Role of heterogeneity of Kir2.1 channel protein expression in the genesis and stability of cardiac arrhythmias

Previous studies have postulated an important role for the inwardly rectifying potassium current (IK1) in controlling the dynamics of electrophysiological spiral waves responsible for ventricular tachycardia and fibrillation. In this study, we developed a novel tissue model of cultured neonatal rat ventricular myocytes (NRVMs) with uniform or heterogeneous Kir2.1 expression achieved by lentiviral transfer to elucidate the role of IK1 in cardiac arrhythmogenesis. Immunohistochemistry, Trypan Blue exclusion, Annexin V binding followed by flow cytometry, and Terminal transferase dUTP Nick End Labeling (TUNEL) assays revealed that lentiviral transduction had no adverse cytotoxic effects on NRVM cultures. Using immunohistochemistry and Western blot, we characterized our cultures for Kir2.1, cardiac troponin I, actin and connexin43 expression levels. Whole-cell patch clamping of Kir2.1-overexpressed NRVMs showed increased IK1 density, hyperpolarized resting membrane potential and increased action potential upstroke velocity compared with GFP-transduced NRVMs. Opposite results were observed in Kir2.1-suppressed NRVMs. Optical mapping of uniformly Kir2.1 gene-modified monolayers showed changes in conduction velocity (CV) and action potential duration (APD) compared with non-transduced and empty vector-transduced monolayers, but functional reentrant waves could not be induced. In monolayers with islands of altered Kir2.1 expression, CV and APD of the transduced and non-transduced regions were similar to those of the uniformly transduced and non-transduced monolayers, respectively. Functional reentrant waves could be induced in monolayers with heterogeneous IK1 expression. The waves were anchored to regions of Kir2.1 overexpression and remained stable, but dropped in frequency and meandered away from regions of Kir2.1 suppression. In monolayers with an inverse pattern of IK1 heterogeneity, stable high frequency spiral waves were present with IK1 overexpression, whereas lower frequency, meandering spiral waves were observed with IK1 suppression. Our study provides direct evidence for the contribution of IK1 heterogeneity to the genesis and stability of spiral waves and highlights the potential importance of IK1 as an anti-arrhythmia target.