Constitutively active AMPKγ2 causes WPW syndrome and kidney injury 1 Physiological Expression of AMPKγ2 Mutation Causes Wolff-Parkinson-White Syndrome and Induces Kidney Injury in Mice

Mutations of AMPKγ2 subunit, N488I (AMPKγ2) and R531G (AMPKγ2), are associated with Wolff-Parkinson-White (WPW) syndrome, a cardiac disorder characterized by ventricular pre-excitation in humans. Cardiacspecific transgenic overexpression of human AMPKγ2 or AMPKγ2 leads to constitutive AMPK activation and the WPW phenotype in mice. However, overexpression of these mutant proteins also caused profound, nonphysiological increase in cardiac glycogen, which might abnormally alter the true phenotype. To investigate whether physiological levels of AMPKγ2 or AMPKγ2 mutation cause WPW syndrome and metabolic changes in other organs, we generated two knock-in mouse lines on the C57BL/6N background harboring mutations of human AMPKγ2 and AMPKγ2, respectively. Similar to the reported phenotypes of mice overexpressing AMPKγ2 or AMPKγ2 in the heart, both lines developed WPW syndrome and cardiac hypertrophy, however these effects were independent of cardiac glycogen accumulation. Compared with AMPKγ2 mice, AMPKγ2 http://www.jbc.org/cgi/doi/10.1074/jbc.M116.738591 The latest version is at JBC Papers in Press. Published on September 12, 2016 as Manuscript M116.738591 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 6, 2017 hp://w w w .jb.org/ D ow nladed from Constitutively active AMPKγ2 causes WPW syndrome and kidney injury 2 and AMPKγ2 mice exhibited reduced body weight, fat mass, and liver steatosis when fed with a high-fat diet (HFD). Surprisingly, AMPKγ2 but not AMPKγ2 mice fed with a HFD exhibited severe kidney injury characterized by glycogen accumulation, inflammation, apoptosis, cyst formation, and impaired renal function. These results demonstrate that expression of AMPKγ2 and AMPKγ2 mutations at physiological levels can induce beneficial metabolic effects but that is accompanied by WPW syndrome. Our data also reveal an unexpected effect of AMPKγ2 in the kidney, linking lifelong constitutive activation of AMPK to a potential risk for kidney dysfunction in the context of a HFD. These AMPK-engineered mouse models provide new tools for investigating the mechanisms leading to WPW syndrome, the roles of AMPK in kidney function and disease, and the consequences of physiological AMPK activation on mammalian metabolism and physiology. AMPK is an energy sensor that functions to maintain energy homeostasis in response to changes in the ratio of AMP to ATP in the cell (1). AMPK is a heterotrimeric complex composed of catalytic α subunits (α1 and α2), regulatory β (β1 and β2), and γ subunits (γ1, γ2, and γ3) (2). The AMPK γ subunit has 3 AMP binding sites, two sites exhibit reversible and one site irreversible binding (3,4). AMPK is activated by its upstream kinase LKB1 via phosphorylation of Thr172 of α subunit (5-7). Upon binding of AMP to AMPK γ subunit, AMPK can be further allosterically activated (8,9). AMPK is reported to regulate multiple metabolic functions in different organs, such as protein synthesis, lipid metabolism, insulin receptor signaling pathway, hepatic gluconeogenesis, glucose transport in skeletal muscle, cardiac function, and kidney development etc (8,10). To date, AMPKγ2 (Prkag 2) is the only subunit recognized to be associated with disease-causing mutations. Human genetic studies reported association of AMPKγ2 mutations and Wolff-Parkinson-White (WPW) syndrome, a cardiac defect characterized by intermittent or persistent ventricular preexcitation in sinus rhythm and arrhythmia and sometimes accompanied by paroxysmal tachycardia and cardiac hypertrophy (11,12). In total, 11 mutations of and 1 insertion in the AMPKγ2 subunit have been identified that are correlated to WPW syndrome (13). Of these mutations / insertion, AMPKγ2 (Asn to Ile mutation at amino acid 488) and AMPKγ2 (Arg to Gly mutation at amino acid 531) have been intensively studied (14-17). Both AMPKγ2 and AMPKγ2 mutations are believed to generate constitutively active AMPK complexes. AMP can further enhance AMPK activity upon binding to AMPKγ2 (16,17). In contrast, AMPKγ2, a mutation within the irreversible AMP binding region, lacks responsiveness to AMP as it has lost its ability to bind AMP (3,18,19). Transgenic mice with cardiac-specific overexpression of human AMPKγ2 and AMPKγ2 at more than 20-fold over endogenous AMPKγ2 levels exhibit dramatic glycogen accumulation, cardiac hypertrophy, and ventricular pre-excitation compared to mice overexpressing human AMPKγ2 at similar levels (16-18). The effect of the AMPKγ2 mutation in the regulation of cardiac-specific ion channels and on early onset of pre-excitation and atrial fibrillation in the WPW patients suggested that AMPKγ2 mutations can cause abnormalities of cardiac conduction system during development of the heart (20). In a recent report, Kim et al reported that eliminating glycogen storage in AMPKγ2 transgenic mice by genetic inhibition of glucose6-phosphate–stimulated glycogen synthase activity prevented pre-excitation in mice but did not affect cardiac hypertrophy, indicating a glycogen-induced abnormality in cardiac electrical conductance but a glycogenindependent effect on cardiac hypertrophy in WPW syndrome (21). In order to investigate how AMPKγ2 and AMPKγ2 mutations affect cardiac function and metabolism, we generated two knock-in mice harboring single mutation of AMPKγ2 or AMPKγ2. The current study was undertaken to understand the causality of by gest on Sptem er 6, 2017 hp://w w w .jb.org/ D ow nladed from Constitutively active AMPKγ2 causes WPW syndrome and kidney injury 3 AMPKγ2 and AMPKγ2 mutations expressed at physiological levels on the WPW syndrome and explore whether they affect whole-body metabolism and function of other organs. We hypothesize that these mutations not only affect cardiac function but also whole-body metabolism, especially those major metabolic organs, such as liver, skeletal muscle, and kidney. We describe our initial findings in this paper.