(Received for publication, January 6, 1995; and in revised form, March 16, 1995)
From the
We determined whether high fatty acid oxidation rates during
aerobic reperfusion of ischemic hearts could be explained by a decrease
in malonyl-CoA levels, which would relieve inhibition of carnitine
palmitoyltransferase 1, the rate-limiting enzyme involved in
mitochondrial uptake of fatty acids. Isolated working rat hearts
perfused with 1.2 mM palmitate were subjected to 30 min of
global ischemia, followed by 60 min of aerobic reperfusion. Fatty acid
oxidation rates during reperfusion were 136% higher than rates seen in
aerobically perfused control hearts, despite the fact that cardiac work
recovered to only 16% of pre-ischemic values. Neither the activity of
carnitine palmitoyltransferase 1, or the IC
Fatty acids are a major fuel of the heart, with fatty acid
oxidation normally providing 60-70% of the hearts energy
requirements (1, 2, 3, 4) . In the
presence of high circulating levels of fatty acids, fatty acid
oxidation increases and accounts for almost all the hearts ATP
production(1, 3) . Clinically, high circulating levels
of fatty acids are commonly seen following a myocardial infarction (5, 6, 7) or during and following cardiac
surgery(8, 9) . Reperfusion of ischemic hearts with
high levels of fatty acids results in a rapid recovery of fatty acid
oxidation(10, 11, 12) , with 90-100% of
ATP production being derived from fatty acid oxidation. This
over-reliance on fatty acid oxidation has a detrimental effect on
functional recovery of hearts following severe
ischemia(13, 14) , with several lines of evidence
suggesting that fatty acid inhibition of glucose oxidation contributes
to this functional
depression(11, 13, 15, 16, 17) .
In addition to the level of circulating fatty acid concentration,
workload is another important determinant of myocardial fatty acid
oxidation rates. Normally, a close correlation exists between cardiac
work and fatty acid oxidation, with fatty acid oxidation increasing and
decreasing in parallel with increases and decreases in cardiac work (18) . However, following severe ischemia in rat hearts, fatty
acid oxidation rates are high, even though mechanical function is
markedly depressed(10, 11, 20) . This
suggests that normal control of fatty acid oxidation is altered in the
post-ischemic heart. Since fatty acid oxidation accounts for the
majority of oxygen consumption following ischemia(19) , this
uncoupling of fatty acid oxidation from cardiac work probably
contributes to the poor cardiac efficiency seen during reperfusion (i.e. an increase in oxygen consumed/unit of cardiac
work)(10, 20) . The mechanism responsible for the
deregulation of fatty acid oxidation during reperfusion is not clear.
Recent studies have shown malonyl-CoA to be an important regulator
of fatty acid oxidation in the heart(21, 22) .
Malonyl-CoA is a potent inhibitor of carnitine palmitoyltransferase 1
(CPT 1),
Malonyl-CoA is produced from acetyl-CoA carboxylase, which
catalyzes the carboxylation of acetyl-CoA to form
malonyl-CoA(24, 25, 26) . Both a 280-kDa
isoform (ACC 280) and a 265-kDa isoform (ACC 265) are present in the
heart, with ACC 280
predominating(21, 27, 28) . Regulation of ACC
in tissues such as liver and adipose tissue have been well
characterized (see (24, 25, 26) for
reviews). It is now apparent that a major kinase responsible for
short-term inactivation of ACC is a novel 5`-AMP-dependent protein
kinase (AMPK)(29, 30) , which phosphorylates Ser-79 of
ACC 265(31) . Liver AMPK is stimulated both by 5`-AMP and by
phosphorylation by an AMPK
kinase(29, 30, 32) , and is thought to be
activated under conditions of metabolic stress (i.e. ATP
depletion and AMP accumulation)(29, 33) . To date,
AMPK activity has not been extensively characterized in the heart,
although mRNA levels for the catalytic subunit of AMPK is abundant in
heart tissue(34) . It is also not clear to what degree cardiac
ACC is controlled by phosphorylation.
In this study, we determined
whether a decrease in malonyl-CoA levels and/or an increase in CPT 1
activity was responsible for the high rates of fatty acid oxidation in
the reperfused ischemic heart. We also determined the effects of
ischemia and reperfusion on the activity of both ACC and AMPK.
The perfusion protocol
involved: (a) a 60-min aerobic perfusion, (b) a
30-min aerobic perfusion followed by a 30-min period of global no-flow
ischemia, or (c) a 30-min aerobic perfusion followed by a
30-min period of global no-flow ischemia and a subsequent 60-min period
of aerobic reperfusion. Heart rate (HR), peak systolic pressure (PSP),
developed pressure (
CoA esters were extracted from the powdered tissue
using 6% perchloric acid, as described previously(21) . The CoA
esters were separated and quantified using a previously described high
performance liquid chromatography procedure(35) . 5`-AMP levels
from the perchloric acid extract were determined using previously
described methodology(17) .
Acetyl-CoA carboxylase activity in the 6% PEG
8000 fraction was determined using the
[
AMPK was assayed in the 6% PEG
8000 fraction by following the incorporation of
CPT 1 activity was
determined by the method of Bremer(38) . The mitochondrial
preparations (35-40 µg) were preincubated with various
concentrations of malonyl-CoA (0-1 µM) in an
incubation mixture containing 75 mM KCl, 50 mM mannitol, 25 mM HEPES (pH 7.3), 2 mM NaCN, 0.2
mM EGTA, 1 mM dithiothreitol, and 1% bovine serum
albumin (essentially fatty acid free) at 30 °C for 3 min. Following
this period, 0.1 µCi of L-[methyl-
Malonyl-CoA decarboxylase was assayed by the method of Svoronos and
Kumar(39) , coupled to the method of Constantin-Teodosiu et
al.(40) . The mitochondrial preparations (25-50
µg) were incubated in 210 µl of reaction mixture containing 0.1 M Tris-HCl (pH 8.0), 0.5 mM dithiothreitol, and 1
mM malonyl-CoA at 37 °C for 10 min. The reaction was
stopped by adding 40 µl of 0.5 M perchloric acid. The
solution was then neutralized with 2.2 M KHCO
Figure 1:
Graph showing the
recovery of cardiac work in isolated working rat hearts reperfused
following ischemia. Values are the mean ± S.E. of 20 hearts.
Hearts were perfused for 30 min under aerobic conditions, followed by
30 min of global no flow ischemia, and 60 min of aerobic reperfusion.
*, significantly different from pre-ischemic
values.
Figure 2:
Graph showing the inhibition of carnitine
palmitoyltransferase 1 by malonyl-CoA in aerobic, ischemic, and
reperfused ischemic hearts. Values are the mean ± S.E. of 3
aerobic hearts, 4 ischemic hearts, and 3 reperfused ischemic hearts.
Hearts were perfused for either a 60-min aerobic period, a 30-min
period of aerobic perfusion followed by 30 min of ischemia, or a 30-min
aerobic period followed by 30 min of ischemia and 60 min of aerobic
reperfusion. Hearts were then cut down and mitochondria were
immediately isolated as described under ``Experimental
Procedures.'' Mitochondrial CPT 1 activity was measured as
described under ``Experimental Procedures.'' *, significantly
different from aerobic hearts.
Figure 3:
Graph showing basal and citrate-dependent
acetyl-CoA carboxylase activity in aerobic, ischemic, and reperfused
ischemic hearts. Values are the mean ± S.E. of 8 aerobic hearts,
8 ischemic hearts, and 8 hearts reperfused following ischemia. Hearts
were frozen following either a 60-min aerobic period, a 30-min period
of aerobic perfusion followed by 30 min of ischemia, or a 30-min
aerobic period followed by 30 min of ischemia and 60 min of aerobic
reperfusion. Acetyl-CoA carboxylase was isolated and the activity
measured as described under ``Experimental Procedures.'' *,
significantly different from comparable conditions in aerobic hearts. t, significantly different from comparable conditions in
ischemic hearts.
To
rule out the possibility that the decrease in ACC activity seen in
reperfused ischemic hearts was due to a loss of ACC protein, the
relative amount of ACC in aerobic and reperfused hearts was compared
using Western blot analysis (Fig. 4). Since ACC is a
biotin-containing enzyme, blotting with streptavidin-peroxidase can be
utilized for determination of the relative protein content. Fig. 4shows the presence of bands at 280 and 265 kDa
corresponding to the ACC 280 and ACC 265 isozymes,
respectively(21, 27, 28) . The intensity of
these two bands were not different between aerobic and reperfused
ischemic hearts. These results suggest that the reduction of ACC
activity is not due to a decrease in enzyme content, but rather to a
direct modification of enzyme activity.
Figure 4:
Graph showing acetyl-CoA carboxylase
isoform distribution and content in aerobic and reperfused ischemic
hearts. ACC was extracted from hearts as described under
``Experimental Procedures.'' SDS-polyacrylamide gel
electrophoresis was performed followed by transfer to nitrocellulose
membranes. Peroxidase-labeled streptavidin was used to detect the
relative content of ACC 280 and ACC 265 (streptavidin recognizes the
biotin-containing group of carboxylases). Each lane represents an
individual heart frozen at the end of the 60-min aerobic period, or at
the end of the 60-min period of reperfusion following ischemia.
Pyruvate carboxylase and propionyl-CoA carboxylase are two
biotin-containing enzymes that are also recognized by
streptavidin.
Figure 5:
Graph showing basal and 5`-AMP dependence
of AMPK activity in aerobic, ischemic, and reperfused ischemic hearts.
Values are the mean ± S.E. of 8 aerobic hearts, 8 ischemic
hearts, and 8 reperfused ischemic hearts. Hearts were frozen after
either a 60-min aerobic period, a 30-min period of aerobic perfusion
followed by 30 min of ischemia, or a 30-min aerobic period followed by
30 min of ischemia and 60 min of aerobic reperfusion. AMPK was isolated
and the activity measured as described under ``Experimental
Procedures.'' *, significantly different from comparable
conditions in aerobic hearts.
Under normal physiological conditions a close positive
correlation exists between cardiac work and fatty acid
oxidation(18) . This ensures that the demand of acetyl-CoA for
the tricarboxylic acid cycle is matched by the supply of acetyl-CoA
from fatty acid
As shown in this study (Table 2), as well as in previous
studies (10, 11, 12, 19, 20) , the
close coupling normally seen between fatty acid oxidation and cardiac
work is not apparent during reperfusion of ischemic hearts. The
uncoupling of fatty acid oxidation from mechanical function was
accompanied by a significant decrease in cardiac efficiency during
reperfusion (i.e. cardiac work per oxygen consumed). The
results from this study suggests that a dramatic drop in malonyl-CoA
levels during reperfusion of ischemic rat hearts is primarily
responsible for the high rates of fatty acid oxidation. CPT 1 activity
and the sensitivity of CPT 1 to inhibition by malonyl-CoA were
essentially unaffected by either ischemia or reperfusion. In contrast,
a significant decrease in ACC activity occurred during reperfusion. A
decrease in ACC activity in conjunction with maintained malonyl-CoA
decarboxylase activity could explain the dramatic drop in malonyl-CoA
levels seen during reperfusion of ischemic hearts. The observation that
low ACC activity could be reversed in vitro by addition of 10
mM citrate suggests that the enyzme was in the phosphorylated
inhibited state during reperfusion. We hypothesize that activation of
AMPK during ischemia and reperfusion is primarily responsible for
phosphorylating and inhibiting ACC during reperfusion. A hypothetical
scheme outlining this is shown in Fig. 6. Support for this
proposed scheme is discussed below.
Figure 6:
Scheme showing proposed mechanism by which
ischemia stimulates fatty acid oxidation in the reperfused ischemic
heart. Myocardial ischemia results in an increase in 5`-AMP levels.
This can either directly activate AMPK, or enhance the phosphorylation
of AMPK by an AMPK kinase. AMPK remains activated throughout the
immediate reperfusion period, resulting in a phosphorylation and
inhibition of ACC during reperfusion. The decrease in ACC activity in
combination with a maintained malonyl-CoA degradation (possibly by
malonyl-CoA decarboxylase) will result in a decrease in malonyl-CoA
levels during reperfusion. This will relieve malonyl-CoA inhibition of
CPT 1, resulting in an increase in fatty acid oxidation. High fatty
acid oxidation rates will decrease cardiac efficiency and contribute to
ischemic injury during reperfusion of ischemic
hearts.
In liver, the activity of CPT
1 and the sensitivity of CPT 1 to malonyl-CoA inhibition can change
dramatically under various physiological or pathological conditions,
such as diabetes or fasting (23) and maturation(46) .
In contrast to the liver, heart CPT 1 activity and the sensitivity to
malonyl-CoA inhibition does not change in hearts from diabetic or
fasted rats(47) . Furthermore, although fatty acid oxidation
rates increase dramatically in the newborn rabbit heart(48) ,
we have observed that both CPT 1 activity and the sensitivity of CPT 1
to malonyl-CoA inhibition do not change(43) . Rather, a
decrease in malonyl-CoA levels appears to be primarily responsible for
the increase in fatty acid oxidation in the newborn heart. As shown in Fig. 2, CPT 1 activity and the sensitivity to inhibition by
malonyl-CoA in adult rat hearts were also not significantly affected by
ischemia and/or reperfusion. In dog hearts subjected to regional low
flow ischemia, mitochondria from the ischemic region do show a decrease
in the sensitivity of CPT 1 to inhibition by malonyl-CoA(49) .
This was attributed to an increase in the activity of a CPT 1 expressed
which was less sensitive to malonyl-CoA, and a decrease in the activity
of CPT 1 which was more sensitive to malonyl-CoA. Whether these two
activities were related to the 88- and 82-kDa isoforms, respectively,
was not determined. The reason for the differences between the dog
study and the present study are not clear. It was also not clear if the
alterations in CPT 1 sensitivity to malonyl-CoA seen in regionally
ischemic dog hearts remained if the tissue were reperfused. Regardless,
our data suggest that in rat hearts it is the change in the actual
levels of malonyl-CoA, as opposed to direct alterations in the
characteristics of CPT 1, that are primarily responsible for the
increase in fatty acid oxidation rates observed during reperfusion of
ischemic rat hearts.
It would be expected that deregulation of CPT 1
during reperfusion should result in an increased contribution of
endogenous triacylglycerols as a source of fatty acids for oxidation.
In a previous study (50) we found that the absolute
contribution of triacylglycerol as a source of fatty acids for
In liver it appears that AMPK is the principle kinase involved in
the phosphorylation control of ACC(29, 30) . To date,
AMPK has not been extensively characterized in heart tissue.
Witter's group has shown that the heart contains abundant mRNA
for the catalytic subunit of AMPK, although measured AMPK activity in
the heart was low when compared to liver(34) . Using a PEG 8000
extraction technique previously established in Hardie's
laboratory we observed abundant AMPK activity in heart, which rivals
the activity found in liver.
AMPK is not only
directly activated by 5`-AMP, but also secondary to phosphorylation by
AMPK kinase(29, 30) . Weekes et al.(32) have recently purified liver AMPK kinase and showed that
AMPK kinase is activated by 5`-AMP. Thus elevation of 5`-AMP levels can
activate AMPK activity by dual mechanisms whose effects are
synergistic. We propose that the activation of AMPK at the end of
ischemia occurred secondary to the dramatic increase in 5`-AMP that
occurred during ischemia, either due to a direct activation of AMPK or
secondary to phosphorylation of AMPK by AMPK kinase. During
reperfusion, the activity of AMPK remained high, even though 5`-AMP
levels returned to pre-ischemic values. This suggests that
phosphorylation of AMPK by AMPK kinase may be partially responsible for
the activation of AMPK in the ischemic and reperfused hearts. However,
to date the presence of AMPK kinase, or whether AMPK is phosphorylated
by AMPK kinase in ischemic and reperfused ischemic hearts remains to be
determined. It is also possible that AMP levels in the cytoplasmic
compartment remain elevated during reperfusion, resulting in a
continuation of AMPK stimulation. In this study, we only measured
overall tissue levels of AMP, which is not an accurate marker of free
cytoplasmic levels of AMP. Cytoplasmic phosphorylation potential is
decreased in stunned myocardium during
reperfusion(52, 53) , which suggests that cytoplasmic
AMP levels remain elevated. As shown in Fig. 1, a considerable
degree of stunning was observed in our hearts. As a result, it is
possible that a direct AMP activation of AMPK persists into
reperfusion. Unfortunately, it is still not clear how much AMP is
necessary to stimulate cardiac AMPK activity. Studies by Hardie's
group using purified liver AMPK found that the K
The reason why fatty acid inhibition of glucose
oxidation is deleterious to the recovery of mechanical function
following reperfusion of ischemic myocardium is uncertain. Recent
studies have suggested that the activation of pyruvate dehydrogenase
complex is critical for functional recovery following
ischemia(52, 53) . If pyruvate dehydrogenase complex
activation is inhibited by high concentrations of fatty acid during
reperfusion, this could lead to a decreased aqueous cytoplasmic
phosphorylation potential(52, 53) . Alternatively, a
decrease in glucose oxidation could uncouple glycolysis from glucose
oxidation, leading to an increase the production of H
Since high fatty acid oxidation rates are
detrimental to reperfusion recovery of ischemic hearts, then decreased
levels of malonyl-CoA during reperfusion have the potential to
contribute to the detrimental effects of fatty acids. As a result, it
stands to reason that modification of the pathways involved in
malonyl-CoA production and the interaction of malonyl-CoA with CPT 1
have pharmacological potential in the protection of the reperfused
ischemic heart. It has already been determined that inhibition of CPT 1
will overcome the detrimental effects of fatty
acids(11, 13) . It remains to be determined whether
increasing malonyl-CoA levels during reperfusion secondary to: (a) an inhibition of AMPK, (b) a stimulation of ACC,
or (c) an inhibition of malonyl-CoA decarboxylase, will
decrease fatty acid oxidation rates and improve functional recovery.
value of
malonyl-CoA for carnitine palmitoyltransferase 1 were altered in
mitochondria isolated from aerobic, ischemic, or reperfused ischemic
hearts. Levels of malonyl-CoA were extremely low at the end of
reperfusion compared to levels seen in aerobic controls, as was the
activity of acetyl-CoA carboxylase, the enzyme which produces
malonyl-CoA. The activity of 5`-AMP-activated protein kinase, which has
been shown to phosphorylate and inactivate acetyl-CoA carboxylase in
other tissues, was significantly increased at the end of ischemia, and
remained elevated throughout reperfusion. These results suggest that
accumulation of 5`-AMP during ischemia results in an activation of
AMP-activated protein kinase, which phosphorylates and inactivates ACC
during reperfusion. The subsequent decrease in malonyl-CoA levels will
result in accelerated fatty acid oxidation rates during reperfusion of
ischemic hearts.
(
)a key enzyme involved in fatty acid
transport into the mitochondrial matrix(23) . We recently
demonstrated a close relationship between cardiac malonyl-CoA levels
and fatty acid oxidation rates in aerobically perfused working rat
hearts(21) . This raises the possibility that either a decrease
in malonyl-CoA levels, an increase in CPT 1 activity, or a decrease in
the sensitivity of CPT 1 to malonyl-CoA inhibition may contribute to
the high fatty acid oxidation rates seen during reperfusion of ischemic
hearts.
Heart Perfusions
Male Wistar rats (250-300
g) were anesthetized with an injection of sodium pentobarbital (60
mg/kg intraperitoneally). Hearts were subsequently excised from
unconscious animals, the aorta cannulated, and a retrograde perfusion
with Krebs-Henseleit buffer (pH 7.4, gassed with 95% O and
5% CO
, 37 °C) initiated. During this initial perfusion,
the hearts were trimmed of excess tissue, and both the pulmonary artery
and the opening of the left atrium cannulated. Following a 10-min
Langendorff washout period, hearts were switched to the working mode.
Perfusate was delivered from the oxygenator into the left atrium at an
11.5 mm Hg preload, and was ejected from spontaneously beating hearts
against an 80 mm Hg hydrostatic afterload(11) . Perfusate
consisted of 100 ml of re-circulated Krebs-Henseleit buffer (pH 7.4,
gassed with 95% O
and 5% CO
, 37 °C)
containing 1.2 mM [1-
C]palmitate, 3%
bovine serum albumin, 11 mM glucose, 2.5 mM free
Ca
, and 100 microunits/ml insulin (bovine, regular
Connaught-Nova, Willowdale, Ontario, Canada).
P), cardiac output, aortic flow, coronary
flow, and O
consumption were measured as described
previously(13, 17, 21) .
Measurement of Palmitate Oxidation
Steady state
rates of palmitate oxidation were measured in aerobic or reperfused
ischemic hearts by quantitatively collecting CO
produced from hearts perfused with 1.2 mM
[1-
C]palmitate (approximately 50,000 dpm/ml
buffer). Collection of
CO
released as a gas in
the oxygenation chamber and the
CO
trapped in
the NaHCO
in the perfusate was performed as described
previously(11, 13) . Sampling was performed at 10-min
intervals throughout the 60-min aerobic perfusion, or at 10-min
intervals during reperfusion of previously ischemic
hearts(11) .
Tissue Analysis
At the end of the perfusions, the
hearts were freeze-clamped with Wollenberger tongs cooled to the
temperature of liquid nitrogen. Frozen ventricular tissue was weighed
and powdered in a mortar and pestle cooled to the temperature of liquid
nitrogen. A portion of the powdered tissue was used to determine the
dry-to-wet ratio.
Extraction and Measurement of Acetyl-CoA Carboxylase and
5`-AMP-activated Protein Kinase Activity
Approximately 200 mg of
frozen tissue was homogenized, using a Tekmar homogenizer, for 30 s at
4 °C in 0.4 ml of buffer containing 50 mM Tris-HCl (pH
7.5), 0.25 M mannitol, 1 mM EGTA, 1 mM EDTA,
1 mM dithiothreitol, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride,
1 mM benzamidine, and 4 µg/ml soybean trypsin inhibitor.
The homogenate was then centrifuged at 14,000 g for 20
min at 4 °C, and the resultant supernatant made up to 2.5% (w/v)
polyethylene glycol 8000 (PEG 8000) using a stock 25% (w/v) PEG 8000
solution. The solution was stirred for 10 min, the precipitate removed
by centrifugation (10,000
g for 10 min), and the
supernatant made up to 6% PEG 8000. After stirring and centrifugation
as before, the pellet was washed with a 6% PEG 8000/homogenizing buffer
and resuspended in 100 mM Tris-HCl (pH 7.5), 50 mM
sodium fluoride, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.02%
sodium azide, 1 mM benzamidine, 4 µg/ml soybean trypsin
inhibitor, and 10% glycerol. Protein content was measured using the BCA
method(36) .
C]bicarbonate fixation assay (37) . The
assay mixture contained 60.6 mM Tris acetate (pH 7.5), 1 mg/ml
bovine serum albumin, 1.3 µM 2-mercaptoethanol, 2.1 mM ATP, 1.1 mM acetyl-CoA, 5 mM magnesium acetate,
18.2 mM NaH
CO
(approximately 1000
dpm/nmol), and 25 µg of the 6% PEG 8000 pellet. Following a 2-min
incubation at 37 °C, in the absence or presence of 10 mM citrate, the reaction was stopped by adding 25 µl of 10%
perchloric acid, then centrifuged at 2000
g for 20
min. Radioactivity of supernatant was determined using standard liquid
scintillation counting procedures.
P into a
synthetic peptide (termed SAMS peptide) with the amino acid sequence
HMRSAMSGLHLVKRR(29, 37) . The assay was performed in a
25-µl total volume containing 40 mM HEPES-NaOH (pH 7.0),
80 mM NaCl, 8% glycerol, 0.8 mM EDTA, 200 µM SAMS peptide, 0.8 mM dithiothreitol, 5 mM MgCl
, 200 µM [
-
P]ATP (400-600 dpm/pmol), and
6-8 µg of the 6% PEG 8000 pellet. The assay was performed in
the absence or presence of 200 µM 5`-AMP at 30 °C for
5 min. The reaction was initiated by the addition of
[
P]ATP/Mg. At the end of the incubation,
15-µl aliquots were removed and spotted on 1
1-cm square of
phosphocellulose paper (P81, Whatman), which were subsequently placed
into 500 ml of 150 mM H
PO
. These
papers were washed 4 times for 30 min with 150 mM H
PO
, and then washed 20 min with acetone.
The papers were then dried and placed in vials containing 4 ml of
scintillant. Radioactivity was determined using standard liquid
scintillation procedures.
Mitochondrial Isolation and Measurement of Carnitine
Palmitoyltransferase 1 and Malonyl-CoA Decarboxylase Activity
A
series of hearts were perfused under conditions identical to those
described above except that radiolabeled
[C]palmitate was not included in the perfusate.
At the end of the aerobic perfusion, the ischemic perfusion, or the
reperfusion period, hearts were rapidly cut down and mitochondria
isolated as described previously(21) .
H]carnitine was added
to a final L-carnitine concentration of 200 µM,
and the incubation was continued for a further 6 min. The reaction was
stopped by adding 100 µl of 10 N HCl. The
[
H]palmitoyl carnitine was extracted with butanol
and counted using standard liquid scintillation procedures.
and
centrifuged at 10,000
g for 3 min. The acetyl-CoA
formed by malonyl-CoA decarboxylase was determined by following the
conversion of acetyl-CoA to [
C]citrate in the
presence of [
C]oxaloacetate and citrate
synthase. [
C]Oxaloacetate was initially formed
by a transamination reaction utilizing aspartate aminotransferase and
[
C]aspartate, as described by
Constantin-Teodosiu et al.(40) . Following the
reaction, sodium glutamate and aspartate aminotransferase were used to
remove excess [
C]oxaloacetate after the citrate
synthase reaction by transaminating unreacted
[
C]oxaloacetate back to
[
C]aspartate. Dowex (50W-8X, 100-200 mesh)
was then used to separate [
C]aspartate from
[
C]citrate. Acetyl-CoA content of supernatant
samples was quantified by comparison to acetyl-CoA standard curves.
Statistical Analysis
The unpaired t test
was used for the determination of statistical difference of group
means. Analysis of variance followed by the Neuman-Keuls test was used
in the determination of statistical difference in groups containing
three sample populations. A value of p < 0.05 was
considered as significant. All data are presented as mean ± S.E.
Heart Function in the Hearts Reperfused After Global
Ischemia
Functional recovery of hearts reperfused after the
30-min period of global ischemia is shown in Fig. 1. In the
hearts reperfused with the buffer containing 11 mM glucose and
1.2 mM palmitate, cardiac work recovered to a maximum of 30%
of preischemic values over the 60-min reperfusion period. Mechanical
function in aerobic hearts and reperfused ischemic hearts is summarized
in Table 1. Impaired cardiac work was reflected by decreases in
both cardiac output and peak systolic pressure. In addition, heart
rate, P, and rate pressure product were all depressed during
reperfusion. Although cardiac work was only 16% of aerobic values at 60
min of reperfusion, oxygen consumption recovered to 48% of initial
aerobic values. As a result, a significant decrease in cardiac
efficiency (cardiac work/O
consumed) was seen during
reperfusion.
Palmitate Oxidation Rates during Reperfusion of Ischemic
Hearts
Palmitate oxidation rates in aerobic and reperfused
ischemic hearts are shown in Table 2. As previously
observed(4, 11) , cumulative rates of palmitate
oxidation were linear in both the aerobic hearts and during the 60-min
period of aerobic reperfusion (data not shown). Despite the observation
that cardiac work was markedly impaired during reperfusion, palmitate
oxidation rates were significantly higher during reperfusion than rates
seen in aerobically perfused nonischemic hearts. Previous studies by
Neely's group have shown that fatty acid oxidation in aerobically
perfused hearts is closely correlated to the work performed by the
hearts(18) . We therefore also normalized palmitate oxidation
rates for both cardiac work and oxygen consumption (Table 2).
During reperfusion, a dramatic increase in palmitate oxidized per unit
of work and per O consumed was observed, suggesting that
the normal relationship between fatty acid oxidation and both cardiac
work and oxygen uptake are dramatically altered in the reperfused
ischemic heart.
CPT 1 Activity in Aerobic, Ischemic, and Reperfused
Ischemic Hearts
To determine if an alteration in CPT 1 activity
could explain the high rates of fatty acid oxidation during reperfusion
of ischemic hearts, we isolated mitochondria from fresh hearts cut down
at the end of the aerobic period, at the end of the ischemic period, or
at the end of the reperfusion period. The effects of malonyl-CoA on CPT
1 activity is shown in Fig. 2. In the absence of malonyl-CoA,
CPT 1 activity was not dramatically different between any of the
experimental groups. As a result, it is unlikely that an increase in
CPT 1 activity accounts for the high fatty acid oxidation rates during
reperfusion of ischemic hearts. The sensitivity of CPT 1 to malonyl-CoA
inhibition also did not differ between the experimental groups. The
IC values of malonyl-CoA for CPT 1 were calculated as 33.2
± 3.7, 30.9 ± 0.4, and 27.4 ± 2.3 nM in
aerobic, ischemic, and reperfused ischemic hearts, respectively. These
data demonstrate that it is unlikely that alterations in either CPT 1
activity or alterations in the sensitivity of CPT 1 to inhibition by
malonyl-CoA are responsible for the high fatty acid oxidation rates
seen in reperfused ischemic hearts.
Malonyl-CoA Levels in Aerobic, Ischemic, and Reperfused
Ischemic Hearts
Since the regulation of CPT 1 by malonyl-CoA did
not change in reperfused ischemic hearts, we addressed the possibility
that a decrease in myocardial levels of malonyl-CoA may be responsible
for the high fatty acid oxidation rates seen during reperfusion. Levels
of malonyl-CoA and acetyl-CoA in the hearts are shown in Table 3.
We determined the levels of these CoA ester in ventricular tissue
frozen at the end of aerobic, ischemic, and reperfusion periods. At the
end of ischemia, malonyl-CoA levels had dropped to 38.5% of the levels
observed in aerobic hearts. By the end of reperfusion, a further
dramatic drop in malonyl-CoA levels occurred, such that levels were
only 1.1% of levels observed in aerobic hearts. As a result, inhibition
of CPT 1 by malonyl-CoA was likely to be decreased during reperfusion
in the intact heart.
In a previous study we observed that acetyl-CoA
levels were positively correlated with malonyl-CoA levels in the
aerobic heart(21) . As shown in Table 3, a significant
decrease in acetyl-CoA levels was seen in hearts frozen at the end of
ischemia, which may have accounted for the decrease in malonyl-CoA
levels seen in these hearts. However, no further decrease in acetyl-CoA
levels was observed at the end of reperfusion, even though a further
dramatic decrease in malonyl-CoA occurred.
Acetyl-CoA Carboxylase Activity in Aerobic, Ischemic, and
Reperfused Hearts
To explore the potential explanations for the
dramatic decrease in malonyl-CoA levels in reperfused ischemic hearts,
the activity and relative content of ACC were determined in extracts
obtained from frozen ventricular tissue obtained from these hearts. Fig. 3shows ACC activity in the PEG 8000 precipitate obtained
from aerobic, ischemic, and reperfused ischemic hearts. Since citrate
has been shown to increase the activity of the phosphorylated and
inhibited form of ACC, we also measured ACC activity in the presence of
10 mM citrate. At the end of ischemia, no significant
difference in basal ACC activity was seen compared to aerobic hearts
(9.6 ± 2.0 and 9.0 ± 3.2
nmolmin
mg protein, respectively).
However, at the end of the reperfusion period, ACC activity dropped to
2.3 ± 1.6 nmol
min
mg
protein
, which was significantly lower than ACC
activity seen in either aerobic or ischemic hearts.
As shown in Fig. 3, ACC activity in both aerobic and ischemic hearts was
stimulated approximately 2-fold by 10 mM citrate. In contrast,
10 mM citrate resulted in a 5.3-fold increase in ACC activity
in reperfused ischemic hearts. This is consistent with ACC being in a
phosphorylated and inhibited state at the end of reperfusion.
5`-AMP-dependent Protein Kinase Activity in Aerobic,
Ischemic, and Reperfused Ischemic Hearts
Several lines of
evidence suggest that AMPK is a key kinase involved in the
phosphorylation inhibition of liver ACC
activity(29, 30, 33, 37) . Since
AMPK activity is stimulated by 5`-AMP, which increases in the heart
during ischemia, we measured AMPK activity in the 6% PEG 8000
precipitate of aerobic, ischemic, and reperfused ischemic hearts. As
shown in Fig. 5, AMPK activity in the absence of added 5`-AMP
was significantly elevated in both the ischemic and reperfused ischemic
hearts, compared to aerobically perfused hearts. Addition of 200
µM AMP to the incubation medium resulted in an increase in
AMPK activity in all experimental groups, with the significant increase
in AMPK remaining in reperfused ischemic hearts compared to aerobic
hearts.
Increases in 5`-AMP have been previously demonstrated to
facilitate the phosphorylation and activation of liver
AMPK(29, 32) . We therefore measured 5`-AMP levels in
aerobic, ischemic, and reperfused ischemic hearts. In aerobic hearts,
AMP levels were 2.4 ± 0.3 µmolg dry
weight
. AMP levels increased to 18.4 ± 1.4
µmol
g dry weight
by the end of ischemia,
but dropped back to 2.4 ± 0.5 µmol
g dry
weight
by the end of reperfusion.
Malonyl-CoA Degradation in Aerobic, Ischemic, and
Reperfused Hearts
Another possibility for the decrease in the
level of malonyl-CoA during reperfusion is an accelerated rate of
malonyl-CoA degradation in the heart. However, to date very little is
known as to how malonyl-CoA is degraded in the heart. We have recently
characterized the activity of a malonyl-CoA decarboxylase which is
present in intact rat heart mitochondria.(
)We
therefore measured malonyl-CoA decarboxylase activity in mitochondria
isolated from aerobic, ischemic, and reperfused ischemic hearts. Rates
were 38.0 (n = 2), 27.6 ± 2.1 (n = 3), and 48.1 ± 8.6 (n = 3)
nmol
min
mg protein
in
aerobic, ischemic, and reperfused ischemic hearts, respectively. As a
result, malonyl-CoA decarboxylase activity was at least maintained, and
may even have been slightly increased in reperfused ischemic hearts.
Maintaining malonyl-CoA decarboxylase activity coupled with a dramatic
decrease in ACC activity may be responsible for the large decrease in
malonyl-CoA levels seen in the reperfused ischemic heart.
-oxidation. Within the mitochondria, flux through
-oxidation is determined to a large extent by the levels of the
products and substrates of the
-oxidation spiral (see (41) for review). It is also becoming apparent that cytoplasmic
ACC also has an important role in regulating the initial transport of
fatty acids into the
mitochondria(21, 22, 42, 43) . In
conjunction with the mitochondrial acetylcarnitine
transferase-acetylcarnitine translocase system, a rise in acetyl-CoA
levels is associated with an increase in malonyl-CoA
levels(21) . This results in a decrease in fatty acid
-oxidation and a decrease in acetyl-CoA production. Presumably,
under conditions of high work, when tricarboxylic acid cycle activity
increases and acetyl-CoA levels drop, a parallel decrease in
malonyl-CoA levels would be expected to occur, resulting in an
acceleration of both fatty acid oxidation and acetyl-CoA production.
Regulation of Fatty Acid Oxidation in the Heart by Malonyl-CoA
Inhibition of CPT 1
CPT 1 has long been recognized as the key
regulatory enzyme in the mitochondrial uptake of fatty
acids(23) . McGarry's group has recently demonstrated
that the heart expresses two isoforms of CPT 1, 88 and 82 kDa. The
82-kDa isoform, which predominates in heart, is thought to confer the
high sensitivity of cardiac CPT 1 to inhibition by
malonyl-CoA(44, 45) .
-oxidation did not change, although mechanical function of hearts
during reperfusion was significantly depressed. As a result, endogenous
triacylglycerol fatty acid oxidation normalized for mechanical function
was increased by 78% during reperfusion, supporting the conclusion that
overall fatty acid oxidation normalized for cardiac work dramatically
increases during reperfusion following ischemia.
Acetyl-CoA Carboxylase Production of
Malonyl-CoA
Although the importance of malonyl-CoA in regulating
cardiac CPT 1 activity has long been recognized, the importance of ACC
as the source of malonyl-CoA has only been recently characterized. The
predominance of ACC 280 in heart, as well as other tissues with a high
capacity for fatty acid oxidation, has implicated this isoform of ACC
in the regulation of fatty acid oxidation(28) . Recent studies
from our laboratory (21, 43) and others (22) have provided support for this concept. We have shown that
the supply of acetyl-CoA to ACC is important in the control of
malonyl-CoA production(21) . A decrease in acetyl-CoA levels
may partly explain the drop in malonyl-CoA levels observed at the end
of ischemia (Table 3). However, it is clear that other factors
must be involved in the decrease in ACC activity observed during
reperfusion of ischemic hearts. In liver, phosphorylation of ACC is
important in the acute allosteric regulation of ACC
265(24, 25, 26) . It has not been determined
whether ACC 280 that predominates in cardiac tissue is controlled by
phosphorylation, although indirect evidence to support this does exist.
Awan and Saggerson (22) used isolated myocytes to demonstrate
that insulin and norepinephrine regulate fatty acid oxidation by
changing malonyl-CoA levels in the hearts. Both of these hormones are
important in the phosphorylation control of ACC in liver. We have also
recently shown that insulin and glucagon have an important role in
regulating ACC activity and fatty acid oxidation in the newborn
heart(43) .
Phosphorylation Control of ACC by AMPK
Acute
phosphorylation control of ACC activity has been well documented in
liver and white adipose
tissue(24, 25, 26, 27) . ACC 265 can
be phosphorylated in vitro by a variety of kinases (AMPK,
cAMP-dependent protein kinase, casein kinase, and protein kinase C) at
multiple phosphorylation sites (see (24, 25, 26) for reviews). Recent evidence
has convincingly demonstrated that phosphorylation of Ser-79 by AMPK
and phosphorylation of Ser-1200 by cAMP-dependent protein kinase are
critical for inactivation of ACC 265 in vivo(31) .
Phosphorylation of Ser-79 by AMP-dependent protein kinase results in a
decrease in V and an increase in citrate
dependence of ACC 265
activity(24, 25, 26, 27) .
Phosphorylation of liver ACC 280 by cAMP-dependent protein
kinase(51) , as well as activation of ACC 280 in vitro by citrate(27) , suggests that ACC 280 is regulated by a
mechanism similar to ACC 265. However, only a partial amino acid
sequence of ACC 280 is presently known, and it is unclear whether
phosphorylation of ACC 280 regulates its activity in vivo. The
low specific activity and high citrate dependence of ACC at the end of
reperfusion following ischemia is consistent with phosphorylation
inactivation of ACC. Whether preferential involvement of ACC 280 or ACC
265 occurs in this process has not been elucidated from this study.
(
)While the
function of AMPK in the heart has not been determined, one obvious role
is the phosphorylation control of ACC. This is supported by the
observation that during reperfusion of ischemic hearts low ACC activity
was accompanied by an increase in AMPK activity.
of AMPK for AMP is 1.4 and 14 µM in the
presence of 0.2 and 2 mM ATP, respectively(30) . While
we did not measure myocardial ATP levels in the present study, we have
previously demonstrated in heart perfused under identical conditions
that ATP drops from 16.0 ± 0.5 µmol
g dry
weight
in aerobic hearts to 6.3 ± 0.7
µmol
g dry weight
at the end of
ischemia(15) . During reperfusion, ATP increases to 10.7
± 1.6 µmol
g dry weight
. Whether
these changes in [ATP] are of significance in the kinetic
relationship between AMP and cardiac AMPK remain to be determined. This
absolute value of ATP roughly translates to a concentration range of
ATP from 8 mM in aerobic hearts, 3 mM at the end of
ischemia, and 5 mM at the end of reperfusion. If cardiac AMPK
behaves similar to liver, it suggests that changes in [ATP]
concentrations are without major effect on AMPK activity during and
following ischemia.
Malonyl-CoA Degradation in the Heart
It is clear
from this study, as well as from previous
studies(21, 22, 43) , that malonyl-CoA levels
can rapidly decrease in isolated perfused hearts. While ACC appears to
be the primary source of malonyl-CoA, the fate of malonyl-CoA in the
heart is not clear. Unlike liver, fatty acid synthase does not appear
to be a major fate of malonyl-CoA in heart. Awan and Saggerson (22) have suggested that malonyl-CoA may be involved in fatty
acid elongation in isolated myocytes. However, it is unlikely that the
dramatic drop in malonyl-CoA levels during reperfusion of ischemic
hearts can be explained by an increase in fatty acid elongation
activity. An increase in energy requiring anabolic processes would
appear unlikely in the metabolically compromised myocardium seen during
reperfusion of ischemic hearts. An alternate fate of malonyl-CoA is by
decarboxylation back to acetyl-CoA. An earlier study by Kim and
Kolattukudy (54) identified the presence of an
immunoprecipitable malonyl-CoA decarboxylase activity in heart
mitochondria. We have also recently characterized the activity of
malonyl-CoA decarboxylase in heart, and found that heart mitochondria
have an active malonyl-CoA decarboxylase activity.(
)Approximately 25% of total activity can be measured
in intact mitochondria, suggesting that malonyl-CoA decarboxylase has
access to cytoplasmic malonyl-CoA. We propose that a malonyl-CoA
decarboxylase present on the outer mitochondrial membrane may be
responsible for the conversion of malonyl-CoA back to acetyl-CoA in the
cytoplasm of the heart. As a result, the decrease in ACC activity
coupled to a normal malonyl-CoA decarboxylase activity can explain the
low levels of malonyl-CoA observed during reperfusion of ischemic
hearts.
The Consequence of High Rates of Fatty Acid Oxidation in
the Reperfused Ischemic Heart
Several lines of evidence have
shown that an over-reliance on fatty acid oxidation as a source of
energy production is detrimental to functional recovery of severely
ischemic
hearts(10, 11, 13, 14, 15, 16, 17) .
This detrimental effect of fatty acids can be attributed to an
inhibition of glucose
oxidation(11, 13, 14, 15, 16, 17) .
The increase in the mitochondrial acetyl-CoA/CoA and
NADH/NAD ratios caused by fatty acid oxidation lead to
an inhibition of the pyruvate dehydrogenase complex(55) .
Compounds which stimulate glucose oxidation directly by stimulating the
pyruvate dehydrogenase complex, such as
dichloroacetate(15, 17) , or indirectly such as CPT 1
inhibitors(11, 13) , improve functional recovery
following ischemia.
from glycolytically derived ATP (see (56) for review).
Thus, an over-reliance on fatty acid oxidation could lead to a greater
production of H
. This greater production of
H
not only has the potential to directly inhibit
cardiac contractility(57) , but can also decrease cardiac
efficiency (58) .
Summary
This study demonstrates that ACC activity
decreases during reperfusion of previously ischemic hearts. Decreased
malonyl-CoA production, coupled with a maintained malonyl-CoA
decarboxylase activity, appears to be responsible for the decreased
malonyl-CoA levels during reperfusion, resulting in a significant
increase in fatty acid oxidation rates. We also demonstrate that the
heart has a significant amount of AMPK activity, which is activated
during ischemia and remains activated during reperfusion of ischemic
hearts. We propose that phosphorylation of ACC by AMPK is primarily
responsible for the inhibition of ACC during reperfusion.
P, developed pressure; SAMS, the synthetic
peptide substrate with the amino acid sequence HMRSAMSGLHLVKRR used to
assay AMPK; PEG, poly(ethylene) glycol.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.