Download Acute hibernation decreases myocardial pyruvate carboxylation and

Am J Physiol Heart Circ Physiol
281: H1613–H1620, 2001.
Acute hibernation decreases myocardial pyruvate
carboxylation and citrate release
ASHISH R. PANCHAL,1 BLANDINE COMTE,2 HAZEL HUANG,1 BASIL DUDAR,1
BRIDGETTE ROTH,1 MARGARET CHANDLER,1 CHRISTINE DES ROSIERS,3
HENRI BRUNENGRABER,2 AND WILLIAM C. STANLEY1,2
Departments of 1Physiology and Biophysics and 2Nutrition, Case Western Reserve
University, Cleveland, Ohio 44106-4970; and 3Department of Nutrition, University
of Montreal, Montreal, Quebec H3C 3Y7, Canada
Received 3 April 2001; accepted in final form 25 May 2001
THE HIBERNATING MYOCARDIUM is defined by reversible myocardial contractile dysfunction due to reduced coronary
flow and decreased oxygen supply to the myocardium
(26). The hallmarks of hibernation are the retention of
viable myocardial tissue with residual mitochondrial
functions such as pyruvate and fatty acid oxidation, electron-transport-chain flux, and oxidative phosphorylation
to generate ATP (1, 29). Little is known about the function of the citric acid cycle (CAC) in myocardium during
hibernation. Ischemia in isolated perfused rat hearts
causes an increase in the net efflux of the CAC intermediate succinate (15, 24, 25). Although this suggests a net
loss of CAC intermediates during ischemia, it is not clear
whether ischemia results in depletion of the pool of CAC
intermediates (11, 15, 24). The tissue content of CAC
intermediates is small compared with the flux through
the cycle, and loss of CAC intermediates from the cycle
must be balanced by the entry of intermediates into the
CAC if the pool size is to be maintained (see Fig. 1). With
normal myocardial blood flow, the loss of CAC intermediates is balanced by the entry of newly synthesized
intermediates into the cycle; this process is termed
anaplerosis (7, 8, 11, 14, 21). The effects of acute myocardial hibernation on the rate of anaplerosis and the tissue
content of CAC intermediates are not known.
Pyruvate carboxylation is a major anaplerotic pathway in normal myocardium in vivo (21) and generates
malate and oxaloacetate (OAA) via malic enzyme and
pyruvate carboxylase, respectively (2, 8, 23, 35, 36) (see
Fig. 1). We recently developed a method to measure in
the heart of anesthetized swine the rate of pyruvate
carboxylation and decarboxylation using [U-13C3]lactate
and [U-13C3]pyruvate tracers and mass isotopomer analysis of tissue pyruvate and citrate (21). In isolated rat
hearts and in vivo swine myocardium, we found that
pyruvate carboxylation accounted for 2.5–8% of the citrate synthase flux (7, 8, 21, 39). We also found that the
rate of pyruvate carboxylation was not significantly altered when the rate of pyruvate decarboxylation (i.e., flux
through pyruvate dehydrogenase) was inhibited by
⬎90% by infusion of octanoate. Thus constitutive pyruvate carboxylation appears to be essential for normal
cardiac function as was demonstrated in isolated working
rat hearts where there was a dramatic fall in ventricular
power when pyruvate carboxylation was pharmacologically inhibited (28). Constitutive pyruvate carboxylation
balances the loss of CAC intermediates (11, 15, 21, 39)
including citrate (21, 39). Under aerobic conditions, citrate efflux from rat, swine, and human hearts ranges
from 5 to 20 nmol䡠g⫺1 䡠min⫺1 (21, 38, 39). Net citrate
Address for reprint requests and other correspondence: W. C.
Stanley, Dept. of Physiology and Biophysics, School of Medicine,
Case Western Reserve Univ., 10900 Euclid Ave. Cleveland, OH
44106-4970 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
cardiac; citric acid cycle; dehydrogenase; metabolism; ischemia
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0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
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Panchal, Ashish R., Blandine Comte, Hazel Huang,
Basil Dudar, Bridgette Roth, Margaret Chandler,
Christine Des Rosiers, Henri Brunengraber, and William C. Stanley. Acute hibernation decreases myocardial
pyruvate carboxylation and citrate release. Am J Physiol
Heart Circ Physiol 281: H1613–H1620, 2001.—In the wellperfused heart, pyruvate carboxylation accounts for 3–6% of
the citric acid cycle (CAC) flux, and CAC carbon is lost via
citrate release. We investigated the effects of an acute reduction in coronary flow on these processes and on the tissue
content of CAC intermediates. Measurements were made in
an open-chest anesthetized swine model. Left anterior descending coronary artery blood flow was controlled by a
extracorporeal perfusion circuit, and flow was decreased by
40% for 80 min to induce myocardial hibernation (n ⫽ 8). An
intracoronary infusion of [U-13C3]lactate and [U-13C3]pyruvate
was given to measure the entry of pyruvate into the CAC
through pyruvate carboxylation from the 13C-labeled isotopomers of CAC intermediates. Compared with normal coronary
flow, myocardial hibernation resulted in parallel decreases of
65% and 79% in pyruvate carboxylation and net citrate release
by the myocardium, respectively, and maintenance of the CAC
intermediate content. Elevation of the arterial pyruvate concentration by 1 mM had no effect. Thus a 40% decrease in coronary
blood flow resulted in a concomitant decrease in pyruvate carboxylation and citrate release as well as maintenance of the
CAC intermediates.
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PYRUVATE CARBOXYLATION IN MYOCARDIUM DURING HIBERNATION
Fig. 1. Scheme of the incorporation of pyruvate into the
citric acid cycle (CAC) via pyruvate carboxylation and
decarboxylation. Arrows going toward the CAC are
anaplerotic fluxes (feeding in); arrows going out are
CAC intermediate release.
MATERIALS AND METHODS
Chemicals. Chemicals, enzymes, and coenzymes were purchased from Boehringer Mannheim (Indianapolis, IN) and
Sigma-Aldrich (Milwaukee, WI). [2H6]succinic acid, [U-13C3]
lactate, and [U-13C3]pyruvate were obtained from Isotec (Miamisburg, OH). The derivatization agent N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide was supplied by Regis
AJP-Heart Circ Physiol • VOL
Chemical (Morton Grove, IL). Intralipid solution was obtained
from Baxter Healthcare (Deerfield, IL).
Experimental model. A previously described in vivo technique was used to deliver 13C-labeled substrates directly into
the left anterior descending (LAD) coronary artery of swine
(31, 32). Overnight-fasted domestic swine (weight 27–38 kg)
of either sex were sedated with Telazol (6 mg/kg im), anesthetized with pentobarbital sodium (25 mg/kg ⫹ 5
mg 䡠 kg⫺1 䡠 h⫺1 iv), intubated via a tracheotomy, and ventilated to maintain arterial blood gas values in the normal
range (PO2 ⬎ 100 mmHg, PCO2 of 35–45 mmHg, and pH of
7.35–7.45). A 7-Fr high-fidelity pressure transducer catheter
(Millar; Houston, TX) was positioned in the left ventricle via
the carotid artery. The animal was then heparinized (300
U/kg bolus ⫹ 150 U 䡠 kg⫺1 䡠 h⫺1 iv) and infused with a 20%
triglyceride emulsion (Intralipid 20%, 0.3 ml 䡠 kg⫺1 䡠 h⫺1 iv) to
increase plasma free fatty acids (FFA) to 0.6 mM (32). Coronary blood flow in the anterior wall was controlled by an
extracorporeal circuit as previously described (31, 32). The
anterior interventricular vein was cannulated to collect venous blood samples from the perfusion territory of the LAD.
The coronary perfusion pump flow was adjusted to give an
interventricular venous Hb saturation of 35–40% (31, 32).
Experimental protocols. Pigs were subjected to acute myocardial hibernation induced by decreasing the blood flow to
the LAD bed by 40% (see Fig. 2). Pyruvate carboxylation and
decarboxylation were measured with an intracoronary infusion of [U-13C3]lactate and/or [U-13C3]pyruvate for 60 min,
with subsequent analysis of myocardial tissue for 13C isotopomers of pyruvate and CAC intermediates by gas chromatography-mass spectrometry (GC-MS) as previously described (21). Stock solutions of 99% [U-13C3]lactate and/or
99% [U-13C3]pyruvate were directly infused into the LAD
perfusion circuit at a rate of 6.5 ␮l/ml of LAD blood flow (see
Fig. 2). Pyruvate carboxylation was measured in hibernating
myocardium under either near-normal arterial lactate and
pyruvate concentrations (HIB group, n ⫽ 8), or with elevated
arterial pyruvate concentration (HIB ⫹ PYR group, n ⫽ 8). In
HIB animals, the concentrations of [U-13C3]lactate and
[U-13C3]pyruvate in the infusate were 154 and 15.4 mM,
respectively, so that the lactate and pyruvate concentrations
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efflux from swine myocardium amounts to ⬃20% of the
rate of pyruvate carboxylation (21).
The extent of pyruvate carboxylation and citrate
efflux in the hibernating or ischemic myocardium is not
known. Opie (20) reported a 33% decrease in myocardial citrate content after 30 min of severe ischemia (an
⬃90% reduction in flow) in dogs; however, the actual
rate of citrate efflux from the heart was not measured.
Evidence for a role of CAC intermediate depletion in
postischemic cardiac dysfunction was suggested by improved postischemic functional recovery when anaplerotic substrates (pyruvate, glutamate, or propionate)
were administered to isolated rodent hearts (4, 6, 27,
28, 37). The functional benefits of elevated pyruvate
concentration suggest that anaplerotic pyruvate carboxylation may play an important role in the correction
of metabolic abnormalities during conditions of stress
such as hibernation.
The goals of the present study were to: 1) examine
the effects of hibernation on the content of CAC intermediates in the heart; 2) measure pyruvate carboxylation, pyruvate decarboxylation, and citrate efflux in the
myocardium during hibernation; and 3) determine the
effects of pharmacological concentrations of pyruvate
on the hibernating myocardium. We used [U-13C3]
lactate and [U-13C3]pyruvate and isotopomer analysis
of tissue pyruvate and citrate to measure pyruvate
carboxylation and decarboxylation in the well-characterized swine model of acute myocardial hibernation
(1, 18, 29).
PYRUVATE CARBOXYLATION IN MYOCARDIUM DURING HIBERNATION
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Fig. 2. Experimental protocol was divided into equilibration and experimental periods. At time ⫺20, blood
flow from the left anterior descending
(LAD) coronary artery was reduced by
40%. Infusion of 13C isotopic substrates
began at time 0 and continued to the
end of the protocol. A large punch biopsy was taken at time 60 for measurement of pyruvate decarboxylation and
carboxylation. Arrows designate sampling of arterial and venous blood and
cardiovascular recordings. HIB, hibernating myocardium under near-normal arterial lactate and pyruvate concentrations; HIB ⫹ PYR, hibernating
myocardium with elevated arterial
pyruvate concentration.
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perfusion bed. Mass isotopomers of metabolites containing
0-n 13C atoms are identified as Mi, where i ⫽ 0, 1,. . .,n. The
relative rates of pyruvate carboxylation and decarboxylation
were calculated as previously described (7) from 1) the M3
enrichment of tissue pyruvate, and 2) the M2 and M3 enrichments of the acetyl-CoA and OAA moieties of citrate, respectively (21). Mass isotopomers were adjusted for the natural
abundance measured in myocardial tissue samples from pigs
(n ⫽ 4) that were not infused with labeled substrates (5, 9).
The measured enrichment of the M3 OAA moiety of citrate
was corrected for 1) the fraction of M3 OAA molecules coming
from some citrate isotopomers metabolized in the CAC and 2)
the dilution of 13C in the CAC, as described in detail by
Comte and co-workers (Eqs. 8–10 in Ref. 7). The absolute
rates of pyruvate carboxylation and decarboxylation were
calculated from the relative rates of pyruvate carboxylation
and decarboxylation and the absolute rate of CAC flux. The
latter was calculated from the myocardial oxygen consumption (MV̇O2) and the stoichiometric relationships between
oxygen consumption and citrate formation from fat and carbohydrate as previously described (21).
Statistical analysis. Data are presented as means ⫾ SE.
The hemodynamic variables were compared between the two
protocols using repeated-measures ANOVA. Statistical significance was determined using paired and unpaired t-tests
as appropriate.
RESULTS
Cardiovascular parameters. There were no significant changes over the course of the experiment or
between groups at any time point in peak systolic LV
pressure, peak LV dP/dt, heart rate (see Fig. 3), or LV
end-diastolic pressure (data not shown). In the HIB
and HIB ⫹ PYR groups, MV̇O2 decreased by ⬃38%
after 80 min of flow reduction (see Fig. 3).
Metabolite concentrations and enrichments. With the
onset of flow reduction, there was a metabolic switch
from net lactate uptake to lactate production (see
Fig. 4).
The arterial blood citrate concentrations were 56 ⫾ 3
and 56 ⫾ 2 ␮M in the HIB and HIB ⫹ PYR groups,
respectively, and did not change from the beginning to
the end of the protocol. Net citrate release by the
myocardium decreased significantly (P ⬍ 0.05) from
the preischemic control period (11.3 ⫾ 3.8 and 12.4 ⫾
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in LAD blood were raised by 1.0 and 0.1 mM, respectively. In
the HIB ⫹ PYR group, the concentration of [U-13C3]pyruvate
in the infusate was 154 mM, so that the pyruvate concentration in LAD blood was raised by 1.0 mM. Left ventricular
pressure, end-diastolic pressure, peak first derivative of left
ventricular (LV) pressure with time (dP/dt), heart rate, and
arterial and venous blood samples were taken at all sample
times (see Fig. 2). Plasma samples were stored at ⫺80°C
until further analysis. At the end of each protocol, large
punch biopsies (3 g) of the LAD and circumflex (CFX) beds
were quickly taken, freeze-clamped, and stored at ⫺80°C
until analysis. We have previously shown that the CFX
biopsy receives normal myocardial blood flow and thus serves
as control tissue (18, 31). The heart was excised, and black
ink was infused down the right and left main coronary
arteries to identify the LAD perfusion bed, which was dissected and weighed (37.1 ⫾ 2.7 g).
Analytic methods. The concentrations of plasma FFA,
blood glucose, lactate, pyruvate, and citrate, as well as tissue
lactate, pyruvate, and malate were determined using spectrophotometric enzymatic assays (3, 33, 40). Tissue pyruvate
concentrations were measured immediately after homogenization in neutralized perchloric acid extracts to prevent loss
of pyruvate from freeze-thaw (40). Tissue concentrations of
ATP and ADP were measured using the ATP Bioluminescent
Assay Kit (Sigma-Aldrich).
Isotopic enrichments of plasma lactate and pyruvate were
determined from the GC-MS analysis of the corresponding
tert-butyldimethylsilyl (TBDMS) derivatives as previously
described (7, 8, 21). The mass isotopomer distribution of
tissue lactate, pyruvate, citrate, succinate, fumarate, and
malate and the OAA moiety of citrate were also assayed as
TBDMS derivatives (7, 8, 15, 39). Analyses were performed
on a Hewlett-Packard 5890 Series II GC with an HP-5 capillary column (length 50 m, inside diameter 0.2 mm, and film
thickness 0.3 ␮m) coupled to a mass-selective detector (model
5970). Helium gas flow in the capillary column was 0.8–1.0
ml/min. Individual enrichments are averages of two or three
GC-MS injections. The tissue concentrations of citrate, succinate, and fumarate were also assayed by GC-MS as previously described (7, 21).
Enzyme activity of malic enzyme was measured using the
methods described by Lin and Davis (17). Activity of pyruvate carboxylase was measured as per a modification of the
original method by Struck and colleagues (34).
Calculations. The myocardial blood flow was calculated as
the LAD perfusion pump flow divided by the mass of the LAD
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PYRUVATE CARBOXYLATION IN MYOCARDIUM DURING HIBERNATION
5). Thus myocardial hibernation resulted in an ⬃80%
reduction in citrate release by the heart.
The arterial plasma FFA level was stable for the HIB
and HIB ⫹ PYR groups at 0.54 ⫾ 0.05 and 0.56 ⫾ 0.06
mM, respectively. Net FFA uptake values during the
Fig. 3. Left ventricular (LV) peak systolic pressure (A), peak first
derivative of LV pressure with time (dP/dt, B), heart rate (C), and
myocardial oxygen consumption (D).
2.1 nmol 䡠 g⫺1 䡠 min⫺1 for HIB and HIB ⫹ PYR groups,
respectively) to the hibernation period (2.5 ⫾ 2.3 and
1.4 ⫾ 2.1 nmol 䡠 g⫺1 䡠 min⫺1 for HIB and HIB ⫹ PYR
groups, respectively) in both the HIB and HIB ⫹ PYR
groups. The rate of citrate release during hibernation
was significantly lower than our previously published
values (21) from animals subjected to the same isotope
infusion but with normal coronary blood flow (see Fig.
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Fig. 5. Rate of citrate release (A) and pyruvate carboxylation (B).
Data for the aerobic values are from previous work (21); *P ⬍ 0.05.
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Fig. 4. Net myocardial lactate production from ⫺40 to 0 min. *P ⬍
0.05 compared with samples at ⫺40 to ⫺25 min.
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PYRUVATE CARBOXYLATION IN MYOCARDIUM DURING HIBERNATION
Table 1. Tissue pyruvate and lactate concentrations
in tissue from left anterior descending artery beds
Table 3. Tissue concentration of citric acid cycle
intermediates in LAD and CFX beds
Tissue Concentration, ␮mol/g
wet wt
Group
HIB
HIB ⫹ PYR
Lactate
2.29 ⫾ 0.12
2.00 ⫾ 0.12
Pyruvate
0.17 ⫾ 0.02
0.19 ⫾ 0.02
Tissue Concentration, ␮mol/g wet wt
Group
15.5 ⫾ 2.3
11.4 ⫾ 1.9
Values are means ⫾ SE. HIB, hibernating conditions; PYR, high
arterial pyruvate.
Table 2. Tissue concentrations of ATP and ADP
and ratio of ATP/ADP in left anterior descending
and circumflex artery beds
Succinate
Fumarate
Malate
ATP
ADP
ATP/ADP
3.71 ⫾ 0.29
3.88 ⫾ 0.31
1.16 ⫾ 0.19
1.42 ⫾ 0.28
4.08 ⫾ 0.90
4.50 ⫾ 1.64
3.68 ⫾ 0.36
3.93 ⫾ 0.34
1.19 ⫾ 0.26
1.30 ⫾ 0.13
4.35 ⫾ 1.05
3.34 ⫾ 0.51
Values are means ⫾ SE. LAD, left anterior descending artery;
CFX, circumflex artery.
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1.61 ⫾ 0.075 0.098 ⫾ 0.008 0.109 ⫾ 0.009 0.165 ⫾ 0.016
1.41 ⫾ 0.074 0.093 ⫾ 0.004 0.104 ⫾ 0.005 0.153 ⫾ 0.015
1.55 ⫾ 0.114 0.112 ⫾ 0.011 0.115 ⫾ 0.007 0.170 ⫾ 0.016
1.40 ⫾ 0.105 0.096 ⫾ 0.010 0.107 ⫾ 0.005 0.151 ⫾ 0.008
Values are means ⫾ SE.
The tissue contents of citrate, succinate, fumarate,
and malate were not different between the LAD and
CFX beds, nor was there a difference between the HIB
and HIB ⫹ PYR groups for a given bed (see Table 3).
These values are similar to those previously measured
in aerobic animals and aerobic animals with elevated
pyruvate (21). Thus hibernation induced by a 40%
reduction in LAD flow did not result in a change in the
concentration of the CAC intermediates.
Pyruvate carboxylation and decarboxylation. The relative rates of pyruvate decarboxylation and carboxylation were calculated as previously described (7, 21).
There were no changes in pyruvate decarboxylation
between the groups (see Table 4) nor were these values
different from values obtained in aerobic hearts (21).
The relative rates of pyruvate carboxylation during
hibernation were unaffected by elevated arterial pyruvate concentrations; however, values from hibernating
myocardium were 50% lower than those previously
found in aerobic and aerobic ⫹ pyruvate animals (see
Table 4). Because there was not a significant change in
pyruvate decarboxylation, the ratio of carboxylation to
decarboxylation was significantly decreased by hibernation in both groups (see Table 4).
Absolute rates of pyruvate decarboxylation and carboxylation were calculated from the rate of CAC flux,
which was estimated from MV̇O2 as described previously (21). The rates of CAC flux were similar between
the two groups (1.06 ⫾ 0.08 and 1.14 ⫾ 0.12
␮mol 䡠 g⫺1 䡠 min⫺1 for the HIB and HIB ⫹ PYR groups,
respectively). The absolute rates of pyruvate decarboxylation (which is the flux through pyruvate dehydrogeTable 4. Relative fluxes through pyruvate
decarboxylation and carboxylation per citric
acid cycle flux and ratios of pyruvate
carboxylation to decarboxylation
Tissue Concentration, ␮mol/g
wet wt
HIB
LAD
CFX
HIB ⫹ PYR
LAD
CFX
HIB
LAD
CFX
HIB ⫹ PYR
LAD
CFX
Relative Flux, %
Group
PD/CAC
PC/CAC
PC/PD
Source
Aerobic
HIB
Aerobic ⫹ PYR
HIB ⫹ PYR
41.5 ⫾ 4.8
46.1 ⫾ 1.9
34.3 ⫾ 3.3
41.2 ⫾ 1.7
4.7 ⫾ 0.7
2.5 ⫾ 0.3*
5.7 ⫾ 0.6
2.7 ⫾ 0.5†
12.1 ⫾ 2
5.5 ⫾ 0.7*
17.7 ⫾ 3
6.5 ⫾ 1.2†
Ref. 21
This investigation
Ref. 21
This investigation
Values are means ⫾ SE. PD, pyruvate decarboxylation; CAC, citric
acid cycle; PC, pyruvate carboxylation. * P ⬍ 0.05 vs. aerobic group;
† P ⬍ 0.05 vs. aerobic ⫹ PYR group.
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equilibration period were 0.12 ⫾ 0.02 and 0.12 ⫾ 0.03
␮mol 䡠 g⫺1 䡠 min⫺1 for the HIB and HIB ⫹ PYR animals,
respectively, and were not different at the end of the
protocol (0.09 ⫾ 0.01 and 0.09 ⫾ 0.03 ␮mol 䡠 g⫺1 䡠 min⫺1
for the HIB and HIB ⫹ PYR groups, respectively). The
rates of glucose uptake were similar at the end of the
protocol compared with the equilibration period for the
HIB animals (0.31 ⫾ 0.06 and 0.51 ⫾ 0.20
␮mol 䡠 g⫺1 䡠 min⫺1, respectively) and the HIB ⫹ PYR
animals (0.26 ⫾ 0.04 and 0.47 ⫾ 0.19 ␮mol 䡠 g⫺1 䡠 min⫺1,
respectively).
Tissue metabolites. Tissue lactate and pyruvate content and the lactate-to-pyruvate ratio were similar
between the two groups (see Table 1) and were not
different from previously published values from normal-flow animals subjected to the same isotope-infusion protocol (21). Thus despite lactate production at
the onset of flow reduction, we observed normal lactate
and lactate-to-pyruvate ratios after 80 min of reduced
flow. Furthermore, the concentrations of ATP and ADP
and the ATP-to-ADP ratio were unchanged between
the hibernating LAD and CFX beds or between the two
groups for a given bed (see Table 2), which confirms the
observation of Schulz et al. (29) that there are normal
ATP levels after ⬃80 min of moderate flow reduction in
swine myocardium.
The tissue M3 enrichments of lactate and pyruvate
were not different between the two groups and were
the same as previously published values with normal
myocardial blood flow (21). There were no detectable
M1 enrichments of pyruvate or lactate in either group,
which demonstrates that there was no significant decarboxylation of malate to form pyruvate.
Group
Citrate
Lactate/Pyruvate
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DISCUSSION
The key finding of this study is that a 40% reduction
in MV̇O2 results in a decrease in both pyruvate carboxylation and citrate efflux with the maintenance of tissue levels of CAC intermediates. The absolute rate of
pyruvate carboxylation decreased by 65% when there
was only a 38% drop in MV̇O2 (see Fig. 3). If the
decrease in malate and/or OAA supply to the CAC via
pyruvate carboxylation were not matched by a decrease in the rate of the net efflux of CAC intermediates from the cycle, then the concentration of CAC
intermediates in the myocardium would decrease. Hibernation had no effect on the tissue contents of citrate, succinate, fumarate, or malate, which suggests
that the decrease in citrate release reflects a decrease
in other efflux pathways for CAC intermediates, thus
preserving the integrity of the CAC and the production
of reducing equivalents, electron-transport-chain flux,
and ATP formation, albeit at decreased rates.
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An acute reduction in coronary blood flow of ⬃40%
results in an initial acceleration of glycolysis, a decrease in ATP, and a subsequent resetting of the metabolism to better match the reduced oxygen supply (1,
10, 22, 29). After 60–90 min of coronary blood flow
reduction, the myocardium is in a relative state of
hibernation with restored ATP content and reduced
lactate production (1, 10, 29). The results of the present
investigation extend the understanding of the acute
phase of myocardial hibernation and demonstrate that
the heart adjusts its metabolism to maintain the tissue
content of CAC intermediates by a decrease in pyruvate carboxylation and citrate release. Thus the mitochondria conserve the constituents of the CAC. This is
reflected in the greater degree of carbon recycling in
the CAC as assessed by mass isotopomer analysis. It is
important to note that prolonged hibernation may result in impaired CAC function, as recently suggested
by Schulz and colleagues (30), who observed a progressive decrease in MV̇O2 during the course of 24 h of
hibernation (an ⬃40% reduction in LAD flow) in a
similar swine model.
It has been suggested that with reduced coronary
blood flow the rate of anaplerosis does not match the
rate of CAC intermediate efflux, and there is a significant depletion of CAC intermediates (11). This is
clearly not the case in acutely hibernating swine myocardium (see Table 3). As noted above, the decrease in
anaplerotic pyruvate carboxylation matched with a
decrease in net citrate efflux suggests that the CAC
intermediate release is balanced by anaplerosis during
hibernation. We measured net citrate efflux values
from the heart of 2.5 and 1.4 nmol 䡠 g⫺1 䡠 min⫺1, which
account for 10% and 6% of the absolute pyruvate carboxylation flux for HIB and HIB ⫹ PYR animals,
respectively. This suggests that a decrease in the efflux
of other metabolites such as succinate (15) plays a role
in maintaining the total pool size of CAC intermediates. It is important to note that net citrate release
may underestimate the true rate of citrate loss from
the CAC. Cytosolic ATP-citrate lyase could cleave citrate into OAA and acetyl-CoA (12); therefore citrate
loss from the CAC may be greater than the net citrate
release and account for a larger fraction of the pyruvate carboxylation unless the carbon of OAA returns to
the mitochondria.
Another possible site of release from the CAC is the
decarboxylation of malate by malic enzyme (13). However, the energetics of the reaction toward decarboxylation are unfavorable (35), and ex vivo experiments
suggest that malic enzyme is mainly a carboxylating
enzyme (8, 36, 39). In addition, we observed no M1
labeling of pyruvate with either normal flow (21) or
with hibernation, which indicates that malate was not
being decarboxylated by malic enzyme to produce pyruvate.
Myocardial hibernation did not affect the relative
contribution of pyruvate decarboxylation (via pyruvate
dehydrogenase) to the CAC flux (see Table 4). This
confirms the work of Liedtke (16), which used 14Clabeled substrate to demonstrate that a 60% reduction
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nase) were similar between the HIB and HIB ⫹ PYR
groups (488 ⫾ 44 and 457 ⫾ 47 nmol 䡠 g⫺1 䡠 min⫺1, respectively) and our previously published values from
aerobic myocardium (21). The absolute rates of pyruvate carboxylation were again similar between the HIB
and HIB ⫹ PYR groups; however, these fluxes were
decreased by 65% compared with previously published
data from aerobic and aerobic ⫹ pyruvate animals (see
Fig. 5) (21).
Analysis of the mass isotopomer distribution of the
OAA moiety of citrate and succinate allow one to calculate the fraction of the OAA pool that is recycled
after one turn of the CAC. The fraction that is lost has
two components: loss of CAC intermediates from the
cell, and isotopic exchanges between metabolites of the
CAC and related compounds such as glutamate, glutamine, and aspartate. In the present investigation,
the extent of recycling of citrate through the CAC was
72 ⫾ 3 and 70 ⫾ 4% for the HIB and HIB ⫹ PYR
animals, respectively. These are significantly higher
than our previously published values obtained during
normal flow aerobic conditions (60 ⫾ 4% and 56 ⫾ 4%
for normal and high-pyruvate conditions, respectively).
Thus there is greater recycling of CAC carbon during
hibernation as confirmed by the decrease in citrate
release (see Fig. 5).
Malic enzyme and pyruvate carboxylase activity. Owing to the lower rate of pyruvate carboxylation in
hibernating myocardium, we examined the possibility
that there was a decrease in the activity of malic
enzyme and/or pyruvate carboxylase. The activity of
malic enzyme was unchanged between the LAD perfusion bed (840 ⫾ 50 nmol 䡠 g⫺1 䡠 min⫺1) and the CFX bed
(830 ⫾ 50 nmol 䡠 g⫺1 䡠 min⫺1) in the HIB group. There
was also no difference between the LAD and CFX beds
in the activity of pyruvate carboxylase (32 ⫾ 7 and
31 ⫾ 9 nmol 䡠 g⫺1 䡠 min⫺1, respectively) in the HIB
group. Similar values were obtained in the HIB ⫹ PYR
group.
PYRUVATE CARBOXYLATION IN MYOCARDIUM DURING HIBERNATION
The authors thank Dr. D. Kerr and M. Lusk of the Department of
Pediatrics, Case Western Reserve University for assistance with the
pyruvate carboxylase assay and F. David and T. McElfresh for
technical assistance in conducting this study.
This work was supported by National Institutes of Health Grants
HL-58653 (to W. C. Stanley), HL-59219 (to H. Brunengraber), and
HL-07887, and Grant-in-Aid 005031N from the American Heart
Association National Center (to W. C. Stanley).
REFERENCES
1. Arai AE, Pantely GA, Anselone CG, Bristow J, and Bristow
JD. Active downregulation of myocardial energy requirements
during prolonged moderate ischemia in swine. Circ Res 69:
1458–1469, 1991.
AJP-Heart Circ Physiol • VOL
2. Attwood PV. The structure and the mechanism of action of
pyruvate carboxylase. Int J Biochem Cell Biol 27: 231–249, 1995.
3. Bergmeyer HU. Method of Enzymatic Analysis: Metabolites 2.
Weinhein: VCH Verlagsgesellschaft, 1989.
4. Bittl JA and Shine KI. Protection of ischemic rabbit myocardium by glutamic acid. Am J Physiol Heart Circ Physiol 245:
H406–H412, 1983.
5. Brunengraber H, Kelleher JK, and Des Rosiers C. Applications of mass isotopomer analysis to nutrition research. Annu
Rev Nutr 17: 559–596, 1997.
6. Bunger R, Mallet RT, and Hartman DA. Pyruvate-enhanced
phosphorylation potential and inotropism in normoxic and postischemic isolated working heart. Near-complete prevention of
reperfusion contractile failure. Eur J Biochem 180: 221–233,
1989.
7. Comte B, Vincent G, Bouchard B, and Des Rosiers C.
Probing the origin of acetyl-CoA and oxaloacetate entering the
citric acid cycle from the 13C labeling of citrate released by
perfused rat hearts. J Biol Chem 272: 26117–26124, 1997.
8. Comte B, Vincent G, Bouchard B, Jette M, Cordeau S, and
Des Rosiers C. A 13C mass isotopomer study of anaplerotic
pyruvate carboxylation in perfused rat hearts. J Biol Chem 272:
26125–26131, 1997.
9. Des Rosiers C, Di Donato L, Comte B, Laplante A, Marcoux C, David F, Fernandez CA, and Brunengraber H.
Isotopomer analysis of citric acid cycle and gluconeogenesis in
rat liver. Reversibility of isocitrate dehydrogenase and involvement of ATP-citrate lyase in gluconeogenesis. J Biol Chem 270:
10027–10036, 1995.
10. Fedele FA, Gewirtz H, Capone RJ, Sharaf B, and Most AS.
Metabolic response to prolonged reduction of myocardial blood
flow distal to a severe coronary artery stenosis. Circulation 78:
729–735, 1988.
11. Gibala MJ, Young ME, and Taegtmeyer H. Anaplerosis of
the citric acid cycle: role in energy metabolism of heart and
skeletal muscle. Acta Physiol Scand 168: 657–665, 2000.
12. Huang WY and Kummerow FA. Cholesterol and fatty acid
synthesis in swine. Lipids 11: 34–41, 1976.
13. Jeffrey FM, Storey CJ, Sherry AD, and Malloy CR. 13C
isotopomer model for estimation of anaplerotic substrate oxidation via acetyl-CoA. Am J Physiol Endocrinol Metab 271: E788–
E799, 1996.
14. Kornberg HL. Anaplerotic sequences and their role in metabolism. In: Essays in Biochemistry, edited by Campbell PN and
Marshall RD. London: Academic, 1966, p. 1–31.
15. Laplante A, Vincent G, Poirier M, and Des Rosiers C.
Effects and metabolism of fumarate in the perfused rat heart. A
13
C mass isotopomer study. Am J Physiol Endocrinol Metab 272:
E74–E82, 1997.
16. Liedtke AJ. Alterations of carbohydrate and lipid metabolism
in the acutely ischemic heart. Prog Cardiovasc Dis 23: 321–336,
1981.
17. Lin RC and Davis EJ. Malic enzymes of rabbit heart mitochondria. Separation and comparison of some characteristics of a
nicotinamide adenine dinucleotide-preferring and a nicotinamide adenine dinucleotide phosphate-specific enzyme. J Biol
Chem 249: 3867–3875, 1974.
18. Mazer CD, Cason BA, Stanley WC, Shnier CB, Wisneski
JA, and Hickey RF. Dichloroacetate stimulates carbohydrate
metabolism but does not improve systolic function in ischemic
pig heart. Am J Physiol Heart Circ Physiol 268: H879–H885,
1995.
19. McNulty PH, Sinusas AJ, Shi CQ, Dione D, Young LH,
Cline GC, and Shulman GI. Glucose metabolism distal to a
critical coronary stenosis in a canine model of low-flow myocardial ischemia. J Clin Invest 98: 62–69, 1996.
20. Opie LH. Effects of regional ischemia on metabolism of glucose
and fatty acids. Relative rates of aerobic and anaerobic energy
production during myocardial infarction and comparison with
effects of anoxia. Circ Res 38: I52–I74, 1976.
21. Panchal AR, Comte B, Huang H, Kerwin T, Darvish A, Des
Rosiers C, Brunengraber H, and Stanley WC. Partitioning
of pyruvate between oxidation and anaplerosis in swine hearts.
Am J Physiol Heart Circ Physiol 279: H2390–H2398, 2000.
281 • OCTOBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 15, 2017
in LAD flow in swine decreases CAC flux but does not
alter the relative contributions of fatty acids and carbohydrates to oxidative metabolism. This same observation was made using 13C-labeled glucose and NMR
analysis in dogs subjected to a 30% reduction in coronary flow for 3–4 h (19). Thus with acutely hibernating
myocardium, there is no change in the contribution of
carbohydrate to oxidative metabolism.
The elevated pyruvate concentrations in the HIB ⫹
PYR group did not elicit an increase in tissue pyruvate,
pyruvate decarboxylation, or pyruvate carboxylation
(see Tables 1 and 4; Fig. 5) or an increase in tissue M3
enrichment of pyruvate (data not shown). The lack of
effect of elevated arterial pyruvate may be attributed
to a low uptake of pyruvate into the myocardium.
Studies infusing pyruvate at higher concentrations (5
mM) have reported an increased contribution of pyruvate to citrate after ischemia and improved contractile
function (6, 37). In this study, with an arterial pyruvate concentration of 1.21 mM, we did not see any
effect of pyruvate on pyruvate carboxylation or decarboxylation compared with an arterial pyruvate concentration of 0.28 mM. Further experimentation will be
necessary to determine whether higher arterial concentrations of pyruvate will increase the contribution
of pyruvate to citrate during hibernation.
The present results suggest that there is a fine interregulation among the rates of CAC flux, pyruvate
carboxylation, and release of CAC intermediates and
the CAC pool size; however, the biochemical mechanisms are unclear. We did not observe a decrease in the
in vitro activities of malic enzyme and/or pyruvate
carboxylase, which suggests that there was not stable
covalent modification of these enzymes, though it is
possible that the effect of in vivo modification was lost
when measured in vitro at near maximal activity.
Hibernation does not appear to cause gross changes in
some of the regulators of malic enzyme or pyruvate
carboxylase such as pyruvate, NADP⫹/NADPH, ATP,
ADP, malate, and acetyl-CoA (see Tables 1–3) (21, 32).
Thus the biochemical mechanisms for the decrease in
pyruvate carboxylation remain to be elucidated.
In conclusion, the acute hibernation response is accompanied by a decrease in both pyruvate carboxylation and citrate efflux and the maintenance of tissue
levels of CAC intermediates. Thus the integrity of the
CAC is maintained during the early phase of myocardial hibernation.
H1619
H1620
PYRUVATE CARBOXYLATION IN MYOCARDIUM DURING HIBERNATION
AJP-Heart Circ Physiol • VOL
32.
33.
34.
35.
36.
37.
38.
39.
40.
extraction but not glucose uptake. Am J Physiol Heart Circ
Physiol 262: H91–H96, 1992.
Stanley WC, Hernandez LA, Spires D, Bringas J, Wallace S, and McCormack JG. Pyruvate dehydrogenase activity and malonyl-CoA levels in normal and ischemic swine
myocardium: effects of dichloroacetate. J Mol Cell Cardiol 28:
905–914, 1996.
Stanley WC, Lopaschuk GD, Hall JL, and McCormack JG.
Regulation of myocardial carbohydrate metabolism under normal and ischemic conditions. Potential for pharmacological interventions. Cardiovasc Res 33: 243–257, 1997.
Struck E, Ashmore J, and Wieland O. Pyruvate carboxylase
activity and glucose production in isolated perfused rat liver.
Enzymol Biol Clin (Basel) 7: 38–52, 1966.
Sundqvist KE, Heikkila J, Hassinen IE, and Hiltunen JK.
Role of NADP⫹ (corrected)-linked malic enzymes as regulators of
the pool size of tricarboxylic acid-cycle intermediates in the
perfused rat heart. Biochem J 243: 853–857, 1987.
Sundqvist KE, Hiltunen JK, and Hassinen IE. Pyruvate
carboxylation in the rat heart. Role of biotin-dependent enzymes.
Biochem J 257: 913–916, 1989.
Tejero-Taldo MI, Caffrey JL, Sun J, and Mallet RT. Antioxidant properties of pyruvate mediate its potentiation of ␤-adrenergic inotropism in stunned myocardium. J Mol Cell Cardiol
31: 1863–1872, 1999.
Thomassen AR, Nielsen TT, Bagger JP, and Henningsen
P. Myocardial exchanges of glutamate, alanine, and citrate in
controls and patients with coronary artery disease. Clin Sci
(Lond) 64: 33–40, 1983.
Vincent G, Comte B, Poirier M, and Des Rosiers C. Citrate
release by perfused rat hearts: a window on mitochondrial
cataplerosis. Am J Physiol Endocrinol Metab 278: E846–E856,
2000.
Williamson JR and Corkey BE. Assays of intermediates of the
citric acid cycle and related compounds by fluorometric enzyme
methods. In: Methods in Enzymology. New York: Academic,
1969, p. 434–516.
281 • OCTOBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 15, 2017
22. Pantely GA, Malone SA, Rhen WS, Anselone CG, Arai A,
Bristow J, and Bristow JD. Regeneration of myocardial phosphocreatine in pigs despite continued moderate ischemia. Circ
Res 67: 1481–1493, 1990.
23. Peuhkurinen KJ and Hassinen IE. Pyruvate carboxylation
as an anaplerotic mechanism in the isolated perfused rat heart.
Biochem J 202: 67–76, 1982.
24. Peuhkurinen KJ, Takala TE, Nuutinen EM, and Hassinen
IE. Tricarboxylic acid cycle metabolites during ischemia in isolated perfused rat heart. Am J Physiol Heart Circ Physiol 244:
H281–H288, 1983.
25. Pisarenko OI, Studneva IM, Shulzhenko VS, and Kapelko
VI. Relations between the energy state of the myocardium and
release of some products of anaerobic metabolism during underperfusion. Pflügers Arch 416: 434–441, 1990.
26. Rahimtoola SH. A perspective on the three large multicenter
randomized clinical trials of coronary bypass surgery for chronic
stable angina. Circulation 72: 123–135, 1985.
27. Russell RR III, Mommessin JI, and Taegtmeyer H. Propionyl-L-carnitine-mediated improvement in contractile function of
rat hearts oxidizing acetoacetate. Am J Physiol Heart Circ
Physiol 268: H441–H447, 1995.
28. Russell RR III and Taegtmeyer H. Pyruvate carboxylation
prevents the decline in contractile function of rat hearts oxidizing acetoacetate. Am J Physiol Heart Circ Physiol 261: H1756–
H1762, 1991.
29. Schulz R, Guth BD, Pieper K, Martin C, and Heusch G.
Recruitment of an inotropic reserve in moderately ischemic myocardium at the expense of metabolic recovery. A model of shortterm hibernation. Circ Res 70: 1282–1295, 1992.
30. Schulz R, Post H, Neumann T, Gres P, Luss H, and Heusch
G. Progressive loss of perfusion-contraction matching during
sustained moderate ischemia in pigs. Am J Physiol Heart Circ
Physiol 280: H1945–H1953, 2001.
31. Stanley WC, Hall JL, Stone CK, and Hacker TA. Acute
myocardial ischemia causes a transmural gradient in glucose