HCN as a Biological Pacemaker

In sick sinus syndrome, the primary pacemaker oscillates between excessively high and excessively low heart rates. In AV block, life is sustained by an excessively low spontaneous rate which originates in the ventricular conducting system. In both these cases the current approach to therapy is the implantation of an electronic pacemaker to guarantee a constant physiologic rate consistent with a normal lifestyle. However, even the most up-to-date electronic versions are not immediately sensitive to changes in physiologic state induced by activation of the sympathetic and parasympathetic nervous systems. It is for this reason that current pacemaker therapy represents palliation rather than cure.

Subsequent to the cloning of the members of the HCN gene family in the latter part of the 1990s, it became possible to consider replacing the palliation of the electronic pacemakers with a biological cure (Rosen et al. 2004). If HCN family members could be delivered to the appropriate target regions of the heart (atrium for sick sinus syndrome, and the conducting system for AV block) then it would be possible to create a new primary pacemaker responsive to changes in input from the autonomic nervous system.

The initial approaches to create an HCN-based biological pacemaker used an adenoviral delivery system. The adenoviral vector contained the murine HCN2 gene. The virus was delivered either by injection into the wall of canine left atrium (Qu et al. 2003) or via catheter into the ventricular conducting system (Plotnikov et al. 2004). Evidence for a new biological pacemaker was demonstrated several days after injection by anesthetizing the animal and stimulating both vagi to cause sinus standstill. Vagal escape rhythms could be demonstrated in both studies originating near the sites of injection. After the in vivo studies were completed, cells were isolated from the injection sites and studied with the whole-cell patch-clamp technique. The transfected cells [which could be identified by green fluorescent protein (GFP), which was used as a reporter gene], expressed an If-like current which was between one and three orders of magnitude larger than If recorded from non-transfected myocytes.

As encouraging as these initial successes were, there are significant drawbacks with adenoviral gene delivery. Since the plasmid is not incorporated into the genome, the effect is transient, rarely lasting more than 6 weeks. Second, there is also the risk of allergic reaction, which has limited adenovi-ral approaches in prior clinical trials for other disease processes. Alternative viruses from the retroviral family are incorporated into the genome but come with an increased associated risk of neoplasia. In 2004 Potapova et al. (2004) employed an altogether different approach to deliver the HCN genes to the cardiac syncytium. They took human mesenchymal stem cells (hMSCs) and incorporated the same HCN2 gene by electroporation. After demonstrating high levels of expression of an If-like current in the transfected hMSCs, they injected one million of these transfected cells into the left epicardium and studied the animals 3-10 days later in the same manner described for viral delivery. Spontaneous vagal escape rhythms at roughly 60 beats per minute were observed which mapped to the site of the injection. This rate was significantly higher than those observed in sham animals which received hMSCs containing only enhanced GFP. Although the injected cells were not a stable cell line, separate experiments taking advantage of a neomycin resistance cassette in the original plasmid suggested that the electroporated cells could be selected by growth on antibiotic and continue to express the transfected proteins for at least 3 months.

As encouraging as these initial attempts appear to be, both the long-term reliability and safety of these pacemakers must be demonstrated before they can be considered a clinical tool.

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