Glucose Metabolism and Energy Homeostasis in Mouse Hearts Overexpressing Dominant Negative α2 Subunit of AMP-activated Protein Kinase

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that plays a pivotal role in regulating cellular metabolism for sustaining energy homeostasis under stress conditions. Activation of AMPK has been observed in the heart during acute and chronic stresses, but its functional role has not been completely understood because of the lack of effective activators and inhibitors of this kinase in the heart. We generated transgenic mice (TG) with cardiac-specific overexpression of a dominant negative mutant of the AMPK α2 catalytic subunit to clarify the functional role of this kinase in myocardial ischemia. In isolated perfused hearts subjected to a 10-min ischemia, AMPK α2 activity in wild type (WT) increased substantially (by 4.5-fold), whereas AMPK α2 activity in TG was similar to the level of WT at base line. Basal AMPK α1 activity was unchanged in TG and increased normally during ischemia. Ischemia stimulated a 2.5-fold increase in 2-deoxyglucose uptake over base line in WT, whereas the inactivation of AMPK α2 in TG significantly blunted this response. Using 31P NMR spectroscopy, we found that ATP depletion was accelerated in TG hearts during no-flow ischemia, and these hearts developed left ventricular dysfunction manifested by an early and more rapid increase in left ventricular end-diastolic pressure. The exacerbated ATP depletion could not be attributed to impaired glycolytic ATP synthesis because TG hearts consumed slightly more glycogen during this period of no-flow ischemia. Thus, AMPK α2 is necessary for maintaining myocardial energy homeostasis during ischemia. It is likely that the functional role of AMPK in myocardial energy metabolism resides both in energy supply and utilization. AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that plays a pivotal role in regulating cellular metabolism for sustaining energy homeostasis under stress conditions. Activation of AMPK has been observed in the heart during acute and chronic stresses, but its functional role has not been completely understood because of the lack of effective activators and inhibitors of this kinase in the heart. We generated transgenic mice (TG) with cardiac-specific overexpression of a dominant negative mutant of the AMPK α2 catalytic subunit to clarify the functional role of this kinase in myocardial ischemia. In isolated perfused hearts subjected to a 10-min ischemia, AMPK α2 activity in wild type (WT) increased substantially (by 4.5-fold), whereas AMPK α2 activity in TG was similar to the level of WT at base line. Basal AMPK α1 activity was unchanged in TG and increased normally during ischemia. Ischemia stimulated a 2.5-fold increase in 2-deoxyglucose uptake over base line in WT, whereas the inactivation of AMPK α2 in TG significantly blunted this response. Using 31P NMR spectroscopy, we found that ATP depletion was accelerated in TG hearts during no-flow ischemia, and these hearts developed left ventricular dysfunction manifested by an early and more rapid increase in left ventricular end-diastolic pressure. The exacerbated ATP depletion could not be attributed to impaired glycolytic ATP synthesis because TG hearts consumed slightly more glycogen during this period of no-flow ischemia. Thus, AMPK α2 is necessary for maintaining myocardial energy homeostasis during ischemia. It is likely that the functional role of AMPK in myocardial energy metabolism resides both in energy supply and utilization. The AMP-activated protein kinase (AMPK) 1The abbreviations used are: AMPK, AMP-activated protein kinase; TG, transgenic; WT, wild type; PCr, phosphocreatine; HA, hemagglutinin; 2-DG, 2-deoxyglucose; HPLC, high pressure liquid chromatography; pHi, intracellular pH; ANOVA, analysis of variance. functions as a fuel gauge, such that when the cell is exposed to stresses associated with energy depletion, it switches off ATP-utilizing pathways and switches on ATP-generating pathways to restore energy homeostasis (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1281) Google Scholar, 2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar). This kinase is activated by decreases in ATP/AMP and in phosphocreatine (PCr)/creatine through Thr172 phosphorylation by one or more upstream kinases (AMPK kinase) and through allosteric modification by AMP (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1281) Google Scholar, 2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar). AMPK is a heterotrimeric protein consisting of a catalytic subunit (α) and two regulatory subunits (β and γ) (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1281) Google Scholar, 3Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.P. Witters L.A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Each subunit has two or more different isoforms; the α1 subunit is widely expressed, whereas the α2 subunit is expressed primarily in liver, heart, and skeletal muscle (2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar, 4Stapleton D. Woollatt E. Mitchelhill K.I. Nicholl J.K. Fernandez C.S. Michell B.J. Witters L.A. Power D.A. Sutherland G.R. Kemp B.E. FEBS Lett. 1997; 409: 452-456Crossref PubMed Scopus (108) Google Scholar, 5Stapleton D. Mitchelhill K.I. Gao G. Widmer J. Michell B.J. Teh T. House C.M. Fernandez C.S. Cox T. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; 271: 611-614Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). It has been suggested that AMPK regulates glucose and fatty acid metabolism in striated muscles (6Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar, 7Ruderman N.B. Saha A.K. Vavvas D. Witters L.A. Am. J. Physiol. 1999; 276: E1-E18Crossref PubMed Google Scholar). Studies from our groups and others showed increased AMPK activity during acute and chronic stresses, such as hypoxia and exercise in skeletal muscle and ischemia and pressure overload in the heart (8Hayashi T. Hirshman M.F. Fujii N. Habinowski S.A. Witters L.A. Goodyear L.J. Diabetes. 2000; 49: 527-531Crossref PubMed Scopus (382) Google Scholar, 9Fujii N. Hayashi T. Hirshman M.F. Smith J.T. Habinowski S.A. Kaijser L. Mu J. Ljungqvist O. Birnbaum M.J. Witters L.A. Thorell A. Goodyear L.J. Biochem. Biophys. Res. Commun. 2000; 273: 1150-1155Crossref PubMed Scopus (290) Google Scholar, 10Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar, 11Tian R. Musi N. D'Agostino J. Hirshman M.F. Goodyear L.J. Circulation. 2001; 104: 1664-1669Crossref PubMed Scopus (262) Google Scholar, 12Kudo N. Gillespie J.G. Kung L. Witters L.A. Schulz R. Clanachan A.S. Lopaschuk G.D. Biochim. Biophys. Acta. 1996; 1301: 67-75Crossref PubMed Scopus (218) Google Scholar). Activation of AMPK in the heart is associated with enhanced glucose uptake and glycolysis (10Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar, 11Tian R. Musi N. D'Agostino J. Hirshman M.F. Goodyear L.J. Circulation. 2001; 104: 1664-1669Crossref PubMed Scopus (262) Google Scholar, 13Russell 3rd, R.R. Bergeron R. Shulman G.I. Young L.H. Am. J. Physiol. 1999; 277: H643-H649PubMed Google Scholar). As glycolysis is a major source of ATP during ischemia, stimulation of glucose uptake and glycolysis by AMPK in the ischemic heart is consistent with the overall function of this enzyme in restoring cellular energy levels during stress. To establish a causal role of AMPK for these stress responses, however, inhibition of AMPK is required during stress. This has not been possible because of the lack of a specific inhibitor of AMPK in the heart. Furthermore, the inability to block AMPK activation during ischemia makes it difficult to test whether AMPK also functions to preserve energy by reducing ATP consumption by the heart during ischemia. In the present study, we sought to inhibit AMPK activity by generating transgenic mice (TG) overexpressing a dominant negative mutant of the AMPK α2 catalytic subunit in the heart. This approach led to a selective inhibition of AMPK α2 activity in the heart. Here we report that AMPK α2 mediates critical cellular responses in maintaining energy homeostasis in the ischemic heart. Generation of Transgenic Mice—A full-length cDNA of rat AMPK α2 subunit was tagged at the 5′ end with a HA epitope. The aspartic acid at residue 157 was changed to alanine to render the catalytic subunit inactive (14Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (499) Google Scholar, 15Woods A. Azzout-Marniche D. Foretz M. Stein S. Lemarchand P. Ferre P. Foufelle F. Carling D. Mol. Cell. Biol. 2000; 20: 6704-6711Crossref PubMed Scopus (360) Google Scholar). The TG mice with cardiac-specific overexpression of the mutant α2 subunit were generated by injecting the recombinant DNA construct, regulated by a mouse α-myosin heavy chain promoter, into fertilized Friend virus B-type mouse oocytes. Transgenic mouse founders were identified by the polymerase chain reaction-based method, and transgene expression was confirmed by Western blotting of the HA tag. Three transgenic lines were established. Mice from F1 and F2 generations were used for this study, and TG mice were compared with their wild type (WT) littermates. All the procedures were approved by the Institutional Animal Care and Use Committee of the Harvard Medical School. Isolated Perfused Heart Experiments—Hearts were perfused in the Langendorff mode with phosphate-free Krebs-Henseleit buffer containing (in millimoles/liter) NaCl (118), NaHCO3 (25Bergeron R. Russell 3rd, R.R. Young L.H. Ren J.M. Marcucci M. Lee A. Shulman G.I. Am. J. Physiol. 1999; 276: E938-E944Crossref PubMed Google Scholar), KCl (5.3), CaCl2 (2.5), MgSO4 (1.2), EDTA (0.5), glucose (10Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar), and pyruvate (0.5) as described previously (16Tian R. Abel E.D. Circulation. 2001; 103: 2961-2966Crossref PubMed Scopus (184) Google Scholar). All hearts were perfused with a constant perfusion pressure of 80 mm Hg, and the left ventricular function was continuously monitored using a water-filled balloon (16Tian R. Abel E.D. Circulation. 2001; 103: 2961-2966Crossref PubMed Scopus (184) Google Scholar). Fig. 1 illustrates the protocols for isolated perfused heart experiments. After stabilization, one base-line 31P NMR spectrum was collected (16Tian R. Abel E.D. Circulation. 2001; 103: 2961-2966Crossref PubMed Scopus (184) Google Scholar), and one-half of the hearts were subjected to a 10-min no-flow ischemia and the other half to a 10-min normal perfusion. During ischemia, four consecutive 2-min 31P NMR spectra were collected to monitor the dynamic changes in high energy phosphate content. At the end of the 10-min period, a subgroup of hearts was freeze-clamped for biochemical analysis, and the rest were reperfused with a buffer in which glucose was replaced with 5 mm 2-deoxyglucose (2-DG). Five consecutive 4-min 31P NMR spectra were collected for determination of the time-dependent accumulation of 2-DG-phosphate. The rate of glucose uptake was estimated by the slope of the fitted line as described previously (11Tian R. Musi N. D'Agostino J. Hirshman M.F. Goodyear L.J. Circulation. 2001; 104: 1664-1669Crossref PubMed Scopus (262) Google Scholar, 17Abel E.D. Kaulbach H.C. Tian R. Hopkins J.C. Duffy J. Doetschman T. Minnemann T. Boers M.E. Hadro E. Oberste-Berghaus C. Quist W. Lowell B.B. Ingwall J.S. Kahn B.B. J. Clin. Invest. 1999; 104: 1703-1714Crossref PubMed Scopus (272) Google Scholar). During 2-DG perfusion, 1.2 mm KH2PO4 and5mm pyruvate were supplied to replenish the intracellular inorganic phosphate pool and to maintain ATP synthesis. AMPK Activity Assay and Western Blotting—Freeze-clamped heart samples were homogenized as described previously, and lysates were used for AMPK activity assays and for Western blotting (18Musi N. Fujii N. Hirshman M.F. Ekberg I. Froberg S. Ljungqvist O. Thorell A. Goodyear L.J. Diabetes. 2001; 50: 921-927Crossref PubMed Scopus (308) Google Scholar). AMPK activity was measured after immunoprecipitating 200 μg of protein using antibodies made against the amino acid sequences 339–358 of rat AMPK α1, 352–366 of α2, and 2–16 of both α1 and α2 (pan-α) (18Musi N. Fujii N. Hirshman M.F. Ekberg I. Froberg S. Ljungqvist O. Thorell A. Goodyear L.J. Diabetes. 2001; 50: 921-927Crossref PubMed Scopus (308) Google Scholar). The kinase reaction was done using synthetic peptide with sequence HMR-SAMSGLHLVKRR as substrate, and AMPK activity is expressed as incorporated ATP (picomoles) per mg of protein per min (19Davies S.P. Carling D. Hardie D.G. Eur. J. Biochem. 1989; 186: 123-128Crossref PubMed Scopus (373) Google Scholar). Western blotting was done with antibodies against AMPK α1, α2, pan-α, HA (Roche Diagnostics), GLUT1, GLUT4 (Chemicon, Intl., Inc., Temecula, CA), SERCA2 (Affinity BioReagents, Golden, CO), and Na+/Ca2+ exchanger (Swant, Bellinzona, Switzerland). HPLC Measurements and Glycogen Assay—Freeze-clamped tissues were used for determination of myocardial content of adenine nucleotides, and by a HPLC as previously Ingwall J.S. J. Clin. Invest. PubMed Scopus Google Scholar). glycogen content was by the of glucose from glycogen by of an to glycogen and glucose Biochem. PubMed Scopus Google Scholar). content in the was measured using a and ATP content by HPLC was to an intracellular content of and a protein content of of J. Mol. Cell. 20: Full Text PDF PubMed Scopus Google Scholar). The of for WT or TG hearts was used to the ATP of the base-line 31P NMR of other were using the of their to the ATP and intracellular was by the of inorganic phosphate to Ingwall J.S. J. Clin. Invest. PubMed Scopus Google Scholar). The of and in the ischemic hearts were from spectra of Each the of four from a of in from WT and TG hearts were compared by test or during ischemia and 2-DG perfusion were compared by All the were with and a of was α2 in of AMPK mutant α2 subunit of AMPK was expressed in TG hearts as by the of protein The expression of mutant α2 subunit an of the AMPK α2 to the HA The of a AMPK α2 in the TG hearts that the AMPK α2 protein is replaced by the mutant protein subunit protein is and has a B.E. J. Kemp B.E. Witters L.A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), subunits that were by the mutant protein and to with and subunits likely to be In to the changes in AMPK α2, the of AMPK α1 was unchanged in the TG hearts AMPK α2 activity was in the TG hearts at base line by Ischemia increased AMPK α2 activity by in the WT whereas the activation of AMPK α2 in the TG hearts was blunted The AMPK α1 activity in TG hearts was not different from that of WT hearts at base and it increased normally in to ischemia that the dominant negative transgenic approach used in specific inhibition of AMPK α2 activity in the heart. the activation of AMPK α2 during ischemia led to a in AMPK activity in the heart that AMPK α2 is a major to AMPK activity in the heart. and of the TG the base-line of the mice used for this was in the and heart TG and WT The TG mice were and was observed to 1 not ventricular function at base line was similar for TG and WT that inhibition of AMPK α2 activity not function during normal perfusion. subjected to ischemia, however, the TG hearts showed a more rapid increase in left ventricular end-diastolic pressure This is because of a in the of in TG The protein levels for SERCA2 and Na+/Ca2+ exchanger were unchanged in TG hearts 1 and Na+/Ca2+ for WT and TG, in The rapid increase in left ventricular end-diastolic pressure is consistent with the that ATP depletion was accelerated during ischemia in TG hearts a of in these and base-line 1 in a changes in high energy phosphate content during ischemia were monitored by 31P NMR and in Fig. of and not different in WT and TG in both groups during the was in TG hearts after min of ischemia, whereas a but for more in WT The rate and of ATP depletion was also accelerated in TG by 80 and from the in TG and WT The (by during ischemia. The significantly in both but was in WT hearts in TG hearts at the end of ischemia and ATP and accumulation of adenine and during ischemia measured by content of adenine was similar in WT and TG hearts during base-line perfusion. At the end of a 10-min ischemia, ATP content in TG and WT hearts by 80 and from the base-line Thus, the by HPLC was consistent with the NMR ATP in the TG hearts during ischemia in a for content of and compared with WT of adenine nucleotides, and in the mouse in a base-line perfusion, TG hearts showed normal of 2-DG uptake and normal glycogen content The protein content of and two glucose in the heart, was also unchanged in TG hearts that AMPK α2 has a on glucose metabolism in hearts under content of of and ATP generated by in a Fig. and GLUT4 protein content in the heart. The of glucose in WT and TG hearts was by as described under per In WT ischemia a 2.5-fold increase in the rate of 2-DG In 2-DG uptake increased by in TG hearts after ischemia, a in the to ischemia Thus, the activation of AMPK α2 in the ischemic heart significantly blunted the increase in myocardial glucose During no-flow ischemia, glycogen is the for glycolysis that To glycogen consumption during ischemia, we the in myocardial glycogen content and after ischemia ATP from glycogen consumption was estimated that glucose from glycogen of glycogen content in TG hearts at the end of ischemia was WT and ATP generated during ischemia was slightly in TG that accelerated ATP depletion in TG hearts during ischemia is not because of impaired ATP major in this overexpressing the dominant negative α2 subunit of AMPK in mouse hearts in inhibition of AMPK α2 the activation of AMPK α2 glucose uptake in the heart. inhibition of AMPK α2 activity during ischemia to an accelerated depletion of ATP and exacerbated because of increased energy that AMPK α2 regulates energy metabolism in the ischemic hearts by both energy supply and of AMPK α2 is the of the AMPK In this study, we sought to inhibit AMPK activity by generating transgenic mice with cardiac-specific overexpression of a mutant of the α2 subunit (14Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (499) Google Scholar, 15Woods A. Azzout-Marniche D. Foretz M. Stein S. Lemarchand P. Ferre P. Foufelle F. Carling D. Mol. Cell. Biol. 2000; 20: 6704-6711Crossref PubMed Scopus (360) Google Scholar). overexpression of this mutant α2 subunit in and selective of the α2 subunit the α1 subunit AMPK α1 activity in TG hearts unchanged and normally during ischemia. We found that AMPK measured by with an against was by two the that AMPK α2 the of AMPK activity under these conditions. In a transgenic mouse described previously, in which a different of α2 subunit was in skeletal muscle J. J.T. O. M. Birnbaum M.J. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), the that both subunits were replaced by the This the two the that skeletal muscle from muscle in subunit of AMPK inhibition of AMPK α2 activity in the heart, as observed in our a to function of AMPK in the heart. AMPK α2 and has as a of increased glucose uptake in to energy activation of AMPK by in increased glucose uptake in both skeletal and muscle in an 3rd, R.R. Bergeron R. Shulman G.I. Young L.H. Am. J. Physiol. 1999; 277: H643-H649PubMed Google Scholar, R. Russell 3rd, R.R. Young L.H. Ren J.M. Marcucci M. Lee A. Shulman G.I. Am. J. Physiol. 1999; 276: E938-E944Crossref PubMed Google Scholar, T. Hirshman M.F. W.W. Goodyear L.J. Diabetes. 1998; PubMed Scopus Google Scholar). Furthermore, a increased AMPK activity and enhanced glucose uptake has been observed under a of stress (8Hayashi T. Hirshman M.F. Fujii N. Habinowski S.A. Witters L.A. Goodyear L.J. Diabetes. 2000; 49: 527-531Crossref PubMed Scopus (382) Google Scholar). ischemia also glucose uptake through an Chen W. E. C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, 3rd, R.R. R. M.J. Ren J. Shulman G.I. Young L.H. Circulation. 1998; PubMed Scopus Google Scholar, S. N. M. Res. 1999; PubMed Scopus Google Scholar), the role of AMPK in this is the of a causal AMPK activation and increased glucose uptake during ischemia has been by the lack of an effective AMPK inhibitor for the heart. Using the transgenic approach to AMPK we the that AMPK α2 plays a critical role in glucose also that AMPK α2 is not the for glucose The that AMPK TG hearts a in glucose uptake the that AMPK α1 is also in the of glucose uptake in the heart. study, however, not the that other in to AMPK also for glucose It has been suggested that AMPK to enhanced glycolysis during ischemia by the enzyme for the synthesis of a of glycolysis (10Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). In this study, we found a of glycogen in TG hearts during no-flow ischemia, that AMPK α2 activity is not required for glycolysis under our conditions. the AMPK activity increased in the ischemic TG hearts to the activation of AMPK α1, it is possible that this increase is to and In be and activated by other kinases such as protein kinase C. Rider M.H. Hue L. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar). Furthermore, glycolysis is also regulated by the of adenine and intracellular Mol. Biol. Google Scholar, L.H. The from to Circulation. Scholar). accelerated depletion of ATP and in TG hearts increased glycolytic the role of glycolysis for myocardial during ischemia, it is that for stimulation of of AMPK α2 in during found normal content of high energy phosphate and adenine in the TG hearts under base-line conditions. This with the normal glucose metabolism in TG the that the function of AMPK is to cellular to stress. hearts to ischemia, we observed a more rapid of ATP in TG This that AMPK α2 plays a critical role in sustaining energy homeostasis in the exacerbated ATP be by ATP synthesis the no-flow ischemia in this not of energy other our likely increased ATP consumption by TG hearts during ischemia. It has been that in the AMPK energy consumption by off synthetic in the cell (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1281) Google Scholar, 2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar, 15Woods A. Azzout-Marniche D. Foretz M. Stein S. Lemarchand P. Ferre P. Foufelle F. Carling D. Mol. Cell. Biol. 2000; 20: 6704-6711Crossref PubMed Scopus (360) Google Scholar). It has not been AMPK mediates energy in the heart. The of energy consumed by the heart the reaction Res. PubMed Scopus Google Scholar, Physiol. Rev. PubMed Scopus Google Scholar). During our ischemic the heart in 1 ATP depletion in TG hearts under this likely increased ATP consumption for for maintaining metabolism and homeostasis Res. PubMed Scopus Google Scholar, Physiol. Rev. PubMed Scopus Google Scholar). this we found a in a accumulation of intracellular in TG This is in to the that of ATP and glycogen is accelerated in TG which to increased during ischemia. Thus, it is likely that the TG heart is more in our that AMPK α2 plays a role in energy in the ischemic heart, by the In inactivation of AMPK α2 accelerated ATP depletion and early of myocardial and to glucose uptake in to ischemia. that AMPK plays a critical role in sustaining energy homeostasis and myocardial during ischemia, by cellular functions for both energy supply and utilization. We for