Published online before print March 26, 2007, doi: 10.1161/CIRCULATIONAHA.106.659391
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(Circulation. 2007;115:1839-1850.)
© 2007 American Heart Association, Inc.
Arrhythmia/Electrophysiology |
Mechanistic Role of If Revealed by Induction of Ventricular Automaticity by Somatic Gene Transfer of Gating-Engineered Pacemaker (HCN) Channels
From the Stem Cell Program and Department of Cell Biology and Human Anatomy, University of California, Davis (T.X., C.-W.S., D.K.L., R.A.L.); Department of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong (C.-P.L., H.-F.T., R.A.L.); and Institute of Pediatric Regenerative Medicine, Shriners Hospital for Children of North America, Sacramento, Calif (R.A.L.).
Correspondence to Ronald Li, PhD, University of California, Room 650, Shriners Hospital, 2425 Stockton Blvd, Sacramento, CA 95817. E-mail ronaldli{at}ucdavis.edu
Received December 16, 2005; accepted January 16, 2007.
Background— Although If, 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 If, 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-
, and Ad-CGI-HCN1-Ins, which mediate ectopic expression of GFP alone, WT, EVY235-7, and Ins HCN1 channels, respectively; EVY235-7 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-, and Ad-CGI-HCN1-Ins, but not control Ad-CGI, transduction of LVCMs led to robust expression of If with comparable densities when fully open (
–22 pA/pF at –140 mV; P>0.05) but distinctive activation profiles (V1/2=–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-–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-–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-–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 If. Correlation analysis revealed the specific biophysical parameters required for If 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.
Key Words: genes • ion channels • pacemakers • sinoatrial node • tissue engineering