Abstract 376: In Vitro Evaluation of a Chimeric HCN-based Biological Pacemaker Predicts in vivo Function
Gene- and cell-based strategies to deliver the HCN2 gene that encodes an If-like current have been employed to produce biological pacemakers in canine heart. We hypothesized that a chimeric murine HCN212 channel incorporating the S1-S6 core transmembrane domain of HCN1 would manifest fast HCN1 kinetics while the cytoplasmic NH2 and COOH termini from HCN2 would maintain the strong HCN2 cAMP response, resulting in increased biological pacemaker rate and preserved adrenergic response. HCN212 and HCN2 channels were expressed in newborn rat ventricle cells using adenoviruses. HCN212 channels had a current density, voltage dependence (mean V1/2= −69.2 mV, s=13.5 mV; n=10), and cAMP modulation (10 mV shift) similar to those of HCN2 channels but with ~2x faster activation/ deactivation kinetics. Basal spontaneous beating was faster in HCN212 than in HCN2 myocytes, and when adjusted for basal rate adrenergic agonists induced a similar rate increase for both constructs. However, spontaneous activity exhibited a bursting pattern more often (36%) in HCN212 than in HCN2 cultures (23%). The constructs were then tested in vivo: HCN2 (n=6) or HCN212 (n=6) virus was injected into LBB and complete AV block induced via RF ablation. An electronic pacemaker was implanted, at VVI 45 bpm. On day 11–14, epinephrine (Epi; 1–2 μg/kg/min) was infused to evaluate adrenergic responsiveness. Maximal rate determined on 24h monitoring was 168±16 bpm (±SEM) in HCN212 and 97±3 bpm in HCN2 (p<0.05). In addition, HCN212 exhibited fast, bursting VT with QRS configuration pace-mapped to the injection site and maximal rate=285±37 bpm; HCN2 did not exhibit bursting VT. Epi increased heart rate 48±11% (from 57±5 to 83±6 bpm) in HCN2 and 72±17% (45±3 to 79±11 bpm) in HCN212 (p<0.05). In 6/6 HCN212 and 3/6 HCN2 dogs Epi was terminated due to HR increase >50% (p<0.05). In the other 3 HCN2 dogs Epi was terminated because of VT occurrence or failure to induce 50% HR increase after the maximum dose. The faster basal rate and arrhythmias seen with HCN212 in vivo are consistent with the in vitro results, suggesting genetic manipulation can be used to modify biological pacemaker properties and that myocyte cultures can be used to prescreen potential HCN constructs as biological pacemakers.