows by
⬃50% and ⬃85–90%, respectively (Fig. 3B), while fH decreased significantly from 14.1 ⫾ 1.7 to ⬃10 min⫺1 (Fig. 5A).
A new steady state in fH was reached after 0.42 h, after which
there were no significant changes for the remainder of the ⬃3-h
experiment.
Prolonged anoxia: phosphorous metabolite, pHi and dG/d␰
changes. Prolonged anoxia affected the myocardial phosphorous metabolite content, pHi and dG/d␰. At 5°C, the temporal
Table 1. Effect of temperature and anoxia exposure (2.85 h at 21°C and 11 days at 5°C) on turtle myocardial high-energy
phosphate metabolism, pHi and energetic state
21°C
ATP, ␮mol/g
PCr, ␮mol/g
pHi, units
Total Pi, ␮mol/g
Effective Pi2⫺, ␮mol/g
dG/d␰, kJ/mol
ADPf, ␮mol/g
AMPf, nmol/g
PME,␮mol/g
PDE, ␮mol/g
5°C
Normoxia
Anoxia
Normoxia
Anoxia
2.9⫾0.3
5.8⫾0.9
7.40⫾0.10
3.2⫾0.8
2.4⫾0.4
⫺59.6⫾1.0
0.02⫾0.004
0.08⫾0.03
2.2⫾0.3
3.7⫾0.9
2.4⫾0.2
1.8⫾0.3*
7.01⫾0.04*
7.7⫾0.6*
4.8⫾0.4*
⫺52.5⫾0.9*
0.03⫾0.003*
0.41⫾0.07*
3.5⫾0.4*
3.9⫾0.9
1.5⫾0.2†
8.4⫾0.5†
7.66⫾0.06†
4.1⫾0.7
3.0⫾0.4
⫺53.8⫾0.7†
0.06⫾0.004†
5.56⫾1.70†
6.0⫾0.5†
9.7⫾0.5†
0.7⫾0.1*
3.2⫾0.7*
7.12⫾0.04*
8.8⫾0.6*
5.7⫾0.3*
⫺47.4⫾1.0*
0.12⫾0.05
8.36⫾4.94
7.9⫾0.7*
10.1⫾0.5
Values are expressed as means ⫾ SE (n ⫽7 for 21°C and 5°C Normoxia, 6 for 21°C Anoxia and 5 for 5°C Anoxia). ATP: adenosine triphosphate; PCr;
phosphocreatine; pHi: intracellular pH; Pi: inorganic phosphate; dG/d␰: free energy of ATP hydrolysis; ADPf, free adenosine diphosphate; AMPf, free adenosine
monophosphate; PME, phosphomonoester; PDE, phosphodiester. *Significant differences are P ⬍ 0.05 between Normoxia and Anoxia at each acclimation
temperature. †Significant differences for normoxia are P ⬍ 0.05 between 21°C and 5°C.
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IN VIVO ENERGETICS OF THE ANOXIC TURTLE HEART
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exposed to 6 h of anoxia, had clearly reached plateaus for PCr,
Pi, pHi, and dG/d␰ by ⬃1.7 h of anoxia (Fig. 5).
After 11 days of anoxia at 5°C, PCr was reduced by 62%,
total Pi was increased by 2.1-fold, effective P2⫺
was nearly
i
doubled, ATP was decreased by 53%, pHi was reduced by 0.54
units, and dG/d␰ was decreased by 6.4 kJ/mol (Tables 1 and 2).
The respective 1.8- and 1.5-fold increases in ADPf and AMPf
did not reach statistical significance (Fig. 4, E and F; Table 1).
PME increased by 1.3-fold during anoxia, but PDE content did
not change (Table 1).
After 2.85 h of anoxia at 21°C, PCr was reduced by 69%,
total Pi was increased by 2.4-fold, effective P2⫺
was doubled,
i
pHi was reduced by 0.39 units, and dG/d␰ was decreased by 7.1
kJ/mol (Tables 1 and 2). The minor (17%) decrease in ATP
was not statistically significant (Fig. 5D; Tables 1 and 2), but
ADPf and AMPf increased significantly by 1.7- and 4.9-fold,
respectively (Fig. 5, E and F; Table 1). Similar to 5°C turtles,
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Fig. 2. Typical flow-weighted MR images of the central blood vessels of a 5°C
turtle during normoxia (A) and following ⬃12 h of anoxia exposure (B). The
magnitude of blood flow through each vessel is proportional to mean signal
intensity. Note the absence of flow in the right and left pulmonary arteries and
reduced blood flow in the right and left aortic arches with anoxia exposure. The
open circle in the top-right corner of B represents the typical placement of our
noise region of interest (see Supplemental Material in the online version of this
article). V, ventricle; R, right atria; L; left atria, RPA, right pulmonary artery;
LPA, left pulmonary artery; RAo, right aortic arch; LAo, left aortic arch; Bc;
brachiocephalic artery.
change in PCr, Pi, ATP, pHi and dG/d␰ was clearly asymptotic
(Fig. 4, B–D, G and H), suggesting that, following an initial
disrupted state upon the onset of anoxia, a new steady state was
established within ⬃3 h to 3 days (depending on the variable)
of the 11-day anoxic exposure. Likewise, at 21°C, the changes
in PCr, Pi, pHi, and dG/d␰ over the ⬃3 h of anoxia also
appeared to be asymptotic (Fig. 5, B, C, G, and H), suggesting
a new energetic steady state was approached within 0.9 to 1.7 h
at this higher temperature. Indeed, one 21°C turtle, which was
AJP-Regul Integr Comp Physiol • VOL
Fig. 3. Chronological changes of aortic and pulmonary blood flows in 5°C (A)
and 21°C turtles (B) during prolonged anoxia exposure, as determined by MRI
(present study) and by surgically implanted flow probes (data adapted from
Ref. 63 for left aortic flows and Ref. 29 for pulmonary flow). Please note the
different timescale between temperature acclimation groups. Values are expressed as means ⫾ SE; n ⫽ 5–7.
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Fig. 4. Chronological changes of fH, myocardial phosphorous metabolites (PCr; total Pi,
effective P2⫺
i ; ATP, ADPf, and AMPf), cardiac
pHi and cardiac dG/d␰ in 5°C turtles during 11
days of anoxia exposure. For each variable,
statistically significant differences (P ⬍ 0.05)
are indicated by lines above or below the traces.
Solid lines indicate statistical significance from
normoxic control (t ⫽ 0). Dotted lines indicate
statistical significance from the final recording
time (i.e., day 11). C: statistical significance
lines for total Pi and effective P2⫺
are above and
i
below the traces, respectively. Arrows indicate
the anoxia exposure time temporally equivalent
(assuming a Q10 of 2) to the 2.85-h anoxia exposure
at 21°C. Values are means ⫾ SE; n ⫽ 5–7.
T2
PME increased by 1.6-fold during the ⬃3-h anoxia exposure,
but no change occurred in PDE content (Table 1).
To compare the changes in myocardial high-energy phosphate metabolism and energetic state at 5 and 21°C (Table 2),
we assumed that the metabolic processes obey a Q10 of 2, so
that the temperature difference of 16°C corresponds to a
3.2-fold difference in time. Using this approach, we found that
2.85 h of anoxia at 21°C equals 9.1 h (i.e., 0.38 day) of anoxia
at 5°C (indicated by arrows in Fig. 4). This comparison
revealed similar relative decreases of ATP and identical decreases of pHi for 21°C and 5°C turtles. Even so, 21°C turtles
AJP-Regul Integr Comp Physiol • VOL
exhibited a greater relative depletion of PCr and greater relative accumulation of both total Pi and effective P2⫺
i . Consequently, the decrease in dG/d␰ was greater at 21°C than 5°C.
Despite certain quantitative similarities in the perturbation of
cardiac energy states at both temperatures, only at 5°C did the
anoxic depression of cardiac activity closely reflect changes of
myocardial PCr, effective P2⫺
i , ATP, pHi and dG/d␰ (Figs. 3
and 4). Indeed, plotting 5°C anoxic fH and aortic blood flow
against changes in effective P2⫺
i , pHi, or dG/d␰ revealed that
anoxic cardiac performance was tightly matched with the
changes in effective P2⫺
i , pHi, and dG/d␰, and excellent linear
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Fig. 5. Chronological changes of fH, myocardial phosphorous metabolites (PCr; total
Pi, effective P2⫺
i ; ATP, ADPf and AMPf),
cardiac pHi, and cardiac dG/d␰ in 21°C turtles during 2.85 h of anoxia exposure. For
each variable, statistically significant differences (P ⬍ 0.05) are indicated by lines above
or below the traces. Solid lines indicate statistical significance from normoxic control
(t ⫽ 0). Dotted lines indicate statistical significance from the final recording time (i.e.,
2.85 h). C: statistical significance indications
refer to both total Pi and effective P2⫺
i . Open
symbols are expressed as means ⫾ SE; n ⫽
5–7. Gray symbols are data from one turtle
that was exposed to anoxia for 6 h. Values
from this individual are included in the presented mean data.
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regressions were obtained (P ⬍ 0.001; r2 values ⱖ 0.81) (Fig. 6).
It should be noted that unlike for aortic blood flow (Fig. 6,
D–F), the close coordination of fH and cardiac energetic status
was not immediately prevalent upon the onset of anoxia.
Immediately following the commencement of anoxia, fH of
5°C turtles increased from ⬃4 to ⬃6 min⫺1, where it remained
relatively stable until 1.7 h (Figs. 4A; 6, A–C). Concurrently,
AJP-Regul Integr Comp Physiol • VOL
measures of cardiac energetic status either remained stable for
a minimum of 15 min to a maximum of 1.7 h before showing
signs of changing (i.e., ATP, PCr, total Pi, dG/d␰, ADPf, and
AMPf) or immediately changed values (i.e., pHi, and effective
P2⫺
i ) (Figs. 4 and 6). At 1.7 h of anoxia, the bradycardia typical
of anoxic turtles was initiated and, near this time point, the
alterations in PCr, total Pi, effective P2⫺
i , pHi, and dG/d␰
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Table 2. Comparison of the effect of anoxia on myocardial
high-energy phosphate metabolism, pHi, and energetic state
between 21°C and 5°C turtles
21°C
ATP
PCr
pHi, units
Total Pi
Effective Pi2⫺
dG/d␰, kJ/mol
5°C
2.85 h of Anoxia
9 h of Anoxia
11 Days of Anoxia
⫺0.17⫻
⫺0.69⫻
⫺0.39
⫹2.4⫻
⫹2⫻
⫺7.1
⫺0.13⫻
⫺0.51⫻
⫺0.39
⫹1.9⫻
⫹1.8⫻
⫺5.3 kJ/mol
⫺0.53⫻
⫺0.62⫻
⫺0.54
⫹2.1⫻
⫹1.9⫻
⫺6.4 kJ/mol
Magnitudes of change were calculated between normoxic values and those
at the indicated time points of anoxia exposure. Nine hours of anoxia exposure
at 5°C is assumed to be temporally equivalent (assuming a Q10 of 2) to the
2.85 h anoxia exposure duration at 21°C (see text for details).
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became significantly different from control normoxic values
(Fig. 4). Thus, for fH, 1.7 h was the initial time used in the
linear regression analyses.
In contrast to 5°C turtles, fH and aortic blood flow responses
to anoxia at 21°C were not correlated with any measure of
cardiac energy status (Figs. 3 and 5). Instead, fH and aortic
blood flow stabilized within 0.5 h of the commencement of
anoxia, whereas PCr, Pi, pHi, and dG/d␰ continued to either
increase or decrease until 0.90 –1.77 h of exposure, as evidenced by the values not being statistically similar to the 2.85 h
values until this time. As a result, neither fH nor aortic blood
flow was correlated with effective P2⫺
i , pHi, or dG/d␰ (Fig. 7).
DISCUSSION
Our primary objective was to address whether decreased
cardiac performance during oxygen deprivation correlates to
disruptions in cardiac energy metabolism. This important basic, clinical, and pathophysiological question has been extremely difficult to study in mammals because both cardiac
metabolism and power output dissipate rapidly when oxygen is
unavailable. We circumvented this quandary by combining in
vivo 31P-NMR spectroscopy and MRI to simultaneously measure cardiac energetics and performance during prolonged
anoxia with anoxia-tolerant turtles. The turtle lent itself for this
study because cardiac metabolism and power output decline
slowly and reach new steady states during anoxia, allowing for
an excellent time resolution of biochemical and functional
parameters. Moreover, the absence of autonomic cardiac control in 5°C turtles, but its presence in 21°C turtles (30, 67),
enabled the roles of cardiac metabolism and autonomic control
in anoxic cardiac depression to be separated in vivo. We
conclude that a causal relationship exists between anoxic
cardiac cellular energy status and cardiac performance when
autonomic control was absent at 5°C, but not in the presence of
autonomic cardiac control at 21°C.
Critique of methods. The conclusions drawn regarding the
relationship between turtle cardiac energetic components and
cardiac performance are based on the assumption that MRI
measurements of fH and aortic blood flow (i.e., Qsys) faithfully
reflect cardiac work output. We are confident of this approach
because numerous previous in vivo studies have shown anoxic
turtle POsys to be closely coordinated to Qsys and fH (29, 31, 67,
69) (see Supplemental Fig. 1 in the online version of this
article), and our MRI techniques provided reliable fH and aortic
AJP-Regul Integr Comp Physiol • VOL
blood flow measurements. Specifically, our reported stable
anoxic fH of ⬃1 min⫺1 at 5°C and 10 min⫺1 at 21°C and the
depression of aortic blood flow by 80 –90% at 5°C and ⬃50%
at 21°C are very similar to previous measurements with implanted blood flow probes (29 –31, 67, 69) (Figs. 3–5). Indeed,
at both acclimation temperatures, the relationship between
cardiac energetic components and cardiac performance was
very similar independent of whether fH or aortic blood flow
was examined, albeit for a notable distinction during the initial
1.7 h of the 11-day anoxia exposure at 5°C (Figs. 6 and 7).
Considering the fact that fH and Qsys are not necessarily
regulated by the same cellular mechanisms, we feel the overall
consistency and redundancy in our findings add credence to the
notion that changes in cardiac energetic components likely play
an important role in mediating the overall depression in cardiac
performance in cold, anoxic turtles.
Correlation of turtle cardiac energetic components and
cardiac performance. The present study strongly suggests that
alterations of effective P2⫺
i , pHi, and/or dG/d␰ may play
important roles for the depression of cardiac activity in freshwater turtles during prolonged anoxia at 5°C, but not at 21°C
(Figs. 6 and 7). The disparate findings between 21°C and 5°C
could reflect the different roles of the autonomic cardiac
control system. At 21°C, autonomic cardiac control is an
important regulator of anoxic cardiac status (30, 31). It is, thus,
foreseeable that parasympathetic cholinergic cardiac inhibition
decreases cardiac activity faster than the potential negative
effects of cellular perturbations, whereas sympathetic adrenergic cardiac stimulation serves to maintain cardiac activity in
the face of the negative effects of increased effective P2⫺
i ,
reduced pHi, and dG/d␰. For example, negative chronotropic
and inotropic effects of extracellular acidosis, and the associated intracellular acidosis, on the turtle myocardium (75, 76)
can be offset with adrenergic stimulation in vitro (54, 64 – 66,
83). In contrast, at 5°C, when autonomic cardiac control is
severely blunted (30), the negative effects of cellular perturbations on cardiac performance likely predominate.
The decreased pHi during prolonged anoxia is a potential
candidate for mediating the depression of anoxic cardiac performance at 5°C. For mammalian hearts, it is well established
that acidosis negatively affects cardiac chronotopy and inotropy. The negative chronotropic effects arise from the direct
effect of protons on sinoatrial (60. 61) and atrioventricular
node electrophysiology (13). The negative inotropic effects
result from the inhibitory effect of protons on the numerous
proteins involved in Ca2⫹ cycling, as well as on the sensitivity
of troponin C to Ca2⫹ (reviewed by Refs. 52 and 82). For the
turtle myocardium, acidosis has been shown to slow the maximum rate of force development during contraction in warmacclimated hearts (64, 65) and reduce twitch force in coldacclimated hearts (54). Thus, it is highly probable that decreased pHi contributes to the anoxic cardiac downregulation at
5°C via negative inotropic effects. In contrast, other findings
suggest that reduced pHi may not be important in reducing fH
of 5°C anoxic turtles. This is because cold-acclimation appears
to precondition the turtle heart and improve its chronotropic
tolerance to anoxia and the accompanying acidosis (66). Specifically, spontaneous fH of 5°C-acclimated turtles only slows
during a combination of anoxia, acidosis, and hyperkalemia
(66), whereas these extracellular changes, individually as well
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Fig. 6. Relationships between fH (A–C) and
aortic blood flow (D–F) and myocardial effective P2⫺
(A, D), pHi (B, E), and dG/d␰ (C, F)
i
of 5°C turtles exposed to 11 days of anoxia.
Vertical black arrows indicate control normoxic (t ⫽ 0) values. For the fH data (A–C),
vertical gray arrows indicate the initial time
(t ⫽ 1.7 h) utilized in linear regression analyses (see RESULTS). Values are expressed as
means ⫾ SE; n ⫽ 5–7.
as collectively, do slow the spontaneous fH of warm-acclimated
hearts (18, 35, 59, 66, 75–77, 79, 83).
The elevation of unbound effective P2⫺
during prolonged
i
anoxia at 5°C could also depress cardiac activity. In mammals, elevated Pi diminishes the fraction of activated actinmyosin cross-bridges, reduces the calcium sensitivity of
troponin C (22, 56), and has been argued to be the major
determinant of hypoxic contractile failure (6, 16, 26, 81).
Likewise, contraction force and calcium sensitivity of turtle
atrial tissue at high temperature decreases with increased Pi
(39). However, increased ADP concentration counteracts
the negative inotropic effects of Pi (39). In the present study,
normoxic myocardial ADPf was 3-fold greater at 5°C than at
21°C (Table 1). The elevated ADPf could perhaps serve as
a preparatory defense strategy to counteract the inevitable
accumulation of Pi associated with the utilization of the large PCr
store during winter anoxia. Furthermore, the negative effects of Pi
on the turtle myocardium will also be counteracted by the
intracellular acidosis that would protonate some of the accumulating P2⫺
i .
Fig. 7. Relationships between fH (A–C) and
aortic blood flow (D–F) and myocardial effective P2⫺
(A, D), pHi (B, E), and dG/d␰ (C,
i
F) of 21°C turtles exposed to 2.85 h of
anoxia. Vertical black arrows indicate control
normoxic (t ⫽ 0) values. Values are expressed as means ⫾ SE; n ⫽ 5–7.
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IN VIVO ENERGETICS OF THE ANOXIC TURTLE HEART
The strong correlations between dG/d␰ and fH and dG/d␰
aortic blood flow during prolonged anoxia at 5°C suggest
dG/d␰ influences cardiac performance (Fig. 6, C and F).
Decreased dG/d␰ is reasoned to impair cardiac activity because
cardiomyocytes require a high phosphorylation potential (i.e.,
ratio of [ATP]/[ADPf][Pi]), and hence, a favorable dG/d␰ to
drive ATPase-dependent reactions (33). Therefore, a decrease
of dG/d␰ below the energy level required for any particular
ATP-consuming process involved in cardiac contraction and
repeated action potential generation could diminish cardiac
performance (40). Nonetheless, experimental support for this
theory is contradictory in mammals (41, 42, 49), but it has been
proposed that a reduced dG/d␰ decreases cardiac performance
by diminishing Ca2⫹ pumping capacity of the sarcoplasmic
reticulum (SR), which ultimately leads to less Ca2⫹ that can be
released from the SR to activate the contractile system upon
excitation (23). A similar mechanism seems unlikely for the
turtle because the SR plays a very minor role in their beat-tobeat cardiac Ca2⫹ cycling (19, 20). However, it remains to be
studied whether SR Ca2⫹ cycling is enhanced at low temperature in the turtle, as in cold-acclimated fish (Oncorhynchus
mykiss) (74). Obviously, much future work is needed to fully
elucidate how decreased dG/d␰ is manifested as a reduction in
turtle cardiac performance.
The initial rise in fH during the first 1.7 h of anoxia at 5°C
did not correlate with key cardiac energetic components,
whereas the fall in aortic blood flow was immediately correlated to changes in energetic components (Fig. 6). If fH is
viewed as the superior surrogate for cardiac work, the initial
lack of correlation may be the result of a transient stress
response, similar to that displayed by some ectothermic vertebrates at the onset of hypoxia exposure (5, 57), overriding the
effects of changes in high-energy phosphate metabolism on
cardiac performance. Alternatively, the discrepancy could indicate that the inherently different cellular mechanisms regulating cardiac chronotropy and inotropy have dissimilar sensitivities to disruptions of cellular energetic components. Specifically, our data suggest that processes involved in regulating
Qsys are more sensitive than those involved in regulating fH. In
this regard, it is interesting to note that turtle dG/d␰ in normoxia at 21°C (⫺59.6 kJ/mol) and 5°C (⫺53.8 kJ/mol) falls
below the ⫺63.5 kJ/mol deemed critical of cardiac contraction
in the mammalian heart (81). The lower dG/d␰ in the turtle is
less than that expected solely by the temperature difference,
implying that the critical cardiac dG/d␰ may vary between
turtles and mammals and that the physiological processes
underlying cardiac contraction of the turtle heart may operate
with an inherently lower free-energy requirement. This may
possibly be related to the considerably lower cardiac power
output in turtle compared with mammals. On the other hand,
dG/d␰ of the anoxic turtle heart never dropped below the
critical value of ⫺45 kJ/mol advanced by Kammermeier et al.
(41), indicating the possibility of a common anoxic critical
cardiac dG/d␰ among vertebrates.
Anoxic cardiac energetic status. Beyond describing the
temporal relationship between cardiac high-energy phosphate
metabolism and performance, the magnitude and time-course
of change in myocardial high-energy phosphates, pHi, and
dG/d␰ during anoxia lead to three inferences of turtle anoxic
cardiac energetics.
AJP-Regul Integr Comp Physiol • VOL
First, anoxic turtles reorganized their cellular energetic state
to a new, but lower steady state within hours, taking slightly
longer at the colder temperature. This phenomenon is clearly
reflected by the asymptotic change of high-energy phosphates,
pHi and dG/d␰, during the onset of anoxia and their subsequent
stability (Figs. 4 and 5). Such reorganization requires that
cardiac energy-consuming processes be reduced during anoxia.
At the organismal level, the anoxic turtle drastically reduces
ATP demand by decreasing metabolic rate (28, 34). In liver
and brain, this metabolic depression involves suppression of
protein turnover by “translational arrest,” a reduction of transmembrane ion movement via “channel arrest” and a reduction
of electrical activity of brain cells by “spike arrest” (reviewed
by Refs. 32, 36, 51, 72). Anoxic depression of resting cardiac
metabolic rate also contributes to decreased cardiac ATP demand (53), but the reduction in mechanical work, driven
largely by anoxic bradycardia, represents the primary energysaving mechanism (17, 29, 55, 71). Thus, in line with the
depression of whole body metabolism, a 6- to 22-fold reduction in
POsys lowers cardiac ATP demand well below the capacity for
cardiac anaerobic ATP generation (17, 29). At the cellular level,
translational arrest has been documented in the warm, anoxic
turtle ventricle (4), but anoxic channel arrest does not seem to
occur (70, 71). Ventricular ␤-adrenergic receptor density, nevertheless, is down-regulated in anoxia, rendering their heart
less sensitive to adrenergic stimulation (30, 66) in spite of the
very high levels of circulating catecholamines (43). In mammalian hearts, inotropic agents increase performance and counteract cardiac failure during hypoxia. However, by augmenting
cardiac energetic costs, they ultimately increase, rather than
decrease, heart failure mortality (33). Thus, the blunting of
autonomic control in cold-acclimated turtles may be critical to
conserving energy.
Secondly, our data reveal that the creatine kinase equilibrium played an important role in supplying ATP during early
phases of anoxia at both temperatures. As shown previously for
anoxic turtle heart and brain (38, 76, 79, 80), the reduction of
PCr during anoxia was mirrored by a rise of total Pi (Figs. 4
and 5). In this regard, the higher content of PCr at 5°C in
normoxia may be a preparatory response for winter hibernation. On the other hand, depletion of a large PCr store would
increase Pi, which may reduce contractility (39), but, as discussed above, cold turtles appear to have strategies to counteract increased P2⫺
during prolonged anoxia. Likewise, the
i
greater abundance of PME and PDE at 5°C than 21°C in
normoxia (Table 1) may also be a preparatory response for
winter anoxia. PDEs are greater in hearts of anoxia-tolerant
turtles compared with anoxia-intolerant species, and their lysophospholipase inhibitory function has been hypothesized to
be important for tolerance to anoxia and ischemia (78).
The third inference relates to important similarities and
differences between temperatures. In some regard, the changes
in high-energy phosphate metabolism were very similar between temperatures when a Q10 of 2–3 is taken into consideration (Table 2). ATP and pHi, for example, were altered by
identical amounts after ⬃3 h of anoxia at 21°C and 9 h of
anoxia at 5°C. However, Pi increased, while PCr and dG/d␰
decreased, faster at 21°C compared with 5°C. This situation
likely arises because the turtle heart exhibits inverse thermal
acclimation; a phenomenon in which physiological processes
not only passively decrease with cold temperature, but are
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IN VIVO ENERGETICS OF THE ANOXIC TURTLE HEART
additionally actively downregulated to further minimize ATP
consumption (28). Thus, cold acclimation induces an active
depression of cardiac activity that both decreases ATP demand
for mechanical work (28, 29, 67, 69) and extensively modifies
the electrophysiology, which may further reduce energetic
costs (70). Consequently, cold-acclimated turtle hearts are
apparently primed to function under low energy conditions,
which may translate to smaller reductions of PCr and dG/d␰
and lesser accumulation of Pi at 5°C. Despite this cold-induced
metabolic preparation, cardiac ATP was reset to 50% of the
normoxic value by the 3rd day of anoxia at 5°C and remained
at this level for a further 8 days (Fig. 4H). This finding clearly
signifies that cardiac ATP does not have to be maintained at
control normoxic levels for successful and prolonged anoxia
survival.
Perspectives and Significance
In 1929, the Danish physiologist and Nobel laureate August
Krogh wrote, “For such a large number of problems, there will
be some animal of choice or a few such animals on which it can
be most conveniently studied” (45). Here, we affirm Krogh’s
principle. By combining comparative physiology with modern
in vivo 31P-NMR spectroscopy and MRI measurement techniques, we have provided the first in vivo evidence for a close,
long-term coordination of cardiac function with high-energy
phosphate metabolism during an extended period of oxygen
deprivation in a vertebrate. By comparison, decades of investigation of oxygen-starved mammalian hearts have not unequivocally demonstrated such a correlation. Further, we discovered that although turtle cardiac energetic status is initially
disrupted with the onset of anoxia, energetic status is relatively
rapidly reset to a new, reduced steady state during prolonged
anoxia exposure at both high and low temperature. Again, the
detection of such a phenomenon was only possible because of
our choice of study species. Combined, these findings stress the
worthiness of the comparative approach to important physiological questions of basic and biomedical importance. Anoxiarelated diseases such as stroke and heart infarction are major
causes of death, and invaluable insights could be gained by
deciphering how the champions of vertebrate anoxia tolerance
(i.e., the freshwater turtle and the crucian carp, Carassius
carassius, which possess the unique ability to retain normal
cardiac performance during prolonged anoxia; 68) have solved
the problem of living long term without oxygen. Further, the
role of autonomic control clearly needs to be investigated in
whether it overrides the relationship between cellular energy
status and cellular performance in mammals in similar ways as
it does in turtles. Future examination is also needed to determine whether the correlation between cardiac performance and
energetic status persists upon reoxygenation.
ACKNOWLEDGMENTS
This research was supported by Natural Sciences and Engineering Research
Council of Canada grants to A. P. Farrell and J. A. W. Stecyk, a Company of
Biologists Travel Fund, and the Journal of Experimental Biology Traveling
Fellowship to J. A. W. Stecyk, a Response of Higher Life to Change grant
(within MARCOPOLI) to H.-O. Pörtner, and Danish Research Council funding to T. Wang. Special thanks to Rolf Wittig for his postprocessing of
31
P-NMR spectra and flow-weighted images.
Present address of Jonathan A. W. Stecyk: Physiology Programme, Department of Molecular Biosciences, University of Oslo, P.O. Box 1041, NO-0316
Oslo Norway.
AJP-Regul Integr Comp Physiol • VOL
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JOBNAME: AUTHOR QUERIES PAGE: 1 SESS: 1 OUTPUT: Thu Jul 16 20:26:56 2009
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AUTHOR QUERIES
AUTHOR PLEASE ANSWER ALL QUERIES
1
AQ1— Present addresses of authors are placed at the end of the Acknowledgments section.
Footnote 2 has been deleted from the author/ affiliation line.
AQ2— Please note that the author list in the abstract line represents the form in which these
names will appear in many online databases, such as the NCBI/NIH/NLM Pubmed database.
Check this carefully, be sure there are no misrepresentations. Please make a note on the proof,
if any corrections are needed.
AQ3— To aid the reader, could you please identify the section heading where this is discussed,
rather than using “see below”?
AQ4 — Please note that AJP style now allows many common abbreviations to be used without the
traditional explicit definition. This list is published in some issues of AJP and includes such
items as “NMR”, and many others.
AQ5— APS style does not permit the use of italics for common Latin terms.
AQ6 — “dG/d␰ ” stated as meant?
AQ7— In Fig. 2B, RA and LA are not defined in the caption. Should they instead be R and L, for
right atria and left atria, respectively?
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