P3826Gene therapy for cardiac conduction system dysfunction in heart failure
Authors: Stuart L , Oh I Y , Wang Y , Nakao S , Starborg T , Yanni-Gerges J , Kitmitto A , Dobrzynski H , Cartwright E J , Oceandy D , Boyett M R 
Published: October 2019

Abstract

Background and purpose
Heart failure (HF) is characterised by generalised dysfunction of the cardiac conduction system (CCS). Ion channel and structural remodelling in the CCS have been widely 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. Free running PFs were processed for serial block face scanning electron microscopy (SBF-SEM). Manual contrast-based segmentation techniques were used on IMOD software to determine the 3D cellular ultrastructure. 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 K+ channel subunit expressed throughout the CCS, or the HCN4 promoter, a key pacemaker ion channel, to target the sinus node. The strong ubiquitous cytomegalovirus (CMV) promoter was used as a positive control. Adenovirus was produced using via transfection into the 293A cell line for viral packaging and amplification.

Results
Purkinje cells (PCs) formed a central core within PFs, encapsulated by an extensive collagen matrix. PCs were uninucleated and spindle shaped with an irregular membrane. Gap junctions were abundant and distributed along the lateral surface of cells, and there was a trend towards decreased expression in HF (p=0.0526, n=3 cells analysed per group). Hypertrophy and nuclear membrane breakdown were evident in HF PCs, the latter facilitating mitochondrial entry.
Using the CMV-GFP adenoviral construct, abundant GFP expression was conferred in ex vivo sinus node tissue, isolated sinus node myocytes, and neonatal ventricular rat cardiomyocytes (NRCMs). The KCNE1 promoter conferred relatively high GFP expression in NRCMs, greater than that from the HCN4 promoter. In isolated sinus node myocytes, the HCN4 promoter conferred greater transgene expression than in NRCMs. In ex vivo sinus node tissue, only the CMV construct was capable of driving significant GFP expression. Notably, expression was largely confined to the sinus node, with only sparse expression detected in the surrounding atrial muscle.

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. Preliminary data suggest that 1.2 kb and 0.8 kb fragments of the HCN4 promoter are capable of driving sinus node specific transgene expression. Further tests are warranted to confirm the utility of these promoters to express therapeutic transgenes, such as miR sponges to competitively inhibit miR activity in vitro and in vivo.

Acknowledgement/Funding
The British Heart Foundation