Objective: Besides structural remodeling, pathological cardiac hypertrophy (PCH) also results in electrical remodeling and is associated with lethal ventricular arrhythmias. We investigated in an in vitro model the possibility to prevent the pro-arrhythmic electrophysiological changes accompanying PCH by using a member of the nuclear factor of activated T cells (NFAT) family of transcription factors as they are important regulators of cardiac ion channel gene expression. Materials and Methods: Neonatal rat ventricular cardiomyocytes (nrCMCs) were isolated from 2-day-old Wistar rats and cultured as confluent monolayers. At day 2, cells were transduced with lentiviral vectors encoding either the eGFP alone (LV.eGFP; control vector) or together with a mutant version of NFAT3 (LV.NFAT3.eGFP). Pathological hypertrophy was induced by 24-hour treatments with 100 μM phenylephrine (PE) at day 3 and 8. The pathological nature of the hypertrophy was investigated by analysis of cell surface area (CSA), total protein content, capacitance and Cx43 and SERCA2A levels. Electrophysiological properties, including conduction velocity (CV), action potential duration (APD) and APD dispersion were analysed by optical mapping. Results: NFAT3 expression and predominant nuclear localization was confirmed by western blot. PE treatment of nrCMC cultures increased mean CSA and total protein content and capacitance by ~50%. Expression levels of all Nav1.5, Cx43, SERCA2A and Cav1.2 proteins were reduced upon PE treatment and recovered by NFAT3 gene transfer. Local 1-Hz stimulation of the nrCMC cultures resulted in 41.8% arrhythmias in the PEtreated nrCMCs transduced with LV.eGFP (n=141) while PE-treated nrCMCs transduced with LV.NFAT3.eGFP showed only 2.8% arrhythmias (n=139). Following 1-Hz pacing, PEtreated nrCMCs transduced with LV.eGFP showed prolongation of repolarization. To investigate the underlying mechanism of arrhythmia attenuation by NFAT3, L–type Ca2+ channel gating properties were studied. PE-treated nrCMCs transduced with LV.GFP showed increase in L–type Ca2+ current density (ICaL of 10.7 ± 1.2 pA/pF vs 4.0 ± 0.5 pA/pF in PEtreated nrCMCs transduced with LV.NFAT3.GFP). Conclusion: Increase in ICaL is the possible mechanism of arrhythmias in PCH. NFAT3 gene transfer can counteract the PEinduced arrhythmias and therefore represent an interesting target for the development of novel therapies to reduce the detrimental aspects of PCH.

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