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(Circulation. 2003;107:485.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Dominant-Negative Suppression of HCN Channels Markedly Reduces the Native Pacemaker Current I_f and Undermines Spontaneous Beating of Neonatal Cardiomyocytes

Fikret Er, MD; Robert Larbig; Andreas Ludwig, MD; Martin Biel, MD; Franz Hofmann, MD; Dirk J. Beuckelmann, MD; Uta C. Hoppe, MD

From the Department of Medicine III, University of Cologne (F.E., R.L., D.J.B., U.C.H.); the Institut für Pharmakologie und Toxikologie, Technische Universität München (A.L., F.H.); and the Department of Pharmacy, Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München (M.B.), Germany.

Correspondence to Uta C. Hoppe, MD, Department of Medicine III, University of Cologne, Joseph-Stelzmann-Straße 9, 50924 Cologne, Germany. E-mail uta.hoppe{at}uni-koeln.de

	Abstract

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Background— The pacemaker current I_f contributes to spontaneous diastolic depolarization of cardiac autonomic cells. In heterologous expression, HCN channels exhibit a hyperpolarization-activated inward current similar to I_f. However, the links between HCN genes and native I_f are largely inferential, and it remains unknown whether I_f is essential for cardiac pacing.

Methods and Results— To clarify this situation, we generated a GYG_402–404AYA pore mutation of HCN2, which rendered the channel nonfunctional and suppressed wild-type HCN2 in a dominant-negative manner in Chinese hamster ovary cells. In addition, HCN2-AYA suppressed I_HCN4 in a dominant-negative manner when coexpressed with wild-type HCN4, indicating that the 2 isoforms HCN2 and HCN4 are able to coassemble to form heteromultimeric complexes. Given that HCN2 and HCN4 are the dominant HCN mRNA transcripts in neonatal rat ventricle, we expressed HCN2-AYA in neonatal cardiocytes using adenoviral gene transfer to test the effect of HCN suppression on native I_f. I_f density was indeed reduced markedly, from 7.8±1.6 pA/pF (n=13) in control cells to 0.3±0.2 pA/pF (n=11) in HCN2-AYA–infected cells when measured at -130 mV (P<0.001). To probe the effect of HCN on cardiac pacing, we infected spontaneously beating neonatal monolayers with adenoviral vectors expressing wild-type and mutant HCN channels. Infection with HCN2 and HCN4 accelerated the beating rate significantly, to 230.5±8.6 bpm (n=12) and 223.5±12.3 bpm (n=10), respectively, compared with control cultures (83.4±4.5 bpm, n=13, P<0.001). Conversely, HCN2-AYA completely undermined spontaneous pacing of neonatal cardiocytes.

Conclusions— HCN channels are the major molecular component of native I_f and are critical for spontaneous beating of neonatal cardiomyocytes.

Key Words: ion channels • pacemakers • viruses • gene therapy • electrophysiology

	Introduction

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The pacemaker current I_f is present in both cardiac autonomic and nonautonomic tissue. In pacemaker regions and spontaneously beating neonatal cardiocytes, I_f is believed to contribute physiologically to spontaneous diastolic depolarization, whereas in diseased ventricular myocardium, this current might play a role in arrhythmogenesis.^1–3 However, whether I_f is essential for pacemaker activity or instead modulates the beating frequency of autonomic myocardial cells has been controversial over the years.^4–7 Four HCN gene family members have been cloned, 3 of which are present in heart (HCN1, HCN2, and HCN4), with varying message levels in different cardiac regions.^8–11 Heterologous expression of these channel subunits results in a hyperpolarization-activated inward current with similar although not identical properties compared with native I_f. This led to the hypothesis that HCN genes might be the molecular correlate of the native pacemaker current. By functional interference, it has been proposed that the isoforms HCN1 and HCN2 coassemble to form heteromeric channel complexes like voltage-gated K⁺ channels.^12,13 Very recently, coassembly between these 2 subunits has been shown more directly by use of the yeast 2-hybrid system¹⁴ and a dominant-negative construct.¹⁵ Detailed understanding of the molecular structure and function of the pacemaker channel is critical to any future therapeutic modulation of this current in myocardium.

Because the links between HCN channels and cardiac I_f are still largely inferential, we sought to confirm HCN as a critical molecular component of I_f by overexpressing an HCN construct carrying a dominant-negative mutation in cardiomyocytes via viral gene transfer and looking for changes in native I_f. Second, we aimed to determine the functional relevance of I_f in spontaneously beating neonatal cardiomyocytes. We generated a GYG_402–404AYA pore mutation of HCN2, which not only rendered the channel nonfunctional but also suppressed wild-type HCN2 and wild-type HCN4 in a dominant-negative manner. Overexpression of this channel construct in neonatal ventriculocytes markedly reduced native I_f and undermined spontaneous beating activity. Our results demonstrate the critical role of HCN channels in I_f current and in automaticity of neonatal cardiomyocytes.

	Methods

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Plasmid Construction and Adenovirus Preparation
The adenovirus shuttle vectors pAdEGI, pAdCGI, and pAdC-DBEcR and the expression plasmid pCGI-Kv1.3AYA have been described.^16,17 The full-length coding sequence of mHCN2 was cloned into the multiple cloning sites of pAdCGI and pAdEGI to generate pAdCGI-HCN2 and pAdEGI-HCN2, respectively. The point mutation AYA was introduced into the pore region (GYG_402–404) of HCN2 by site-directed mutagenesis, creating the vectors pAdCGI-HCN2AYA and pAdEGI-HCN2AYA, respectively. The full-length coding sequence of hHCN4 was cloned into the multiple cloning sites of pAdCGI and pAdEGI to give pAdCGI-HCN4 and pAdEGI-HCN4, respectively. Adenovirus vectors were generated as previously described.^17–19

Transient Transfections
Twenty-four hours before transfection, Chinese hamster ovary (CHO)-K1 cells (ATCC CCL 61, American Type Culture Collection) were seeded at a density of 2.0x10⁵ per 35 mm. Cells were cotransfected with 0.5 µg/well plasmid DNA of wild-type channels (as indicated) and 1 µg/well plasmid DNA of mutant channels (as indicated) with Lipofectamine Plus (Life Technologies) as directed by the manufacturer. After 4 hours, transfection media were replaced with normal growth media.

Myocyte Isolation and Adenovirus Infection
A standard trypsin dissociation method was used to prepare ventricular myocytes of 1- to 2-day-old neonatal rats.²⁰ Action potential studies were conducted on 4- to 6-day-old monolayer cultures. For voltage-clamp experiments, 3- to 5-day-old monolayer cultures were dispersed by trypsin and replated at a low density to study isolated cells within 2 to 8 hours. Infection of neonatal cells was performed 1 to 3 days after plating at a multiplicity of infection of 15 to 100 pfu/cell. Cells were incubated for 4 hours at 37°C, after which the infection medium was replaced with culture medium. Expression was induced by addition of ponasterone A 10 µmol/L (Invitrogen) for 36 to 60 hours.

Electrophysiology
Experiments were carried out with the use of standard microelectrode whole-cell patch-clamp techniques^16,21 with an Axopatch 200B amplifier (Axon Instruments) while sampling at 10 kHz and filtering at 2 kHz. Current recordings were performed at room temperature (21°C to 23°C), and action potential measurements were done at 36±0.5°C. The recording bath solution contained (in mmol/L) NaCl 135, KCl 5, CaCl₂ 2, glucose 10, MgCl₂ 1, HEPES 10; pH was adjusted to 7.4 with NaOH. For I_f recordings of cardiomyocytes, [K⁺]_o was increased to 100 mmol/L, and BaCl₂ 2 mmol/L, CdCl₂ 200 µmol/L, and 4-aminopyridine 4 mmol/L were added to block I_K1, I_CaL, and I_to, respectively. The micropipette electrode solution was composed of (in mmol/L): K-glutamate 130, KCl 15, NaCl 5, MgCl₂ 1, HEPES 10, and Mg-ATP 5; pH was adjusted to 7.3 with KOH. Borosilicate microelectrodes had tip resistances of 2 to 4 M when filled with the internal recording solution.

I_f/I_HCN size was measured as the difference between the instantaneous current at the beginning of a hyperpolarizing step ranging from -50 to -150 mV in 10-mV increments and the steady-state current at the end of hyperpolarization for 2.45 to 3 sec, as previously described.² Fast-current inactivation was achieved by a depolarization pulse to 20 mV. For the calculation of activation curves, specific current conductances were normalized to the maximal current conductance to give g/g_max. Boltzmann distributions were fitted to these normalized values: g/g_max=1/{1+exp[(V_1/2-V_m)/S]}, where V_m is the membrane voltage, V_1/2 is the voltage at half-maximal activation, and S is a slope factor at V_m=V_1/2. In HCN2-AYA–infected myocytes, action potentials were initiated by short depolarizing current pulses (2 ms, 500 to 800 pA). A xenon arc lamp was used to view enhanced green fluorescent protein (EGFP) at 488/530 nm (excitation/emission). Pooled data are presented as mean±SEM. Comparisons between groups were performed with 1-way ANOVA. Probability values of P<0.05 were deemed significant.

	Results

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HCN2-AYA Suppresses Wild-Type HCN2 and Wild-Type HCN4 in a Dominant-Negative Manner
Various mutant K⁺ channels have been reported to exert a dominant-negative effect when coassembling with wild-type subunits.^16,17 In analogy to the structure of K⁺ channels, we speculated that the double mutation AYA in the pore region (GYG_402–404) of HCN2 would alter channel function and would result in a dominant-negative ion channel construct. To test this hypothesis, CHO cells were cotransfected with wild-type HCN2/4 and HCN2-AYA, with wild-type HCN2/4 and a nonfunctional unrelated Kv1.3 construct carrying a similar pore mutation (Kv1.3-AYA, to exclude any unspecific effects of HCN2-AYA), or with HCN2-AYA and Kv1.3-AYA. All vectors were bicystronic, also expressing EGFP under control of a single CMV promoter for easy identification of transfected cells.

Overexpression of HCN2 and HCN4 resulted in time- and voltage-dependent inward currents on hyperpolarization, consistent with previous reports of heterologous expression of these ion channel subunits (Figure 1, A and B).^8,9 Mean current densities at -130 mV were 33.8±4.2 pA/pF (n=16) and 21.3±2.5 pA/pF (n=14) for I_HCN2 and I_HCN4, respectively. V_1/2 for I_HCN2 and I_HCN4 was -82.6±1.0 and -98.8±1.1 mV, respectively, and the slope factor S was 10.8±0.6 and 11.5±1.0 mV, respectively. In contrast, expression of HCN2-AYA did not yield any measurable hyperpolarization-activated inward current (n=9) (Figure 1C). This indicated that the pore mutation AYA completely undermined HCN2 channel function, which is in agreement with a very recently reported pore mutation of HCN1.¹⁵

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of HCN2-AYA on wild-type HCN2 and wild-type HCN4 heterologously expressed in CHO cells. Overexpression of HCN2 (A) and HCN4 (B) resulted in time- and voltage-dependent inward currents on hyperpolarization. C, Conversely, expression of HCN2-AYA did not yield any measurable hyperpolarization-activated inward current. Representative current recordings (D) and mean current densities measured at -130 mV (F) show that HCN2-AYA significantly decreased IHCN2 in a dominant-negative manner when mutant was coexpressed with wild-type HCN2 channels. In addition, HCN2-AYA suppressed IHCN4 in a dominant-negative manner when coexpressed with wild-type HCN4, as illustrated by original current recordings (E) and mean current densities measured at -130 mV (G).

To probe the effect of HCN2-AYA on wild-type HCN2, both channel constructs were coexpressed. Representative current recordings (Figure 1D) and mean current density (3.3±0.9 pA/pF at -130 mV, n=11; Figure 1F) show that I_HCN2 was significantly decreased by HCN2-AYA (P<0.001), confirming the dominant-negative properties of the HCN2 pore mutation. In addition, HCN2-AYA suppressed I_HCN4 in a dominant-negative manner when coexpressed with wild-type HCN4 (mean current density at -130 mV, 14.2±1.8 pA/pF, n=21, P=0.025) (Figure 1, E and G). This indicated that the 2 isoforms HCN2 and HCN4 are also able to coassemble to form heteromultimeric complexes.

HCN2-AYA Suppresses Native I_f in Neonatal Cardiomyocytes
HCN genes are thought to underlie the pacemaker current I_f. In neonatal rat ventricular myocytes, the dominant HCN mRNA transcripts are HCN2 and HCN4.¹⁰ Considering our observations in CHO cells, overexpression of a construct carrying HCN2-AYA should reduce or eliminate native I_f if HCN channels are indeed the major molecular components of this cardiac current. Therefore, I_f current recordings were performed in neonatal control ventriculocytes and in myocytes expressing HCN2-AYA. For increased expression efficiency, viral gene transfer techniques were used to deliver our genes of interest with bicystronic adenoviral vectors, which express the HCN gene and EGFP under the control of an ecdysone-inducible promoter.²² Ecdysone responsiveness was conferred by coinfection with the receptor virus AdC-DBEcR and stimulation with the ecdysone analogue ponasterone A.¹⁶ I_f density was indeed reduced markedly, from 7.8±1.6 pA/pF (n=13) in control cells to 0.3±0.2 pA/pF (n=11) in HCN2-AYA–infected cells, when measured at -130 mV (P<0.001) (Figure 2, A through C). These results confirm that HCN channels are a critical component of native I_f.

View larger version (18K): [in this window] [in a new window]   Figure 2. HCN2-AYA suppressed native If in neonatal cardiomyocytes, whereas expression of HCN2 and HCN4 resulted in a robust hyperpolarization-activated inward current. Representative original current traces of native If recorded in a noninfected neonatal myocyte (A) and in an HCN2-AYA–infected cell (B) demonstrate dominant-negative effect of HCN2-AYA on native If. Mean If current density measured at -130 mV was almost completely suppressed by HCN2-AYA compared with noninfected cells (C). Conversely, infection with HCN2 (D) and HCN4 (E) resulted in high current densities of IHCN2 and IHCN4 in neonatal cardiomyocytes. Pulse protocol identical to Figure 1.

HCN Channels Are Crucial for Spontaneous Beating of Neonatal Myocytes
In recent years, it has been controversial whether I_f is the major current responsible for spontaneous diastolic depolarization of pacemaker cells and neonatal tissue.^1,5 Given our results that HCN channels are the dominant molecular component of I_f, we therefore probed the functional effect of HCN channel overexpression and dominant-negative suppression on spontaneous beating activity of monolayer cultures of neonatal cardiocytes.

Control cultures beat spontaneously with a mean rate of 83.4±4.5 bpm (n=13). Cycle length tended to vary from beat to beat (Figure 3A). Maximal diastolic potential (MDP) was -63.6±3.2 mV. Infection of neonatal cells with the adenoviral vectors AdEGI-HCN2 and AdEGI-HCN4 resulted in high HCN current levels (Figure 2, D and E). Mean current densities of I_HCN2 and I_HCN4 at -130 mV were 32.9±5.1 pA/pF (n=11) and 25.1±4.5 pA/pF (n=8), respectively. Enhanced expression of HCN2 and HCN4 accelerated beating frequency to 230.5±8.6 bpm (n=12, P<0.001 versus control) and 223.5±12.3 bpm (n=10, P<0.001 versus control), respectively, and resulted in a more regular rhythm and marked diastolic depolarization (Figure 3, B, C, and E). Consistent with a very recent report of HCN2 overexpression,²⁰ we observed a reduction of MDP in HCN2-infected (-45.4±2.2 mV) and HCN4-infected (-48.7±1.6 mV) cells compared with controls (P<0.01) (Figure 3F). Conversely, HCN2-AYA–expressing cultures did not generate any spontaneous beating activity despite a similar MDP (-59.3±2.8 mV, n=11) versus control cultures (P=NS) (Figure 3, E and F). Figure 3D illustrates a representative recording of an action potential that was induced artificially by a short depolarizing current pulse in an HCN2-AYA–infected myocyte. Diastolic potentials in some of these cells were unstable, exhibiting irregular minor depolarizations, which, however, were not sufficient to reach threshold and initiate spontaneous action potentials (Figure 3D). These observations indicate a critical role of HCN channels for automaticity of neonatal cardiomyocytes.

View larger version (21K): [in this window] [in a new window]   Figure 3. Effect of HCN overexpression and dominant-negative suppression on spontaneous activity of neonatal ventricle cultures. Representative original recordings of spontaneous action potentials (A through C) and mean data (E, F) illustrate that overexpression of HCN2 and HCN4 significantly accelerated beating rate, resulted in a more regular rhythm, and reduced MDP compared with control monolayer cultures. Conversely, HCN2-AYA infection undermined spontaneous beating activity (E) despite an unchanged MDP compared with control (F). (D) In some HCN2-AYA–infected cells, action potentials artificially induced by a depolarizing pulse were followed by an unstable diastolic potential with minor depolarizations, which, however, were not sufficient to reach threshold and to initiate spontaneous action potentials.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Overexpression of dominant-negative constructs in native cells has been a useful tool to dissect the contributions of various ion channel genes and different native ionic currents to excitability and repolarization.^16,17,19 In the present study, we used this strategy to confirm for the first time the identity of HCN genes as the major molecular component of native cardiac I_f by introducing a dominant-negative HCN construct into cardiomyocytes to cripple I_f. Furthermore, our results clearly indicate that HCN channels are critical for the generation of spontaneous action potentials in neonatal cardiomyocytes.

Similar to selective K⁺ channels, HCN channels contain a GYG pore motif, despite their permeability for both Na⁺ and K⁺.^8,9 We mutated this pore region to generate an HCN2-AYA construct. This mutation rendered the ion channel completely nonfunctional, consistent with a very recent report of an HCN1 pore mutation that also disrupted channel function.¹⁵ This indicates that in HCN channels, the GYG pore motif has a critical role for permeation properties comparable to that in K⁺ channels. In heterologous expression, HCN2-AYA not only suppressed wild-type HCN2 current, which confirmed its dominant-negative effect in homomeric complexes but also reduced wild-type HCN4 current in a dominant-negative manner. These results indicate that HCN2 and HCN4 can coassemble to form heteromeric complexes. Thus far, heteromultimerization has been proposed only for the isoforms HCN1 and HCN2. Tandem heterodimers and coexpression of HCN1 and HCN2 yielded intermediate electrophysiological phenotypes that cannot be explained by a simple addition of individual isoform properties at any population proportional ratio.^12,13 More recently, interaction of these 2 subunits has been suggested in use of a dominant-negative construct and the 2-yeast hybrid system.^14,15 Our observations now extend evidence that different HCN isoforms can coassemble and may suffice to explain the regional diversity of pacemaker current in cardiac tissue.

In neonatal ventricle, HCN2 and HCN4 are the dominant HCN isoforms on the basis of RNase protection.¹⁰ Because in heterologous expression, our HCN2-AYA vector was able to suppress both HCN2 and HCN4 wild-type channels in a dominant-negative manner, this channel construct was considered useful to test the effect of HCN suppression on native I_f current in neonatal cardiomyocytes. Indeed, we could demonstrate almost complete reduction of native I_f after infection with HCN2-AYA, indicating a dominant-negative suppression of native HCN channels in neonatal cells. This demonstrates that HCN subunits are the major determinants of cardiac I_f. The remaining minor I_f current recorded in HCN2-AYA–expressing cells can be attributed to incomplete elimination of I_f because of some functional homotetramers that are made up of only wild-type subunits. Alternatively, incomplete elimination may result from competition between expression of suppressive gene products and turnover of preexisting functional channel proteins.

There has been a great deal of controversy about the functional contribution of I_f to cardiac pacing. Some authors speculate that I_f is essential for spontaneous diastolic depolarization, whereas others postulate that I_f only modulates the beating frequency of autonomic cardiac cells.^4–7,23 Overexpression of HCN2 and HCN4 markedly accelerated the spontaneous beating rate of neonatal cardiomyocyte monolayers, demonstrating the ability of HCN channels to modify pacing frequency. More importantly, however, HCN channels and native I_f were found to be critical for spontaneous action potential generation revealed by HCN/I_f "knockout" in HCN2-AYA–infected cultures. The diastolic potential of some HCN2-AYA–expressing cells was unstable, exhibiting minor depolarizations. These irregular depolarizations might have been a result of the small remaining I_f current, which was not sufficient to drive the potential toward threshold or might reflect the balance of other ionic currents (ie, I_K, I_Ca) involved in pacemaker activity.^4,5,23

Given the obvious importance of HCN channels in cardiac pacing, it seems plausible that mutations in HCN genes might cause bradycardia or sick sinus syndrome in patients, although our observations in neonatal cardiomyocytes cannot readily be generalized to sinus node cells. In any event, our HCN constructs may prove useful to further determine the physiological role of I_f and HCN channels for cardiac pacing in the sinoatrial node and for arrhythmogenesis in diseased adult ventricular myocardium.^1–3 Detailed molecular insight into the contribution of this diastolic current to cardiac pacing might facilitate the rational design of blockers or activator drugs and of gene therapy strategies to modify cardiac automaticity.

	Acknowledgments

 
This study was supported by a grant from the Bundesministerium für Bildung und Forschung (ZMMK TV74), by a grant from the Deutsche Forschungsgemeinschaft (Ho 2146/2-1), and by the Marga und Walter Boll-Stiftung. We thank Nadine Henn for skillful technical assistance.

Received August 26, 2002; revision received October 8, 2002; accepted October 8, 2002.

	References

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