A PRKAG2 mutation causes biphasic changes in myocardial AMPK activity and does not protect against ischemia

Biochemical and Biophysical Research Communications 360 (2007) 381–387 www.elsevier.com/locate/ybbrc

A PRKAG2 mutation causes biphasic changes in myocardial AMPK activity and does not protect against ischemia Sanjay K. Banerjee a, Ravi Ramani a, Samir Saba a, Jennifer Rager a, Rong Tian b, Michael A. Mathier a, Ferhaan Ahmad a,c,* a

Cardiovascular Institute, Department of Medicine, University of Pittsburgh, 200 Lothrop Street, Scaife Hall, S558, Pittsburgh, PA 15213, USA b Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA 02115, USA c Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15213, USA Received 6 June 2007 Available online 19 June 2007

Abstract Dominant mutations in the c2 regulatory subunit of AMP-activated protein kinase (AMPK), encoded by the gene PRKAG2, cause glycogen storage cardiomyopathy. We sought to elucidate the effect of the Thr400Asn (T400N) human mutation in a transgenic mouse (TGT400N) on AMPK activity, and its ability to protect the heart against ischemia–reperfusion injury. TGT400N hearts had markedly vacuolated myocytes, excessive accumulation of glycogen, hypertrophy, and preexcitation. Early activation of myocardial AMPK, followed by depression, and then recovery to wild-type levels was observed. AMPK activity correlated inversely with glycogen content. Partial rescue of the phenotype was observed when TGT400N mice were crossbred with TGa2DN mice, which overexpress a dominant negative mutant of the AMPK a2 catalytic subunit. TGT400N hearts had greater infarct sizes and apoptosis when subjected to ischemia–reperfusion. Increased AMPK activity is responsible for glycogen storage cardiomyopathy. Despite high glycogen content, the TGT400N heart is not protected against ischemia–reperfusion injury.  2007 Elsevier Inc. All rights reserved. Keywords: Glycogen cardiomyopathy; AMPK; Transgenic mouse model; Cardiac hypertrophy; Myocardial ischemia–reperfusion

AMP-activated protein kinase (AMPK), a heterotrimeric protein composed of a catalytic a subunit and regulatory b and c subunits, is activated under conditions of energy depletion manifested by increased cellular AMP levels [1,2]. AMPK modulates glucose uptake and glycolysis [1,3]. We and others have found dominant mutations in the c2 regulatory subunit, encoded by the gene PRKAG2, to cause cardiac hypertrophy and increased risk of sudden cardiac death [4–6]. PRKAG2 mutations produce a distinctive cardiac histopathology characterized by enlarged myocytes with vacuoles containing glycogen derivatives [6]. * Corresponding author. Address: Cardiovascular Institute, Department of Medicine, University of Pittsburgh, 200 Lothrop Street, Scaife Hall, S558, Pittsburgh, PA 15213, USA. Fax: +1 412 647 4227. E-mail address: [email protected] (F. Ahmad).

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.06.067

In addition, patients frequently manifest electrophysiological abnormalities such as ventricular preexcitation, atrial fibrillation, and progressive development of atrioventricular conduction block [4–6]. The association between PRKAG2 mutations and glycogen cardiomyopathy has been confirmed in three transgenic (TG) mouse models [7–9]. However the effect of these mutations on AMPK activity remains controversial. Whereas the TGN488I mouse was reported to have an increase in AMPK activity [7], other investigators found a decrease in activity in TGR302Q [8] and TGR531G [9] mice. To resolve this discrepancy and to uncover the mechanisms by which PRKAG2 defects cause disease, we constructed a transgenic mouse with the PRKAG2 Thr400Asn (T400N) mutation (TGT400N), previously identified in humans [6]. Because of our demonstration that stored glycogen in

382

S.K. Banerjee et al. / Biochemical and Biophysical Research Communications 360 (2007) 381–387

TGN488I hearts can be utilized during exercise stress [10], we also tested whether glycogen in TGT400N hearts confers protection against ischemia–reperfusion injury. Materials and methods Generation of transgenic mice (TGT400N). A T400N cDNA was generated from human cardiac RNA by PCR mutagenesis, and inserted into a pBluescript based vector with the mouse a-myosin heavy chain (aMHC) promoter [11], a highly active cardiac myocyte specific promoter (Fig. 1A). The transgenic vector was linearized with Bam HI, size-fractionated, purified, and microinjected into fertilized FVB mouse oocytes at the University of Pittsburgh Transgenic and Chimeric Mouse Facility. Transgenic founders were identified by Southern blot analyses. Offspring of founders were genotyped by PCR amplification of the transgene using two sets of primer pairs—SPKG2 (5 0 -CCGCTCCTCCTCCAAAGAGT-3 0 ) and ASPKG3 (5 0 -GCAATGTTGTGGTACGTTCC-3 0 ) both within the

T400N

A

C → A 1289 ATG

Human PRKAG2 cDNA

TGA

91

1754

α MHC Promoter

hGH

SalI

BamHI

BamHI

WT

B

AAAA

TGT400N

TGT400N Lines

1

2

3

4

5

Prkag1 Prkag2

WT

C TGT400N Lines

TGT400N 1

2

AMPK γ 2 (75 kDa)

PRKAG2 cDNA; and MHC F1 (5 0 -CGGCACTCTTAGCAAACCTC-3 0 ) within the vector backbone 5 0 of the cDNA, and MHC R1 (5 0 -TTCT GGCTGGCATTTTTCTT-3 0 ) within the cDNA. FVB background TGa2DN mice, which overexpress a dominant negative mutant of the AMPK a2 catalytic subunit and have low myocardial AMPK activity, have been previously described [12]. Double transgenic mice (TGT400N/TGa2DN) were obtained by crossbreeding. Transgenic mouse lines with the wildtype PRKAG2 have been generated previously by three laboratories [7–9] and demonstrate only a mild phenotype intermediate between wild-type (WT) and mutant lines. Because our focus was on the mechanisms of glycogen cardiomyopathy, which is not seen by mere overexpression of a cardiac protein, and on the effect of excess glycogen on response to ischemia, we did not use these mouse lines in the current study. RNA and protein analyses. Northern blots and quantitative analysis of PRKAG1 and PRKAG2 RNA expression were performed as described [7]. Western blots for a and c2 subunits of AMPK were performed using 30 lg total cardiac protein from WT and TGT400N mice with antibodies specific for the a and c2 subunits (Cell Signaling). Glycogen content, AMPK activity, and AMPK phosphorylation. Glycogen content was determined by the amyloglucosidase digestion method [10] and total basal AMPK activity was assayed as described [7,10]. We measured myocardial AMPK activity and glycogen levels in TGT400N and WT hearts at ages 2 days, and 1, 2, 4, 8, 12, and 20 weeks. Levels of Thr172 phosphorylated AMPK a subunit (P-AMPK) were determined by Western blotting using 10 lg total cardiac protein with antibody specific for P-AMPK (Cell Signaling). Echocardiography and electrocardiography (ECG). Transthoracic echocardiography was performed every 4 weeks at ages 4–20 weeks after sedation with tribromoethanol (125 mg/kg IP) using a VisualSonics Vevo 770 machine with a 45 MHz transducer [10]. Left ventricular end diastolic (LVEDD) and end systolic (LVESD) chamber dimensions and anterior wall thickness (LVAWT) were obtained from M-mode tracings. LV fractional shortening (FS) was calculated as (LVEDD-LVESD)/ LVEDD · 100%. ECGs were performed as described [7,10]. Histopathology. Hearts were excised, washed in PBS, and weighed. Cardiac tissue was treated as described previously [7] for staining with either hematoxylin and eosin (H&E) or periodic acid–Schiff (PAS) for glycogen. Cardiac ischemia and reperfusion. Six to eight week old mice were anesthetized with tribromoethanol (125 mg/kg IP) and subjected to in vivo ischemia for 30 min by ligation of the left anterior descending coronary artery (LAD), followed by 48 h reperfusion [13]. The LAD was then reoccluded and injected with 1 ml 1.0% Evans blue (Sigma) through the jugular vein to delineate nonischemic tissue. The heart was excised and cut into four transverse slices. Slices were stained for 15 min with 1.5% 2,3,5triphenyltetrazolium chloride (TTC) (Sigma) and placed in 2.5% formaldehyde to determine infarct area under a microscope. Left ventricular (LV) area, area at risk (AAR), and infarct area (IA) were determined by computerized planimetry using Image J software. Five micrometer heart sections were assessed for apoptosis by TUNEL staining using ApopTag Peroxidase Kits (Millipore). Data analysis. Results are expressed as means ± SEM. Differences between pairs of mouse genotypes were assessed by Student’s t-test. A p < 0.05 was considered significant.

AMPK α (62 kDa) Fig. 1. Generation of transgenic mouse. (A) Transgene construct containing human PRKAG2 cDNA with the T400N missense mutation at nucleotide 1289, under the control of the aMHC promoter and terminated by the human growth hormone 3 0 UTR (hGH) and polyadenylation signal (AAAA). (B) Northern blots using Prkag1 and Prkag2 cRNA probes to assess expression in WT and TGT400N hearts. Prkag1 expression was similar in both WT and TGT400N hearts. (C) Western blots using AMPK a and c2 subunit antibodies to assess protein expression in WT and TGT400N hearts. PRKAG2 RNA and the c2 subunit protein were expressed only in TGT400N hearts.

Results PRKAG2 expression PRKAG2 RNA expression was evident at different levels in four of five TGT400N lines (Fig. 1B), whereas similar expression of Prkag1 RNA was observed in both WT and TGT400N lines. Initial characterization demonstrated glycogen cardiomyopathy in transgenic lines 1 through 4. Line 1, demonstrating the highest levels of expression,

S.K. Banerjee et al. / Biochemical and Biophysical Research Communications 360 (2007) 381–387

383

Table 1 Heart weight/body weight ratio and cardiac glycogen content in TGT400N and TGT400N/TGa2DN mice 1 Week WT Heart weight/body weight (n = 6) Cardiac glycogen content (lg/mg wet weight) (n = 3) *  

2 Weeks T400N

WT

TG

*

0.0060 ± 0.0002 0.0072 ± 0.0001

4 Weeks T400N

TG

WT *

0.0047 ± 0.0003 0.0074 ± 0.0003

TGT400N

TGT400N/TGa2DN *

0.0043 ± 0.0002 0.0157 ± 0.0007 0.31 ± 0.05

31.01 ± 4.07*

0.0086 ± 0.0002*,  16.82 ± 3.81*, 

p < 0.001 vs. WT. p < 0.01 vs. TGT400N..

was used for further studies. Transgene expression was further confirmed by Western blotting of the c2 and a subunits of AMPK. Only TGT400N hearts expressed AMPK c2 while the expression of AMPK a appeared similar in both WT and TGT400N hearts (Fig. 1C). Cardiac mass and histology The heart weight to body weight ratio was elevated beginning at age 1 week and was almost four times higher at age 4 weeks in TGT400N compared to WT mice (Table 1). H&E staining of myocardial sections showed large vacuoles in TGT400N myocytes throughout the ventricles (Fig. 2B). These myocytes were filled with PAS-positive material indicating glycogen (Fig. 2D). The vacuoles and PAS-positive material were absent in WT myocytes (Figs. 2A and C). Echocardiography and electrocardiography Left ventricular anterior wall thickness (LVAWT) was significantly increased in TGT400N hearts compared with WT at all ages evaluated (4, 8, 12, 16, and 20 weeks) (Table 2). However, LVAWT in TGT400N mice appeared to be maximal at age 8 weeks and then progressively declined to age 20 weeks. Progressive LV dilation and contractile dysfunction was observed beginning between ages 8 and 12 weeks. LV end diastolic diameter (LVEDD) in TGT400N mice was increased significantly (p < 0.01) compared to WT mice at ages 12, 16, and 20 weeks. LV dilation was accompanied by contractile impairment. At 20 weeks of age, FS in TGT400N mice was reduced to one-third that of WT. Similar to humans, TGT400N mice manifested ventricular preexcitation as evidenced by the presence of delta waves, a short PR interval (17 ± 3 vs. 48 ± 6 ms in WT; p < 0.01) and a wide QRS (28 ± 4 vs. 19 ± 2 ms in WT; p < 0.05) (n = 3/group). Evolution of AMPK activity, AMPK phosphorylation, and myocardial glycogen content over time A biphasic pattern in AMPK activity was observed in TGT400N mice relative to WT littermates over time (Fig. 3A). At 2 days of age, we observed a small but significant increase (1.1 times) in myocardial AMPK activity of

TGT400N mice relative to WT. AMPK activity rose further in TGT400N hearts at 1 and 2 weeks (1.4 and 1.5 times relative to WT, respectively). In contrast, we observed significant reductions in AMPK activity at ages 4, 8, and 12 weeks, (0.54, 0.52, and 0.41 times relative to WT, respectively). Finally, at 20 weeks of age, AMPK activity returned to WT levels. Parallel to these changes in AMPK activity, increased levels of P-AMPK were observed at age 2 weeks in TGT400N hearts relative to WT, followed by decreased levels at age 4 and 8 weeks, and ending in normal levels at age 20 weeks (Fig. 3C). There was an inverse correlation between myocardial AMPK activity and glycogen content (Figs. 3A and B). Myocardial glycogen levels were similar between TGT400N and WT mice at age 2 days, but rose progressively in TGT400N mice from age 1 week to age 8 weeks. However, by age 20 weeks, glycogen levels in TGT400N hearts had reverted towards WT levels. Partial normalization of phenotype in TGT400N/TGa2DN mice TGa2DN mice, carrying a dominant negative AMPK catalytic a2 subunit, were crossbred with TGT400N mice. At age 4 weeks, both the heart weight/body weight ratio and the glycogen content in TGT400N/TGa2DN hearts were significantly reduced to one half those of TGT400N hearts (Table 1). Echocardiographic studies in 4-, 12-, and 20week-old mice demonstrated a reduction in LVAWT and LVEDD and an improvement in FS in TGT400N/TGa2DN mice relative to TGT400N (Table 2). Excess glycogen does not protect against myocardial ischemia–reperfusion injury Representative photographs of myocardial tissues after staining with Evans blue dye to delineate AAR and 2,3,5triphenyltetrazolium chloride to delineate IA in TGT400N and WT mice are shown in Fig. 4A. The ratio of AAR to LV area was the same in TGT400N and WT mice. However, the ratios of IA to AAR and IA to LV area were more than three times greater in TGT400N relative to WT mice (Fig. 4B). TGT400N ischemia–reperfused hearts exhibited a larger area of necrosis and greater neutrophil infiltration compared to WT hearts after H&E stain (Fig. 4A).

384

S.K. Banerjee et al. / Biochemical and Biophysical Research Communications 360 (2007) 381–387

Fig. 2. Histopathology of WT (A) and TGT400N (B) hearts stained with hematoxylin and eosin, and of WT (C) and TGT400N (D) hearts stained with PAS. Large vacuoles were abundant throughout the TGT400N myocardium, containing PAS-positive glycogen.

A higher proportion of TUNEL-positive cells were present in the myocardium of TGT400N compared to WT mice (Fig. 4A). Myocardial P-AMPK levels were assessed by immuno blot following ischemia–reperfusion. Decreased levels of P-AMPK protein were observed in TGT400N hearts (Fig. 4C), consistent with lower AMPK activity. Discussion This study describes a novel murine model of glycogen cardiomyopathy caused by the human PRKAG2 T400N mutation. Mutant mice develop all the characteristic features of glycogen cardiomyopathy, i.e., left ventricular hypertrophy, glycogen deposition in the heart, and ventricular preexcitation. The hypertrophy manifests itself at an early age, and leads to ventricular remodeling characterized by dilation and impaired contractility at 8–12 weeks. Previously characterized transgenic mouse models with R302Q, N488I, and R531G mutations have a similar phenotype despite being reported to have dissimilar changes in AMPK activity. TGN488I mice showed an increase AMPK activity [7], whereas the TGR302Q and TGR531G mice showed a decrease in the level of AMPK activity [8,9]. The TGT400N mouse had evidence of early elevation of AMPK activity starting from age 2 days to 2 weeks. This initial overactivity of AMPK was followed by a reduction in AMPK activity below WT levels at ages 4–12 weeks, and finally normalization at age 20 weeks. This variation in AMPK activity correlated well with the variation in the level of Thr172 phosphorylation of the a subunit of AMPK (P-AMPK) over the same time period. The

relationship between the level of phosphorylation of the a subunit with AMPK activity has also been observed by other investigators [14,15]. Taken together, our data strongly suggest that there is a biphasic response of AMPK activity to mutations in PRKAG2. Our results provide an explanation for the discrepant findings concerning AMPK activity in past studies. Consistent with the present work, Arad et al. detected activation of AMPK from 1-weekold TGN488I hearts [7]; whereas Davies et al. reported a decrease in AMPK activity at 4 and 8 weeks in TGR531G hearts [9]. We observed an inverse correlation between AMPK activity and cardiac glycogen content. AMPK activity reached a trough when glycogen levels were at a peak, and returned to normal as glycogen levels declined (Figs. 3A and B). As in the TGT400N model, decreased glycogen content at age 20 weeks was also reported in the TGN488I and TGR531G models [9,16]. In skeletal muscle, AMPK activity is inversely correlated with the level of glycogen [17]. Interestingly, the b subunit isoforms of AMPK contain a glycogen-binding domain [18]. This domain may play a role in downregulating AMPK activity in response to cardiac glycogen. All these observations suggest that AMPK activity could be reduced in presence of high glycogen levels. The basis for the eventual reduction in cardiac glycogen levels at 20 weeks of age in TGT400N mice is unclear. We speculate that myocardial energetic demands may change as the heart fails, necessitating greater glycogen mobilization. Our results suggest that glycogen cardiomyopathy results from an initial inappropriate activation of AMPK.

AMPK Activity (% of Control)

385

WT

200

*

150

TG T400N

*

*

100

*

*

*

50 0 2 Days

1 Week

2 Weeks

4 Weeks

8 Weeks

12 Weeks

20 Weeks

Age

Glycogen Content (Fold Over Control)

†

LVAWT, left ventricular anterior wall thickness; LVEDD, left ventricular end diastolic diameter; FS, fractional shortening; HR, heart rate. n = 4–7/group. * p < 0.01 vs. WT.   p < 0.01 vs. TGT400N.

0.89 ± 0.03 1.64 ± 0.20* 0.97 ± 0.02 1.26 ± 0.04* 1.23 ± 0.07* 3.17 ± 0.11 4.66 ± 0.32* 3.14 ± 0.11 5.15 ± 0.41* 3.74 ± 0.15  48 ± 3 16 ± 2* 47 ± 1 16 ± 2* 25 ± 1*,  506 ± 28 416 ± 38 398 ± 16 374 ± 29 387 ± 17 0.81 ± 0.02 1.80 ± 0.20* 0.99 ± 0.09 1.72 ± 0.10* 1.26 ± 0.13  3.18 ± 0.12 2.96 ± 0.13 3.06 ± 0.18 4.07 ± 0.31* 2.62 ± 0.25  40 ± 2 38 ± 4 40 ± 2 21 ± 3* 54 ± 5*,  376 ± 21 368 ± 26 416 ± 16 378 ± 16 342 ± 11 LVAWT(mm) 0.76 ± 0.04 1.81 ± 0.26* 1.37 ± 0.08*,  LVEDD(mm) 2.72 ± 0.11 2.47 ± 0.18 2.67 ± 0.09 FS (%) 37 ± 5 57 ± 6* 63 ± 3* HR(bpm) 435 ± 4 403 ± 23 411 ± 25

WT WT WT TGT400N WT

8 Weeks

TGT400N/ TGa2DN TGT400N WT

4 Weeks

Table 2 Echocardiographic parameters in wild-type, TGT400N and TGT400N/TGa2DN mice

12 Weeks

TGT400N

TGT400N/ TGa2DN

16 Weeks

TGT400N

20 Weeks

TGT400N

TGT400N/ TGa2DN

S.K. Banerjee et al. / Biochemical and Biophysical Research Communications 360 (2007) 381–387

WT

†

100

TG T400N

80 60 40

†

20

†

*

0 2 Days

1 Week

2 Weeks

4 Weeks

8 Weeks

20 Weeks

Age

Age (weeks)

1

2

4

8

20

WT TG WT TG WT TG WT TG WT TG P-AMPK Loading control

Fig. 3. Effect of the PRKAG2 T400N mutation on AMPK activity, cardiac glycogen content, and Thr172 phosphorylated AMPK a subunit. (A) Activation of myocardial AMPK in TGT400N hearts relative to WT was observed at 2 days to 2 weeks of age, followed by reduction at ages 4 to 12 weeks, and recovery to WT levels at age 20 weeks. n = 3–6/group. *p < 0.05 versus WT. (B) Glycogen content in TGT400N hearts as a multiple of that in age-matched WT. A progressive increase in TGT400N cardiac glycogen content occurred from age 1 week, reaching a maximum at 4 weeks, and declining by 20 weeks. n = 3/group. *p < 0.05 and  p < 0.001 versus WT. (C) Levels of Thr172 phosphorylated AMPK a subunit were increased in TGT400N relative to WT hearts at age 2 weeks, decreased at 4 and 8 weeks, and normalized at 20 weeks. A Coomassie blue stained protein band (lower panel) was used as a loading control.

To further confirm this hypothesis, we replicated in TGT400N mice a genetic strategy we had previously used in TGN488I mice [10]. The phenotype of TGT400N mice was attenuated in the presence of a dominant-negative AMPK a catalytic subunit. Thus, the phenotype of the T400N mutation, as that of the N488I mutation [10], is caused by an initial inappropriate activation of AMPK. Hence, reducing AMPK activity at a very early stage can reduce phenotype and restore cardiac physiology to nearly normal. A unique feature of the glycogen storage disease phenotype caused by PRKAG2 mutations is that the heart sustains its ability to use glycogen [10,16]. Therefore, we hypothesized that the stored glycogen in TGT400N heart may be protective during ischemia–reperfusion injury. Inconsistent with the hypothesis, increased infarct size, necrosis and apoptosis were observed in TGT400N hearts following ischemia–reperfusion injury. AMPK is normally upregulated during ischemia and likely confers myocardial protection [19]. In contrast, TGT400N hearts, with low

386

S.K. Banerjee et al. / Biochemical and Biophysical Research Communications 360 (2007) 381–387

A

WT

TGT400N

B WT

TTC

1 mm

Percent Relative Size

TG T400N

*

90 80 70 60 50 40 30 20 10 0

* AAR/LV

H&E

IA/LV

IA/AAR

20 µm

C

WT

TGT400N

TUNEL P-AMPK

20 µm

Loading Control

Fig. 4. Effect of the T400N mutation on ischemia–reperfusion injury. (A) Representative myocardial tissues from WT (left) and TGT400N mice at 48 h after ischemia–reperfusion. Upper panel. The nonischemic area is indicated by blue or black, the non-infarcted area at risk (AAR) by red, and the infarct area (IA) by white. The IA (arrowheads) was more extensive in TGT400N mice. Middle panel. Severe necrosis and neutrophil infiltration (arrowheads) was present in TGT400N (H&E staining). Lower panel. Representative photographs of TUNEL-stained heart sections showed greater apoptosis (arrowheads) in TGT400N hearts. (B) Quantification of infarct size in WT and TGT400N mice. AAR/LV, ratio of area at risk to left ventricular area[20]; IA/AAR, ratio of infarct area to AAR; IA/LV, ratio of IA to LV. IA/AAR and IA/LV were increased more than three-fold in TGT400N relative to WT mice. n = 5/group. *p < 0.001 versus WT. (C) Increased P-AMPK in heart tissues from WT relative to TGT400N mice at 48 h after ischemia–reperfusion. A Coomassie blue stained protein band (lower panel) was used as a loading control.

baseline AMPK activity at the age of the ischemia–reperfusion studies and lower induction of P-AMPK, responded more poorly, despite the availability of a greater amount of glycogen. We have previously shown that P-AMPK does not respond to stress in TGN488I mice because of loss of sensitivity to AMP [14]. This study further underscores the importance of AMPK activity in protection against ischemia. Other cardiomyopathic processes, in addition to the effects of AMPK and glycogen, may contribute to the greater injury. In summary, we demonstrate for the first time a biphasic change in AMPK activity and glycogen content in TGT400N hearts. Thus, the time at which AMPK activity was measured in other studies may explain the discrepant results obtained. Moreover, excess glycogen fails to confer protection against myocardial injury secondary to ischemia and reperfusion. Reducing AMPK activity at an early stage might be a strategy for treating this fatal disease.

Acknowledgments We thank Jeffrey Robbins, Ph.D., Cincinnati Children’s Hospital, for the aMHC vector. Funding was provided by an American Heart Association Scientist Development Grant (FA), the Hillgrove Foundation (SKB), and the University of Pittsburgh Cardiovascular Institute (Director,

Barry London, M.D., Ph.D.) (FA). FA is a Doris Duke Charitable Foundation Clinical Scientist. References [1] B.E. Kemp, K.I. Mitchelhill, D. Stapleton, B.J. Michell, Z.P. Chen, L.A. Witters, Dealing with energy demand: the AMP-activated protein kinase, Trends Biochem. Sci. 24 (1999) 22–25. [2] D.G. Hardie, Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status, Endocrinology 144 (2003) 5179–5183. [3] P.C. Cheung, I.P. Salt, S.P. Davies, D.G. Hardie, D. Carling, Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding, Biochem. J. 346 (Pt 3) (2000) 659–669. [4] M.H. Gollob, M.S. Green, A.S. Tang, T. Gollob, A. Karibe, A.S. Ali Hassan, F. Ahmad, R. Lozado, G. Shah, L. Fananapazir, L.L. Bachinski, R. Roberts, Identification of a gene responsible for familial Wolff–Parkinson–White syndrome, N. Engl. J. Med. 344 (2001) 1823–1831. [5] E. Blair, C. Redwood, H. Ashrafian, M. Oliveira, J. Broxholme, B. Kerr, A. Salmon, I. Ostman-Smith, H. Watkins, Mutations in the gamma(2) subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis, Hum. Mol. Genet. 10 (2001) 1215–1220. [6] M. Arad, D.W. Benson, A.R. Perez-Atayde, W.J. McKenna, E.A. Sparks, R.J. Kanter, K. McGarry, J.G. Seidman, C.E. Seidman, Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy, J. Clin. Invest. 109 (2002) 352–357.

S.K. Banerjee et al. / Biochemical and Biophysical Research Communications 360 (2007) 381–387 [7] M. Arad, I.P. Moskowitz, V.V. Patel, F. Ahmad, A.R. Perez-Atayde, D.B. Sawyer, M. Walter, G.H. Li, P.G. Burgon, C.T. Maguire, D. Stapleton, J.P. Schmitt, X.X. Guo, A. Pizard, S. Kupershmidt, D.M. Roden, C.I. Berul, C.E. Seidman, J.G. Seidman, Transgenic mice overexpressing mutant PRKAG2 define the cause of Wolff–Parkinson–White syndrome in glycogen storage cardiomyopathy, Circulation 107 (2003) 2850–2856. [8] J.S. Sidhu, Y.S. Rajawat, T.G. Rami, M.H. Gollob, Z. Wang, R. Yuan, A.J. Marian, F.J. DeMayo, D. Weilbacher, G.E. Taffet, J.K. Davies, D. Carling, D.S. Khoury, R. Roberts, Transgenic mouse model of ventricular preexcitation and atrioventricular reentrant tachycardia induced by an AMP-activated protein kinase loss-offunction mutation responsible for Wolff-Parkinson-White syndrome, Circulation 111 (2005) 21–29. [9] J.K. Davies, D.J. Wells, K. Liu, H.R. Whitrow, T.D. Daniel, R. Grignani, C.A. Lygate, J.E. Schneider, G. Noel, H. Watkins, D. Carling, Characterization of the role of gamma2 R531G mutation in AMP-activated protein kinase in cardiac hypertrophy and WolffParkinson-White syndrome, Am. J. Physiol. Heart Circ. Physiol. 290 (2006) H1942–H1951. [10] F. Ahmad, M. Arad, N. Musi, H. He, C. Wolf, D. Branco, A.R. Perez-Atayde, D. Stapleton, D. Bali, Y. Xing, R. Tian, L.J. Goodyear, C.I. Berul, J.S. Ingwall, C.E. Seidman, J.G. Seidman, Increased alpha2 subunit-associated AMPK activity and PRKAG2 cardiomyopathy, Circulation 112 (2005) 3140–3148. [11] J. Gulick, A. Subramaniam, J. Neumann, J. Robbins, Isolation and characterization of the mouse cardiac myosin heavy chain genes, J. Biol. Chem. 266 (1991) 9180–9185. [12] Y. Xing, N. Musi, N. Fujii, L. Zou, I. Luptak, M.F. Hirshman, L.J. Goodyear, R. Tian, Glucose metabolism and energy homeostasis in mouse hearts overexpressing dominant negative alpha2 subunit of AMP-activated protein kinase, J. Biol. Chem. 278 (2003) 28372– 28377. [13] R. Ramani, M. Mathier, P. Wang, G. Gibson, S. Togel, J. Dawson, A. Bauer, S. Alber, S.C. Watkins, C.F. McTiernan, A.M. Feldman,

[14]

[15]

[16]

[17]

[18]

[19]

[20]

387

Inhibition of tumor necrosis factor receptor-1-mediated pathways has beneficial effects in a murine model of postischemic remodeling, Am. J. Physiol. Heart Circ. Physiol. 287 (2004) H1369–H1377. L. Zou, M. Shen, M. Arad, H. He, B. Lofgren, J.S. Ingwall, C.E. Seidman, J.G. Seidman, R. Tian, N488I mutation of the gamma2subunit results in bidirectional changes in AMP-activated protein kinase activity, Circ Res. 97 (2005) 323–328. S.R. Hamilton, D. Stapleton, J.B. O’Donnell Jr., J.T. Kung, S.R. Dalal, B.E. Kemp, L.A. Witters, An activating mutation in the gamma1 subunit of the AMP-activated protein kinase, FEBS Lett. 500 (2001) 163–168. I. Luptak, M. Shen, H. He, M.F. Hirshman, N. Musi, L.J. Goodyear, J. Yan, H. Wakimoto, H. Morita, M. Arad, C.E. Seidman, J.G. Seidman, J.S. Ingwall, J.A. Balschi, R. Tian, Aberrant activation of AMP-activated protein kinase remodels metabolic network in favor of cardiac glycogen storage, J. Clin. Invest. (2007). J.F. Wojtaszewski, S.B. Jorgensen, Y. Hellsten, D.G. Hardie, E.A. Richter, Glycogen-dependent effects of 5-aminoimidazole-4-carboxamide (AICA)-riboside on AMP-activated protein kinase and glycogen synthase activities in rat skeletal muscle, Diabetes 51 (2002) 284–292. G. Polekhina, A. Gupta, B.J. Michell, B. van Denderen, S. Murthy, S.C. Feil, I.G. Jennings, D.J. Campbell, L.A. Witters, M.W. Parker, B.E. Kemp, D. Stapleton, AMPK beta subunit targets metabolic stress sensing to glycogen, Curr. Biol. 13 (2003) 867–871. R.R. Russell 3rd, J. Li, D.L. Coven, M. Pypaert, C. Zechner, M. Palmeri, F.J. Giordano, J. Mu, M.J. Birnbaum, L.H. Young, AMPactivated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury, J. Clin. Invest. 114 (2004) 495–503. J. Li, X. Hu, P. Selvakumar, R.R. Russell 3rd, S.W. Cushman, G.D. Holman, L.H. Young, Role of the nitric oxide pathway in AMPK-mediated glucose uptake and GLUT4 translocation in heart muscle, Am. J. Physiol. Endocrinol. Metab. 287 (2004) E834–E841.