Activation of AMPK - and -isoform complexes in the intact ischemic rat heart

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1 Am J Physiol Heart Circ Physiol 291: H1927 H1934, First published April 28, 2006; doi: /ajpheart Activation of AMPK - and -isoform complexes in the intact ischemic rat heart Ji Li, 1 David L. Coven, 1 Edward J. Miller, 1 Xiaoyue Hu, 1 Martin E. Young, 2 David Carling, 3 Albert J. Sinusas, 1 and Lawrence H. Young 1 1 Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut; 2 Children s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas; and 3 Cellular Stress Group, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom Submitted 10 March 2006; accepted in final form 26 April 2006 Li, Ji, David L. Coven, Edward J. Miller, Xiaoyue Hu, Martin E. Young, David Carling, Albert J. Sinusas, and Lawrence H. Young. Activation of AMPK - and -isoform complexes in the intact ischemic rat heart. Am J Physiol Heart Circ Physiol 291: H1927 H1934, First published April 28, 2006; doi: /ajpheart AMP-activated protein kinase (AMPK) plays a key role in modulating cellular metabolic processes. AMPK, a serine-threonine kinase, is a heterotrimeric complex of catalytic -subunits and regulatory - and -subunits with multiple isoforms. Mutations in the cardiac 2-isoform have been associated with hypertrophic cardiomyopathy and pre-excitation syndromes. However, physiological regulation of AMPK complexes containing different subunit isoforms is not well defined and is important for an understanding of the function of this signaling pathway in the intact heart. We evaluated the kinase activity associated with heart AMPK complexes containing specific - and -subunit isoforms of AMPK in an in vivo rat model of regional ischemia. Left coronary artery occlusion activated the immunoprecipitated 1-isoform (6-fold, P 0.01) and 2-isoform (9-fold, P 0.01) in the ischemic left ventricle compared with sham controls. The degree of -subunit activation depended on the extent of ischemia and paralleled echocardiographic contractile dysfunction. The regulatory 1- and 2-isoforms were expressed in the heart. The 1- and 2-isoforms coimmunoprecipitated with 1- and 2-isoforms in proportion to -subunit content. 1-Isoform immunocomplexes accounted for 70% of AMPK activity and AMPK phosphorylation (Thr 172 ) in hearts. Ischemia similarly increased AMPK activity associated with the 1- and 2-isoform complexes threefold (P 0.01 for each). Thus AMPK catalytic 1- and 2-isoforms are activated by regional ischemia in vivo in the heart, irrespective of the regulatory 1-or 2-isoforms to which they are complexed. Despite the pathophysiological importance of 2-isoform mutations, 1-isoform complexes account for most of the AMPK activity in the ischemic heart. adenosine monophosphate-activated protein kinase; ischemia; subunits; isoforms ADENOSINE MONOPHOSPHATE-ACTIVATED protein kinase (AMPK), a serine-threonine kinase, has an important role in the regulation of cellular metabolism, ion channels, and gene expression (16, 17, 21, 22, 43). AMPK is activated by increases in the AMP-to-ATP ratio (AMP/ATP) and, as such, is a key signaling pathway during cellular metabolic or energetic stress in the heart (45). AMPK is a heterotrimeric complex consisting of a catalytic -subunit and regulatory - and -subunits (5). AMP binds to the regulatory -subunit of AMPK (36), leading to allosteric activation while also rendering the catalytic -subunit more amenable to phosphorylation and activation by upstream AMPK kinases (3, 18). AMPK has emerged as an important regulator of fatty acid oxidation and glucose uptake in the heart (23, 34), as well as in skeletal muscle (30, 42). AMPK inactivates acetyl-coa carboxylase, decreasing malonyl-coa levels and, thus, enhancing carnitine palmitoyltransferase I activity and fatty acid oxidation (23). AMPK also increases cellular glucose uptake by causing translocation of GLUT4 transporters to the sarcolemma (34) and activates phosphofructokinase-2, which accelerates glycolysis (26). In most tissues, two isoforms of the catalytic subunit, 1 and 2, are present and may be differentially activated in response to stress. During exercise, there is greater activation of the catalytic 2 -isoform in skeletal muscle (14) and the heart (8), which may reflect a greater sensitivity of the catalytic 2 - isoform to changes in AMP concentration (36). On the other hand, in isolated skeletal muscles in vitro, parallel activation of 1 - and 2 -isoforms occurs during hypoxia and contraction (19). In the isolated perfused mouse heart, there is twofold activation of the 1 -isoform and a threefold activation of the 2 -isoform during ischemia (35). However, the extent to which the cardiac -subunit isoforms are activated in vivo is unclear and might be important because of potentially different physiological effects of the -subunit isoforms. For instance, the 2 -isoform appears to be more important in the regulation of glucose transport in skeletal muscle (20) and the heart (44). Whether the two isoforms contribute to the cardioprotective action of AMPK against injury and apoptosis during ischemiareperfusion is uncertain (35, 38). Mutations in the PRKAG2 gene, coding for the 2 -isoform, are associated with familial hypertrophic cardiomyopathy, Wolff-Parkinson-White syndrome, and cardiac glycogen overload (1, 4, 15). A similar phenotype is found in mouse hearts containing the 2 -isoform mutations (1, 11, 39). The mechanism responsible for glycogen accumulation remains undefined, with conflicting data on the extent to which AMPK is active in these models. In hearts with the N488I mutation, AMPK basal activity is increased, but AMPK activation is impaired (46). In contrast, basal AMPK activity is decreased in the case of the R302Q mutation (39) and is normal in R531G Address for reprint requests and other correspondence: L. H. Young, Section of Cardiovascular Medicine, 3 FMP, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT ( lawrence.young@yale.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact /06 $8.00 Copyright 2006 the American Physiological Society H1927

2 H1928 hearts before the development of glycogen overload (11). Basic molecular studies indicate diminished activation of AMPK complexes containing -subunit mutations in response to AMP (10). However, interpretation of the in vivo results is complicated by the potential inhibitory effects of glycogen overload on AMPK activation in hearts with the established phenotype. Little is known about the physiological function and regulation of AMPK activity associated with specific -subunit isoforms in the intact heart and their response to ischemia. Data suggest that basal AMPK activity is associated primarily with 1 -isoform complexes (7), but it is uncertain how complexes containing the different -subunit isoforms respond to physiological stress, e.g., during ischemia. Therefore, the primary objectives of the present study were to 1) evaluate the kinase activity associated with specific - and -subunit isoforms of AMPK in the ischemic heart in vivo, 2) determine whether there is preferential association of specific - and -subunit isoforms in heart AMPK complexes, and 3) examine whether activation of AMPK parallels the extent of ischemic contractile dysfunction. The results indicate that ischemia activates catalytic 1 - and 2 -isoforms similarly in 1 - and 2 -isoformcontaining AMPK complexes and that 1 -isoform complexes account for most of the AMPK activity in the ischemic heart. MATERIALS AND METHODS Experimental animals. Male Sprague-Dawley rats ( g body wt) were allowed ad libitum access to standard chow and water before the experiments. All procedures were approved by the Yale University Animal Care and Use Committee. In vivo regional ischemia. The rats were anesthetized by an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg), intubated, and mechanically ventilated with 100% oxygen for the duration of the procedure. After 10 min of stabilization, a baseline transthoracic echocardiogram was obtained (see below). A left thoracotomy was performed, and a suture was placed around the left anterior descending coronary artery (LAD) at a proximal, intermediate, or distal location and ligated to produce regional ischemia (28). The chest wall incision was approximated, and an echocardiogram was obtained 10 min after ligation of the coronary artery. The heart was then rapidly excised, and the left ventricular (LV) free wall (not including the intraventricular septum) and right ventricle (RV) were immediately freeze clamped and stored in liquid nitrogen until analysis. Tissue samples and echocardiographic data were also obtained from control sham-operated animals, in which a suture was placed around the LAD, but the LAD was not ligated. Core body temperature was maintained throughout the procedure with a heating pad. Assessment of LV function. LV function was assessed by transthoracic echocardiography. Two-dimensional images of the LV were obtained in the short-axis view at the level of the papillary muscles with a 12-MHz probe and an Agilent Sonos 5500 instrument. The images were stored on optical disk and analyzed by two blinded independent observers for regional wall motion abnormalities. Each of six standard circumferential LV segments was assigned a wall motion score: 0 for normal, 1 for mildly hypokinetic, 2 for moderately hypokinetic, 3 for akinetic, and 4 for dyskinetic. The segment scores were summed for calculation of an overall score. Myocardial perfusion defect. For determination of the extent of the LV myocardium that was rendered ischemic during coronary artery occlusion, separate rats were similarly subjected to 10 min of proximal LAD occlusion. The hearts were immediately excised and perfused retrogradely via a cannula inserted into the aorta, initially with buffered saline to remove blood and then with monastral blue dye, which stained the regions of the myocardium with intact perfusion. Five 2-mm-thick cross sections were cut along the heart s long axis. The slices were stained with 2,3,5-triphenyltetrazolium chloride to exclude the presence of overt myocardial necrosis. Digital photos were analyzed to determine the percentage of the total LV that was not stained with blue dye as a measure of the ischemic area during coronary occlusion. Measurement of hemodynamics. In an additional group of rats, a transmyocardial Millar 1.5-F pressure transducer was introduced into the RV for 1 min to obtain baseline heart rate and systolic and diastolic pressure measurements. The pressure transducer was then inserted into the LV, and measurements were recorded at baseline and for 10 min after proximal LAD occlusion. The pressure transducer was reinserted into the RV for a final measurement. Therefore, each animal served as its own control for RV and LV hemodynamic parameters during baseline and ischemic periods. Real-time RT-PCR. Total RNA was isolated from rat hearts with use of TRIzol reagent (Invitrogen, Carlsbad, CA) and treated with RNase-free DNase I. For quantitation of the mrna levels of AMPK - and -subunit isoforms, real-time quantitative RT-PCR was performed using a SYBR Green quantitative RT-PCR kit (Qiagen, Valencia, CA) according to the manufacturer s instructions. For comparison of the amplification efficiency of - and -subunit isoform genes, standard curves were generated from serial fourfold dilutions of cdna derived from 100 ng of total RNA. The slope of the curves indicated equal amplification efficiency for each of the - and -subunit isoform genes. Final expression levels of AMPK subunit isoform mrna were normalized to GAPDH mrna content. The following primer pairs were used for the AMPK subunit isoforms: 5 -GCCGGAATTCATGTCTAAGTCTCTG-3 (forward) and 5 -TGCTGCCAAATTAATCACATCAAAC-3 (reverse) for 1, 5 -AGAATAGTGAGAGCCGAGGTCCATC-3 (forward) and 5 -CTCTTTCTATTGCAGCCAGTGTTCA-3 (reverse) for 2, 5 -GAGAACCTGTGCTATACGGGGAGTC-3 (forward) and 5 -GCACAGTCGGGCAAGAACAG-3 (reverse) for 3, 5 -TTAAACCCACAGAAATCCAAACAC-3 (forward) and 5 -CTTCGCACACGCAAATAATAGG-3 (reverse) for 1, and 5 -GTGGTGTTATCCTGTATGCCCTTCT-3 (forward) and 5 - CTGTTTAAACCATTCATGCTCTCGT-3 (reverse) for 2. AMPK assays. The AMPK activity associated with specific catalytic subunits was examined after immunoprecipitation with rabbit pan- subunit- or sheep 1- and 2-isoform-specific antibodies, as previously described (8, 25, 35). The AMPK activity associated with specific isoforms of the regulatory -subunits was assessed after immunoprecipitation with antibodies to the 1-isoform (PENEHSQETPESNS) or 2- isoform (LTPAGAKQKETETE) sequence, as previously reported (7). Immunoprecipitates were assayed for AMPK activity in homogenization buffer containing 0.8 mm DTT, 0.2 mm AMP, 5 mm MgCl 2, and 0.2 mm [ 32 P]ATP, with 0.2 mm AMPK substrate SAMS peptide, as previously described (25, 35). Cell culture. Adult cardiomyocytes were isolated from male Wistar rats and cultured on laminin-coated plates, as described previously (12). Cardiomyocytes were cultured in glutamine-free DMEM supplemented with 4% FCS, 1 mm pyruvate, 4 mm NaHCO 3, 8.6 mm HEPES (ph 7.3), 5 mm creatine, 2 mm L-carnitine, 5 mm taurine, 5 mm glucose, 10 M cytosine -D-arabinofuranoside, 100 IU/ml penicillin, and 100 g/ml streptomycin at 37 C. The cardiomyocytes were allowed 2 h to attach to the plates, and only the attached cells were utilized for further analysis. Human microvascular endothelial cells (EAhy 926, a kind gift of Dr. C. J. Edgell, University of North Carolina, Chapel Hill, NC) (13) were maintained in DMEM supplemented with 10% FCS, 100 IU/ml penicillin, 100 g/ml streptomycin, 2 mm glutamine, and 2% HAT (hypoxanthine, aminopterin, and thymidine) at 37 C. Immunoblotting. Proteins were combined with Laemmli sample buffer and subjected to SDS-PAGE. After transfer to polyvinylidene difluoride membranes, the proteins were immunoblotted, detected with enhanced chemiluminescence, and quantified by densitometry of

3 H1929 autoradiographs, as previously described (8, 35). Immunoblots were performed with sheep 1-, 2-, and 1-isoform (7) and 2-isoform (40) antibodies (kind gift of Dr. B. Kemp) at a dilution of 1:2,000. Rabbit polyclonal antibodies against phosphorylated (Thr 172 ) AMPK and AMPK pan- -subunit antibody were purchased from Cell Signaling (Beverly, MA). Rabbit polyclonal antibodies against 1-, 2-, 1-, and 2-isoforms were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Statistical analysis. Values are means SE. Data were analyzed by two-tailed, unpaired Student s t-test and Welch s corrected t-test when indicated (GraphPad Software, San Diego, CA). Differences were considered significant at P RESULTS Contractile dysfunction and AMPK activation during regional ischemia. During proximal LAD ligation, echocardiography revealed the development of LV anterolateral hypokinesis, indicative of ischemic contractile dysfunction (Fig. 1A). No regional wall motion abnormality was detected in sham-operated (control) animals (Fig. 1A). Perfusion of excised ischemic hearts with blue dye showed a clearly delineated large nonstaining anterolateral region extending from the base to the apex of the LV, affecting 40% of the total LV myocardium (Fig. 1B). Coronary artery occlusion resulted in an initial mild reduction in LV systolic pressure (P 0.05 vs. baseline), which remained 15% reduced at the end of 10 min of ischemia (Table 1). Heart rate remained stable during the ischemic period. LV diastolic pressure tended to increase Fig. 1. Left ventricular (LV) contractile dysfunction and perfusion abnormalities during regional ischemia. A: regional LV contractile function assessed by echocardiography after 10 min of proximal left anterior descending coronary artery (LAD) occlusion. Representative short-axis images at end systole and end diastole show anterolateral hypokinesis between arrows. B: excised ischemic hearts perfused with monastral blue dye to delineate extent of hypoperfusion. Cross-sectional slices from a representative heart demonstrate normal blue dye perfusion to intraventricular septum and right ventricle (RV) but lack of perfusion to anterolateral-anteroapical region of the ischemic LV free wall (red). Free wall stains red with 2,3,5-triphenyltetrazolium chloride stain, indicating absence of overt myocardial necrosis in the ischemic region. during ischemia, reflecting decreased LV contractility, but RV pressure was unchanged (Table 1). There was progressively less regional contractile dysfunction with occlusion of the intermediate or distal portion of the LAD than with moreproximal occlusion (Fig. 2A). Total AMPK activity was initially measured in pan- -subunit immunoprecipitates of the anterolateral LV free wall, which was rapidly excised and freeze clamped. The degree of AMPK activation (Fig. 2B) and phosphorylation at Thr 172 (Fig. 2C) paralleled the extent of ischemia in the LV. There was little effect of distal occlusion, which excluded nonspecific AMPK activation related to suture ligation. Table 1. Heart rates and ventricular pressures during regional ischemia Baseline 1 min 3 min 5 min 10 min Heart rate, beats/min LV pressure, mmhg Systolic * 73 7* 83 7* 85 6* Diastolic RV pressure, mmhg Systolic 43 5 NP NP NP 43 8 Diastolic 15 2 NP NP NP 13 1 Values are means SE (n 3 6). Hearts rates and ventricular pressures were measured during 10 min of in vivo regional ischemia. LV, left ventricle; RV, right ventricle; NP, not performed. *P 0.05 vs. baseline.

4 H1930 Fig. 2. AMP-activated protein kinase (AMPK) activation related to extent of LV ischemia. A: LV regional contractile dysfunction assessed by echocardiography during 10 min of proximal (n 7), intermediate (mid, n 4), or distal (n 3) LAD occlusion. Sham (control) animals (n 5) had no wall motion abnormalities. B: ischemic LV free wall homogenate total AMPK activity expressed as fold activation vs. control. C: representative immunoblots of LV free wall homogenate phosphorylated (Thr 172 ) AMPK (p-ampk) or pan- -subunit (AMPK ). Phosphorylated AMPK was quantified relative to total amount of AMPK. Values are means SE. *P 0.05 vs. sham control. P 0.01 vs. sham control. Activation of AMPK catalytic 1 - and 2 -isoforms in ischemic LV. To assess the specific activation of complexes containing the catalytic 1 - and 2 -isoforms during regional ischemia, AMPK activity and Thr 172 phosphorylation were measured in 1 - and 2 -isoform immunoprecipitates from the anterolateral LV after proximal LAD or sham occlusion (Fig. 3). There was an increase in kinase activity associated with the 1 -isoform (6-fold, P 0.01) and 2 -isoform (9-fold, P 0.01) in ischemic compared with control hearts (Fig. 3A). AMPK 2 -isoform activity was two- to threefold greater than Fig. 3. Activation and phosphorylation of AMPK 1- and 2-isoforms in ischemic LV. A: AMPK activity associated with immunoprecipitated catalytic 1- and 2-isoforms in LV free wall after 10 min of proximal LAD occlusion (n 11) or sham control (n 5). Values are means SE. *P 0.01 vs. control. P 0.01 vs. 1. B: representative immunoblots using phosphorylated (Thr 172 ) or pan- -subunit AMPK antibody demonstrate phosphorylation and amount of immunoprecipitated (IP) 1- and 2-isoforms. 1 -isoform activity in control (P 0.01) and ischemic (P 0.01) hearts. In addition, the amount of -subunits was two- to threefold greater in 2 - than in 1 -isoform immunoprecipitates, as assessed on immunoblots using a pan- -subunit antibody that recognizes a common epitope on both -subunit isoforms (Fig. 3B). Immunoblotting the supernatants of the 1 - and 2 -isoform immunoprecipitates demonstrated that each was efficient and selective for its own specific isoform (data not shown). Ischemia stimulated phosphorylation of the 1 - and 2 -isoforms, as shown by immunoblotting of heart immunoprecipitates with phosphorylated (Thr 172 ) AMPK antibody (Fig. 3B). The increase in phosphorylation paralleled the increase in AMPK activity in these isoform complexes. -Subunit isoform expression, binding to AMPK -subunits, and activation in ischemic LV. The catalytic -subunit isoforms are bound in heterotrimeric AMPK complexes to regulatory -subunits, which play an important role in modulating -subunit activation, AMP responsiveness, and physiological function (3, 9). The cardiac expression of mrna for the - and -subunit isoforms was initially examined using real-time RT-PCR with specific - and -subunit isoform primers. The results revealed a 7.7-fold greater amount of 2 - than 1 - isoform mrna in the heart (P 0.01; Fig. 4A). The expression level of 1 -isoform mrna was 1.5-fold higher than that of 2 -isoform mrna (P 0.01; Fig. 4B), whereas there was no evidence of 3 -isoform mrna in the heart. Although rat quadriceps skeletal muscle had similar -subunit isoform expression, mrna for all three -subunit isoforms, including 3, was present (Fig. 4, A and B). Although the predominant cell type in the intact heart is the cardiomyocyte, additional cell types, including a large number of endothelial cells, are also present. To examine whether the AMPK expression observed in the intact heart reflected myocyte isoform expression, isolated rat cardiomyocytes were prepared from nonischemic hearts (12). Immunoblots of cardiomyocyte lysates confirmed the PCR results and demonstrated that the 2 - and 1 -isoforms predominate in these cells (Fig. 4C). We also examined AMPK isoform expression in a human endothelial cell line (13). Immunoblots of endothelial

5 H1931 cells demonstrated selective 2 - and 1 -isoform expression (Fig. 4C). However, we were unable to detect the 1 -isoform in the endothelial cells by immunoblotting (Fig. 4C), although 1 -isoform mrna was demonstrated in endothelial cells by RT-PCR (data not shown). We next examined whether there might be preferential interaction between specific - and -subunit isoforms in the intact heart by immunoblotting 1 -or 2 -isoform immunoprecipitates with 1 - and 2 -isoform antibodies. The results showed that 1 - and 2 -isoforms were each bound to catalytic 1 - as well as 2 -isoforms in control and ischemic hearts (Fig. 4D). The bridging 1 - and 2 -isoforms were also present in the heart, and each appeared to bind to the catalytic 1 - and 2 -isoforms (Fig. 4D). A greater amount of 1 -, 2 -, 1 -, and Fig. 4. AMPK - and -subunit isoform expression and association in ischemic heart. mrna expression of - and -subunit isoforms (A and B) in isolated rat cardiomyocytes (CM), heart (H), or skeletal muscle (SM) quantified by real-time RT-PCR. Values were normalized to GAPDH expression and then compared with 2-isoform (A) or 1-isoform (B) mrna expression in cardiomyocytes. Products of predicted molecular weight were identified by agarose gel electrophoresis. Skeletal muscle RNA was used as a positive control for 3-isoform expression. Values are means SE (n 4). *P 0.01 vs. 2 (A) or 1 (B). C: expression of - and -subunit isoforms in rat cardiomyocytes and endothelial cells [EC, human macrovascular endothelial cells (EAhy 926)]. D: association of -, -, and -subunits in heart. Heart homogenates were immunoprecipitated with 1- or 2-isoform antibodies, subjected to SDS- PAGE, and immunoblotted with 1-, 2-, 1-, and 2-isoform antibodies to assess association of -, -, and -subunit isoforms. 2 -isoforms was associated with the 2 - than with the 1 - isoform, consistent with a greater amount of -subunit in 2 -isoform immunoprecipitates, as detected using the pan- subunit antibody (Fig. 4D). To assess the effects of ischemia on 1 - and 2 -isoformcontaining complexes, we performed immunoblots of 1 - and 2 -isoform immunoprecipitates with 1 -, 2 -, 1 -, and 2 - isoform antibodies. Catalytic 1 - and 2 -isoforms were each found in 1 -and 2 -isoform complexes in control and ischemic hearts (Fig. 5A). In addition, bridging 1 - and 2 -isoforms were each bound to 1 - and 2 -isoforms (Fig. 5A). The amount of - and -subunits associated with 1 -isoform complexes was greater than that associated with 2 -isoform complexes. In control hearts, the amount of phosphorylated AMPK (Fig. 5A) Fig. 5. AMPK -subunit isoform activation in ischemic heart. A: association of -subunits with - and -subunits in heart homogenates immunoprecipitated with 1-or 2-isoform antibodies, subjected to SDS-PAGE, and immunoblotted with 1-, 2-, 1-, and 2-isoform antibodies. B: AMPK activity associated with 1- and 2-isoform-containing complexes after immunoprecipitation with -subunit isoform-specific antibodies in LV free wall after 10 min of proximal LAD occlusion (n 11) or sham operation (control, n 5). Values are means SE. *P 0.01 vs. control. P 0.05 vs. 1 control; P 0.05 vs. 1 ischemia.

6 H1932 and AMPK enzymatic activity (Fig. 5B) was greater in 1 - than in 2 -isoform complexes, consistent with prior observations that the 1 -isoform accounts for most of the basal activity in the intact rat heart (7). Furthermore, during coronary occlusion, 1 - and 2 -isoform-associated AMPK activity increased twoto threefold (Fig. 5B), demonstrating that the -subunit isoforms are similarly activated by ischemia in the intact heart. The 1 -isoform immunoprecipitates accounted for 70% of total AMPK activity in the ischemic heart (P 0.05 vs. 2 ). Similarly, a greater amount of Thr 172 -phosphorylated AMPK was found in the 1 -isoform immunoprecipitates (Fig. 5A), further indicating that 1 -isoform complexes account for most of the AMPK activity in the ischemic LV. DISCUSSION This study assessed the regulation of AMPK complexes containing different isoforms of the AMPK catalytic -subunits and regulatory -subunits in the intact ischemic rat heart. The results demonstrate that ischemia activates 1 - and 2 - isoforms and that the -subunits are phosphorylated when associated with 1 -or 2 -isoform-containing complexes. The 2 -isoform appears to predominate in the rat heart, on the basis of higher mrna expression in intact hearts and isolated cardiomyocytes. The 2 -isoform-containing complexes also constitute 75% of the AMPK activity during regional ischemia in vivo. The findings also indicate that the 1 -isoform predominates in the rat heart, on the basis of the level of mrna expression, the amount of -subunits associated with the 1 -isoform compared with the 2 -isoform, and the higher AMPK activity contained within 1 -isoform-containing complexes. There does not appear to be any preferential association between specific - and -subunit isoforms in the intact heart, on the basis of the findings of the immunoprecipitation experiments. The results also indicate that 1 - and 2 -isoformcontaining AMPK complexes are subject to physiological regulation during ischemia but that 70% of AMPK activity is associated with the 1 -isoform-containing complexes. Activation of the 1 - and 2 -isoforms occurred during regional myocardial ischemia. These in vivo findings are consistent with prior results in isolated perfused hearts subjected to global ischemia (6, 35, 44) and suggest that not only the 2 -isoform, but also the 1 -isoform, is activated in response to severe metabolic stress. Similar 1 -isoform activation has been observed in skeletal muscle during in vitro contraction, hypoxia, and metabolic inhibition (19, 33). In contrast, during exercise, the 1 -isoform is activated less than the 2 -isoform in the heart (8, 31) and skeletal muscle (14), perhaps reflecting the lower sensitivity of the 1 - than the 2 -isoform complexes to smaller increases in AMP concentration (36). Heart and skeletal muscle contain multiple cell types, including myocytes, endothelial cells, smooth muscle cells, and fibroblasts. Although myocytes predominate by mass, endothelial cells are abundant in number and exclusively express the 1 -isoform of AMPK (37). Endothelial cell AMPK activity is known to be increased during oxidative stress and hypoxia (24, 32). Although these experiments do not directly address whether activation of heart endothelial cell AMPK occurred during ischemia, it is likely that endothelial cells contributed to the 1 -isoform activity measured basally and during ischemia. However, isolated cardiomyocytes also demonstrated AMPK 1 -isoform mrna and protein expression, indicating that 1 -isoform activity in the heart does not solely reflect endothelial cell AMPK. Interestingly, transgenic mice expressing a kinase-dead form of the 2 -isoform in myocytes demonstrate partially reduced heart 1 -isoform content and 1 -isoformassociated activity during ischemia (35). 1 -Isoform expression in heart endothelial cells would not be affected by transgenic myocyte expression of the 2 -kinase-dead isoform. However, the 2 -kinase isoform might displace the native 1 -isoform from AMPK complexes in cardiomyocytes, leading to its degradation and accounting for the decreased 1 -isoform activity (35). Although 1 - and 2 -isoforms are activated during in vivo ischemia, whether they have distinct or overlapping physiological actions in the ischemic heart remains uncertain. In noncardiac cells, the two -subunit isoforms appear to have different subcellular locations, with the 2 -isoform partially localized to the nucleus and the 1 -isoform distributed in the cytoplasm (27, 36, 41). Stimulation of glucose transport also seems to be primarily due to the 2 -isoform in skeletal muscle (20). AMPK has additional important actions in the ischemic heart after reperfusion, regulating fatty acid oxidation through the phosphorylation and inactivation of acetyl-coa carboxylase (23). Finally, AMPK-deficient transgenic mice exhibit increased myocardial necrosis and apoptosis in the setting of low-flow ischemia-reperfusion (35, 44), indicating that AMPK has a cardioprotective effect in the heart. The isoform specificity and mechanisms responsible for these effects are also not well understood and are of interest. Heart AMPK activity was associated primarily with the 1 -isoform and the remainder with the 2 -isoform. There was no evidence for 3 -isoform expression or activity in the heart, consistent with previous findings (7) and contrasting with skeletal muscle, where the 3 -isoform has an important role (2, 7, 29). We observed that the AMPK activity associated with the 1 - and 2 -isoforms increased during regional ischemia, indicating that 1 - and 2 -isoform complexes are physiologically regulated in the heart. Furthermore, coimmunoprecipitation experiments indicated that there was no preferential binding of specific 1 - or 2 -isoforms to the catalytic 1 - or 2 -isoforms in the in vivo heart under ischemic conditions, consistent with prior studies in normal rat hearts (7). Further understanding of the regulation of 2 -isoforms in the heart is of interest in view of observations that mutations in the human PRKAG2 gene (encoding the 2 -isoform) lead to hypertrophic cardiomyopathy and the Wolff-Parkinson-White syndrome (1, 4, 15). These abnormalities appear to be attributable to cardiac glycogen overload, although the mechanism responsible for glycogen accumulation remains elusive. Different mutations have different effects on AMPK activity: baseline AMPK activity is increased in mouse hearts expressing the N488I mutation (46) but decreased in those expressing the R302Q mutation (39). Interestingly, AMPK activity is normal in R531G hearts before the development of glycogen overload, but the AMPK activity is reduced when the mice age (11). Activation of the N488I mutation appears to be diminished compared with normal hearts after ischemic stress (46). When expressed in isolated cells, similar mutations in the cystathionine -synthase domains of the 2 -isoform, which bind AMP, render the AMPK complex relatively insensitive to AMP (10). The present findings indicating that normal 2 -

7 isoform-containing complexes are physiologically activated support the possibility that impaired activation of AMPK containing 2 -isoform mutations might lead to compensatory changes in other pathways mediating glucose transport and/or glycogen deposition. Thus this study demonstrates that activation of 1 - and 2 -isoforms occurs in AMPK complexes containing regulatory 1 -or 2 -isoforms in an in vivo model of regional ischemia. Whether specific AMPK isoforms have distinct downstream targets, subcellular localization, or function during ischemiareperfusion remains to be determined. GRANTS This work was supported by National Heart, Lung, and Blood Institute Grants R01 HL and T32 HL and National Research Service Award HL REFERENCES 1. Arad M, Moskowitz IP, Patel VV, Ahmad F, Perez-Atayde AR, Sawyer DB, Walter M, Li GH, Burgon PG, Maguire CT, Stapleton D, Schmitt JP, Guo XX, Pizard A, Kupershmidt S, Roden DM, Berul CI, Seidman CE, and Seidman JG. 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