Abstract 186: Biopacemakers Induced by Gene Transfer of a Dual-Functional Kir2.1 Mutant

Abstract

Previous efforts to engineer biopacemakers have focused either on overexpression of HCN, or on suppression of I K1 to liberate endogenous pacemaker activity. Here we report a novel strategy designed to convert I K1 into a nonselective “leak” current. The idea is to combine the key virtues of HCN (leak current) and I K1 suppression (destabilization of the resting potential) in a single gene construct. We focused on the human Kir2.1ER mutation (E138R and R148E, KER) which can work as not only a functional non-selective channel in homotetramers but also as a dominant-negative suppressor for the Kir2.X gene family in heart. The plasmid CMV-KER-IRES-wtKir2.1 coexpresses the KER mutant and wild-type Kir2.1. In HEK cells transduced with CMV-KER-IRES-wtKir2.1, barium sensitive hyperpolarization-activated inward current (−5.5 pA/pF at −80mV) could be detected (n=5) with a reversal potential of −35.1± 2.1 mV in normal Tyrode external solution. These data demonstrate that overexpression of KER undermines the ability of Kir to produce functional I K1 channels, while producing a cation-nonselective leak current. To probe the consequences of KER in primary tissues, we created the adenovirus AdKER in which KER expression is driven by the CMV promoter. Isolated guinea-pig myocytes transduced with AdKER exhibited spontaneous action potential oscillations with high reproducibility (n=10). Electrocardiograms performed 3 days after AdKER injection into guinea pig hearts showed idioventricular rhythms in virus injected-animals, while no such rhythms were seen in control (AdGFP) animals (n=6 each). These results show that KER mutants, if expressed in ventricular muscle, produce biopacemakers in vitro and in vivo . This study provides proof of principle for the notion that biological pacemakers can be readily induced by a single gene designed to destabilize I K1 and to create a leak current.