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

Background 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. Methods 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. Results 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. Conclusion 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.