PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of springeropenLink to Publisher's site
Pflugers Archiv
 
Pflugers Arch. 2010 August; 460(3): 593–601.
Published online 2010 June 16. doi:  10.1007/s00424-010-0851-z
PMCID: PMC2903684

Atrial arrhythmogenicity in aged Scn5a+/[increment]KPQ mice modeling long QT type 3 syndrome and its relationship to Na+ channel expression and cardiac conduction

Abstract

Recent studies have reported that human mutations in Nav1.5 predispose to early age onset atrial arrhythmia. The present experiments accordingly assess atrial arrhythmogenicity in aging Scn5a+/[increment]KPQ mice modeling long QT3 syndrome in relationship to cardiac Na+ channel, Nav1.5, expression. Atrial electrophysiological properties in isolated Langendorff-perfused hearts from 3- and 12-month-old wild type (WT), and Scn5a+/[increment]KPQ mice were assessed using programmed electrical stimulation and their Nav1.5 expression assessed by Western blot. Cardiac conduction properties were assessed electrocardiographically in intact anesthetized animals. Monophasic action potential recordings demonstrated increased atrial arrhythmogenicity specifically in aged Scn5a+/ΔKPQ hearts. These showed greater action potential duration/refractory period ratios but lower atrial Nav1.5 expression levels than aged WT mice. Atrial Nav1.5 levels were higher in young Scn5a+/ΔKPQ than young WT. These levels increased with age in WT but not Scn5a+/ΔKPQ. Both young and aged Scn5a+/ΔKPQ mice showed lower heart rates and longer PR intervals than their WT counterparts. Young Scn5a+/ΔKPQ mice showed longer QT and QTc intervals than young WT. Aged Scn5a+/ΔKPQ showed longer QRS durations than aged WT. PR intervals were prolonged and QT intervals were shortened in young relative to aged WT. In contrast, ECG parameters were similar between young and aged Scn5a+/ΔKPQ. Aged murine Scn5a+/ΔKPQ hearts thus exhibit an increased atrial arrhythmogenicity. The differing Nav1.5 expression and electrocardiographic indicators of slowed cardiac conduction between Scn5a+/ΔKPQ and WT, which show further variations associated with aging, may contribute toward atrial arrhythmia in aged Scn5a+/ΔKPQ hearts.

Keywords: LQT3 syndrome, Atrial arrhythmogenicity, Genetically modified mice, Na channels, Age, Arrhythmia, Sodium channel, Mouse, Electrophysiology, Excitation

Introduction

Atrial arrhythmia, of which the most common form is atrial fibrillation (AF), is particularly common in the elderly. Genetically normal patients show a median age of onset of AF of 75 years with ~70% of such patients aged between 65 and 85 years [13]. There have been recent studies implicating an involvement of cardiac ion channels in the development of AF [57]. Thus, long QT syndrome (LQTS) patients show an earlier than normal onset of AF [20], typically at age ~50 ± 14 years [9]. In particular, variants of the SCN5A gene encoding the cardiac Na+ channel (Nav1.5) have been clinically associated with the presence of AF. A study resequencing the SCN5A coding regions reported that 6% of 375 patients with AF showed variations in SCN5A [9]. Roles for Nav1.5 in the development of atrial arrhythmia are further implicated by the SCN5A polymorphism, H558R, prevalent in 20% of the population, also being a risk factor for AF [8].

Of the >200 such SCN5A mutations reported [40], the gain of function mutation Scn5a+/ΔKPQ involves deletion of the three conserved amino acids, KPQ1505–1507. It has been clinically associated with long QT syndrome type 3 (LQT3). This in turn is associated with a potentially fatal ventricular arrhythmogenic tendency that increases with age and that becomes evident within the first four decades of life [50, 51]. Loss of function mutations in SCN5A are associated with Brugada syndrome (BrS), a cardiac disorder characterized by an elevated ST segment in electrocardiographic (ECG) waveform [18]. Furthermore, a proportion of patients that harbor various losses and gain of function SCN5A mutations all demonstrate AF. Thus, 10–30% of BrS patients show increased propensities to atrial fibrillation [4, 26, 31, 32]. In contrast, Benito et al. [3] described a family, with eight members across three generations, showing both early onset AF and LQT3 associated with a mutant SCN5A gene involving the Y1795C mutation. Similarly, early onset AF in a Japanese family has been related to a novel gain-of-function, M1875T, mutation in SCN5A [45].

Such similarities in clinical outcome as a result of differing SCN5A mutations are consistent with reports of overlap syndrome in patients with LQT3 and BrS; conversely, some mutations can be associated with more than one phenotype in particular patients [42, 45]. For example, a single Na+ channel mutation involving deletion of lysine 1500, ΔK1500, is associated with not only LQTS but also BrS and conduction system disease [16]. Also the insertion D1795 induces both LQTS and BrS and has been shown to result in a 62% reduction of channel expression [2]. Finally, clinical phenotypes that overlap with those observed in BrS have also been reported to occur in patients carrying the SCN5A+/ΔKPQ mutation [35].

The physiological relationships between the underlying SCN5A+/ΔKPQ mutation and such related ventricular arrhythmic phenotypes have been investigated using a murine Scn5a+/ΔKPQ model [44]. Murine systems have also been used to study atrial arrhythmogenicity [25, 48]. However, very few experimental studies have related SCN5A mutations to alterations in atrial arrhythmogenicity. One such report described a reduced atrial arrhythmogenicity in Scn5a+/ΔKPQ murine hearts modeling LQT3 syndrome [10]. However, it did not investigate the effects of aging. Nevertheless, this is an important factor contributing to the variable penetrance of LQT3 [15].

This study accordingly proceeds to explore for the development of atrial arrhythmogenic properties with aging, comparing these to action potential waveform and refractory periods, Nav1.5 expression, and electrocardiographic properties in Scn5a+/[increment]KPQ and WT mice for the first time. We demonstrated that Nav1.5 protein expression was affected by age and genotype in a manner that would be compatible with a phenotypic overlap and attributed these findings to evidence for a compromised Nav1.5 function.

Methods

Experimental animals Wild-type (WT) and Scn5a+/[increment]KPQ mice, with an inbred 129/Sv genetic background, were housed in cages at 21 ± 1°C with 12 h light/dark cycles. The mice, studied at ages of either 3 months (young) and 12 months (aged), were killed by cervical dislocation, in compliance with the UK Animals (Scientific Procedures) Act 1986.

Isolation, cannulation, and perfusion of mouse hearts For the purpose of Na+ channel extraction and experiments on isolated Langendorff preparations, hearts were excised and immediately placed in ice-cold bicarbonate Krebs–Henseleit (KH) buffer solution containing (mM): 119 NaCl, 25 NaHCO3, 4.0 KCl, 1.2 KH2PO4, 1.0 MgCl2, 1.8 CaCl2, 10 glucose, and 2.0 sodium pyruvate (pH 7.4). The KH buffer was bubbled with 95% O2/5% CO2 (British Oxygen Company, Manchester, UK) for 20 min prior to addition of 1.8 mM CaCl2. The bubbling with 95% O2/5% CO2 was continued during the experiments themselves. A 2-mm length of the aorta was cannulated and held in place with an aneurysm clip. The hearts were perfused with KH buffer warmed to 37°C until they regained a healthy pink appearance and resumed spontaneous activity. Perfusion was continued for a further 10 min before either electrophysiological studies or protein extraction.

Monophasic action potential recording Hearts were excised and cannulated as described above, and AV nodes were ablated. Left atrial monophasic action potentials (MAPs) were recorded using a miniaturized MAP electrode tip (Linton Instruments, Harvard Apparatus, UK). A platinum stimulating electrode (1 mm inter-pole spacing) was placed on the right atrium. Square wave stimuli (Grass S48 stimulator, Grass-Telefactor, Slough, UK) of 2 ms duration and ×1.5 threshold were applied at a cycle length of 125 ms for 10 min until MAP waveforms had stable baselines, rapid upstroke phases, and smooth repolarization phases. Action potential durations were determined from regular pacing. Programmed electrical stimulation (PES) was used to assess arrhythmogenicity and refractoriness. This consisted of cycles of stimulus trains of eight S1 beats delivered at 8 Hz at ×1.5 threshold followed by an S2 extrastimulus, at progressively shorter S1S2 coupling intervals until the S2 no longer elicited an action potential. Arrhythmia was defined as three or more consecutive premature atrial waveforms.

Nav1.5 protein preparation After perfusion, left atrial appendages were removed and clamp-frozen. The tissue was homogenized in liquid nitrogen with a mortar and pestle on dry ice. Homogenates were resuspended in a solution consisting of (in mM) 50 Tris–HCl, 10 NaCl, 320 sucrose, 5 EDTA, and 2.5 EGTA (pH 7.4) and placed into Eppendorf tubes and gently resuspended with a pipette tip. Once thawed, but when still cool, the samples were placed on ice. Samples were blade homogenized for 20 s each, rinsing the blade thoroughly between samples. The preparation was lysed with 1% Triton X100 and rotated for 1 h at 4°C. After incubation, the samples were subjected to centrifugation at 12,000×g and 4°C. The supernatant was decanted into a sample loading buffer (NuPAGE, Invitrogen, Paisley, UK) to obtain a 1:4 dilution. Samples were boiled at 70°C for 10 min, flash frozen and stored at −80°C.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots Five micrograms of total protein was loaded per lane (NuPAGE gels) as was measured in triplicate with Bradford Assay (Bio-Rad Laboratories Ltd, Herts, UK) alongside a molecular weight marker (SeeBlue Plus prestained standard, Paisley, UK). Duplicate gels were subjected to Imperial Blue Protein stain (Thermo Scientific, Rockford, IL, USA) to confirm equal loading. NuPAGE electrophoresis tanks were used to run sodium dodecyl sulfate polyacrylamide gel electrophoresis at 100 mA for 3 h. Protein bands were transferred onto nitrocellulose membrane by a NuPAGE blotting system for 2 h. Membranes were blocked overnight in 5% milk–Tris-buffered saline–1% Tween, followed by several rinses prior to incubation with Nav1.5 antibody (1:500, ASC005, Caltag Medisystems, Alomone, Israel). The membranes were incubated with secondary antibody conjugated with horseradish peroxidase from Sigma-Aldrich (Poole, Dorset, UK) and rinsed five times. Western blot development was performed with Amersham ECL reagents (Amersham Biosciences, Amersham, Bucks, UK).

Electrophysiological study Mice were anesthetized with Avertin (Sigma) 240 mg/kg at a dose rate of 0.1 ml/10 g body weight. Injection was given intraperitoneally with a 27G hypodermic needle into the left peritoneal cavity. Lead II ECG recordings were acquired using LabChart software (ADI Instruments, Chalgrove, Oxfordshire, UK). This incorporated algorithms for determinations of ECG parameters, including RR intervals, P wave durations, PR intervals, QRS intervals and QT durations. It also included QTc intervals derived from the QT interval corrected for variations in the RR interval [19, 33]

Data analysis and statistics Data was acquired using a model 1401 interface, analysed with Spike version 5.2 (Cambridge Electronic Design, Cambridge, UK) and are presented as means ± standard errors of the means. Image J was used for densitometry (NIH, Bethesda, MD, USA). Comparisons were performed by a one-way analysis of variance (ANOVA) or Fisher’s exact test where appropriate, with SPSS software (SPSS UK, Woking, Surrey, UK). For all tests, young and aged mice of the same genotype were compared followed by tests between WT and Scn5a+/[increment]KPQ of the same age. Statistical significance was assumed at P < 0.05.

Results

The Scn5a+/[increment]KPQ mutation abolishes normal increases in atrial refractory periods with age and results in high action potential duration/atrial effective refractory period (APD/AERP) ratios

Figure 1 shows representative traces of atrial MAPs from young (3 months old) WT (a), young Scn5a+/ΔKPQ (b), aged (12 month) WT (c), and aged Scn5a+/ΔKPQ (d) atria, which show fast upstroke and smooth repolarization phases. APDs were measured from the interval between upstroke peak and 90% repolarization. WT and Scn5a+/ΔKPQ atrial preparations showed similar action potential durations (Fig. 2a). Thus, there were no significant differences in APD between young and aged hearts of the same genotype or between WT and Scn5a+/ΔKPQ hearts of the same age.

Fig. 1
Atrial monophasic action potentials (MAPs). Representative traces of hearts young (3 months old) WT (a), young Scn5a+/ΔKPQ (b), aged (12 month) WT (c) and aged Scn5a+/ΔKPQ (d) paced at 8 Hz
Fig. 2
Action potential durations (APDs) and atrial effective refractory periods (AERPS) (a) APD90 values of young WT, young Scn5a+/ΔKPQ, aged WT, and aged Scn5a+/ΔKPQ atria. b The effect of age and Scn5a+/ΔKPQ mutation on AERPs in WT ...

Figure 2b demonstrates that the AERP was prolonged in aged WT compared to young WT (P < 0.001), consistent with previous findings in murine hearts that atrial refractory periods increase with advancing age. This would be expected to offset any arrhythmogenic effects of corresponding decreases in conduction velocity known to occur with age [22, 23, 30]. In contrast, young and aged Scn5a+/ΔKPQ atria showed similar AERPs. Young WT and young Scn5a+/ΔKPQ hearts showed indistinguishable AERPs. However, AERPs in aged WT hearts were significantly prolonged compared to aged Scn5a+/ΔKPQ (P < 0.01; n = 7 in each group).

Figure 2c summarizes APD/AERP ratios, previously used to assess atrial arrhythmogenicity [10], derived from the APD and AERP findings summarized above. Young Scn5a+/ΔKPQ hearts showed increased APD/AERP ratios compared to young WT hearts (P < 0.05). However, whereas APD/AERP ratios decreased with age in WT (P < 0.01), these remained the same in Scn5a+/ΔKPQ hearts, with values approaching unity and significantly high ratios in aged Scn5a+/ΔKPQ relative to aged WT (P < 0.001). This was consistent with the increased arrhythmic tendency found specifically in aged Scn5a+/ΔKPQ observed in the experiments that followed.

Significant increases in atrial arrhythmogenic tendency are specific to aged Scn5a+/ΔKPQ mice

The presence or otherwise as well as the frequency of arrhythmic tendency in both WT and Scn5a+/ΔKPQ atrial preparations were compared using PES in isolated heart Langendorff-perfused hearts. The latter involved impositions of extrasystolic S2 stimuli at the end of trains of eight pacing S1 stimuli. The S1 stimuli were delivered at 8 Hz, and the S2 stimuli were delivered at S1S2 intervals that were progressively decremented by 1 ms with each successive pacing cycle. The protocols were terminated when hearts either became refractory or went into self-terminating arrhythmias. Figure 3a shows typical results from an aged Scn5a+/ΔKPQ heart in the course of such a procedure. Figure 3b exemplifies typical action potentials at the end of such a procedure showing refractoriness (arrowed). Figure 3c shows episodes of arrhythmias (arrowed).

Fig. 3
Assessment for arrhythmogenicity during S1S2 pacing of the left atria. A representative trace from an aged Scn5a+/ΔKPQ heart, paced at 8 Hz (S1), delivered at ×1.5 threshold (a). An example of atrial refractoriness with programmed ...

Table 1 summarizes the results of such experiments in which the observed incidences of atrial arrhythmia in young and aged, WT and Scn5a+/ΔKPQ hearts were compared using Fisher's exact tests, assuming significance with P < 0.05. It demonstrates that both young and aged WT hearts did not show significant incidences of arrhythmia. Similarly, both young WT and young Scn5a+/ΔKPQ showed indistinguishable incidences of arrhythmogenesis. In contrast, aged Scn5a+/ΔKPQ hearts showed significantly greater incidences of arrhythmia compared to both young Scn5a+/ΔKPQ (P < 0.001) and aged WT (P < 0.001).

Table 1
Incidence of atrial arrhythmia

The Scn5a+/ΔKPQ mutation results in abnormal changes in Na+ channel expression levels with age

Expression levels of the cardiac Na+ channel, Nav1.5, from atria of young and aged WT and Scn5a+/ΔKPQ mice were examined by Western blotting of randomized blinded samples. Nav1.5 was extracted from WT and Scn5a+/ΔKPQ hearts, and Western blots were performed with 5 μg of total protein per lane. Figure 4a shows blots of Nav1.5 obtained from atrial tissue of the young WT (labeled +/+; n = 3) and Scn5a+/ΔKPQ hearts (labeled +/k; n = 4 respectively). Figure 4b shows antibody specific binding to murine Nav1.5 from aged WT (n = 4) and Scn5a+/ΔKPQ (n = 4) hearts. Figure 4c shows the results of densitometric analysis that provided a relative quantification of Nav1.5.

Fig. 4
Cardiac Na+ channel, Nav1.5, expression. Western blots of Nav1.5 obtained from atrial tissue of young (3 months) WT (labeled +/+) and Scn5a+/ΔKPQ hearts (labeled +/k) (n = 3 and 4, respectively). Five micrograms of total ...

The young WT mice showed lower levels of Nav1.5 expression than did the aged WT, indicating increases in expression with aging (P < 0.01). In contrast, both young and aged Scn5a+/ΔKPQ atria contained similar levels of Nav1.5. Comparisons between genotypes similarly demonstrated that young Scn5a+/ΔKPQ mice showed higher Nav1.5 expression than did young WT (P < 0.05). Conversely, aged Scn5a+/ΔKPQ atria showed decreased Nav1.5 expression compared to aged WT (P < 0.05). Thus, Scn5a+/ΔKPQ atria failed to show the age dependent expression patterns demonstrated in WT mice.

Scn5a+/[increment]KPQ hearts show altered conduction properties

Figure 5 shows typical results from experiments that compared in vivo ECG features of WT and Scn5a+/[increment]KPQ hearts in intact mice anesthetized with Avertin. Each recording period followed a 10-min period permitting the preparation to stabilize and lasted 5 min. The PQRST complexes in the lead II ECG readings confirmed normal sequences of atrial activation and conduction, atrioventricular (AV) conduction and ventricular depolarization as well as recovery in all hearts whether in young (a) or aged WT (b), or young (c) or aged Scn5a+/ΔKPQ (d). No spontaneous arrhythmia was observed in any of the ECG recordings.

Fig. 5
Representative ECG recordings from young WT (a), young Scn5a+/ΔKPQ (b), aged WT (c), and aged Scn5a+/ΔKPQ (d) hearts respectively

Table 2 summarizes differences in electrocardiographic parameters and therefore in cardiac pacing and conduction function, expressed as means ± SEMs, between the different murine populations examined. It also summarizes the results of ANOVA tests of the electrocardiographic parameters, sorted by the single variables of genotype and age. This was followed by statistical tests for significant differences when these findings were sorted by both these variables. These demonstrated the following differences in pacemaker function and intracardiac conduction that corroborated the present findings, extending some of them to ventricular properties of the heart. Thus, (1) the Scn5a+/ΔKPQ mutation results in an inhibited SA node function. Thus, RR intervals were greater in young Scn5a+/ΔKPQ compared to young WT mice and aged Scn5a+/ΔKPQ compared to the aged WT mice. (2) Scn5a+/ΔKPQ and WT mice showed similar action potential durations as reflected in their P wave durations in agreement with MAP results. (3) AV conduction as assessed by PR intervals was consistently slower in Scn5a+/ΔKPQ than WT, when comparisons were made by age. It was also slower in aged Scn5a+/ΔKPQ compared to aged WT. (4) Intraventricular conduction, reflected in QRS intervals, was prolonged in the aged Scn5a+/ΔKPQ compared to aged WT but was similar between young Scn5a+/ΔKPQ and young WT. Finally, (5) ventricular action potential durations, assessed by QT intervals and QTc intervals, were longer in the young Scn5a+/ΔKPQ compared to the young WT hearts. Such intervals were similar between young and aged Scn5a+/ΔKPQ but increased with age in WT.

Table 2
ECG parameters compared in WT and Scn5a+/ΔKPQ

Discussion

The present experiments compared changes in basic electrophysiological properties and their relationship to the development or otherwise of atrial arrhythmogenic properties in aging murine Scn5a+/ΔKPQ and WT hearts. They extend previous reports on atrial [10] and ventricular [41, 44] arrhythmogenicity in Scn5a+/ΔKPQ hearts that, however, did not explore effects of aging on these properties. An initial determination of the normal changes in WT provided controls against which to assess the presence or absence of abnormal changes in the Scn5a+/ΔKPQ hearts. MAP records were first obtained from Langendorff-perfused hearts to explore for arrhythmogenic properties in response to both regular and extrasystolic imposed stimulation. These properties were then compared with the features of action potential recovery as reflected in their durations and refractory periods. The presence or absence of arrhythmogenicity was then related to the results of Western blot determinations of cardiac Na+ channel (Nav1.5) expression levels performed for the first time in the Scn5a+/ΔKPQ system. These in turn were related to electrocardiographic assessments of in vivo cardiac conduction properties in anesthetized animals.

The MAP studies in isolated Langendorff-perfused hearts assessed the frequencies of atrial arrhythmogenesis in young and aged, WT and Scn5a+/ΔKPQ, hearts using a PES procedure. They demonstrated for the first time that aged Scn5a+/ΔKPQ hearts showed an increased atrial arrhythmogenicity compared to any of the other three groups. This took place despite an absence of any detectable differences in atrial action potential durations to 90% recovery (APD). The latter finding rules out arrhythmogenic mechanisms involving action potential recovery previously implicated in the ventricular arrhythmogenicity shown by Scn5a+/ΔKPQ on earlier occasions [46]. Whereas refractory periods in young WT and young Scn5a+/ΔKPQ were closely similar, they sharply increased with age in WT, consistent with previous reports [23]; however, they did not do so in Scn5a+/ΔKPQ. Consequently, Scn5a+/ΔKPQ hearts showed shorter refractory periods and greater APD/AERP ratios than aged WT. Both of these have been previously introduced as indices of ventricular [41] as well as atrial [10] arrhythmogenicity, respectively. These results were thus consistent with the higher inducibility of atrial arrhythmias by programmed atrial stimulation that was observed in aged Scn5a/ΔKPQ mice.

These findings were then compared with results from complementary examinations of Nav1.5 protein expression in the experimental groups. Such Western blot studies directly measure specific protein levels as opposed to indirect measures involving messenger RNA (mRNA). These demonstrated that Nav1.5 channel expression increased with age and genotype, complementing previous electrophysiological reports of altered cardiac Ca2+ and K+ channel expression with age in WT [11, 21]. Thus, young WT showed relatively low levels of Nav1.5 expression, but these markedly increased with normal aging. In contrast, young Scn5a+/ΔKPQ showed higher Nav1.5 expression than young WT but then showed no further increases with age. Aged Scn5a+/ΔKPQ consequently showed substantially lower Nav1.5 expression than aged WT.

The latter findings were compatible with the final, electrocardiographic, results. These similarly revealed genotype- and age-specific phenotypic alterations in intact anesthetized WT and Scn5a+/ΔKPQ mice. Firstly, young Scn5a+/ΔKPQ mice showed longer QT and QTc intervals than did young WT, consistent with the LQT3 condition. Secondly, even young WT and young Scn5a+/ΔKPQ mice differed in those ECG features that bore on atrial pacemaker and AV conduction properties. Young and aged Scn5a+/ΔKPQ mice had lower heart rates and slower AV conduction than their respective WT counterparts. In addition, aged Scn5a+/ΔKPQ mice showed depressed intra-ventricular conduction relative to aged WT. Finally, WT and Scn5a+/ΔKPQ also showed differing alterations with age. Thus, while PR and QT intervals change with age in WT mice, this was not evident in Scn5a+/ΔKPQ hearts.

Comparisons between these findings were consistent with a hypothesis in which small APD/AERP ratios together with high levels of Nav1.5 expression might balance the arrhythmic effects of a slowing of conduction with age. This could be related to a progressive interstitial fibrosis that has been shown to occur in the atria of humans [12, 43] and animals [1, 17]; the latter includes mice with mutations involving Scn5a [39]. Such fibrosis is also associated with clinical AF [24, 29]. In addition, localized alterations in AERP shown to increase with age would be expected to accentuate dispersions of intra-atrial refractoriness [49]. All these processes would be expected to cause a heterogeneous slowing of atrial conduction and consequent age-related increases in atrial arrhythmic tendency.

In such a situation, the observed increases in Nav1.5 could protect against atrial arrhythmia in aged WT. Thus, WT hearts were not arrhythmogenic: APD/AERP ratios were smaller for aged WT than young WT, while there was a higher Nav1.5 expression in aged WT relative to young WT. In contrast, aged Scn5a+/ΔKPQ were more arrhythmogenic than the remaining groups. This was consistent with their higher APD/AERP ratios and reduced Nav1.5 expression levels relative to aged WT. Finally, these results additionally relate the greater arrhythmogenecity of aged Scn5a+/ΔKPQ compared to young Scn5a+/ΔKPQ, despite similar APD/AERP ratios and AERP values, to their failure to increase Nav1.5 expression with age unlike WT. Thus, despite possessing a gain of function mutation, aged Scn5a+/ΔKPQ atria showed reduced Nav1.5 expression levels and high APD/AERP ratios. This may have resulted in an atrial arrhythmogenicity that was prevented by the increase in Nav1.5 expression with age shown by the WT.

Such changes could also involve altered expression of a range of genes and ion channels, besides Nav1.5. Thus, ventricular mRNA expression profiles in studies of human BrS associated with Na+ channelopathy suggested remodeled K+ and Ca2+ in addition to Na+-channel expression [14]. For example, their reduced expression of Kv4.3 encoding the principal Ito subunit, increased transcript expression of Nav2.1, and increased 2P-channel expression would tend to compensate for the Nav1.5 underexpression observed in BrS-affected individuals. Conditions of atrial fibrillation or tachycardia results in reductions of transient outward K+ current (Ito), downregulation of ICaL pore-forming α-subunit mRNA, and increased expression of Kir2.1 mRNA and protein [36]. Furthermore, there is evidence associating BrS with such atrial remodeling [47].

In demonstrating that aged Scn5a+/ΔKPQ mice show an increased propensity to atrial arrhythmia, the findings in this study thus recapitulate clinical situations of phenotypic overlap between LQTS and BrS. A loss of Nav1.5 function may reflect haploinsufficiency and impaired intracellular trafficking reducing surface level expression [38]. The reduction in Nav1.5 protein expression observed here is compatible with such a possibility. This in turn may be the cause of a situation resembling the case of the Brugada mouse model in which a loss of Na+ channel expression results in both ventricular [37] and atrial arrhythmogenic properties [27, 28]. Such a notion would be compatible with the electrocardiographic evidence for reduced pacemaker activity and delayed conduction in both young and aged Scn5a+/ΔKPQ relative to WT hearts. These also parallel reduced SA node activity and slowed AV conduction that has been reported in BrS patients [34]. Taken together, these findings could therefore account for phenotypic overlaps between LQT3 and BrS patients [42] besides demonstrating the existence of a complex dynamic interplay between aging, ion channel expression, and cardiac tissue excitability.

Acknowledgements

This study was supported by grants from the Wellcome Trust and the British Heart Foundation.

Conflicts of interest The authors declare that they have no conflict of interest.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

References

1. Anyukhovsky EP, Sosunov EA, Plotnikov A, Gainullin RZ, Jhang JS, Marboe CC, Rosen MR. Cellular electrophysiologic properties of old canine atria provide a substrate for arrhythmogenesis. Cardiovasc Res. 2002;54:462–469. doi: 10.1016/S0008-6363(02)00271-7. [PubMed] [Cross Ref]
2. Baroudi G, Chahine M. Biophysical phenotypes of SCN5A mutations causing long QT and Brugada syndromes. FEBS Lett. 2000;487:224–228. doi: 10.1016/S0014-5793(00)02360-7. [PubMed] [Cross Ref]
3. Benito B, Brugada R, Perich RM, Lizotte E, Cinca J, Mont L, Berruezo A, Tolosana JM, Freixa X, Brugada P, Brugada J. A mutation in the sodium channel is responsible for the association of long QT syndrome and familial atrial fibrillation. Heart Rhythm. 2008;5:1434–1440. doi: 10.1016/j.hrthm.2008.07.013. [PubMed] [Cross Ref]
4. Bordachar P, Reuter S, Garrigue S, Caï X, Hocini M, Jaïs P, Haïssaguerre M, Clementy J. Incidence, clinical implications and prognosis of atrial arrhythmias in Brugada syndrome. Eur Heart J. 2004;25:879–884. doi: 10.1016/j.ehj.2004.01.004. [PubMed] [Cross Ref]
5. Brundel BJJM, Henning RH, Kampinga HH, Gelder IC, Crijns HJGM. Molecular mechanisms of remodeling in human atrial fibrillation. Cardiovasc Res. 2002;54:315–324. doi: 10.1016/S0008-6363(02)00222-5. [PubMed] [Cross Ref]
6. Brundel BJJM, Gelder IC, Henning RH, Tuinenburg AE, Wietses M, Grandjean JG, Wilde A, Gilst WH, Crijns HJGM. Alterations in potassium channel gene expression in atria of patients with persistent and paroxysmal atrial fibrillation: differential regulation of protein and mRNA levels for K+ channels. J Am Coll Cardiol. 2001;37:926–932. doi: 10.1016/S0735-1097(00)01195-5. [PubMed] [Cross Ref]
7. Brundel BJJM, Gelder IC, Henning RH, Tieleman RG, Tuinenburg AE, Wietses M, Grandjean JG, Gilst WH, Crijns HJGM. Ion channel remodeling is related to intraoperative atrial effective refractory periods in patients with paroxysmal and persistent atrial fibrillation. Circulation. 2001;103:684–690. [PubMed]
8. Chen LY, Ballew JD, Herron KJ, Rodeheffer RJ, Olson TM. A common polymorphism in SCN5A is associated with lone atrial fibrillation. Clin Pharmacol Ther. 2007;81:35–41. doi: 10.1038/sj.clpt.6100016. [PMC free article] [PubMed] [Cross Ref]
9. Darbar D, Kannankeril PJ, Donahue BS, Kucera G, Stubblefield T, Haines JL, George AL, Jr, Roden DM. Cardiac sodium channel (SCN5A) variants associated with atrial fibrillation. Circulation. 2008;117:1927–1935. doi: 10.1161/CIRCULATIONAHA.107.757955. [PMC free article] [PubMed] [Cross Ref]
10. Dautova Y, Zhang Y, Sabir I, Grace AA, Huang CL-H. Atrial arrhythmogenesis in wild-type and Scn5a+/Δ murine hearts modelling LQT3 syndrome. Pflügers Arch Eur J Physiol. 2009;458:1432–2013. [PMC free article] [PubMed]
11. Dun W, Boyden P. Aged atria: electrical remodeling conducive to atrial fibrillation. J Interv Card Electrophysiol. 2009;25:9–18. doi: 10.1007/s10840-008-9358-3. [PubMed] [Cross Ref]
12. Everett TH, 4th, Olgin JE. Atrial fibrosis and the mechanisms of atrial fibrillation. Heart Rhythm. 2007;4:S24–S27. doi: 10.1016/j.hrthm.2006.12.040. [PMC free article] [PubMed] [Cross Ref]
13. Fuster V, Rydén L, Cannom D, Crijns H, Curtis A, Ellenbogen K, Halperin J, Heuzey J, Kay G, Lowe J, Olsson S, Prystowsky E, Tamargo J, Wann S, Smith SJ, Jacobs A, Adams C, Anderson J, Antman E, Halperin J, Hunt S, Nishimura R, Ornato J, Page R, Riegel B, Priori S, Blanc J, Budaj A, Camm A, Dean V, Deckers J, Despres C, Dickstein K, Lekakis J, McGregor K, Metra M, Morais J, Osterspey A, Tamargo J, Zamorano J. ACC/AHA/ESC 2006 guidelines for the management of patients with atrial fibrillation: a report of the American College of Cardiology/American Heart Association Task Force on practice guidelines and the European Society of Cardiology Committee for practice guidelines (writing committee to revise the 2001 guidelines for the management of patients with atrial fibrillation): developed in collaboration with the European Heart Rhythm Association and the Heart Rhythm Society. Circulation. 2006;114:e257–e354. doi: 10.1161/CIRCULATIONAHA.106.177292. [PubMed] [Cross Ref]
14. Gaborit N, Wichter T, Varro A, Szuts V, Lamirault G, Eckardt L, Paul M, Breithardt G, Schulze-Bahr E, Escande D, Nattel S, Demolombe S. Transcriptional profiling of ion channel genes in Brugada syndrome and other right ventricular arrhythmogenic diseases. Eur Heart J. 2009;30:487–496. doi: 10.1093/eurheartj/ehn520. [PubMed] [Cross Ref]
15. Goldenberg I, Moss AJ, Bradley J, Polonsky S, Peterson DR, McNitt S, Zareba W, Andrews ML, Robinson JL, Ackerman MJ, Benhorin J, Kaufman ES, Locati EH, Napolitano C, Priori SG, Qi M, Schwartz PJ, Towbin JA, Vincent GM, Zhang L. Long-QT syndrome after age 40. Circulation. 2008;117:2192–2201. doi: 10.1161/CIRCULATIONAHA.107.729368. [PubMed] [Cross Ref]
16. Grant AO, Carboni MP, Neplioueva V, Starmer F, Memmi M, Napolitano C, Priori SG. Long QT syndrome, Brugada syndrome, and conduction system disease are linked to a single sodium channel mutation. J Clin Investig. 2002;110:1201–1209. [PMC free article] [PubMed]
17. Hayashi H, Wang C, Miyauchi Y, Omichi C, Pak HN, Zhou S, Ohara T, Mandel WJ, Lin SF, Fishbein MC, Chen PS, Karagueuzian HS. Age-related increase to inducible atrial fibrillation in the rat model. J Cardiovasc Electrophysiol. 2002;13:801–808. doi: 10.1046/j.1540-8167.2002.00801.x. [PubMed] [Cross Ref]
18. Hedley PL, Jørgensen P, Schlamowitz S, Moolman-Smook J, Kanters JK, Corfield VA, Christiansen M. The genetic basis of Brugada syndrome: a mutation update. Hum Mutat. 2009;30:1256–1266. doi: 10.1002/humu.21066. [PubMed] [Cross Ref]
19. Jeevaratnam K, Zhang Y, Guzadhur L, Duehmke RM, Lei M, Grace AA, Huang CL (2010) Differences in sino-atrial and atrio-ventricular function with age and sex attributable to the Scn5a mutation in a murine cardiac model. Acta Physiol. PMID 20331542 [PubMed]
20. Johnson JN, Tester DJ, Perry J, Salisbury BA, Reed CR, Ackerman MJ. Prevalence of early-onset atrial fibrillation in congenital long QT syndrome. Heart Rhythm. 2008;5:704–709. doi: 10.1016/j.hrthm.2008.02.007. [PubMed] [Cross Ref]
21. Jones SA, Boyett MR, Lancaster MK. Declining into failure: the age-dependent loss of the L-type calcium channel within the sinoatrial node. Circulation. 2007;115:1183–1190. [PubMed]
22. Kistler P, Sanders P, Fynn S, Stevenson I, Spence S, Vohra J, Sparks P, Kalman J. Electrophysiologic and electroanatomic changes in the human atrium associated with age. J Am Coll Cardiol. 2004;44:109–116. doi: 10.1016/j.jacc.2004.03.044. [PubMed] [Cross Ref]
23. Kojodjojo P, Kanagaratnam P, Markides V, Davies DW, Peters N. Age-related changes in human left and right atrial conduction. J Cardiovasc Electrophysiol. 2006;17:120–127. doi: 10.1111/j.1540-8167.2005.00293.x. [PubMed] [Cross Ref]
24. Kostin S, Klein G, Szalay Z, Hein S, Bauer EP, Schaper J. Structural correlate of atrial fibrillation in human patients. Cardiovasc Res. 2002;54:462–469. doi: 10.1016/S0008-6363(02)00273-0. [PubMed] [Cross Ref]
25. Kovoor P, Wickman K, Maguire C, Pu W, Gehrmann J, Berul C, Clapham D. Evaluation of the role of I(KACh) in atrial fibrillation using a mouse knockout model. J Am Coll Cardiol. 2001;37:2136–2143. doi: 10.1016/S0735-1097(01)01304-3. [PubMed] [Cross Ref]
26. Kusano K, Taniyama M, Nakamura K, Miura D, Banba K, Nagase S, Morita H, Nishii N, Watanabe A, Tada T, Murakami M, Miyaji K, Hiramatsu S, Nakagawa K, Tanaka M, Miura A, Kimura H, Fuke S, Sumita W, Sakuragi S, Urakawa S, Iwasaki J, Ohe T. Atrial fibrillation in patients with Brugada syndrome relationships of gene mutation, electrophysiology, and clinical backgrounds. J Am Coll Cardiol. 2008;51:1169–1175. doi: 10.1016/j.jacc.2007.10.060. [PubMed] [Cross Ref]
27. Lei M, Goddard C, Liu J, Léoni A-L, Royer A, Fung SSM, Xiao G, Ma A, Zhang H, Charpentier F, Vandenberg JI, Colledge WH, Grace AA, Huang CL-H. Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene, SCN5A. J Physiol. 2005;567:387–400. doi: 10.1113/jphysiol.2005.083188. [PubMed] [Cross Ref]
28. Lei M, Huang CLH, Zhang Y. Genetic Na+ channelopathies and sinus node dysfunction. Prog Biophys Mol Biol. 2008;98(2–3):171–178. doi: 10.1016/j.pbiomolbio.2008.10.003. [PubMed] [Cross Ref]
29. Li D, Fareh S, Leung TK, Nattel S. Promotion of atrial fibrillation by heart failure in dogs: atrial remodeling of a different sort. Circulation. 1999;100:87–95. [PubMed]
30. Maguire C, Bevilacqua L, Wakimoto H, Gehrmann J, Berul C. Maturational atrioventricular nodal physiology in mouse. J Cardiovasc Electrophysiol. 2000;11:557–564. doi: 10.1111/j.1540-8167.2000.tb00009.x. [PubMed] [Cross Ref]
31. Makiyama T, Akao M, Shizuta S, Doi T, Nishiyama K, Oka Y, Ohno S, Nishio Y, Tsuji K, Itoh H, Kimura T, Kita T, Horie M. A novel SCN5A gain-of-function mutation M1875T associated with familial atrial fibrillation. J Am Coll Cardiol. 2008;52:1326–1334. doi: 10.1016/j.jacc.2008.07.013. [PubMed] [Cross Ref]
32. Makiyama T, Akao M, Tsuji K, Doi T, Ohno S, Takenaka K, Kobori A, Ninomiya T, Yoshida H, Takano M, Makita N, Yanagisawa F, Higashi Y, Takeyama Y, Kita T, Horie M. High risk for bradyarrhythmic complications in patients with Brugada syndrome caused by SCN5A gene mutations. J Am Coll Cardiol. 2005;46:2100–2106. doi: 10.1016/j.jacc.2005.08.043. [PubMed] [Cross Ref]
33. Mitchell GF, Jeron A, Koren G. Measurement of heart rate and Q-T interval in the conscious mouse. Am J Physiol. 1998;274:H747–H751. [PubMed]
34. Morita H, Kusano-Fukushima K, Nagase S, Fujimoto Y, Hisamatsu K, Fujio H, Haraoka K, Kobayashi M, Morita ST, Nakamura K, Emori T, Matsubara H, Hina K, Kita T, Fukatani M, Ohe T. Atrial fibrillation and atrial vulnerability in patients with Brugada syndrome. J Am Coll Cardiol. 2002;40:1437–1444. doi: 10.1016/S0735-1097(02)02167-8. [PubMed] [Cross Ref]
35. Moss A, Windle J, Hall W, Zareba W, Robinson J, McNitt S, Severski P, Rosero S, Daubert J, Qi M, Cieciorka M, Manalan A. Safety and efficacy of flecainide in subjects with Long QT-3 syndrome (DeltaKPQ mutation): a randomized, double-blind, placebo-controlled clinical trial. Ann Noninvasive Electrocardiol. 2005;10:59–66. doi: 10.1111/j.1542-474X.2005.00077.x. [PubMed] [Cross Ref]
36. Nattel S, Burstein B, Dobrev D. Atrial remodeling and atrial fibrillation: mechanisms and implications. Circulation Arrhythm Electrophysiol. 2008;1:62–73. doi: 10.1161/CIRCEP.107.754564. [PubMed] [Cross Ref]
37. Papadatos GA, Wallerstein PMR, Ratcliff R, Head CE, Huang CLH, Saumarez RC, Colledge WH, Grace AA. Slowed conduction and ventricular tachycardia following targeted disruption of the cardiac sodium channel Scn5a. Proc Nat Acad Sci. 2002;99:6210–6215. doi: 10.1073/pnas.082121299. [PubMed] [Cross Ref]
38. Pfahnl AE, Viswanathan PC, Weiss R, Shang LL, Sanyal S, Shusterman V, Kornblit C, London B, Dudley JSC. A sodium channel pore mutation causing Brugada syndrome. Heart Rhythm. 2007;4:46–53. doi: 10.1016/j.hrthm.2006.09.031. [PMC free article] [PubMed] [Cross Ref]
39. Royer A, Veen TA, Bouter S, Marionneau C, Griol-Charhbili V, Leoni AL, Steenman M, Rijen HV, Demolombe S, Goddard CA, Richer C, Escoubet B, Jarry-Guichard T, Colledge WH, Gros D, Bakker JM. Mouse model of SCN5A-linked hereditary Lenegre’s disease: age-related conduction slowing and myocardial fibrosis. Circulation. 2005;111:1738–1746. doi: 10.1161/01.CIR.0000160853.19867.61. [PubMed] [Cross Ref]
40. Ruan Y, Liu N, Priori SG. Sodium channel mutations and arrhythmias. Nat Rev Cardiol. 2009;6:337–348. doi: 10.1038/nrcardio.2009.44. [PubMed] [Cross Ref]
41. Sabir I, Li L, Jones V, Goddard CA, Grace AA, Huang CLH. Criteria for arrhythmogenicity in genetically-modified Langendorff-perfused murine hearts modelling the congenital long QT syndrome type 3 and the Brugada syndrome. Pflügers Arch Eur J Physiol. 2008;455:637–651. [PMC free article] [PubMed]
42. Schwartz PJ, Priori SG, Spazzolini C, Moss AJ, Vincent GM, Napolitano C, Denjoy I, Guicheney P, Breithardt G, Keating MT, Towbin JA, Beggs AH, Brink P, Wilde AAM, Toivonen L, Zareba W, Robinson JL, Timothy KW, Corfield V, Wattanasirichaigoon D, Corbett C, Haverkamp W, Schulze-Bahr E, Lehmann MH, Schwartz K, Coumel P, Bloise R. Genotype–phenotype correlation in the long-QT syndrome: gene-specific triggers for life-threatening arrhythmias. Circulation. 2001;103:89–95. [PubMed]
43. Spach MS, Dolber PC. Relating extracellular potentials and their derivatives to anisotropic propagation at a microscopic level in human cardiac muscle. Evidence for electrical uncoupling of side-to-side fiber connections with increasing age. Circ Res. 1986;58:356–371. [PubMed]
44. Stokoe KS, Thomas G, Goddard CA, Colledge WH, Grace AA, Huang CL-H. Effects of flecainide and quinidine on arrhythmogenic properties of SCN5A+/Δ murine hearts modelling long QT syndrome 3. J Physiol. 2006;578:69–84. doi: 10.1113/jphysiol.2006.117945. [PubMed] [Cross Ref]
45. Takeru M, Masaharu A, Satoshi S, Takahiro D, Kei N, Yuko O, Seiko O, Yukiko N, Keiko T, Hideki I, Takeshi K, Toru K, Minoru H. A novel SCN5A gain-of-function mutation M1875T associated with familial atrial fibrillation. J Am Coll Cardiol. 2008;52:1326–1334. doi: 10.1016/j.jacc.2008.07.013. [PubMed] [Cross Ref]
46. Thomas G, Killeen MJ, Grace AA, Huang CLH. Pharmacological separation of early afterdepolarizations from arrhythmogenic substrate in delta KPQ Scn5a murine hearts modelling human long QT 3 syndrome. Acta Physiol. 2008;192:505–517. doi: 10.1111/j.1748-1716.2007.01770.x. [PMC free article] [PubMed] [Cross Ref]
47. Toh N, Morita H, Nagase S, Taniguchi M, Miura D, Nishii N, Nakamura K, Ohe T, Kusano KF, Ito H. Atrial electrophysiological and structural remodeling in high-risk patients with Brugada syndrome: assessment with electrophysiology and echocardiography. Heart Rhythm. 2010;7:218–224. doi: 10.1016/j.hrthm.2009.10.035. [PubMed] [Cross Ref]
48. Wakimoto H, Maguire CT, Kovoor P, Hammer PE, Gehrmann J, Triedman JK, Berul CI. Induction of atrial tachycardia and fibrillation in the mouse heart. Cardiovasc Res. 2001;50:463–473. doi: 10.1016/S0008-6363(01)00264-4. [PubMed] [Cross Ref]
49. Xu D, Murakoshi N, Tada H, Igarashi M, Sekiguchi Y, Aonuma K. Age-related increase in atrial fibrillation induced by transvenous catheter-based atrial burst pacing: an in vivo rat model of inducible atrial fibrillation. J Cardiovasc Electrophysiol. 2010;21:88–93. doi: 10.1111/j.1540-8167.2009.01591.x. [PubMed] [Cross Ref]
50. Zareba W, Moss AJ, Locati EH, Lehmann MH, Peterson DR, Hall WJ, Schwartz PJ, Vincent GM, Priori SG, Benhorin J, Towbin JA, Robinson JL, Andrews ML, Napolitano C, Timothy K, Zhang L. Modulating effects of age and gender on the clinical course of long QT syndrome by genotype. J Am Coll Cardiol. 2003;42:103–109. doi: 10.1016/S0735-1097(03)00554-0. [PubMed] [Cross Ref]
51. Zareba W, Moss AJ, Schwartz PJ, Vincent GM, Robinson JL, Priori SG, Benhorin J, Locati EH, Towbin JA, Keating MT, Lehmann MH, Hall WJ, Andrews ML, Napolitano C, Timothy K, Zhang L, Medina A, MacCluer JW, The International Long QTSRRG Influence of the genotype on the clinical course of the long-qt syndrome. N Engl J Med. 1998;339:960–965. doi: 10.1056/NEJM199810013391404. [PubMed] [Cross Ref]

Articles from Springer Open Choice are provided here courtesy of Springer