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Am J Physiol Heart Circ Physiol 291: H2875-H2883, 2006. First published July 28, 2006; doi:10.1152/ajpheart.01032.2005
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Role of the {alpha}2-isoform of AMP-activated protein kinase in the metabolic response of the heart to no-flow ischemia

Elham Zarrinpashneh,1 Karla Carjaval,2 Christophe Beauloye,1 Audrey Ginion,1 Philippe Mateo,2 Anne-Catherine Pouleur,1 Sandrine Horman,3 Sophie Vaulont,4 Jacqueline Hoerter,2 Benoit Viollet,4 Louis Hue,3 Jean-Louis Vanoverschelde,1 and Luc Bertrand1

1Division of Cardiology, School of Medicine, 3Hormone and Metabolic Research Unit, Université catholique de Louvain and Christian de Duve Institute of Cellular Pathology, Brussels, Belgium; and 2Institut National de la Santé et de la Recherche Médicale, Unité 769, Université Paris-Sud, and IFR-141, Châtenay-Malabry; and 4Unité 567 Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 8104 Centre National de la Recherche Scientifique, René Descartes University, Institut Cochin, Department of Endocrinology, Metabolism. and Cancer, Paris, France

Submitted 29 September 2005 ; accepted in final form 13 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
AMP-activated protein kinase (AMPK) is a major sensor and regulator of the energetic state of the cell. Little is known about the specific role of AMPK{alpha}2, the major AMPK isoform in the heart, in response to global ischemia. We used AMPK{alpha}2-knockout (AMPK{alpha}2–/–) mice to evaluate the consequences of AMPK{alpha}2 deletion during normoxia and ischemia, with glucose as the sole substrate. Hemodynamic measurements from echocardiography of hearts from AMPK{alpha}2–/– mice during normoxia showed no significant modification compared with wild-type animals. In contrast, the response of hearts from AMPK{alpha}2–/– mice to no-flow ischemia was characterized by a more rapid onset of ischemia-induced contracture. This ischemic contracture was associated with a decrease in ATP content, lactate production, glycogen content, and AMPKbeta2 content. Hearts from AMPK{alpha}2–/– mice were also characterized by a decreased phosphorylation state of acetyl-CoA carboxylase during normoxia and ischemia. Despite an apparent worse metabolic adaptation during ischemia, the absence of AMPK{alpha}2 does not exacerbate impairment of the recovery of postischemic contractile function. In conclusion, AMPK{alpha}2 is required for the metabolic response of the heart to no-flow ischemia. The remaining AMPK{alpha}1 cannot compensate for the absence of AMPK{alpha}2.

glycogen; glycolysis; acetyl-CoA carboxylase

ADENOSINE 5'-MONOPHOSPHATE-ACTIVATED protein kinase (AMPK), a ubiquitous serine/threonine protein kinase, senses the energy state of the cell. AMPK is a heterotrimeric enzyme consisting of a catalytic ({alpha}) and two regulatory (beta and {gamma}) subunits. Different isoforms for each of these subunits have been identified (43). In the heart, the catalytic {alpha}2-subunit is predominant and represents 60–70% of total AMPK activity; the remaining activity is due to the {alpha}1-subunit (9). Control of AMPK activity is complex and involves allosteric stimulation by AMP as well as AMP-dependent phosphorylation at Thr172, a residue located in the activation loop of the {alpha}-subunit (15). Several protein kinases responsible for this phosphorylation have been identified: LKB1 (33, 39, 50) and the Ca2+/calmoldulin-dependent protein kinase kinase (16, 24, 49). The rapid rise in AMP concentration during an ischemic episode explains the activation of cardiac AMPK under this pathological condition (21, 22). Moreover, AMPK can also be activated by antidiabetic drugs (12, 55), osmotic stress (11), leptin (35), and adiponectin (53), probably via AMP-independent pathways. In hearts subjected to ischemia-reperfusion, AMPK can be considered a metabolic master switch (21, 42, 54). Indeed, once activated, AMPK phosphorylates several downstream targets, switching on ATP-generating pathways and switching off ATP-consuming biosynthetic pathways, thereby moderating the negative effects of ischemia-reperfusion on the heart's energy balance (14). During ischemia, AMPK promotes glycolysis by a dual mechanism: 1) it increases glucose uptake by stimulating translocation of GLUT4 transporters to the sarcolemmal membrane (37) and 2) it indirectly stimulates 6-phosphofructo-1-kinase (PFK-1) activity by phosphorylating and activating the heart isoform of 6-phosphofructo-2-kinase, the enzyme that synthesizes fructose 2,6-bisphosphate, a potent PFK-1 stimulator (34). During early reperfusion, AMPK is involved in the dominance of fatty acid oxidation over glucose oxidation. AMPK phosphorylates and inactivates acetyl-CoA carboxylase (ACC), thereby decreasing the concentration of malonyl-CoA, the inhibitor of fatty acid transport into mitochondria, and increasing fatty acid oxidation (31). Recent studies also implicate AMPK in the inhibition of protein synthesis during anaerobic conditions. Indeed, AMPK inactivates eukaryotic elongation factor 2 (eEF2) by phosphorylating and activating eEF2 kinase, the upstream kinase responsible for phosphorylation and inactivation of eEF2 (6, 17, 18). Moreover, AMPK plays a role in regulation of the mammalian target of rapamycin (mTOR), a protein kinase controlled by hormonal and nutritional status and involved in cell growth (2). It has been recently shown that AMPK can inactivate mTOR by direct phosphorylation of the enzyme (5) and phosphorylation of the tuberous sclerosis complex 2, an upstream regulator of mTOR (25). In previous studies, the metabolic role of AMPK has most often been evaluated by use of mitochondrial poisons, such as oligomycin, or pharmacological AMPK activators, such as 5-aminoimidazole 4-carboxamide 1beta,D-ribofuranoside or antidiabetic drugs. The results of these studies should be interpreted with caution, because these agents are not totally specific for AMPK. Recently, transgenic mice overexpressing a dominant-negative form (K45R mutation) of AMPK{alpha}2 (DN-K45R) in the heart have been studied during low-flow ischemia (38). These mice are characterized by a loss of AMPK{alpha}1 and AMPK{alpha}2 stimulation, glucose uptake, and fatty acid oxidation during ischemia-reperfusion. Accordingly, hearts of these mice exhibit impaired recovery of left ventricular (LV) function during reperfusion. In the present study, we used mice in which only the catalytic AMPK{alpha}2 gene was inactivated (AMPK{alpha}2–/– mice) (48). These mice allowed us 1) to study the specific role of AMPK{alpha}2 in the control of heart metabolism during no-flow ischemia and 2) to compare the data obtained with these AMPK{alpha}2–/– mice with those reported in other transgenic models.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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This study was approved by the Animal Research Committee at Université catholique de Louvain and conformed to the American Heart Association Guidelines for Use of Animals in Research.

Echocardiographic analysis. Two-dimensional echocardiography was performed with a Sonos 7500 system equipped with a 15-MHz linear-array transducer (Phillips Medical System, Eindhoven, The Netherlands). LV function was assessed in tribromoethanol-anesthetized (Avertin, Fluka; 0.3 mg/g body wt ip) mice from short-axis images of the LV obtained at the level of the papillary muscles. LV internal areas, as well as the anterior wall thickness, were measured at end diastole (ED) and end systole (ES). Fractional area changes (FAC) and the systolic thickening of the anterior wall (AWT) were computed using the following equations

Formula

Formula

Perfusion protocols. For metabolic measurements, hearts from 3-mo-old mice anesthetized with ketamine (Ketalar, Pfizer; 0.1 mg/g body wt ip)-xylazine (Rompun, Bayer; 0.01 mg/g body wt ip) were retrogradely perfused (32) at 37°C at a constant pressure of 75 mmHg with a Krebs-Henseleit buffer (in equilibrium with a 95% O2-5% CO2 gas phase) containing 1.5 mM CaCl2, 11 mM glucose, and 1.1 mM mannitol. After 15 min of equilibration, the hearts were subjected to 10 min of normoxia or no-flow ischemia. During ischemia, the hearts were maintained at 37°C. At the end of the procedure, the hearts were freeze clamped and stored at –80°C for further analysis.

For LV functional measurements, the hearts were retrogradely perfused as previously described (13). Briefly, the hearts were perfused at constant pressure (75 mmHg) with Krebs-Henseleit buffer (95% O2-5% CO2, 37°C) containing 1.8 mM calcium, 11 mM glucose, and 1.1 mM mannitol. After initiation of the retrograde perfusion, a compliant latex balloon was inserted into the LV chamber. LV ED pressure was set at 5–8 mmHg by adjustment of the volume of the LV balloon. Heart rate, LV pressure, and coronary flow were constantly monitored throughout the experiments. After 15 min of equilibrium, the hearts were subjected to 30 min of no-flow ischemia and 45 min of reperfusion.

Enzyme assays and phosphorylation state measurements. The frozen hearts were homogenized (Ultra-Turrax) at 0°C in 10 vol (vol/wt) of homogenization buffer as previously described (34), and the supernatants (10,000 g, 30 min) were stored at –80°C. Total AMPK activity was assayed in the presence of 0.2 mM AMP in a 10% (wt/vol) polyethylene glycol 6,000 fraction (34). AMPK{alpha}1- and AMPK{alpha}2-specific activities were measured after immunoprecipitation (34, 51) with anti-AMPK{alpha}1 and anti-AMPK{alpha}2 antibodies. One unit of AMPK activity corresponds to 1 nmol of product formed per minute under the assay conditions. Phosphorylation states of AMPK and ACC were monitored by immunoblots with anti-phosphorylated Thr172 AMPK and anti-phosphorylated Ser227 ACC2 antibodies, respectively. Protein levels of AMPK subunits were monitored by immunoblots with anti-AMPK{alpha}1, anti-AMPK{alpha}2, anti-AMPKbeta2, and anti-pan AMPKbeta antibodies. Band intensities of immunoblot films were quantitated by scanning and processing with the ImageJ (1.33 for Mac OS X) program.

Metabolites and protein measurements. AMP, ADP, and ATP contents were measured in neutralized perchloric acid extracts of the frozen hearts after their separation by high-performance liquid chromatography (47). Lactate was measured enzymatically in extracts described previously (3). For evaluation of glycogen content, frozen hearts were homogenized in 10 vol (vol/wt) of 1 M KOH. The homogenates were heated (80°C, 15 min), chilled on ice, neutralized (0.25 vol of 3.3 M acetic acid, pH 6, 10 min), and then centrifuged (5,000 g, 4 min). Glycogen content was measured in the supernatants as previously described (23). Protein was estimated by the method of Bradford, with bovine serum albumin as a standard.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Cardiac phenotype of AMPK{alpha}2–/– mice. Generation of AMPK{alpha}2–/– mice and their phenotype have been previously described (48). Size and weight of hearts from AMPK{alpha}2–/– and wild-type (WT) mice were similar (data not shown). Moreover, histological studies revealed no morphological abnormalities in any of the hearts (data not shown). Table 1 shows results of the echocardiographic examinations. In contrast to the results obtained in dominant-negative AMPK transgenic mice (38), LV functional parameters were not significantly different between hearts from AMPK{alpha}2–/– and WT mice. In particular, no signs of LV hypertrophy, LV dilation, or heart failure were noted.


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  		Table 1. Echocardiographic analysis

 
Expression of {alpha}- and beta-subunits of AMPK in AMPK{alpha}2–/– mice. As expected, immunoblot analysis of myocardial protein extracts of hearts from AMPK{alpha}2–/– mice revealed no significant amount of the native {alpha}2-isoform of AMPK, whereas the level of AMPK{alpha}1 was comparable to that in WT mice (Fig. 1). In contrast to skeletal muscle and liver, a band corresponding to a truncated and inactive AMPK{alpha}2 (resulting from deletion of amino acids 189–260 matching the AMPK catalytic domain) could clearly be identified on the immunoblots (Fig. 1).


Figure 1
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  		Fig. 1. Western blot of AMP-activated protein kinase (AMPK){alpha}1, AMPK{alpha}2, AMPKbeta2, and pan AMPKbeta proteins in perfused hearts from wild-type (WT, +/+), AMPK{alpha}2-knockout (AMPK{alpha}2–/–, –/–), and AMPK{alpha}2+/– (+/–) mice during normoxia (N) and ischemia (I). Total eukaryotic elongation factor 2 (eEF2) was used as loading control (data not shown).

 
AMPKbeta, which binds to AMPK{alpha} and AMPK{gamma} and, therefore, is essential for formation of the heterotrimer (7, 14), has been recently shown to be involved in binding of AMPK to glycogen (20, 36, 40). Immunoblots revealed a dramatic decrease in AMPKbeta2, the major beta-subunit in the heart (44), in AMPK{alpha}2–/– mice (Fig. 1). Immunodetection with an anti-pan AMPKbeta antibody showed the same profile, eliminating possible compensation by an increase in AMPKbeta1 (Fig. 1).

AMPK activity during normoxia and ischemia. The effect of ischemia on AMPK activity was evaluated in Langendorff-perfused hearts from AMPK{alpha}2–/– mice subjected to 10 min of normoxia or no-flow ischemia. In WT mice, no-flow ischemia induced a fivefold increase in AMPK{alpha}2 activity (Fig. 2A). As expected, in hearts from AMPK{alpha}2–/– mice, no AMPK{alpha}2 activity could be detected during normoxia and ischemia. Because the same antibody was used for activity measurement and immunoblotting, these results indicate that the truncated form of AMPK{alpha}2, visible in Fig. 1, is indeed inactive. In contrast, AMPK{alpha}1 activity was not different during normoxia and increased four- to fivefold after 10 min of ischemia in WT and AMPK{alpha}2–/– mice (Fig. 2B). Finally, the absence of the active AMPK{alpha}2 resulted in a significant decrease (50%) in the activation of total AMPK by no-flow ischemia, as measured by polyethylene glycol fractionation (Fig. 2C). Similarly, it induced a significant decrease in total AMPK phosphorylation at Thr172 (data not shown).


Figure 2
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  		Fig. 2. Cardiac AMPK{alpha}2 (A), AMPK{alpha}1 (B), and total AMPK (C) activities during normoxia (open bars) and ischemia (solid bars) in hearts from WT and AMPK{alpha}2–/– mice. Values are means ± SE of ≥7 hearts. *P < 0.05; **P < 0.01 (unpaired t-test).

 
Nucleotide content. Cardiac AMPK activation during ischemia has been shown to result from an increase in the AMP-to-ATP ratio (21, 22). A clear correlation between the level of AMPK activation and the increase in the AMP-to-ATP ratio after 10 min of ischemia has indeed been reported in WT animals (3, 34). To evaluate whether this relation is preserved in hearts from AMPK{alpha}2–/– mice, the nucleotide content of perfused hearts was measured during normoxia and ischemia. In WT mice, no-flow ischemia induced a threefold increase in AMP content, whereas ATP concentration was slightly, but not significantly, diminished (Fig. 3), thereby causing a threefold increase in the AMP-to-ATP ratio (Fig. 3; P < 0.05, unpaired t-test) as previously described in rat heart (3). In hearts from AMPK{alpha}2–/– mice subjected to ischemia, these changes were even more pronounced (Fig. 3), inasmuch as the AMP content increased 10-fold and the ATP content decreased 5-fold, resulting in an 80-fold increase in the AMP-to-ATP ratio.


Figure 3
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  		Fig. 3. AMP and ATP contents and AMP-to-ATP ratio in perfused hearts from WT and AMPK{alpha}2–/– mice during normoxia (open bars) and ischemia (solid bars). Total amount of adenine nucleotides of each animal was measured (not shown), and no significant differences were observed between WT and AMPK{alpha}2–/– mice. Values are means ± SE of ≥6 hearts. *P < 0.05; **P < 0.01 (unpaired t-test).

 
Lactate and glycogen content. To evaluate possible mechanisms underlying the decrease in ATP content in hearts of AMPK{alpha}2–/– mice subjected to ischemia, glycogen and lactate cardiac tissue levels were measured during normoxia and ischemia. In WT mice, ischemia resulted in a 4-fold decrease in glycogen content and a 15-fold increase in lactate concentration (Fig. 4). In contrast, glycogen content was fourfold lower in hearts from AMPK{alpha}2–/– mice than from WT mice during normoxia and was barely detectable after ischemia. Accordingly, lactate accumulation was three times less in AMPK{alpha}2–/– than in WT mice.


Figure 4
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  		Fig. 4. Glycogen and lactate contents of perfused hearts from WT and AMPK{alpha}2–/– mice during normoxia (open bars) and ischemia (solid bars). Values are means ± SE of ≥8 hearts. **P < 0.01 (unpaired t-test).

 
LV function. The evolution of LV functional parameters during equilibration, ischemia, and reperfusion is shown in Figs. 5 and 6. During normoxia, the rate-pressure product, representing the index of contractility, was similar among WT and AMPK{alpha}2–/– mice (Fig. 6). During no-flow ischemia, ischemic contracture developed in both groups (Fig. 5). However, the onset of contracture and the time to its maximal amplitude were reduced three- and twofold, respectively, in AMPK{alpha}2–/– mice compared with WT mice. Peak diastolic pressure was also significantly higher in hearts from AMPK{alpha}2–/– than WT mice.


Figure 5
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  		Fig. 5. Sensitivity of heart function to no-flow ischemia. Perfused hearts of WT (open bars) and AMPK{alpha}2–/– (solid bars) mice were subjected to 15 min of normoxia (15 min) followed by no-flow ischemia. Heart function was followed during the entire period of perfusion (up to 31 min). A: left ventricular pressure recording from a representative experiment. B: time corresponding to beginning of ischemic contracture and time and amplitude of maximal contracture [maximal end-diastolic pressure (EDP)]. Values are means ± SE of 3 hearts. **P < 0.01 (unpaired t-test).

 

Figure 6
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  		Fig. 6. Time-dependent response of function parameters during ischemia-reperfusion protocol. After equilibration in normoxic conditions, perfused heart of WT ({circ}) and AMPK{alpha}2–/– (bullet) mice were subjected to 30 min of no-flow ischemia (from –30 to 0 min) followed by 45 min of reperfusion (from 0 to 45 min). Heart rate, left ventricular systolic pressure, end-diastolic pressure, and coronary flow were measured online. Contractility was estimated as rate-pressure product. Oxygen consumption was measured online from the difference in oxygen content between incoming (aortic) and outgoing (pulmonary artery) perfusate and expressed in µmol O2·min–1·g wet wt–1.

 
Although the onset of ischemia-induced contracture is more rapid in hearts from AMPK{alpha}2–/– mice, both groups are characterized by the same functional recovery on reperfusion (Fig. 6). Indeed, the resulting decrease in rate-pressure product and oxygen consumption was similar in WT and AMPK{alpha}2–/– mice, whereas coronary flow was not statistically modified in either group of animals.

Downstream targets of AMPK. To determine whether the absence of myocardial AMPK{alpha}2 impairs phosphorylation of downstream substrates of AMPK during ischemia, we measured ACC phosphorylation in hearts perfused during normoxia and ischemia. ACC, one of the first AMPK substrates identified, was clearly phosphorylated (80% increase) during ischemia in WT mice (Fig. 7). In hearts from AMPK{alpha}2–/– mice, ischemia also induced ACC phosphorylation, although it was three to five times less than in WT mice. Accordingly, the ACC phosphorylation state in ischemic hearts from AMPK{alpha}2–/– mice was two times less than in normoxic WT mice (Fig. 7), despite the presence of AMPK{alpha}1 in the AMPK{alpha}2–/– mice.


Figure 7
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  		Fig. 7. Acetyl-CoA carboxylase (ACC) phosphorylation (Ser227) state of perfused hearts from WT and AMPK{alpha}2–/– mice during normoxia (open bars) and ischemia (solid bars). Immunoblots represent results from densitometric scanning of all immunoblots. Values are means ± SE of ≥6 hearts. *P < 0.01 vs. normoxia; #P < 0.01 vs. control (unpaired t-test). eEF2 was used as loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Cardiac phenotype of AMPK{alpha}2–/– mice. We have shown that the absence of AMPK{alpha}2 does not result in any detectable morphological or functional changes in the heart. Indeed, heart size and echocardiographic and hemodynamic parameters under basal conditions were similar among hearts from WT and AMPK{alpha}2–/– mice. In contrast, in DN-K45R transgenic mice, a smaller heart and a statistically significant decrease in cardiac contractility (decreased maximum rate of pressure increase and fractional wall thickening) were recently reported (38). Although this remains speculative, the differences between DN-K45R and AMPK{alpha}2–/– mice could be related to the absence of AMPK{alpha}1 activation in DN-K45R mice or the development of untoward side effects provoked by overexpression of a dominant-negative form of AMPK.

AMPK{alpha} and AMPKbeta expression. Hearts from AMPK{alpha}2–/– mice are characterized by the presence of a normal AMPK{alpha}1 content and a small, although significant, amount of the truncated and inactive AMPK{alpha}2. In contrast, in skeletal muscle of the same animal, AMPK{alpha}1 compensation has been found, but truncated AMPK{alpha}2 has not (27).

Nevertheless, in the heart, the combined presence of AMPK{alpha}1 and residual truncated AMPK{alpha}2 is not sufficient to prevent the decrease in AMPKbeta2 content. This suggests that the presence of a normal amount and a normal length of AMPK{alpha}2 is mandatory for the presence of a sufficient amount of AMPKbeta2 in the heart. Analysis of AMPKbeta2 content in dominant-negative transgenic mice would be interesting.

AMPK activation and adenine nucleotide content. Despite an apparently normal functional phenotype under normoxic conditions, hearts from AMPK{alpha}2–/– mice were more susceptible than WT hearts to development of contracture during no-flow ischemia. As a result of the absence of AMPK{alpha}2, total AMPK activation after 10 min of no-flow ischemia was reduced by one-half in hearts from AMPK{alpha}2–/– mice compared with WT mice. This reduced AMPK activation can hardly be explained by a smaller increase in the AMP-to-ATP ratio, because, in the end, this ratio increased much more in hearts from AMPK{alpha}2–/– mice. In WT hearts, such an AMP-to-ATP ratio can be achieved only after a more prolonged (20-min) period of ischemia (3). Moreover, we previously showed that a higher AMPK activation is stimulated in WT hearts by a metabolic poison, such as oligomycin, than by ischemia and that this increase in AMPK activation is correlated with a higher AMP-to-ATP ratio (34). Together, these data suggest that the correlation between the AMP-to-ATP ratio and AMPK activation established in WT hearts is modified in hearts from AMPK{alpha}2–/– mice. One explanation for this difference could be that AMPK{alpha}1 is less sensitive than AMPK{alpha}2 to AMP (41).

Glycolysis, glycogen, and AMPKbeta. Our data indicate that the ATP depletion characteristic of ischemic hearts from AMPK{alpha}2–/– mice most probably results from a reduction in ATP production by glycolysis as measured by lactate production. Indeed, ATP production is known to be correlated to glucose utilization by anaerobic glycolysis, the sole energy-providing pathway in the absence of oxygen (21, 22). Although several mechanisms could account for the decrease in ATP production, including the loss of the stimulating effect of AMPK on glycolysis [via activation of PFK-2 (34)], our data suggest that preischemic glycogen depletion (5 and 20 µm/g of protein in hearts from AMPK{alpha}2–/– and WT mice, respectively) was the most likely cause of the decrease in lactate production.

There are several potential explanations for the lower glycogen content in hearts from AMPK{alpha}2–/– mice during normoxia. 1) Defective cardiomyocytes from AMPK{alpha}2–/– mice, which are less able to take up exogenous glucose, may have less glycogen-storing capacity than WT cardiomyocytes. Previous studies have indeed shown that AMPK activation is important for translocation of the glucose transporter GLUT4 to the plasma membrane (37). Moreover, this mechanism is indeed deficient in hearts from AMPK{alpha}2–/– mice and contributes to their reduced ability to increase exogenous glucose uptake as, for instance, during normoxia and low-flow ischemia (unpublished observations). 2) Several recent studies have shown that AMPK is physically linked to glycogen, probably via AMPKbeta (20, 36, 40). Although the exact role of this glycogen-AMPK interaction remains to be elucidated, it is somehow tempting to hypothesize that the decrease in AMPKbeta2 in hearts from AMPK{alpha}2–/– mice interfered with the ability of AMPK to bind glycogen and, thereby, contributed to disruption of its glycogen-storing capabilities. Alteration of glycogen content, a hallmark of several AMPK isoform mutations, reinforces this hypothesis (7, 36, 54) and further suggests that the mere decrease in the level of AMPKbeta2 in hearts from AMPK{alpha}2–/– mice could explain the perturbation of glycogen storage. 3) We cannot exclude the possibility that a modification of glycogen synthase or glycogen phosphorylase activity contributed as well. Indeed, recently, it was reported that AMPK{alpha}2 is a glycogen synthase kinase in skeletal muscle (26).

Very recently, we studied the effect of cardiac deletion of LKB1, one of the putative upstream AMPK kinases, on ischemia-induced AMPK activation and nucleotide content (39). Under the same experimental protocol, the absence of LKB1 in the heart induced total abolition of AMPK{alpha}2 activity during normoxia and ischemia, whereas AMPK{alpha}1 activity was partially decreased (50% compared with WT animals). So, in terms of AMPK activity, a more dramatic phenotype was induced in hearts from LKB1–/– than from AMPK{alpha}2–/– mice. In contrast to hearts from AMPK{alpha}2–/– mice, the increase in the AMP-to-ATP ratio during ischemia is, however, only slightly affected by the lack of LKB1 (39). This implies that the presence of AMPK{alpha}2 protein, which is not affected by the absence of LKB1, is more important than its activity in preservation of ATP and, probably, glycogen content.

Our study was performed with glucose as the sole energy-providing substrate. We intentionally perfused hearts without fatty acids or insulin, two molecules usually found in vivo, to avoid any possible interference due to the nature (type of fatty acids) and concentration of these molecules. It has been shown that fatty acids and insulin can modify AMPK activity (3, 4, 10, 19). Furthermore, it is difficult to mimic the in vivo situation, inasmuch as fatty acid concentration considerably fluctuates in vivo (42). One could, however, argue that our glucose-only perfusion protocol could have resulted in partial energy starvation and, thereby, contributed to artificially reduced preischemic glycogen levels. However, this is unlikely, because normal normoxic levels of glycogen and nonmodified ischemic ATP levels were found in WT hearts (Figs. 3 and 4). It would nonetheless be interesting to compare our results with those of similar studies performed with different substrates or during low-flow ischemia.

Early LV contracture but normal postischemic contractile function recovery. In isolated perfused hearts, prolonged ischemia almost invariably results in the appearance of ischemic contracture. This rise in ED pressure is due to the lack of ATP at the myofibrillar level, which maintains the cross bridges in the attached state (46). In the present study, ischemic contracture appeared four times more rapidly in hearts from AMPK{alpha}2–/– mice from WT mice, probably as a result of a faster and more important decrease in ATP content, in contrast to a more ancient transgenic dominant-negative AMPK{alpha}2 (D157A mutation) mouse (52). In these mouse hearts, which were perfused following a protocol similar to that used in this study (heart perfused without fatty acids or insulin and subjected to 10 min of no-flow ischemia), ischemic contracture and ATP depletion appeared only slightly more rapidly than in WT hearts. There are several possible explanations for the different behavior of D157A transgenic mice and AMPK{alpha}2–/– mice. 1) AMPK{alpha}2 activity was completely blunted in our model, whereas significant activity persisted in the D157A transgenic mice. 2) Glycogen content was markedly reduced in AMPK{alpha}2–/– mice, whereas it was unaffected in D157A transgenic mice. 3) As mentioned above, AMPKbeta2 content was reduced in our model.

Nevertheless, hearts from AMPK{alpha}2–/– mice were characterized by a faster appearance of ischemic contracture and less stimulation of anaerobic glycolysis. We (45) and others (1, 28) previously demonstrated that the rate of glycolysis during ischemia was the most important determinant of the ischemic contracture. The present study of hearts from AMPK{alpha}2–/– mice confirms the previously established relation between glycolytic rate and ischemic contracture. In contrast, the usual relation between intensity of ischemic contracture and impairment of postischemic contractility is clearly not present in the AMPK{alpha}2–/– mouse model. Indeed, AMPK{alpha}2 deletion did not aggravate the contractile consequences induced by no-flow ischemia, notwithstanding acceleration of the ischemic contracture. Similar observations were also made under low-flow ischemia (unpublished observations). The same dichotomy between acceleration of ischemic contracture and recovery of contractile function has been found in WT animals after preconditioning (29, 30). Our results seem to reveal a dual effect of AMPK{alpha}2: a positive effect (e.g., glycogen storage and stimulation of glucose) during normoxia and ischemia and a negative effect during reperfusion. Several hypotheses could explain this putative deleterious effect of AMPK during reperfusion. 1) AMPK, via ACC inactivation, is known to increase postischemic fatty acid oxidation. This fatty acid oxidation induces uncoupling of glycolysis and glucose oxidation and, therefore, participates in myocardial reperfusion injury (42). Even if our perfusion protocol (with glucose as the sole substrate) excludes a role for exogenous fatty acid, the remaining endogenous fatty acid could participate in this deleterious effect, which should be less pronounced in hearts from AMPK{alpha}2–/– mice, characterized by a lower ACC phosphorylation (see below). 2) The energetic cost of contractility has been found to be modified in hearts from AMPK{alpha}2–/– mice (unpublished observations). This could also be a factor in the postischemic recovery of contractile function that characterizes these hearts.

Our results are different from those reported by Russell et al. (38) from DN-K45R transgenic mouse hearts, because 1) they used fatty acid as the alternative substrate, 2) the remaining AMPK{alpha}1 in our model could compensate for the absence of AMPK{alpha}2, and 3) overexpression of a dominant-negative form of AMPK can induce side effects (see above).

Metabolic consequences under normoxia and ischemia. Hearts from AMPK{alpha}2–/– mice are also characterized by reduced phosphorylation of ACC during normoxia and no-flow ischemia. Even if AMPK{alpha}1 is still present and is probably responsible for the remaining stimulation of ACC phosphorylation during ischemia, the level of phosphorylated ACC after 10 min of ischemia in hearts from AMPK{alpha}2–/– mice was smaller than the level during normoxia in control hearts. It is likely that the general decrease in ACC phosphorylation in hearts from AMPK{alpha}2–/– mice would reduce the stimulation of fatty acid oxidation during reperfusion, as in DN-K45R transgenic mice (38).

In conclusion, our results demonstrate that the absence of AMPK{alpha}2 is responsible for several alterations in myocardial metabolism during normoxia and ischemia: a reduced ability to store glycogen and stimulate glycolysis, an impaired regulation of ATP homeostasis, and a decrease in the stimulation of fatty acid oxidation. The presence of AMPK{alpha}1 does not compensate for the absence of AMPK{alpha}2. Despite an apparent worse metabolic adaptation during ischemia, the absence of AMPK{alpha}2 does not exacerbate the impairment of postischemic recovery of contractile function. This is the first study that reveals a specific role for one of the two catalytic {alpha}-subunits of AMPK in the heart.


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 GRANTS
REFERENCES
 
This work is supported in part by Fonds National de la Recherche Scientifique et Médicale (Belgium) Grant 3.4568.05, European Commission Grant QLG1-CT-2001-01488, the Institut National de la Santé et de la Recherche Médicale (France), and a grant from the Fondation de France. L. Bertrand is a Research Associate and A.-C. Pouleur is a Research Fellow of the Fonds National de la Recherche Scientifique (Belgium). E. Zarrinpashneh is supported by the Fonds Spéciaux de Recherche, Université Catholique de Louvain. K. Carjaval is supported by a grant from the French Association Against Myopathy.


   ACKNOWLEDGMENTS
 
D. G. Hardie (Dundee, Scotland) is acknowledged for the generous gift of antibodies. We thank B. Belge for participation in the initiation of this work, M. Decloedt and V. Race for expert technical assistance, and R. Ventura-Clapier for interest in and critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Bertrand, Division of Cardiology, Université catholique de Louvain, Ave. Hippocrate, 55, CARD5550, B-1200 Brussels, Belgium (e-mail: bertrand{at}card.ucl.ac.be)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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