Download Differential effects of heptanoate and hexanoate on myocardial citric

J Appl Physiol 100: 76 – 82, 2006.
First published September 1, 2005; doi:10.1152/japplphysiol.00255.2005.
Differential effects of heptanoate and hexanoate on myocardial citric acid
cycle intermediates following ischemia-reperfusion
Isidore C. Okere,1 Tracy A. McElfresh,1 Daniel Z. Brunengraber,1 Wenjun Martini,1,2 Joseph P. Sterk,1
Hazel Huang,1 Margaret P. Chandler,1 Henri Brunengraber,2 and William C. Stanley1,2
Departments of 1Physiology and Biophysics and 2Nutrition, Case Western Reserve University, Cleveland, Ohio
Submitted 4 March 2005; accepted in final form 30 August 2005
anaplerosis; fatty acids; heart; metabolism; mitochondria; pyruvate
dehydrogenase
required to support cardiac function
is dependent on sufficient citric acid cycle (CAC) flux. Normal
CAC flux is sustained by a small pool of intermediates with a
rapid turnover. Under normal aerobic conditions, there is a
physiological loss of some CAC intermediates, i.e., citrate,
␣-ketoglutarate (␣-KG), succinate, fumarate, and malate (5, 8,
16, 21, 25, 26, 42). This loss of CAC intermediates is matched
by the entry of intermediates from outside the cycle, a process
termed anaplerosis (14), from pyruvate, glutamate, or propionyl-CoA (Fig. 1) (6, 8, 22, 26, 28). The necessity of constant
anaplerosis in the normal heart is evident by the decrease in
THE ATP FORMATION THAT IS
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: wcs4
@case.edu).
76
mechanical work by hearts perfused with buffer containing
acetoacetate as the only fuel. When an anaplerotic substrate
such as pyruvate, lactate, or propionate was added to the
perfusate, in addition to acetoacetate, the mechanical function
of the heart improved immediately (30, 31).
In vitro studies suggest that, during postischemic reperfusion, there is inhibition of the activity of the CAC enzymes
isocitrate dehydrogenase, aconitase, and ␣-KG dehydrogenase,
causing impairment in CAC flux and reduction in mitochondrial ATP generation (12, 19, 24, 32). Additional evidence
suggests that there is excessive loss of CAC intermediates in
the reperfused heart and that infusion of anaplerotic substrates
improves the mechanical function of the heart (11, 31, 40, 41).
Elevating arterial pyruvate concentrations reduces infarct size
and improves contractile recovery following ischemia, possibly through stimulation of anaplerosis via pyruvate carboxylation (20, 36). It has been suggested that treatment with propionate improves cardiac metabolic and contractile function with
ischemia and/or reperfusion (9, 17). Our laboratory recently
demonstrated that propionate is efficiently converted to the
CAC intermediate succinyl-CoA in perfused rat hearts and in
live pig hearts (22, 28).
In patients with deficiencies in the ␤-oxidation of long-chain
fatty acids, supplementation of the diet with the triglyceride of
heptanoate [which generates propionyl-CoA after two cycles of
␤-oxidation (Fig. 1)] resulted in a dramatic clinical improvement in cardiac and skeletal muscle function that is not observed with octanoate supplementation (29). As these patients
often present with high activities of plasma creatine kinase
(reflecting increased cell permeability), it was postulated that
the beneficial effect of heptanoate resulted from its anaplerotic
property. One can thus hypothesize that acute treatment with
heptanoate during and after myocardial ischemia could be
cardioprotective due to enhanced anaplerotic flux. On the other
hand, treatment with the medium-chain fatty acids hexanoate
or octanoate results in an increase in the myocardial content of
some CAC intermediates (26, 39), despite the absence of a
direct anaplerotic effect. The differential effects of even-numbered and odd-numbered medium-chain fatty acids on cardiac
function, substrate metabolism, and the myocardial content of
the various CAC intermediates during ischemia-reperfusion
have not been addressed.
The goal of the present investigation was to assess whether
treatment with the anaplerotic medium-chain fatty acid heptanoate would improve contractile function during ischemia
and reperfusion. Studies were performed in anesthetized pigs
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Okere, Isidore C., Tracy A. McElfresh, Daniel Z. Brunengraber, Wenjun Martini, Joseph P. Sterk, Hazel Huang, Margaret P. Chandler, Henri Brunengraber, and William C. Stanley. Differential effects of heptanoate and hexanoate on myocardial
citric acid cycle intermediates following ischemia-reperfusion.
J Appl Physiol 100: 76 – 82, 2006. First published September 1, 2005;
doi:10.1152/japplphysiol.00255.2005.—In the normal heart, there is
loss of citric acid cycle (CAC) intermediates that is matched by the
entry of intermediates from outside the cycle, a process termed
anaplerosis. Previous in vitro studies suggest that supplementation
with anaplerotic substrates improves cardiac function during myocardial ischemia and/or reperfusion. The present investigation assessed
whether treatment with the anaplerotic medium-chain fatty acid heptanoate improves contractile function during ischemia and reperfusion. The left anterior descending coronary artery of anesthetized pigs
was subjected to 60 min of 60% flow reduction and 30 min of
reperfusion. Three treatment groups were studied: saline control,
heptanoate (0.4 mM), or hexanoate as a negative control (0.4 mM).
Treatment was initiated after 30 min of ischemia and continued
through reperfusion. Myocardial CAC intermediate content was not
affected by ischemia-reperfusion; however, treatment with heptanoate
resulted in a more than twofold increase in fumarate and malate, with
no change in citrate and succinate, while treatment with hexanoate did
not increase fumarate or malate but increased succinate by 1.8-fold.
There were no differences among groups in lactate exchange, glucose
oxidation, oxygen consumption, and contractile power. In conclusion,
despite a significant increase in the content of carbon-4 CAC intermediates, treatment with heptanoate did not result in improved mechanical function of the heart in this model of reversible ischemiareperfusion. This suggests that reduced anaplerosis and CAC dysfunction do not play a major role in contractile and metabolic
derangements observed with a 60% decrease in coronary flow followed by reperfusion.
CITRIC ACID CYCLE INTERMEDIATES IN REPERFUSED HEARTS
77
trolled blood flow to the LAD, with blood supplied from the femoral
artery. The LAD pump flow was adjusted to give an interventricular
venous oxygen saturation of 35– 45%. Arterial blood gases were
maintained in the normal range (PO2 ⱖ 100 Torr, PCO2 35– 45 Torr,
and pH 7.35–7.45). Left ventricular (LV) pressure was measured with
a high-fidelity manometer-tipped catheter (Millar Instruments). Regional segment length was measured in duplicate in the LAD bed
using sonomicrometry, and anterior wall contractile function was
assessed from the LV pressure-segment length loop area (3).
Experimental Protocols
subjected to a 60% reduction in coronary blood flow for 1 h
followed by 30 min of reperfusion, with heptanoate treatment
administered during the last 30 min of ischemia and during
reperfusion. Heptanoate-treated pigs were compared with nonischemic hearts, ischemic-reperfused hearts treated with saline,
and ischemic-reperfused hearts treated with hexanoate, a nonanaplerotic medium-chain fatty acid. We hypothesized that
treatment with heptanoate would selectively increase the myocardial content of four carbon CAC intermediates (reflective of
greater anaplerosis from propionyl-CoA) and improve cardiac
function during ischemia and reperfusion. In addition, we
assessed the incorporation of heptanoate into the CAC through
anaplerosis and ␤-oxidation (Fig. 1) using an intracoronary
infusion of [13C]heptanoate under normal flow condition.
METHODS
Domestic pigs (30 – 40 kg) were randomly assigned to four groups:
a saline-treated control group (Con) (n ⫽ 9), a heptanoate group (Hep)
(n ⫽ 10), a hexanoate group (Hex) (n ⫽ 7), and a nonischemic group
(n ⫽ 7/group). All personnel involved in performance of the animal
experiments or biochemical analysis were blinded to treatment. The
experimental solutions were prepared freshly each day by an independent investigator not involved in the animal experimentation.
Studies were conducted in accordance with the Guide for the Care and
Use of Laboratory Animals (NIH publication Number 85–23) and the
Institutional Animal Care and Use Committee at Case Western Reserve University.
Surgical Preparation
The experimental preparation has been previously described in
detail (3, 26). Briefly, animals were sedated with Telazol (6 mg/kg
im), anesthetized with isoflurane (5%), intubated, and maintained on
isoflurane (0.75–1.5%) and ketamine (3 mg 䡠 kg⫺1 䡠 h⫺1 iv). The heart
was exposed via a midline sternotomy, and the animal was heparinized (200 U/kg bolus ⫹ 100 U 䡠 kg⫺1 䡠 h⫺1 iv). A cannula was placed
in the anterior interventricular vein to collect venous blood samples
from the perfusion zone of the left anterior descending coronary artery
(LAD). An extracorporeal perfusion circuit via roller pumps conJ Appl Physiol • VOL
Ischemia-Reperfusion Protocol
After the instrumentation was completed, LAD blood flow was
adjusted to give interventricular venous oxygen saturation of 35–
45%. A continuous infusion of [U-14C]glucose (0.2 ␮Ci/min) was
initiated 30 min before ischemia into the proximal end of the coronary
perfusion line at a rate of 0.1 ml/min. Regional myocardial ischemiareperfusion was induced in the LAD perfusion bed by reducing the
LAD flow by 60% for 60 min, followed by 30 min of reperfusion at
the preischemic LAD flow. Arterial and interventricular venous samples were drawn 5 and 3 min before ischemia; at 5, 20, 26, 40, 50, and
57 min of ischemia; and at 7, 20, and 30 min of reperfusion. From 30
min of ischemia to the end of the protocol, animals were treated with
an infusion of either 1) saline (Con); 2) sodium heptanoate (Hep) (50
mM; Sigma-Aldrich); or 3) sodium hexanoate (Hex) (50 mM; SigmaAldrich) in NaCl at 308 mosmol/kgH2O into the perfusion circuit at 8
␮l/min for every milliliter of blood flow in the LAD perfusion circuit.
This infusion rate was aimed to result in concentration of hexanoate
and heptanoate of 0.4 mM in LAD arterial blood. Dilution of anterior
interventricular venous blood with blood not derived from the LAD
was measured by using a constant infusion of indocyanide green dye
(0.3 mg/min for 5 min) into the LAD perfusion line under aerobic
conditions (0.15 mg/ml for 5 min) during ischemia with substrate
perfusion and (0.3 mg/ml for 5 min) during reperfusion (3). All blood
samples were analyzed for the concentrations of oxygen, lactate,
glucose, plasma free fatty acids, heptanoate, hexanoate concentrations, and 14CO2. Heart rate, LV pressure, and segment length were
recorded online (3). At the end of the protocol, a transmural myocardial biopsy was taken from the anterior LV free wall, rapidly freezeclamped in large steel tongs precooled with liquid nitrogen, and stored
at ⫺80°C for subsequent analysis for the myocardial content of
lactate, glycogen, ATP, and CAC intermediates.
For the nonischemic group, the animals were anesthetized and
surgically prepared as described above but with no flow restriction or
substrate perfusion during the protocol. Transmural myocardial punch
biopsies were taken after 55 min from the LV free wall and freezeclamped as above.
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Fig. 1. Schematic depiction of the metabolism of odd- and even-chain fatty
acids in the heart. CPT, carnitine palmatoyl transferase; ␣-KG, ␣-ketoglutarate.
We first performed studies by using the swine preparation described above to assess the incorporation of heptanoate into the CAC
through anaplerosis via propionyl-CoA formation from carbons 5, 6,
and 7 of heptanoate and subsequent carboxylation to methylmalonylCoA and isomerization to succinyl-CoA, and to acetyl-CoA through
␤-oxidation (Fig. 1). An infusion of unlabeled heptanoate (50 mM as
a sodium salt in NaCl at 308 mosmol/kgH2O) was started into the
coronary bypass circuit at a rate that imposed an inflowing concentration in the LAD artery of 0.25 mM. After 20 min, the infused
substrate was switched to either [5,6,7-13C3]heptanoate (n ⫽ 2) or
[1-13C]heptanoate from Isotec (Miamisburg, OH) (n ⫽ 6) at the same
concentration, which was infused for an additional 60 min. At the end
of the experiment, a biopsy of the LAD perfusion bed was rapidly
freeze clamped and subsequently analyzed by GC-MS for the enrichment of citrate, succinate, fumarate, and malate with 13C, as previously described (22). The raw data were corrected for natural enrichment of heavy isotopomers using GC-MS data from pigs not receiving
13
C isotope infusion (1, 10).
78
CITRIC ACID CYCLE INTERMEDIATES IN REPERFUSED HEARTS
Table 1. Mass isotopomer distribution of citric acid cycle
intermediates in the left anterior descending coronary artery
bed myocardium following 60 min of infusion with [5,6,713
C3]heptanoate or [1-13C]heptanoate
Citrate
Succinate
Fumarate
Malate
[5,6,7-13C3]Heptanoate M3
[1-13C]Heptanoate M1
5.5
3.2
4.8
4.6
6.2⫾1.3
2.8⫾0.5
2.3⫾0.5
1.8⫾0.5
centrations and uptakes over time and among treatment groups. A
one-way ANOVA was used for the comparison of tissue contents
measured in the terminal myocardial biopsy. All values are reported as
means ⫾ SE, with a 0.05 level of significance.
RESULTS
Preliminary Studies with [13C]Heptanoate
Values are in %; n ⫽ 2 for [5,6,7-13C3]heptanoate and n ⫽ 6 for [1-13C]heptanoate (means ⫾ SE). M, mass of the natural 12C molecule; M1, M with an
additional mass unit from 13C; M3, M with 3 additional mass units.
Analytic Methods
Ischemia-Reperfusion Studies
Hemodynamic measurements and regional contractile function. Myocardial ischemia resulted in decreased regional contractile function without a significant change in heart rate or
LV peak systolic pressure (Table 2). Contractile function
improved during reperfusion but did not return to preischemic
values. There were no significant differences among treatment
groups.
Metabolic measurements. Myocardial oxygen consumption
was not different among groups under nonischemic conditions
(2.41 ⫾ 0.21, 2.87 ⫾ 0.18, and 2.23 ⫾ 0.28 ␮mol䡠g⫺1 䡠min⫺1
in Con, Hep, and Hex groups, respectively), decreased by 49%
during ischemia, and was restored to 78% of nonischemic
values by 30 min of reperfusion. There were no differences
Calculations
The myocardial net uptake (␮mol 䡠 g⫺1 䡠 min⫺1) of lactate and
free fatty acids was calculated as the product of the arterial and
coronary venous substrate concentration difference and myocardial
blood flow. The rate of glucose oxidation (␮mol 䡠 g⫺1 䡠 min⫺1) was
calculated as the product of myocardial blood flow (ml 䡠 g⫺1 䡠 min⫺1)
and the release of either 14CO2 (disintegrations 䡠 min⫺1 䡠 ml⫺1) into the
coronary vein, divided by the arterial specific radioactivity of glucose
(disintegrations 䡠 min⫺1 䡠 ␮mol⫺1) (3, 43, 44). The interventricular venous concentration of 14CO2 was corrected for dilution of blood (⬃
10%) derived from coronary arteries other than the LAD by dividing
the measured values by the ratio of venous to arterial plasma green
dye concentration (3). Myocardial blood flow (ml 䡠 g⫺1 䡠 min⫺1) was
measured from the calibrated pump flow of the coronary perfusion
line and the weight of the LAD perfusion bed (3). The LV pressure
segment length loop area times heart rate was used as an index of
anterior wall external power (3).
Statistical Analysis
A three-way ANOVA was used to compare all changes in hemodynamic function, plasma fatty acids, and glucose and lactate conJ Appl Physiol • VOL
Table 2. Cardiac function
Groups
Nonischemic
Ischemia
Con
Hep
Hex
82.6⫾5.1
83.8⫾2.3
80.7⫾3.1
78.9⫾4.8
80.5⫾1.8
75.9⫾4.0
Con
Hep
Hex
106.7⫾7.9
120.5⫾5.2
101.1⫾5.5
118.⫾7.8
123.6⫾6.5
100.2⫾7.5
Ischemia ⫹ Substrate
Reperfusion
Peak left ventricular pressure, mmHg
80.8⫾4.5
82.5⫾2.6
74.9⫾4.3
80.9⫾4.1
80.9⫾2.6
80.5⫾4.9
Heart rate, beats/min
116.7⫾8.0
125.2⫾7.2
109.2⫾7.2
116.4⫾10.4
133.1⫾9.3
127.8⫾7.5
Left ventricular anterior wall power (as % of nonischemic)
Con
Hep
Hex
100
100
100
50.9⫾6.6*
58.4⫾6.7*
37.7⫾3.6*
45.4⫾7.7*
52.0⫾6.3*
35.3⫾2.4*
63.2⫾9.1*
74.0⫾6.8*
49.1⫾5.8*
Values are means ⫾ SE; n ⫽ 9 for control group (Con), n ⫽ 10 for
heptanoate group (Hep), and n ⫽ 7 for hexanoate group (Hex). *P ⬍ 0.05
compared with the normal flow preischemic period with the treatment group.
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Blood levels of glucose and lactate were measured by using
enzymatic spectrophotometric assays. Plasma-free fatty acids were
assayed by using enzymatic spectrophotometric assays (Wako Chemicals, Richmond, VA), which measures both medium- and long-chain
fatty acids, and thus includes heptanoate and hexanoate concentrations. The concentration of 14CO2 and the specific radioactivity of
[14C]glucose in blood were measured as previously described (3).
Tissue lactate and glycogen content were measured by using enzymatic spectrophotometric methods; ATP content of the tissue was
determined by the luciferase assay (3). The myocardial activity of
pyruvate dehydrogenase (PDH) was measured in the terminal biopsy
by using [2-14C]pyruvate, as previously described (38).
CAC intermediates were analyzed on an Agilent 5973 mass spectrometer, equipped with an Agilent 6890 gas chromatograph, using a
HP-5MS 5% phenyl methyl siloxane fused silica capillary column (60
m, 250-␮m inner diameter, 0.25-␮m film thickness), according to
previously published methods (7, 34). The internal standards were
[2,2,4,4-2H4]citrate, [2,2,3,3-2H4]succinate, [U-13C4]fumarate, [2,3,32
H3]malate, and ␣-keto-[U-13C5]glutarate (␣-KG). The latter standard
was generated by the transamination of [U-13C5]glutamate, as previously described (15). For the studies with [5,6,7-13C3]heptanoate or
[1-13C]heptanoate, the enrichment on CAC intermediates was corrected for the background natural abundance of 13C, as previously
described (26). The enrichment of CAC intermediates is expressed
relative to the natural abundance, with M set to the natural abundance,
and M⫹1 and M⫹3 meaning 1 or 3 additional mass units. Free CoA
and acetyl-CoA levels were measured by using a radioenzymatic
method (2).
In the pigs perfused with [1-13C]heptanoate (which enters
the CAC with the conversion of 1-13C-acetyl-CoA to citrate),
there was clear incorporation of label into citrate, and the
expected fall in enrichment from citrate to succinate clearly
demonstrating that carbon-1 of heptanoate enters the CAC via
␤-oxidation and not through anaplerosis. An estimate of the
enrichment of mitochondrial acetyl-CoA can be calculated by
deducting the M1 enrichment of oxaloacetate from that of M1
citrate and multiplying the result by two [since one heptanoate
molecule yields two acetyl-CoA groups: (6.2 ⫺ 2.3) ⫻ 2 ⫽
7.8%]. In the experiments with [5,6,7-13C3]heptanoate, there
was a clear enrichment of M3 succinate, which is the approximate percentage of the succinate molecules derived from
anaplerosis from heptanoate through the propionyl-CoA pathway (Table 1) (22). Taken together, these results demonstrate
that, even at low arterial concentrations (0.25 mM), heptanoate
is readily metabolized by the myocardium and enters the CAC
via both ␤-oxidation and anaplerosis.
79
CITRIC ACID CYCLE INTERMEDIATES IN REPERFUSED HEARTS
Table 3. Myocardial metabolite content and pyruvate
dehydrogenase activity in the nonischemic control
group and in the groups subjected to ischemia
followed by reperfusion
Ischemia ⫹ Reperfusion
Lactate, ␮mol/g wet
wt
Glycogen, ␮mol/g
wet wt
ATP, ␮mol/g protein
ADP, ␮mol/g protein
Acetyl-CoA, ␮mol/g
wet wt
Free CoA, nmol/g
wet wt
Pyruvate
dehydrogenase
activity, % of total
Nonischemic
Con
Hep
Hex
1.8⫾0.23
2.4⫾0.5
3.1⫾0.6
2.3⫾0.3
25.1⫾3.9
36.1⫾3.3
8.5⫾0.9
10.2⫾1.7*
24.8⫾2.7*
7.9⫾1.1
10.6⫾1.9*
27.8⫾4.6*
8.7⫾1.2
12.0⫾2.0*
21.2⫾1.7*
8.2⫾1.3
5.6⫾0.4
8.7⫾1.1*
3.2⫾0.4†
11.3⫾2.1
40.4⫾3.5
31.0⫾5.5
20.6⫾5.2
30.4⫾16.5
29.7⫾4.0
47.7⫾5.1*
41.9⫾5.4
42.6⫾3.8
Values are means ⫾ SE; n ⫽ 7 for the nonischemic group, n ⫽ 9 for Con,
n ⫽ 10 for Hep, and n ⫽ 7 for Hex. *P ⬍ 0.05 compared with nonischemic
group; †P ⬍ 0.05 compared with Con and Hex groups.
group, but there were no differences among the Con, Hep, and
Hex groups. There was no difference in myocardial ADP
content (Table 3). The myocardial acetyl-CoA content was
increased by ischemia-reperfusion compared with normal aerobic conditions and was significantly reduced by heptanoate
treatment (Table 3). Free CoA content was not different among
groups.
Ischemia-reperfusion resulted in a significant increase in the
percent activation of PDH in the Con group compared with the
nonischemic group (Table 3); however, there were no differences among the Con, Hep, and Hex groups. In addition, there
was no difference in the maximal activity of PDH among
treatment groups (data not shown).
The myocardial content of CAC intermediates was not significantly different between ischemia-reperfusion Con group compared with normal aerobic myocardium (Table 4, Fig. 3), as we
have previously reported in a swine model with 1 h of total
LAD occlusion with 2-h reperfusion (36). Treatment with
heptanoate during ischemia and reperfusion resulted in a significant reduction in ␣-KG and increases in fumarate and
malate content, but no change in citrate or succinate. On the
other hand, compared with the Con group, treatment with
hexanoate resulted in a significant decrease in ␣-KG and an
increase in succinate, with no change in citrate, fumarate, and
Table 4. Myocardial citric acid cycle intermediate content
Fig. 2. Net rate of fatty acid uptake by the myocardium. Values are means ⫾
SE. *P ⬍ 0.05, and †P ⬍ 0.01 compared with the saline group.
J Appl Physiol • VOL
Ischemia ⫹ Reperfusion
Citric Acid Cycle
Intermediates
Nonischemic
Con
Hep
Hex
Citrate
␣-KG
Succinate
Fumarate
Malate
Sum
412⫾41
26.3⫾6.0
213⫾41
59.8⫾6.1
142⫾18
852⫾93
515⫾98
21.6⫾2.9
231⫾52
56.4⫾8.1
127⫾20
952⫾152
433⫾42
14.0⫾2.1*
256⫾25
143⫾18‡
327⫾39‡
1174⫾108
569⫾51
12.3⫾1.8*
430⫾67†
79⫾10
160⫾19
1220⫾142
Values are means ⫾ SE in nmol/g wet wt. ␣-KG, ␣-ketoglutarate. *P ⬍ 0.05
compared with Con group. †P ⬍ 0.05 compared with Con and Hep groups.
‡P ⬍ 0.05 compared with Con and Hex groups.
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among groups in myocardial oxygen consumption during ischemia or reperfusion. Arterial free fatty acid concentration
was not different among groups under nonischemic condition
or during the first 30 min of ischemia but was significantly
elevated in the Hep and Hex groups compared with the Con
group during the ischemic treatment period (0.649 ⫾ 0.078,
0.816 ⫾ 0.149, and 0.479 ⫾ 0.096 mM, respectively) and
during reperfusion (0.849 ⫾ 0.126, 0.983 ⫾ 0.115, and
0.496 ⫾ 0.122 mM, respectively). The net uptake of fatty acids
was not different among groups during ischemia before treatment; however, it was significantly higher in the Hep and Hex
groups during ischemia and reperfusion (Fig. 2).
There were no differences among treatment groups in
myocardial net lactate uptake. Ischemia resulted in a switch
from positive to negative lactate uptake (⫺0.225 ⫾ 0.073,
⫺0.365 ⫾ 0.087, and ⫺0.291 ⫾ 0.169 ␮mol䡠g⫺1 䡠min⫺1 in
Con, Hep, and Hex groups, respectively), which persisted into
the ischemic treatment period (⫺0.175 ⫾ 0.084, ⫺0.152 ⫾
0.064, and ⫺0.250 ⫾ 0.163 ␮mol䡠g⫺1 䡠min⫺1, respectively).
Reperfusion resulted in a reversal back to net lactate uptake
(0.160 ⫾ 0.063, 0.164 ⫾ 0.057, and 0.106 ⫾ 0.015
␮mol䡠g⫺1 䡠min⫺1, respectively). The rate of glucose oxidation
was not affected by ischemia or reperfusion within each treatment group, and it was not different among the three treatment
groups under aerobic conditions (0.048 ⫾ 0.014, 0.059 ⫾
0.016, and 0.068 ⫾ 0.036 ␮mol䡠 g⫺1 䡠min⫺1 for the Con, Hep,
and Hex groups, respectively) during ischemia before treatment (0.056 ⫾ 0.013, 0.077 ⫾ 0.027, and 0.053 ⫾ 0.021
␮mol䡠g⫺1 䡠min⫺1), during ischemia with treatment (0.068 ⫾
0.021, 0.091 ⫾ 0.024, and 0.040 ⫾ 0.016 ␮mol䡠g⫺1 䡠min⫺1),
and during reperfusion (0.048 ⫾ 0.012, 0.064 ⫾ 0.014, and
0.045 ⫾ 0.011 ␮mol䡠g⫺1 䡠min⫺1, respectively).
Myocardial lactate content was not different between the
normal nonischemic group and the Con ischemic/reperfused
group, and there were no differences among the Con, Hep, and
Hex groups (Table 3). Myocardial glycogen content was significantly reduced in the Con ischemic/reperfused compared
with the normal nonischemic group, and there were no
differences among the Con, Hep, and Hex groups. Myocardial ATP content was significantly reduced in the Con
ischemic/reperfused compared with the normal nonischemic
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CITRIC ACID CYCLE INTERMEDIATES IN REPERFUSED HEARTS
malate contents compared with the Con group (Table 4). The
sum of the concentrations of the measured CAC intermediates
was increased significantly in the Hep group relative to the Con
group (Table 4), whereas there was no difference between the
Hep and Hex groups.
DISCUSSION
The present study found that, despite exerting significant
effects on myocardial CAC intermediate content, treatment
with heptanoate did not improve contractile function during
ischemia and reperfusion. Myocardial uptake of heptanoate
resulted in more than twofold increase in fumarate and malate
but did not alter myocardial lactate exchange or glucose oxidation or improve regional anterior wall contractile power. On
the other hand, the nonanaplerotic medium-chain fatty acid
hexanoate did not affect fumarate and malate levels but increased succinate by 1.8-fold. Thus, although the heart readily
took up either heptanoate or hexanoate, either one differentially increased CAC intermediate content but did not result in
improved contractile function or energetics. These findings
suggest that, with a 60% flow reduction followed by reperfusion, impaired CAC function or depletion of CAC intermediates does not contribute to ischemic or postischemic dysfunction.
The present findings support the concept that mitochondrial
substrate oxidation, specifically the function of the CAC,
effectively adapts to short-term ischemia. As our laboratory
has previously reported in a swine model with 1 h of total LAD
occlusion and 2 h of reperfusion (36), in the present study there
were no significant changes in the levels of CAC intermediates
in myocardium subjected to ischemia-reperfusion compared
with normal flow conditions. It is important to note, however,
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Fig. 3. Myocardial content of citric acid cycle intermediates expressed as a
percentage of the nonischemic group. I/R, subjected to ischemia and reperfusion; CIT, citrate; SUC, succinate; FUM, fumarate; MAL, malate. Values are
means ⫾ SE, expressed as nmol/g. *P ⬍ 0.05 compared with saline group;
**P ⬍ 0.05 compared with saline and heptanoate groups; †P ⬍ 0.05 compared
with saline and hexanoate groups.
that there can be a disconnection between changes in anaplerotic flux through the propionyl-CoA-generating pathway
without affecting the myocardial content of CAC intermediates
(22, 26, 36). The results of the present investigation show that
increasing anaplerotic flux and increasing CAC intermediate
content do not provide a clear benefit in ischemia-reperfusion.
There were several differences in the effects of hexanoate
and heptanoate in the profile of myocardial metabolites. The
lower concentration of acetyl-CoA and the trend to lower free
CoA with heptanoate vs. hexanoate treatment probably reflect
the trapping of CoA in the intermediates in the propionyl-CoA
pathway (13). In regard to the CAC, we observed a significant
reduction in ␣-KG content in the Hep and Hex groups compared with the normal group or the saline-treated ischemiareperfusion group. In contrast, Comte et al. (5) observed higher
myocardial ␣-KG concentrations in isolated rat hearts perfused
with octanoate compared with oleate under aerobic conditions,
but no difference when the hearts were stressed with low-flow
ischemia. While the data from the present study demonstrate a
clear decrease in ␣-KG under treatment with medium-chain
fatty acids during ischemia-reperfusion, the cause of this effect
is not clear. Among the four carbon intermediates of the CAC,
heptanoate had no effect on succinate concentration but significantly increased fumarate and malate (Fig. 3). This is
surprising since one would expect that stimulation of the
propionyl-CoA pathway would also raise succinate concentration. On the other hand, hexanoate elevated only succinate
content. The present study would have been strengthened if
isotopically labeled heptanoate and hexanoate were used to
trace their route of metabolism and quantify their contribution
to CAC flux. In any case, while the mechanisms for the
changes in the concentration of CAC intermediates are not
clear, one must keep in mind that we assessed whole tissue and
not the mitochondrial compartment. While it is likely that most
of the myocardial CAC intermediate pool resides in the mitochondria, it is possible that the changes we observed reflect
alterations in the extramitochondrial pool.
Glucose oxidation was unaffected by ischemia or reperfusion in all groups, and PDH activity was elevated in myocardium subjected to ischemia-reperfusion compared with nonischemic hearts (Table 3). Previous in vivo studies in dogs and
pigs found no impairment in glucose oxidation or PDH activity
with a 50 –70% decrease in LAD blood flow or with reperfusion compared with normal flow conditions (23, 27, 33, 35).
The present investigation extends these findings to show that
supplementation with medium-chain fatty acids does not affect
either glucose oxidation or PDH activity during reperfusion.
These results run counter to work from isolated buffer perfused
rodent hearts, where there is a consistent reduction in glucose
oxidation during reperfusion following global no-flow ischemia in the presence of fatty acids (18, 37). Churchill et al. (4)
recently found reduced PDH activity following no-flow ischemia in isolated rat hearts perfused in the absence of insulin with
10 mM glucose as the sole substrate. The discrepancies
between the ex vivo studies and the present investigation are
likely due to the severity of ischemia and the lack of physiological conditions (e.g., normal arterial concentrations of
substrates, oxygen, and hormones, and high coronary buffer
flow during reperfusion) in the in vitro studies. In addition,
we measured PDH activity in rapidly frozen tissue, while
Churchill et al. assayed activity following tissue homogeniza-
CITRIC ACID CYCLE INTERMEDIATES IN REPERFUSED HEARTS
tion and centrifugation to isolated mitochondria, which would
likely affect the phosphorylation state of the PDH complex.
In conclusion, we observed that acute treatment with the
anaplerotic medium-chain fatty acid heptanoate resulted in a
significant increase in CAC intermediates but did not improve
contractile function during ischemia or reperfusion. In addition, a 60% reduction in coronary flow followed by reperfusion
had no effect on the myocardial content of CAC intermediates.
Taken together, these results suggest that depletion of CAC
intermediates does not play a major role in the functional and
metabolic derangements observed in acute ischemia and reperfusion.
ACKNOWLEDGMENTS
The authors thank Dr. Radu Iancu and Julie Rennison for assistance.
GRANTS
This research was supported by National Institutes of Health Grants
HL-074237 and DK-35543.
1. Brunengraber H, Kelleher JK, and des Rosiers C. Applications of mass
isotopomer analysis to nutrition research. Annu Rev Nutr 17: 559 –596,
1997.
2. Cederblad G, Carlin JI, Constantin-Teodosiu D, Harper P, and
Hultman E. Radioisotopic assays of CoASH and carnitine and their
acetylated forms in human skeletal muscle. Anal Biochem 185: 274 –278,
1990.
3. Chandler MP, Huang H, McElfresh TA, and Stanley WC. Increased
nonoxidative glycolysis despite continued fatty acid uptake during demand-induced myocardial ischemia. Am J Physiol Heart Circ Physiol 282:
H1871–H1878, 2002.
4. Churchill EN, Murriel CL, Chen CH, Mochly-Rosen D, and Szweda
LI. Reperfusion-induced translocation of delta PKC to cardiac mitochondria prevents pyruvate dehydrogenase reactivation. Circ Res 97: 78 – 85,
2005.
5. Comte B, Vincent G, Bouchard B, Benderdour M, and des Rosiers C.
Reverse flux through cardiac NADP(⫹)-isocitrate dehydrogenase under
normoxia and ischemia. Am J Physiol Heart Circ Physiol 283: H1505–
H1514, 2002.
6. 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.
7. Des Rosiers C, Fernandez CA, David F, and Brunengraber H. Reversibility of the mitochondrial isocitrate dehydrogenase reaction in the perfused rat liver. Evidence from isotopomer analysis of citric acid cycle
intermediates. J Biol Chem 269: 27179 –27182, 1994.
8. des Rosiers C, Lloyd S, Comte B, and Chatham JC. A critical perspective of the use of (13)C-isotopomer analysis by GCMS and NMR as
applied to cardiac metabolism. Metab Eng 6: 44 –58, 2004.
9. Di Lisa F, Menabo R, Barbato R, and Siliprandi N. Contrasting effects
of propionate and propionyl-L-carnitine on energy-linked processes in
ischemic hearts. Am J Physiol Heart Circ Physiol 267: H455–H461, 1994.
10. Fernandez CA, des Rosiers C, Previs SF, David F, and Brunengraber
H. Correction of 13C mass isotopomer distributions for natural stable
isotope abundance. J Mass Spectrom 31: 255–262, 1996.
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. Janero DR and Hreniuk D. Suppression of TCA cycle activity in the
cardiac muscle cell by hydroperoxide-induced oxidant stress. Am J Physiol
Cell Physiol 270: C1735–C1742, 1996.
13. Kasumov T, Martini WZ, Reszko AE, Bian F, Pierce BA, David F,
Roe CR, and Brunengraber H. Assay of the concentration and (13)C
isotopic enrichment of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA by gas chromatography-mass spectrometry. Anal Biochem 305:
90 –96, 2002.
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.
J Appl Physiol • VOL
15. Laplante A, Comte B, and des Rosiers C. Assay of blood and tissue
oxaloacetate and alpha-ketoglutarate by isotope dilution gas chromatography-mass spectrometry. Anal Biochem 224: 580 –587, 1995.
16. Laplante A, Vincent G, Poirier M, and des Rosiers C. Effects and
metabolism of fumarate in the perfused rat heart. A 13C mass isotopomer
study. Am J Physiol Endocrinol Metab 272: E74 –E82, 1997.
17. Liedtke AJ, Hacker T, Renstrom B, and Nellis SH. Anaplerotic effects
of propionate on oxidations of acetate and long-chain fatty acids. Am J
Physiol Heart Circ Physiol 270: H2197–H2203, 1996.
18. Lopaschuk GD, Wambolt RB, and Barr RL. An imbalance between
glycolysis and glucose oxidation is a possible explanation for the detrimental effects of high levels of fatty acids during aerobic reperfusion of
ischemic hearts. J Pharmacol Exp Ther 264: 135–144, 1993.
19. Lucas DT and Szweda LI. Declines in mitochondrial respiration during
cardiac reperfusion: age-dependent inactivation of alpha-ketoglutarate
dehydrogenase. Proc Natl Acad Sci USA 96: 6689 – 6693, 1999.
20. Mallet RT. Pyruvate: metabolic protector of cardiac performance. Proc
Soc Exp Biol Med 223: 136 –148, 2000.
21. Malloy CR, Sherry AD, and Jeffrey FM. Evaluation of carbon flux and
substrate selection through alternate pathways involving the citric acid
cycle of the heart by 13C NMR spectroscopy. J Biol Chem 263: 6964 –
6971, 1988.
22. Martini WZ, Stanley WC, Huang H, des Rosiers C, Hoppel CL, and
Brunengraber H. Quantitative assessment of anaplerosis from propionate in
pig heart in vivo. Am J Physiol Endocrinol Metab 284: E351–E356, 2003.
23. Myears DW, Sobel BE, and Bergmann SR. Substrate use in ischemic
and reperfused canine myocardium: quantitative considerations. Am J
Physiol Heart Circ Physiol 253: H107–H114, 1987.
24. Nulton-Persson AC and Szweda LI. Modulation of mitochondrial function by hydrogen peroxide. J Biol Chem 276: 23357–23361, 2001.
25. Panchal AR, Comte B, Huang H, Dudar B, Roth B, Chandler M, des
Rosiers C, Brunengraber H, and Stanley WC. Acute hibernation
decreases myocardial pyruvate carboxylation and citrate release. Am J
Physiol Heart Circ Physiol 281: H1613–H1620, 2001.
26. 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.
27. Renstrom B, Nellis SH, and Liedtke AJ. Metabolic oxidation of glucose
during early myocardial reperfusion. Circ Res 65: 1094 –1101, 1989.
28. Reszko AE, Kasumov T, Pierce BA, David F, Hoppel CL, Stanley WC,
des Rosiers C, and Brunengraber H. Assessing the reversibility of the
anaplerotic reactions of the propionyl-CoA pathway in heart and liver.
J Biol Chem 278: 34959 –34965, 2003.
29. Roe CR, Sweetman L, Roe DS, David F, and Brunengraber H.
Treatment of cardiomyopathy and rhabdomyolysis in long-chain fat oxidation disorders using an anaplerotic odd-chain triglyceride. J Clin Invest
110: 259 –269, 2002.
30. Russell RR III and Taegtmeyer H. Changes in citric acid cycle flux and
anaplerosis antedate the functional decline in isolated rat hearts utilizing
acetoacetate. J Clin Invest 87: 384 –390, 1991.
31. 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.
32. Sadek HA, Humphries KM, Szweda PA, and Szweda LI. Selective
inactivation of redox-sensitive mitochondrial enzymes during cardiac
reperfusion. Arch Biochem Biophys 406: 222–228, 2002.
33. Schoder H, Knight RJ, Kofoed KF, Schelbert HR, and Buxton DB.
Regulation of pyruvate dehydrogenase activity and glucose metabolism in
post-ischaemic myocardium. Biochim Biophys Acta 1406: 62–72, 1998.
34. Sharma N, Okere IC, Brunengraber DZ, McElfresh TA, King KL,
Sterk JP, Huang H, Chandler MP, and Stanley WC. Regulation of
pyruvate dehydrogenase activity and citric acid cycle intermediates during
high cardiac power generation. J Physiol 562: 593– 603, 2005.
35. 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.
36. Stanley WC, Kivilo KM, Panchal AR, Hallowell PH, Bomont C,
Kasumov T, and Brunengraber H. Post-ischemic treatment with dipyruvyl-acetyl-glycerol decreases myocardial infarct size in the pig. Cardiovasc Drugs Ther 17: 209 –216, 2003.
37. Stanley WC, Lopaschuk GD, Hall JL, and McCormack JG. Regulation of myocardial carbohydrate metabolism under normal and ischaemic
100 • JANUARY 2006 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.1 on May 8, 2017
REFERENCES
81
82
CITRIC ACID CYCLE INTERMEDIATES IN REPERFUSED HEARTS
conditions. Potential for pharmacological interventions. Cardiovasc Res
33: 243–257, 1997.
38. Sterk JP, Stanley WC, Hoppel CL, and Kerner J. A radiochemical
pyruvate dehydrogenase assay: activity in heart. Anal Biochem 313:
179 –182, 2003.
39. Sundqvist KE, Vuorinen KH, Peuhkurinen KJ, and Hassinen IE.
Metabolic effects of propionate, hexanoate and propionylcarnitine in
normoxia, ischaemia and reperfusion. Does an anaplerotic substrate protect the ischaemic myocardium? Eur Heart J 15: 561–570, 1994.
40. Thomassen A, Nielsen TT, Bagger JP, Pedersen AK, and Henningsen
P. Antiischemic and metabolic effects of glutamate during pacing in
patients with stable angina pectoris secondary to either coronary artery
disease or syndrome X. Am J Cardiol 68: 291–295, 1991.
41. Thomassen AR. Myocardial uptake and effects of glutamate during
non-ischaemic and ischaemic conditions. A clinical study with special
reference to possible interrelationships between glutamate and myocardial
utilization of carbohydrate substrates. Dan Med Bull 39: 471– 488, 1992.
42. 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.
43. Wisneski JA, Gertz EW, Neese RA, Gruenke LD, Morris DL, and
Craig JC. Metabolic fate of extracted glucose in normal human myocardium. J Clin Invest 76: 1819 –1827, 1985.
44. Wisneski JA, Gertz EW, Neese RA, and Mayr M. Myocardial metabolism of free fatty acids. Studies with 14C-labeled substrates in humans.
J Clin Invest 79: 359 –366, 1987.
Downloaded from http://jap.physiology.org/ by 10.220.33.1 on May 8, 2017
J Appl Physiol • VOL
100 • JANUARY 2006 •
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