Background and objectives: Heart failure (HF) is characterised by generalised dysfunction of the cardiac conduction system (CCS). Ion channel and structural remodelling in the CCS have been demonstrated in animal models of cardiovascular disease. As Purkinje fibres (PFs) are minute strands of tissue, little is known about their ultrastructure and remodelling in disease. Furthermore, given the role for microRNAs (miRs) in CCS molecular remodelling, we aimed to develop a tissue specific method for delivering therapeutic transgenes, such as miR sponges. Methods: New Zealand rabbits were used for PF ultrastructural studies. HF was induced via pressure and volume overload as previously described(1). Free running PFs were stained and embedded for serial block face scanning electron microscopy (SBF-SEM). Manual contrast-based segmentation techniques were used on IMOD software to determine the 3D ultrastructure of free running PFs in control and HF animals. To target transgene expression to the CCS, adenoviral plasmids were cloned expressing a GFP reporter gene. GFP transcription was placed under control of the KCNE1 promoter, a potassium channel subunit expressed throughout the CCS, or the HCN4 promoter, a key pacemaker ion channel, to target the SAN. Adenovirus was then produced using Gateway Cloning Technology (Thermo-Fisher Scientific) in combination with the ViraPower Adenoviral Expression system (Invitrogen) and transfected into the 293A cell line for amplification. For in vivo use, all plasmids were also cloned into AAV packaging plasmids. Results: Purkinje cells (PCs) formed a central core within PFs, encapsulated by an extensive collagen matrix. 3D reconstructions revealed a central core of longitudinally running cells, running parallel to the direction of the fibre. PCs were uninucleated and spindle shaped with an irregular membrane, and an average length of 108µm, diameter 15µm and volume of 7965µm3 (n=9). Gap junctions were abundant and distributed along the lateral surface of cells, and there was a trend towards decreased expression in HF (Figure 1B, p=0.0526, n=3 cells analysed per group). Hypertrophy (figure 1B) and nuclear membrane breakdown were evident in HF PCs, the latter facilitating mitochondrial entry. Testing the various promoter constructs for therapeutic gene delivery to the CCS is ongoing in vitro using isolated neonatal rat cardiomyocytes and ex vivo using SAN tissue isolated from Wistar rats. These tests will show the efficacy of transgene expression under each promoter as well as tissue specificity. All adenoviral plasmids were successfully cloned, verified by restriction digest and Sanger sequencing. Conclusions: SBF-SEM revealed ultrastructure of free running PFs in situ, and uncovered novel structural changes in HF that are likely to be pro-arrhythmic. The efficacy of our proposed gene therapy approach in targeting the CCS will be further elucidated in the coming months whilst testing the final viral expression constructs.