Connexin43 silencing in myofibroblasts prevents arrhythmias in myocardial cultures: role of maximal diastolic potential

Abstract

Arrhythmogenesis in cardiac fibrosis remains incompletely understood. Therefore, this study aims to investigate how heterocellular coupling between cardiomyocytes (CMCs) and myofibroblasts (MFBs) affects arrhythmogeneity of fibrotic myocardial cultures. Potentially, this may lead to the identification of novel anti-arrhythmic strategies. Co-cultures of neonatal rat CMCs and MFBs in a 1:1 ratio were used as a model of cardiac fibrosis, with purified CMC cultures as control. Arrhythmogeneity was studied at day 9 of culture by voltage-sensitive dye mapping. Heterocellular coupling was reduced by transducing MFBs with lentiviral vectors encoding shRNA targeting connexin43 (Cx43) or luciferase (pLuc) as control. In fibrotic cultures, conduction velocity (CV) was lowered (11.2 ± 1.6 cm/s vs. 23.9 ± 2.1 cm/s; P < 0.0001), while action potential duration and ectopic activity were increased. Maximal diastolic membrane potential (MDP) of CMCs was less negative in fibrotic cultures. In fibrotic cultures, (n = 30) 30.0% showed spontaneous re-entrant tachyarrhythmias compared with 5% in controls (n = 60). Cx43 silencing in MFBs made the MDP in CMCs more negative, increased excitability and CV by 51% (P < 0.001), and reduced action potential duration and ectopic activity (P < 0.01), thereby reducing re-entry incidence by 40% compared with pLuc-silenced controls. Anti-arrhythmic effects of Cx43 down-regulation in MFBs was reversed by depolarization of CMCs through I(k1) inhibition or increasing extracellular [K(+)]. Arrhythmogeneity of fibrotic myocardial cultures is mediated by Cx43 expression in MFBs. Reduced expression of Cx43 causes a more negative MDP of CMCs. This preserves CMC excitability, limits prolongation of repolarization and thereby strongly reduces the incidence of spontaneous re-entrant tachyarrhythmias.