Recently, we found that repolarization heterogeneities between subepicardial and midmyocardial cells can form a substrate for reentrant ventricular arrhythmias in failing myocardium. We hypothesized that the mechanism responsible for maintaining transmural action potential duration heterogeneities in heart failure is related to intercellular uncoupling from downregulation of cardiac gap junction protein connexin43 (Cx43). With the use of the canine model of pacing-induced heart failure, left ventricles were sectioned to expose the transmural surface (n = 5). To determine whether heterogeneous Cx43 expression influenced electrophysiological function, high-resolution transmural optical mapping of the arterially perfused canine wedge preparation was used to measure conduction velocity (θ_TM), effective transmural space constant (λ_TM), and transmural gradients of action potential duration (APD). Absolute Cx43 expression in failing myocardium, quantified by confocal immunofluorescence, was uniformly reduced (by 40 ± 3%, P < 0.01) compared with control. Relative Cx43 expression was heterogeneously distributed and lower (by 32 ± 18%, P < 0.05) in the subepicardium compared with deeper layers. Reduced Cx43 expression in heart failure was associated with significant reductions in intercellular coupling between transmural muscle layers, as evidenced by reduced θ_TM (by 18.9 ± 4.9%) and λ_TM (by 17.2 ± 1.4%; P < 0.01) compared with control. Heterogeneous transmural distribution of Cx43 in failing myocardium was associated with lower subepicardial θ_TM (by 12 ± 10%) and λ_TM (by 13 ± 7%), compared with deeper transmural layers (P < 0.05). APD dispersion was greatest in failing myocardium, and the largest transmural APD gradients were consistently found in regions exhibiting lowest relative Cx43 expression. These data demonstrate that reduced Cx43 expression produces uncoupling between transmural muscle layers leading to slowed conduction and marked dispersion of repolarization between epicardial and deeper myocardial layers. Therefore, Cx43 expression patterns can potentially contribute to an arrhythmic substrate in failing myocardium.