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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

1995; Elsevier BV; Volume: 270; Issue: 29 Linguagem: Inglês

10.1074/jbc.270.29.17513

ISSN

1083-351X

Autores

Naomi Kudo, Amy Barr, Rick L. Barr, Snehal Desai, Gary D. Lopaschuk,

Tópico(s)

Metabolism and Genetic Disorders

Resumo

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 IC50 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. 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 IC50 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. Fatty acids are a major fuel of the heart, with fatty acid oxidation normally providing 60-70% of the hearts energy requirements (1Bing R.J. Physiol. Rev. 1965; 45: 171-213Crossref PubMed Scopus (231) Google Scholar, 2Challoner D. Steinberg D. Am. J. Physiol. 1966; 210: 280-286Crossref PubMed Scopus (107) Google Scholar, 3Neely J.R. Morgan H.E. Physiol. Rev. 1974; 36: 413-459Crossref Scopus (991) Google Scholar, 4Saddik M. Lopaschuk G.D. J. Biol. Chem. 1991; 266: 8162-8170Abstract Full Text PDF PubMed Google Scholar). In the presence of high circulating levels of fatty acids, fatty acid oxidation increases and accounts for almost all the hearts ATP production(1Bing R.J. Physiol. Rev. 1965; 45: 171-213Crossref PubMed Scopus (231) Google Scholar, 3Neely J.R. Morgan H.E. Physiol. Rev. 1974; 36: 413-459Crossref Scopus (991) Google Scholar). Clinically, high circulating levels of fatty acids are commonly seen following a myocardial infarction (5Oliver M.F. Kurien V.A. Greenwood T.W. Lancet. 1968; 1: 710-715Abstract PubMed Google Scholar, 6Opie L.H. Am. J. Cardiol. 1975; 36: 938-953Abstract Full Text PDF PubMed Scopus (245) Google Scholar, 7Mueller H.S. Ayres S.T. Am. J. Cardiol. 1978; 42: 363-371Abstract Full Text PDF PubMed Scopus (56) Google Scholar) or during and following cardiac surgery(8Svensson S. Svedjeholm R. Ekroth R. Milocco I. Nilsson F. Sabel G.S. William-Olsson G. J. Thorac. Cardiovasc. Surg. 1990; 99: 1063-1073Abstract Full Text PDF PubMed Google Scholar, 9Lopaschuk G.D. Collins-Nakai R. Olley P.M. Montague T.J. McNeil G. Gayle M. Ryan T. Penkoske P. Yeung L. Finegan B.A. Am. Heart J. 1994; 128: 61-67Crossref PubMed Scopus (120) Google Scholar). Reperfusion of ischemic hearts with high levels of fatty acids results in a rapid recovery of fatty acid oxidation(10Liedtke A.J. DeMaison L. Eggleston A.M. Cohen L.M. Nellis S.H. Circ. Res. 1988; 62: 535-542Crossref PubMed Scopus (201) Google Scholar, 11Lopaschuk G.D. Spafford M.A. Davies N.J. Wall S.R. Circ. Res. 1990; 66: 546-553Crossref PubMed Scopus (216) Google Scholar, 12Görge G. Chatelain P. Schaper J. Lerch R. Circ. Res. 1991; 68: 1681-1692Crossref PubMed Scopus (58) Google Scholar), 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(13Lopaschuk G.D. Wall S.R. Olley P.M. Davies N.J. Circ. Res. 1988; 63: 1036-1043Crossref PubMed Scopus (219) Google Scholar, 14Lopaschuk G.D. Saddik M. Barr R. Huang L. Barker C. Muzyka R.A. Am. J. Physiol. 1992; 263: E1046-E1053PubMed Google Scholar), with several lines of evidence suggesting that fatty acid inhibition of glucose oxidation contributes to this functional depression(11Lopaschuk G.D. Spafford M.A. Davies N.J. Wall S.R. Circ. Res. 1990; 66: 546-553Crossref PubMed Scopus (216) Google Scholar, 13Lopaschuk G.D. Wall S.R. Olley P.M. Davies N.J. Circ. Res. 1988; 63: 1036-1043Crossref PubMed Scopus (219) Google Scholar, 15McVeigh J.J. Lopaschuk G.D. Am. J. Physiol. 1990; 259: H1070-H1085Google Scholar, 16Broderick T.L. Quinney H.A. Barker C.C. Lopaschuk G.D Circulation. 1990; 87: 972-981Crossref Google Scholar, 17Lopaschuk G.D. Wambolt R.B. Barr R.L. J. Pharmacol. Exp. Therap. 1993; 264: 135-144PubMed Google Scholar). 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 (18Neely J.R. Whitmer K.M. Mochizuki S. Circ. Res. 1976; 38: I-22-I-30Google Scholar). However, following severe ischemia in rat hearts, fatty acid oxidation rates are high, even though mechanical function is markedly depressed(10Liedtke A.J. DeMaison L. Eggleston A.M. Cohen L.M. Nellis S.H. Circ. Res. 1988; 62: 535-542Crossref PubMed Scopus (201) Google Scholar, 11Lopaschuk G.D. Spafford M.A. Davies N.J. Wall S.R. Circ. Res. 1990; 66: 546-553Crossref PubMed Scopus (216) Google Scholar, 20Benzi R.H. Lerch R. Circ. Res. 1992; 71: 567-576Crossref PubMed Scopus (97) Google Scholar). 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(19Renstrom B. Nellis S.H. Liedtke A.J. Circ. Res. 1989; 65: 1094-1101Crossref PubMed Scopus (54) Google Scholar), 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)(10Liedtke A.J. DeMaison L. Eggleston A.M. Cohen L.M. Nellis S.H. Circ. Res. 1988; 62: 535-542Crossref PubMed Scopus (201) Google Scholar, 20Benzi R.H. Lerch R. Circ. Res. 1992; 71: 567-576Crossref PubMed Scopus (97) Google Scholar). 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(21Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar, 22Awan M.M. Saggerson E.D. Biochem. J. 1993; 295: 61-66Crossref PubMed Scopus (169) Google Scholar). Malonyl-CoA is a potent inhibitor of carnitine palmitoyltransferase 1 (CPT 1),1 1The abbreviations used are: CPTcarnitine palmitoyltransferaseCoAcoenzyme AACCacetyl-CoA carboxylaseACC 265the 265-kDa isoform of ACCACC 280the 280-kDa isoform of ACCAMPK5'AMP-activated protein kinaseHRheart ratePSPpeak systolic pressureΔPdeveloped pressureSAMSthe synthetic peptide substrate with the amino acid sequence HMRSAMSGLHLVKRR used to assay AMPKPEGpoly(ethylene) glycol. 1The abbreviations used are: CPTcarnitine palmitoyltransferaseCoAcoenzyme AACCacetyl-CoA carboxylaseACC 265the 265-kDa isoform of ACCACC 280the 280-kDa isoform of ACCAMPK5'AMP-activated protein kinaseHRheart ratePSPpeak systolic pressureΔPdeveloped pressureSAMSthe synthetic peptide substrate with the amino acid sequence HMRSAMSGLHLVKRR used to assay AMPKPEGpoly(ethylene) glycol. a key enzyme involved in fatty acid transport into the mitochondrial matrix(23McGarry J.D. Woeltje K.F. Kuwajima M. Foster D.W. Diabetes/Metabol. Revs. 1989; 5: 271-284Crossref PubMed Scopus (288) Google Scholar). We recently demonstrated a close relationship between cardiac malonyl-CoA levels and fatty acid oxidation rates in aerobically perfused working rat hearts(21Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar). 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. carnitine palmitoyltransferase coenzyme A acetyl-CoA carboxylase the 265-kDa isoform of ACC the 280-kDa isoform of ACC 5'AMP-activated protein kinase heart rate peak systolic pressure developed pressure the synthetic peptide substrate with the amino acid sequence HMRSAMSGLHLVKRR used to assay AMPK poly(ethylene) glycol. carnitine palmitoyltransferase coenzyme A acetyl-CoA carboxylase the 265-kDa isoform of ACC the 280-kDa isoform of ACC 5'AMP-activated protein kinase heart rate peak systolic pressure developed pressure the synthetic peptide substrate with the amino acid sequence HMRSAMSGLHLVKRR used to assay AMPK poly(ethylene) glycol. Malonyl-CoA is produced from acetyl-CoA carboxylase, which catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA(24Hardie D.G. Prog. Lipid Res. 1989; 28: 117-146Crossref PubMed Scopus (147) Google Scholar, 25Kim K.-H. Lopes-Casillas F. Bai D.H. Luo X. Pape M.E. FASEB J. 1989; 3: 2250-2256Crossref PubMed Scopus (172) Google Scholar, 26Wakil S.J. Stoops J.K. Joshi V.C. Annu. Rev. Biochem. 1983; 52: 537-579Crossref PubMed Google Scholar). Both a 280-kDa isoform (ACC 280) and a 265-kDa isoform (ACC 265) are present in the heart, with ACC 280 predominating(21Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar, 27Thampy K.G. J. Biol. Chem. 1989; 264: 17631-17634Abstract Full Text PDF PubMed Google Scholar, 28Bianchi A. Evans J.L. Iverson A.J. Nordlund A.-C. Watts T.D. Witters L.A. J. Biol. Chem. 1990; 265: 1502-1509Abstract Full Text PDF PubMed Google Scholar). Regulation of ACC in tissues such as liver and adipose tissue have been well characterized (see (24Hardie D.G. Prog. Lipid Res. 1989; 28: 117-146Crossref PubMed Scopus (147) Google Scholar, 25Kim K.-H. Lopes-Casillas F. Bai D.H. Luo X. Pape M.E. FASEB J. 1989; 3: 2250-2256Crossref PubMed Scopus (172) Google Scholar, 26Wakil S.J. Stoops J.K. Joshi V.C. Annu. Rev. Biochem. 1983; 52: 537-579Crossref PubMed Google Scholar) 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)(29Hardie D.G. Biochim. Biophys. Acta. 1992; 1123: 231-238Crossref PubMed Scopus (156) Google Scholar, 30Carling D. Clarke P.R. Zammit V.A. Hardie D.G. J. Biochem. 1989; 29: 129-136Google Scholar), which phosphorylates Ser-79 of ACC 265(31Ha J. Daniel S. Broyles S.S. Kim K.-H. J. Biol. Chem. 1994; 269: 22162-22168Abstract Full Text PDF PubMed Google Scholar). Liver AMPK is stimulated both by 5'-AMP and by phosphorylation by an AMPK kinase(29Hardie D.G. Biochim. Biophys. Acta. 1992; 1123: 231-238Crossref PubMed Scopus (156) Google Scholar, 30Carling D. Clarke P.R. Zammit V.A. Hardie D.G. J. Biochem. 1989; 29: 129-136Google Scholar, 32Weekes J. Hawley S.A. Corton J. Shugar D. Hardie D.G. Eur. J. Biochem. 1994; 219: 751-757Crossref PubMed Scopus (67) Google Scholar), and is thought to be activated under conditions of metabolic stress (i.e. ATP depletion and AMP accumulation)(29Hardie D.G. Biochim. Biophys. Acta. 1992; 1123: 231-238Crossref PubMed Scopus (156) Google Scholar, 33Sato R. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9261-9265Crossref PubMed Scopus (139) Google Scholar). 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(34Aquan K. Scott J. See C.G. Sarkar N.H. Gene (Amst.). 1994; 149: 345-350Crossref PubMed Scopus (28) Google Scholar). 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. 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% O2 and 5% CO2, 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(11Lopaschuk G.D. Spafford M.A. Davies N.J. Wall S.R. Circ. Res. 1990; 66: 546-553Crossref PubMed Scopus (216) Google Scholar). Perfusate consisted of 100 ml of re-circulated Krebs-Henseleit buffer (pH 7.4, gassed with 95% O2 and 5% CO2, 37°C) containing 1.2 mM [1-14C]palmitate, 3% bovine serum albumin, 11 mM glucose, 2.5 mM free Ca2+, 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 (ΔP), cardiac output, aortic flow, coronary flow, and O2 consumption were measured as described previously(13Lopaschuk G.D. Wall S.R. Olley P.M. Davies N.J. Circ. Res. 1988; 63: 1036-1043Crossref PubMed Scopus (219) Google Scholar, 17Lopaschuk G.D. Wambolt R.B. Barr R.L. J. Pharmacol. Exp. Therap. 1993; 264: 135-144PubMed Google Scholar, 21Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar). Steady state rates of palmitate oxidation were measured in aerobic or reperfused ischemic hearts by quantitatively collecting 14CO2 produced from hearts perfused with 1.2 mM [1-14C]palmitate (approximately 50,000 dpm/ml buffer). Collection of 14CO2 released as a gas in the oxygenation chamber and the 14CO2 trapped in the NaHCO3 in the perfusate was performed as described previously(11Lopaschuk G.D. Spafford M.A. Davies N.J. Wall S.R. Circ. Res. 1990; 66: 546-553Crossref PubMed Scopus (216) Google Scholar, 13Lopaschuk G.D. Wall S.R. Olley P.M. Davies N.J. Circ. Res. 1988; 63: 1036-1043Crossref PubMed Scopus (219) Google Scholar). Sampling was performed at 10-min intervals throughout the 60-min aerobic perfusion, or at 10-min intervals during reperfusion of previously ischemic hearts(11Lopaschuk G.D. Spafford M.A. Davies N.J. Wall S.R. Circ. Res. 1990; 66: 546-553Crossref PubMed Scopus (216) Google Scholar). 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(21Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar). The CoA esters were separated and quantified using a previously described high performance liquid chromatography procedure(35King M.T. Reiss P.D. Cornell N.W. Methods Enzymol. 1988; 166: 70-79Crossref PubMed Scopus (60) Google Scholar). 5'-AMP levels from the perchloric acid extract were determined using previously described methodology(17Lopaschuk G.D. Wambolt R.B. Barr R.L. J. Pharmacol. Exp. Therap. 1993; 264: 135-144PubMed Google Scholar). 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 εg/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 εg/ml soybean trypsin inhibitor, and 10% glycerol. Protein content was measured using the BCA method(36Smith P.K. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18512) Google Scholar). Acetyl-CoA carboxylase activity in the 6% PEG 8000 fraction was determined using the [14C]bicarbonate fixation assay (37Witters L.A. Kemp B. J. Biol. Chem. 1992; 267: 2864-2867Abstract Full Text PDF PubMed Google Scholar). The assay mixture contained 60.6 mM Tris acetate (pH 7.5), 1 mg/ml bovine serum albumin, 1.3 εM 2-mercaptoethanol, 2.1 mM ATP, 1.1 mM acetyl-CoA, 5 mM magnesium acetate, 18.2 mM NaH14CO3 (approximately 1000 dpm/nmol), and 25 εg 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 εl 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 32P into a synthetic peptide (termed SAMS peptide) with the amino acid sequence HMRSAMSGLHLVKRR(29Hardie D.G. Biochim. Biophys. Acta. 1992; 1123: 231-238Crossref PubMed Scopus (156) Google Scholar, 37Witters L.A. Kemp B. J. Biol. Chem. 1992; 267: 2864-2867Abstract Full Text PDF PubMed Google Scholar). The assay was performed in a 25-εl total volume containing 40 mM HEPES-NaOH (pH 7.0), 80 mM NaCl, 8% glycerol, 0.8 mM EDTA, 200 εM SAMS peptide, 0.8 mM dithiothreitol, 5 mM MgCl2, 200 εM [γ-32P]ATP (400-600 dpm/pmol), and 6-8 εg of the 6% PEG 8000 pellet. The assay was performed in the absence or presence of 200 εM 5'-AMP at 30°C for 5 min. The reaction was initiated by the addition of [32P]ATP/Mg. At the end of the incubation, 15-εl 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 H3PO4. These papers were washed 4 times for 30 min with 150 mM H3PO4, 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. A series of hearts were perfused under conditions identical to those described above except that radiolabeled [14C]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(21Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar). CPT 1 activity was determined by the method of Bremer(38Bremer J. Biochim. Biophys. Acta. 1981; 665: 628-631Crossref PubMed Scopus (179) Google Scholar). The mitochondrial preparations (35-40 εg) were preincubated with various concentrations of malonyl-CoA (0-1 εM) 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 εCi of L-[methyl-3H]carnitine was added to a final L-carnitine concentration of 200 εM, and the incubation was continued for a further 6 min. The reaction was stopped by adding 100 εl of 10 N HCl. The [3H]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(39Svoronos S. Kumar S. Comp. Biochem. Physiol. 1988; 90: 179-185Google Scholar), coupled to the method of Constantin-Teodosiu et al.(40Constantin-Teodosiu D. Cederblad G. Hultman E. Anal. Biochem. 1991; 198: 347-351Crossref PubMed Scopus (136) Google Scholar). The mitochondrial preparations (25-50 εg) were incubated in 210 εl 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 εl of 0.5 M perchloric acid. The solution was then neutralized with 2.2 M KHCO3 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 [14C]citrate in the presence of [14C]oxaloacetate and citrate synthase. [14C]Oxaloacetate was initially formed by a transamination reaction utilizing aspartate aminotransferase and [14C]aspartate, as described by Constantin-Teodosiu et al.(40Constantin-Teodosiu D. Cederblad G. Hultman E. Anal. Biochem. 1991; 198: 347-351Crossref PubMed Scopus (136) Google Scholar). Following the reaction, sodium glutamate and aspartate aminotransferase were used to remove excess [14C]oxaloacetate after the citrate synthase reaction by transaminating unreacted [14C]oxaloacetate back to [14C]aspartate. Dowex (50W-8X, 100-200 mesh) was then used to separate [14C]aspartate from [14C]citrate. Acetyl-CoA content of supernatant samples was quantified by comparison to acetyl-CoA standard curves. 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. 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, ΔP, 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/O2 consumed) was seen during reperfusion.Tabled 1 Open table in a new tab Palmitate oxidation rates in aerobic and reperfused ischemic hearts are shown in Table 2. As previously observed(4Saddik M. Lopaschuk G.D. J. Biol. Chem. 1991; 266: 8162-8170Abstract Full Text PDF PubMed Google Scholar, 11Lopaschuk G.D. Spafford M.A. Davies N.J. Wall S.R. Circ. Res. 1990; 66: 546-553Crossref PubMed Scopus (216) Google Scholar), 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(18Neely J.R. Whitmer K.M. Mochizuki S. Circ. Res. 1976; 38: I-22-I-30Google Scholar). 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 O2 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.Tabled 1 Open table in a new tab 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 IC50 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. 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.Tabled 1 Open table in a new tab In a previous study we observed that acetyl-CoA levels were positively correlated with malonyl-CoA levels in the aerobic heart(21Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar). 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. 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. 3 shows ACC

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