AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 292: H140-H148, 2007. First published August 18, 2006; doi:10.1152/ajpheart.00424.2006
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AMPK and metabolic adaptation by the heart to pressure overload

Michael F. Allard,1 Hannah L. Parsons,1 Ramesh Saeedi,1 Richard B. Wambolt,1 and Roger Brownsey2

1James Hogg iCapture Centre for Cardiovascular and Respiratory Research, Department of Pathology and Laboratory Medicine, University of British Columbia-St. Paul's Hospital; and 2Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada

Submitted 27 April 2006 ; accepted in final form 15 August 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Accelerated glycolysis in hypertrophied hearts may be a compensatory response to reduced energy production from long-chain fatty acid oxidation with 5'-AMP-activated protein kinase (AMPK) functioning as a cellular signal. Therefore, we tested the hypothesis that enhanced fatty acid oxidation improves energy status and normalizes AMPK activity and glycolysis in hypertrophied hearts. Glycolysis, fatty acid oxidation, AMPK activity, and energy status were measured in isolated working hypertrophied and control hearts from aortic-constricted and sham-operated male Sprague-Dawley rats. Hearts from halothane (3–4%)-anesthetized rats were perfused with KH solution containing either palmitate, a long-chain fatty acid, or palmitate plus octanoate, a medium-chain fatty acid whose oxidation is not impaired in hypertrophied hearts. Compared with control, fatty acid oxidation was lower in hypertrophied hearts perfused with palmitate, whereas it increased to similar values in both groups with octanoate plus palmitate. Glycolysis was accelerated in palmitate-perfused hypertrophied hearts and was normalized in hypertrophied hearts by the addition of octanoate. AMPK activity was increased three- to sixfold with palmitate alone and was reduced to control values by octanoate plus palmitate. Myocardial energy status improved with the addition of octanoate but did not differ between groups. Our findings, particularly the correspondence between glycolysis and AMPK activity, provide support for the view that activation of AMPK is responsible, in part, for the acceleration of glycolysis in cardiac hypertrophy. Additionally, they indicate myocardial AMPK is activated by energy state-independent mechanisms in response to pressure overload, demonstrating AMPK is more than a sensor of the heart's energy status.

adenosine 5'-monophosphate-activated protein kinase; energy metabolism; cardiac function

AMP-ACTIVATED PROTEIN KINASE (AMPK), which has a key role in the control and regulation of energy metabolism (19, 20, 24), is activated at the cellular level in the heart and other tissues by physiological or pathological metabolic stresses that deplete cellular high-energy phosphates, including ATP and phosphocreatine (11, 19, 39). In general terms, AMPK activation inhibits cellular processes that utilize ATP and stimulates those that produce ATP. Activation of AMPK in the heart can stimulate both fatty acid and glucose catabolism, resulting in increased production of ATP (54). AMPK activation in the heart leads to phosphorylation and inhibition of acetyl-CoA carboxylase (ACC) (14, 34), resulting in a reduction in malonyl-CoA levels (26). Reduction in malonyl-CoA, which inhibits carnitine palmitoyltransferase-1, a key enzymatic step that controls entry of long-chain fatty acids into the mitochondria, is responsible for the stimulation of fatty acid oxidation (26, 39). With respect to glucose catabolism, translocation of glucose transporters to the sarcolemma and enhanced rates of glucose uptake (38, 39, 53), as well as stimulation of glycolysis due to phosphorylation and activation of phosphofructokinase-2 (33), accompany AMPK activation in the heart.

Catabolism of glucose by glycolysis is accelerated in hearts hypertrophied in response to long-standing pressure overload (3, 27, 35, 48). Acceleration of glycolysis arises as a result of increased delivery of glucose via sarcolemmal glucose transport proteins and increased flux through key enzymes in the glycolytic pathway, such as phosphofructokinase-1 (3, 35, 46). The increased rates of glycolysis in hypertrophied hearts cannot be accounted for by significant changes in expression of relevant glycolytic enzymes but rather by nontranscriptional control mechanisms (4, 35, 41). Accordingly, it has been proposed that a decreased myocardial energy reserve in cardiac hypertrophy is responsible for the acceleration of glycolysis, with activation of AMPK playing an important role in the control of key metabolic enzymes and proteins (35, 46).

The mechanisms responsible for the reduced myocardial energy reserve have not yet been fully defined for hearts with compensated hypertrophy. A decreased capacity and velocity of the creatine kinase reaction and, therefore, flux through this pathway are believed to be responsible for the reduced energy reserve in failing hearts (36, 45) and could possibly play a role in hypertrophied hearts without evidence of failure, although this remains to be proven. Alternatively, decreased energy production from long-chain fatty acid oxidation, which also occurs in pressure-overload cardiac hypertrophy (3, 27, 48), may be responsible for an impaired myocardial energy status. If the latter is true, increased energy production arising from accelerated rates of glycolysis can be considered a compensatory response to reduced energy production from fatty acid catabolism, with activation of AMPK playing a significant role in the control mechanisms.

In this study, we tested the hypothesis that enhanced energy production from fatty acid oxidation would improve myocardial energy status and lower glycolytic rates by reducing the activation state of AMPK. Isolated working hearts from sham-operated and aortic-constricted rats perfused either with palmitate, a long-chain fatty acid, or with a combination of palmitate and octanoate, a medium-chain fatty acid whose oxidation is not impaired in hypertrophied hearts (15), were compared.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

A mild pressure-overload left ventricular hypertrophy was produced in male Sprague-Dawley rats by constriction of the suprarenal abdominal aorta with a metallic clip (0.4-mm diameter) at 3 wk of age (3). In sham-operated control rats, the aorta was isolated, but not clipped. Experiments were performed 8 wk after surgery. In a subset of animals, hearts were removed from anesthetized (3–4% halothane), sham-operated, and aortic-constricted rats 8 wk after surgery (N = 4 per group) and placed in 10% neutral buffered formalin. Regional heart weights, including left ventricle plus septum, right ventricle, and atria, were determined subsequently. Food and water were administrated ad libitum. These experiments were approved by the institutional committee on the use of laboratory animals in research and conform with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH Publication No. 85–23, Revised 1996).

Isolated Heart Preparation and Perfusion Protocol

Hearts from halothane (3–4%)-anesthetized rats were isolated and perfused as working preparations with Krebs-Henseleit (KH) solution under normoxic nonischemic conditions at a left atrial preload of 11.5 mmHg and an aortic afterload of 80 mmHg, in a closed recirculating system with oxygenated (95% O2-5% CO2) KH solution, maintained at 37°C, as described (3, 41). Hearts were all perfused under comparable conditions to avoid potentially confounding effects of exposure to different afterloads. Although hearts from aortic-constricted rats are exposed to elevated afterloads in vivo (41), we have shown that adjusting afterload to normalize coronary flow per gram in isolated working hypertrophied hearts does not significantly influence the functional or metabolic outcomes observed (50).

Two series of perfusions were performed in each group. The first series of hearts was perfused with KH solution containing a long-chain fatty acid, 1.2 mM [1-14C]- or [9,10-3H]palmitate, prebound to fatty acid-free albumin (3%), together with 5.5 mM glucose, 0.5 mM lactate, and 100 mU/l insulin. The second series of hearts was perfused with KH solution containing both a long-chain fatty acid, 0.6 mM [9,10-3H]palmitate, and a medium-chain fatty acid, 1.2 mM [1-14C]octanoate, as well as 5.5 mM glucose, 0.5 mM lactate, and 100 mU/l insulin. The total concentration of exogenous fatty acid in each series yields an equivalent amount of acetyl-CoA, assuming complete beta-oxidation in mitochondria. Concentrations of insulin and palmitate in the high physiological range were used to ensure that supply of glucose and fatty acid to the heart was not limited, based on previous work in which it was found that lower concentrations of palmitate are associated with a depletion of endogenous substrate stores (22, 40). Additional parallel perfusions in each series were performed in which the KH solution contained tracer amounts of [5-3H]/[U-14C]glucose in place of radiolabeled fatty acids to measure glycolysis and glucose oxidation, respectively.

A pressure transducer (Viggo-Spectramed, Oxnard, CA) inserted in the afterload line was used to measure heart rate and peak systolic pressure. Cardiac output and aortic flow were measured via external flow probes (Transonic Systems, Ithaca, NY) on the left atrial preload and aortic afterload lines, respectively. External work performed by the heart was expressed as "rate-pressure product," the product of heart rate and peak systolic pressure, and "hydraulic work," the product of cardiac output and peak systolic pressure. Hearts were perfused for 30 min during which perfusate and gas samples were taken every 10 min. At the end of perfusion, hearts were clamped with tongs that had been cooled to the temperature of liquid nitrogen and stored in cryovials at –70°C until they were used for biochemical and other analyses or determination of wet-to-dry tissue weight ratio.

Myocardial Substrate Utilization Rates

Oxidation of palmitate, octanoate, and glucose was determined by quantitative collection of 14CO2 released from 14C-labeled fatty acids or [14C]glucose as a gas and dissolved in the perfusate as [14C]bicarbonate (3, 29). Rates of glycolysis and palmitate oxidation were determined by quantitatively measuring the rate of 3H2O released into the perfusate from [5-3H]glucose or [9,10-3H]palmitate, respectively, as described (3, 31). Rates of palmitate oxidation determined using [14C]- or [3H]palmitate yield comparable results (data not shown). It should be noted that these rates refer to catabolic rates of exogenous substrates, as the contribution of endogenous substrates, such as glycogen and triglyceride, was not taken into account. Fatty acid and glucose oxidation rates are expressed as nanomoles of carbon equivalents produced per minute per gram of dry weight, calculated from substrate oxidation rates, assuming 6 arise from glucose, 16 arise from palmitate, and 8 arise from octanoate. In a similar manner, rates of glycolysis are expressed as carbon equivalents per minute per gram dry heart weight.

AMPK Activity

Isoform-specific ({alpha}1 and {alpha}2) AMPK activity was determined after immunoprecipitation from myocardial homogenates, essentially as described with minor modifications (9). Briefly, 100 mg of frozen myocardium were homogenized in buffer containing 50 mM Tris, 0.25 M mannitol, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM Na4P2O7, and 1 mM DTT. After centrifugation, supernatant containing ~500 µg of protein was incubated with isoform-specific anti-{alpha}1 or anti-{alpha}2 AMPK antibodies (Upstate, Charlottesville, VA) bound to protein A-sepharose. The immunoprecipitate was washed/recentrifuged three times at 4°C with AMPK resuspension buffer containing 100 mM Tris, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM Na4P2O7, 1 mM DTT, 10% glycerol, and 0.12% Triton. Activity of AMPK in the immunprecipitate was measured by determining the incorporation of 32P into the synthetic AMARA peptide (25). The extent of phosphorylation of ACC was also determined by immunoblot analysis as a further means to assess AMPK activity using a previously described method (30). ACC is a downstream target of AMPK and can thus be used as a marker of AMPK activation. Protein concentration was measured using the bicinchoninic acid method (Sigma Chemical, procedure no. TPRO-562).

Myocardial Metabolites and PDH Activity

Adenine nucleotides and creatine phosphate were determined in perchloric acid extracts of frozen ventricular tissue by high-performance liquid chromatography (30). Since high-performance liquid chromatography measures the total amount of AMP in tissue extracts, with the functionally relevant free AMP representing a small fraction of the total, concentration of free AMP was determined using the creatine kinase and adenylate kinase equilibrium reactions, as previously described (35). Myocardial glycogen content was determined following extraction from frozen ventricular tissue with 30% KOH, ethanol precipitation, and acid hydrolysis of glycogen (22). Total lipids were extracted from frozen ventricular tissue following a chloroform/methanol extraction (8). Triglyceride content was determined using a colorimetric method (Roche Hitachi, Indianapolis, IN). Malonyl-CoA content was measured as previously described (29). Pyruvate dehydrogenase (PDH) activity was determined in homogenates of frozen ventricular tissue by measuring [14C]citrate synthesis from [14C]oxaloacetate and PDH complex-derived acetyl-CoA (32).

Content of Calcium Calmodulin Kinase Kinase Isoforms-{alpha} and -beta

Myocardial content of {alpha}- and beta-isoforms of calcium calmodulin kinase kinase (CaMKK), recently recognized as upstream AMPK kinases (21, 23, 24, 52), was determined by immunoblot analysis (41). Briefly, samples of frozen ventricular tissue homogenate (containing 100 µg total protein) were solubilized by boiling in reducing sample buffer, separated by electrophoresis on 10% SDS-polyacrylamide gels, and transferred by electroblotting to a nitrocellulose membrane. After nonspecific blocking, the blots were probed overnight with primary antibodies against the {alpha}- and beta-isoforms of CaMKK (Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with secondary antibody, the signal was detected by an enhanced chemiluminescence-based detection system and was quantitated by densitometry.

Statistical Analysis

Data are expressed as means ± SE. Weight data were analyzed by one-way ANOVA, whereas other parameters were analyzed by two-way ANOVA for other parameters. Post hoc tests with Bonferroni adjustment were applied to determine the source of differences among groups. A corrected P value >0.05 was considered as nonsignificant (NS).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Morphological Data

Heart weight of aortic-constricted rats (2.18 ± 0.04 g, N = 43) was ~25% greater than that of sham-operated rats (1.74 ± 0.03 g, N = 38, P < 0.05), while body weight did not differ (477.3 ± 6.8 vs. 476.6 ± 7.1 g, P = NS). There were no significant differences between corresponding palmitate or octanoate-palmitate-perfused groups (data not shown). Weight of the left ventricle plus septum was significantly increased in aortic-constricted rats (1.56 ± 0.07 g, N = 4) compared with sham-operated rats (1.11 ± 0.01 g, N = 4, P < 0.05), but weights of the right ventricle (0.35 ± 0.02 vs. 0.31 ± 0.01 g, N = 4, P = NS) and atria (0.35 ± 0.02 vs. 0.31 ± 0.01 g, N = 4, P = NS) did not differ between the two groups.

Heart Function

In the presence of palmitate, the function of hypertrophied hearts, including cardiac output, rate-pressure product, and hydraulic work, was significantly lower than in nonhypertrophied hearts (Table 1). Hypertrophied and nonhypertrophied heart function was improved by perfusion with the combination of octanoate and palmitate, compared with hearts perfused with palmitate as the sole exogenous fatty acid. Furthermore, contractile function of hypertrophied hearts was no longer lower than that of nonhypertrophied hearts in the presence of octanoate plus palmitate; in fact, peak systolic pressure and hydraulic work were greater in hypertrophied hearts than in nonhypertrophied hearts when octanoate was also present. Rates of coronary flow were lower in hypertrophied hearts than in nonhypertrophied hearts in perfusions with palmitate, but this difference was abolished when octanoate was also present.


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  		Table 1. Function of hearts perfused with palmitate or palmitate and octanoate

 
Substrate Use

Fatty acid oxidation. With palmitate as the sole exogenous fatty acid source, oxidation of palmitate and, therefore, overall rates of fatty acid oxidation were lower in hypertrophied hearts than in nonhypertrophied hearts (Fig. 1). Overall rates of fatty acid oxidation increased to similar values in both groups in the presence of octanoate and palmitate, with no differences in rates of octanoate or palmitate oxidation between hypertrophied and nonhypertrophied hearts. The inclusion of octanoate led to a lowering of palmitate oxidation in nonhypertrophied hearts but was not associated with any significant lowering of the already diminished rates of palmitate oxidation in hypertrophied hearts.


Figure 1
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  		Fig. 1. Fatty acid oxidation in hypertrophied (hypertrophy) and nonhypertrophied (control) hearts perfused with palmitate (palmitate) or palmitate and octanoate (palmitate/octanoate). Open bar, palmitate oxidation. Shaded bar, octanoate oxidation. *Significantly different from nonhypertrophied hearts, P < 0.05. {dagger}Significantly different from corresponding hearts perfused with palmitate, P < 0.05. n = 5–12 per group.

 
Glycolysis. When palmitate was provided as the sole exogenous fatty acid source, rates of glycolysis were accelerated in hypertrophied hearts compared with nonhypertrophied hearts (Fig. 2). In contrast, rates of glycolysis were normalized in hypertrophied hearts perfused with octanoate and palmitate.


Figure 2
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  		Fig. 2. Glycolysis in hypertrophied (hypertrophy) and nonhypertrophied (control) hearts perfused with palmitate (palmitate) or palmitate and octanoate (palmitate/octanoate). *Significantly different from nonhypertrophied hearts, P < 0.05. {dagger}Significantly different from corresponding hearts perfused with palmitate, P < 0.05. n = 5–12 per group.

 
Glucose oxidation. Glucose oxidation did not differ significantly between hypertrophied and nonhypertrophied hearts with palmitate alone or with a combination of octanoate and palmitate in the perfusate (Fig. 3). Oxidation of glucose actually increased somewhat in hearts perfused with octanoate and palmitate compared with those perfused with palmitate alone, although this difference was significant only in nonhypertrophied hearts. The profile of substrate catabolism described above persists when cardiac workload is taken into account (data not shown).


Figure 3
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  		Fig. 3. Glucose oxidation in hypertrophied (hypertrophy) and nonhypertrophied (control) hearts perfused with palmitate (palmitate) or palmitate and octanoate (palmitate/octanoate). {dagger}Significantly different from nonhypertrophied hearts, P < 0.05. n = 5–12 per group.

 
Isoform-Specific AMPK Activity

Isoform-specific AMPK activity was significantly increased in palmitate-perfused hypertrophied hearts compared with corresponding nonhypertrophied hearts. Specifically, {alpha}1-AMPK activity was increased three- to fourfold, whereas {alpha}2-AMPK activity was increased nearly sixfold (Fig. 4). Isoform-specific AMPK activity was normalized in hypertrophied hearts perfused in the presence of octanoate plus palmitate. Phosphorylation of ACC was increased in palmitate-perfused hypertrophied hearts compared with corresponding nonhypertrophied hearts (Fig. 5). In keeping with changes in isoform-specific AMPK activity, phosphorylation of ACC was higher in hypertrophied than in nonhypertrophied hearts perfused with palmitate, and this difference was abolished following perfusion with octanoate plus palmitate.


Figure 4
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  		Fig. 4. Activity of {alpha}1 (A) and {alpha}2 (B) AMP-activated protein kinase (AMPK) activity in hypertrophied (hypertrophy) and nonhypertrophied (control) hearts perfused with palmitate (palmitate) or palmitate and octanoate (palmitate/octanoate). *Significantly different from nonhypertrophied hearts, P < 0.05. {dagger}Significantly different from corresponding hearts perfused with palmitate, P < 0.05. n = 6 per group.

 

Figure 5
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  		Fig. 5. Representative immunoblots of phospho-acetyl-CoA carboxylase (phosphoACC; top) and total acetyl-CoA carboxylase (ACC; bottom) in hypertrophied (hypertrophy) and nonhypertrophied (control) hearts perfused with palmitate (palmitate) or palmitate and octanoate (palmitate/octanoate). Each lane represents a single heart. Densitometric analysis is expressed in arbitrary units as the ratio of phosphoACC and ACC. n = 3–4 per group. *Significantly different from nonhypertrophied hearts, P < 0.05.

 
Myocardial Metabolites and PDH Activity

High-energy phosphate content, including ratios of AMP to ATP and Cr to PCr, was not different between hypertrophied and nonhypertrophied hearts of either perfusion group (Table 2). Notably, ATP was higher, whereas ADP, AMP, and AMP-to-ATP ratio were lower, in hearts perfused with octanoate and palmitate compared with those perfused with palmitate alone. Free AMP did not differ between hypertrophied and nonhypertrophied hearts perfused with either palmitate (0.59 ± 0.18 vs. 0.39 ± 0.12 µmol/l, N = 6 per group, P = NS) or octanoate and palmitate combined (1.09 ± 0.38 vs. 0.62 ± 0.21 µmol/l, N = 7 per group, P = NS). Tissue content of glycogen and triglyceride did not differ between hypertrophied and nonhypertrophied hearts perfused with either palmitate alone or a combination of octanoate and palmitate. Glycogen and triglyceride contents were, however, higher in hearts perfused with octanoate and palmitate, reaching significance for glycogen in nonhypertrophied hearts and triglycerides in hypertrophied hearts. Malonyl-CoA content did not differ between nonhypertrophied and hypertrophied hearts perfused with palmitate (29.2 ± 3.2 vs. 29.7 ± 1.1 nmol/g dry wt, N = 6 per group, P = NS) or with octanoate and palmitate (25.5 ± 1.2 vs. 23.1 ± 1.9 nmol/g dry wt, N = 5 to 6 per group, P = NS). Activity of PDH was highest in hypertrophied hearts perfused with palmitate alone, with no significant differences among the other groups. Total PDH, measured by either activity assay or on the basis of expression of protein subunits, has previously been shown not to differ between hypertrophied and nonhypertrophied rat hearts in this model of cardiac hypertrophy (32).


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  		Table 2. Myocardial metabolites and pyruvate dehydrogenase activity in hearts perfused with palmitate or palmitate and octanoate

 
Content of CaMKK

Content of the beta-isoform of CaMKK did not differ significantly between hypertrophied and nonhypertrophied hearts (Fig. 6), regardless of whether hearts were perfused with palmitate alone or palmitate and octanoate combined (data not shown), whereas the {alpha}-isoform of CaMKK was not detected in either group. Failure to detect the {alpha}-isoform of CaMKK in rat heart by immunoblot analysis has been reported previously (5).


Figure 6
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  		Fig. 6. Representative immunoblot of calcium calmodulin kinase kinase beta (CaMKK-beta) in hypertrophied (hypertrophy) and nonhypertrophied (control) hearts perfused with palmitate. Each lane represents a single heart. Densitometric analysis is expressed in arbitrary density units. n = 4 per group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we found that accelerated rates of glycolysis in hypertrophied rat hearts are normalized by provision of both long-chain (palmitate) and medium-chain (octanoate) fatty acids as substrates, a combination that elevates fatty acid oxidation and results in rates in hypertrophied hearts similar to those in nonhypertrophied hearts. Elevated activity of AMPK in hypertrophied hearts is also reduced to values observed in nonhypertrophied hearts, an outcome that provides strong support for the concept that AMPK is involved in alterations in glucose use in cardiac hypertrophy. Of additional interest, we found that AMPK is activated in hypertrophied hearts in the absence of measurable changes in cellular energy state, indicating that AMPK is activated by energy state-independent mechanism(s) in hearts exposed to pressure overload.

Accelerated rates of glycolysis have been consistently observed in models of pressure-overload cardiac hypertrophy in rodents, including rats (3, 27, 35, 48) (Fig. 2) and mice (49). Some have suggested that an increased capacity of glycolytic enzymes is responsible for the acceleration of glycolysis in this setting, based primarily on data obtained in other animal models (6, 37). However, we (4, 41) and others (35, 46) have found that alteration in expression or capacity of relevant proteins and enzymes does not readily account for the changes in glycolytic flux observed in rodent models of cardiac hypertrophy, indicating other mechanisms, including sarcolemmal translocation of glucose transporters and activation of relevant enzymes by allosteric or covalent modification, are responsible. Using a thoracic aortic constriction model of cardiac hypertrophy in rats, Tian and colleagues nicely show that changes in glycolysis occur in association with a decreased myocardial energy status and activation of AMPK (35, 46) and propose that glycolysis is accelerated in cardiac hypertrophy as a response to the impaired energy status with AMPK as a key signaling intermediary. The findings in the present study and, specifically, the correspondence between changes in glycolytic rates and AMPK activity (Figs. 2, 4, and 5) are entirely in keeping with the view that AMPK plays a role in the acceleration of glycolysis in hypertrophied hearts. Of additional significance, we demonstrate that this relationship exists in working hearts perfused with a mixture of substrates, including physiological long-chain fatty acids.

AMPK is a heterotrimeric complex comprised of {alpha}-catalytic and beta- and {gamma}-regulatory subunits (19, 20, 24), whose activity is increased when cellular high-energy phosphates, including ATP and phosphocreatine, are depleted. Activation of AMPK involves phosphorylation of Thr-172 within the activation loop of the {alpha}-catalytic subunit by upstream AMPK kinase(s) and direct allosteric effects of elevated AMP-to-ATP and creatine-to-phosphocreatine ratios. LKB1, a tumor suppressor originally identified as the gene mutated in Peutz-Jeghers Syndrome, is now recognized as a major upstream kinase involved in phosphorylation and activation of AMPK in response to elevations in AMP-to-ATP ratios in the heart (42) and other tissues (20). More recently, it has been recognized that AMPK can also be activated by a calcium-dependent pathway involving CaMKK as an upstream AMPK kinase (21, 23, 24, 52) and long-chain fatty acids (9, 16), modes of AMPK activation independent of changes in energy status.

In the present study, we observed, for the first time, that AMPK is activated in hypertrophied hearts in the absence of any measurable reduction in energy status (Figs. 4 and 5, Table 2), including no decline in creatine phosphate or elevation of free AMP content in hypertrophied hearts, indicating that AMPK can be activated in the hypertrophied heart by energy status-independent as well as energy status-dependent (35) mechanisms. The exact mechanism(s) and upstream kinase(s) responsible for this activation remain to be determined. However, it is interesting to speculate that energy status-dependent activation of AMPK is mediated by LKB1, while energy status-independent activation occurs by other mechanism(s), possibly by a calcium-dependent pathway downstream from Gq-coupled receptors (24, 51) and involved in the development of cardiac hypertrophy (13). In this regard, differences in expression of CaMKK were not observed between hypertrophied and nonhypertrophied hearts (Fig. 6), indicating that, if this pathway participates in the activation of AMPK in hypertrophied hearts, it does so independent of changes in protein expression. Future studies beyond the scope of the present investigation are required to fully assess the potential role of CaMKK in this setting.

Long-chain fatty acids and CoA esters might also act as important modulators of AMPK, based on recent studies showing that AMPK in isolated hearts and skeletal muscle cells can be activated by long-chain fatty acids (9, 16), while others found that long-chain CoA esters can inhibit AMPK activity (44). Given that long-chain fatty acid catabolism is significantly altered in hypertrophied hearts, it is conceivable that such changes are responsible for or contribute to the activation of AMPK that we observed. Moreover, a differential effect of long-chain vs. medium-chain fatty acids on AMPK activation, as observed by others (44), may be a factor involved in the reduction in AMPK activity in hypertrophied hearts perfused with palmitate and octanoate combined. As with CaMKK, studies in the future will be needed to investigate this area. With these comments in mind, AMPK may be considered as more than simply a sensor of the energy state of the heart and may function more broadly as a mediator of extracellular signals and as a metabolic sensor, potentially being involved in the early adaptive responses of the heart to pressure overload. The roles of long-chain fatty acids, as well as CaMKK in the control of AMPK, require future investigation.

With palmitate as the sole fatty acid in the perfusate, rates of fatty acid oxidation were lower in hypertrophied hearts than nonhypertrophied hearts (Fig. 1). As with the acceleration of glycolysis, a reduction in long-chain fatty acid oxidation has been consistently observed in models of cardiac hypertrophy (3, 27, 35, 48, 49). Reduced expression of oxidative enzymes and fatty acid uptake/transport proteins, as well as low levels of myocardial carnitine, a cofactor required for long-chain fatty acid transport into mitochondria, have been proposed as being responsible (43). Presumably, these changes result in a limitation in the oxidation of long-chain fatty acids in hypertrophied hearts that prevents an increase in oxidative rates with activation of AMPK, as would be expected to occur in a normal heart.

Perfusion with both long-chain and medium-chain fatty acids increased overall rates of fatty acid oxidation in hypertrophied and nonhypertrophied hearts, primarily by increasing the contribution of medium-chain fatty acids, effectively normalizing fatty acid oxidation in hypertrophied hearts (Fig. 1). The enhanced rates of fatty acid oxidation caused by the addition of octanoate to the perfusate were accompanied by improvements in contractile function (Table 2), a finding most notable in hypertrophied hearts. Beneficial functional effects of medium-chain fatty acids combined with long-chain fatty acids in isolated working hypertrophied hearts have been previously observed, especially in the presence of an adrenergic stress (27). Interestingly, malonyl-CoA did not differ among groups, a finding that likely reflects the complexity of factors controlling the activities of ACC and malonyl-CoA decarboxylase. For example, ACC activity is controlled covalently by phosphorylation and allosterically by activators, such as citrate, and inhibitors, such as long-chain fatty acyl-CoA (7). Because of the inclusion of long-chain fatty acids in all perfusion conditions in the studies reported here, it is reasonable to expect that feedback control of ACC by long-chain acyl-CoA provided sufficient allosteric suppression of ACC that would make it difficult to discern any further effects of AMPK activation on malonyl-CoA content of the hearts. In addition, since the major ACC isoform in the heart is believed to be targeted to mitochondria (1, 18), it is conceivable that localized changes in malonyl-CoA may have occurred that were not reflected in total tissue malonyl-CoA.

Measured glucose oxidation rates and PDH activity are discordant under both perfusion conditions. In palmitate-perfused hearts, rates of glucose oxidation are similar in hypertrophied and nonhypertrophied hearts, even though PDH activity is higher in extracts from hypertrophied hearts (Table 2, Fig. 2). This finding is consistent with previous work in our laboratory (28, 32) and likely reflects a limitation of glucose oxidation in hypertrophied hearts, a limitation that is convincingly demonstrated when glucose oxidation is substantially increased (28). The discordance between PDH activity and glucose oxidation in hearts perfused with octanoate and palmitate may actually be more apparent than real, largely because the contribution of glucose derived from glycogen was not taken into account. Myocardial glycogen makes a significant contribution to energy production in heart perfused with long-chain fatty acids, especially via oxidation of glucose from glycogen (2, 17, 22), with oxidation rates of endogenous glucose ~80% of those from exogenous glucose in both hypertrophied and nonhypertrophied hearts (2). In hearts perfused with octanoate and palmitate, rates of degradation of glycogen and oxidation of endogenous glucose were likely extremely small or minimal, such that overall glucose oxidation actually decreased, in keeping with increased fatty acid oxidation and decreased PDH activity. That oxidation of exogenous glucose increased in the second series of perfusions is likely related to the greater workload achieved, a situation known to selectively enhance oxidation of glucose (10) and possibly to differences in the type of fatty acid supplied.

Energy status was significantly improved in both hypertrophied and nonhypertrophied hearts by the presence of octanoate (Table 2), a result that was associated with a reduction in glycolysis and AMPK activity. Given that high ATP concentrations are known to inhibit phosphofructokinase (12), a key control step in glycolysis, and AMPK activation (20), the improved energy status likely contributed to the reduced rates of glycolysis and AMPK activity in hypertrophied hearts, with increased fatty acid oxidation also contributing to the reduction in glycolysis. That the changes occurred only in hypertrophied hearts suggests that glycolysis and AMPK activity are already near basal values in nonhypertrophied hearts perfused with palmitate alone. Of additional interest, these data indicate that improvements in energy status can overcome energy status-independent factors causing AMPK activation in the hypertrophied heart.

Our data also indicate that the mechanisms responsible for activation of AMPK in the setting of pressure-overload cardiac hypertrophy are dependent on the specific experimental model used, as well as the severity of hypertrophy and stage at which the hearts are studied. The abdominal aortic constriction model used in the present investigation results in a mild form of cardiac hypertrophy (~25% increase above sham-operated control). The absence of significant differences in weights of right ventricle and atria between sham-operated and aortic-constricted rats indicates that the hypertrophied hearts are in a fully compensated stage at a time when the heart is not yet energetically compromised. In contrast, the thoracic aortic constriction model used by Tian and colleagues (35, 47) produces a moderate degree of cardiac hypertrophy (~35–50% increase above sham-operated control). That atrial weight is increased in this model suggests the presence of some degree of ventricular dysfunction, a functional effect accompanied by a reduced myocardial energy status.

Taken together, these data indicate that myocardial AMPK is activated by at least two separate, and apparently interacting, pathways in response to pressure overload (Fig. 7). In the early, compensated stage or in mild forms of cardiac hypertrophy, AMPK is activated by energy status-independent mechanisms, where it may participate in the adaptive response of the heart to pressure overload by modulating substrate catabolism. Since fatty acid oxidation is inherently impaired in hypertrophied hearts, an acceleration of glycolysis is the major catabolic alteration possible in response to AMPK activation. In later stages of disease or more severe degrees of cardiac hypertrophy, a reduced myocardial energy status likely also contributes to the catabolic changes seen, with AMPK participating as a metabolic sensor and key cellular signal.


Figure 7
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  		Fig. 7. Diagram summarizing energy state-dependent and -independent activation of AMPK in the heart in response to pressure overload and the effects on glycolysis. In this scheme, alterations in cellular energy state can influence energy state-independent activation of AMPK and glycolysis. LCFA, long-chain fatty acids or corresponding long-chain acyl-CoA esters. LKB1, Peutz-Jegher Sydrome protein. Solid line indicates activation; dotted line indicates inhibition.

 

   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Canadian Institutes of Health Research (CIHR). R. Saeedi is a recipient of a CIHR-Canada Graduate Scholarship doctoral research award.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. F. Allard, James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, Rm 166, St. Paul's Hospital, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6 (e-mail: mallard{at}mrl.ubc.ca)

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
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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