Cardiovascular Research

Cardiovascular Research 2005 66(3):472-481; doi:10.1016/j.cardiores.2005.02.011

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Copyright © 2005, European Society of Cardiology

Sinus node dysfunction and hyperpolarization-activated (HCN) channel subunit remodeling in a canine heart failure model

Stephen Zicha^a,b, María Fernández-Velasco^c, Giuseppe Lonardo^a, Nathalie L'Heureux^a and Stanley Nattel^a,b,*

^aDepartments of Medicine and Research Center, Montreal Heart Institute and University of Montreal, 5000 Belanger St E, Montreal, Quebec, Canada H1T 1C8
^bDepartment of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada
^cUniversidad Complutense, Madrid, Spain

^* Corresponding author. Departments of Medicine and Research Center, Montreal Heart Institute and University of Montreal, 5000 Belanger St E, Montreal, Quebec, H1T 1C8, Canada. Tel.: +1 514 376 3330x3990; fax: +1 514 376 1355. Email address: stanley.nattel{at}icm-mhi.org

Received 22 December 2004; revised 9 February 2005; accepted 16 February 2005

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions Acknowledgments References

 
Background: The hyperpolarization-activated cation current I_f contributes significantly to sinoatrial node pacemaker function and possibly to ectopic arrhythmogenesis. Little is known about the expression of corresponding hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits in normal hearts and HCN remodeling by diseases, like congestive heart failure (CHF), associated with disturbances of cardiac rhythm.

Methods and results: We assessed expression of HCN1, 2 and 4 in normal mongrel dogs and dogs subjected to 2-week ventricular tachypacing-induced CHF. Competitive RT-PCR, Western blot and immunohistochemistry were used to quantify HCN subunit mRNA and protein expression in the right atrium (RA) and sinoatrial node. CHF approximately doubled sinus node recovery time, indicating suppressed sinus node pacemaker function. HCN expression under control conditions was HCN4>HCN2>>HCN1. HCN2 and HCN4 expression was greater at both protein and mRNA levels in sinoatrial node than RA. CHF significantly decreased sinus node HCN expression at both mRNA and protein levels (HCN2 by 78% and 82%; HCN4 by 42% and 77%, respectively). RA HCN2 expression was unaltered by CHF, but HCN4 was significantly upregulated (by 209%).

Conclusions: HCN4 is the dominant subunit in canine sinoatrial node and RA; strong sinus node HCN expression likely contributes to its pacemaker function; downregulation of HCN4 and HCN2 expression contribute to CHF-induced sinus node dysfunction; and upregulation of atrial HCN4 may help to promote atrial arrhythmia formation. These findings provide novel information about the molecular basis of normal and disease-related impairments of cardiac impulse formation.

KEYWORDS Pacemaker current; Sinoatrial node; Congestive heart failure; Ion channels; Remodeling

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This article is referred to in the Editorial by W.R. Giles (pages 430–432) in this issue.

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions Acknowledgments References

 
Normal cardiac rhythm is controlled by the activity of the sinoatrial node (SAN). The hyperpolarization-activated cation current, known as I_f (based on its initial historical appellation of "funny current"), contributes significantly to phase 4 diastolic depolarization and is particularly prominent in the SAN [1–4]. I_f is a voltage-gated current with many unique features, including an ability to carry both K⁺ and Na⁺ with a reversal potential around –20 mV [2], and activation by polarization to negative voltages [3,4]. Sympathetic nervous system stimulation accelerates cardiac rate via β-adrenergic increases in intracellular cAMP, which shift the voltage-dependence of I_f activation to more positive potentials [5,6]. Increases in I_f can lead to enhanced automaticity and ectopic rhythm formation.

Despite the importance of I_f in cardiac electrophysiology, its molecular composition was only recently elucidated [7]. The family of voltage-gated HCN subunits, including HCN1-4, encodes I_f in many excitable tissues. Only HCN1 [8], HCN2 [9] and HCN4 [10] are expressed in the heart. Few studies have examined the expression profiles of these subunits in the SAN and other regions of the heart [8,10–16]. Furthermore, most of these studies were performed in rodents, which may have different ion-channel subunit dependence compared to larger mammals.

Congestive heart failure (CHF) is a common cause of sudden arrhythmic death and is an important risk factor for atrial fibrillation (AF) [17]. Abnormalities in SAN function are common in CHF and may contribute to bradyarrhythmic death [17]. CHF impairs single sinus-node cell automaticity by downregulating I_f [18]. There is no information available about CHF-induced remodeling of the HCN subunits underlying I_f.

This study examined the expression profiles of HCN subunits in the SAN and right atrium (RA) of dogs and their modification by CHF.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions Acknowledgments References

 
2.1. CHF model
Seven dogs were outfitted with custom-modified pacemakers (Medtronic) as previously reported [19]. Briefly, pacemakers were inserted in subcutaneous pockets in the necks and attached to pacing leads inserted into the right ventricular apex under fluoroscopy. After 24 h for recovery, ventricular tachypacing was initiated at 240 bpm and maintained for 2 weeks. The dogs were then confirmed to have CHF based on clinical signs and hemodynamic findings, sinus-node function was assessed under morphine (2 mg/kg s.c.) and -chloralose (100 mg/kg IV) anesthesia, and then dogs were euthanized with an -chloralose overdose. The hearts were removed and the RA free wall and SAN isolated. The SAN was identified as a whitish endocardial region near the junction of the RA free wall and the atrial appendage near the crista terminalis, with subsequent histological confirmation. The RA free wall and SAN were removed (avoiding epicardial arteries), cleaned in Tyrode solution containing (mmol/L): NaCl 136, KCl 5.4, MgCl₂ 1, CaCl₂ 2, NaH₂PO₄ 0.33, HEPES 5 and dextrose 10, pH 7.35 (NaOH), then flash-frozen in liquid-N₂ and stored at –80 °C. Seven non-paced dogs served as controls. All animal handling procedures adhered to the guidelines of the Canadian Council on Animal Care, were approved by the Montreal Heart Institute Animal Research Ethics Committee and conformed to the guidelines of the National Institutes of Health.

2.2. Sinus-node recovery time changes
Bipolar pacing and recording hook electrodes were inserted into the RA appendage. The baseline cardiac rate (assumed to represent sinus-node rate when standard ECG criteria for sinus rhythm were met) was measured before and after suppression of vagal and β-adrenergic influences by administering nadolol (0.5 mg/kg IV) and severing the vagus nerves in the neck. The RA was then paced at a cycle length of 250 or 300 ms for 30 s. The post-pacing interval until the first sinus escape beat was recorded and subtracted from the pre-pacing spontaneous cycle length to obtain the corrected sinus-node recovery time (SNRT_C).

2.3. RNA isolation
RNA was isolated from 0.1 to 1.0 g samples using Trizol reagent (Invitrogen) followed by chloroform extraction and isopropanol precipitation. Genomic DNA was eliminated by incubating in DNase I (0.1 U/µL, 37 °C) for 30 min followed by acid-phenol-chloroform extraction. RNA was quantified by spectrophotometric absorbency at 260 nm, purity confirmed by A₂₆₀/A₂₈₀ ratio and integrity evaluated by ethidium bromide staining on a denaturing agarose gel. RNA samples were stored at –80 °C in RNAsecure Resuspension Solution (Ambion).

2.4. PCR primers and the synthesis of the RNA mimic
Gene-specific primers (GSPs) for competitive RT-PCR were designed based on previously-published cDNA sequences for HCN1, HCN2 and HCN4, with specificity confirmed with BLAST and FASTA (Table 1). Resulting PCR products were sequenced to ensure subunit-specificity. The canine-specific sequences obtained from cardiac mRNA are registered in GenBank as AY686750 [GenBank] (HCN2) and AY686751 [GenBank] (HCN4). Chimeric primer pairs for RNA mimic synthesis were constructed with rabbit cardiac -actin sequences flanked by the same GSPs. An 8-nucleotide sequence, GGCCGCGG, corresponding to the 3'-end of the T7 promoter, was conjugated to the 5'-end of each forward primer. First-strand cDNA (synthesized by reverse transcription with canine ventricular mRNA samples) was used as a template for subsequent PCR amplification steps with chimeric primer pairs. The resulting cDNA-mimic contained a 460-bp -actin sequence flanked at the 5'-end by the sense GSP sequence and an 8-bp T7 promoter sequence at the 3'-end flanking the antisense GSP sequence. Products were gel-purified with the QIAquick Gel Extraction Kit (Qiagen). The RNA-mimic (internal standard) was created by in vitro transcription (mMESSAGE MACHINE, Ambion). The product was incubated with RNase-free DNase I (30 min, 37 °C) to eliminate cDNA contamination, followed by phenol/chloroform extraction and isopropanol precipitation. Mimic size and concentration were determined by migration on a denaturing RNA gel alongside markers of known molecular weight and pre-determined RNA concentrations to create a standard curve.

View this table: [in this window] [in a new window]   Table 1 Primer sequences used for competitive RT-PCR

 
2.5. Competitive RT-PCR
RNA-mimic samples were added with serial 10-fold dilutions to reaction mixtures containing 1-µg sample RNA. RNA was denatured at 65 °C (15 min). RT was conducted in a 20-µL reaction mixture containing reaction buffer (10 mmol/L Tris–HCl, pH 8.3, 50 mmol/L KCl), 2.5 mmol/L MgCl₂, 1 mmol/L dNTPs (Roche), 3.2 µg random primers p(dN)₆ (Roche), 5 mmol/L DTT, 50 U RNase inhibitor (Promega), and 200 U M-MLV reverse transcriptase (Gibco-BRL). First-strand cDNAs were synthesized at 42 °C (1 h) and remaining enzymes heat-deactivated (99 °C, 5 min).

First-strand cDNA from the RT step was used as a template in 25-µL reaction mixtures including 10 mmol/L Tris–HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 1 mmol/L dNTPs, 0.5 µmol/L GSPs, 0.625 mmol/L DMSO and 2.5 U of Taq Polymerase (Gibco-BRL). Reactions were hot-started at 93 °C for 3 min of denaturing, followed by 30 amplification cycles (93 °C, 30 s [denaturing]; 55 °C, 30 s [annealing]; 72 °C, 30 s [extension]). A final 72 °C extension step was performed for 5 min. RT-negative controls were obtained for all RT-PCR reactions to exclude genomic contamination.

PCR products were visualized under UV light with ethidium-bromide staining in 1.5% agarose gels. Images were captured with a Nighthawk camera, and band intensity determined with Quantity One software. A DNA Mass Marker (100 ng) was used to determine the size and quantity of DNA bands, and to create standard curves in each experiment for absolute quantification.

2.6. Western blot studies
Membrane protein was extracted from tissue samples with 5-mmol/L Tris–HCl (pH 7.4), 2-mmol/L EDTA, 5-µg/mL leupeptin, 10-µg/mL benzamidine, and 5-µg/mL soybean trypsin inhibitor, followed by tissue homogenization. All procedures were performed at 4 °C. Membrane proteins were fractionated on 8% SDS-polyacrylamide gels and transferred electrophoretically to Immobilon-P polyvinylidene fluoride membranes (Millipore) in 25-mmol/L Tris-base, 192-mmol/L glycine and 5% methanol at 0.09 mA for 18 h. Membranes were blocked in 5% non-fat dry milk (Bio-Rad) in TTBS (Tris–HCl 50-mmol/L, NaCl 500-mmol/L; pH 7.5, 0.05% Tween-20) for 2 h (room temperature) and then incubated with primary antibody (1:500 dilution) in 5% non-fat dry milk in TTBS for 4 h at room temperature. All antibodies were purchased from Alomone Labs: HCN2 catalogue #APC-030 and HCN4 catalogue #APC-052. Membranes were washed 3 times in TTBS, reblocked in 5% non-fat dry milk in TTBS (15 min) and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5000) in 5% non-fat dry milk in TTBS (40 min). They were subsequently washed 3 times in TTBS and once in TBS (same as TTBS but without Tween-20). Signals were obtained with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). Band densities were determined with a laser-scanning densitometer (PDI 420oe) and Quantity One software (PDI). Protein loading was controlled by probing all Western blots with antiGAPDH antibody (Research Diagnostics Incorporated) and normalizing ionchannel proteinband intensity to that of GAPDH. Blots with antibody preincubated with antigenic peptide served as negative controls.

2.7. Immunohistochemistry
Tissue samples were obtained in the same manner as described above, placed in 2-methylbutane (Sigma) which was pre-cooled to –80 °C, and then stored before sectioning. Samples were prepared for cryosectioning by covering with Optimal Cutting Temperature (OCT) embedding medium. Serial 14-µm sections were cut with a Leica CM1900 cryostat at –20 °C and then allowed to dry on slides before storage at –80 °C. Tissue sections were fixed in a 4% paraformaldehyde solution (pH 7.2) before permeabilization and blocking in 2% NDS and 0.5% Triton for 1 h at room temperature. Primary antibodies (HCN2, HCN4 same as for Western blotting, connexin 43 from Research Diagnostics Inc.) were diluted 1:100 in 1% NDS, 0.1% Triton and incubated overnight at 4 °C. Tissue sections were then washed three times with 1 x PBS before incubation with the fluorochrome-conjugated secondary antibody (Alexa Fluor 555 donkey anti-mouse for Connexin 43, Alexa Fluor 647 donkey anti-rabbit for HCN, Molecular Probes) in 1% normal donkey serum (NDS) and 0.1% Triton for 1 h at room temperature in the dark. Slides were washed again before mounting using 0.2% DABCO in glycerol and coverslip (22–25 mm², thickness #1), and sealed with nail polish.

2.8. Data analysis
All data are expressed as mean ± SEM. Each biochemical determination was performed on an individual heart: n-values represent the number of hearts studied. Western-blot band intensities are expressed as OD units corresponding to densitometric band-intensity following background subtraction, divided by GAPDH-signal intensity for the same sample. HCN-subunit mRNA levels are expressed as absolute concentrations (attomol/µg total RNA). Statistical comparisons were performed with ANOVA and Student's t-test with Bonferroni's correction. A 2-tailed p<0.05 was taken to indicate statistical significance.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions Acknowledgments References

 
3.1. SAN recovery time and in vivo measurements
The mean sinus cycle length was shorter in CHF dogs than controls prior to vagotomy and nadolol, reflecting a faster baseline heart rate (Table 2); however, this was likely due to enhanced sympathetic and or reduced vagal tone in CHF, because after vagotomy and nadolol administration the sinus cycle length was longer (heart rate slower) in CHF, indicating reduced intrinsic sinus-node automaticity. Fig. 1A and B show RA-electrogram recordings during the protocol used to calculate the SNRT_c. The left panels show baseline measurements before the start of RA-appendage overdrive pacing. The right panel shows the recording just before the end of tachypacing, as well as the delay to the first spontaneous post-pacing beat. The mean data in Fig. 1C demonstrate that the SNRTc more than doubled in CHF hearts compared to control hearts (n=7/group, p<0.05). The QTc interval was prolonged in CHF dogs (Table 2), compatible with known effects on repolarizing currents and action potential duration [17].

View this table: [in this window] [in a new window]   Table 2 In vivo measurements

 

View larger version (36K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 SNRT measurements in the canine model of CHF. (A) Atrial-electrogram recordings from a control (CTL) dog. Baseline sinus-node cycle-length measurements were taken before pacing-onset at a cycle length of 300 ms ("pacing start"). The right panel shows the last paced cycles before pacing was stopped ("pacing end"). The SNRT was the first post-pacing sinus-node interval. The corrected SNRT (SNRTc) was calculated as SNRT minus the pre-pacing sinus-node cycle length. (B) Representative atrial-electrogram recordings from a CHF dog. Same format as (A). (C) Mean ± SEM SNRTc at 250 and 300 ms pacing cycle lengths.

 
3.2. HCN-subunit mRNA measurements
While HCN2 and HCN4 mRNA were readily detected by RT-PCR in canine RA and sinus-node tissue, HCN1 was not detected in either region (Fig. 2, arrow indicates expected size). In contrast, HCN1 was readily detected in dog brain (Fig. 2, lanes 5 and 6). Fig. 3A shows representative competitive RT-PCR gels for HCN2 on control RA and sinus-node samples. For all gels, lane 0 contains the DNA Mass Ladder used to create the standard curve for the absolute quantification of PCR products. Lanes 1 through 5 contain 2 ng, 200 pg, 20 pg, 2 pg, and 0.2 pg of mimic RNA, respectively. The competition between mimic and target PCR products can be seen as the intensity of the mimic decreases while that of the target increases from left to right. The point of mimic-target equality reflects the sample mRNA concentration and is quantified based on graphs of ln([target]/[mimic]) versus ln([mimic]) as previously described [20], with linear regression used to determine the absolute concentration of target mRNA. The approximate point of identity is evident from the gels, and for control SAN was to the left of RA, indicating larger concentrations. Fig. 3B shows representative RT-PCR gels on CHF samples. Compared to panel A, the point of target-mimic identity for RA is unchanged but that for SAN has moved to the right, indicating down-regulation. Under control conditions, the HCN2 mRNA concentration in the SAN (7.6 ± 1.8 attomol/µg total RNA) is over 6 times larger than that in the RA (1.2 ± 0.5, n=7/group, p<0.05, Fig. 3C). CHF does not alter the RA HCN2 mRNA concentration (1.1 ± 0.3 attomol/µg total RNA), but decreases sinus-node HCN2 expression to <1/3 of control values (2.7 ± 1.5, n=6/group, P<0.05).

View larger version (52K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Standard RT-PCR to identify HCN1 transcripts in dog samples. No HCN1 was detected in cardiac samples (first 4 lanes). The primers generated products of the expected size from brain (lanes 5–6). The last 2 lanes are reverse transcriptase-negative controls.

 

View larger version (40K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 HCN2 competitive RT-PCR. (A) Representative gels from competitive RT-PCR on control (CTL) RA and sinus-node samples. (B) Competitive RT-PCR with CHF RA (upper panel) and sinus-node (lower panel) tissue. (C) Mean ± SEM HCN2 subunit expression.

 
HCN4-subunits were more abundant than HCN2 in all regions, averaging ~14 times greater concentration in control RA and ~4 times greater in control SAN. Fig. 4A shows examples of HCN4 competitive RT-PCR in control samples. The concentrations of mimic used in all HCN4 gels were: (from left to right) 2 ng, 200 pg, 20 pg, 2 pg and 0.2 pg. Representative gels for CHF are shown in Fig. 4B. The point of target-mimic identity for SAN is to the left of that for RA in control, indicating greater HCN4 expression. In the presence of CHF, the identity point moves to the left for RA but to the right for sinus-node tissue, indicating RA HCN4 up-regulation and sinus-node down-regulation respectively. The mean data in Fig. 4C show that mean sinus-node HCN4 concentrations (29.1 ± 5.0 attomol/µg total RNA) were about twice those in RA (16.8 ± 3.0, n=7/group, p<0.05). CHF increased RA HCN4 mRNA expression several-fold to 112.3 ± 35.5 attomol/µg total RNA (n=5, p<0.05) while decreasing expression in the SAN to 5.1 ± 1.0 attomol/µg total RNA (p<0.05).

View larger version (41K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 HCN4 competitive RT-PCR. (A) Representative gels from competitive RT-PCR experiments for HCN4 in control. (B) Examples of HCN4 competitive RT-PCR for CHF samples. (C) Mean ± SEM HCN4 expression.

 
3.3. HCN-subunit protein expression
Fig. 5A shows representative Western blots from a membrane probed with anti-HCN2 antibody. The bands detected at the expected molecular mass (~50 kDa, arrow) disappeared when the primary antibody was pre-incubated with the antigen against which it was raised (+CA lanes). Corresponding GAPDH bands from the same samples are shown at the bottom. Overall, HCN2 protein was more strongly expressed in SAN (6.7 ± 1.5 arbitrary OD units, Fig. 5B) compared to RA (3.2 ± 0.9, n=6/group, p<0.05). CHF had no effect on RA HCN2 protein expression (2.5 ± 0.8), but significantly reduced SAN expression by >40%, to 3.9 ± 0.2.

View larger version (57K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Western-blot analyses. (A) Representative blot probed with anti-HCN2. Last 2 lanes: samples probed with primary antibody that had been pre-incubated with antigenic peptide. (B) Mean ± SEM HCN2 protein-expression data. (C) Representative Western blots obtained with anti-HCN4 antibody. A band at the expected molecular mass of 120 kDa was eliminated by antigen pre-incubation. A second, lower molecular-weight band was not suppressed by antigen pre-incubation, indicating that it was non-specific. Bottom: GAPDH signals on the same samples. (D) Mean ± SEM HCN4 protein expression data.

 
Representative anti-HCN4 blots are shown in Fig. 5C. The band detected at the expected molecular mass (~120 kDa, arrow) disappeared when antibody was pre-incubated with antigenic peptide. Overall, there was more than twice as much HCN4 protein in the SAN (0.29 ± 0.06 arbitrary OD units, Fig. 5D) compared to the RA (0.11 ± 0.03, n=6/group, p<0.05). CHF increased RA HCN4 expression ~2 fold (0.23 ± 0.05), but decreased SAN HCN4 expression by ~75%, to 0.07 ± 0.008 arbitrary OD units.

The identity of SAN tissue localization for immunoblotting was confirmed by histological analysis. However, as an additional confirmation of the localization of the HCN distribution and changes with CHF, immunohistochemistry was used to assess the relative expression patterns of HCN2 and HCN4 in RA and SAN (Figs. 6 and 7). Connexin 43 was used as negative marker for SAN since it has been demonstrated previously that this subunit is not expressed in the SAN [21]. In both Figs. 6 and 7, the upper left panel shows myocardial staining with an anti-connexin 43 antibody and the lower left with an anti-HCN subunit antibody, while the lower right panel shows these two images superimposed. A dark field image is presented in the upper right panel. In control tissues, HCN2 expression is greatest in the SAN regions, devoid of connexin 43 staining (Fig. 6A). The difference between the HCN2 signal intensities in the RA and SAN is reduced under CHF conditions because of a decrease in the intensity of SAN HCN2 staining (Fig. 6B), consistent with the downregulation of HCN2 in the SAN and lack of change in the RA indicated by competitive RT-PCR and Western blotting results. Similarly, for control preparations the HCN4 protein expression signal is strongest in connexin 43-lacking SAN regions (Fig. 7A). For tissues from dogs with CHF, HCN4 signal intensity increased in regions with strong connexin 43 expression (RA), making them at least as strong as in connexin 43-lacking SAN tissues, in which HCN4 intensity appeared to decrease (Fig. 7B). These results confirm the observations obtained with Western blot and competitive RT-PCR techniques that HCN2 and 4 expression are stronger in SAN than in RA, that HCN2 and HCN4 down-regulate in the SAN with CHF, that HCN4 up-regulates in the RA with CHF and that the ratio of SAN to RA HCN4 expression is reduced by CHF.

View larger version (91K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Immunohistochemical analysis of HCN2 protein expression. (A) Representative immunohistochemistry of SAN/RA region using CTL tissue. Upper left: Tissue stained with anti-connexin 43 to identify SAN. Lower left: Same tissue double-stained with anti-HCN2. Lower right: Superimposed images. Upper right: Dark field image. (B) Similar organization of panels to (A), but for a CHF tissue. Similar results were seen in 3 preparations of each type.

 

View larger version (90K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Immunohistochemical analysis of HCN4 protein expression. (A) Immunohistochemical staining of SAN/RA region with CTL tissue. Layout as in Fig. 6A. (B) Similar to (A), but with tissue from a dog with CHF. Similar results were seen in 3 preparations of each type.

 

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions Acknowledgments References

 
In this study, we evaluated the expression of HCN subunits in the normal and remodeled SAN and RA. Our results indicate that HCN4 is the primary subunit, that HCN2 likely contributes as well, and that HCN1 is not expressed. Greater HCN subunit expression in the SAN is a candidate to contribute to its specialized pacemaking function. CHF remodels HCN subunit expression non-congruently in SAN versus RA, with SAN HCN down-regulation and RA up-regulation. SAN HCN downregulation in CHF may underlie previously-described I_f decreases in this condition that contribute to sinus-node dysfunction [3], and RA HCN upregulation possibly predisposes to ectopic impulse formation.

4.1. Comparison with previous studies of HCN expression
Since the first cloning of the HCN family subunits which underlie I_f [7,9–11,14], there have been relatively few studies examining their expression profiles. To our knowledge, this is the first study to compare the mRNA and protein expression of HCN subunits in the SAN and RA under normal and CHF conditions. In agreement with our findings, HCN1 was not detected in whole heart samples from humans [12]. However, HCN1 mRNA has been detected in the SAN of rabbits and mice [8,13,16]. The activation kinetics of human SAN I_f [4] more closely resemble those of HCN2 or HCN4 than HCN1 [8,10]. The possible participation of heteromeric HCN channels [22,23] complicates analysis of the relationship between native currents and currents carried by individual subunits. Our findings are consistent with mRNA evidence of HCN4 predominance in mouse [13] and rabbit [16] SAN. HCN4 mRNA is more strongly expressed in rabbit SAN than atrium [10]. We are not aware of studies of HCN expression changes in animal models of CHF. Gene microarray analysis points to HCN4 mRNA upregulation in the failing human ventricle [24].

4.2. Possible functional and clinical significance
Intrinsic sinus-node automaticity is impaired in rabbits with CHF [25], as well as in the dog in the present study. Recent clinical observations indicate similar changes in SAN function in humans with CHF [26]. Decreased I_f underlies CHF-induced reductions in sinus-node automaticity [18]. Reduced sinus-node automaticity may have a protective effect in CHF, at least in part by preventing delayed afterdepolarization-related triggered activity [17,25]. On the other hand, bradyarrhythmias contribute to sudden death in CHF [27], and may favor the induction of early afterdepolarization-related tachyarrhythmias caused by repolarization impairment due to outward-current downregulation [28,29]. In this study, we provide evidence which suggests that the molecular basis of sinus-node I_f impairment is reduced expression of HCN2 and HCN4 subunits. Biological pacemakers based on regional HCN-overexpression are a promising tool for inadequate cardiac rhythmicity [30], and may prove useful for CHF patients with clinically significant sinus-node dysfunction related to HCN-subunit downregulation.

Our study is the first of which we are aware of HCN subunit composition in the canine heart and the first of HCN subunit remodeling in CHF. Knockout studies in mice suggest that HCN2 is important in establishing the diastolic potential, whereas HCN4 constitutes the bulk (~80%) of I_f and contributes importantly to pacemaker function and its adrenergic regulation [31].

AF is a common and problematic condition, and present therapy is suboptimal [32]. CHF is one of the most common clinical causes of atrial tachyarrhythmias, particularly AF [33]. The basic mechanisms underlying AF are under active investigation and there is hope of developing novel mechanistically based therapeutic approaches [32]. Although reentrant mechanisms have traditionally been considered to underlie AF, recent evidence points to ectopic impulse formation as playing a potentially-important role [34]. Stambler et al. have described ectopic atrial tachyarrhythmias in dogs with CHF [35] and atrial HCN mRNA expression has been reported to correlate with left-atrial pressure, AF occurrence and atrial ectopic beat frequency in man [36]. Our study suggests atrial HCN4-upregulation as a potential molecular mechanism for enhanced atrial ectopic-beat formation in states associated with atrial dilation. It would be interesting to assess the potential value of HCN current inhibitors, such as zatebridine and ivabradine, in clinical and experimental atrial tachyarrhythmias associated with CHF. Caution might, however, be needed in the face of SAN HCN downregulation and the risk of excessive bradycardic responses.

4.3. Potential limitations
Although we confirmed the presence of SAN histologically in the corresponding tissue samples used for RT-PCR and Western blotting, it is impossible to obtain pure SAN tissue in such specimens and there is inevitably some RA tissue in the periphery. In addition, the SAN is a complex structure and there may be heterogeneity within it. The immunohistochemical studies are valuable in this regard in confirming the principal observations regarding HCN distribution directly in a histologically defined preparation.

SAN pacemaking function is complex and determined not only by the hyperpolarization-activated, time-dependent inward current carried by I_f, but also by the behaviour of a variety of other inward (like T- and L-type Ca²⁺ currents, I_CaT and I_CaL) and outward currents (like slow and rapid delayed-rectifier currents, I_Kr and I_Ks) [31]. We limited our analysis in the present study to HCN subunits that constitute I_f, based on strong evidence that I_f downregulation plays a key role in SAN dysfunction caused by CHF [18] and that intact HCN4 is essential for pacemaker action potentials in the embryonic heart [37]. A detailed analysis of all of the potential contributors to pacemaking in the normal and CHF canine SAN, including voltage clamp analyses of I_f, I_Ks, I_Kr, I_CaT and I_CaL and molecular studies of changes in underlying subunits like KCNQ1, KCNE1, KCNH2, KCNE2, CACNA1C, CACNA1D, CACNA1G, CACNA1H, CACN1I, CACNA2D2, CACNB1 and CACNB2 would be of interest but goes beyond the scope of the present study. CHF produces complex neuroendocrine changes, including alterations in atrial and brain natriuretic peptides, changes in adrenergic receptor number, function and stimulation, changed concentrations of arginine vasopressin, angiotensin-II and aldosterone, etc. [38]. Many of these could affect SAN function, but were not evaluated in the present study.

	5. Conclusions

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions Acknowledgments References

 
Regional HCN-subunit expression parallels and may serve to maintain physiological sinus-node pacemaker dominance in the dog. CHF-induced remodeling of HCN-subunit expression may be an important contributor to the abnormal sinus-node function and atrial dysrhythmias that typically occur in CHF. Therapies that target HCN expression and/or HCN-based currents may be an interesting approach to treating CHF-related dysrhythmias.

	Acknowledgments

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions Acknowledgments References

 
The authors thank Louis Villeneuve and Evelyn Landry for technical assistance and France Thériault for secretarial help. Funding was provided by the Canadian Institutes of Health Research, the Quebec Heart and Stroke Foundation, and the Mathematics of Information Technology and Complex Systems (MITACS) Network of Centers of Excellence. Stephen Zicha was supported by a "Fonds de la recherche en santé de Québec (FRSQ)" graduate-studentship.

	Notes

 
Time for primary review 18 days

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions Acknowledgments References

 

1. Brown H.F., DiFrancesco D., Noble S.J. How does adrenaline accelerate the heart? Nature (1979) 280:235–236.[CrossRef][Medline]
2. Ho W.K., Brown H.F., Noble D. High selectivity of the I(f) channel to Na+ and K+ in rabbit isolated sinoatrial node cells. Pflugers Arch (1994) 426:68–74.[CrossRef][ISI][Medline]
3. DiFrancesco D. Characterization of single pacemaker channels in cardiac sino-atrial node cells. Nature (1986) 324:470–473.[CrossRef][Medline]
4. DiFrancesco D. Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol (1993) 55:455–472.[CrossRef][ISI][Medline]
5. Bois P., Renaudon B., Baruscotti M., Lenfant J., DiFrancesco D. Activation of f-channels by cAMP analogues in macropatches from rabbit sino-atrial node myocytes. J Physiol (1997) 501:565–571.[Abstract/Free Full Text]
6. DiFrancesco D., Tortora P. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature (1991) 351:145–147.[CrossRef][Medline]
7. Santoro B., Grant S.G., Bartsch D., Kandel E.R. Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels. Proc Natl Acad Sci U S A (1997) 94:14815–14820.[Abstract/Free Full Text]
8. Moroni A., Gorza L., Beltrame M., Gravante B., Vaccari T., Bianchi M.E., et al. Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current I(f). J Biol Chem (2001) 276:29233–29241.[Abstract/Free Full Text]
9. Vaccari T., Moroni A., Rocchi M., Gorza L., Bianchi M.E., Beltrame M., et al. The human gene coding for HCN2, a pacemaker channel of the heart. Biochim Biophys Acta (1999) 1446:419–425.[Medline]
10. Ishii T.M., Takano M., Xie L.H., Noma A., Ohmori H. Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J Biol Chem (1999) 274:12835–12839.[Abstract/Free Full Text]
11. Ludwig A., Zong X., Jeglitsch M., Hofmann F., Biel M. A family of hyperpolarization-activated mammalian cation channels. Nature (1998) 393:587–591.[CrossRef][Medline]
12. Ludwig A., Zong X., Stieber J., Hullin R., Hofmann F., Biel M. Two pacemaker channels from human heart with profoundly different activation kinetics. EMBO J (1999) 18:2323–2329.[CrossRef][ISI][Medline]
13. Moosmang S., Stieber J., Zong X., Biel M., Hofmann F., Ludwig A. Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues. Eur J Biochem (2001) 268:1646–1652.[ISI][Medline]
14. Santoro B., Liu D.T., Yao H., Bartsch D., Kandel E.R., Siegelbaum S.A., et al. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell (1998) 93:717–729.[CrossRef][ISI][Medline]
15. Seifert R., Scholten A., Gauss R., Mincheva A., Lichter P., Kaupp U.B., et al. Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis. Proc Natl Acad Sci U S A (1999) 96:9391–9396.[Abstract/Free Full Text]
16. Shi W., Wymore R., Yu H., Wu J., Wymore R.T., Pan Z., et al. Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues. Circ Res (1999) 85:e1–e6.[Abstract/Free Full Text]
17. Janse M.J. Electrophysiological changes in heart failure and their relationship to arrhythmogenesis. Cardiovasc Res (2004) 61:208–217.[Abstract/Free Full Text]
18. Verkerk A.O., Wilders R., Coronel R., Ravesloot J.H., Verheijck E.E. Ionic remodeling of sinoatrial node cells by heart failure. Circulation (2003) 108:760–766.[Abstract/Free Full Text]
19. Cha T.J., Ehrlich J.R., Zhang L., Shi Y.F., Tardif J.C., Leung T.K., et al. Dissociation between ionic remodeling and ability to sustain atrial fibrillation during recovery from experimental congestive heart failure. Circulation (2004) 109:412–418.[Abstract/Free Full Text]
20. Zicha S., Moss I., Allen B., Varro A., Papp J., Dumaine R., et al. Molecular basis of species-specific expression of repolarizing K+ currents in the heart. Am J Physiol, Heart Circ Physiol (2003) 285:H1641–H1649.[Abstract/Free Full Text]
21. ten Velde I., de Jonge B., Verheijck E.E., van Kempen M.J., Analbers L., Gros D., et al. Spatial distribution of connexin 43, the major cardiac gap junction protein, visualizes the cellular network for impulse propagation from sinoatrial node to atrium. Circ Res (1995) 76:802–811.[Abstract/Free Full Text]
22. Altomare C., Terragni B., Brioschi C., Milanesi R., Pagliuca C., Viscomi C., et al. Heteromeric HCN1-HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node. J Physiol (2003) 549:347–359.[Abstract/Free Full Text]
23. Ulens C., Tytgat J. Functional heteromerization of HCN1 and HCN2 pacemaker channels. J Biol Chem (2001) 276:6069–6072.[Abstract/Free Full Text]
24. Borlak J., Thum T. Hallmarks of ion channel gene expression in end-stage heart failure. FASEB J (2003) 17:1592–1608.[Abstract/Free Full Text]
25. Opthof T., Coronel R., Rademaker H.M., Vermeulen J.T., Wilms-Schopman F.J., Janse M.J. Changes in sinus node function in a rabbit model of heart failure with ventricular arrhythmias and sudden death. Circulation (2000) 101:2975–2980.[Abstract/Free Full Text]
26. Sanders P., Kistler P.M., Morton J.B., Spence S.J., Kalman J.M. Remodeling of sinus node function in patients with congestive heart failure: reduction in sinus node reserve. Circulation (2004) 110:897–903.[Abstract/Free Full Text]
27. Faggiano P., d'Aloia A., Gualeni A., Gardini A., Giordano A. Mechanisms and immediate outcome of in-hospital cardiac arrest in patients with advanced heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol (2001) 87:655–657.[CrossRef][ISI][Medline]
28. Nuss H.B., Kaab S., Kass D.A., Tomaselli G.F., Marban E. Cellular basis of ventricular arrhythmias and abnormal automaticity in heart failure. Am J Physiol (1999) 277:H80–H91.[ISI][Medline]
29. Li G.R., Lau C.P., Ducharme A., Tardif J.C., Nattel S. Transmural action potential and ionic current remodeling in ventricles of failing canine hearts. Am J Physiol, Heart Circ Physiol (2002) 283:H1031–H1041.[Abstract/Free Full Text]
30. Plotnikov A.N., Sosunov E.A., Qu J., Shlapakova I.N., Anyukhovsky E.P., Liu L., et al. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation (2004) 109:506–512.[Abstract/Free Full Text]
31. Stieber J., Hofmann F., Ludwig A. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc Med (2004) 14:23–28.[CrossRef][ISI][Medline]
32. Nattel S., Khairy P., Roy D., Thibault B., Guerra P., Talajic M., et al. New approaches to atrial fibrillation management: a critical review of a rapidly evolving field. Drugs (2002) 62:2377–2397.[CrossRef][ISI][Medline]
33. Nattel S. New ideas about atrial fibrillation 50 years on. Nature (2002) 10:219–226.
34. Ehrlich J.R., Nattel S., Hohnloser S.H. Atrial fibrillation and congestive heart failure: specific considerations at the intersection of two common and important cardiac disease sets. J Cardiovasc Electrophysiol (2002) 13:399–405.[CrossRef][ISI][Medline]
35. Fenelon G., Shepard R.K., Stambler B.S. Focal origin of atrial tachycardia in dogs with rapid ventricular pacing-induced heart failure. J Cardiovasc Electrophysiol (2003) 14:1093–1102.[CrossRef][ISI][Medline]
36. Lai L.P., Su M.J., Lin J.L., Tsai C.H., Lin F.Y., Chen Y.S., et al. Measurement of funny current (I(f)) channel mRNA in human atrial tissue: correlation with left atrial filling pressure and atrial fibrillation. J Cardiovasc Electrophysiol (1999) 10:947–953.[ISI][Medline]
37. Stieber J., Herrmann S., Feil S., Loster J., Feil R., Biel M., et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci U S A (2003) 100:15235–15240.[Abstract/Free Full Text]
38. Kjaer A., Appel J., Hildebrandt P., Petersen C.L. Basal and exercise-induced neuroendocrine activation in patients with heart failure and in normal subjects. Eur J Heart Fail (2004) 6:29–39.[CrossRef][ISI][Medline]

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