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

Tian Xue, Chung Wah Siu, Deborah Lieu, Chu Pak Lau, Hung Fat Tse, Ronald A. Li

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

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-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 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-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 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.

Original languageEnglish (US)
Pages (from-to)1839-1850
Number of pages12
JournalCirculation
Volume115
Issue number14
DOIs
StatePublished - Apr 2007

Fingerprint

Cyclic Nucleotides
Cardiac Myocytes
Membrane Potentials
Genes
Action Potentials
Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels
Bioengineering
Sinoatrial Node
Adenoviridae
Adrenergic Agents
Guinea Pigs
Phenotype

Keywords

  • Genes
  • Ion channels
  • Pacemakers
  • Sinoatrial node
  • Tissue engineering

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Mechanistic role of If revealed by induction of ventricular automaticity by somatic gene transfer of gating-engineered pacemaker (HCN) channels. / Xue, Tian; Siu, Chung Wah; Lieu, Deborah; Lau, Chu Pak; Tse, Hung Fat; Li, Ronald A.

In: Circulation, Vol. 115, No. 14, 04.2007, p. 1839-1850.

Research output: Contribution to journalArticle

Xue, Tian ; Siu, Chung Wah ; Lieu, Deborah ; Lau, Chu Pak ; Tse, Hung Fat ; Li, Ronald A. / Mechanistic role of If revealed by induction of ventricular automaticity by somatic gene transfer of gating-engineered pacemaker (HCN) channels. In: Circulation. 2007 ; Vol. 115, No. 14. pp. 1839-1850.
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abstract = "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-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 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-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 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.",
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TY - JOUR

T1 - Mechanistic role of If revealed by induction of ventricular automaticity by somatic gene transfer of gating-engineered pacemaker (HCN) channels

AU - Xue, Tian

AU - Siu, Chung Wah

AU - Lieu, Deborah

AU - Lau, Chu Pak

AU - Tse, Hung Fat

AU - Li, Ronald A.

PY - 2007/4

Y1 - 2007/4

N2 - 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-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 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-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 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.

AB - 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-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 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-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 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.

KW - Genes

KW - Ion channels

KW - Pacemakers

KW - Sinoatrial node

KW - Tissue engineering

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