Sinus node dysfunction (SND) is a major clinically relevant disease that is associated with sudden cardiac death and is responsible for >50% of surgical implantations of permanent pacemakers per year worldwide. Although the pathologies of acquired SND have been studied in detail, little is known about the molecular and cellular mechanisms that cause congenital SND. This article reports for the first time that mice lacking the pacemaker channel HCN1 display congenital SND characterized by bradycardia, sinus dysrhythmia, prolonged sinoatrial node recovery time, increased sinoatrial conduction time, and recurrent sinus pauses. As a consequence of SND, HCN1-deficient mice display a severely reduced cardiac output. Our study has important clinical impact. Because HCN1 is expressed in the human sinoatrial node, this channel may be added to the list of candidate genes associated with human sinoatrial node dysfunction. Mutations in HCN1 could be well tolerated and thus could be relevant for human disease. HCN1-selective blockers have been considered for the treatment of epileptic disorders, chronic pain, and depression. Given the functional importance of HCN1 for cardiac pacemaking, selective HCN1 blockers should be carefully tested with respect to potential adverse effects on sinoatrial node function. In conclusion, we propose that HCN1 stabilizes the leading pacemaker region within the sinoatrial node and hence is crucial for stable heart rate and regular beat-to-beat variation. Furthermore, we suggest that HCN1-deficient mice may be a valuable genetic disease model for human SND.
Sick Sinus Syndrome in HCN1-Deficient Mice
Abstract
Background—
Sinus node dysfunction (SND) is a major clinically relevant disease that is associated with sudden cardiac death and requires surgical implantation of electric pacemaker devices. Frequently, SND occurs in heart failure and hypertension, conditions that lead to electric instability of the heart. Although the pathologies of acquired SND have been studied extensively, little is known about the molecular and cellular mechanisms that cause congenital SND.
Methods and Results—
Here, we show that the HCN1 protein is highly expressed in the sinoatrial node and is colocalized with HCN4, the main sinoatrial pacemaker channel isoform. To characterize the cardiac phenotype of HCN1-deficient mice, a detailed functional characterization of pacemaker mechanisms in single isolated sinoatrial node cells, explanted beating sinoatrial node preparation, telemetric in vivo electrocardiography, echocardiography, and in vivo electrophysiology was performed. On the basis of these experiments we demonstrate that mice lacking the pacemaker channel HCN1 display congenital SND characterized by bradycardia, sinus dysrhythmia, prolonged sinoatrial node recovery time, increased sinoatrial conduction time, and recurrent sinus pauses. As a consequence of SND, HCN1-deficient mice display a severely reduced cardiac output.
Conclusions—
We propose that HCN1 stabilizes the leading pacemaker region within the sinoatrial node and hence is crucial for stable heart rate and regular beat-to-beat variation. Furthermore, we suggest that HCN1-deficient mice may be a valuable genetic disease model for human SND.
Introduction
The heartbeat is initiated and maintained by the generation of spontaneous action potentials in pacemaker cells of the sinoatrial node (SAN) region. A hallmark of sinoatrial pacemaker action potentials is the presence of a slow diastolic depolarization phase after repolarization. The slow diastolic depolarization causes the cell membrane, which typically maintains a resting membrane potential of about −60 mV,1,2 to reach the threshold potential and consequently fire the next action potential. Over the last years, the molecular basis of the ionic currents involved in SAN action potential has been analyzed in humans,3 rabbits,4 and mice.5 Of particular interest are ion channels and Ca2+-handling proteins, which are essential for the slow diastolic depolarization and thus for autonomous electric activity of the heart.6–8 Mutations in ion channels in humans (Nav1.5, HCN4) and the knockout of ion channels in mice (Cav1.3, Cav3.1, HCN4, and HCN2) have been linked to sick sinus syndrome or bradycardia.7,9–13 Among these channels, HCN (hyperpolarization-activated cyclic nucleotide-gated) channels, which are the molecular correlate of hyperpolarization-activated current (If),14 are considered to be of particular importance. Three of the 4 members of the HCN channel family (HCN1, HCN2, and HCN4) have been identified in pacemaker cells. Quantitatively, in all vertebrates studied so far, HCN4 underlies the major fraction of SAN If, amounting to ≈70% to 80% of the total If. HCN4 is essential for the formation of mature pacemaker cells during embryogenesis.15 Moreover, analysis of human HCN4 channelopathies16 and genetic mouse models1,17,18 (see also the work by Herrmann et al9 and Hoesl et al10) suggests that this channel plays an important role in autonomic control of heart rate. Mice deficient in HCN2 display mild cardiac dysrhythmia, whereas autonomic control of heart rate is preserved in these mice.11,19 In contrast to HCN4 and HCN2, the role of HCN1 in heart has not yet been examined. HCN1 was originally cloned from mouse brain.20 Indeed, analysis of HCN1 knockout (KO) mice revealed that this channel is involved in the control of numerous neuronal functions, including the control of rhythmic activity in neuronal circuits, the control of the resting membrane potential, and dendritic integration.21,22 However, there is also increasing evidence that HCN1 is expressed in heart. In the rabbit SAN, for example, RNase-protection assay and immunolabeling data23,24 indicate significant expression of HCN1. On the basis of heterologous expression data and electrophysiological studies, it was speculated that HCN1, together with HCN4, contributes to native If in rabbit SAN.25 Finally, recent studies identified profound expression of HCN1 protein in mouse cardiac conduction system.19,26 Taken together, these findings suggest that HCN1 may be involved in pacemaker function. In the present study, we addressed this important question by analyzing the HCN1-deficient mouse line. To this end, we performed telemetric in vivo ECG recordings, in vivo electrophysiology, echocardiography and electrophysiological experiments in the intact beating SAN preparation and in isolated sinoatrial pacemaker cells. We show that mice lacking the pacemaker channel HCN1 display congenital SAN dysfunction characterized by bradycardia accompanied by low cardiac output, sinus dysrhythmia, and recurrent sinus pauses.
Clinical Perspective on p 2594
Methods
Animals
HCN1-deficient (HCN1−/−) mice were obtained from The Jackson Laboratory (B6;129-HCN1tm2Kndl/J27; Bar Harbor, ME) and maintained on a mixed C57BL/6N and 129/SvJ background. Six- to 12-week-old HCN1−/− mice and wild-type (WT) littermates derived from heterozygous breeding pairs were randomly assigned to the experimental procedures. Care was taken that at least 1 WT littermate was tested with 1 of its HCN1−/− littermates in a given test series. Each group consisted of animals taken from at least 3 different litters. All animal studies were approved by the Regierung von Oberbayern, were in accordance with German laws on animal experimentation, and were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Effort was taken to keep the number of animals at a minimum.
Morphology
Hearts from 12-week-old WT (n=5) and HCN1−/− (n=6) mice were removed and fixed in 4% paraformaldehyde for 2 hours and incubated in sucrose at 4°C overnight. Then, 16-μm cryosections were stained with hematoxylin and eosin according to standard protocols.
Echocardiography
Echocardiographic images were obtained with an ultrasound imaging system for rodents (Vevo 2100, FUJIFILM VisualSonics, Toronto, ON, Canada) using the 22- to 55-MHz transducer (MS550D) of the system. Twelve WT and 12 HCN1−/− mice were analyzed.
SAN Whole-Mount Dissection
The hearts of 6- to 12-week-old litter-matched mice were put in a dish filled with Tyrode III solution, and the SAN region was cut out along the superior vena cava and crista terminalis. The dissected SANs were pinned on a silicone block and rinsed with Tyrode III solution.
Western Blot
For protein isolation, mouse SAN and atrial tissues of WT (n=6) and HCN1−/− (n=6) mice were homogenized. After heating at 95°C for 15 minutes followed by centrifugation at 1000g for 10 minutes to remove cell debris, the resulting supernatant was used in Western blot analysis as previously described.28 The following antibodies were used: mouse anti-HCN1 (1:1000; Abcam, Cambridge, UK), rat anti-HCN4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HCN2 L (1:500),11 and mouse anti-tubulin (1:2000; Dianova, Hamburg, Germany).
Immunofluorescence Whole-Mount SAN
Tissues of WT (n=5) and HCN1−/− (n=5) mice were stained with rat monoclonal antibody to HCN4 (1:100; Santa Cruz Biotechnology) and rabbit monoclonal antibody to HCN1 (1:100; Alomone Labs, Jerusalem, Israel). As secondary antibodies, Alexa488-conjugated anti-rat (1:400; Invitrogen, Karlsruhe, Germany) and Alexa555-conjugated anti-rabbit (1:400; Invitrogen) antibodies were used.
Electrophysiological Recordings in Single SAN Cells
SAN cells were isolated from 15 HCN1−/− and 16 WT mice as described previously.29
Microelectrode Recordings in Whole-Mount SAN
Spontaneous action potentials were recorded from isolated SAN preparations of WT (n=3) and HCN1−/− (n=4) mice by using 3 M KCl microelectrodes at 30.5±0.5°C unless stated otherwise.
Telemetric ECG Recordings in Mice
Telemetry and analyses of heart rate and heart rate variability (HRV) of WT (n=3) and HCN1−/− (n=4) mice were performed as described.30
In Vivo Electrophysiological Study
The electrophysiological analysis was performed in 9 WT and 11 HCN1−/− mice as described.31
For details on the materials and methods used, please see the online-only Data Supplement.
Statistical Analyses
Data are expressed as mean±SEM. Data were analyzed by the Grubb outlier test, and 2 outliers were excluded from the whole data analysis. Subsequently, we confirmed normal distribution by Kolmogorov-Smirnov, D’Agostino & Pearson omnibus, and Shapiro-Wilk normality tests (Graph Pad Prism 5.0). If not stated otherwise, groups were compared by 2-sample t tests uncorrected for multiple testing. For multiple comparisons, in case of factorial design, 2-way ANOVAs (genotype, parameter/treatment/time) with or without dependent measures were used as indicated, followed by the Newman-Keuls post hoc test if appropriate (StatSoft Statistica 5). In cases of experiments with small sample sizes (telemetric in vivo experiments and SAN whole-mount and isolated hearts), we additionally performed nonparametric tests (Mann-Whitney U tests). Relative genotype differences were assessed by expressing data as a percentage of the mean WT value (in case of independent data sets) or the individual basal level (in case of dependent data sets). Missing data led to a list-wise exclusion of the respective data set. All P values were 2 sided except for the nonparametric analyses, in which 1-sided tests were used because, on the basis of our own findings, we expected a decrease (or no change) in heart rate (single cells, echocardiography, SAN whole mount, in vivo electrophysiological study) or an increase in heart rate fluctuation (or no change; in vivo electrophysiological study). Values of P≤0.05 were accepted as statistically significant.
Results
HCN1 Is a Major Component of If in the SAN
To dissect the particular role of HCN1 channels in sinoatrial pacemaking, we analyzed a mouse line in which HCN1 was globally deleted.27 A microarray analysis of the SAN (WT, n=3; HCN1−/−, n=3; Tables I and II in the online-only Data Supplement) confirmed that the expression levels of ion channels or proteins that were shown to contribute to sinoatrial pacemaker and action potential are not altered in response to the global deletion of HCN1. Importantly, the transcripts for the major depolarizing and repolarizing ion channels are unchanged in the HCN1−/− mice. In line with the unaffected gene expression profile, an echocardiographic analysis demonstrated a normal cardiac structure, diastolic function, and systolic function of HCN1−/− (n=12) and WT (n=12) mice (Figure 1 and Table III in the online-only Data Supplement). In addition, cardiac slice preparation confirmed the finding that cardiac morphology is unchanged. Together, these results indicate that compensatory remodeling or changes in gene expression profile are not a relevant issue in the HCN1−/− heart.
We first investigated the expression of HCN channels in cardiac tissue using specific antibodies (Figure 2). A 120-kDa band corresponding to the mature glycosylated HCN1 protein was present in the SAN of WT mice but was absent in HCN1−/− mice (Figure 2A). A 115-kD band that was detected by the antibody represents an unspecific band because it was present in both WT and HCN1−/− mice. In line with this interpretation, no truncated HCN1 protein has been reported in another study using this HCN1−/− mouse line.27 Comparable amounts of HCN4 protein were detected in SANs of WT and HCN1−/− mice. In contrast, neither HCN4 nor HCN1 was detectable in the right atrium. The HCN2 protein was below the detection limit in SAN and atrium.
Using whole-mount immunohistochemistry, we found overlapping expression of HCN1 and HCN4 in the central region of the SAN (Figure 2B and 2C). At higher magnification, it was evident that both channels are expressed within single pacemaker cells of the central region of the SAN (Figure 2D). Given the robust HCN1 expression in the SAN, we next asked to what extent HCN1 contributes to If in single isolated primary pacemaker cells. We developed an optimized protocol for the isolation of central pacemaker cells and focused on 2 major subtypes of primary sinoatrial cells, spindle cells and elongated cells, which make up 90% to 95% of nonatrial pacemaker cells (Figure 3A). In both cell types, hyperpolarizing voltage steps activated robust If in WT mice (Figure 3B). Deletion of HCN1 reduced the amplitude of If by 36±14% in spindle cells (n=18; P<0.05 versus 100±16% in WT [n=15]) and by 40±19% in elongated cells (n=22; P<0.05 vs. 100±16% in WT [n=20]; Figure 3C). Deletion of HCN1 also dramatically slowed the activation kinetics of If (Figure 3D–3F). The activation time course of If from WT mice required a sum of 2 exponentials for an adequate fit, whereas If from HCN1−/− mice was generally well fit by a single exponential. The slower kinetics of residual If in HCN1−/− mice was similar to that of cloned HCN4 channels.32 These findings indicate that HCN1 and HCN4 are the major determinants of If in spindle and elongated SAN pacemaker cells.
Reduced Beating Frequency in Isolated Pacemaker Cells of HCN1−/− Mice
We next investigated the contribution of HCN1 to the generation of spontaneous action potentials in isolated sinoatrial pacemaker cells using current clamp recordings. Isolated pacemaker cells from either WT or HCN1−/− mice fired rhythmic, spontaneous pacemaker potentials (Figure 4A). Deletion of HCN1 shifted the resting membrane potential to more hyperpolarized potentials in spindle and elongated cells (WT: −59.9±1.3 mV, n=11; HCN1−/−: −63.9±1.1 mV, n=8; P<0.05). Furthermore, in HCN1−/− mice, the firing rate was significantly reduced by 13±4% compared with WT mice (100±5%; WT, n=19; KO, n=18; P<0.05; Table IVA in the online-only Data Supplement). Some recordings were performed in the presence of submaximal (2 nmol/L) and saturating (10 µmol/L) doses of isoproterenol (Figure 4B). In these experiments, the maximal firing rate was consistently lower in HCN1−/− mice compared with WT mice (Table IVA in the online-only Data Supplement). The relative increase in the firing rate induced by isoproterenol was the same in the 2 groups of mice (Table IVA in the online-only Data Supplement).
Pronounced Fluctuations of the Beat-to-Beat Interval in Intact SAN Preparations of HCN1−/− Mice
We used an explanted SAN preparation to investigate how the effects observed on the level of isolated pacemaker cells influence the properties of the cellular network of the SAN. Like isolated sinoatrial pacemaker cells, the intact SAN preparation from either WT or HCN1−/− mice fired spontaneous pacemaker potentials (Figure 4C). The firing rate was significantly reduced in HCN1−/− mice by 29±2% compared with WT mice (100±7%; WT, n=3; KO, n=4; P<0.05; Table IVB in the online-only Data Supplement). A comparable reduction in the beating rate by 20.2±2.8% was observed in isolated perfused hearts (WT, 100±6 %; WT, n=5; KO, n=6; P<0.05; Table IVC in the online-only Data Supplement), indicating that deletion of HCN1 leads to a reduction in the intrinsic heart rate and thus to bradycardia originating within the SAN. In agreement with the findings in isolated SAN cells, the maximal firing rate in the presence of low doses of isoproterenol was consistently lower in HCN1−/− than in WT preparations (Figure 4D and Table IVB in the online-only Data Supplement). The firing rate of HCN1−/− SAN preparations increased by the same relative amount in the presence of isoproterenol compared with WT SAN preparations (Table IVB in the online-only Data Supplement).
In WT SANs, the interval between consecutive beats was regular in the absence and in the presence of isoproterenol (Figure 4E and 4F). In contrast, unlike in the WT, the interbeat interval rapidly fluctuated (range, 200–400 milliseconds) in HCN1−/− preparations (Figure 4E). In the presence of 10 µmol/L isoproterenol, these rapid fluctuations were still significantly higher than in WT mice (Table IVD in the online-only Data Supplement). To rule out the possibility that the irregular firing rate of the HCN1−/− SAN resulted from a lower overall firing rate, we compared the WT and HCN1−/− nodes at similar slow firing rates. Because in WT nodes slow rates were not achieved under normal recording conditions (30.5±0.5°C), we intentionally slowed the rate by cooling the SAN to 28°C (Figure 4E). Under these conditions, the firing rate was still very regular. These findings strongly suggest that the observed increase in beat-to-beat fluctuation in HCN1−/− mice is intrinsic to the SAN itself. To confirm this hypothesis, we determined SAN function using atrial pacing of isolated right atrial preparation containing the SAN. Sinus node automaticity was assessed by analyzing the sinus node recovery time (SNRT) in WT (n=13) and HCN1−/−(n=9) mice. The preparation was continuously paced for 10 seconds at 10 Hz. Before and after pacing, normal spontaneous sinus cycles were recorded. SNRT is defined as the duration of the return cycle, which is the interval from the last paced atrial activation to the first postpacing spontaneous beat (Figure 5A).There was a clear increase in SNRT in HCN1−/− mice compared with WT mice. This difference was also evident after adjustment for the cycle length (corrected SNRT; Figure 5B and Table V in the online-only Data Supplement).
SAN Dysfunction in HCN1−/− Mice
The rapidly fluctuating interbeat intervals of HCN1−/− SANs, together with the increased SNRT, are highly suggestive of sinus node dysfunction. To test for this possibility, we recorded in vivo telemetric long-term ECG in freely moving WT (n=3) and HCN1−/− (n=4) mice (Figure 6). In long-term measurements over 72 hours, HCN1−/− mice revealed a marked sinus bradycardia (Figure 6A) characterized by lower mean heart rates by 16±2% compared with the WT (100±2%; WT, n=3; KO, n=4; Table VIA in the online-only Data Supplement and Figure 6B) and more frequent episodes of low heart rate in HCN1−/− mice compared with controls (Figure 6C). Marked sinus bradycardia was also observed in an echocardiographic analysis (Table III in the online-only Data Supplement). The lower overall heart rates in WT and HCN1−/− mice in echocardiographic analysis compared with ECG telemetry are attributable to isoflurane anesthesia.33 Bradycardia in HCN1−/− mice was not attributable to an increase in resting phases because HCN1−/− and WT mice displayed the same activity and resting behavior. In addition, heart rates >600 bpm were almost absent in HCN1−/− mice (Figure 6C). The minimum, mean, and maximum heart rates were shifted to lower values in the HCN1−/− compared with the WT mice. In contrast, the dynamic range of heart rate regulation (relative differences between minimal and maximal heart rates) and the relative degree of heart rate regulation (maximal heart rate/minimal heart rate) were comparable between the 2 groups of animals (Table VIB in the online-only Data Supplement). The reduction was consistently observed in HCN1−/− mice during 12-hour periods of low and high activity (Figure 6D and 6E). On β-adrenergic stimulation achieved by intraperitoneal injection of isoproterenol (0.1 mg/kg), the maximal heart rate was lower in HCN1−/− than in WT preparations. The heart rate in HCN1−/− mice increased by the same relative amount in the presence of isoproterenol compared with WT mice (Figure 6B and Table VIA in the online-only Data Supplement). Injection of carbachol (0.15 mg/kg) reduced the heart rate to the same level in WT and HCN1−/− mice (Figure 6B and Table VIA in the online-only Data Supplement). Besides bradycardia and reduced maximum heart rate, the ECG of HCN1−/− mice revealed periods of recurrent sinus pauses, which were in some cases accompanied by escape beats (Figure 6F and 6G). Atrioventricular conduction was normal in HCN1−/− mice compared with WT mice (PQ interval: WT, 36.0±2.9 milliseconds; KO, 37.6±0.8 milliseconds).
Increased Heart Rate Variability in HCN1−/− Mice
To further investigate the dynamics of heart rate oscillations, we performed an HRV analysis. To assess variability of the heart with respect to time, we determined the mean RR interval (Figure IA in the online-only Data Supplement), standard deviation of all RR intervals, and root mean square of the difference of successive RR intervals during a 2-hour time period during low physical activity (time domain parameters; Figure IB in the online-only Data Supplement). In WT mice, these parameters were consistent with only narrow fluctuations of the RR intervals, indicating a relatively stable heart rhythm. In line with this finding, Poincaré plots of WT mice displayed a low beat-to-beat dispersion that is characteristic for a stable heart rate, leading to an elliptical configuration (Figure 7A). In contrast, in HCN1−/− mice, time domain parameters of HRV were markedly increased, consistent with dramatic fluctuations of the RR interval and a markedly increased HRV (Figure IB in the online-only Data Supplement). Accordingly, Poincaré plots determined for HCN1−/− mice displayed a broad comet-shaped pattern caused by high beat-to-beat dispersion typically observed in SAN dysfunction (Figure 7B).
For frequency domain analysis of HRV, we compared the HRV of WT and HCN1−/− mice during a phase of relatively low heart rate (average heart rate, ≈400 bpm) corresponding to a RR interval of 150 milliseconds and during a phase of relatively high heart rate (average heart rate, ≈600 bpm) corresponding to an average RR interval of 100 milliseconds. Tachograms of WT mice showed only a slight variation of the RR interval (Figure 7C). In the absence of SAN pathology, as in WT mice, these beat-to-beat fluctuations in heart rate are generated by the opposing effects of the sympathetic and parasympathetic nervous systems. In contrast to WT mice, HCN1−/− mice displayed broad fluctuations of the RR interval, consistent with a markedly increased HRV (Figure 7D). These fluctuations were more pronounced during phases of slow heart rate (Figure II in the online-only Data Supplement). The pronounced increase in HRV in HCN1−/− mice compared with WT controls is reflected in the HRV spectra (Figure 7E and 7F and Figure IIA in the online-only Data Supplement). The spectra were subdivided into 3 frequency ranges: high frequency (1.5–4 Hz), low frequency (0.4–1.5 Hz), and very low frequency (<0.4 Hz). In contrast to the HRV spectra of WT mice (Figure 7E), the HRV spectra of HCN1−/− mice had a markedly increased power in all frequency bands during periods of low heart rate (Figure 7F and Figure II in the online-only Data Supplement). The heart rate fluctuations of HCN1−/− animals were in the same range as observed in the intact isolated SAN preparation (Figure 4E). This finding indicates that the SAN is the origin of pronounced increased heart rate fluctuations rather than the autonomic nervous system.
To analyze the SAN function in vivo, an intracardiac electrophysiological study was performed (WT, n=9; KO, n=11). In HCN1−/− mice, SNRT and SNRT corrected for the spontaneous cycle lengths were decreased at pacing cycles ranging from 110 to 80 milliseconds compared with WT mice (Figure 8 and Table VIIA in the online-only Data Supplement). In line with similar experiments in the literature, at the fastest pacing cycle, the SNRT decreased in WT and HCN1−/− mice.34 The increased SNRT in HCN1−/− mice is in line with the in vitro microelectrode experiments in explanted atrial preparations containing the SAN (Figure 5) and confirms a delayed impulse formation within the SAN of HCN1−/−. In addition, premature atrial stimulation revealed a prolonged sinoatrial conduction time (WT: 16.1±1.9 milliseconds, n= 9; HCN1−/−: 28.34±2.4 milliseconds, n=10; P≤0.01; Figures III and IV and Table VIIA in the online-only Data Supplement). We also calculated a range for the sinoatrial conduction time to account for the baseline sinus dysrhythmia, which was present in HCN1−/− mice (Table VIIB in the online-only Data Supplement). To this end, we used the longest and shortest spontaneous sinus cycle lengths. The sinoatrial conduction time range was 13.6 to 17.5 milliseconds (n=9) for WT mice and 25.5 to 38.8 milliseconds (n=10) for HCN1−/− mice, indicating that the difference in the sinoatrial conduction time is independent of changes in sinus cycle lengths. This result is consistent with impaired impulse propagation and with a sinoatrial exit block in HCN1−/−and fits the overall clinical picture of sinus node dysfunction. There was no difference in the refractory periods of the atrium, atrioventricular node, and ventricle and in the Wenckebach points between control and HCN1−/− mice (Table VIIC and VIID and Figure V in the online-only Data Supplement).
Finally, we analyzed the hemodynamic consequences of the observed cardiac phenotype in vivo. Echocardiographic in vivo testing revealed that the bradycardia of HCN1−/− mice leads to a hemodynamically relevant reduction in cardiac output to 69% compared with WT mice (Table III in the online-only Data Supplement). The stroke volume was not different between the 2 groups of mice. This indicates that there is no compensation for the reduced heart rate, at least on the level of the stroke volume. In vivo pressure measurements revealed no difference in systolic and diastolic blood pressures between the 2 groups of mice (systolic pressure: WT, 118±5 mm Hg versus KO, 114±3 mm Hg; diastolic pressure: WT, 74±3 mm Hg versus KO, 67±8 mm Hg; WT, n=6; KO, n=3). Together, these results suggest that the reduced heart rate is the primary cause of the decreased cardiac output in HCN1−/− mice.
Discussion
Here, we show that HCN1 channels make up a physiologically relevant component of If in the SAN. If was reduced by ≈30% in HCN1−/− SAN cells, and the activation kinetics of the remaining If was significantly slowed down. Our results also suggest that HCN1 is functionally important in spindle and elongated primary pacemaker cells of the central SAN. The main cardiac phenotype of the HCN1−/− mouse is a pronounced sinus node dysfunction characterized by impaired impulse formation and sinoatrial conduction, resulting in bradycardia, sinus arrhythmia, and recurrent sinus pauses. Hemodynamically, these changes lead to a relevant reduction in cardiac output.
Several mechanisms may contribute to this complex phenotype. First, a reduction in If density is an important factor that could contribute to or explain the bradycardia observed in HCN1−/− mice. Heterologous expression studies and analysis of neuronal hyperpolarization-activated currents predominantly carried by HCN1 revealed that the HCN1 channel activates with much faster kinetics and at more positive voltages than other HCN channel types, including HCN4. Deletion of HCN1 would lead to a remaining If that is reduced and largely determined by HCN4. Therefore, one would expect that the activation curve of If in HCN1−/− mice is shifted to more hyperpolarized potentials compared with WT mice. Moreover, HCN1 is only slightly upregulated, if at all, when the cAMP concentration is increased from low-micromolar to millimolar cAMP concentrations. Our finding that in HCN1−/− mice chronotropic competence is almost preserved is in line with that property. This particular biophysical profile implies that a major fraction of the HCN1 channels will be opened at rest. Thus, 1 function of HCN1 channels may be to provide a basal depolarizing current in the central region of the SAN that facilitates opening of the other channels, contributing to the slow diastolic depolarization. This mechanism would well explain the bradycardia resulting from HCN1 deletion.
In addition to bradycardia, HCN1−/− mice revealed pronounced sinus dysrhythmia characterized by dramatic fluctuations in the heart rate and recurrent sinus pauses. Similar fluctuations of the beat-to-beat interval were observed in intact SAN preparations. The pronounced heart rate fluctuations not only were observed in in vivo telemetric ECG recordings and experiments using intact SAN preparations that were performed with small sample sizes (WT, n=3; HCN1−/−, n=4) but also were consistently found in a third and independent set of experiments, an in vivo electrophysiological study that was performed with a larger sample size (WT, n=9; HCN1−/−, n=11). The presence of heart rate fluctuations in in vivo experiments and in intact SAN preparations favors the SAN as the possible origin of the observed dysrhythmia and virtually excludes autonomic modulation of the SAN. In line with this conclusion, SNRT was increased in HCN1−/− mice, indicating that the sinus dysrhythmia and the sinus pauses are caused by a failure of impulse formation in the SAN. In addition, we provide evidence for a prolonged sinoatrial conduction. The prolonged sinoatrial conduction time in HCN1−/− mice could result from a more negative maximal diastolic potential, which increases the distance to the threshold at which an action potential is generated. In this situation, more current and more time are required for a cell to charge the cell membrane of an adjacent cell to reach the threshold potential for an action potential and therefore slows the action potential conduction. This effect outweighs the competing effect of a more negative maximal diastolic potential to increase the availability of L-type Ca2+ channels and voltage-gated Na+ channels, which increase dV/dt and thus the conduction velocity. Our results suggest that cardiac HCN channels, in addition to impulse formation, are important for cardiac excitability and impulse propagation.
The SND of HCN1−/− mice could develop in the extensively distributed cellular network of the intact SAN itself. Within the SAN, a frequent exchange of dominance among multiple pacemakers coincides with changes in heart rate and beat-to-beat cycle lengths.35 A stable monofocal position of the leading pacemaker could account for stable heart rate observed in WT animals. In contrast, disorganized shift of the leading pacemaker focus without any predominant direction or competing multiple pacemakers could be responsible for enhanced beat-to-beat variability and pronounced sinus node dysrhythmia and sinus pauses in HCN1−/− mice. Such a mechanism has been shown for SAN dysrhythmia.36 It is quite possible that in WT mice HCN1 channel activity at the resting membrane potential increases the membrane conductance, stabilizes 1 leading pacemaker focus, and thus decreases beat-to-beat fluctuations in the heart. In addition, HCN1 channels could protect the SAN from excess hyperpolarizing electrotonic loading by the surrounding atrium, which could significantly slow pacemaking37 and could impair sinoatrial conduction. Such a role has been postulated for If in the SAN.37 Strands of atrial myocytes have been shown to extend into the central SAN.38 It is possible that HCN1 plays an important role in protecting the central SAN from the hyperpolarization at these atrio-sinoatrial contact sites. Sinus node dysrhythmia and recurrent sinus pauses were most pronounced at low heart rates and were nearly absent at the high heart rates observed during high physical activity or after pharmacological stimulation by isoproterenol. These findings suggest that a fully functional If is of particular relevance under these vulnerable conditions.
In humans, SND is a relevant disease that affects 1 in 600 cardiac patients >65 years of age and is responsible for >50% of surgical implantations of permanent pacemakers per year worldwide.35,39 SAN dysfunction commonly occurs in the setting of heart failure and arterial hypertension but also is a genetic disease. Because HCN1 is expressed in the human SAN,3,40,41 this channel may be added to the list of candidate genes associated with human SAN dysfunction. Mutations in HCN1 could be well tolerated and thus could be relevant for human disease. HCN1-selective blockers have been considered for the treatment of epileptic disorders, chronic pain, and depression.42,43 Given the functional importance of HCN1 for cardiac pacemaking, selective HCN1 blockers should be carefully tested with respect to potential adverse effects on SAN function.
Acknowledgments
We thank Dr Robert Fischer for help during the setup of the in vivo intracardiac measurements and for the extensive discussions during the preparation of the manuscript. We thank Katrin Roetzer for excellent technical assistance.
Clinical Perspective
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© 2013 American Heart Association, Inc.
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Received: 16 May 2013
Accepted: 27 September 2013
Published online: 11 November 2013
Published in print: 17 December 2013
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This study was supported by a research scholarship for S.I.H. Hassan according to the Bayerische Eliteförderungsgesetz. Dr Fenske was supported by the LMU Mentoring program. S.C. Krause was supported by Deutsche Forschungsgemeinschaft.
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