Mechanistic Role of I f Revealed by Induction of Ventricular Automaticity by Somatic Gene Transfer of Gating-Engineered Pacemaker (HCN) Channels

Abstract

Background— Although I f , encoded by the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channel gene family, is known to be functionally important in pacing, its mechanistic action is largely inferential and indeed somewhat controversial. To dissect in detail the role of I f , we investigated the functional consequences of overexpressing in adult guinea pig left ventricular cardiomyocytes (LVCMs) various HCN1 constructs that have been engineered to exhibit different gating properties. Methods and Results— We created the recombinant adenoviruses Ad-CMV-GFP-IRES (CGI), Ad-CGI-HCN1, Ad-CGI-HCN1-ΔΔΔ, and Ad-CGI-HCN1-Ins, which mediate ectopic expression of GFP alone, WT, EVY235-7ΔΔΔ, and Ins HCN1 channels, respectively; EVY235-7ΔΔΔ and Ins encode channels in which the S3–S4 linkers have been shortened and lengthened to favor and inhibit opening, respectively. Ad-CGI-HCN1, Ad-CGI-HCN1-ΔΔΔ, and Ad-CGI-HCN1-Ins, but not control Ad-CGI, transduction of LVCMs led to robust expression of I f with comparable densities when fully open (≈−22 pA/pF at −140 mV; P >0.05) but distinctive activation profiles (V 1/2 =−70.8±0.6, −60.4±0.7, and −87.7±0.7 mV; P <0.01, respectively). Whereas control (nontransduced or Ad-CGI–transduced) LVCMs were electrically quiescent, automaticity (206±16 bpm) was observed exclusively in 61% of Ad-HCN1-ΔΔΔ–transduced cells that displayed depolarized maximum diastolic potential (−60.6±0.5 versus −70.6±0.6 mV of resting membrane potential of control cells; P <0.01) and gradual phase 4 depolarization (306±32 mV/s) that were typical of genuine nodal cells. Furthermore, spontaneously firing Ad-HCN1-ΔΔΔ–transduced LVCMs responded positively to adrenergic stimulation ( P <0.05) but exhibited neither overdrive excitation nor suppression. In contrast, the remaining 39% of Ad-HCN1-ΔΔΔ–transduced cells exhibited no spontaneous action potentials; however, a single ventricular action potential associated with a depolarized resting membrane potential and a unique, incomplete “phase 4–like” depolarization that did not lead to subsequent firing could be elicited on simulation. Such an intermediate phenotype, similarly observed in 100% of Ad-CGI-HCN– and Ad-CGI-HCN1-Ins–transduced LVCMs, could be readily reversed by ZD7288, hinting at a direct role of I f . Correlation analysis revealed the specific biophysical parameters required for I f to function as an active membrane potential oscillator. Conclusions— Our results not only contribute to a better understanding of cardiac pacing but also may advance current efforts that focus primarily on automaticity induction to the next level by enabling bioengineering of central and peripheral cells that make up the native sinoatrial node.