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.