Funny Current Downregulation and Sinus Node Dysfunction Associated With Atrial Tachyarrhythmia
A Molecular Basis for Tachycardia-Bradycardia Syndrome
Background— Sinoatrial node (SAN) dysfunction is frequently associated with atrial tachyarrhythmias (ATs). Abnormalities in SAN pacemaker function after termination of ATs can cause syncope and require pacemaker implantation, but underlying mechanisms remain poorly understood. This study examined the hypothesis that ATs impair SAN function by altering ion channel expression.
Methods and Results— SAN tissues were obtained from 28 control dogs and 31 dogs with 7-day atrial tachypacing (400 bpm). Ionic currents were measured from single SAN cells with whole-cell patch-clamp techniques. Atrial tachypacing increased SAN recovery time in vivo by ≈70% (P<0.01), a change which reflects impaired SAN function. In dogs that underwent atrial tachypacing, SAN mRNA expression (real-time reverse-transcription polymerase chain reaction) was reduced for hyperpolarization-activated cyclic nucleotide-gated subunits (HCN2 and HCN4) by >50% (P<0.01) and for the β-subunit minK by ≈42% (P<0.05). SAN transcript expression for the rapid delayed-rectifier (IKr) α-subunit ERG, the slow delayed-rectifier (IKs) α-subunit KvLQT1, the β-subunit MiRP1, the L-type (ICaL) and T-type (ICaT) Ca2+-current subunits Cav1.2 and Cav3.1, and the gap-junction subunit connexin 43 (were unaffected by atrial tachypacing. Atrial tachypacing reduced densities of the HCN-related funny current (If) and IKs by ≈48% (P<0.001) and ≈34% (P<0.01), respectively, with no change in voltage dependence or kinetics. IKr, ICaL, and ICaT were unaffected. SAN cells lacked Ba2+-sensitive inward-rectifier currents, irrespective of AT. SAN action potential simulations that incorporated AT-induced alterations in If accounted for slowing of periodicity, with no additional contribution from changes in IKs.
Conclusions— AT downregulates SAN HCN2/4 and minK subunit expression, along with the corresponding currents If and IKs. Tachycardia-induced remodeling of SAN ion channel expression, particularly for the “pacemaker” subunit If, may contribute to the clinically significant association between SAN dysfunction and supraventricular tachyarrhythmias.
Received April 30, 2008; accepted October 17, 2008.
It is well-established that sinoatrial node (SAN) dysfunction is common in patients with atrial fibrillation (AF) and can lead to syncopal attacks after AF termination, a condition often called the tachycardia-bradycardia syndrome.1,2 Although abnormalities of SAN structure have been noted in patients with AF,3 there is increasing evidence of a reversible component related to SAN remodeling caused by rapid atrial tachyarrhythmias. SAN dysfunction is commonly noted 1 day after electrical cardioversion in patients with lone AF.4 Although this finding was originally believed to be due to the intrinsic electrophysiological abnormalities that characterize the condition, Elvan et al5 demonstrated in an elegant study that electrically induced AF causes SAN dysfunction in dogs, with SAN abnormalities becoming reversed within a week after AF termination. SAN dysfunction noted after termination of chronic atrial flutter was also found to reverse itself over several weeks,6 which supports the notion that atrial tachyarrhythmias lead to reversible SAN dysfunction in humans. Subsequently, Hocini et al7 demonstrated that when AF patients show prolonged sinus pauses on AF termination, successful AF ablation is followed by marked recovery in SAN function indices.
Clinical Perspective p 1585
Despite extensive accumulating evidence for atrial tachyarrhythmia-induced SAN dysfunction, the underlying mechanisms have remained unclear. Atrial tachyarrhythmias, including AF, cause substantial remodeling of the ionic current properties of atrial cardiomyocytes, which causes action potential abbreviation that increases vulnerability to AF induction and maintenance.8 It is quite conceivable that SAN ionic current changes induced by AF lead to the depressed SAN function that characterizes the tachycardia-bradycardia syndrome.
The present study tested the hypothesis that sustained atrial tachycardia alters ionic current properties in SAN cardiomyocytes, thereby causing SAN dysfunction. We first studied changes in the expression of SAN ion channel subunits that resulted from 1 week of atrial tachypacing (ATP) at 400 bpm, a recognized paradigm of AF-related atrial tachycardia remodeling.8,9 We then developed the necessary methods to isolate canine SAN cardiomyocytes and performed voltage-clamp studies to characterize the effects of ATP on their ionic currents. Our results implicate alterations of the funny current, If, in atrial tachyarrhythmia-induced SAN dysfunction.
Animal Handling and Tachypacing Protocol
Animal care procedures were consistent with National Institutes of Health guidelines and were approved by the animal research ethics committee of the Montreal Heart Institute. Adult mongrel dogs (weight 25 to 35 kg; Laka Inc, Saint-Basile-le-Grand, Quebec, Canada) were instrumented with a unipolar right-atrial (RA) lead attached to a pacemaker programmed to provide 1 week of RA pacing at 400 bpm. Ventricular rate control was ensured by radiofrequency ablation-induced AV block, with a right ventricular demand pacemaker set at 80 bpm. ATP dogs (n=31) were compared with control dogs (n=28) that were similarly instrumented but with the RA pacemaker inactivated. At the time the animals were euthanized, SAN cardiomyocytes were isolated for electrophysiological study, and SAN and RA free-wall (RAFW) tissue samples were collected, snap-frozen in liquid N2, and stored at −80°C.
Sinus Node Recovery Time Changes
On study days, dogs were anesthetized (morphine 2 mg/kg SC; α-chloralose 120 mg/kg IV load, 29.25 mg · kg−1 · h−1) and mechanically ventilated. Bipolar pacing and recording hook electrodes were inserted into the RA appendage. The baseline sinus rate was measured after suppression of potentially confounding vagal and β-adrenergic influences by administration of nadolol (0.5 mg/kg IV) and severing of the vagus nerves in the neck.2 The RA was then paced at cycle lengths (CLs) of 250 or 300 ms for 1 minute. The corrected sinus node recovery time (SNRTc) was obtained from the interval from the last paced atrial activation to the first sinus escape beat, minus the prepacing spontaneous CL.
RNA Extraction and TaqMan Real-Time Polymerase Chain Reaction
RNA was isolated from tissue samples by guanidine thiocyanate-phenol-chloroform extraction, then treated with DNase (RNeasy mini kit, Qiagen, Valencia, Calif), quantified, and subjected to quality control by microelectrophoresis on polyacrylamide gels (Agilent 2100 Bioanalyzer, Agilent Technologies Inc, Santa Clara, Calif).1 DNA contamination was excluded by reverse-transcription-negative polymerase chain reaction (PCR). First-strand complementary DNA was synthesized from 2 μg of total RNA with a high-capacity cDNA archive kit (Applied Biosystems, Foster City, Calif). Real-time quantitative PCR was performed with either 6-carboxy-fluorescein-labeled fluorogenic TaqMan primers and probes (assay-by-design) with TaqMan universal master mix (Applied Biosystems) or custom primers (Invitrogen, Carlsbad, Calif) with SYBR Green master mix (Applied Biosystems; sequences provided in the Table). Fluorescence signals were detected with the Stratagene Mx3000P sequence-detection system (Stratagene, La Jolla, Calif) in duplicate, normalized to the reference (18S ribosomal RNA, Applied Biosystems), and quantified with MxPro QPCR software (Stratagene).
SAN Cardiomyocyte Isolation
An RA preparation containing the SAN region was perfused at ≈10 mL/min via the right coronary artery for cardiomyocyte isolation.9 The preparation was first perfused with 2 mmol/L Ca2+-containing Tyrode solution until all leaking coronary artery branches were ligated, followed by Ca2+-free Tyrode solution for 15 minutes. Then, Ca2+-free Tyrode solution that contained collagenase (110 U/mL CLS II collagenase; Worthington Biochemical, Lakewood, NJ) and 0.1% bovine serum albumin was used to perfuse the preparation for ≈40 minutes. The SAN region was identified as a whitish endocardial zone near the junction between the superior vena cava and the RA appendage. Dispersed cells were stored in a high-K+ storage solution.
SAN Cellular Electrophysiology
Currents were recorded with whole-cell patch clamping at 36±0.5°C as described previously.9 Potential SAN cardiomyocytes were identified on the basis of distinct morphologies (fine, elongated spindlelike or spider-shaped cells; see supplemental Figure I). Only cells presenting If, which was never seen in atrial cardiomyocytes, were selected for SAN cell current recording. Borosilicate glass electrodes had tip resistances between 2.0 and 4.0 MΩ when filled. Compensated series resistances and capacitive time constants averaged 3.4±0.3 MΩ and 289±64 μs, respectively. SAN cell capacitance averaged 80±5 pF (n=30) for control dogs and 85±3 pF (n=44) for tachypaced dogs. Original recordings are presented in terms of absolute current amplitude, but mean data are shown as current density (pA/pF). Junction potentials averaged 15.0±0.7 mV and were corrected only for resting-potential measurements. Resting potentials averaged −52±1 mV in control and −53±1 mV in tachypaced SAN cells (n=14 and 17 cells, respectively, from 3 dogs each).
The cell-storage solution contained (in mmol/L) KCl 20, KH2PO4 10, dextrose 10, mannitol 40, L-glutamic acid 70, β-OH-butyric acid 10, taurine 20, EGTA 10, and 0.1% bovine serum albumin (pH 7.3, KOH). Tyrode (extracellular) solution contained (in mmol/L) NaCl 136, KCl 5.4, MgCl2 1, NaH2PO4 0.33, HEPES 5, and dextrose 10 (pH 7.35, NaOH), with CaCl2 of 1 mmol/L for If recording and 2 or 0 mmol/L for cell isolation. The internal solution for If and K+-current recording contained (in mmol/L) K-aspartate 110, KCl 20, MgCl2 1, MgATP 5, Li-GTP 0.1, HEPES 10, Na-phosphocreatine 5, and EGTA 5 (pH 7.3, KOH). Ba2+ (1 mmol/L)-sensitive current was used to assess inward-rectifier K+ currents as described previously.9 For IKs recording, nifedipine (5 μmol/L), 4-aminopyridine (2 mmol/L), dofetilide (1 μmol/L), and atropine (200 nmol/L) were added to suppress ICaL, Ito, IKr, and 4-aminopyridine-dependent muscarinic K+ currents. For IKr recording, the same solutions were used as for IKs, except dofetilide was not included and the IKs blocker HMR 1556 (0.5 μmol/L) was added. The external solution for ICa recording contained (in mmol/L) tetraethylammonium chloride 136, CsCl 5.4, CaCl2 2, MgCl2 0.8, HEPES 10, and dextrose 10 (pH 7.4, CsOH). Niflumic acid (50 μmol/L) was added to inhibit Ca2+-dependent Cl− current . The internal solution for ICa recording contained (in mmol/L) CsCl 120, TEA-Cl 20, MgCl2 1, MgATP 5, Li-GTP 0.1, EGTA 10, and HEPES 10 (pH 7.3, CsOH). Unless otherwise specified, chemicals were obtained from Sigma Chemicals (St Louis, Mo).
Transmembrane Potential Simulations
The Kurata model of the rabbit SAN cell action potential10 was modified to produce a spontaneous rate similar to that in dogs in the present study (the sustained inward current [Ist] was set to zero) and implemented in C++ on an AMD64 processor-based computer (AMD, Sunnyvale, Calif). Model implementation used a variable time-step algorithm (Runge-Kutta-Merson fourth-order integration scheme) with maximum relative tolerance of 10−6. Simulations with each parameter value set were run for 100 seconds. The last 2 seconds of simulation were then analyzed to compare the effects of the observed degrees of If and IKs remodeling on SAN activity.
Data are expressed as mean±SEM. Repeated-measures 2-way ANOVA and Bonferroni-adjusted t tests were used for statistical comparisons of current-voltage relations. Reverse-transcription PCR data (nonrepeated measures) were also analyzed by 2-way ANOVA. When ANOVA revealed a statistically significant interaction, Bonferroni-adjusted comparisons were performed to compare individual group means by multiplying each probability value by the number of comparisons. In the absence of significant interactions, statistical data are presented only in terms of main effects (region [SAN versus RA] or condition [control versus ATP]). An unpaired Student t test was used to compare spontaneous CL between control and ATP-remodeled dogs. Analyses of ionic currents controlled for dog of origin as a variable to avoid weighting results from different dogs by the number of cells studied. A 2-tailed probability value <0.05 was considered statistically significant. Clampfit 9.0 (Axon Instruments, Foster City, Calif) and GraphPad Prism 3.0 (GraphPad, San Diego, Calif) software were used for data analysis.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
SAN Recovery Times In Vivo
Figure 1A illustrates RA electrogram recordings used to calculate the SNRTc. The left panel shows baseline after vagotomy and nadolol administration, whereas the right panel shows recordings just before and after the end of tachypacing. There was a clear delay to the emergence of the first spontaneous postpacing beat, which was enhanced after 1 week of ATP. After vagotomy and nadolol administration, the sinus CL was longer in ATP dogs than in control (P<0.05), which indicates reduced intrinsic SAN automaticity (Figure 1B). SNRTc was substantially prolonged in ATP dogs versus controls (Figure 1C).
Ion Channel Subunit Expression
ATP causes atrial electrical remodeling, which alters the mRNA expression of ion channel subunits.8 Quantitative reverse-transcription PCR was used to investigate ATP-induced changes in mRNA expression profiles and to compare SAN and RAFW expression patterns to support tissue-identification validity (Figure 2).
The mRNA expression levels of hyperpolarization-activated cyclic nucleotide-gated subunits (HCN2 and HCN4) were enriched in control SAN tissue versus RAFW (≈5.5-fold and 12-fold, respectively, P<0.001 for both; Figures 2A and 2B). There was a statistically significant interaction between region (RAFW and SAN) and condition (control versus ATP) in expression of both HCN2 (F=5.67, dfn=1, dfd=34, P=0.023) and HCN4 (F=6.07, dfn=1, dfd=34, P=0.019), which indicates that region is a determinant of the response to ATP. ATP reduced SAN expression of both HCN2 (by 56%, P<0.05) and HCN4 (59%, P<0.05). There was no interaction between region and condition for the putative If β-subunit MiRP1 (Figure 2C), but there was a significant main effect of region (F=15.70, dfn=1, dfd=30, P=0.0004), with greater expression in SAN than in RAFW, which was unchanged by tachypacing.
Expression of the α-subunits corresponding to IKr (ERG; Figure 2D) and IKs (KvLQT1; Figure 2E) was similar for RAFW versus SAN tissues, and neither was significantly altered by ATP. The IKs β-subunit minK (Figure 2F) was more strongly expressed in SAN than in RAFW (main effect of region: F=12.42, dfn=1, dfd=33, P=0.0013) and was globally reduced by ATP (main effect of condition: F=11.48, dfn=1, dfd=33, P=0.0018).
The L-type Ca2+ current (ICaL) α-subunit Cav1.2 (main effect of region: F=22.95, dfn=1, dfd=32, P<0.0001; Figure 2G) and T-type Ca2+ current (ICaT) α-subunit Cav3.1 (main effect of region: F=10.25, dfn=1, dfd=33, P=0.0030; Figure 2H) were both more strongly expressed in SAN than in RAFW. Neither subunit was significantly affected by ATP.
There was a significant interaction between region and condition for connexin 43 (Cx43) expression (F=26.21, dfn=1, dfd=32, P<0.0001; Figure 2I). In control tissue, Cx43 was expressed ≈3.4-fold more in RAFW than in SAN (P<0.001). Although ATP had no effect on SAN Cx43 expression, tachypacing downregulated Cx43 by 48% (P<0.001) in RAFW.
Expression of the reference gene, which encoded 18S ribosomal RNA, was comparable among groups (RAFW control 6.38±0.47; RAFW ATP 6.75±0.63; SAN control 6.68±0.67; SAN ATP 6.77±0.31).
Ionic currents were selected for measurement on the basis of the mRNA data. The 2 currents that showed significant subunit expression changes (If and IKs) were recorded, along with 2 Ca2+ currents believed to play important roles in SAN pacemaking4 that showed no significant alteration in α-subunit expression (ICaL and ICaT) and 1 K+ current that showed no mRNA change (IKr). Figure 3A shows representative recordings of If in control and ATP cells. Both time-dependent activating (Figure 3B) and tail-current components of If were significantly reduced by ATP. For example, at a step voltage of −140 mV, If was reduced from −10.8±1.0 pA/pF in control cells to −6.0±1.0 pA/pF in ATP cells (P<0.001). If activation kinetics were well fitted by biexponential relations and were not affected by ATP (Figure 3C), showing similar fast (τfast) and slow (τslow) activation time constants, of the order of 50 to 200 and 250 to 1000 ms, respectively, over the full voltage range. To analyze steady state activation voltage dependence, If tail currents on repolarization to −140 mV were normalized by the maximum tail-current value and plotted as a function of the preceding step potential. Tachypacing did not significantly affect the activation variable at different voltages (Figure 3D). The V1/2 and slope-constant values obtained from Boltzmann fits of data in each experiment averaged −72.4±3.0 and −8.3±1.1 mV, respectively, for control cells and −76.4±3.3 and −7.6±1.3 mV, respectively, for ATP cells.
We also recorded Ba2+-sensitive K+ currents in SAN cells to assess their constitutive acetylcholine-regulated (IKACh) and background inward-rectifier (IKl) expression phenotype and to evaluate possible ATP-induced inward-rectifier current upregulation of the type previously observed in atrial cells.8 Supplemental Figures IIA and IIB show such current recordings from 1 SAN cell before and after exposure to 1 mmol/L Ba2+. Consistent with very limited IKl expression in the SAN region, Ba2+ had no clear effect on the currents, and the Ba2+-sensitive currents obtained by digital subtraction (supplemental Figure IIC) were negligible. Mean Ba2+-sensitive current-voltage density relations in SAN cells are illustrated in supplemental Figure IID and contrasted with corresponding results in RA cardiomyocytes. Unlike SAN cells, atrial cardiomyocytes showed clear Ba2+-sensitive currents with current-voltage relations typical of inward-rectifier K+ currents. These results support the characteristic ion channel properties of the canine SAN cells that we studied, because lack of IKl is characteristic of most mammalian SAN cells.11 In addition, we recorded Ba2+-sensitive currents in SAN cells isolated from ATP dogs. As shown by the results in supplemental Figure IID, no significant Ba2+-sensitive currents were detected, which indicates the absence of inward-rectifier current (IKl or constitutive IKACh) upregulation by ATP in SAN cells.
The slow delayed-rectifier current IKs plays important roles in SAN pacemaking in most species,11 and IKs β-subunit minK gene expression was downregulated. Figure 4A shows recordings of IKs in control and ATP cells. Both the step (Figure 4B) and tail (Figure 4C) currents were significantly reduced by ATP. For example, the IKs step-current density at 60 mV was reduced from 12.3±0.7 pA/pF in control to 8.8±0.7 pA/pF in ATP cells (P<0.001), whereas the tail current density was reduced from 2.9±0.3 pA/pF in control to 1.9±0.2 pA/pF in ATP (P<0.01). Voltage dependence of IKs activation (tail-current analysis) was not altered by tachypacing (Figure 4D), with control and ATP cells showing similar mean V1/2 values, which averaged 10.2±2.1 and 13.5±2.4 mV, respectively. The time courses of both step-current activation on depolarization to 60 mV and tail-current deactivation on repolarization from 60 to −40 mV were biexponential. Time constants corresponding to both the slow and fast components of step-current activation and tail-current deactivation showed no significant differences between control and ATP values, as illustrated in Figure 4E.
Results of rapid delayed-rectifier current recordings are shown in Figure 5. IKr tail currents were recorded during 4-second repolarizing pulses to −40 mV after a 2-second activating pulse to voltages between −40 and 70 mV. As shown in Figures 5A and 5B, IKr tail currents were small in canine SAN cells, both from dogs without and with ATP. Figure 5C shows mean tail-current density-voltage relations, which were unchanged by ATP.
Ca2+ currents are important in SAN pacemaking,11 and atrial tachyarrhythmias have been shown to change atrial ICaL, with both transcriptional and posttranscriptional mechanisms having been implicated.8 Accordingly, we compared ICaL and ICaT in SAN cells from control and ATP dogs. Original ICaL recordings are shown in Figure 6A. ICaL densities were comparable between ATP and control cells (Figure 6B). For example, at 10 mV, ICaL density averaged −4.6±0.6 pA/pF in control and −4.1±0.7 pA/pF in ATP cells, respectively. Original recordings corresponding to total ICa (including both ICaL and ICaT components) obtained on depolarization from −90 to −20 mV and recordings from the same cells that reflect ICaL without a contribution from ICaT (obtained by depolarization from −50 to −20 mV) are shown in Figure 6C. T-type current was obtained by subtracting currents recorded with a holding potential of −50 mV from current recorded with a holding potential of −90 mV, as described previously.9 ICaT was not present in all SAN cells but was found in a large and similar proportion (≈70%) of both control and ATP cells. No significant change in ICaT (Figure 6D) current density-voltage relations was produced by ATP; for example, ICaT at −20 mV was −1.4±0.4 for control versus −1.3±0.2 pA/pF for ATP cells.
Transmembrane Potential Simulations
Simulation of SAN cell action potentials provided the spontaneous activity shown by the blue curves in Figure 7. The results of different combinations of If and IKs remodeling are superimposed in specific colors. Remodeling of IKs alone was simulated by reproducing the same mean density decrease (35%) obtained in voltage-clamp recordings and did not change spontaneous SAN cell periodicity (CL 407.8 ms in control versus 407.7 ms with reduced IKs). A 50% decrease in If slowed periodic activity by increasing the CL ≈9%, which was a change of the same order as but slightly less than the increase in spontaneous CL seen with ATP (13.8%) in the absence of autonomic influences (vagotomy/nadolol). Simulation of the decrease in If and IKs together did not appreciably alter the slowing effect of simulated ATP compared with If effects alone (CL 443.6 ms with If reduction alone versus 443.4 ms with combined IKs/If reduction).
We have completed a detailed analysis of the functional and gene expression changes for selected ion channel subunits of SAN cells isolated from dogs subjected to 1 week of atrial tachycardia remodeling. The results indicate significant changes in the expression of specific subunits involved in SAN pacemaking, with alterations in If appearing to be particularly important for associated SAN dysfunction.
Mechanisms Underlying Reversible SAN Dysfunction in Tachycardia-Bradycardia Syndrome
Early studies implicated anatomic structural abnormalities in SAN dysfunction associated with atrial tachyarrhythmias, which suggests a fixed SAN dysfunction substrate1,3; however, several subsequent lines of evidence have pointed to an important functional, and potentially reversible, component. Elvan et al5 showed that electrically sustained AF over 2 to 6 weeks induced SAN dysfunction in parallel with atrial remodeling and that significant SAN recovery occurred within 1 week of AF cessation. These observations were confirmed by studies that showed that SAN dysfunction due to atrial tachycardia remodeling was fully reversed 4 weeks after tachycardia cessation.12 Termination of chronic atrial flutter in humans is followed by progressive improvement in SNRTc abnormalities over 3 weeks, which supports the applicability of the experimental findings to clinical tachyarrhythmias.6 Paroxysmal AF patients with prolonged (>3 seconds) sinus pauses on AF termination show progressive improvements in sinus node function after AF ablation, with a clinical evolution that indicates an absence of clinically significant SAN disease.7 The results obtained in the present study provide a potential ionic current mechanism to explain these experimental and clinical observations, based on atrial tachycardia-induced remodeling of SAN ion channel expression and function. If contributes to cellular automaticity by depolarizing cells toward their threshold potential, whereas IKs can contribute by accelerating phase 3 repolarization and advancing the time when the cell begins spontaneous phase 4 depolarization. Our mathematical modeling analyses suggest that the If changes caused by atrial tachycardia remodeling largely account for the associated SAN dysfunction. The lack of a significant role for IKs changes is likely due to the very positive activation potential for this current,9 which is not attained by SAN cells with their low resting potential and limited overshoot.11
Relationship to Previous Studies of Atrial Tachycardia Remodeling and Disease-Related SAN Dysfunction
Atrial ionic current remodeling due to sustained atrial tachycardia has been evaluated in detail. The principal changes include downregulation of ICaL8,9,13,14 and Ito8,9,14,15 and upregulation of inward-rectifier K+ currents.8,14–18 Atrial delayed-rectifier K+ current function is not altered by atrial tachycardia.8,9 Changes in atrial If function have not been described in atrial tachycardia remodeling. The profile of atrial tachycardia-induced SAN remodeling differs substantially from changes seen at the atrial level, being dominated by alterations in HCN subunits and If function, along with statistically significant changes in minK expression and IKs density. The tachycardia-induced downregulation of ICaL and upregulation of inward-rectifier K+ currents that are believed to be of great functional importance at the atrial level8 are not observed in SAN cells.
mRNA profiling in the present study showed some interesting differences between ATP-induced remodeling in SAN and RAFW. HCN subunits were downregulated only in SAN tissue, and Cx43 was downregulated only in RAFW. The IKs β-subunit minK was downregulated in both SAN and RAFW. Although we found SAN IKs to be downregulated by ATP in the present study, previous reports have not described corresponding changes in atrial tissue.9 Cav1.2 mRNA expression was not altered by ATP in either SAN or RAFW. The SAN result is consistent with unchanged SAN ICaL in the present study, but the atrial findings are discrepant with results of previous investigations of ATP-induced atrial remodeling.8 The reason for this discrepancy in atrial Cav1.2 mRNA changes is unclear and may relate to technical factors or the site of atrial sampling, but a detailed experimental analysis goes beyond the scope of the present study.
The basis for the differential atrial tachycardia remodeling response of atrial cardiomyocyte ionic currents versus those in SAN cells is unclear. Although we were unable to identify previous studies of SAN cell ion-channel remodeling with atrial tachycardia, Verkerk et al19 have analyzed in detail the changes in SAN ionic currents caused by congestive heart failure in rabbits with chronic pressure and volume overload. They found changes quite similar to those we noted here, with decreases in If and IKs and lack of change in the other currents they studied. The alterations that they observed were of the same order that we saw but were slightly smaller: ≈40% versus ≈50% decrease in If and ≈20% versus ≈35% decrease in IKs. They also performed mathematical modeling to assess the relative importance of If and IKs changes to altered SAN automaticity, and like us, they concluded that If downregulation is the principal contributor. Verkerk et al19 did not examine the molecular basis of the SAN ionic current remodeling they observed with congestive heart failure, but we subsequently studied HCN subunit expression changes in a canine ventricular-tachypaced congestive heart failure model and observed downregulation of HCN2 and HCN4 mRNA.20 The similarities in SAN ionic current changes that occur with congestive heart failure-induced and ATP-related remodeling are striking and may suggest a characteristic SAN ionic current response to pathological insults. A possible explanation for the lack of changes in SAN ICaL and inward-rectifier K+ currents could be that SAN cells are not fired at such high frequencies as atrial cardiomyocytes during AF, owing to SAN slow-channel properties that cause entry block into the central SAN and limit follow frequencies.21
Novelty and Potential Significance
The present study is the first of which we are aware to study changes in SAN ion channel subunit expression and ionic current function with atrial tachycardia remodeling. Our results provide novel insights into the fundamental mechanisms at the ionic and molecular level responsible for a clinically important phenomenon, the SAN dysfunction that is associated with atrial tachyarrhythmias. The importance of this problem has been underlined in a recent detailed review of SAN physiology in relation to sick sinus syndrome, with an absence of information about the underlying molecular/ionic basis being evident.22 The observation that HCN subunit downregulation underlies SAN dysfunction in the present experimental model of tachycardia-bradycardia syndrome, as it does in experimental congestive heart failure,19,20 provides further rationale for the development of cell/gene therapy approaches that involve HCN subunit expression enhancement for the management of clinical bradyarrhythmia syndromes.23
The present study is also the first to the best of our knowledge to study the properties of ionic currents in the canine SAN. The rabbit has been the species most commonly used for SAN cell isolation and study, but the dog has clear advantages in terms of widespread availability of clinically relevant pathological models. Kwong et al24 isolated cells of various morphologies from canine SAN preparations and described spider- and spindle-shaped cells as having unique connexin distribution properties that suggested a primary role in pacemaking function. The same group subsequently isolated cells with these morphologies from rabbit SANs and showed that they have prominent If-like currents, which are larger for the spider-type cells.25 In the present study, we confirmed the prominent If shown by these cell types in canine SAN, which contrasts with the lack of If that we noted in atrial cells. The present studies thus provide further evidence for the pacemaker-cell phenotype specialization of spider and spindle cells in the dog. Further studies of SAN cell pathophysiology in other canine models of human cardiac disease would be of potential interest.
The SAN origin of isolated cells and tissue preparations is always difficult to confirm with certainty. We isolated cells for study on the basis of SAN localization in the dog that we identified in previous studies,20 and we used well-described morphological criteria24,25 to define SAN-derived spider and spindle cells. The SAN preparations that we used for real-time PCR quantification of ion channel subunits also had subunit distribution properties typical of SAN: greater mRNA expression-levels of HCN2, HCN4, and MiRP1 subunits and lower expression levels of Cx43 than RA tissue.20,26
Although the present results are compelling evidence for a contribution of HCN/If downregulation to ATP-induced SAN dysfunction, we cannot exclude the possibility that other changes may contribute as well. We did not study the properties of all channels, ion transporters, and ion-handling systems in SAN tissue. In particular, there is recent evidence for an important contribution of sarcoplasmic reticulum Ca2+ uptake and release processes to cardiac pacemaking.27 Thus, changes in important components of the cellular Ca2+-handling machinery, including, for example, the Na+,Ca2+ exchanger, sarcoplasmic reticulum Ca2+ ATPase (SERCA), the ryanodine receptor, calsequestrin, and phospholamban, could have been changed by ATP and could contribute to altered SAN automaticity. A role for other ionic currents cannot be excluded, including Cl− currents such as the Ca2+-dependent, swelling-induced, and cAMP-regulated Cl− current; K+ currents such as the Ca2+-dependent K+ current; various 2-pore, 4-transmembrane domain channels; and nonselective cation channels. In addition, adrenergic and cholinergic regulation importantly modify If function and SAN automaticity. We cannot exclude a role for ATP-related changes in autonomic and associated G-protein-coupled regulation of If or other currents controlling SAN function. Nevertheless, we have succeeded in identifying congruent ionic current and channel subunit mRNA changes that are consistent with previous studies of SAN pathological remodeling and that on the basis of an ion-current-based SAN mathematical model account for a substantial portion of the SAN slowing that we observed. Finally, although the present results implicate HCN/If remodeling in ATP-induced SAN dysfunction, we did not study the underlying molecular mechanisms, which would be an appropriate objective for future studies.
The authors thank Nathalie L'Heureux and Chantal St-Cyr for technical assistance and France Thériault for secretarial support.
Sources of Funding
This study was supported by the Canadian Institutes of Health Research (Award MOP 44365), the Quebec Heart and Stroke Foundation, the Mathematics of Information Technology and Complex Systems (MITACS) Network of Centers of Excellence, and the European-North American Atrial Fibrillation Research Alliance (ENAFRA) network award from Fondation Leducq. Dr Burstein received a Canadian Institutes of Health Research (CIHR) MD/PhD studentship.
Kumagai K, Akimitsu S, Kawahira K, Kawanami F, Yamanouchi Y, Hiroki T, Arakawa K. Electrophysiological properties in chronic lone atrial fibrillation. Circulation. 1991; 84: 1662–1668.
Elvan A, Wylie K, Zipes DP. Pacing-induced chronic atrial fibrillation impairs sinus node function in dogs: electrophysiological remodeling. Circulation. 1996; 94: 2953–2960.
Sparks PB, Jayaprakash S, Vohra JK, Kalman JM. Electrical remodeling of the atria associated with paroxysmal and chronic atrial flutter. Circulation. 2000; 102: 1807–1813.
Hocini M, Sanders P, Deisenhofer I, Jais P, Hsu LF, Scavee C, Weerasoriya R, Raybaud F, Macle L, Shah DC, Garrigue S, Le Metayer P, Clementy J, Haissaguerre M. Reverse remodeling of sinus node function after catheter ablation of atrial fibrillation in patients with prolonged sinus pauses. Circulation. 2003; 108: 1172–1175.
Nattel S, Maguy A, Le Bouter S, Yeh YH. Arrhythmogenic ion-channel remodeling in the heart: heart failure, myocardial infarction, and atrial fibrillation. Physiol Rev. 2007; 87: 425–456.
Yue L, Feng J, Gaspo R, Li GR, Wang Z, Nattel S. Ionic remodeling underlying action potential changes in a canine model of atrial fibrillation. Circ Res. 1997; 81: 512–525.
Kurata Y, Hisatome I, Imanishi S, Shibamoto T. Dynamical description of sinoatrial node pacemaking: improved mathematical model for primary pacemaker cell. Am J Physiol Heart Circ Physiol. 2002; 283: H2074–H2101.
Van Wagoner DR, Pond AL, Lamorgese M, Rossie SS, McCarthy PM, Nerbonne JM. Atrial L-type Ca2+ currents and human atrial fibrillation. Circ Res. 1999; 85: 428–436.
Bosch RF, Zeng X, Grammer JB, Popovic K, Mewis C, Kühlkamp V. Ionic mechanisms of electrical remodeling in human atrial fibrillation. Cardiovasc Res. 1999; 44: 121–131.
Van Wagoner DR, Pond AL, McCarthy PM, Trimmer JS, Nerbonne JM. Outward K+ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circ Res. 1997; 80: 772–781.
Dobrev D, Graf E, Wettwer E, Himmel HM, Hála O, Doerfel C, Christ T, Schüler S, Ravens U. Molecular basis of downregulation of G-protein-coupled inward rectifying K+ current (I(K,ACh) in chronic human atrial fibrillation: decrease in GIRK4 mRNA correlates with reduced IK,ACh and muscarinic receptor-mediated shortening of action potentials. Circulation. 2001; 104: 2551–2557.
Dobrev D, Friedrich A, Voigt N, Jost N, Wettwer E, Christ T, Knaut M, Ravens U. The G protein-gated potassium current IK,ACh is constitutively active in patients with chronic atrial fibrillation. Circulation. 2005; 112: 3697–3706.
Cha TJ, Ehrlich JR, Chartier D, Qi XY, Xiao L, Nattel S. Kir3-based inward rectifier potassium current: potential role in atrial tachycardia remodeling effects on atrial repolarization and arrhythmias. Circulation. 2006; 113: 1730–1737.
Verkerk AO, Wilders R, Coronel R, Ravesloot JH, Verheijck EE. Ionic remodeling of sinoatrial node cells by heart failure. Circulation. 2003; 108: 760–766.
Zicha S, Fernández-Velasco M, Lonardo G, L'Heureux N, Nattel S. Sinus node dysfunction and hyperpolarization-activated (HCN) channel subunit remodeling in a canine heart failure model. Cardiovasc Res. 2005; 66: 472–481.
Kirchhof CJ, Allessie MA. Sinus node automaticity during atrial fibrillation in isolated rabbit hearts. Circulation. 1992; 86: 263–271.
Dobrzynski H, Boyett MR, Anderson RH. New insights into pacemaker activity: promoting understanding of sick sinus syndrome. Circulation. 2007; 115: 1921–1932.
Kwong KF, Schuessler RB, Green KG, Laing JG, Beyer EC, Boineau JP, Saffitz JE. Differential expression of gap junction proteins in the canine sinus node. Circ Res. 1998; 82: 604–612.
Wu J, Schuessler RB, Rodefeld MD, Saffitz JE, Boineau JP. Morphological and membrane characteristics of spider and spindle cells isolated from rabbit sinus node. Am J Physiol Heart Circ Physiol. 2001; 280: H1232–H1240.
Yu H, Wu J, Potapova I, Wymore RT, Holmes B, Zuckerman J, Pan Z, Wang H, Shi W, Robinson RB, El Maghrabi MR, Benjamin W, Dixon J, McKinnon D, Cohen IS, Wymore R. MinK-related peptide 1: a beta subunit for the HCN ion channel subunit family enhances expression and speeds activation. Circ Res. 2001; 88: E84–E87.
Sinoatrial node dysfunction is frequently associated with atrial tachyarrhythmias, and patients with the combination are said to suffer from the relatively common tachycardia-bradycardia syndrome. Abnormalities in sinus node pacemaker function on termination of atrial tachyarrhythmias such as atrial fibrillation can cause syncope and require pacemaker implantation, but the underlying mechanisms remain poorly understood. There is evidence from clinical and experimental studies that suggests that a significant component of sinus node dysfunction in patients with the tachycardia-bradycardia syndrome may actually be caused by supraventricular tachyarrhythmia and may be reversible if the tachyarrhythmia is controlled. The present study examined the hypothesis that very rapid atrial tachyarrhythmias can cause ion channel downregulation in the sinus node, thereby causing abnormal sinus node function. Dogs subjected to atrial tachypacing at 400 bpm for 7 days showed prolonged sinus node recovery time, which indicates sinus node dysfunction. Ion channel subunit messenger RNA expression was measured in sinus node tissue and showed downregulation by atrial tachycardia of 2 specific types of subunits: Those underlying the funny current, which is known to be particularly important in cardiac pacemaking activity, and an accessory subunit involved in the slow delayed-rectifier K+ channel. Patch-clamp studies on sinus node cells isolated from control dogs and dogs subjected to atrial tachypacing confirmed the specific downregulation of funny current and slow delayed-rectifier K+ current with atrial tachycardia. These alterations were incorporated in a mathematical model of sinus node electrical activity, which suggested that the funny current changes were the principal factor in sinus node suppression by atrial tachycardia. Our results provide insights into the molecular mechanisms underlying clinically significant bradycardic complications of this common and important clinical syndrome.
↵*The first 3 authors contributed equally to this work.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.789677/DC1.