Abstract 186: Biopacemakers Induced by Gene Transfer of a Dual-Functional Kir2.1 Mutant
Previous efforts to engineer biopacemakers have focused either on overexpression of HCN, or on suppression of IK1 to liberate endogenous pacemaker activity. Here we report a novel strategy designed to convert IK1 into a nonselective “leak” current. The idea is to combine the key virtues of HCN (leak current) and IK1 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 IK1 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 IK1 and to create a leak current.