©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
High Rates of Fatty Acid Oxidation during Reperfusion of Ischemic Hearts Are Associated with a Decrease in Malonyl-CoA Levels Due to an Increase in 5`-AMP-activated Protein Kinase Inhibition of Acetyl-CoA Carboxylase (*)

(Received for publication, January 6, 1995; and in revised form, March 16, 1995)

Naomi Kudo (§) , Amy J. Barr , Rick L. Barr , Snehal Desai , Gary D. Lopaschuk (¶)

From the Cardiovascular Disease Research Group, Lipid and Lipoprotein Research Group, Department of Pediatrics and Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Alberta T5G 2S2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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),()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.

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.


EXPERIMENTAL PROCEDURES

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).

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 (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.

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) .

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) .

Acetyl-CoA carboxylase activity in the 6% PEG 8000 fraction was determined using the [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 NaHCO (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.

AMPK was assayed in the 6% PEG 8000 fraction by following the incorporation of 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 HPO. These papers were washed 4 times for 30 min with 150 mM HPO, 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) .

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-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.

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 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.


RESULTS

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.


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.





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.


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.



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 nmolminmg protein, respectively). However, at the end of the reperfusion period, ACC activity dropped to 2.3 ± 1.6 nmolminmg protein, which was significantly lower than ACC activity seen in either aerobic or ischemic 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.



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.

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.



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.


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.



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 µmolg dry weight by the end of ischemia, but dropped back to 2.4 ± 0.5 µmolg 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) nmolminmg 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.


DISCUSSION

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 -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.

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.



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) .

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 -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.

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.()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.

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 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 µmolg dry weight in aerobic hearts to 6.3 ± 0.7 µmolg dry weight at the end of ischemia(15) . During reperfusion, ATP increases to 10.7 ± 1.6 µmolg 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.

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 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) .

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.

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.


FOOTNOTES

*
This work was supported in part by a grant from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Post-doctoral fellow of the Heart and Stroke Foundation of Canada.

Medical Research Council of Canada Scientist and a Senior Scholar of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: 423 Heritage Medical Research Centre, University of Alberta, Edmonton Alberta, Canada T6G 2S2. Tel.: 403-492-2170; Fax: 403-492-9753; glopasch{at}gpu.srv.ualberta.ca

The abbreviations used are: CPT, carnitine palmitoyltransferase; CoA, coenzyme A; ACC, acetyl-CoA carboxylase; ACC 265, the 265-kDa isoform of ACC; ACC 280, the 280-kDa isoform of ACC; AMPK, 5`AMP-activated protein kinase; HR, heart rate; PSP, peak systolic pressure; P, developed pressure; SAMS, the synthetic peptide substrate with the amino acid sequence HMRSAMSGLHLVKRR used to assay AMPK; PEG, poly(ethylene) glycol.

S. Desai and G. D. Lopaschuk, manuscript in preparation.

N. Kudo, L. Kung, L. A. Witters, R. Schulz, A. S. Clanachan, and G. D. Lopaschuk, manuscript in preparation.

S. Desai and G. D. Lopaschuk, unpublished observations.


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