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Am J Physiol Heart Circ Physiol 293: H3456-H3464, 2007. First published September 28, 2007; doi:10.1152/ajpheart.00936.2007
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The AMPK {gamma}1 R70Q mutant regulates multiple metabolic and growth pathways in neonatal cardiac myocytes

Karalyn D. Folmes,1 Lee A. Witters,2 Michael F. Allard,3 Martin E. Young,4 and Jason R. B. Dyck1

1Cardiovascular Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada; 2Dartmouth Medical School, Hanover, New Hampshire; 3James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, Department of Pathology and Laboratory Medicine, University of British Columbia-St Paul's Hospital, Vancouver, British Columbia, Canada; and 4Children's Nutrition Research Center, Baylor College of Medicine, Houston, Texas

Submitted 13 August 2007 ; accepted in final form 25 September 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although mutations in the {gamma}-subunit of AMP-activated protein kinase (AMPK) can result in excessive glycogen accumulation and cardiac hypertrophy, the mechanisms by which this occurs have not been well defined. Because >65% of cardiac AMPK activity is associated with the {gamma}1-subunit of AMPK, we investigated the effects of expression of an AMPK-activating {gamma}1-subunit mutant ({gamma}1 R70Q) on regulatory pathways controlling glycogen accumulation and cardiac hypertrophy in neonatal rat cardiac myocytes. Whereas expression of {gamma}1 R70Q displayed the expected increase in palmitate oxidation rates, rates of glycolysis were significantly depressed. In addition, glycogen synthase activity was increased in cardiac myocytes expressing {gamma}1 R70Q, due to both increased expression and decreased phosphorylation of glycogen synthase. The inhibition of glycolysis and increased glycogen synthase activity were correlated with elevated glycogen levels in {gamma}1 R70Q-expressing myocytes. In association with the reduced phosphorylation of glycogen synthase, glycogen synthase kinase (GSK)-3β protein and mRNA levels were profoundly decreased in the {gamma}1 R70Q-expressing myocytes. Consistent with GSK-3β negatively regulating hypertrophy via inhibition of nuclear factor of activated T cells (NFAT), the dramatic downregulation of GSK-3β was associated with increased nuclear activity of NFAT. Together, these data provide important new information about the mechanisms by which a mutation in the {gamma}-subunit of AMPK causes altered AMPK signaling and identify multiple pathways involved in regulating both cardiac myocyte metabolism and growth that may contribute to the development of the {gamma} mutant-associated cardiomyopathy.

adenosine 5'-monophosphate-activated protein kinase; glycogen; metabolism; glycogen synthase kinase-3β

AMP-ACTIVATED PROTEIN KINASE (AMPK) is a heterotrimeric serine/threonine kinase consisting of a catalytic {alpha}-subunit and regulatory β- and {gamma}-subunits. Of these two regulatory subunits, the {gamma}-subunit is thought to "sense" the cellular energy state by virtue of its ability to bind ATP and AMP (10). In most mammalian cell types, AMPK is activated as a means to maintain adequate ATP levels during times of metabolic stress (22). In the heart, activation of AMPK by ischemia is associated with increased glucose uptake (37) and stimulation of ATP production via both increased glycolysis (33) and fatty acid oxidation (38). In addition, AMPK activation has been suggested to be involved in the regulation of muscle glycogen levels (5). However, the effects of AMPK activation on the pathways regulating myocardial glycogen levels are controversial. For example, in the stressed heart, AMPK activation has been shown to partition glucose away from glycogen synthesis in favor of glycolysis (24). In contrast, absence of the AMPK {alpha}2-subunit resulted in reduced myocardial glycogen storage (44). Consistent with AMPK playing a central role in glycogen metabolism, activating mutations in one of the isoforms of the {gamma}-subunit isoforms of AMPK found in the heart ({gamma}2 N488I) results in a dramatic increase in cardiac glycogen levels (2, 3), indicating that AMPK activation either promotes glycogen synthesis or inhibits glycogenolysis. Interestingly, a separate mutation in the {gamma}2-subunit ({gamma}2 R302Q) also promotes glycogen accumulation in the heart, yet this mutation has been reported to be an inactivating mutation of AMPK (12, 40). Therefore, although the role of AMPK in the regulation of myocardial glucose uptake, glycolysis, and fatty acid oxidation appears to be well established, the mechanism by which AMPK activation controls myocardial glycogen levels has yet to be clearly defined.

Since the {gamma}-subunits of AMPK are important regulators of AMPK activity, considerable research effort has focused on characterizing these subunits. To date, three isoforms of the AMPK {gamma}-subunit have been identified, each with its own expression profile (10). The {gamma}1-subunit is ubiquitously expressed, whereas {gamma}2 is found at high levels in both the heart and the brain and {gamma}3 is expressed almost exclusively in the skeletal muscle (10). All {gamma}-subunits contain two tandem Bateman domains that are sites of allosteric regulation by adenosine-containing nucleotides (7, 39). Studies involving the mutations within the Bateman domains of the {gamma}-subunits have shown that the mutations alter either the basal activation of AMPK and/or the response of AMPK to AMP or ATP (20, 39, 45). These mutations are thought to mimic the allosteric activation of AMP and may make the AMPK holoenzyme a better substrate for upstream kinases such as LKB1, leading to increased phosphorylation of AMPK{alpha} and activation of the enzyme (45). One of the better understood mutations within the CBS domain of the {gamma}-subunit is the {gamma}1 R70Q mutation, which results in increased basal AMPK activity in a number of cell types (20) as well as in intact skeletal muscle (6). As such, expression of the {gamma}1 R70Q mutation may provide an alternative genetic means to activate AMPK in the cardiac myocyte as well as potentially providing insight into the effects of {gamma} mutations on the cellular function of AMPK.

Utilizing the {gamma}1 R70Q mutation as a mechanism to activate AMPK in the cardiac myocyte has several advantages over the more commonly used methods such as the nonspecific AMPK agonists or the constitutively active truncated/mutated form of AMPK{alpha} (31). Interestingly, the AMPK-truncated mutant does not activate AMPK in the neonatal rat cardiac myocyte in our hands as it does in other cell types such as the hepatocyte (Ref. 23 and unpublished data). In addition, using the {gamma}1 R70Q mutation to genetically activate AMPK also may provide insights into how mutations in the {gamma}-subunits cause a cardiomyopathy characterized by 1) ventricular preexcitation and 2) hypertrophy and excessive glycogen accumulation (2, 8, 18). Currently, the mechanisms that promote the latter of these cardiac abnormalities in patients with {gamma}2 mutations are not well understood, although some contributing pathways have recently been identified (32). In addition, transgenic mouse models of {gamma}2-subunit mutations recapitulate the human condition, indicating that this is not a polygenic phenotype but the result of a single mutation (3, 40). Despite the obvious importance of the {gamma}2-subunit in controlling AMPK activity, cardiac glycogen accumulation, and cardiac hypertrophy, the AMPK complexes containing the {gamma}1-subunit account for >65% of the AMPK activity in the heart (30). As such, it is possible that expression of a {gamma}1 mutant may induce a more dramatic phenotype than that observed in the {gamma}2 mutant-expressing mice. Although a transgenic mouse model of the {gamma}1 R70Q mutation has been developed, the cDNA for the {gamma}1 R70Q mutation is driven by the skeletal muscle {alpha}-actin promoter, resulting in only very low-level expression in the hearts of these animals (6). Thus it is not surprising that these mice do not display cardiac glycogen accumulation (6) or myocardial hypertrophy (unpublished data). What effect, if any, higher level of expression of the {gamma}1 R70Q mutation in cardiac myocytes has on AMPK activity, cardiac energy metabolism, glycogen accumulation, or cardiac hypertrophy is currently unknown.

Therefore, the aims of this study were to 1) characterize the metabolic effects of expression of the AMPK-activating {gamma}1 R70Q mutation in cardiac myocytes, 2) determine whether expression of the {gamma}1 R70Q mutation in cardiac myocytes recapitulates aspects of the phenotype observed with expression of the {gamma}2 mutations, and 3) identify early molecular signaling events that may contribute to {gamma} mutation-induced glycogen accumulation and/or cardiac hypertrophy. Since glycogen accumulation and/or cardiac hypertrophy occurs in hearts expressing mutated versions of the {gamma}-subunit, activation of AMPK via expression of the {gamma}1 R70Q mutation should provide valuable insight into the mechanisms involved in regulating the metabolic pathways and signaling events that contribute to development of the cardiac phenotype observed in patients with {gamma}-subunit mutations.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care. The University of Alberta complies with National Institutes of Health animal care guidelines and adheres to the principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences. The University of Alberta approved the experimental procedures involving animals presented in this study.

Cell culture. Cardiac myocytes were isolated from 1- to 3-day-old neonatal rat hearts and plated at a density of 2.0 x 106 cells/plate as previously described (26).

Cell treatment. After 18 h of culture, neonatal rat cardiac myocytes were cultured in serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham's (HyClone) containing 50 µg/ml gentamicin (Invitrogen), 1x insulin/transferrin/sodium selenite (ITS)+3 liquid media supplement (Sigma), and 10 µM cytosine β-D-arabinofuranoside (Sigma) to prevent the growth of fibroblasts. For viral infections, neonatal rat cardiac myocytes were infected with Ad.GFP, Ad.AMPK{gamma}1wt, or Ad.AMPK{gamma}1R70Q adenovirus at the multiplicity of infection (MOI) of 20 as previously described (26). For nuclear factor of activated T cells (NFAT) experiments, cells were infected with Ad.NFAT-Luc-Promoter adenovirus (Seven Hills Bioreagents) at the MOI of 10, along with the above viruses. This construct expresses multiple NFAT binding sites linked to the luciferase promoter gene via the {alpha}-myosin heavy chain promoter (43). Twenty-four hours after infection at 37°C in 5% CO2, cells were treated with 1 mM 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) or 10 µM phenylephrine in fresh serum-free medium for 24 h at 37°C where indicated. For metabolic rate experiments, cells were incubated with 1 µCi/ml D-[5-3H]glucose (Perkin Elmer Life Sciences) in serum-free DMEM/F-12 Ham's or with 1.2 mM palmitate containing 0.5 µCi/ml [9,10-3H-(N)]palmitic acid (Perkin Elmer Life Sciences) prebound to 3% fatty acid-free BSA (Sigma) for 3 h. Forty-eight hours after infection at 37°C in 5% CO2, cells were harvested as described in the appropriate protocol below.

Measurement of metabolic rates. Following treatment with radiolabeled substrate, an aliquot of the cell medium was removed and the vapor transfer method was utilized to determine metabolic rates essentially as previously described (16). Following sampling of the medium, cells were scraped and lysed as described by Kovacic et al. (26). The protein concentration of the supernatant was then determined with the Bradford protein assay (Bio-Rad), and radioactivity counts were standardized to protein content.

Measurement of AMPK activity. The AMPK {gamma}1-isoform was immunoprecipitated from 100 µg of protein from cell homogenates with anti-{gamma}1 antibody (Kinasource) and subjected to an AMPK peptide substrate activity assay as described previously (27).

Measurement of NFAT activity. Cells were lysed, and luminescence was detected using the Luciferase Assay System (Promega) according to the manufacturer's instructions. Luminescence counts were standardized to protein content.

Measurement of glycogen content and glycogen synthesis rates. Cells were treated for 24 h with 1 µCi/ml D-[5-3H]glucose and harvested in 1x PBS with protease inhibitor mixture, phosphatase inhibitor mixture I, and 1 mM dithiothreitol and centrifuged at 800 g for 10 min at 4°C, and the cell pellet was kept. Glycogen in the cell pellet was converted to glucose by reacting with 4 M H2SO4. The glucose concentration was determined using a Sigma glucose analysis kit and was standardized to cell pellet mass, and the rate of glycogen synthesis was determined by measuring the number of micromoles of D-[5-3H]glucose incorporated into glycogen during the 3 h treatment time.

Measurement of glycogen synthase activity. Activity was measured in cell homogenates as percent activity in the presence of 0.25 mM glucose 6-phosphate (G6P) over 15 mM G6P, essentially as described (19).

Measurement of protein synthesis. Myocytes were plated in 12-well plates, and 24 h after infection, they were treated with 1 µCi/ml [3H]phenylalanine for 24 h at 37°C. Preparation of precipitates was performed as previously described (17). Briefly, cells were washed in 1x phosphate-buffered saline (PBS) and incubated with 10% trichloroacetic acid for 1 h at 4°C to precipitate the proteins. The precipitates were washed with 95% ethanol and then harvested in 1 M NaOH. The resulting solution, which contained the trichloroacetic acid-insoluble fraction, was neutralized with 1 M HCl, and the radioactivity was counted in a liquid scintillation counter.

Quantitative reverse transcriptase-polymerase chain reaction analysis. Cells were pelleted in 1x PBS as described above. RNA extraction and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) of samples were performed as previously described (42). 5'-GAGAACGCAGTGCTTTTCGA-3', 5'-TCATCCCCTGTCACCTTCG-3', and 5'-FAM-AGGTGGCCAACAAGGTGGGTGGCAT-TAMRA-3' were used as the forward primer, reverse primer, and probe for muscle glycogen synthase (gys1), respectively. 5'-GCACCGAGAGGCTCTCAGAT-3', 5'-GGAAAAGCCCTGCTCAGTGT-3', and 5'-FAM-TCTGGCATGCTGGTAATATCTGCCCA-TAMRA-3' were used as the forward primer, reverse primer, and probe for liver glycogen synthase (gys2), respectively. 5'-GGTGTGGATCAGTTGGTGGA-3', 5'-TGCCTTGATTTGAGGGAATTT-3', and 5'-FAM-AAAGGTCCTAGGAACACCAACAAGGGAGCA-TAMRA-3' were used as the forward primer, reverse primer, and probe for GSK-3β (gsk-3β), respectively.

Immunoblot analysis. Cells were harvested and protein concentration determined as described in Measurement of metabolic rates. Boiled cell homogenate samples were subjected to SDS-PAGE in 5, 8, or 10% acrylamide gels and transferred onto nitrocellulose as previously described (41). Membranes were blocked in 5% milk-1x Tris-buffered saline (TBS)-0.1% Tween 20 and then immunoblotted at 1:1,000 dilution (unless otherwise specified) with either rabbit anti-phospho-AMPK{alpha} (Thr172), rabbit anti-AMPK{alpha}, rabbit anti-GSK-3β, rabbit anti-phospho-glycogen synthase (Ser641/645), rabbit anti-glycogen synthase, goat anti-actin, rabbit anti-phospho-acetyl CoA carboxylase (Ser79) antibody, or peroxidase-labeled streptavidin (1:500 dilution) in 5% BSA-1x TBS-0.1% Tween 20 overnight at 4°C. After washing, the membranes were incubated with peroxidase-conjugated goat anti-rabbit or donkey anti-goat secondary antibody in 5% milk-1 x TBS-0.1% Tween 20, with the exception of peroxidase-labeled streptavidin-incubated immunoblots. Signals were visualized using the Amersham Biosciences enhanced chemiluminescence Western blotting detection system.

Statistical analysis. All data are means ± SE. Student's t-test was used for the determination of statistical significance. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of {gamma}1 R70Q activates AMPK and increases palmitate oxidation rates in neonatal rat cardiac myocytes. Using our model system, we showed that expression of {gamma}1 R70Q in the cardiac myocyte leads to a twofold increase (P < 0.01) in phosphorylation of AMPK{alpha} on Thr172 compared with the wild-type {gamma}1-subunit (Fig. 1A). Since phosphorylation of Thr172 has been associated with activation of the AMPK, we immunoprecipitated the {gamma}1-subunit and measured the associated AMPK activity. Consistent with the increased phosphorylation of AMPK{alpha} at Thr172, AMPK activity was significantly increased in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (P < 0.05, Fig. 1B).


Figure 1
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  		Fig. 1. Expression of the {gamma}1 R70Q mutation in neonatal rat cardiac myocytes increases AMP-activated protein kinase (AMPK) activity and phosphorylation of acetyl-CoA carboxylase (ACC) compared with the wild-type {gamma}1-subunit ({gamma}, WT). A: representative immunoblot and densitometry of cell homogenates probed with anti-phospho-AMPK{alpha} (Thr172) and anti-AMPK{alpha} antibodies (n = 4–8). B: AMPK activity was measured in {gamma}1 immunoprecipitated cell homogenates in units of picomoles of [{gamma}-32P]ATP incorporated per minute (n = 4). C: representative immunoblot and densitometry of cell homogenates probed with anti-phospho-ACC antibody and peroxidase-labeled streptavidin (n = 11). All data are normalized to Ad.GFP. Asterisks denote statistical significance as indicated. {gamma}1 wt, wild-type {gamma}1-subunit.

 
To confirm the intracellular activation of AMPK, we examined the phosphorylation status of both isoforms of acetyl-CoA carboxylase (ACC{alpha}, ACCβ). As expected with activation of AMPK, phosphorylation of both the ACC isoforms was increased more than twofold (P < 0.01) in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (Fig. 1C). In accordance with phosphorylation and inhibition of ACC, there was nearly a doubling of palmitate oxidation rates in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (P < 0.001, Fig. 2A). This increase in palmitate oxidation was comparable to that seen with the AMPK activator AICAR (1.89- and 1.96-fold increase vs. adenoviral control for Ad{gamma}1R70Q and AICAR, respectively).


Figure 2
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  		Fig. 2. Palmitate oxidation rates are increased, glycolytic rates are decreased, and glycogen content is increased in neonatal rat cardiac myocytes expressing the {gamma}1 R70Q mutation. Metabolic rates were determined using tritiated substrates and expressed as fold change in nanomoles of palmitate oxidized per gram of protein per minute (typical rates of 300–800 nmol·g–1·min–1, n = 4; A) or nanomoles of glucose metabolized per gram of protein per minute (typical rates of 2,500–9,500 nmol·g–1·min–1, n = 4; B). Glycogen synthesis rates are expressed as fold change in nanomoles of D-[5-3H]glucose incorporated into glycogen per gram of cells (typical rates of 130–560 nmol/g, n = 4; C). Glycogen content is expressed as fold change in micromoles of glucose from glycogen per gram of cells (n = 4; D). Data are normalized to Ad.GFP. Asterisks denote statistical significance as indicated.

 
Expression of {gamma}1 R70Q modulates glucose handling in neonatal rat cardiac myocytes. In contrast to the predicted effects of increased AMPK activity on glycolysis, glycolytic rates were dramatically decreased in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (P < 0.001, Fig. 2B). In addition, there was a more than twofold increase in both the rates of glycogen synthesis and total glycogen content in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (P < 0.05 and P < 0.005, Fig. 2, C and D, respectively). Consistent with this elevated level of glycogen, there was a significant increase in glycogen synthase activity (P < 0.05, Fig. 3A) and a reduction in the ratio of inhibitory phosphorylation of glycogen synthase at serine residues 641/645 to total glycogen synthase protein (P < 0.05, Fig. 3B), along with elevated levels of glycogen synthase protein (P < 0.05, Fig. 3B) in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit. On the basis of the increase in glycogen synthase protein levels, we measured glycogen synthase transcript levels and found that muscle glycogen synthase (gys1) levels were increased nearly 2.5-fold in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (P < 0.01, Fig. 3C). However, unlike the upregulation of the liver isoform of glycogen synthase (gys2) observed in the muscle-specific {gamma}1 R70Q transgenic model (6), we found no change in the transcript level of gys2 in cardiac myocytes expressing the {gamma}1 R70Q mutation (data not shown).


Figure 3
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  		Fig. 3. Glycogen synthase activity is increased in neonatal rat cardiac myocytes expressing the {gamma}1 R70Q mutation resulting from increased glycogen synthase protein levels and reduced phosphorylation. A: glycogen synthase activity is expressed as fold change in percent activation compared with stimulation with 15 mM glycogen-6-phosphate (n = 4–8). B: representative immunoblot and densitometry of cell homogenates probed with anti-phospho-glycogen synthase (Ser641/645), anti-glycogen synthase, and anti-actin antibodies (n = 5–6). C: transcript levels of gys1 were determined by quantitative RT-PCR and normalized to the housekeeping gene cyclophilin (n = 5–6). Data are normalized to Ad.GFP. Asterisks denote statistical significance as indicated.

 
Expression of {gamma}1 R70Q decreases GSK-3β expression and induces NFAT translocation to the nucleus in neonatal rat cardiac myocytes. Because the ratio of phosphorylated to total glycogen synthase was decreased in cells expressing the {gamma}1 R70Q mutation, we investigated the kinase responsible for phosphorylation at these sites, GSK-3β. Since phosphorylation of GSK-3β at Ser9 is inversely correlated with its activity, we examined the phosphorylation of GSK-3β at this residue in cell homogenates using immunoblot analysis. Surprisingly, GSK-3β phosphorylation was not different in any of the groups when normalized to total GSK-3β protein levels (data not shown). However, GSK-3β protein levels were decreased more than 50% in cells expressing the {gamma}1 R70Q subunit mutation compared with the wild-type {gamma}1-subunit (P < 0.01, Fig. 4A). This appears to be specific to the mutation, since activation of AMPK in neonatal rat cardiac myocytes via either treatment with phenformin or expression of the LKB1/MO25/STRAD complex does not significantly alter GSK-3β protein levels compared with control cells (phenformin treatment: 1.19 ± 0.55-fold increase, n = 3; LKB1/MO25/STRAD treatment: 1.14 ± 0.39-fold increase, n = 3). The reduction in GSK-3β protein in cells expressing the {gamma}1 R70Q subunit mutation was likely due to decreased mRNA levels, since gsk-3β transcript content was also reduced nearly 50% in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (P < 0.01, Fig. 4B).


Figure 4
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  		Fig. 4. Nuclear factor of activated T cells (NFAT)-directed transcriptional activity is increased due to a reduction of glycogen synthase kinase (GSK)-3β in neonatal rat cardiac myocytes expressing the {gamma}1 R70Q mutation. A: representative immunoblot and densitometry of cell homogenates probed with anti-GSK-3β and anti-actin antibodies (n = 7). B: transcript levels of gsk-3β were determined by quantitative RT-PCR and normalized to the housekeeping gene cyclophilin (n = 5–6). C: NFAT transcriptional activity was measured as luciferase activity per gram of protein. Data are normalized to Ad.GFP (n = 4–5). Asterisks denote statistical significance as indicated.

 
Although GSK-3β plays a major role in the regulation of glycogen synthesis, it also regulates cardiac hypertrophic growth by phosphorylating the transcription factor NFAT, thereby preventing NFAT activation of the hypertrophic gene program (1, 21). Given this role of GSK-3β, we determined whether expression of the {gamma}1 R70Q mutation had an effect on NFAT signaling. To do this, we utilized an adenovirus expressing the luciferase reporter gene under transcriptional control of an NFAT-responsive promoter (43). Because adenoviral DNA must be delivered to the nucleus to access the transcriptional machinery, only nuclear NFAT can induce transcription of luciferase mRNA, leading to the eventual production of luciferase protein. As predicted by the downregulation of GSK-3β, activity of the NFAT-driven reporter construct was significantly increased in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (P < 0.05, Fig. 4C). Although there was no observable change in cell size at 48 h of expression in any of the groups, this is not surprising given that expression of the {gamma}1 R70Q mutation is likely a very subtle hypertrophic stimulus compared with phenylephrine treatment. However, although not significantly different, protein synthesis rates were increased 1.31 ± 0.25-fold compared with control cells, suggesting a subtle induction of hypertrophic growth. In summary, our data indicate that NFAT levels are increased in the nucleus in cells expressing the {gamma}1 R70Q mutation, which is a prerequisite for NFAT activation of the hypertrophic gene program (reviewed in Ref. 15).


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
To examine the early signaling events involved in regulating glycogen accumulation and cardiac hypertrophic growth induced by {gamma} mutations, we acutely expressed the {gamma}1 R70Q mutation in the neonatal rat cardiac myocyte. We chose to express this mutant subunit for a number of reasons: 1) the AMPK complexes containing the {gamma}1-subunit account for >65% of the AMPK activity in the heart (30), suggesting an important role for the {gamma}1-subunit in the cardiac myocyte; 2) the {gamma}1 R70Q mutation provided us with a genetic means to activate AMPK; and 3) the {gamma}1 R70Q mutation is homologous to the {gamma}2 R302Q mutation and as such may potentially provide insight into the general effects of {gamma} mutations on the intramyocellular function of AMPK. In addition, expressing this mutant in the neonatal rat cardiac myocyte is particularly relevant given that many of the {gamma} mutations induce symptoms in mice and humans at a very young age (reviewed in Ref. 4). Moreover, acute expression of the {gamma}1 R70Q mutation allows us to study the early signaling events of the {gamma} mutation on AMPK activity in the absence of dramatic glycogen accumulation and/or hypertrophy that are present in the transgenic mice.

Consistent with previous studies in different cell types and in skeletal muscle (6, 20), we have shown that expression of {gamma}1 R70Q in the cardiac myocyte results in AMPK activation. Indeed, expression of {gamma}1 R70Q leads to a twofold increase in phosphorylation of AMPK{alpha} Thr172, with no change in {alpha}-subunit protein levels (data not shown) and a significant increase in {gamma}1-subunit-associated AMPK activity, compared with the wild-type {gamma}1-subunit (Fig. 1, A and B). As a confirmation of the intracellular activation of AMPK, the phosphorylation status of both ACC{alpha} and ACCβ (downstream targets of activated AMPK in the heart; Ref. 13) was increased more than twofold in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (Fig. 1C). Because inhibition of ACC generally results in increased rates of fatty acid oxidation (38), we measured the rate of palmitate oxidation in the cardiac myocyte. Cells expressing the {gamma}1 R70Q mutation had nearly twice the palmitate oxidation rates of the wild-type {gamma}1-subunit (Fig. 2A). This increase in palmitate oxidation was similar to that of the AMPK activator AICAR and further supports the view that the {gamma}1 R70Q mutation results in activation of AMPK in the cardiac myocyte.

Since AMPK activation also has been shown to have dramatic effects on glucose handling (33, 36), we further characterized the effects of this mutation on glucose metabolism. In contrast to classic AMPK activation by pharmacological activators or ischemia/hypoxia (33, 34), AMPK activation induced by the {gamma}1 R70Q mutation did not increase glycolysis. In fact, glycolytic rates were drastically reduced in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (Fig. 2B), suggesting that the effect is specific to the mutation-mediated increase in AMPK activity. Because glucose has two major fates in the cardiac myocyte (either metabolized via the glycolytic pathway or stored as glycogen), activation of AMPK via this mutation has the potential to direct exogenous glucose away from glycolysis in favor of glycogen synthesis. Indeed, there was a more than twofold increase in both glycogen synthesis rates and total glycogen content in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (Fig. 2, C and D). These findings are consistent with previous studies showing that {gamma}-subunit mutations result in glycogen accumulation in both the skeletal muscle and the heart (2, 18, 35). Although the increase in glycogen levels in our model is modest compared with transgenic mice expressing {gamma}2 mutations (i.e., up to 30-fold more glycogen than controls), we are comparing only 48 h of expression to the lifetime of the transgenic animal (3). However, the advantage of our model is that we can directly assess the effect of the mutation on AMPK activity and the functional consequences of this activation in the absence of confounding variables such as profound glycogen accumulation and cardiac hypertrophy observed in the transgenic mice (3, 40).

Whereas the dramatic depression in glycolytic rates may be one mechanism by which these {gamma} mutations promote further glycogen accumulation, we also demonstrated that there was a significant increase in glycogen synthase activity, a reduction in inhibitory phosphorylation of glycogen synthase, and elevated glycogen synthase protein and mRNA expression (Fig. 3, A–C). Furthermore, GSK-3β protein and mRNA levels were both decreased by more than 50% in cells expressing the {gamma}1 R70Q mutation compared with the wild-type {gamma}1-subunit (Fig. 4, A and B). This effect appears to be specific to the mutated {gamma}-subunit, since pharmacological activation of AMPK has no effect on GSK-3β protein level and may, in fact, result in activation of GSK-3β (11, 14, 25). Therefore, the increase in glycogen synthase activity observed with expression of the AMPK {gamma}1 R70Q mutation was likely due to a combination of upregulation of glycogen synthase itself and reduced inhibition via GSK-3β.

Interestingly, the finding that GSK-3β protein levels were decreased with the expression of the {gamma}1 R70Q mutation also suggests that this mutation can modify pathways involved in regulating cardiac hypertrophic growth (1, 21). Consistent with this, we have shown that depressed GSK-3β activity induced by expression of the {gamma}1 R70Q mutation increased nuclear NFAT activity (Fig. 4C), which is a prerequisite for NFAT activation of the hypertrophic gene program (reviewed in Ref. 15). Again, it appears that this effect is not a result of simple activation of AMPK, since AICAR induced a significant decrease in NFAT transcriptional activity (data not shown), consistent with prior studies indicating that AMPK is a negative regulator of hypertrophy (9, 29). Although there was no observed hypertrophic growth associated with 48 h of {gamma}1 R70Q mutant expression, protein synthesis trended to increase, and this mutation may induce a more subtle stimulation of the hypertrophic program that may not be as easily observed as with other inducers of hypertrophy such as phenylephrine. Therefore, with longer expression of the mutation, induction of hypertrophic growth would likely be observed. Thus, although it has been suggested that the hypertrophy associated with AMPK {gamma}-subunit mutations in the hearts of humans and transgenic mice is due to glycogen accumulation alone (2), these results suggest that this may not be the case, particularly early in disease development. Together, our data clearly implicate the {gamma}1 R70Q mutation-mediated increase in AMPK activity as altering a number of metabolic circuits regulating glucose metabolism, glycogen accumulation, and hypertrophic growth.

Based on the results of this study, we conclude that expression of the AMPK-activating {gamma}1 R70Q mutation results in a rerouting of exogenous glucose toward glycogen synthesis as well as an activation of the rate-limiting enzyme glycogen synthase, leading to glycogen accumulation in neonatal rat cardiac myocytes. In addition, cells expressing the mutated {gamma}-isoform have increased nuclear activity of the prohypertrophic transcription factor NFAT (summarized in Fig. 5). Many of the effects induced by the {gamma}1 R70Q mutation occurred at the transcriptional level, indicating long-term changes. Since GSK-3β is an important negative regulator of both glycogen synthesis and hypertrophy, a long-term reduction in GSK-3β content of the magnitude observed upon expression of a {gamma}1 R70Q mutation could lead to excessive glycogen accumulation and hypertrophy, both of which are hallmarks of the cardiomyopathy associated with mutations in the {gamma}2-subunit of AMPK. Since AMPK is known to interact with various transcription factors and transcriptional coactivators (reviewed in Ref. 28), it is probable that the mutation alters either AMPK substrate specificity or subcellular localization, thereby promoting increased or inappropriate interaction with transcription factor(s)/coactivator(s) that control glycogen-related genes. Whether our findings using the {gamma}1 R70Q mutation define the precise mechanisms responsible for the cardiomyopathy observed in humans and mice expressing the {gamma}2 mutations is currently unknown. A recent study using the {gamma}2 N488I transgenic mice also showed reduced flux of exogenous glucose through glycolysis and increased glycogen synthase activity and protein expression at 7 wk of age (32). However, in contrast to our model system, the former study reported an increase in glycogen synthase phosphorylation. Although we cannot provide evidence to explain this discrepancy, it is likely due to the differences in the effects of the two mutants of the {gamma}-subunit of AMPK and/or the differences in experimental models. In addition, our study shows a striking downregulation of GSK-3β, which relieves inhibition of glycogen synthase, leading to glycogen accumulation, whereas Luptak et al. (32) defined a pathway in which increased expression and activity of UDP-linked glucose pyrophosphorylase (UDPG-PPL) facilitated the observed increase in glycogen content. Whether UDPG-PPL plays a role in our model system was not investigated.


Figure 5
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  		Fig. 5. Schematic of {gamma}1 R70Q-induced effects in the cardiac myocyte. Expression of {gamma}1 R70Q has effects on metabolism, glycogen accumulation, and growth control in the cardiac myocyte. These effects, especially those on glycogen accumulation and hypertrophy, may help to explain the molecular mechanisms involved in the development of AMPK {gamma}-subunit mutation-associated cardiomyopathy.

 
Although there have been no discoveries of human mutations in the AMPK {gamma}1-subunit, this may be due to the fact that {gamma}1 is ubiquitously expressed in all tissues, and a mutation inducing effects of the magnitude seen in this study, especially during development, may not be compatible with life. However, this possibility has yet to be examined. Nevertheless, in addition to increased glucose uptake and UDPG-PPL activity defined in a recent publication (32), our study provides important new information as to the mechanisms by which a mutation in the {gamma}-subunit of AMPK causes altered AMPK signaling. In addition, this study has identified multiple pathways involved in regulating both cardiac myocyte metabolism and growth that are influenced by mutations in the {gamma}-subunits of AMPK and that may contribute to the development of the {gamma} mutant-associated cardiomyopathy in humans.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Canada (to J. R. B. Dyck), National Institutes of Health (NIH) Grant HL-074259 (to M. E. Young), and NIH Grant DK-35712 (to L. A. Witters). K. D. Folmes was funded by PhD graduate studentships provided by the Alberta Heritage Foundation for Medical Research (AHFMR) and the Natural Sciences and Engineering Research Council. J. R. B. Dyck is an AHFMR Senior Scholar and a Canada Research Chair in Molecular Biology of Heart Disease and Metabolism.


   ACKNOWLEDGMENTS
 
We acknowledge the expert technical support of Suzanne Kovacic, Amy Barr, and Hannah Parsons.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. B. Dyck, 474 Heritage Medical Research Centre, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2S2 (e-mail: jason.dyck{at}ualberta.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.


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 ABSTRACT
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 DISCUSSION
 GRANTS
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