Ph.D Thesis

Ph.D StudentBen Jehuda Ronen
SubjectElectrophysiological Abnormalities and CRISPR
Gene Editing of PRKAG2 and DMD Patients'
Induced Pluripotent Stem Cell-Derived
DepartmentDepartment of Biotechnology
Supervisors PROFESSOR EMERITUS Ofer Binah


Mutations in the PRKAG2 gene encoding the γ-subunit of adenosine monophosphate-kinase (AMPK) cause hypertrophic cardiomyopathy (HCM) and familial-Wolff-Parkinson-White syndrome (WPW). Patients carrying the R302Q mutation in PRKAG2 present sinus bradycardia, escape rhythms, ventricular pre-excitation, supraventricular tachycardia and atrioventricular block. This mutation affects AMPK activity and increases glycogen storage in cardiomyocytes. The link between glycogen storage, WPW, HCM and arrhythmias remains unknown. To investigate the pathological changes caused by the PRKAG2 mutation we tested the hypothesis that the patient’s induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) display clinical aspects of the disease. Using the CRISPR technology we corrected the mutation and generated isogenic control iPSC-CMs. Action potentials were recorded from spontaneously firing and paced cardiomyocytes using the patch clamp technique. PRKAG2-mutated iPSC-CMs exhibited abnormal firing patterns, delayed afterdepolarizations (DADs), triggered arrhythmias and augmented Beat Rate Variability (BRV). Importantly, the CRISPR correction eliminated the electrophysiological abnormalities, the augmented glycogen storage and cardiomyocyte hypertrophy. PRKAG2-mutated iPSC-CMs displayed functional and structural abnormalities, which were abolished by correcting the mutation in the patient's iPSCs using the CRISPR technology.

            Duchenne muscular dystrophy (DMD), an X-linked progressive muscle degenerative disease, results in death by the 3rd decade of life due to respiratory or cardiac failure. DMD is caused by mutations in the dystrophin gene leading to loss of protein, destabilization of dystrophin glycoprotein complex (DGC), and sarcolemma instability. Eventually, degenerated muscle is replaced by fibrous tissue, leading to loss of function. To investigate the cardiac manifestation of DMD, we generated iPSCs and iPSC-CMs from two DMD patients: a male and a female manifesting carrier. We analyzed dystrophin gene and protein expression and used current and voltage clamp to record action potentials and ion currents, respectively. Our major finding were: (1) Next generation sequencing (NGS) demonstrated DMD female iPSCs underwent X chromosome reactivation (XCR), and the iPSC-CM population displayed a mosaic X chromosome expression of wild type (WT) and mutated alleles. (2) DMD iPSC-CMs presented low spontaneous firing rate, arrhythmias, and prolonged action potential duration (APD). (3) DMD female iPSC-CMs displayed increased BRV. (4) DMD male iPSC-CMs manifested decreased pacemaker current (If) density, and DMD female and male iPSC-CMs showed increased L-type calcium current (ICa,L) density. In summary, Dystrophin-mutated iPSC-CMs express key molecular and functional features of DMD, thus supporting the validity of this experimental model.