Influence of PRKAG2 gene G100S novel mutation on glycogen storage and calcium homeostasis in cardiomyocytes

Abstract

Objective To investigate the possible mechanism by which PRKAG2 G100S,a novel mutation recently found in a Chinese family with Wolff-Parkinson-White(WPW) syndrome,induces cardiac hypertrophy.Methods The recombinant adenovirus containing human PRKAG2 G100S was constructed using Invitrogen's Gateway TM system and was used to infect rat embryonic H9c2 cardiomyocytes and cardiomyocytes of neonatal SD rats using EGFP as the reporter gene;Western blotting analysis was used to determine PRKAG2 protein expression.The intracellular free Ca2 + was determined in the H9c2 cells before and 48 h after infected with Ad-EGFP,Ad-PRKAG2 or Ad-PRKAG2 G100S by incubating with Rohd-2/AM.The glycogen contents in neonatal SD rat cardiomyocytes were determined by PAS 48-72 h after infection.Results Bright green fluorescence was observed in the cultured H9c2 cells and neonatal SD rat cardiomyocytes 48 h after infected with Ad-PRKAG2 G100S,and PRKAG2 protein expression was identified with PRKAG2 monoclonal antibody.Compared with wild-type PRKAG2 and EGFP groups,intercellular free Ca2 + concentration in H9c2 cells of Ad PRKAG2 G100S group had no noticeable changes 48 h after infection,and the concentrations were not significantly different in Ad PRKAG2 G100S group before and 48 h after infection.PAS revealed evident glycogen storage in the neonatal SD rat cardiomyocytes in PRKAG2 G100S group 48 h after infection.Conclusion Our findings indicate that myocardial glycogenosis might contribute to the pathogenesis of cardiac hypertrophy in patients with PRKAG2 G100S mutation,and the imbalance of calcium homeostasis in cardiomyocytes might not be involved in the pathogenesis.