Electrophysiological remodeling involving gap junctions has been demonstrated in failing hearts and may contribute to intercellular uncoupling, delayed conduction, enhanced arrhythmias and vulnerability to sudden death in patients with heart failure. Recently, we showed that failing human hearts exhibit marked increases in Cx45 expression in addition to previously documented decreases in Cx43 expression. Each of these changes results in reduced gap junctional coupling. The objective of the present study was to examine functional consequences of increased Cx45 in cardiac gap junctions. Transgenic mice with cardiac-selective overexpression of the developmentally downregulated cardiac connexin, connexin45 (Cx45OEs), were subjected to in vivo electrophysiology studies (anesthetized) using an intracardiac catheter to induce ventricular arrhythmias, and ambulatory ECG monitoring (unanesthetized) to detect spontaneous arrhythmias. Hearts were analyzed by rt-PCR and Taqman, immunostaining, immunoblotting, and echocardiography. Lucifer yellow and neurobiotin dye transfer was used to assess coupling in transgenic and control myocyte cultures. Cx45 mRNA was 2 orders of magnitude greater in Cx45OEs. Cx45 immunoreactive signal at gap junctions increased 2-fold and total Cx45 protein by immunoblotting increased 25% in Cx45OEs compared to nontransgenic littermate controls. Functionally, Cx45OEs exhibited more inducible ventricular tachycardia than controls, but did not exhibit any other functional or structural derangements as assessed by echocardiography. Ventricular myocytes isolated from Cx45OEs exhibited diminished intercellular transfer of Lucifer yellow dye and increased transfer of neurobiotin consistent with altered cell-to-cell communication. Thus, increased myocardial expression of Cx45 results in remodeling of intercellular coupling and greater susceptibility to ventricular arrhythmias in vivo.