The cardiac His-Purkinje system conducts electrical activity throughout the ventricles and, in heart failure, electrical conduction within the His-Purkinje network is impaired and about 36% of patients show left bundle branch conduction block. Intercellular gap junctions conduct the electrical signals from one cardiomyocyte to another and these junctions are composed of proteins known as connexins (Cx). We have developed a rabbit model of congestive heart failure (CHF) that is induced by combined pressure and volume overload1 and investigated expression of connexins and Ca2+-handling proteins in the Purkinje fibres (PF), Purkinje fibre-left ventricular junction (JPF), left ventricle (LV), and papillary muscle (PM). Frozen rabbit ventricular tissues were sectioned at -18˚C using a cryostat (Leica CM 3050S). Tissue sections were of 20 µm thickness. Every fifth section was stained with Masson’s trichrome to identify structures. Others were used for immunohistochemistry, in which sections were labelled for Cx40 and Cx43 and Ca2+-handling proteins: Na+-Ca2+ exchanger (NCX1), ryanodine receptor (RyR2) and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a). Appropriate primary and fluorophore-containing secondary antibodies were used and labelling was visualised by confocal microscopy (Zeiss LSM5 Pascal). High magnification images were obtained and the fluorescence signals were semi-quantified using Volocity software (Improvision). SigmaPlot software was used for statistical analysis while one-way ANOVA for statistical comparison. Tissue from three control and three CHF animals was used and the work was carried out in accordance with UK Home Office regulations. Histology staining with Masson’s trichrome was used to identify tissue sections with intact PF, JPF, LV and PM regions for immunolabelling. Expression of Cx43 and Cx40 was identified at the intercellular junctions, NCX1 labelled cell membranes, and RyR2 and SERCA2a labelled the sarcoplasmic reticulum. We observed significantly higher expression of Cx40 in the PF (+12%) and JPF (+11%) regions compared to LV (P<0.05) in control tissues. In CHF, Cx40 expression decreased in PF and JPF to a level that was no longer significantly different from LV and PM. Reduced Cx40 levels in CHF suggest slower conduction velocities in these tissues. Cx43 labelling was uniform in all control tissues. In CHF, Cx43 levels in PF showed a small increase (5%, P<0.1 vs. LV in CHF). NCX1 expression in CHF was increased in PF by 36% (P<0.05 vs. control), but remained unaffected in LV and PM. RyR2 expression in PM was reduced by 17% in CHF (P<0.1 vs. control); elsewhere RyR2 remained unaffected. SERCA2 levels were slightly reduced in all tissues.