Development of a new model of cardiac hypertrophy with pro-arrhythmic features to study the role of hypertrophy in arrhythmogenesis

Abstract

Purpose: Different factors may trigger the occurrence of arrhythmias in diseased hearts including fibrosis, cardiomyocyte hypertrophy and inflammation. This makes it difficult to establish the relative contribution of each of them to the occurrence of arrhythmias. In this study, we developed an in vitro model of pathological cardiac hypertrophy to study its role in the induction of arrhythmias independent of fibrosis and inflammation. Phorbol 12-myristate 13-acetate (PMA) is a diacylglycerol (DAG)-like activator of protein kinase C (PKC) that not only promotes myocyte hypertrophy but also decrease sarcoplasmic reticulum calcium ATPase (SERCA2) gene expression and impairs the calcium transient. Since pressure overload is associated with DAG production and PKC activation in cardiomyocytes, PMA-induced hypertrophy may be a good model to study hypertension-induced myocardial hypertrophy. Methods: Neonatal rat ventricular cardiomyocytes (nrCMCs) were isolated from 2-day-old animals and cultured as confluent monolayers. To inhibit proliferation of cardiac fibroblasts, cultures were treated with mitomycin-C, 1 day after cell isolation. Pathological hypertrophy was induced by 24-hour treatments with 500 nM PMA at day 4 and day 8 after culture initiation. The pathological nature of the hypertrophic response was confirmed by analysis of cell surface area, protein content and natriuretic peptide type A (NPPA), α-skeletal muscle actin (ASMA) and Serca2a protein levels. Electrophysiological properties of the nrCMCs were analyzed by optical mapping at day 9. Results: The percentage of spontaneous arrhythmias was much higher in the PMA-treated cultures than in the control cultures (46.8 vs 3.2% [p<0.01; n=32]). Local 1-Hz stimulation of the nrCMC cultures resulted in 25% arrhythmias (i.e. triggered activity) in the PMA-treated group (n=12) while the control cells showed no arrhythmias (n=12). Following 1-Hz pacing, PMA-treated nrCMCs showed APD prolongation (494±38.5 vs 242±65.5 ms in control cells [p<0.001; n=32]). Conclusion: A new model of hypertrophy-associated arrhythmias has been developed, which is currently being used to identify and validate specific targets for gene therapeutic interventions with viral vectors.