Activation of AMP-activated Protein Kinase Inhibits Protein Synthesis Associated with Hypertrophy in the Cardiac Myocyte*

A necessary mediator of cardiac myocyte enlargement is protein synthesis, which is controlled at the levels of both translation initiation and elongation. Eukaryotic elongation factor-2 (eEF2) mediates the translocation step of peptide-chain elongation and is inhibited through phosphorylation by eEF2 kinase. In addition, p70S6 kinase can regulate protein synthesis by phosphorylating eEF2 kinase or via phosphorylation of ribosomal protein S6. We have recently shown that eEF2 kinase is also controlled by phosphorylation by AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis. Moreover, the mammalian target of rapamycin has also been shown to be inhibited, indirectly, by AMPK, thus leading to the inhibition of p70S6 kinase. Although AMPK activation has been shown to modulate protein synthesis, it is unknown whether AMPK could also be a regulator of cardiac hypertrophic growth. Therefore, we investi-gated the role of AMPK activation in regulating protein synthesis during both phenylephrine- and Akt-induced

A necessary mediator of cardiac myocyte enlargement is protein synthesis, which is controlled at the levels of both translation initiation and elongation. Eukaryotic elongation factor-2 (eEF2) mediates the translocation step of peptide-chain elongation and is inhibited through phosphorylation by eEF2 kinase. In addition, p70S6 kinase can regulate protein synthesis by phosphorylating eEF2 kinase or via phosphorylation of ribosomal protein S6. We have recently shown that eEF2 kinase is also controlled by phosphorylation by AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis. Moreover, the mammalian target of rapamycin has also been shown to be inhibited, indirectly, by AMPK, thus leading to the inhibition of p70S6 kinase. Although AMPK activation has been shown to modulate protein synthesis, it is unknown whether AMPK could also be a regulator of cardiac hypertrophic growth. Therefore, we investigated the role of AMPK activation in regulating protein synthesis during both phenylephrine-and Aktinduced cardiac hypertrophy. Metformin and 5-aminoimidazole-4-carboxamide 1-␤-D-ribofuranoside were used to activate AMPK in neonatal rat cardiac myocytes. Activation of AMPK significantly decreased protein synthesis induced by phenylephrine treatment or by expression of constitutively active Akt. Activation of AMPK also resulted in decreased p70S6 kinase phosphorylation and increased phosphorylation of eEF2, suggesting that inhibition of protein synthesis involves the eEF2 kinase/eEF2 axis and/or the p70S6 kinase pathway. Together, our data suggest that the inhibition of protein synthesis by pharmacological activation of AMPK may be a key regulatory mechanism by which hypertrophic growth can be controlled.
Cardiac hypertrophy is characterized by an enlargement of the cardiac myocyte that often occurs in response to increased hemodynamic load arising from a variety of conditions, including exercise, hypertension, and valvular heart disease. These morphological changes are adaptive responses that allow the heart to maintain cardiac output. Indeed, exercise-induced hypertrophy is not detrimental to the function of the myocardium and appears to be advantageous (1). However, hypertrophy arising from pathological conditions, such as sustained pressure overload, can become maladaptive and lead to cardiomyopathy, heart failure, and sudden death (see Ref. 2 for review). Although initially adaptive, an alteration in cardiac energy substrate utilization involving a decrease in fatty acid oxidation and an increase in glucose utilization (3,4) can become maladaptive (5). This switch in substrate utilization is also consistent with re-induction of several fetal gene products that regulate metabolism in the hypertrophied heart (see Ref. 5 for review). As a result, the altered metabolic profile found in the hypertrophic heart may play an important role in the development of hypertrophy and/or the progression to heart failure.
At the molecular level, cardiac hypertrophy is characterized by an increase in myocardial cell size, a higher degree of sarcomeric organization, re-activation of the fetal gene program, and changes in gene transcription and translation resulting in enhanced protein synthesis (6). A necessary mediator of cardiac myocyte enlargement is protein synthesis, which is controlled by both translation initiation and peptide-chain elongation (7,8). The eukaryotic elongation factor-2 (eEF2) 1 is responsible for mediating the translocation step of peptide-chain elongation (9). eEF2 is phosphorylated and inhibited by a calcium/calmodulin-dependent protein kinase called eEF2 kinase, which modifies threonine residue 56 (10). Phosphorylation at Thr-56 results in the inactivation of eEF2 by causing a structural alteration that reduces its affinity for the ribosome, thereby preventing its ability to catalyze translocation (11). eEF2 kinase is also subjected to regulation by phosphorylation, and a number of phosphorylation sites have been identified that lead to subsequent activation or inhibition of activity (12)(13)(14)(15). Most recently, we have shown that AMP-activated protein kinase (AMPK), which is a key regulator of cellular energy homeostasis, phosphorylates eEF2 kinase at a novel site in the regulatory domain, serine 398 (16), and activates it. In cardiac myocytes, Horman et al. (17) have shown that AMPK activation leads to increased eEF2 phosphorylation via eEF2 kinase activation, resulting in the inhibition of protein synthe-sis. In addition, AMPK has recently been shown to be involved in modulating the activity of the mammalian target of rapamycin (mTOR) (18). mTOR is a kinase that responds to nutritional status and amino acid availability and is centrally involved in cell growth and proliferation (19,20). Activated mTOR is able to phosphorylate p70S6 kinase (21), which can inactivate eEF2 kinase (22) as well as phosphorylate the 40 S ribosomal protein S6 (23). Recent work has also suggested that AMPK can inhibit mTOR signaling through the phosphorylation of TSC2, an upstream regulator of mTOR (24). Because AMPK may inhibit protein synthesis via a number of different pathways, it is possible that AMPK is also a key regulator of cardiac hypertrophy.
AMPK is a serine/threonine protein kinase, which is activated by cellular stresses that deplete ATP (25). AMPK responds to increases in the AMP/ATP ratio by switching off ATP-consuming pathways and switching on pathways for ATP generation. It is a heterotrimeric protein comprised of a catalytic ␣ subunit and two regulatory subunits, ␤ and ␥ (26,27). The ␣ subunit contains the kinase domain, and phosphorylation at threonine 172 of this subunit results in increased AMPK activity (28). Although recent evidence indicates that AMPK activation inhibits proteins synthesis (17,29) and therefore possibly hypertrophy, AMPK activation has also been shown to correlate with the development of hypertrophy (30,31). For instance, pressure overload-induced hypertrophy has been shown to be associated with increased AMPK activity (30). In addition, AMPK has been linked to familial Wolff-Parkinson-White syndrome and hypertrophic cardiomyopathy via mutations in PRKAG2, the gene encoding the ␥ 2 subunit of AMPK (32)(33)(34). Although much has been learned about AMPK and protein synthesis and/or hypertrophic growth, the involvement of AMPK in the molecular mechanisms regulating cardiac hypertrophy is still unclear.
Although a number of intracellular signaling pathways have been implicated in the complex regulation of the hypertrophic response (see Ref. 35 for review), stimulation of the ␣1-adrenergic receptor with agonists, such as phenylephrine, induces a hypertrophic phenotype in primary cultures of cardiac myocytes (36). As a result, this has become an invaluable cellular model for the study of the hypertrophic response. In addition, the serine/threonine protein kinase Akt has been implicated in cardiac growth associated with physiological hypertrophy (37,38), and forced expression of constitutively activated Akt in the cardiac myocyte can be used as a separate model of cardiac hypertrophy. Furthermore, we have shown that activated Akt decreases the phosphorylation of AMPK at its primary activation site, Thr-172, resulting in an inhibition of activity of AMPK in the heart (39). Although cardiac energy metabolism was the focus in that study, the ability of Akt to negatively regulate AMPK has other implications, including protein translation and the hypertrophic response. Indeed, it is possible that a contributing factor to Akt-induced hypertrophy may be the reduction of a protein synthesis inhibitor such as AMPK. Investigating the role of AMPK in protein synthesis associated with cardiac hypertrophy in both models will allow a better understanding of the complex pathways involved in the hypertrophic response and may enable the development of new therapeutic strategies to prevent or inhibit pathological cardiac hypertrophy and improve heart function.

MATERIALS AND METHODS
Animal Care-The University of Alberta adheres to the principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences and complies with National Institutes of Health animal care guidelines.
Cell Culture-Cardiac myocytes were isolated from the hearts of 1-to 3-day-old neonatal rat pups essentially as described previously (39). The cells were plated on Primeria dishes (Falcon) at a density of 1.0 ϫ 10 6 cells/plate. For measurement of [ 3 H]phenylalanine incorporation, cells were plated on fibronectin-coated 12-well plates (Falcon) at a density of 3.3 ϫ 10 5 cells/well.
Cell Treatment-After 18 h of culture, neonatal rat cardiac myocytes were rinsed twice with serum-free DMEM/F-12 containing 50 g/ml gentamicin and cultured in serum-free DMEM/F-12 containing 50 g/ml gentamicin supplemented with 1ϫ ITSϩ3 liquid media supplement and 10 M cytosine ␤-D-arabinofuranoside to prevent the growth of fibroblasts. After 24 h of culture at 37°C in 5% CO 2 , cells were treated with vehicle (sterile ddH 2 O), 10 M phenylephrine (PE), or 5 mM metformin or 1 mM AICAR in both the presence and absence of 10 M PE in fresh serum-free media for 24 h at 37°C. For viral infections, neonatal rat cardiac myocytes were infected with Ad.GFP or Ad.myrAkt1 (40) adenovirus at the multiplicity of infection (m.o.i.) of 10 immediately after changing to serum free media, as previously described (39). After infection for 4 h cells were treated with vehicle, 5 mM metformin, or 1 mM AICAR in fresh serum-free media for 24 h. For immunoblot analysis purposes, cells were rinsed twice with ice-cold 1ϫ phosphate-buffered saline and 150 l of lysis buffer (20 mM Tris⅐HCL (pH 7.4), 50 mM NaCl, 50 mM NaF, 5 mM Na pyrophosphate, 0.25 M sucrose, 1% Triton X-100, mammalian protease inhibitor mixture, phosphatase inhibitor mixture I, and 1 mM dithiothreitol) was added to each plate after the appropriate treatment time. Cells were scraped and lysed for 10 min on ice and then centrifuged at 800 ϫ g for 10 min at 4°C. The protein concentration of the supernatant was then determined with the Bradford protein assay (Bio-Rad), and samples were subjected to SDS-PAGE and immunoblot analysis. To visualize changes in cell size, additional sets of cells were also treated as above and fixed with 3.7% paraformaldehyde, and fluorescence images were collected following immunocytochemistry using mouse anti-␣ actinin (Sigma) and Texas Red-conjugated donkey anti-mouse antibodies (Jackson Im-munoResearch), as described previously (41).
[ 3 H]Phenylalanine Incorporation-Myocytes were plated in 12-well plates and cultured as described above. [ 3 H]Phenylalanine (1 Ci/ml) was added at the time of drug treatment, and cells were incubated for 24 h at 37°C. Preparation of precipitates to be counted was performed as previously described (42). Briefly, cells were washed three times with ice-cold 1ϫ phosphate-buffered saline and incubated with 10% trichloroacetic acid for 1 h at 4°C to precipitate the proteins. The precipitates were washed twice with 95% ethanol, then dissolved and scraped in 1 M NaOH. The resulting solution, which contained the trichloroacetic acidinsoluble fraction, was neutralized with 1 M HCl, and the radioactivity was counted in a liquid scintillation counter.
Statistical Analysis-All data are presented as means Ϯ S.E. For comparison of three groups, analysis of variance followed by the Bonferroni multiple comparisons test was used for the determination of statistical analysis. A value of p Ͻ 0.05 was considered significant.

Effect of Metformin and AICAR on Protein Synthesis in Phenylephrine-treated Neonatal Rat Cardiac Myocytes-Neonatal
rat cardiac myocytes treated with 10 M phenylephrine displayed a significant 1.7-fold increase in protein synthesis over vehicle-treated cardiac myocytes (Fig. 1A). However, in the presence of 5 mM metformin, a known AMPK activator (44), phenylephrine-induced protein synthesis was significantly reduced (Fig. 1A). Another AMPK activator, AICAR (45), also significantly reduced phenylephrine-induced protein synthesis at a concentration of 1 mM (Fig. 1B) but to a lesser extent than metformin. A qualitative representation of the cell size for these treatments is shown in Fig. 1C.
Effect of Metformin and AICAR on AMPK Activation in Phenylephrine-treated Neonatal Rat Cardiac Myocytes-To confirm that metformin and AICAR treatment resulted in AMPK activation, lysates from neonatal rat cardiac myocytes treated with 10 M phenylephrine in the presence or absence of metformin or AICAR were subjected to immunoblot analysis using an antibody against the ␣ subunit of AMPK (␣-AMPK) phosphorylated at Thr-172. In cardiac myocytes treated with phenylephrine no significant change in ␣-AMPK phosphorylation was seen as compared with vehicle-treated cells ( Fig. 2A). However, the level of phosphorylated ␣-AMPK dramatically increased when cells were treated with either metformin or AICAR, independent of the presence of phenylephrine. The extent of increased ␣-AMPK phosphorylation was not statistically significant between metformin-and AICAR-treated cells (2.67 Ϯ 0.57 versus 1.81 Ϯ 0.62 arbitrary units). With either metformin or AICAR, AMPK is activated 2-to 3-fold, which correlates to a 40 -60% reduction in protein synthesis (Fig. 1, A  and B). Because phenylephrine-induced protein synthesis was not inhibited 100% by these agents, our data suggest that either AMPK needs to be more highly activated to completely inhibit phenylephrine-induced protein synthesis or that other regulatory pathways, independent from AMPK, also contribute to phenylephrine-induced protein synthesis.
Although phosphorylation at Thr-172 is indicative of the activation state of ␣-AMPK, we also immunoblotted with antiphospho-acetyl CoA carboxylase (ACC; Ser-79) antibody to ascertain whether altered phosphorylation of ␣-AMPK had any effects on a downstream AMPK target protein such as ACC (46). In accordance with AMPK activation, ACC phosphorylation was markedly elevated by both metformin and AICAR treatment relative to myocytes treated with vehicle alone (Fig.  2B). Because we have previously shown that there is cross-talk between Akt and AMPK in the cardiac myocyte (39), we also measured the levels of Akt phosphorylation using anti-phospho-Akt (Ser-473) antibody in these treated myocytes (Fig. 2C). Interestingly, AICAR treatment (not metformin) also stimulated the phosphorylation of Akt at its activating phosphorylation site. Although it is unknown why this occurs, AICAR can stimulate the adenosine receptor (47), which has been shown to result in increased Akt phosphorylation in the cardiac myocyte (48). Regardless of the mechanism, AICAR activation of Akt may explain why the observed effect of AICAR on protein synthesis inhibition was less than that of metformin (Fig. 1, A  and B).
Effect of AMPK Activation on p70S6 kinase, eEF2 Kinase, and eEF2 Phosphorylation in Phenylephrine-treated Neonatal Rat Cardiac Myocytes-To better understand the signaling mechanisms that control protein synthesis in phenylephrineinduced cardiac hypertrophy, cell lysates from cardiac myocytes treated as described above were subjected to immunoblot analysis using anti-phospho-p70S6 kinase (Thr-389 and Thr-421/Ser-424) antibodies. Phosphorylation at these sites is closely related to p70S6 kinase activity (49,50). Phenylephrine increased the phosphorylation status of p70S6 kinase (Fig. 3A), which was dramatically reduced in the presence of metformin or AICAR (Fig. 3A). Although both activators of AMPK resulted in decreased p70S6 kinase phosphorylation, AICAR was not as effective in decreasing p70S6 kinase phosphorylation as metformin. One possible explanation for this may be that AICAR also activates Akt (Fig. 2C), which has previously been shown to result in p70S6 kinase phosphorylation (51).
Cell lysates from cardiac myocytes treated as described above were also subjected to immunoblot analysis using antiphospho-eEF2 (Thr-56) and anti-phospho-eEF2 kinase (Ser-365) antibodies. Phenylephrine treatment of myocytes decreased the phosphorylation of eEF2, which is indicative of increased eEF2 activity and consistent with our previous findings (52). In contrast, treatment of cardiac myocytes with metformin or AICAR resulted in a dramatic increase in the phosphorylation of eEF2 at Thr-56 (Fig. 3B), consistent with the idea that phosphorylation by AMPK activates eEF2 kinase. Because phosphorylation of eEF2 at Thr-56 results in a decrease in eEF2 activity and inhibition of protein synthesis (9), the increases seen in response to metformin or AICAR may, at least in part, explain the decrease in protein synthesis we observed in Fig. 1.
Despite elevated p70S6 kinase phosphorylation in phenylephrine-treated myocytes, phosphorylation of eEF2 kinase at Ser-365 was not increased (22). In addition, AMPK activation did not lead to a reduction in eEF2 kinase phosphorylation at Ser-365 in metformin-or AICAR-treated myocytes, either after phenylephrine treatment or in untreated myocytes (Fig. 3C).

Effect of Metformin and AICAR on Protein Synthesis Induced by Forced Expression of Constitutively Active Akt1 in Neonatal
Rat Cardiac Myocytes-Although this report shows that AMPK activation is able to inhibit phenylephrine-induced protein synthesis, its effect on Akt-induced hypertrophy is unknown. Because we have previously shown that a regulatory interaction between Akt and AMPK exists (39), we speculated that Akt might negatively regulate phosphorylation of ␣-AMPK to promote protein synthesis associated with cardiac hypertrophy. To investigate this, neonatal rat cardiac myocytes were cultured and infected with a recombinant adenovirus expressing constitutively active Akt1 (Ad.myrAkt1) or GFP (Ad.GFP) as a control and, in some cases, treated with metformin or AICAR. Cardiac myocytes infected with Ad.myrAkt1 displayed dramatic increases in the expression of total Akt protein levels and a corresponding increase in the level of Akt phosphorylated at Ser-473, indicating increased Akt activity (53) (Fig. 4A). In addition, cardiac myocytes expressing myrAkt1 displayed a significant 1.5-fold increase in protein synthesis over cardiac myocytes infected with the control virus (Fig. 4B). This Aktinduced increase in protein synthesis was significantly reduced by treatment with either metformin or AICAR (Fig. 4B). Qualitative assessment of cell size also suggests that AMPK activation can reduce Akt-induced hypertrophy (Fig. 4C).
Effect of Metformin and AICAR on AMPK Activation in Neonatal Rat Cardiac Myocytes Transduced with Constitutively Active Akt1-To confirm that metformin and AICAR treatment resulted in AMPK activation, lysates from neonatal rat cardiac myocytes transduced with constitutively active Akt1 and subsequently treated with metformin or AICAR were subjected to immunoblot analysis using anti-phospho-␣-AMPK (Thr-172) antibody. Cardiac myocytes infected with Ad.myrAkt1 dis-played reduced levels of ␣-AMPK phosphorylation as compared with control infected cells (Fig. 5A, lanes 1 and 2). With the addition of metformin or AICAR, the inhibitory effect of Ad.myrAkt1 on ␣-AMPK phosphorylation was less pronounced, and phosphorylated ␣-AMPK levels still exceeded basal levels After incubation, all cells were lysed, and proteins were precipitated and counted as described under "Materials and Methods." C, a qualitative representation of the cell size for these treatments is shown in fixed cells by indirect immunofluorescent staining using anti-␣-actinin antibody followed by Texas Red-conjugated anti-mouse antibody. Values are means Ϯ S.E. from at least four independent experiments with each experiment performed in duplicate. *, significantly different from PE treatment (p Ͻ 0.001); #, significantly different from PE treatment (p Ͻ 0.01), assessed by one-way analysis of variance followed by Bonferroni multiple comparisons test. observed in the control cells (Fig. 5A, lanes 4 and 6). Furthermore, this increase in ␣-AMPK phosphorylation corresponded to a marked increase in phosphorylation of ACC at Ser-79 (Fig.  5B). Neither expression of constitutively active Akt1 nor drug treatments had any effect on the overall expression of the ␣ subunit of AMPK protein or either of the two ACC isoforms.
Effect of AMPK Activation on p70S6 Kinase, eEF2 Kinase, and eEF2 Phosphorylation in Neonatal Rat Cardiac Myocytes Transduced with Constitutively Active Akt1-Cell lysates from cardiac myocytes expressing constitutively active Akt1 were subjected to immunoblot analysis using anti-phospho-p70S6 kinase (Thr-389 and Thr-421/Ser-424) antibodies. Expression of constitutively active Akt increased the phosphorylation status of p70S6 kinase (Fig. 6A). In the presence of metformin or AICAR, these Akt-induced changes in phosphorylation of p70S6 kinase were dramatically reduced (Fig. 6A). Cell lysates from cardiac myocytes treated as described above were also subjected to immunoblot analysis using anti-phospho-eEF2 (Thr-56) and anti-phospho-eEF2 kinase (Ser-365) antibodies. Treatment of cardiac myocytes with metformin or AICAR resulted in a dramatic increase in the phosphorylation of eEF2 at Thr-56 (Fig. 6B), indicating eEF2 kinase activation. However, despite elevated p70S6 kinase phosphorylation in Akt-expressing myocytes, changes in eEF2 kinase phosphorylation at Ser-365 in most of the treatment groups was not observed (Fig. 6C). Nonetheless, statistical analysis of phospho-eEF2 kinase im-munoblots normalized to total eEF2 kinase immunoblots revealed that there is a significant increase in eEF2 kinase phosphorylation in the Akt ϩ AICAR group as compared with the GFP ϩ AICAR group (1.81 Ϯ 0.32 versus 1.19 Ϯ 0.15 arbitrary units, p Ͻ 0.05). The reasons for this increase are currently unknown. Neither expression of constitutively active Akt1 nor the drug treatments had any effect on the overall levels of total eEF2 and eEF2 kinase. DISCUSSION Although AMPK activation has been shown to regulate protein synthesis, it was not known whether this regulates cardiac hypertrophic growth. Furthermore, because cardiac myocyte hypertrophy can occur as a result of a number of independent mechanisms, we sought to investigate the role of AMPK activation in regulating protein synthesis during both phenylephrine-and Akt-induced cardiac hypertrophy. With these treatments, cardiac myocytes demonstrated an apparent increase in cell size, which was associated with a significant increase in protein synthesis (Figs. 1 and 4). However, protein synthesis was significantly decreased in the presence of known activators of AMPK, namely metformin and AICAR. Although it is possible that metformin and AICAR may regulate protein synthesis independently of AMPK, these compounds are from two chemically distinct classes of drugs (45, 54) that activate AMPK by different mechanisms (45,55). This supports the idea that the Representative immunoblots from four independent experiments performed using anti-phospho-eEF2 (Thr-56) (B) and anti-phospho-eEF2 kinase (Ser-365) (C) antibodies are also shown. Anti-eEF2 (B) and anti-eEF2 kinase (C) antibodies served as a loading control for total eEF2 protein and eEF2 kinase, respectively. mechanism by which metformin and AICAR inhibit protein synthesis is via AMPK activation. Indeed, both metformin and AICAR dramatically increased ␣-AMPK phosphorylation at Thr-172 and increased phosphorylation of ACC at Ser-79, a direct substrate of AMPK (Figs. 2A, 2B, and 5). In the presence of phenylephrine or constitutively active Akt, both metformin and AICAR were able to activate AMPK, suggesting that phenylephrine or constitutively active Akt do not alter the mecha-nisms by which these agents activate AMPK, although Akt1 does attenuate the effects of metformin and AICAR. Together, these data support the notion that there is a tight association between AMPK and cardiac myocyte growth.
In this report, we provide evidence that AMPK-induced inhibition of protein synthesis may be, in part, mediated via decreased p70S6 kinase activation. Despite this decrease in p70S6 kinase phosphorylation seen in cardiac myocytes treated . Because Ser-365 of eEF2 kinase is phosphorylated by p70S6 kinase, it is surprising that we did not observe an alteration in its phosphorylation. However, Ser-365 of eEF2 kinase is also a substrate for p90 RSK1 (22), and it is possible that p90 RSK1 maintained the phosphorylation of eEF2 kinase despite the inhibition of mTOR signaling. Given the recent data by Cheng et al. (18), it is possible that AMPK directly phosphorylates and inactivates mTOR, resulting in the observed decrease in p70S6 kinase phosphorylation. In addition, Inoki et al. (24) showed that AMPK phosphorylation of TSC2, an upstream regulator of mTOR, enhances TSC2 activity and thus inhibits p70S6 kinase phosphorylation via the mTOR pathway. As decreased p70S6 kinase phosphorylation did not correlate with changes in eEF2 kinase phosphorylation at Ser-365 in eEF2 kinase, it is also possible that p70S6 kinase plays no role in AMPK-induced inhibition of eEF2 but that it controls protein synthesis via an alternative pathway. This may include the phosphorylation of the 40 S ribosomal protein S6, which has also been proposed as a rapamycin-sensitive mechanism by which p70S6 kinase can activate the translation of 5Ј-TOP (tract of oligopyrimidine) mRNAs encoding for ribosomal proteins and elongation factors (56,57). Because this effect contributes to a long term increase in the translational capacity of the cell, AMPK-induced inhibition of p70S6 kinase may contribute to the decreased rate of protein synthesis in these experiments, which were performed over a time period of 24 h. Future studies will investigate the role of the 40 S ribosomal protein S6 in this process.
Although we did not observe changes in phosphorylation of eEF2 kinase at Ser-365, both phenylephrine treatment and Akt overexpression did induce decreased levels of eEF2 phosphorylation (Figs. 3B and 6B) in cardiomyocytes, presumably contributing to increased eEF2 activity and accelerated peptide elongation. Furthermore, the decrease in eEF2 phosphorylation resulting from phenylephrine treatment and Akt overexpression can be completely reversed by AMPK activation (Figs. 3B and 6B). Indeed, our data show that metformin-and AICAR-induced activation of AMPK result in a dramatic increase in eEF2 phosphorylation. This robust response suggests that AMPK activation ensures that little protein synthesis occurs during times of depleted energy supply, which may be important given that protein synthesis consumes a high proportion of the cell's energy. Because eEF2 is the only known substrate for eEF2 kinase (9), it is likely that the direct downstream target of AMPK is eEF2 kinase and not eEF2. Indeed, we have recently shown that AMPK activation results in the phosphorylation and activation of eEF2 kinase (16). This occurs as a result of the increased phosphorylation of eEF2 kinase at a novel site, Ser-398, not previously shown to be regulated by upstream kinases, but now known to be a direct substrate for AMPK (16). It is tempting to speculate that AMPK activation results in increased eEF2 kinase phosphorylation at Ser-398, suggesting that increased eEF2 phosphorylation and decreased protein synthesis are mediated by AMPK . Representative immunoblots of four independent experiments performed using anti-phospho-eEF2 (Thr-56) (B) and anti-phospho-eEF2 kinase (Ser-365) (C) antibodies are also shown. Anti-eEF2 (B) and anti-eEF2 kinase (C) antibodies served as a loading control for total eEF2 protein and eEF2 kinase, respectively. through the phosphorylation and activation of eEF2 kinase. Although cardiac eEF2 kinase does undergo phosphorylation at this site when treated with AMPK (16), the low levels of eEF2 kinase present in cardiomyocytes have so far precluded showing that treatments that activate AMPK actually increase eEF2 kinase phosphorylation.
We have previously shown that Akt activation negatively regulates AMPK activity (39). In addition to confirming the initial observation, the present data also provide further clues as to how Akt may control myocyte growth based on cellular energy status. The ability of Akt to negatively regulate AMPK may partially explain why Akt signaling can regulate cardiac growth during postnatal development (37,38,58). This inactivation of AMPK also suggests another mechanism by which Akt can promote cardiac hypertrophy. That is, it is possible that a contributing factor to Akt-induced hypertrophy may be the reduction of a protein synthesis inhibitor such as AMPK. In contrast to these data, the inhibition of AMPK does not appear to be a necessary component of hypertrophic growth induced by phenylephrine ( Fig. 2A). Indeed, our data do not support the concept that AMPK is a necessary component of all hypertrophic signaling mechanisms. However, our data do show that AMPK activation can exert inhibitory effects on at least two signaling pathways involved in stimulating cardiac hypertrophy. This suggests that at least one functional consequence of the pharmacological activation of AMPK is the inhibition of protein synthesis, which may prevent hypertrophic growth.
Despite the evidence presented in this study, the role of AMPK in cardiac hypertrophy has not yet been clearly defined. In direct opposition to the results found in this study, Tian et al. (30) have shown that increased AMPK activity is associated with the development of pressure-overload induced cardiac hypertrophy. Although the reasons for this discrepancy are currently unknown, it may be due to the fact that AMPK can both regulate, and be regulated by, cardiac energy status. Because the hypertrophic heart displays a switch in substrate utilization involving a decrease in fatty acid oxidation and an increase in glucose utilization, it is likely that the progression of hypertrophy is associated with an energetically compromised heart. If this were the case, then AMPK would become active in response to depleted ATP supply. At this point it is assumed that an increase in AMPK activity would inhibit protein synthesis via activation of eEF2 kinase phosphorylation and subsequent inhibition of eEF2 and/or inhibition of the p70S6 kinase pathway. Therefore, it is tempting to speculate that pharmacological activation of AMPK during the onset of pressure-overload hypertrophy would slow or prevent the progression of hypertrophic growth.
Recent evidence has also linked activation of AMPK with the induction of hypertrophic growth in the heart (31). Transgenic mice overexpressing a PRKAG2 mutation results in increase AMPK activity, which precedes hypertrophic growth (31). However, the elevation of AMPK activity in these transgenic mice also resulted in glycogen accumulation (31), which itself has been shown to cause cardiac hypertrophy (59). Therefore, it is unknown if AMPK activation directly causes hypertrophy or whether it occurs as a result of glycogen accumulation, secondary to AMPK activation. Although our data clearly show that AMPK activation can inhibit protein synthesis associated with two cellular models known to cause myocyte hypertrophy, it is likely that AMPK activation may not be able to inhibit all the pathways known to promote cardiac myocyte growth. This may be the case with glycogen-induced cardiac hypertrophy.
Taken together, our data show that AMPK activation can exert inhibitory effects on at least two signaling pathways involved in stimulating cardiac hypertrophy. Our data also show that a major effect of AMPK activation is to inhibit protein synthesis via the eEF2 kinase/eEF2 signaling axis and/or the p70S6 kinase pathway. Although it is not known whether AMPK activation also inhibits other signaling components involved with hypertrophic growth, the inhibition of protein synthesis by AMPK may be a regulatory mechanism controlling hypertrophic growth. This raises the possibility that pharmacological activation of AMPK can prevent or slow the progression of cardiac hypertrophy in animal models of cardiac hypertrophy or in human patients.