From the
Nuclear Magnetic Resonance Laboratory for
Physiological Chemistry, Cardiovascular Division and
¶Research Division, Joslin Diabetes Center,
Department of Medicine, Brigham and Women's Hospital and Harvard Medical
School, Boston, Massachusetts 02115
Received for publication, April 4, 2003 , and in revised form, May 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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It has been suggested that AMPK regulates glucose and fatty acid metabolism in striated muscles (6, 7). Studies from our groups and others showed increased AMPK activity during acute and chronic stresses, such as hypoxia and exercise in skeletal muscle and ischemia and pressure overload in the heart (812). Activation of AMPK in the heart is associated with enhanced glucose uptake and glycolysis (10, 11, 13). As glycolysis is a major source of ATP during ischemia, stimulation of glucose uptake and glycolysis by AMPK in the ischemic heart is consistent with the overall function of this enzyme in restoring cellular energy levels during stress. To establish a causal role of AMPK for these stress responses, however, inhibition of AMPK is required during stress. This has not been possible because of the lack of a specific inhibitor of AMPK in the heart. Furthermore, the inability to block AMPK activation during ischemia makes it difficult to test whether AMPK also functions to preserve energy by reducing ATP consumption by the heart during ischemia.
In the present study, we sought to inhibit AMPK activity by generating
transgenic mice (TG) overexpressing a dominant negative mutant of the AMPK
2 catalytic subunit in the heart. This approach led to a selective
inhibition of AMPK
2 activity in the heart. Here we report that AMPK
2 mediates critical cellular responses in maintaining energy
homeostasis in the ischemic heart.
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EXPERIMENTAL PROCEDURES |
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Isolated Perfused Heart ExperimentsHearts were perfused in the Langendorff mode with phosphate-free Krebs-Henseleit buffer containing (in millimoles/liter) NaCl (118), NaHCO3 (25), KCl (5.3), CaCl2 (2.5), MgSO4 (1.2), EDTA (0.5), glucose (10), and pyruvate (0.5) as described previously (16). All hearts were perfused with a constant perfusion pressure of 80 mm Hg, and the left ventricular function was continuously monitored using a water-filled balloon (16). Fig. 1 illustrates the protocols for isolated perfused heart experiments. After stabilization, one base-line 31P NMR spectrum was collected (16), and one-half of the hearts were subjected to a 10-min no-flow ischemia and the other half to a 10-min normal perfusion. During ischemia, four consecutive 2-min 31P NMR spectra were collected to monitor the dynamic changes in high energy phosphate content. At the end of the 10-min period, a subgroup of hearts was freeze-clamped for biochemical analysis, and the rest were reperfused with a buffer in which glucose was replaced with 5 mM 2-deoxyglucose (2-DG). Five consecutive 4-min 31P NMR spectra were collected for determination of the time-dependent accumulation of 2-DG-phosphate. The rate of glucose uptake was estimated by the slope of the fitted line as described previously (11, 17). During 2-DG perfusion, 1.2 mM KH2PO4 and5mM pyruvate were supplied to replenish the intracellular inorganic phosphate pool and to maintain ATP synthesis.
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AMPK Activity Assay and Western BlottingFreeze-clamped
heart samples were homogenized as described previously, and lysates were used
for AMPK activity assays and for Western blotting
(18). AMPK activity was
measured after immunoprecipitating 200 µg of protein using antibodies made
against the amino acid sequences 339358 of rat AMPK 1,
352366 of
2, and 216 of both
1 and
2
(pan-
) (18). The kinase
reaction was done using synthetic peptide with sequence HMR-SAMSGLHLVKRR as
substrate, and AMPK activity is expressed as incorporated ATP (picomoles) per
mg of protein per min (19).
Western blotting was done with antibodies against AMPK
1,
2,
pan-
, HA (Roche Diagnostics), GLUT1, GLUT4 (Chemicon, Intl., Inc.,
Temecula, CA), SERCA2 (Affinity BioReagents, Golden, CO), and
Na+/Ca2+ exchanger (Swant, Bellinzona,
Switzerland).
HPLC Measurements and Glycogen AssayFreeze-clamped tissues were used for determination of myocardial content of adenine nucleotides, nucleosides, and purine bases by a HPLC method as reported previously (20). Myocardial glycogen content was determined by measuring the amount of glucose released from glycogen by use of an alkaline extraction to separate glycogen and exogenous glucose (21). Glucose content in the extract was measured using a Sigma assay kit.
Data Analysis and StatisticsMyocardial ATP content obtained by HPLC was converted to [ATP] assuming an intracellular water content of 0.48 ml and a protein content of 0.15 g/g of blotted wet tissue (22). The mean value of [ATP] for WT or TG hearts was used to calibrate the ATP peak area of the base-line 31P NMR spectrum. Concentrations of other metabolites were calculated using the ratio of their peak areas to the ATP peak area, and intracellular pH (pHi) was determined by the chemical shift of inorganic phosphate (Pi) relative to PCr (20). The values of [ATP] and [PCr] in the ischemic hearts were obtained from summed spectra of 34 hearts. Each data point represents the average of four summed results from a total of 13 hearts.
Differences in results obtained from WT and TG hearts were compared by 2-tailed Student's t test or one-way factorial ANOVA. Changes during ischemia and 2-DG perfusion were compared by repeated-measures ANOVA. All the statistical analyses were performed with Statview (Brainpower Inc.), and a value of p < 0.05 was considered significant.
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RESULTS |
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AMPK 2 activity was reduced in the TG hearts at base line by 78%
(Fig. 3A). Ischemia
increased AMPK
2 activity by 4.5-fold in the WT hearts, whereas the
activation of AMPK
2 in the TG hearts was severely blunted
(Fig. 3A). The AMPK
1 activity in TG hearts was not different from that of WT hearts at
base line, and it increased normally in response to ischemia
(Fig. 3B). These
findings suggest that the dominant negative transgenic approach used here
resulted in specific inhibition of AMPK
2 activity in the heart.
Blocking the activation of AMPK
2 during ischemia led to a 65%
reduction in total AMPK activity in the heart
(Fig. 3C), suggesting
that AMPK
2 is a major contributor to ischemia-stimulated AMPK activity
in the heart.
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General Characteristics and Cardiac Function of the TG
MiceTable I summarizes the base-line characteristics of the mice used for this study.
There was no difference in the body weight and heart weight between TG and WT
mice. The TG mice were grossly normal, and no premature death was observed up
to 1 year (data not shown). Left ventricular contractile function at base line
was similar for TG and WT hearts, suggesting that inhibition of AMPK 2
activity does not alter cardiac function during normal perfusion. When
subjected to ischemia, however, the TG hearts showed a more rapid increase in
left ventricular end-diastolic pressure
(Fig. 4, LVEDP). This
is unlikely because of a decrease in the amount of calcium-handling proteins
in TG hearts. The protein levels for SERCA2 and
Na+/Ca2+ exchanger were unchanged in TG
hearts (SERCA2: 31 ± 1 versus 32 ± 2 absorbance units;
and Na+/Ca2+ exchanger: 79 ± 2
versus 76 ± 2 absorbance units for WT and TG, respectively,
n = 4 in each group). The rapid increase in left ventricular
end-diastolic pressure is consistent with the observation that ATP depletion
was accelerated during ischemia in TG hearts (see below), implying a faster
development of myofibril rigor force in these hearts.
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High Energy Phosphate ContentDynamic changes in high energy phosphate content during ischemia were monitored by 31P NMR spectroscopy and shown in Fig. 5. Pre-ischemic values of [PCr], [ATP], and [Pi] are not different in WT and TG hearts. [PCr] decreased rapidly in both groups during ischemia; the PCr peak was no longer detectable in TG hearts after 4 min of ischemia, whereas a very low but detectable PCr peak remained for 2 more minutes in WT hearts. The rate and extent of ATP depletion was also accelerated in TG hearts; [ATP] decreased by 80 and 55% from the pre-ischemic value in TG and WT hearts, respectively (p < 0.05). The [Pi] rose markedly (by 10-fold) during ischemia. The pHi decreased significantly in both groups, but pHi was lower in WT hearts than in TG hearts at the end of ischemia (6.40 ± 0.03 and 6.58 ± 0.05, respectively, p < 0.05).
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Table II shows ATP degradation and accumulation of adenine nucleosides and purine bases during ischemia measured by HPLC. Myocardial content of adenine nucleotides was similar in WT and TG hearts during base-line perfusion. At the end of a 10-min ischemia, ATP content in TG and WT hearts decreased by 80 and 60% from the base-line value, respectively (p < 0.05). Thus, the measurement by HPLC was consistent with the NMR measurement. Increased ATP degradation in the TG hearts during ischemia resulted in a tendency for higher content of AMP, adenosine, and inosine compared with WT (Table II).
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Glucose MetabolismDuring base-line perfusion, TG hearts
showed normal rates of 2-DG uptake (Fig.
6) and normal glycogen content
(Table III). The protein
content of GLUT1 and GLUT4, two primary glucose transporters in the heart, was
also unchanged in TG hearts (Fig.
7). These results suggest that AMPK 2 has a minimum effect
on glucose metabolism in hearts under unstressed conditions.
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In WT hearts, ischemia caused a 2.5-fold increase in the rate of 2-DG
uptake. In contrast, 2-DG uptake increased by only 1.7-fold in TG hearts after
ischemia, representing a 62% reduction in the response to ischemia
(Fig. 6). Thus, blocking the
activation of AMPK 2 in the ischemic heart significantly blunted the
increase in myocardial glucose uptake.
During no-flow ischemia, glycogen is the only substrate for anaerobic glycolysis that generates ATP. To assess glycogen consumption during ischemia, we determined the differences in average myocardial glycogen content before and after ischemia (Table III). ATP produced from glycogen consumption was estimated assuming that each glucose molecule derived from glycogen gave three molecules of ATP. Interestingly, glycogen content in TG hearts at the end of ischemia was lower than WT hearts, and ATP generated during ischemia was slightly greater in TG hearts. These results indicate that accelerated ATP depletion in TG hearts during ischemia is not because of impaired ATP generation via glycogenolysis.
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DISCUSSION |
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Selective Inhibition of AMPK 2
ActivityLittle is known about the isoform-specific characteristics
of the AMPK heterotrimers. In this study, we sought to inhibit AMPK activity
by generating transgenic mice with cardiac-specific overexpression of a
kinase-inactive mutant of the
2 subunit (D157A)
(14,
15). Interestingly,
overexpression of this mutant
2 subunit results in substantial and
selective replacement of the native
2 subunit while leaving the
1 subunit unaffected. Accordingly, AMPK
1 activity in TG hearts
remains unchanged and responds normally during ischemia. We found that total
AMPK activity, measured by immunoprecipitation with an antibody against
pan-
-AMPK was reduced by two thirds, supporting the notion that AMPK
2 contributes the majority of AMPK activity under these conditions. In
a transgenic mouse model described previously, in which a different mutation
of
2 subunit (R45K) was overexpressed in skeletal muscle
(24), the authors reported
that both native
subunits were replaced by the mutant. This apparent
discrepancy between the two models raises the possibility that skeletal muscle
differs from cardiac muscle in subunit composition and/or subcellular
localization of AMPK heterotrimers. Selective inhibition of AMPK
2
activity in the heart, as observed in our model, offers a unique opportunity
to examine isoform-specific function of AMPK in the heart.
AMPK 2 and Glucose MetabolismAMPK has
emerged as a potential mediator of increased glucose uptake in response to
energy depletion. Pharmacological activation of AMPK by
5-aminoimidazole-4-carboxamide ribonucleoside results in increased glucose
uptake in both skeletal and cardiac muscle in an insulin-independent fashion
(13,
25,
26). Furthermore, a close
relationship between increased AMPK activity and enhanced glucose uptake has
been observed under a variety of stress conditions
(8). Because ischemia also
stimulates glucose uptake through an insulin-independent mechanism
(2729),
the role of AMPK in this event is strongly implied. Yet, the effort of
defining a causal relationship between AMPK activation and increased glucose
uptake during ischemia has been hindered by the lack of an effective AMPK
inhibitor for the heart. Using the transgenic approach to prevent AMPK
activation, here we provide the first direct evidence that AMPK
2 plays
a critical role in ischemia-stimulated glucose uptake. Our results also show
that AMPK
2 is not the sole mediator for ischemia-stimulated glucose
uptake. The finding that AMPK
2-specific TG hearts have a partial
reduction in ischemia-stimulated glucose uptake raises the possibility that
AMPK
1 is also important in the regulation of glucose uptake in the
heart. Our study, however, does not rule out the possibility that other
mechanisms in addition to AMPK are also responsible for ischemia-stimulated
glucose uptake.
It has been suggested that AMPK contributes to enhanced glycolysis during
ischemia by activating 6-phosphofructo-2-kinase, the enzyme responsible for
the synthesis of fructose 2,6-bisphosphate, a potent stimulator of glycolysis
(10). In this study, we found
a greater breakdown of glycogen in TG hearts during no-flow ischemia,
suggesting that AMPK 2 activity is not required for stimulating
glycolysis under our experimental conditions. Because the total AMPK activity
increased partially in the ischemic TG hearts due to the activation of AMPK
1, it is possible that this increase is sufficient to activate
6-phosphofructo-2-kinase and hence stimulate glycolysis. In addition,
6-phosphofructo-2-kinase can be phosphorylated and activated by other kinases
such as protein kinase A (30).
Furthermore, glycolysis is also regulated by the concentrations of adenine
nucleotides and intracellular pH
(31,
32). Both accelerated
depletion of ATP and reduced acidosis in TG hearts would favor increased
glycolytic flux. Considering the essential role of glycolysis for myocardial
survival during ischemia, it is conceivable that redundant signaling
mechanisms exist for stimulation of glycolysis.
Role of AMPK 2 in Energy Homeostasis during
IschemiaWe found normal content of high energy phosphate and
adenine nucleotides in the TG hearts under base-line conditions. This
observation, together with the apparent normal glucose metabolism in
unstressed TG hearts, support the notion that the primary function of AMPK is
to regulate cellular response to stress. By subjecting hearts to ischemia, we
observed a more rapid decline of ATP in TG hearts. This finding suggests that
AMPK
2 plays a critical role in sustaining energy homeostasis in
stressed hearts. Importantly, the exacerbated ATP degradation cannot be
explained by decreased ATP synthesis via glycogenolysis. Because the no-flow
ischemia protocol applied in this study does not allow utilization of energy
substrates other than glycogen, our results likely indicate increased ATP
consumption by TG hearts during ischemia.
It has been shown that in the liver AMPK reduces energy consumption by
switching off synthetic reactions in the cell
(1,
2,
15). It has not been
determined if AMPK mediates energy conservation mechanisms in the heart. The
majority of energy consumed by the heart supports contractile function,
i.e. the myosin ATPase reaction
(33,
34). During our ischemic
protocol, the heart stops contracting in less than 1 min. Increased ATP
depletion in TG hearts under this condition likely reflects increased ATP
consumption for noncontractile function, predominantly for maintaining basal
metabolism and ion homeostasis
(33,
34). On this note, we found a
slower decline in pHi indicating a reduced accumulation of
intracellular H+ in TG hearts. This is in contrast to the
observation that degradation of ATP and glycogen is accelerated in TG hearts,
which would lead to increased H+ production during ischemia. Thus,
it is likely that the TG heart is more active in exporting H+.
Taken together, our findings suggest that AMPK 2 plays a role in energy
conservation in the ischemic heart, possibly by modifying the ion transport
process.
In summary, inactivation of AMPK 2 causes accelerated ATP depletion
and early development of myocardial contracture and leads to decreased glucose
uptake in response to ischemia. These findings suggest that AMPK plays a
critical role in sustaining energy homeostasis and myocardial protection
during ischemia, possibly by modulating cellular functions for both energy
supply and utilization.
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FOOTNOTES |
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These authors contributed equally to this work.
|| Supported by a mentor-based award from the American Diabetes
Association.
** Recipient of a Mary K. Iacocca Fellowship at the Joslin Diabetes
Center.
Supported by an American Diabetes Association Research Grant.
Supported by an Established Investigator Award from the American Heart
Association. To whom correspondence should be addressed: NMR Laboratory for
Physiological Chemistry, 221 Longwood Ave., Rm. 252, Boston, MA 02115. Tel.:
617-732-6729; Fax: 617-732-6990; E-mail:
rtian{at}rics.bwh.harvard.edu.
1 The abbreviations used are: AMPK, AMP-activated protein kinase; TG,
transgenic; WT, wild type; PCr, phosphocreatine; HA, hemagglutinin; 2-DG,
2-deoxyglucose; HPLC, high pressure liquid chromatography;
pHi, intracellular pH; ANOVA, analysis of variance.
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ACKNOWLEDGMENTS |
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REFERENCES |
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