AMP Activated Protein Kinase-α2 Deficiency Exacerbates Pressure-Overload–Induced Left Ventricular Hypertrophy and Dysfunction in Mice
AMP activated protein kinase (AMPK) plays an important role in regulating myocardial metabolism and protein synthesis. Activation of AMPK attenuates hypertrophy in cultured cardiac myocytes, but the role of AMPK in regulating the development of myocardial hypertrophy in response to chronic pressure overload is not known. To test the hypothesis that AMPKα2 protects the heart against systolic overload–induced ventricular hypertrophy and dysfunction, we studied the response of AMPKα2 gene deficient (knockout [KO]) mice and wild-type mice subjected to 3 weeks of transverse aortic constriction (TAC). Although AMPKα2 KO had no effect on ventricular structure or function under control conditions, AMPKα2 KO significantly increased TAC-induced ventricular hypertrophy (ventricular mass increased 46% in wild-type mice compared with 65% in KO mice) while decreased left ventricular ejection fraction (ejection fraction decreased 14% in wild-type mice compared with a 43% decrease in KO mice). AMPKα2 KO also significantly exacerbated the TAC-induced increases of atrial natriuretic peptide, myocardial fibrosis, and cardiac myocyte size. AMPKα2 KO had no effect on total S6 ribosomal protein (S6), p70 S6 kinase, eukaryotic initiation factor 4E, and 4E binding protein-1 or their phosphorylation under basal conditions but significantly augmented the TAC-induced increases of p-p70 S6 kinaseThr389, p-S6Ser235, and p-eukaryotic initiation factor 4ESer209. AMPKα2 KO also enhanced the TAC-induced increase of p-4E binding protein-1Thr46 to a small degree and augmented the TAC-induced increase of p-AktSer473. These data indicate that AMPKα2 exerts a cardiac protective effect against pressure-overload–induced ventricular hypertrophy and dysfunction.
Increases of cardiac work resulting from systolic overload necessitate an increase of ATP use in proportion to the increase in left ventricular (LV) systolic wall stress.1,2 In response to chronic systolic overload, cardiac myocyte hypertrophy occurs, characterized by increased protein synthesis, whereas myocardial oxygen consumption and carbon substrate use are increased to accommodate the need for increased energy availability. This initially occurs with no change in high energy phosphate levels, but with the development of pathological hypertrophy and congestive heart failure, ATP levels fall and cytosolic free ADP levels increase (as indicated by a decrease of the myocardial phosphocreatine:ATP ratio).2,3 In this situation, the adenylate kinase reaction can catalyze the reaction of 2 molecules of ADP to produce 1 molecule of ATP and 1 molecule of AMP. An increased AMP:ATP ratio results in activation of the energy stress sensor known as AMP activated protein kinase (AMPK).
AMPK is composed of 1 catalytic α subunit (either α1 or α2) and 2 regulatory subunits (β and γ). AMPKα2 is the dominant catalytic subunit in the heart,3,4 where it is predominantly expressed in cardiac myocytes. AMPK is activated by metabolic stresses that deplete cellular ATP or increase AMP.3,4 Recent studies have demonstrated that activation of AMPK attenuates signaling through the mammalian target of rapamycin (mTOR) pathway, which plays a critical role in activating translational machinery during cell growth or hypertrophy.5 Although genetic deletion of AMPKα26 or AMPKα17 or overexpression of dominant-negative AMPKα has no effect on ventricular mass or function under unstressed conditions, the role of AMPK in the response of the heart to systolic overload has not been directly tested. As AMP:ATP ratios increase during systolic overload, AMPK would be predicted to play a more significant role under these conditions than under basal conditions. Consequently, this study was designed to determine the effect of decreasing AMPK activity by AMPKα2 knockout (KO) on LV mass and function in mice exposed to chronic pressure overload produced by transverse aortic constriction (TAC). Here we report that AMPKα2 KO had no effect on LV structure or function during basal conditions but significantly exacerbated TAC-induced ventricular hypertrophy, fibrosis, and dysfunction, and this was associated with significantly greater increases of p-p70 S6 kinase (p-p70S6K)Thr389, p-S6 ribosomal protein (S6)Ser235, and p-eukaryotic initiation factor 4E (eIF4e)Ser209 in the KO mice as compared with wild-type mice. In addition, we demonstrated that activation of AMPK with either 5-aminoimidazole-4-carboxyamide-β-D-ribofuranoside (AICAR) or metformin or overexpression of constitutively active AMPKα2 attenuated phenylephrine-induced cardiac myocyte hypertrophy and reduced phosphorylation of p70S6K in cultured neonatal rat cardiac myocytes. The data indicate that AMPKα2 negatively regulates pressure-overload–induced ventricular hypertrophy and dysfunction.
Materials and Methods
Heterozygous AMPKα2 KO mice of C57BL6 background (crossed back to C57BL6 mice ≥8 times) were generated as described previously.6,7 The AMPKα2 KO mice and wild-type (WT) littermates generated from the heterozygotes were used as breeders to generate KO and WT mice for the study. This study was approved by the institutional animal care and use committee of the University of Minnesota.
TAC was performed in male WT mice (n=45) and KO mice (n=42) at ≈2 months of age by using the minimally invasive suprasternal approach, as described previously.8–10
LV Function Measurements
Echocardiography was performed 3 weeks after TAC or sham surgery under anesthesia with 1.5% isoflurane by inhalation, as described previously.9,10 LV and aortic hemodynamics were determined by a 1.2F pressure catheter (Scisense Inc) in WT and KO mice under control conditions, as described previously.9
Western Blots and Measurement of Myocardial Fibrosis and Myocyte Hypertrophy
Western blots were performed as described previously.9 Tissue sections (8 μm) from the central portion of the LV were stained with picrosirius red for detection of fibrosis9,10 and fluorescein isothiocyanate–conjugated wheat germ agglutinin (Alexa Fluor-488, Invitrogen) to evaluate myocyte size.9,10 The percentage of fibrosis volume density was quantified on the picrosirius red–stained sections using point counting as described in Unbiased Stereology.11 For the average myocyte size, the mean short diameter and cross-sectional area of ≈120 cells per sample of 4 samples in each group were examined.
Myocardial AMPK Activity
Isoform-specific activities of AMPK were measured as described previously.12 Please see the data supplement available online at http://hyper.ahajournals.org.
Neonatal Rat Cardiomyocyte Isolation and Culture and Cell Area Determination
Neonatal rat cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats by enzymatic digestion13 and separated from nonmuscle cells on a discontinuous Percoll gradient according to a modified protocol from Zhang et al13 with minor modification as described previously.14 Please see the data supplement.
Data and Statistical Analyses
All of the values were expressed as means±SEMs. Statistical significance was defined as P<0.05. Two-way ANOVA was used to test each variable for differences among the treatment groups with SigmaSTAT. If ANOVA demonstrated a significant effect, individual comparisons between groups were made using Tukey’s test.
AMPKα2 KO Has no Effect on LV Function Under Control Conditions
Under control conditions, ventricular mass, lung mass, and the ratio of ventricular mass or lung mass to tibia length were not different between AMPKα2 KO and WT mice matched for age, body weight (24.8±0.4 g in WT mice versus 24.4±0.8 g in KO mice) and gender (Figure 1A and 1B). Histological examination showed no cardiac myocyte hypertrophy and no increase in myocardial fibrosis in the AMPKα2 KO mice. Echocardiographic imaging showed normal LV size and systolic shortening in the KO mice (Table), which are consistent with previous reports demonstrating that disruption of AMPK activity by overexpressing mutated AMPKα2 had no impact on LV structure or function under control conditions in young mice.15,16 However, LV dP/dtmax and LV dP/dtmin were significantly decreased in the KO mice as compared with WT mice, indicating a subtle decrease in contractility, although LV systolic pressure and mean aortic pressure were not different between KO and WT mice under control conditions (Table).
AMPKα2 KO Exacerbates TAC-Induced Myocardial Hypertrophy, Fibrosis, and Dysfunction
In response to TAC for 3 weeks, AMPKα2 KO mice developed significantly greater increases in ventricular weight and the ratio of ventricular weight to body weight or tibia length as compared with WT mice (Figure 1), indicating that AMPKα2 deficiency exacerbated TAC-induced myocardial hypertrophy. In addition, lung weight and the ratio of lung weight to tibia length were significantly greater in KO mice as compared with WT mice, indicating more pulmonary congestion in the KO mice (Figure 1). Furthermore, KO mice had a higher mortality after TAC as compared with WT mice (Figure 1D). The mean body weight and tibia length were not different between WT and KO mice (Table).
Histological analysis demonstrated that TAC resulted in more ventricular fibrosis (Figure 1E) and a greater increase in cardiac myocyte diameter (Figure 1F) in KO mice as compared with WT mice, indicating that the greater ventricular mass in the KO mice after TAC was because of both myocyte hypertrophy and an increase of ventricular fibrosis.
TAC resulted in significantly more impairment of LV systolic function in the KO mice, as demonstrated by a greater reduction of systolic fractional shortening and a significant increase in LV end-systolic diameter as compared with WT mice (Figure 1 and Table). The heart rates were not significantly different between WT and KO groups (Table).
AMPKα2 KO Attenuated Myocardial AMPK Activity and Increased TAC-Induced p-p70S6K-S6 Activation
Total myocardial AMPKα and phosphorylated acetyl coenzyme A (CoA) carboxylase (ACC; p-ACCSer79) were significantly decreased in AMPKα2 KO mice both under control conditions and after TAC as compared with the WT mice (Figure 2A through 2C). Consistent with the decreased myocardial total AMPKα in KO mice, myocardial AMPKα2 activity was diminished in KO mice under both control conditions and after TAC (Figure 2E), whereas AMPKα1 activity was not significantly different under control conditions (7.6±0.2 pmol/mg of protein per minute in WT mice as compared with 8.2±0.3 pmol/mg of protein per minute in KO mice). After 3 weeks of TAC, AMPKα1 activity was significantly increased in AMPKα2 KO mice (8.7±0.42 pmol/mg of protein per minute) as compared with WT mice (7.8±0.22 pmol/mg of protein per minute), indicating a compensatory increase of AMPKα1 in KO mice. The greater increase of AMPKα1 activity and AMPKα1 protein content in the AMPKα2 KO mice after TAC was not able to fully compensate for the AMPKα2 deficiency, as demonstrated by significantly lower levels of p-ACC2 (Figure 2A and 2C) and greater ventricular hypertrophy in the KO mice. In addition, myocardial ANP protein (Figure 2A and 2F) and mRNA (Figure 2G) were significantly greater in the KO mice as compared with WT mice 3 weeks after TAC.
It is reported that activation of AMPK attenuates protein synthesis by inhibiting the mTOR signaling pathway. p70S6K and 4E binding protein-1 (4EBP1) are 2 well-defined downstream targets of mTOR; activation of mTOR increases p70S6KThr389 and p-4EBP1Thr46, which enhances the translation initiation process and protein synthesis. We found that AMPKα2 KO had no significant effect on myocardial total p70S6K, S6, or eIF4e under both control conditions and after TAC. TAC caused a significant increase of total 4EBP1 in WT mice but not in KO mice. Three weeks after TAC, p-p70S6KThr389, p-S6Ser235, p-elf4eSer209, and p-4EBP1Thr46 were increased in both WT and KO mice (Figure 3A and 3B). However, the increases of p70S6KThr389, p-S6Ser235, and p-elf4eSer209 were significantly greater in the AMPKα2 KO animals (Figure 3A and 3B), indicating that AMPKα2 negatively regulates the phosphorylation of p70S6K and its downstream targets. AMPKα2 KO resulted in a small but significant increase of p-4EBP1Thr46 in response to TAC (Figure 3A and 3B).
Eukaryotic elongation factor-2 (eEF2) is responsible for mediating the step of peptide-chain elongation. Phosphorylation of eEF2 at the Thr56 site results in inactivation of eEF2 (or decreased eEF2 activity) and a decrease of protein synthesis.17,18 AMPKα2 KO had no effect on total eEF2 protein content either under control conditions or after TAC. Myocardial p-eEF2Thr56 content was under control conditions, but was not significantly different between WT and KO mice after TAC (Figure 3).
Pressure overload caused an increase of myocardial p-AktSer473, which is generally considered to be an upstream target of AMPK. Interestingly, we found that AMPKα2 KO also significantly augmented the TAC-induced increase of p-AktSer473 (Figure 3A and 3B).
AMPK Activation or Overexpression of Constitutively Active AMPKα2 Attenuated Phenylephrine-Induced Cardiac Myocyte Hypertrophy
We examined the effect of overexpression of constitutively active AMPKα2 or activation of AMPK with AICAR and metformin on phenylephrine-induced hypertrophy of isolated neonatal cardiomyocytes (for the supplementary figure, please see http://hyper.ahajournals.org). Phenylephrine significantly increased the size of the cardiac myocytes over 48 hours of treatment, whereas overexpression of constitutively active AMPKα2 (CA-AMPK; Figure S1), metformin (0.2 to 5.0 mmol/L; Figure S2), and AICAR (0.2 mmol/L; Figure S3) all significantly attenuated phenylephrine-induced cardiac myocyte hypertrophy and an increase of p-p70S6KThr389 (Figure S4 and S5), indicating that activation of AMPK attenuates phenylephrine-induced activation of p70S6K and cardiac myocyte hypertrophy in the cultured cells. Furthermore, to examine whether AMPK acts upstream or downstream of Akt activation and to limit the influence of other signaling pathways activated by AICAR or metformin, we used constitutively active Akt (CA-Akt; myristoylated, 5 plaque-forming units per cell) in combination with CA-AMPK (5 plaque-forming units per cell). Our results demonstrate that p70S6K phosphorylation is activated by CA-Akt, but this activation is significantly blunted by CA-AMPK (Figure S6). In parallel, we examined protein synthesis (H3-leucine incorporation) in response to CA-Akt. Although CA-AMPK had little effect on basal protein synthesis, it significantly reduced CA-Akt–induced protein synthesis (Figure S7). These results together demonstrate that AMPK acts downstream of Akt to reduce mTOR/p70S6K signaling in the in vitro system.
The major new findings of this study are as follows: (1) TAC resulted in more hypertrophy and fibrosis in AMPKα2 KO hearts than in WT hearts, with a greater increase of LV diameter at end systole and a greater decrease of LV ejection fraction (indicative of LV dysfunction); (2) TAC caused greater increases of myocardial p70S6KThr389, p-S6Ser235, p-elf4eSer209, and p-4EBP1Thr46, indicating that AMPKα2 KO augmented the responses of these well-defined downstream targets of mTOR to chronic systolic overload; and (3) AMPKα2 KO resulted in an unanticipated increase of p-AktSer473. To the best of our knowledge, these findings provide the first evidence that AMPKα2 exerts a protective effect against pressure-overload–induced LV remodeling by attenuating the activation of downstream targets of mTOR.
Our finding of greater ventricular hypertrophy in the AMPKα2 KO mice after TAC is consistent with the notion that AMPKα2 negatively regulates protein synthesis.4 This is in agreement with reports19,20 that adiponectin attenuated hypertrophic growth in mice subjected to TAC via activation of AMPK and an AMPK-dependent signaling mechanism. The finding that AMPKα2 KO had no effect on LV structure and function in the present study is consistent with a previous report in which a decrease of myocardial AMPK activity produced by overexpression of mutated AMPKα2 also had no effect on LV function under unstressed conditions.16
The increase of myocardial total-AMPKα, AMPKα1, and p-ACC observed in the WT mice 3 weeks after TAC in the present study is consistent with a previous report that pressure overload produced by clipping the ascending aorta of rats caused increases of myocardial AMPK activity, p-AMPK, and total AMPKα1 protein content.3 The increased myocardial AMPKα1 protein content was not associated with a proportional increase of AMPKα1 activity in these mice, suggesting that some AMPKα1 protein modification might occur to attenuate its activity. The increase in myocardial AMPKα1, total-AMPKα, and p-ACC in the AMPKα2 KO mice after TAC in the present study indicates that compensatory upregulation of AMPKα1 was not sufficient to fully compensate for the loss of AMPKα2. Furthermore, the greater myocardial hypertrophy/fibrosis and dysfunction in the KO animals implies that AMPKα2 contributed to the response by which the heart adjusted to the increased hemodynamic load produced by TAC.
Our finding that AICAR attenuated cardiomyocyte hypertrophy produced by phenylephrine stimulation in cultured rat cardiomyocytes reaffirms the previous observation.18 The finding that constitutively active AMPKα dose-dependently attenuated phenylephrine-induced myocyte hypertrophy is new but also consistent with the findings obtained from activation of AMPK by AICAR or metformin (both of which result in activation of AMPK), as reported previously.18
The rapamycin-sensitive mTOR complex has 2 well-defined primary downstream targets, ribosomal S6 kinase (p70S6K) and eukaryotic initiation factor 4E binding protein (4E-BP1 is the dominant isoform in the heart).5 The increase of 70S6KThr389, p-S6Ser235, and p-4EBP1Thr46 in the mice exposed to TAC indicates mTOR complex activation. Activation of the mTOR signaling pathway exacerbates myocardial hypertrophy,21 whereas inhibition of mTOR signaling with rapamycin attenuates the development of ventricular hypertrophy in mice exposed to ascending aortic banding.21,22 The finding that AMPKα2 deficiency enhanced TAC-induced phosphorylation of a group of downstream targets of mTOR, such as p-70S6KThr389, p-S6Ser235, and p-elf4eSer209, reaffirms the concept that AMPKα2 is a negative regulator of the mTOR/p-70S6K signaling pathway in cardiac myocytes under conditions of hemodynamic overload. The finding that AMPKα2 KO did not affect the phosphorylation of p70S6KThr389, S6Ser235, and elf4eSer209 under control conditions but significantly enhanced the TAC-induced increase of p-p70S6KThr389, p-S6Ser235, and p-elf4eSer209 likely reflects the role of AMPKα2 as a negative regulator of these pathways, particularly under stress conditions. Because it has been demonstrated that upregulation of myocardial p-p70S6KThr389 and p-S6Ser235 activity by expression of either WT or rapamycin-resistant p70S6K causes moderate ventricular hypertrophy, the greater increase of p-p70S6KThr389, p-S6Ser235, and p-elf4eSer209 in the AMPKα2 KO after TAC likely contributed to the increased ventricular hypertrophy in these mice. As compared with the increase of myocardial p-p70S6KThr389 and p-S6Ser235 in the AMPKα2 KO mice after TAC, AMPKα2 KO enhanced the TAC-induced increase of p-4EBP1Thr46 to a relatively smaller degree, indicating that AMPKα2 KO did not equally affect the phosphorylation of all of its downstream targets, suggesting that p-p70S6KThr389 and p-4EBP1Thr46 are differentially regulated. Activation of AMPK18 or rapamycin23 attenuates phenylephrine or endothelin-1–induced protein synthesis, cardiac myocyte hypertrophy, and the increase of p-eEF2Thr56. The unchanged myocardial p-eEF2Thr56 in the AMPKα2 KO mice after TAC in the present study suggests that activation of eEF2 (by a decrease of p-eEF2Thr56) has no effect on the greater hypertrophy in these mice.
Previous studies have demonstrated that chronic pressure overload in response to TAC or myocardial infarction increases myocardial p-AktSer473.24,25 Because AMPK is generally regarded as a downstream target of p-AktSer473, the greater increase of myocardial p-Akt Ser473 in the AMPKα2 KO mice after TAC was not initially anticipated. Our finding that CA-AMPK attenuated a CA-Akt–induced increase of p-p70S6KThr389 and cardiac myocyte hypertrophy suggests that AMPKα2 acts downstream of Akt to reduce mTOR/p70S6K signaling and protein synthesis at least in the in vitro system. Nevertheless, a recent study demonstrated that activation of AMPK with AICAR or metformin increased p-AktSer473 in cultured rat cardiac myocytes,18 indicating that AMPK activity can also regulate p-AktSer473 at least in cultured cardiac myocytes. However, because of the complex physiological role of Akt in ventricular hypertrophy and function, it is not clear whether the increase of myocardial p-AktSer473 in the AMPKα2 KO mice after TAC was a compensatory event or whether it contributed to the hypertrophy and dysfunction in these mice.
AMPK also plays an important role in regulating glucose and fatty acid oxidation under stress conditions. Activation of AMPK causes an increase of p-ACCSer79 to attenuate ACC activity and malonyl-CoA production. A decrease of p-ACCSer79 increases ACC activity to cause an increase of malonyl-CoA,26,27 an endogenous inhibitor of CPT1 (the rate-limiting enzyme for entry of long-chain acyl-CoA into the mitochondria). Recent studies have demonstrated that decreased malonyl-CoA production by ACC2 gene KO not only enhanced fatty acid oxidation but also increased glucose oxidation,28 suggesting that the decreased p-ACCSer79 in AMPKα2 KO mice has the potential to impair myocardial energy production through inhibition of both fatty acid and glucose oxidation by accumulation of malonyl-CoA. Future studies measuring metabolic genes, as well as fatty acid and glucose uptake and oxidation in vivo, will be needed to determine the contribution of abnormalities of myocardial metabolism in the AMPKα2 KO mice.
AMPK plays an important role in regulating energy balance, protein synthesis, and cell growth. AMPK activation enhances fatty acid and glucose metabolism to augment ATP production and attenuates protein synthesis to preserve ATP. Using AMPKα2 KO mice in combination with TAC, we demonstrated that the reduction of myocardial AMPK activity exacerbated the development of ventricular hypertrophy, fibrosis, and dysfunction in response to chronic pressure overload. AMPKα2 KO also significantly augmented the pressure-overload–induced increases of p-p70S6KThr389, p-S6Ser235, p-elf4eSer209, and 4EBP1Thr46. Our findings provide the first direct evidence that AMPKα2 plays an important role in regulating pressure-overload–induced LV remodeling and suggest that regulation of AMPKα2 may be a potential therapeutic approach to attenuate pressure-overload–induced ventricular hypertrophy.
Sources of Funding
This study was supported by National Heart, Lung, and Blood Institute grants HL71790 (to Y.C.) and HL021872 (to R.J.B.) from the National Institutes of Health and by the American Diabetes Association RA-12 (to D.A.B.) and by the EXGENESIS Integrated Project (LSHM-CT-2004-005272) funded by the European Commission (to B.V.). P.Z. and J.F. are supported by Scientist Development Awards from the American Heart Association National Center.
- Received April 11, 2008.
- Revision received May 5, 2008.
- Accepted September 5, 2008.
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