Mutations In PRKAG2 Gene Research Articles

DEVELOPMENT OF AN IN VITRO STUDY MODEL OF PRKAG2 CARDIOMYOPATHY USING HUMAN INDUCED PLURIPOTENT STEM CELLS

<h3>Background</h3> PRKAG2 cardiomyopathy is an autosomal dominant syndrome due to mutation in PRKAG2 gene, which encodes the γ2 subunit of the adenosine monophosphate (AMP)-activated protein kinase (AMPK). PRKAG2 syndrome leads to intracellular cardiomyocyte glycogen storage dysfunctions and clinical conditions that overlap hypertrophic cardiomyopathy, hampering its correct diagnosis. Our group identified a patient with a novel heterozygous missense mutation in PRKAG2 gene (c.1203C>A, p.His401Gln), with a malignant disease phenotype characterized by severe cardiac hypertrophy and cases of sudden death in young relatives. In this regard, induced pluripotent stem cells (iPSC) preserve the donor genome and can be differentiated into specialized cell types, being particularly interesting to study diseases that affect tissues which are difficult to access. Thus, this work aims to establish an in vitro study model of PRKAG2 cardiomyopathy through generation of human iPSC and its subsequent differentiation into patient-specific cardiomyocytes. <h3>Methods</h3> iPSC from patient with PRKAG2 mutation and from a health relative were generated from erythroblasts using CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (containing OCT3/4, KLF4, SOX2 and cMYC as reprogramming factors) and their karyotype was analyzed by qPCR using hPSC Genetic Analysis Kit (Stemcell Technologies). The iPSC strains obtained were differentiated into cardiomyocytes by inducing mesoderma formation with glycogen synthase kinase inhibitor 3-β, CHIR99021 (5 µM), on day 0 and cardiogenic mesoderma formation with the Wnt inhibitor, Wnt-C59 (2 µM), on day 2. On day 30 of culture, cells were stained with periodic acid-Schiff (PAS) to assess cytosolic glycogen storage. <h3>Results</h3> Both iPSC strains have embryonic-like phenotype and normal karyotype. Moreover, preliminary differentiation studies suggest glycogen accumulation in cardiomyocytes derived from the iPSC strain with PRKAG2 mutation. Evaluation of pluripotency factors expression and the ability to differentiate in the 3 embryonic germinative layers are ongoing in iPSC strains. <h3>Conclusion</h3> At the end of the characterization of the generated iPSC and of iPSC-derived cardiomyocytes, is expected that this approach will make possible the establishment of an in vitro study platform to reveal molecular mechanisms triggered by the c.1203C>A mutation that may contribute to the development of new therapies and prognostic evaluation of patients with PRKAG2 syndrome.

Bioenergetic and metabolic aberrations in induced pluripotent stem cell-derived cardiomyocytes generated from PRKAG2-mutated, WPW patient

Abstract Introduction Familial hypertrophic cardiomyopathy (HCM) is caused by over 400 mutations affecting mostly key sarcomere components, such as titin and myosin. However, HCM also results from mutations in non-structural genes such as PRKAG2 which is involved directly in a variety of bioenergetic and metabolic pathways. When the metabolic processes fail to work properly or effectively, structural and functional aberrations resulting in cardiac dysfunction can occur. Thus, mutations in the human PRKAG2 gene lead to HCM, autosomal dominant ventricular pre-excitation - Wolff-Parkinson-White syndrome (WPW), a progressive conduction system disease and vacuolar glycogen accumulation in cardiomyocytes. Purpose To investigate the hypothesis that intervening with the bioenergetic and metabolic consequences of the R302Q mutation in the PRKAG2 gene causing inherited cardiomyopathy, will attenuate the cardiac impairments. Methods We generated mutated and isogenic induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) from a WPW patient carrying the R302Q mutation. As additional control, we used healthy volunteers' iPSC-CMs. Bioenergetic (Oxygen Consumption Rate, OCR) and metabolic status were measured using the Seahorse Flux Analyzer and LC-MS, respectively. To decipher the molecular basis underlying the bioenergetic and metabolic deficits, RNA-seq analysis was performed. Results The OCR results demonstrated in PRKAG2-mutated compared to isogenic and healthy iPSC-CMs, a 2-fold increase in maximal respiration rate and a 3.75-fold increase in spare respiratory capacity, while basal OCR parameters were similar in all groups. Importantly, when treated with the AMPK activator metformin (2.5 [mM]), all the abovementioned OCR parameters were similar in the three groups. RNA-seq analysis demonstrated that of the 553 differently expressed genes (DEGs), and of the 99 DEGs mutually differentially expressed, compared to isogenic and healthy cells, the most relevant altered pathways were glycolysis, carbon metabolism, biosynthesis of amino acids, HIF-1 signaling and fructose and mannose metabolism. These findings are consistent with the LC-MS results demonstrating in PRKAG2-mutated versus isogenic and healthy iPSC-CMs, at least a 3-fold decrease in metabolites linked to the abovementioned pathways: butyryl-carnitine, creatine, docosahexaenoic acid, GMP, IMP, myristoyl-carnitine, palmitoyl-carnitine, succinyl-Cys, UMP, UTP, UDP-GlcNAc. Conclusion PRKAG2-mutated iPSC-CMs exhibit bioenergetic and metabolic aberrations, which contribute to the cardiac pathological aspects of WPW syndrome. Importantly, treatment with the AMPK activator metformin eliminated the bioenergetic abnormalities in the mutated cells, while isogenic and healthy control cells remained unaffected. Based on these novel findings, a new therapeutic modality in WPW patients may be considered. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Israel Science Foundation (ISF)

Molecular Pathogenesis of Familial Wolff-Parkinson-White Syndrome.

Familial Wolff-Parkinson-White (WPW) syndrome is an autosomal dominant inherited disease and consists of a small percentage of WPW syndrome which exhibits ventricular pre-excitation by development of accessory atrioventricular pathway. A series of mutations in PRKAG2 gene encoding gamma2 subunit of 5'AMP-activated protein kinase (AMPK) has been identified as the cause of familial WPW syndrome. AMPK is one of the most important metabolic regulators of carbohydrates and lipids in many types of tissues including cardiac and skeletal muscles. Patients and animals with the mutation in PRKAG2 gene exhibit aberrant atrioventricular conduction associated with cardiac glycogen overload. Recent studies have revealed "novel" significance of canonical pathways leading to glycogen synthesis and provided us profound insights into molecular mechanism of the regulation of glycogen metabolism by AMPK. This review focuses on the molecular basis of the pathogenesis of cardiac abnormality due to PRKAG2 mutation and will provide current overviews of the mechanism of glycogen regulation by AMPK. J. Med. Invest. 65:1-8, February, 2018.

Abstract 243: A Novel PRKAG2 Mutation (K475E): Infantile-onset Hypertrophic Cardiomyopathy Phenotype and Impact on mTOR Signaling

Objective: PRKAG2 encodes the 2 subunit isoform of the 5’ AMP-activated protein kinase (AMPK), a heterotrimeric enzyme with major roles in regulation of energy metabolism in response to cellular stress. Mutations in PRKAG2 have been implicated in a unique hypertrophic cardiomyopathy (HCM) characterized by cardiac glycogen overload and hypertrophy. We identified a novel PRKAG2 mutation in a neonate with prenatal onset of HCM noted on a 27-week prenatal ultrasound. Molecular testing for HCM revealed a de novo PRKAG2 mutation (K475E). We aim to investigate the signaling pathway of mutation in PRKAG2 gene. Methods: HEK-293 and H9C2 cells stably expressing wild type (WT) or K475E FLAG-tagged 2 from a tetracycline-inducible promoter were established. Basal AMPK activities were measured at varying AMP concentrations. Phenformin-stimulated AMPK activity and T172-phosphorylation were also measured. Changes in mTOR signaling pathway were accessed by Western blot. Cells were treated with angiotensin II, rapamycin (RAPA) or both followed by staining or flow cytometry to assess cell area/size. Primary fibroblasts from the K475E patient and non-diseased controls were cultured and compared. Finally, transgenic mice with cardiac-specific overexpression of human wild type (Tg WT ) or K475E (TgK 475E ) were established for further study of the PRKAG2 phenotype by histology and echocardiography. Result: HEK-293 cells: K475E mutation induced a markedly increase in the basal AMPK activity and T172-phosphorylation, reduced sensitivity to AMP in allosteric activation and loss of response to phenformin. H9c2 cells: K475E mutation induced increases in the phosphorylation of p-P70S6K, p-4EBP1 and a hypertrophy phenotype which was reversed by RAPA. Transgenic mice: Tg K475E mice had an early onset hypertrophy and were prone to sudden death. Histology revealed large vacuoles and high glycogen content in Tg K475E myocytes throughout the ventricles. Echocardiography confirmed significantly enlarged heart in Tg K475E mice. Conclusions: We identified a novel infantile-onset PRKAG2 mutation with a severe cardiac phenotype. In vitro study revealed involvement of mTOR signaling pathway and reversal of the hypertrophy phenotype by mTOR inhibitor.

Changes in mTOR/4‐EBP1 pathway induced by a novel mutation in PRKAG2 gene (864.7)

Objective: The genetics of hypertrophic cardiomyopathy (HCM) is heterogeneous. Mutations in PRKAG2 have been implicated in some previous cases of HCM. PRKAG2 encodes the γ2 isoform of AMPK, a heterotrimeric enzyme with major roles in regulation of energy metabolism in response to cellular stress. We have identified a K475E mutation which results in a neonatal HCM phenotype. We have demonstrated that overexpressing the K475E mutant in HEK‐293 cells leads to 1) a markedly increase in the basal phosphorylation of T172 and associated kinase activity; 2) a reduction in the sensitivity to AMP in allosteric activation. The aim of this study was to investigate changes in downstream signaling pathways induced by the mutation.Methods: Primary dermal fibroblast cultures of control and a patient with the K475E mutation were maintained under standard cell culture condition. Cell lysates were subject to Western blotting for downstream factors, including Akt, p70S6K, and 4E‐BP1.Results: K475E mutation results in an increase in the activation of p‐P70S6K as evidenced by increased phosphorylation at both the critical phosphorylation sites (T389 and T421/S424). Fibroblasts from the K475E mutation also show increases in phosphorylation of 4E‐BP1 (S65 and T70).Conclusions: Our study shows K475E mutation leads to activation of a signaling pathway that favors protein synthesis/growth, providing a logical mechanism for the HCM phenotype observed.Grant Funding Source: NIH

A new mutation in PRKAG2 gene causing hypertrophic cardiomyopathy with conduction system disease and muscular glycogenosis

Mutations in the gene encoding the γ2 subunit of AMP-activated protein kinase ( PRKAG2) cause familial cardiac hypertrophy and electrophysiological abnormalities, with glycogen accumulation in the heart of affected patients. The authors describe a 38-year-old man with a new heterozygous PRKAG2 mutation (Ser548Pro) manifesting by hypertrophic cardiomyopathy, severe conduction system abnormalities, and skeletal muscle glycogenosis. Considering those results, PRKAG2 gene could be a potential candidate for unexplained muscle glycogenosis associated with cardiac abnormalities.