Increased ␣2 Subunit–Associated AMPK Activity and
PRKAG2 Cardiomyopathy
Ferhaan Ahmad, MD, PhD*; Michael Arad, MD*; Nicolas Musi, MD; Huamei He, MD, PhD;
Cordula Wolf, MD; Dorothy Branco, BS; Antonio R. Perez-Atayde, MD; David Stapleton, PhD;
Deeksha Bali, PhD; Yanqiu Xing, PhD; Rong Tian, MD, PhD; Laurie J. Goodyear, PhD;
Charles I. Berul, MD; Joanne S. Ingwall, PhD; Christine E. Seidman, MD†; J.G. Seidman, PhD†
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Background—AMP-activated protein kinase (AMPK) regulatory ␥2 subunit (PRKAG2) mutations cause a human
cardiomyopathy with cardiac hypertrophy, preexcitation, and glycogen deposition. PRKAG2 cardiomyopathy is
recapitulated in transgenic mice overexpressing mutant PRKAG2 N488I in the heart (TG␥2N488I). AMPK is a
heterotrimeric kinase consisting of 1 catalytic (␣) and 2 regulatory (␤ and ␥) subunits. Two ␣-subunit isoforms, ␣1 and
␣2, are expressed in the heart; however, the contribution of AMPK utilization of these subunits to PRKAG2
cardiomyopathy is unknown. Mice overexpressing a dominant-negative ␣2 subunit of AMPK (TG␣2DN) provide a tool
for selectively inhibiting ␣2, but not ␣1, subunit-associated AMPK activity.
Methods and Results—In compound-heterozygous TG␥2N488I/TG␣2DN mice, AMPK activity associated with ␣2 but not ␣1
was decreased compared with TG␥2N488I. The TG␣2DN transgene reduced the disease phenotype of TG␥2N488I, partially
or completely normalizing the ECG, cardiac function, cardiac morphology, and exercise capacity in compoundheterozygous mice. TG␥2N488I hearts had normal resting levels of high-energy phosphates and could improve cardiac
performance during exercise. Cardiac glycogen content decreased in TG␥2N488I mice after exercise stress, indicating
availability of the stored glycogen for metabolic utilization. No differences in glycogen-metabolizing enzymes were
observed.
Conclusions—The PRKAG2 N488I mutation causes inappropriate AMPK activation, which leads to glycogen accumulation and conduction system disease. The accumulated glycogen can serve as an energy source, and the animals have
contractile reserve during exercise. Because the dominant-negative ␣2 subunit attenuates the mutant PRKAG2
phenotype, AMPK complexes containing the ␣2 rather than the ␣1 subunit are the primary mediators of the effects of
PRKAG2 mutations. (Circulation. 2005;112:3140-3148.)
Key Words: cardiomyopathy 䡲 exercise 䡲 genetics 䡲 metabolism 䡲 Wolff-Parkinson-White syndrome
M
utations in AMP-activated protein kinase (AMPK)
have been discovered to cause a glycogen storage
cardiomyopathy that ultimately progresses to heart failure1–3
(for reviews, see Gollob4 and Gollob et al5). AMPK, a
heterotrimeric protein composed of ␣, ␤, and ␥ subunits, is a
metabolite-sensing protein kinase that is activated under
conditions of energy depletion manifested by increased cellular AMP levels (reviewed in Hardie6). AMPK is able to
phosphorylate ⬎15 known target proteins and is thought to
regulate energy metabolism by modulating a variety of
Clinical Perspective p 3148
metabolic activities, including glucose transport, stimulation of ␤-oxidation of fatty acids, inactivation of cholesterol synthesis, inhibition of creatine kinase, and transcriptional regulation of several genes.6,7 Mutations in the gene
PRKAG2, encoding the ␥2 subunit of AMPK, have been
demonstrated to produce a distinct cardiomyopathy in
Received March 25, 2005; revision received August 23, 2005; accepted September 12, 2005.
From the Department of Genetics (F.A., M.A., C.E.S., J.G.S.), Harvard Medical School and Howard Hughes Medical Institute, Boston, Mass;
Cardiovascular Institute (F.A.), Department of Medicine, and Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pa; Heart Institute
(M.A.), Sheba Medical Center and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; Research Division (N.M., L.J.G.), Joslin Diabetes
Center, Boston, Mass; NMR Laboratory for Physiological Chemistry (H.H., Y.X., R.T., J.S.I.), Cardiovascular Division, Department of Medicine,
Brigham and Women’s Hospital, Boston, Mass; Department of Cardiology (C.W., D. Branco, C.I.B.), Children’s Hospital, Boston, Mass; Department
of Pathology (A.R.P.-A.), Children’s Hospital, Boston, Mass; Bio21 Molecular Science and Biotechnology Institute (D.S.), University of Melbourne,
Melbourne, Australia; Pediatric Medical Genetics (D. Bali), Duke University Medical Center, Research Triangle Park, NC; and Cardiovascular Division
(C.E.S.), Brigham and Women’s Hospital, Boston, Mass.
*The first 2 authors contributed equally to this work.
†The last 2 authors contributed equally to this work.
Guest Editor for this article was Roberto Bolli, MD.
Correspondence to J.G. Seidman, PhD, Department of Genetics, Harvard Medical School, 77 Ave Louis Pasteur, NRB 256, Boston, MA 02115. E-mail
[email protected]
© 2005 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.105.550806
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Increased AMPK(␣2) in PRKAG2 Cardiomyopathy
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human families characterized by ventricular hypertrophy,
ventricular preexcitation, and progressive conduction system disease. 1–3,8 Mutations recognized thus far are
Exon5:InsLeu, His142Arg, Arg302Gln, Thr400Asn,
Tyr487His, Asn488Ile, and Arg531Gly. The cardiomyopathy associated with PRKAG2 mutations is not associated
with the myocyte and myofibrillar disarray and cardiac
fibrosis characteristic of mutations in genes encoding
sarcomeric proteins. Rather, cardiac hypertrophy results
from formation within the myocytes of vacuoles filled with
glycogen-associated granules.1,9 PRKAG2-mediated cardiomyopathy, unlike other glycogen storage disorders, is
characterized by cardiac glycogen accumulation in the
absence of other remarkable clinical features, such as
skeletal myopathy. The mechanisms by which these
PRKAG2 missense mutations mediate cardiomyopathy and
the accessibility of the accumulated glycogen as an energy
source remain uncertain.
Both cell and murine models of PRKAG2 cardiomyopathy
have been described.1,9 –11 Several in vitro studies suggest that
PRKAG2 cardiomyopathy is caused by increased AMPK
activity.1 Introduction of the Thr400Asn and Asn488Ile
mutations into the yeast homolog of the ␥ subunit, Snf4,
resulted in constitutive activation of the Snf1/Snf4 kinase.1 A
similar Arg70Gln mutation in the ␥1 subunit introduced into
COS7 cells and pulmonary fibroblasts caused markedly
increased AMPK activity, associated with increased phosphorylation of the ␣ subunit Thr172 and increased phosphorylation of 1 of its major substrates, acetyl coenzyme A
carboxylase.9 However, introduction of several mutant
PRKAG2s into mammalian CCL13 cells caused either a
decrease or no change in AMPK activity.10,11 Furthermore,
recent studies by Sidhu and colleagues12 have suggested that
the PRKAG2 Arg302Gln missense mutation inactivates
AMPK and thereby causes glycogen accumulation and cardiac hypertrophy.
TG␥2N488I mice provide a model of PRKAG2 cardiomyopathy because they express a transgene encoding the
Asn488Ile (N488I) mutation, which is expressed only in the
heart under control of the ␣-myosin heavy-chain promoter.13
TG␥2N488I hearts demonstrate elevated AMPK activity, massive glycogen deposition in cardiac myocytes up to 30-fold
above normal, dramatic left ventricular (LV) hypertrophy,
ventricular preexcitation, and sinus node dysfunction. Disruption of the annulus fibrosus by glycogen-filled myocytes is
the anatomic substrate for preexcitation.13,14 TG␥2N488I extracts have markedly elevated AMPK activity,13 and this
increased activity is associated with both the ␣1 and ␣2
subunits.
TG␥2N488I mice provide a unique tool for dissecting the
mechanisms leading to PRKAG2 cardiomyopathy. We demonstrate, using a novel murine stress-echocardiography procedure, that although energy metabolism remains unchanged
in the resting TG␥2N488I heart, the accumulated glycogen in
the TG␥2N488I heart can be mobilized and metabolized during
exercise. Furthermore, we use a genetic approach to confirm
the model that PRKAG2 cardiomyopathy results from hyperactive AMPK, hypothesizing that if the PRKAG2 missense
mutations function by inactivating AMPK, then dominant-
3141
negative ␣2-subunit mutations15 would not alter the phenotype of transgenic mice. Because the transgenic allele
TG␣2DN, which expresses a dominant-negative form of the
AMPK ␣2 catalytic subunit, inactivates AMPK containing
the ␣2 but not the ␣1 subunit,15 we are able to define the
relative contributions of AMPK containing these ␣ subunits.
We conclude that PRKAG2 cardiomyopathy results from
overactive AMPK associated primarily with the ␣2 rather
than the ␣1 subunit.
Methods
Mice
Transgenic mice overexpressing human full-length, wild-type
(TG␥2wt) and N488I (TG␥2N488I) PRKAG2 have been described.13 In
brief, a T3 A substitution was introduced at nucleotide 1553 in the
human PRKAG2 cDNA to alter codon 488 from Asn3 Ile, corresponding to a known human mutation. The transgene is expressed
under control of the strong cardiac-specific ␣-myosin heavy-chain
promoter. Transgenic mice overexpressing a dominant-negative ␣2
subunit of AMPK (TG␣2DN) under control of the same ␣-myosin
heavy-chain promoter were generated as previously reported.15 The
Asp3 Ala substitution at codon 157 in rat PRKAA2 cDNA creates
mutant ␣2 subunits, which bind to ␥2 subunits, producing enzymatically inactive complexes. The ␣2DN polypeptide also contains a
YPYDVPDYA (HA, a synthetic peptide) tag. All mouse studies
were performed in accordance with protocols approved by the
institutional animal care and use committee at Harvard Medical
School.
AMPK Activity
Resting mice were humanely killed by cervical dislocation without
anesthesia, and the heart tissue was immediately frozen in LN2.
Freeze-clamped heart samples were homogenized, and lysates were
used for AMPK activity assays as described.16 Protein (200 ␮g) was
immunoprecipitated with antibodies recognizing amino acids 339 to
358 of rat AMP ␣1 or 352 to 366 of ␣2. The kinase reaction was
performed, in the presence of 0.2 mmol/L AMP, with synthetic
substrate for AMP-activated protein kinase (SAMS) peptide as a
substrate, and AMPK activity was measured as picomoles phosphate
incorporated per milligram protein per minute.
Glycogen Enzyme Activity Assays
Hearts from wild-type, TG␥2wt, and TG␥2N488I mice were excised.
Brancher enzyme or 1,4-␣-glucan branching enzyme [1,4-␣-Dglucan:1,4-␣-D-glucan 6-␣-D-(1,4-␣-D-glucano)transferase; EC
2.4.1.18] and glycogen phosphorylase (1,4-␣-D-glucan:phosphate
␣-D-glucosyltransferase; EC 2.4.1.1) were measured by free phosphate levels with inorganic phosphorus reagent (Roche Diagnostics).17,18 Debrancher enzyme or 4-␣-glucanotransferase (4-␣glucanotransferase 1,4- ␣ - D -glucan:1,4- ␣ - D -glucan 4- ␣ - D glycosyltransferase; EC 2.4.1.25) and glycogen phosphorylase
kinase (ATP:phosphorylase-␤ phosphotransferase; EC 2.7.1.38)
were measured by release of free glucose from Roche glucose
reagent according to a standard protocol.17,19,20
Immunoblots
Antibodies recognizing ␣1, ␣2, and ␥2 subunits of AMPK were
produced as previously described.15,21,22 A lamin B antibody was
used as a loading control (sc-6217, Santa Cruz).
Energetics Assays
Isolated, Langendorff-perfused hearts were freeze-clamped, and
myocardial levels of adenine nucleotides and phosphocreatine were
determined by a high-performance liquid chromatography method as
reported previously.23 31P nuclear magnetic resonance (NMR) spectra were obtained as previously described24 at 161.94 MHz with a
GE-400 wide-bore spectrometer. Hearts were placed in a 10-mm
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November 15, 2005
Cardiac Glycogen and Lactate Levels
Glycogen content was determined by perchloric acid extraction and
amyloglucosidase digestion, followed by determination of glucose
levels with a glucose oxidase kit (Sigma-Aldrich).25 Lactate levels
were determined according to Sigma technical bulletin 826-UV with
the use of NAD, glycine, and lactate dehydrogenase.
Exercise Stress Testing
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Figure 1. Western blots of wild-type, TG␣2DN, TG␥2N488I, and
TG␥2N488I/TG␣2DN cardiac extracts reacted with AMPK ␣1, ␣2,
and ␥2 subunit– and lamin B–specific antibodies. ␥2-subunit
polypeptide expression is dramatically increased in TG␥2N488I
hearts and also increased in mice expressing the ␣2DN allele
(TG␥2N488I/TG␣2DN). TG␣2DN and TG␥2N488I/TG␣2DN mice express
the higher-molecular-weight ␣2 subunit encoded by the ␣2DN
allele rather than the normal ␣2 allele expressed in wild-type
and TG␥2N488I hearts. ␣1-subunit levels are indistinguishable in
all 4 mouse hearts. Anti–lamin B antibody was used to correct
for differences in the amount of total protein extract. Abbreviations are as defined in text.
glass NMR tube and inserted into a custom-made, 1H/31P doubletuned probe situated in an 89-mm bore, 9.4-T superconducting
magnet. The resonance area corresponding to phosphocreatine was
fitted to Lorentzian functions and calculated according to a commercially available program (NMR1).
TABLE 1.
To assess exercise capacity, mice were placed on a treadmill with an
adjustable belt speed and incline and electric shock bars. Mice were
subjected to an endurance protocol as previously described.26 Each
mouse was encouraged to run to the limit of its capacity on this
protocol, and total exercise duration was recorded.
Stress Echocardiography
The treadmill exercise protocol was modified to bring mice rapidly
to maximum exercise capacity. Exercise was initiated at a treadmill
incline of 15° and a speed of 10 m/min. The speed was increased by
2.5 m/min every 3 minutes until each mouse reached its maximum
capacity and appeared exhausted. Mice were acclimated to the stress
protocol by 1 practice exercise session 1 day before stress echocardiography. Echocardiography was performed with an Agilent Sonos
4500 ultrasound machine and a 6- to 15-MHz linear-array transducer
as previously described,23 with the exception that no anesthesia was
used and that immediate postexercise echocardiography was limited
to M-mode views. LV end-diastolic and end-systolic diameters and
wall thicknesses were obtained from M-mode tracings from measurements averaged from 3 separate cardiac cycles. LV fractional
shortening (in percent) was derived from the equation fractional
Characteristics of Wild-Type (WT), TG␣2DN, TG␥2N488I, and TG␥2N488I/TG␣2DN Mouse Hearts
WT
TG␣2DN
TG␥2N488I
TG␥2N488I/
TG␣2DN
P, ␥2N488I
vs WT
P,
␥2N488I/␣2DN
vs ␥2N488I
P,
␥2N488I/␣2DN
vs WT
AMPK activity
No. of mice
2
6
8
␣1 subunit, pmol ATP/mg protein per min
12.7⫾1.2
29.9⫾7.8
23.8⫾10.3
NS
␣2 subunit, pmol ATP/mg protein per min
4.6⫾3.4
14.0⫾4.4
5.3⫾1.9
7⫻10⫺5
Cardiac mass and glycogen content
No. of mice
HW/BW, mg/g
Glycogen/HW, ␮g/mg tissue
9
3
6
6
4.8⫾0.3
4.8⫾0.4
11.7⫾1.8
6.9⫾1.0
1⫻10⫺8
1⫻10⫺5
6⫻10⫺6
6.0⫾1.7
⫺7
⫺4
6⫻10
2⫻10⫺6
6⫻10⫺8
0.01
4⫻10⫺6
1.7⫾0.4
0.9⫾0.2
50.0⫾9.7
2⫻10
Echocardiography at 8 wk
No. of mice
15
4
5
5
LV wall thickness, mm
0.90⫾0.07
0.86⫾0.05
1.54⫾0.28
1.17⫾0.12
LVEDD, mm
2.39⫾0.2
2.62⫾0.4
2.52⫾0.5
2.36⫾0.2
NS
NS
NS
69⫾7
69⫾5
58⫾12
78⫾4
0.01
0.004
0.01
Fractional shortening, %
Echocardiography at 20 wk
7
4
5
5
LV wall thickness, mm
No. of mice
0.86⫾0.09
0.91⫾0.06
1.56⫾0.30
1.06⫾0.05
8⫻10⫺5
0.003
5⫻10⫺4
LVEDD, mm
2.66⫾0.32
2.62⫾0.43
3.69⫾0.96
2.53⫾0.36
0.01
0.02
NS
73⫾11
66⫾7
38⫾21
69⫾7
0.002
0.008
NS
0.004
0.02
NS
Fractional shortening, %
Electrocardiography
No. of mice
PR interval, ms
8
29.5⫾4
6
31.3⫾5
10
21.4⫾7
11
26.4⫾3
HW indicates heart weight; BW, body weight; EDD, end-diastolic diameter; and NS, not significant (P⬎0.05). Other abbreviations are as defined in text.
Double-transgenic mice demonstrate partial or complete normalization of the abnormalities observed in TG␥2N488I mice— cardiac hypertrophy, glycogen deposition,
ventricular preexcitation as manifested by a shortened PR interval on ECG, and at 20 weeks of age, cardiac dilatation and impaired systolic function. Mass ratios were
measured at 5 weeks of age, PR intervals were measured at 10 –16 weeks of age, and echocardiography was performed at 8 and 20 weeks of age, as indicated.
P values reflect comparisons between TG␥2N488I (␥2N488I) vs WT and TG␥2N488I/TG␣2DN (␥2N488I/␣2DN) vs TG␥2N488I (␥2N488I).
Ahmad et al
Increased AMPK(␣2) in PRKAG2 Cardiomyopathy
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Figure 2. Light photomicrographs of
hearts from (A) 8-week-old wild-type, (B)
5-week-old TG␣2DN, (C) 8-week-old
TG␥2N488I, and (D) 20-week-old TG␥2N488I/
TG␣2DN mice stained with hematoxylin
and eosin and electron photomicrographs of hearts from (E) 8-week-old
TG␥2N488I and (F) 20-week-old TG␥2N488I/
TG␣2DN mice. TG␥2N488I/TG␣2DN hearts
demonstrate the absence of glycogenfilled vacuoles, which disrupt the normal
structure of TG␥2N488I hearts. Inset in C
illustrates “spider cells” characteristic of
glycogen infiltration in the myocardium.
Abbreviations are as defined in text.
shortening⫽[(LV end-diastolic diameter⫺LV end-systolic diameter)/LV end-diastolic diameter]⫻100. Echocardiography was completed in unanesthetized animals within 5 minutes before exercise
and within 30 seconds after peak exercise.
Static and Continuous Ambulatory (Holter) ECG
ECG was performed as previously described.14,27 Limb-lead ECGs
were recorded to detect evidence of ventricular preexcitation,
namely, shortened PR intervals and delta waves. In a separate set of
experiments, a telemetry device was implanted to allow continuous
ambulatory ECG (Holter monitoring) and assessment of the chronotropic response to exercise. Mice were exercised to maximum
capacity according to the exercise stress testing protocol described
for stress echocardiography and were monitored for another 10
minutes during recovery.
Data Analysis
All data are presented as mean⫾SD, except as noted in the figure
legends. Comparisons between groups were made with Student’s t
test for pairwise comparisons and ANOVA for multiple
comparisons.
Results
Inhibiting AMPK in TG␥2N488I Mice With ␣2
Dominant-Negative Transgene
We have previously presented evidence suggesting that the
N488I mutation in the ␥2 subunit results in increased AMPK
activity.1,13 Xing et al15 have described a transgenic mouse,
designated TG␣2DN, that expresses a dominant-negative form
of the ␣2 subunit of AMPK. TG␣2DN mice have significantly
reduced ␣2 activity but normal ␣1 activity.15 By mating
TG␣2DN mice with TG␥2N488I transgenic mice, mice carrying
both the TG␥2N488I transgene and the TG␣2DN transgene,15
designated TG␥2N488I/TG␣2DN, were obtained. Levels of
TG␥2N488I polypeptide were the same in TG␥2N488I and
TG␥2N488I/TG␣2DN hearts (Figure 1C). However, the nonfunctional TG␣2DN peptide, which migrates as a highermolecular-weight polypeptide owing to its HA tag, completely replaced the lower-molecular-weight, endogenous,
wild-type ␣2 subunit (Figure 1B). The ␣1-subunit levels in
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November 15, 2005
Figure 3. Representative limb leads from
ECGs of wild-type, TG␣2DN, TG␥2N488I,
and TG␥2N488I/TG␣2DN mice. Whereas
wild-type and TG␣2DN demonstrate normal PR intervals, TG␥2N488I mice demonstrate shortened PR intervals and delta
waves secondary to ventricular preexcitation. TG␥2N488I/TG␣2DN mice demonstrate normalized PR intervals. Abbreviations are as defined in text.
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wild-type, TG␥2N488I, TG␣2DN, and TG␥2N488I/TG␣2DN hearts
were not significantly different (Figure 1A), as suggested
previously.15
We assessed the amount of AMPK activity associated with
the ␣1 and ␣2 subunits in 18-day-old transgenic mice (Table
1). TG␥2N488I/TG␣2DN mice demonstrated a significant reduction in AMP-stimulated, ␣2 subunit–associated AMPK activity, to ⬇37% of that seen in TG␥2N488I mice (Table 1) but no
significant reduction in ␣1-associated AMPK activity compared with TG␥2N488I mice. Consequently, we proceeded to
phenotypic characterization of these mice to define the roles
of the ␣1 and ␣2 subunit–associated AMPK activities.
Cardiac function and morphology were assessed in
TG␥2N488I, TG␣2DN, and TG␥2N488I/TG␣2DN mice. At age 5
weeks, heart-body mass ratios were indistinguishable between wild-type and TG␣2DN mice, whereas TG␥2N488I mice
demonstrated massive cardiac hypertrophy (Table 1). In
contrast, age-matched TG␥2N488I/TG␣2DN mice had nearnormal heart-body mass ratios (Table 1). The heart-body
mass ratio in TG␥2N488I/TG␣2DN mice remained mildly elevated, at a level similar to that found in TG␥2wt mice
(6.9⫾1.0 versus 7.2⫾0.3).13 Echocardiographic studies performed in 8-week-old animals demonstrated a similar significant reduction in LV wall thickness in TG␥2N488I/TG␣2DN
hearts compared with TG␥2N488I hearts (Table 1; 1.17 versus
1.54 mm; P⫽0.01). These differences persisted at age 20
weeks (Table 1).
The reduction in cardiac hypertrophy in TG␥2N488I/TG␣2DN
mice was accompanied by an 8-fold decrease in glycogen
content compared with TG␥2N488I hearts (Table 1). Both light
and electron microscopy confirmed the disappearance of
glycogen-laden vacuoles, the pathological hallmark of
PRKAG2 cardiomyopathy (Figure 2).
TG␥2N488I mice have ventricular preexcitation, manifested
by a short PR interval and delta waves on ECG.13,14 The PR
interval was 24% longer in TG␥2N488I/TG␣2DN mice relative
to TG␥2N488I mice (P⫽0.02), representing normal atrioventricular conduction without ventricular preexcitation (Figure
3 and Table 1). As we previously reported, TG␥2N488I mice
develop LV dilatation and systolic dysfunction, phenomena
not observed in wild-type and TG␥2wt mice.13 However,
TG␥2N488I/TG␣2DN mice demonstrated completely normal LV
end-diastolic diameters and fractional shortening at 20 weeks
of age (Table 1).
The maximal exercise capacity of wild-type, TG␥2wt,
TG␥2N488I, and TG␥2N488I/TG␣2DN mice was assessed to determine the consequences of cardiac morphological and
metabolic changes. Wild-type, TG␥2wt, TG␣2DN, and
TG␥2N488I/TG␣2DN mice demonstrated similar exercise capacities (Figure 4A and data not shown). However, TG␥2N488I
mice demonstrated an exercise capacity approximately half
that of wild-type mice (32.4⫾3.6 versus 58.1⫾6.0 minutes,
P⫽0.001; Figure 4A). This exercise deficit was completely
eliminated in double-transgenic TG␥2N488I/TG␣2DN mice.
Energy Reserve in TG␥2N488I Mice
To determine the physiological basis underlying impaired
exercise capacity in TG␥2N488I mice, we measured resting
cardiac levels of phosphocreatine by NMR and of ATP, ADP,
and AMP by high-performance liquid chromatography and
assessed chronotropic and contractile responses to exercise.
Because defects in glycogen metabolism, as well as cardiomyopathies secondary to mutations in genes encoding sarcomeric proteins, have been found to exhibit altered energetics,24,28,29 we hypothesized that TG␥2N488I mice might have
abnormal energy stores. However, we detected no significant
differences in phosphocreatine, ATP, ADP, and AMP levels
among wild-type, TG␥2wt, and TG␥2N488I in isolated, perfused
hearts from 5-week-old mice (Table 2).
On stress echocardiography, TG␥2N488I mice had lower baseline cardiac contractility (Table 3). However, TG␥2N488I mice
had an enhanced inotropic response to exercise, increased
fractional shortening, and decreased LV end-systolic dimension,
thereby suggesting adequate contractile reserve. Contrasted with
wild-type mice, TG␥2N488I mice demonstrated significantly reduced heart rates at baseline, during exercise, and during
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Increased AMPK(␣2) in PRKAG2 Cardiomyopathy
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tent with the model that stored glycogen was utilized as an
energy source during exercise.
Glycogen Metabolism in TG␥2N488I Hearts
In an attempt to define the mechanism leading to glycogen
accumulation in TG␥2N488I hearts, we assayed the activity of
4 enzymes involved in either glycogen synthesis or glycogenolysis, which are known to cause human glycogen storage
cardiomyopathies when defective. No significant differences
were detected in the activities of brancher enzyme, debrancher enzyme, glycogen phosphorylase, and glycogen
phosphorylase kinase in 5-week-old TG␥2N488I and wild-type
hearts (Table 2).
Discussion
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Figure 4. A, Adult wild-type, TG␥2wt, TG␥2N488I, and TG␥2N488I/
TG␣2DN mice were exercised on a treadmill with stepwise increments in incline and speed. Maximum exercise duration for
each individual mouse and the mean⫾SD for each group are
shown. P vs wild type, 0.001 for TG␥2N488I and 0.302 for
TG␥2N488I/TG␣2DN mice. B, Adult wild-type (䢇, n⫽6) and
TG␥2N488I (䡲, n⫽6) mice were implanted with ambulatory ECG
telemetry devices and exercised. Heart rate responses at baseline, at each stage of exercise, and after recovery (minutes from
the end of exercise) are shown as mean⫾SE. P⫽0.001 for differences between wild-type and TG␥2N488I mice. Abbreviations
are as defined in text.
recovery (Figure 4B; P⫽0.004). Heart rates of wild-type,
TG␥2wt, TG␣2DN, and TG␥2N488I/TG␣2DN mice were not significantly different (data not shown). Therefore, decreased exercise
performance in TG␥2N488I mice appears to be attributable to the
impairment in cardiac contractility and lower heart rates at
baseline and during exercise, but not to defective energetics.
To determine whether the increased glycogen in the cardiac myocytes of TG␥2N488I mice could be metabolized, we
measured cardiac tissue glycogen and lactate levels in hearts
from resting and exercised mice (Table 4). Exercised mice
were humanely killed within 10 seconds after completion of
maximal exercise. Wild-type and TG␥2wt mice demonstrated
significant (at least 40%) decreases in glycogen levels with
exercise. Glycogen levels decreased by ⬇25% in TG␥2N488I
hearts, which represented a very large absolute quantity of
glycogen. Concomitantly, more lactate was produced, consis-
TG␥2N488I mice provide a useful tool for gaining further
insights into the mechanisms by which mutations in the ␥2
subunit of AMPK cause cardiac glycogen accumulation,
preexcitation, cardiomyopathy, and heart failure. Here we
provide evidence that (1) PRKAG2 cardiomyopathy results
from activation of AMPK, which is mediated primarily via
␣2 subunit–associated AMPK, and (2) there is no cardiac
energy depletion in PRKAG2 cardiomyopathy at rest, and the
presence of contractile reserve in response to exercise challenge is observed on stress exercise echocardiography. Despite the pivotal role of AMPK in maintaining energy
metabolism, inappropriate activation of AMPK has minimal
effects on resting cardiac high-energy phosphate levels.
Murine models of hypertrophic cardiomyopathy resulting
from mutations in genes encoding sarcomeric proteins have
demonstrated impaired cellular energetics.24,28 In contrast,
TG␥2N488I mice exhibit normal resting cardiac cellular energetics (Table 2). Thus, although AMPK is a central regulator
of cellular energy levels, the N488I mutation does not perturb
resting cellular energy levels in the heart. Presumably, homeostatic mechanisms preserve normal energetics, at least in
the absence of stress. The functional impairment observed in
these mice is likely the result of glycogen deposition in the
heart rather than energy depletion. The normal resting cellular
energetics observed in TG␥2N488I mice also underscores the
fact that the mechanism of cardiac hypertrophy and cardiomyopathy resulting from this mutation is distinct from the
hypertrophic cardiomyopathy caused by sarcomere protein
gene mutations.
Stress echocardiography allows direct assessment of cardiac function in response to exercise challenge and is a
standard technique for evaluating myocardial ischemia in
humans with coronary heart disease. Although this test has
been found to have prognostic value in patients with hypertrophic or dilated cardiomyopathy, with impaired contractile
response to stress connoting a poorer prognosis,30 few if any
patients with PRKAG2 missense mutations have undergone
this test. To evaluate further cardiac energetics during exercise in TG␥2N488I mice, we developed a stress echocardiography protocol for assessing cardiac function in mice. Stressed
TG␥2N488I mice demonstrated marked improvement in cardiac
function, as fractional shortening increased from 31% to 46%
(P⬍0.0001; Table 3). The improved cardiac function of
exercised TG␥2N488I mice may have resulted from any 1 of
3146
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November 15, 2005
TABLE 2. High-Energy Phosphate Levels and Glycogen Metabolic Enzyme Activity in
Wild-Type (WT), TG␥2WT, and TG␥2N488I Mice
WT
TG␥2WT
TG␥2N488I
Phosphocreatine, mmol/L (n)
18.6⫾1.3 (11)
19.6⫾1.9 (9)
21.8⫾2.2 (10)
ATP, mmol/L (n)
11.0⫾0.5 (4)
10.4⫾0.2 (4)
11.2⫾0.2 (5)
ADP, mmol/L (n)
1.6⫾0.2 (4)
1.7⫾0.3 (4)
2.0⫾0.2 (5)
AMP, mmol/L (n)
0.26⫾0.11 (4)
0.24⫾0.07 (4)
0.22⫾0.04 (5)
Glycogen metabolic enzymes, ␮mol/g tissue per min
Branching enzyme (n)
Debranching enzyme (n)
Glycogen phosphorylase (n)
Glycogen phosphorylase kinase (n)
22⫾7 (2)
33⫾14 (2)
31⫾8 (2)
0.36⫾0.06 (2)
0.32⫾0.06 (2)
0.29⫾0.01 (2)
9.3⫾0.3 (2)
9.3⫾2.2 (2)
8.3⫾3.3 (2)
0.13⫾0.02 (2)
0.09⫾0.00 (2)
0.13⫾0.04 (2)
n indicates No. of mice used in each assay. No differences were found in phosphocreatine as measured by 31P
NMR; in ATP, ADP, and AMP levels as measured by high-performance liquid chromatography; or in activities of 4
enzymes involved in glycogen synthesis or degradation. Enzyme activities are reported as P⬎0.05 for all comparisons
between WT, ␥2WT, and ␥2N488I mice.
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
several mechanisms, including increased glycogen utilization. In contrast, fractional shortening was maximal at rest in
wild-type and TG␥2wt mice and did not improve further with
exercise.
Hypothesizing that improved cardiac function could result
from increased glycogen utilization, we investigated whether
the excess stored glycogen in TG␥2N488I mice could be used
during exercise. TG␥2N488I mice demonstrated a decrease in
cardiac glycogen levels with exercise, indicating that the
excess glycogen in their hearts could be mobilized and
metabolized (Table 4). TG␥2N488I mice also demonstrated an
increase in cardiac lactate, presumably from increased glycogen metabolism. The large decrease in glycogen levels was
even more remarkable, considering that TG␥2N488I mice have
a markedly reduced exercise tolerance. These results raise the
possibility that mobilization of cardiac glycogen allows
improved cardiac function.
We and others have previously suggested that PRKAG2
cardiomyopathy results from inappropriate activation of
AMPK.1,9,13 However, other investigators have proposed
other models, including a biphasic response manifested by
increased resting activity but impaired responsiveness to
metabolic stress.10 –12,31 We have used a genetic approach to
determine whether the principal effect of the N488I mutation
is to increase or decrease AMPK activity. If the phenotypic
effects of this mutation result from an increase in AMPK
␣2-subunit activity, then they should be attenuated or abolished in the presence of a dominant-negative AMPK ␣2
subunit. The features of double-transgenic mice carrying both
mutations (TG␥2N488I/TG␣2DN) are consistent with the model
that PRKAG2 with the N488I mutation inappropriately activates the AMPK ␣2 subunit. In TG␥2N488I/TG␣2DN mice,
normal catalytic ␣2 subunits were replaced by an inactive
form of the ␣2 subunit (Figure 1). As a result, TG␥2N488I/
TG␣2DN mice had a significant reduction in ␣2-, but not ␣1-,
subunit activity, and exhibited normal or near-normal cardiac
function, cardiac morphology, and conduction system function (Table 1 and Figures 2 through 4). Hence, reducing
AMPK-associated ␣2-subunit activity restores cardiac physiology to nearly normal, consistent with the model that in the
presence of the N488I ␥2 subunit, the AMPK ␣2 subunit is
inappropriately active. Moreover, despite the 2-fold increase
in ␣1 subunit–associated AMPK activity observed in
TG␥2N488I mice,13 cardiac hypertrophy was almost completely
TABLE 3. Stress Echocardiography in 8-Week-Old Wild-Type (WT), TG␥2WT, and
TG␥2N488I Mice
WT
No. of mice
TG␥2WT
TG␥2N488I
7
3
6
LVEDD before exercise, mm
2.31⫾0.35
2.69⫾0.12
3.81⫾0.75
LVEDD after exercise, mm
2.25⫾0.26
2.31⫾0.20
3.51⫾0.81
0.6
0.06
0.05
Fractional shortening before exercise, %
75⫾3
73⫾9
31⫾15
Fractional shortening after exercise, %
71⫾7
72⫾3
46⫾10
P LVEDD, before vs after exercise
P fractional shortening, before vs after exercise
0.1
0.7
0.0009
Heart rate before exercise, beats/min
693⫾32
651⫾36
602⫾76
Heart rate after exercise, beats/min
606⫾26
680⫾75
549⫾81
0.008
0.5
P Heart rate, before vs after exercise
0.1
EDD indicates end-diastolic diameter. Other abbreviations are as defined in ext. TG␥2
mice
were able to improve chamber size and contractility, as assessed by fractional shortening, during
exercise.
N488I
Ahmad et al
Increased AMPK(␣2) in PRKAG2 Cardiomyopathy
3147
TABLE 4. Changes in Myocardial Glycogen and Lactate Levels in Response to
Exercise in Wild-Type (WT), TG␥2WT, and TG␥2N488I Mice
WT
TG␥2WT
TG␥2N488I
Resting (n)
1.7⫾0.4 (9)
13.9⫾4.8 (11)
50.0⫾9.7 (6)
After exercise (n)
0.9⫾0.5 (6)
6.8⫾1.6 (8)
36.7⫾14.9 (8)
0.004
0.0005
0.04
Glycogen/HW, ␮g/mg tissue
P resting vs after exercise
Lactate/HW, ␮mol/g tissue
Resting (n)
7.8⫾1.6 (6)
8.1⫾2.8 (5)
9.6⫾0.9 (4)
After exercise (n)
5.4⫾2.3 (5)
10.1⫾1.3 (4)
11.4⫾0.4 (5)
0.03
0.4
0.002
P resting vs after exercise
HW indicates heart weight; n, No. of mice used in each assay. Other abbreviations are as defined
in text.
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
reversed in the presence of a dominant-negative ␣2 subunit,
indicating that hypertrophy is mediated largely through the
effects of mutations in ␣2-subunit activity. Although the
mechanism is uncertain, these data suggest that in vivo
␣2-associated AMPK and ␣1-associated AMPK have different biological targets. This target specificity may be determined directly by the ␣ subunit or by a preference of the ␣
subunit for a particular ␤ or ␥ isoform, which may be
responsible for target specificity.
Although inappropriately active ␣2 subunit–associated
AMPK activity is responsible for the phenotype of this
cardiomyopathy, the mechanism of glycogen accumulation is
still unclear. The activities of branching enzyme, debranching
enzyme, glycogen phosphorylase, and glycogen phosphorylase kinase are unchanged in adult TG␥2N488I hearts (Table 2).
These enzymes are involved in the synthesis or degradation
of glycogen, and deficiencies in each of these enzymes cause
glycogen storage disease. However, regulation of 1 or more
glycogen metabolic enzyme activities must be altered in
PRKAG2 cardiomyopathy.
PRKAG2 cardiomyopathy in TG␥2N488I mice provides a
unique opportunity to investigate the role of AMPK and
glycogen in regulating cardiac energy metabolism. Eventually, cardiac AMPK targets and the mechanisms leading from
AMPK activation to glycogen storage will be defined. Identification of these targets, coupled with an understanding of
the physiological pathways by which glycogen accumulates
in cardiac myocytes, will provide novel therapeutic targets for
a variety of conditions associated with defective cardiac
energetics.
Acknowledgments
This work was supported by the Howard Hughes Medical Institute
(F.A., M.A., C.E.S., J.G.S.), the Canadian Institutes of Health
Research (F.A.), the National Institutes of Health (HL 67970 to R.T.
and HL 52320 and HL 63985 to J.S.I.), the American Heart
Association (R.T.), and the American Diabetes Association (N.M.,
L.G.).
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CLINICAL PERSPECTIVE
We demonstrate that PRKAG2 cardiomyopathy results from activated AMP-dependent protein kinase (AMPK) and that
most of the increased AMPK activity is mediated by ␣2-subunit associated AMPK. Transgenic mice over-expressing
mutant ␥2 AMPK subunit (PRKAG2) N488I (TG␥2N488I) mimic human PRKAG2 cardiomyopathy characterized by cardiac
hypertrophy, pre-excitation and glycogen accumulation. Normally, most cardiac AMPK activity is associated with the ␣1
subunit, however in TG␥2N488I hearts approximately 50% of AMPK activity is associated with ␣1-subunit containing
AMPK and 50% with ␣2-subunit AMPK. To determine which AMPK isoform is responsible for the cardiac phenotypes
we selectively inhibited ␣2-, but not ␣1-, subunit associated AMPK activity by introducing a dominant negative form of
␣2 subunit into TG␥2N488I hearts. PRKAG2 cardiomyopathy was markedly attenuated by dominant negative ␣2 subunit,
concomitant with a significant decrease in ␣2-associated AMPK activity, confirming that inappropriate AMPK activation
causes disease. Further, glycogen stores found in PRKAG2N488I hearts were not associated with energy deficiency and in
fact represent a dynamic pool of glycogen, which can be mobilized during metabolic stress. These results suggest that: (1)
pharmacological inhibition of ␣2 associated AMPK should prevent PRKAG2 cardiomyopathy, and (2) increased AMPK
activity may increase cardiac tolerance to metabolic stress.
Increased α2 Subunit-Associated AMPK Activity and PRKAG2 Cardiomyopathy
Ferhaan Ahmad, Michael Arad, Nicolas Musi, Huamei He, Cordula Wolf, Dorothy Branco,
Antonio R. Perez-Atayde, David Stapleton, Deeksha Bali, Yanqiu Xing, Rong Tian, Laurie J.
Goodyear, Charles I. Berul, Joanne S. Ingwall, Christine E. Seidman and J. G. Seidman
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Circulation. published online November 7, 2005;
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