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.

Roles of hyperpolarization-activated current If in sinoatrial node pacemaking: insights from bifurcation analysis of mathematical models

“…We have suggested that 1) the instability of an equilibrium point (EP) is essentially important for the generation of robust pacemaking without annihilation; 2) I Ca,L is responsible for EP instability and generation of pacemaker activity; 3) I K makes the action potential amplitude larger, eases changes in oscillation frequency during hyperpolarization or depolarization, and plays a pivotal role in preventing bifurcation to quiescence; and 4) I Na contributes to EP instability and robust pacemaking against hyperpolarizing loads in the peripheral SAN. However, the roles of I f in SAN pacemaking, remaining the subject of controversy (27), have not yet been determined by the theoretical approach.I f , encoded by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel gene, is known to contribute to pacemaker depolarization as the pacemaker current (9, 27) and is a key current for engineering of biological pacemakers (43,46,48,53,56). I f contributes to prevention of excess hyperpolarization and excessively slow pacemaking against hyperpolarizing loads (16,27,35), autonomic regulations of spontaneous activity (7), stabilization of pacemaker frequency (16,33,40), and generation of phase 4 depolarization in the periphery of intact SAN, suffering electrotonic influences of the atrium (38).…”

“…I f , encoded by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel gene, is known to contribute to pacemaker depolarization as the pacemaker current (9, 27) and is a key current for engineering of biological pacemakers (43,46,48,53,56). I f contributes to prevention of excess hyperpolarization and excessively slow pacemaking against hyperpolarizing loads (16,27,35), autonomic regulations of spontaneous activity (7), stabilization of pacemaker frequency (16,33,40), and generation of phase 4 depolarization in the periphery of intact SAN, suffering electrotonic influences of the atrium (38).…”

Human Pluripotent Stem Cells in Cardiovascular Research and Regenerative Medicine

“…Therefore, facilitated in vitro maturation is important for the translation of hESC-CMs to the clinic and other applications (such as disease modeling, drug discovery and cardiotoxicity screening). Our group sought to define the immature proarrhythmic electrophysiological properties observed in hESC-CMs by examining the role of different currents in automaticity (Azene et al, 2005;Siu et al, 2006;Xue et al, 2007;Lieu et al, 2008;Chan, Y. C. et al, 2009). I K1 (the inward-rectifier K + current encoded by Kir2.1), which stabilises a negative resting membrane potential, is important for suppressing automaticity and we hypothesize that its absence in hESC-CMs may underlie their immature phenotype.…”

“…Several gene-based approaches have been pursued to induce pacemaker activity in these normally silent cells. Our group took a protein engineering approach to define criteria important for pacing and created a bioengineered construct of HCN (Lesso and Li, 2003;Tse et al, 2006;Xue et al, 2007). This engineeredconstruct was shown to produce pacing in vitro and in vivo (Tse et al, 2006;Xue et al, 2007).…”

“…Our group took a protein engineering approach to define criteria important for pacing and created a bioengineered construct of HCN (Lesso and Li, 2003;Tse et al, 2006;Xue et al, 2007). This engineeredconstruct was shown to produce pacing in vitro and in vivo (Tse et al, 2006;Xue et al, 2007). In a sick sinus syndrome porcine model, pacing of the heart was restored and originated from the site of focal transduction in the left atrium with HCN-construct injection (Tse et al, 2006).…”