Mechanistic role of I(f) revealed by induction of ventricular automaticity by somatic gene transfer of gating-engineered pacemaker (HCN) channels.
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.


RESULTS
We created the recombinant adenoviruses Ad-CMV-GFP-IRES (CGI), Ad-CGI-HCN1, Ad-CGI-HCN1-delta delta delta, and Ad-CGI-HCN1-Ins, which mediate ectopic expression of GFP alone, WT, EVY235-7delta delta delta, and Ins HCN1 channels, respectively; EVY235-7delta delta delta 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-delta delta delta, 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 (approximately = -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-delta delta delta-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-delta delta delta-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-delta delta delta-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.
Xue
Tian
T
Department of Cell Biology and Human Anatomy, University of California, Davis, USA.
Siu
Chung-Wah
CW
Lieu
Deborah K
DK
Lau
Chu-Pak
CP
Tse
Hung-Fat
HF
Li
Ronald A
RA
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002460:Cell Line; D054815:Cyclic Nucleotide-Gated Cation Channels; D005822:Genetic Vectors / Q000494:pharmacology; D006168:Guinea Pigs; D006329:Heart Conduction System / Q000502:physiology; D006352:Heart Ventricles / Q000166:cytology; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009200:Myocardial Contraction / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying / Q000502:physiology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000502:physiology; D026941:Sodium Channel Blockers / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013779:Tetrodotoxin / Q000494:pharmacology; D014161:Transduction, Genetic; D016276:Ventricular Function
D054815:Cyclic Nucleotide-Gated Cation Channels; C578665:HCN1 protein, human; C578666:Hcn1 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D026941:Sodium Channel Blockers; D001464:Barium; D013779:Tetrodotoxin; D000109:Acetylcholine