Maengjo Kim

AMPK isoform expression in the normal and failing hearts.

AMP-activated protein kinase (AMPK) is a master metabolic switch that plays an important role in energy homeostasis at the cellular and whole body level, hence a promising drug target. AMPK is a heterotrimeric complex composed of catalytic α-subunit and regulatory β- and γ-subunits with multiple isoforms for each subunit. It has been shown that AMPK activity is increased in cardiac hypertrophy and failure but it is unknown whether changes in subunit composition of AMPK contribute to the altered AMPK activity. In this study, we determined the protein expression pattern of AMPK subunit isoforms during cardiac development as well as during cardiac hypertrophy and heart failure in mouse heart. We also compared the findings in failing mouse heart to that of the human failing hearts in order to determine whether the mouse heart is a good model of AMPK in human diseases. In mouse developmental hearts, AMPK was highly expressed in the fetal stages and fell back to the adult level after birth. In the failing mouse heart, there was a significant increase in α2, β2, and γ2 subunits both at the mRNA and protein levels. In contrary, we found significant increases in the protein level of α1, β1 and γ2c subunits in human failing hearts with no change in the mRNA level. We also compared isoform-specific AMPK activity in the mouse and human failing hearts. Consistent with the literature, in the failing mouse heart, the α2 complexes accounted for ~2/3 of total AMPK activity while the α1 complexes accounted for the remaining 30-35%. In the human hearts, however, the contribution of α1-AMPK activity was significantly higher (>40%) in the non-failing hearts, and it further increased to 50% in the failing hearts. Thus, the human hearts have a greater amount of α1-AMPK activity compared to the rodent hearts. In summary, the protein level and the isoform distribution of AMPK in the heart change significantly during normal development as well as in heart failure. These observations provide a basis for future development of therapeutic strategies for targeting AMPK.

Mutation in the γ2-Subunit of AMP-activated protein kinase stimulates cardiomyocyte proliferation and hypertrophy independent of glycogen storage

Rationale: AMP-activated protein kinase is a master regulator of cell metabolism and an attractive drug target for cancer and metabolic and cardiovascular diseases. Point mutations in the regulatory &ggr;2-subunit of AMP-activated protein kinase (encoded by Prkag2 gene) caused a unique form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular preexcitation, and glycogen storage. Understanding the disease mechanisms of Prkag2 cardiomyopathy is not only beneficial for the patients but also critical to the use of AMP-activated protein kinase as a drug target. Objective: We sought to identify the pro–growth-signaling pathway(s) triggered by Prkag2 mutation and to distinguish it from the secondary response to glycogen storage. Methods and Results: In a mouse model of N488I mutation of the Prkag2 gene (R2M), we rescued the glycogen storage phenotype by genetic inhibition of glucose-6-phosphate–stimulated glycogen synthase activity. Ablation of glycogen storage eliminated the ventricular preexcitation but did not affect the excessive cardiac growth in R2M mice. The progrowth effect in R2M hearts was mediated via increased insulin sensitivity and hyperactivity of Akt, resulting in activation of mammalian target of rapamycin and inactivation of forkhead box O transcription factor–signaling pathways. Consequently, cardiac myocyte proliferation during the postnatal period was enhanced in R2M hearts followed by hypertrophic growth in adult hearts. Inhibition of mammalian target of rapamycin activity by rapamycin or restoration of forkhead box O transcription factor activity by overexpressing forkhead box O transcription factor 1 rescued the abnormal cardiac growth. Conclusions: Our study reveals a novel mechanism for Prkag2 cardiomyopathy, independent of glycogen storage. The role of &ggr;2-AMP-activated protein kinase in cell growth also has broad implications in cardiac development, growth, and regeneration.

Mutation in the γ2-subunit of AMPK Stimulates Cardiomyocyte Proliferation and Hypertrophy Independent of Glycogen Storage

Rationale: AMP-activated protein kinase is a master regulator of cell metabolism and an attractive drug target for cancer and metabolic and cardiovascular diseases. Point mutations in the regulatory &ggr;2-subunit of AMP-activated protein kinase (encoded by Prkag2 gene) caused a unique form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular preexcitation, and glycogen storage. Understanding the disease mechanisms of Prkag2 cardiomyopathy is not only beneficial for the patients but also critical to the use of AMP-activated protein kinase as a drug target. Objective: We sought to identify the pro–growth-signaling pathway(s) triggered by Prkag2 mutation and to distinguish it from the secondary response to glycogen storage. Methods and Results: In a mouse model of N488I mutation of the Prkag2 gene (R2M), we rescued the glycogen storage phenotype by genetic inhibition of glucose-6-phosphate–stimulated glycogen synthase activity. Ablation of glycogen storage eliminated the ventricular preexcitation but did not affect the excessive cardiac growth in R2M mice. The progrowth effect in R2M hearts was mediated via increased insulin sensitivity and hyperactivity of Akt, resulting in activation of mammalian target of rapamycin and inactivation of forkhead box O transcription factor–signaling pathways. Consequently, cardiac myocyte proliferation during the postnatal period was enhanced in R2M hearts followed by hypertrophic growth in adult hearts. Inhibition of mammalian target of rapamycin activity by rapamycin or restoration of forkhead box O transcription factor activity by overexpressing forkhead box O transcription factor 1 rescued the abnormal cardiac growth. Conclusions: Our study reveals a novel mechanism for Prkag2 cardiomyopathy, independent of glycogen storage. The role of &ggr;2-AMP-activated protein kinase in cell growth also has broad implications in cardiac development, growth, and regeneration.

Activation of γ2-AMPK Suppresses Ribosome Biogenesis and Protects Against Myocardial Ischemia/Reperfusion InjuryNovelty and Significance

Rationale: AMPK (AMP-activated protein kinase) is a heterotrimeric protein that plays an important role in energy homeostasis and cardioprotection. Two isoforms of each subunit are expressed in the heart, but the isoform-specific function of AMPK remains unclear. Objective: We sought to determine the role of &ggr;2-AMPK in cardiac stress response using bioengineered cell lines and mouse models containing either isoform of the &ggr;-subunit in the heart. Methods and Results: We found that &ggr;2 but not &ggr;1 or &ggr;3 subunit translocated into nucleus on AMPK activation. Nuclear accumulation of AMPK complexes containing &ggr;2-subunit phosphorylated and inactivated RNA Pol I (polymerase I)–associated transcription factor TIF-IA at Ser-635, precluding the assembly of transcription initiation complexes for rDNA. The subsequent downregulation of pre-rRNA level led to attenuated endoplasmic reticulum (ER) stress and cell death. Deleting &ggr;2-AMPK led to increases in pre-rRNA level, ER stress markers, and cell death during glucose deprivation, which could be rescued by inhibition of rRNA processing or ER stress. To study the function of &ggr;2-AMPK in the heart, we generated a mouse model with cardiac-specific deletion of &ggr;2-AMPK (cardiac knockout [cKO]). Although the total AMPK activity was unaltered in cKO hearts because of upregulation of &ggr;1-AMPK, the lack of &ggr;2-AMPK sensitizes the heart to myocardial ischemia/reperfusion injury. The cKO failed to suppress pre-rRNA level during ischemia/reperfusion and showed a greater infarct size. Conversely, cardiac-specific overexpression of &ggr;2-AMPK decreased ribosome biosynthesis and ER stress during ischemia/reperfusion insult, and the infarct size was reduced. Conclusions: The &ggr;2-AMPK translocates into the nucleus to suppress pre-rRNA transcription and ribosome biosynthesis during stress, thus ameliorating ER stress and cell death. Increased &ggr;2-AMPK activity is required to protect against ischemia/reperfusion injury. Our study reveals an isoform-specific function of &ggr;2-AMPK in modulating ribosome biosynthesis, cell survival, and cardioprotection.

Transcript variant dictates Prkag2 cardiomyopathy

AMP-activatedprotein kinase (AMPK) is a critical energy sensor and a master regulator of cell metabolism, growth and survival, and therefore an attractive drug target [1–3]. In the cardiovascular system, activation of AMPK in response to stresses has been shown to be cardioprotective [4–7]. AMPK is a heterotrimeric complex composed of a catalytic α-subunit and two regulatory βand γ-subunits, with multiple isoforms for each subunit (α1/α2, β1/β2, and γ1/γ2/γ3, Fig. 1A). Most of the isoforms are widely expressed, except for γ3, which has been shown to be expressed exclusively in skeletal muscle [8–10]. The four cystathione β-synthase (CBS) domains at the C-terminus of the γ subunit are essential for the energy sensing function as they form two pairs of nucleotide binding pockets [11–13]. Intracellular nucleotide concentrations regulate AMPK activity by both allosteric and phosphorylation mechanisms. Binding of AMP/ADP stimulates AMPK activity while binding of ATP inhibits it. The binding of AMP allosterically stimulates the kinase activity. In addition, the binding of AMP or ADP on the γ subunit prevents dephosphorylation and/or promotes phosphorylation of Thr172 of the α subunit, resulting in a substantial increase of AMPK activity [14,15]. ATP inhibits the kinase activity by exchanging for AMP or ADP at the binding sites. Thus, the cellular AMP/ATP or ADP/ATP ratio is an important determinant of AMPK activity. Point mutations of the γ2 isoform (encoded by the Prkag2 gene) cause cardiomyopathy in humans, characterized by cardiac hypertrophy, ventricular pre-excitation and glycogen storage [16–18]. All of the Prkag2 mutations are present within or near the CBS domains (Fig. 1B), and previous studies have shown that the mutations alter the binding affinity of nucleotides, hence abolishing the sensitivity of the kinase to cellular nucleotide concentrations [11,19]. As the intracellular [ATP] (the inhibitor) is an order of magnitude higher than [ADP] and [AMP] (the stimulators), the loss of sensitivity due to the mutation results in a high basal activity [20]. Although significant progress has been made in elucidating the disease mechanisms of Prkag2 cardiomyopathy, it remains puzzling that mutations of the Prkag2 gene cause primarily cardiomyopathy despite the ubiquitous expression of the γ2 isoforms [21–24]. Furthermore, the cardiac-restricted phenotype of Prkag2 mutations cannot be explained by the expression level of the γ2 subunit, since γ2-AMPK accounts for less than 20% of total kinase activity in the adult heart [10]. Understanding the mechanisms responsible for these observations has broad significance for the AMPK field, since the current effort to develop pharmacological activators of AMPK seeks to stimulate kinase activity in a nucleotide independent mode. The study reported by Pinter et al. in this issue of J. Mol. Cell Cardiol. provides novel insights into the mechanism(s) by which Prkag2 mutations may cause cardiac-specific phenotypes [25]. The study analyzed the expression profile of γ-subunits in developing and adult hearts and compared the mouse to man. Three major findings were presented: 1) themajor isoformof theγ-subunits in adult heart (γ1)wasnot expressed

Mutation in the γ2-Subunit of AMP-Activated Protein Kinase Stimulates Cardiomyocyte Proliferation and Hypertrophy Independent of Glycogen StorageNovelty and Significance

Rationale: AMP-activated protein kinase is a master regulator of cell metabolism and an attractive drug target for cancer and metabolic and cardiovascular diseases. Point mutations in the regulatory &ggr;2-subunit of AMP-activated protein kinase (encoded by Prkag2 gene) caused a unique form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular preexcitation, and glycogen storage. Understanding the disease mechanisms of Prkag2 cardiomyopathy is not only beneficial for the patients but also critical to the use of AMP-activated protein kinase as a drug target. Objective: We sought to identify the pro–growth-signaling pathway(s) triggered by Prkag2 mutation and to distinguish it from the secondary response to glycogen storage. Methods and Results: In a mouse model of N488I mutation of the Prkag2 gene (R2M), we rescued the glycogen storage phenotype by genetic inhibition of glucose-6-phosphate–stimulated glycogen synthase activity. Ablation of glycogen storage eliminated the ventricular preexcitation but did not affect the excessive cardiac growth in R2M mice. The progrowth effect in R2M hearts was mediated via increased insulin sensitivity and hyperactivity of Akt, resulting in activation of mammalian target of rapamycin and inactivation of forkhead box O transcription factor–signaling pathways. Consequently, cardiac myocyte proliferation during the postnatal period was enhanced in R2M hearts followed by hypertrophic growth in adult hearts. Inhibition of mammalian target of rapamycin activity by rapamycin or restoration of forkhead box O transcription factor activity by overexpressing forkhead box O transcription factor 1 rescued the abnormal cardiac growth. Conclusions: Our study reveals a novel mechanism for Prkag2 cardiomyopathy, independent of glycogen storage. The role of &ggr;2-AMP-activated protein kinase in cell growth also has broad implications in cardiac development, growth, and regeneration.