Introduction Mutations in the PRKAG2 gene, which encodes the regulatory γ2 subunit of AMP-activated protein kinase, cause human cardiomyopathy characterised by cardiac hypertrophy, ventricular pre-excitation, progressive conduction system disease and myocardial glycogen storage. AMP kinase is a ubiquitous serine/threonine protein kinase that acts as a cellular energy sensor and master regulator of cellular metabolism, activated by stimuli-depleting intracellular ATP. Several transgenic murine models expressing human mutations in the PRKAG2 gene have been utilised to explore the molecular basis for the human phenotype but, until now, have all been overexpressing transgenic models. These transgenic models are confounded by unphysiological overexpression of the γ2 subunit and abnormal splicing. One manifestation of these consequences is that even the wild-type overexpressing transgenic models manifest significant ventricular hypertrophy and upregulation of markers of ventricular stress, eg, brain natriuretic peptide. We report here the generation of the first knock-in model of PRKAG2 cardiomyopathy.
Methods In order to generate a model that more faithfully recapitulates the human disease, a R302Q mutation was introduced into the equivalent position (R299Q) within the orthologous murine PRKAG2 gene through gene targeting. This model allows retention of native expression and feedback regulation via the normal endogenous promoter and splicing mechanisms. The targeting strategy included the insertion of a loxP site together with a FRT flanked neomycin selection cassette within intron 6 upstream of the R299Q mutated exon 7 and a single distal loxP within intron 7 (fig). Founder lines were bred with constitutively expressing Flp recombinase mice to remove the neomycin selection cassette.
Results Mice successfully generated carrying the R299Q mutation in the murine PRKAG2 gene exhibit significant ventricular dysfunction and concentric hypertrophy increasingly apparent with age, as assessed by both gross histology and by 2D-echocardiography when compared with age-matched wild-type controls.
Implications We report here the preliminary findings of the first knock-in, non-overexpressing transgenic model of human PRKAG2 cardiomyopathy designed to recapitulate the human disease more faithfully. Further detailed characterisation of this model is expected to yield significant insights into the underlying mechanisms and phenotype of disease and may shed light on the pathogenesis of more common forms of heart muscle disease.