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RELATED PAPERS |
murine hearts modelling long QT syndrome 3
1 Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK
2 Section of Cardiovascular Biology, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK
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Abstract |
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) murine hearts modelling LQT3. Extra stimuli (S2) following trains of pacing stimuli (S1) applied to the right ventricular epicardium triggered ventricular tachycardia (VT) in 16 out of 28 untreated Scn5a+/ and zero out of 12 WT hearts. Paced electrogram fractionation analysis then demonstrated increased electrogram durations (EGD), expressed as EGD ratios, in arrhythmogenic Scn5a+/ hearts, and prolonged ventricular effective refractory periods in initially non-arrhythmogenic Scn5a+/ hearts. Nevertheless, comparisons of epicardial and endocardial monophasic action potential recordings demonstrated negative transmural repolarization gradients in both groups, giving APD90 values at 90% repolarization of –20.88 ± 1.93 ms (n
= 11) and –16.91 ± 1.43 ms (n
= 23), respectively. Flecainide prevented initiation of VT in 13 out of 16 arrhythmogenic Scn5a+/ hearts, reducing EGD ratio and restoring APD90 to + 7.55 ± 2.24 ms (n
= 9) (P < 0.05). VT occurred in four out of eight non-arrhythmogenic Scn5a+/ hearts in the presence of quinidine, which increased EGD ratio but left APD90 unchanged. In contrast (P < 0.05), WT hearts had positive APD90 values (+ 11.72 ± 2.17 ms) (n
= 20). Flecainide then increased arrhythmic tendency and EGD ratio but conserved APD90; reduced EGD ratios and unaltered APD90 values accompanied the lower arrhythmogenicity associated with quinidine treatment. In addition to the changes in EGD ratio shown by WT hearts, these findings attribute arrhythmogenesis and its modification by flecainide and quinidine to alterations in APD90 in Scn5a+/ hearts. This is consistent with a hypothesis in which incomplete Na+ channel inactivation in Scn5a+/ hearts generates functional substrates dependent on altered refractoriness that cause abnormalities in activation and conduction of subsequent cardiac impulses. Any spatial heterogeneities between the epicardial and endocardial layers would thus cause fragmentation of the activation wavefront and contribute to electrogram spreading.
(Received 27 July 2006;
accepted after revision 3 October 2006;
first published online 5 October 2006)
Corresponding author C. L.-H. Huang: Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK. Email: clh11{at}cam.ac.uk
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Introduction |
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-subunit of the human cardiac voltage-gated Na+ channel are associated with conditions such as the long QT(3) (LQT3) and Brugada syndromes (BrS) (Grant et al. 2002; Shimizu et al. 2005). LQT3 accompanies ‘gain-of-function’ mutations of SCN5A; abnormal prolongation of repolarization due to incomplete Na+ channel inactivation prolongs the plateau phase of the cardiac action potential (AP) and lengthens the electrocardiographic QT interval (Bennett et al. 1995; Kass, 1997; Wang et al. 1995). LQT3 predisposes to ventricular arrhythmias and sudden cardiac death (SCD) particularly at rest or during sleep (Schwartz et al. 2001). In contrast, ‘loss-of-function’ mutations of SCN5A can result in conditions such as BrS in which peak INa is reduced, shortening the plateau phase of the AP and causing ST-segment elevation in the right precordial leads (Alings & Wilde, 1999; Brugada & Roberts, 2001). BrS is also thought to be responsible for a large proportion of the SCDs occurring in the absence of demonstrable structural cardiac disease (Brugada et al. 2003). The existence of such Na+ channel variants has potential implications for the electrophysiological action of specific cardiotropic drugs. For example, both the class Ic agent flecainide (Ramos & O'Leary, 2004) and the class Ia agent quinidine are used in the management of atrial fibrillation (AF) (Page & Roden, 2005; Veloso & de Paola, 2005). However, flecainide binds particularly strongly to activated, mutant Na+ channels such as those implicated in LQT3 and BrS (Abriel et al. 2000; Nagatomo et al. 2000; Liu et al. 2002). In LQT3, flecainide exerts antiarrhythmic effects (Windle et al. 2001; Moss et al. 2005); it shortens the QT interval, normalizes ventricular repolarization and therefore baseline T-wave abnormalities (Benhorin et al. 2000) through heterogeneous effects on AP duration (APD), particularly in the ventricular epicardium (Krishnan & Antzelevitch, 1991). Yet in BrS, flecainide exerts proarrhythmic effects; intravenous administration unmasks the arrhythmogenic phenotype in patients with a concealed form of the condition (Brugada et al. 1999; Priori et al. 2000; Gasparini et al. 2003). In contrast, quinidine causes QT interval prolongation by inhibiting K+ currents, including the transient outward current Ito, thereby slowing ventricular repolarization (Roden et al. 1986). This exacerbates the repolarization abnormalities associated with LQT3 which result in abnormal transmural repolarization gradients and ultimately predispose to torsades de pointes (Roden & Hoffman, 1985; Clancy et al. 2003; Restivo et al. 2004). However, quinidine prevents BrS-related arrhythmias by restoring the balance between the inward INa and outward Ito early in the AP (Alings et al. 2001; Belhassen et al. 2004; Hermida et al. 2004).
The present study demonstrated for the first time contrasting electrophysiological properties of Langendorff-perfused hearts isolated from wild-type (WT) and Scn5a+/ mice lacking the same KPQ residues as LQT3 patients (Nuyens et al. 2001) following treatment with flecainide and quinidine. Use of such genetically modified hearts complements previous models made indirectly to mimic LQT3 by introduction of pharmacological agents (Shimizu & Antzelevitch, 1997; Sicouri et al. 1997). The experiments followed from recent arrhythmogenic characterizations of the Scn5a+/ system that demonstrated its close resemblance to the human phenotype confirming its applicability to the clinical situation (Head et al. 2005). They tested the hypothesis that arrhythmogenesis and its modification by flecainide and quinidine in Scn5a+/ hearts depends on differences in repolarization kinetics through the ventricular myocardial wall, in addition to the altered distribution of conduction velocities associated with re-entrant substrates in WT hearts.
The experiments first determined arrhythmic tendency using a programmed electrical stimulation (PES) technique (Saumarez et al. 2003, 2006
Saumarez & Grace, 2000; Turner et al. 2005) that applied extra-systolic stimuli (S2) following trains of pacing stimuli (S1) at progressively reduced S1–S2 intervals (Head et al. 2005). This approach had also been successfully used to characterize hearts isolated from KCNE1–/– mice modelling LQT5 (Balasubramaniam et al. 2003). Bipolar electrograms recorded from the left ventricular epicardium revealed an arrhythmogenic as opposed to a non-arrhythmogenic phenotype in Scn5a+/ hearts, in which flecainide reduced whereas quinidine increased the incidence of ventricular tachycardia (VT), respectively. Secondly, these PES findings were quantified using an adaptation (Balasubramaniam et al. 2003) of the paced electrogram fractionation analysis (PEFA) procedure used clinically to stratify arrhythmogenic risk in cases of not only hypertrophic cardiomyopathy but also cardiac channelopathy (Saumarez & Grace, 2000; Saumarez et al. 2003, 2006; Turner et al. 2005). This associated arrhythmogenic re-entrant substrates with increases in electrogram duration (EGD) reflecting the distribution or spreading (‘fractionation’) of ventricular myocardial conduction velocities at reduced S1–S2 intervals, in agreement with previous descriptions of murine models of human LQT3 and LQT5 (Balasubramaniam et al. 2003; Head et al. 2005). PEFA also demonstrated that EGDs were reduced when arrhythmic tendency was reduced by pharmacological intervention. Thirdly, epicardial and endocardial monophasic AP recordings identified the presence or absence of abnormal differences in APD with the presence or absence of arrhythmic tendency, whether due to the Scn5a+/ mutation or pharmacological intervention.
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Methods |
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Breeding pairs of heterozygote Scn5a+/ and wild-type (WT) mice with an inbred 129/Sv genetic background were set up, and their offspring weaned accordingly. Mice were kept in plastic cages at room temperature in an animal facility, subjected to 12 h light/dark cycles, and allowed free access to sterile rodent chow and water. All procedures conformed to the UK Animals (Scientific Procedures) Act (1986).
Experimental preparations
Full experimental protocols for the preparation and study of murine hearts have been previously described (Balasubramaniam et al. 2003; Head et al. 2005). Male and female mice (average age 8.05 ± 0.57 months, n = 40) were randomly selected and killed by cervical dislocation (Schedule 1: Animals (Scientific Procedures) Act 1986). Hearts were excised and submerged in ice-cold bicarbonate Krebs–Henseleit 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 severed end of the aorta was cannulated and sutured to a 21-gauge tailor-made cannula prefilled with buffer solution. The preparation was then mounted onto a Langendorff system and perfused retrogradely for not less than 5 min prior to electrophysiological testing at a constant flow rate (2–2.5 ml min–1) (Watson-Marlow Bredel Peristaltic pumps, model 505S, Falmouth, Cornwall, UK) with oxygenated (95% O2, 5% CO2) Krebs–Henseleit buffer solution. The perfusate was filtered through two, 200 µm and 5 µm, membranes (Millipore UK, Watford, UK), and warmed to 37°C by a water-jacketed heat-exchange coil (Techne model C-58A, Cambridge, UK), prior to entering the coronary arterial network. Viable hearts regained a pink colouration and spontaneous rhythmic contractions upon warming. Ischaemic hearts were discarded.
Bipolar electrogram recording
Electrophysiological properties were examined both before and after the administration of pharmacological agents using an established programmed electrical stimulation (PES) technique (Balasubramaniam et al. 2003; Head et al. 2005). Electrograms were recorded from the basal epicardial surface of the left ventricle, using paired platinum recording electrodes (1 mm interpole spacing). Paired platinum stimulating electrodes connected to a Grass S48 stimulator (Grass-Telefactor, UK, Slough, UK) were used to pace the heart high on the right ventricular septum at 1.5–3 times excitation threshold (average stimulus amplitude 3.54 ± 0.13 V, n
= 40) (Tyers et al. 1997; Balasubramaniam et al. 2003, 2004, 2005; Head et al. 2005; Gammage et al. 2006) with square-wave stimuli of 2 ms duration. Stimulation of this strength ensured a consistent 1 : 1 capture for the duration of each experiment. The position of the stimulation and recording electrodes was fixed upon gaining a clear signal, and the distance between them maintained at 1 cm throughout. The heart was paced at a frequency of 10 Hz for 20 min to reach a physiological steady state. Cycles of a decremental, paced electrogram fractionation sequence comprising an eight-beat stimulus (S1) drive train followed by an extra-stimulus (S2) were then applied at 8 Hz and 10 Hz for 
120 s and 100 s, respectively. The S1–S2 interval was reduced by 1 ms between successive drive trains until the preparation became refractory. If VT supervened, pacing was terminated in order to confirm its persistence or otherwise. The resulting bipolar electrograms (BEGs) were amplified, band-pass filtered (30 Hz to 1 kHz) using a Gould 2400S amplifier (Gould-Nicolet Technologies, Ilford, Essex, UK), and digitized using an analog to digital converter at a sampling frequency of 5 kHz (CED 1401plus, Cambridge Electronic Design, Cambridge, UK). Spike2 software (Cambridge Electronic Design) was used to capture and analyse data; electrogram duration (EGD), ventricular effective refractory period (VERP) and conduction latency values were obtained from the resulting ventricular conduction curves where possible.
Figure 1 illustrates typical BEG data obtained in the course of a PES procedure. The S1 and S2 stimulation artefacts are each followed closely by an electrogram response (S1EG, S2EG). Figure 2 illustrates BEG data in conduction curve form derived from responses to extra-systolic stimuli (S2) subjected to an experimental paced electrogram fractionation analysis (PEFA) procedure translated from clinical practice (Saumarez & Grace, 2000; Saumarez et al. 2003, 2006; Turner et al. 2005). The response latencies (ms) of each BEG deflection, defined as the time differences between the extra-stimulus (S2) and the peaks and troughs of the resulting BEG, are plotted against the corresponding S1–S2 interval (ms). The VERP, defined as the shortest S1–S2 interval that elicits an electrogram response in the absence of absolute refractoriness, is given by the abscissa of point a. The EGD describes the degree of spread of conduction velocities at any given S1–S2 interval, and represents the time difference between the first and last BEG peaks. EGD ratios were computed by normalizing the EGD obtained at the shortest S1–S2 interval (a – b) at which the S2 extra-stimulus resulted in a BEG, with the EGD obtained at the longest S1–S2 interval (c – d) to give the expression (a – b)/(c – d). Finally, conduction latency is given as the ordinate of point d.
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Monophasic action potentials (MAPs) were recorded in addition to BEGs using a contact-electrode technique previously described (Franz, 1999; Knollmann et al. 2001, 2002). Recording electrodes were constructed using Teflon-coated 0.25 mm diameter silver wire (99.99% purity), galvanically chlorided to eliminate direct current offset, and placed either on the basal surface of the left ventricular epicardium or the free wall of the endocardium. The latter was achieved by introduction of a recording electrode into the left ventricular cavity through a small access window created using a scalpel on the interventricular septum. The tip of the electrode was rotated until it came to rest against the free wall. As above, paired platinum stimulating electrodes were used to pace the heart high on the right ventricular septum. Custom-made magnetic grips fixed to a metallic platform were used to anchor the electrodes and maintain stable contact pressure.
Epicardial and endocardial MAPs were recorded using the above decremental PES protocol to precipitate arrhythmia in the presence and absence of pharmacological intervention. Most epicardial and endocardial MAPs were acquired from the same heart in which case waveforms were similar. Analysis of MAP waveforms was performed using Spike2 software (Cambridge Electronic Design). MAPs were derived from recordings that satisfied previously established criteria including a rapid upstroke phase with consistent amplitude, a smooth contoured repolarization phase and a stable baseline (Fabritz et al. 2003). The point of maximum positive deflection for each MAP was considered the point of 0% repolarization, and the point of full return to baseline that of 100% repolarization. The intervening waveform was described in terms of action potential duration (APDx) measurements at x
= 90% (APD90), 70% (APD70) and 50% (APD50) repolarization. The effect of heterogeneous repolarization on the difference between epicardial and endocardial AP recovery was expressed empirically as the difference (APDx) between endocardial APDx and epicardial APDx values, i.e. endocardial APDx
– epicardial APDx.
Statistical procedures
Data sets from separate experimental groups (WT and Scn5a+/) were compared using a one-way analysis of variance (ANOVA) for independent samples, whereas results from individual hearts acquired during pharmacological intervention were compared to their respective untreated controls using a one-way ANOVA for correlated samples (Statistical Package for the Social Sciences, SPSS software). APDx values were also compared to a zero APDx. Furthemore, values obtained from hearts treated with flecainide or quinidine were compared to values obtained from untreated hearts under comparable conditions, and APDx values from untreated Scn5a+/ hearts were compared to APDx values from untreated WT hearts. All procedures assumed a significance level of P < 0.05. Results are expressed as mean ±
S.E.M. values.
Drugs
Flecainide and quinidine (Sigma-Aldrich, Poole, UK) were dissolved in doubly distilled water to make 1.0 mM stock solutions. Final drug concentrations were achieved by dilution with buffer solution which was perfused for 15 min prior to the imposition of PES, and maintained throughout. The concentrations used were guided by clinical therapeutic levels (flecainide: 0.2–0.9 mg l–1, i.e. 0.5–2.0 µM; quinidine: 2.0–5.0 mg l–1, i.e. 6.0–20 µM) (Birkett, 1997). Flecainide was stored at 4°C between experiments, and quinidine at room temperature, in darkness to prevent light degradation, in a tightly sealed container.
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Results |
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) murine hearts modelling the human long QT(3) (LQT3) syndrome. Cycles of eight pacing stimuli (S1) were applied at near-physiological 8 Hz and 10 Hz stimulation rates, followed by extra-stimuli (S2) interposed at progressively decrementing S1–S2 intervals. The cardiotropic effects of flecainide and quinidine were then determined. Epicardial bipolar electrograms (BEGs) detected the appearance or otherwise of ventricular tachycardia (VT) in response to S2 extra-stimuli. The resulting data sets were quantified to identify re-entrant substrates. Epicardial and endocardial monophasic action potential (MAP) waveforms were also recorded and quantified to explore for abnormalities of ventricular repolarization associated with the LQT3 phenotype (Antzelevitch et al. 1998).
Arrhythmic properties of Scn5a+/ hearts and the antiarrhythmic effects of flecainide
Table 1 compares the incidence of VT following extra-stimuli (S2) imposed during PES detected by recording BEGs from untreated (control) and (5.0 µM) flecainide or (10 µM) quinidine-treated WT and Scn5a+/ hearts. All hearts showed regular rhythm in the 20 min of S1 pacing applied to confirm preparation stability and viability before PES. However, flecainide and quinidine exerted contrasting effects in WT and Scn5a+/ hearts during PES. Table 1 indicates that all of the 12 untreated WT hearts, the seven WT hearts treated with flecainide and four out of the five WT hearts treated with quinidine remained in regular rhythm. In contrast, 16 out of the 28 untreated Scn5a+/ hearts were arrhythmogenic; the incidence of VT was significant in hearts isolated from both male (6 out of 12) and female (10 out of 16) mice. Therefore, only 12 of the 28 Scn5a+/ hearts remained in regular rhythm, empirically resulting in arrhythmogenic and non-arrhythmogenic groups of Scn5a+/ hearts. Figure 1A illustrates an extracellular BEG voltage trace recorded during the course of a PES procedure from a typical, untreated arrhythmogenic Scn5a+/ heart paced at 8 Hz. The single vertical markers represent S1 stimuli (stim1) and the double vertical markers S2 extra-stimuli (stim2). The trace shows that the extra-stimulus following the two S1 beats initiated an episode of VT that was sustained over six S1 beats.
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hearts. Furthermore, VT was abolished in the presence of flecainide in 13 out of the 16 arrhythmogenic Scn5a+/ hearts. Figure 1B goes on to illustrate a regular rhythm in the arrhythmogenic Scn5a+/ heart recorded during perfusion with (5.0 µM) flecainide; each S1 or S2 stimulation artefact is followed closely by an evoked electrogram (S1EG, S2EG). In contrast to the VT shown in Fig. 1A, regular rhythm persisted following a S2 extra-stimulus that was imposed at exactly the same time interval. In the presence of quinidine, PES induced VT in four out of the eight non-arrhythmogenic Scn5a+/ hearts. Stimulus capture or electrogram generation failed, precluding detailed study in quinidine-treated arrhythmogenic Scn5a+/ hearts. Conduction curve representations of BEG data
Conduction curves were next obtained by application of paced electrogram fractionation analysis (PEFA) to the BEG data; the previous studies on KCNE1–/– and Scn5a+/ mice had used such an approach to determine the presence or absence of arrhythmogenic re-entrant pathways (Balasubramaniam et al. 2003; Head et al. 2005). Figure 2 exemplifies typical conduction curves derived from arrhythmogenic (Fig. 2A) and non-arrhythmogenic (Fig. 2B) Scn5a+/ hearts paced at 8 Hz in the presence (Fig. 2A and B: 
) and absence (Fig. 2A and B: +) of flecainide (5.0 µM: A) and quinidine (10 µM: B). The response latencies (ms) of each BEG deflection, defined as the time differences between the extra-stimulus (S2) and the peaks and troughs of the resulting BEG, are plotted against the corresponding S1–S2 interval (ms), permitting determination of electrogram duration (EGD), ventricular effective refractory period (VERP) and latency values for each data set. Table 2 summarizes these parameters as obtained from untreated WT, non-arrhythmogenic and arrhythmogenic Scn5a+/ hearts (mean ±
S.E.M.). The findings associate arrhythmic tendency with increased electrogram spreading in parallel with the known characteristics of murine LQT3 and LQT5 (KCNE1–/–) models (Balasubramaniam et al. 2003; Head et al. 2005). In addition, untreated arrhythmogenic Scn5a+/ hearts showed prolonged EGD ratios compared to WT controls and their non-arrhythmogenic counterparts. The non-arrhythmogenic Scn5a+/ hearts could only be distinguished from WT hearts by their prolonged VERPs.
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hearts
Conduction curves from WT and Scn5a+/ hearts thus showed contrasting changes in the presence of flecainide and quinidine that correlated closely with their opposing effects on arrhythmic tendency (Table 1). Figure 2A and B goes on to illustrate typical effects of flecainide (A: ) and quinidine (B: ) on conduction curves, compared to results from untreated controls (+). Flecainide (5.0 µM) treatment appeared primarily to increase the conduction latency and VERP but reduce the EGD ratio (defined in methods) of the arrhythmogenic Scn5a+/ heart (Fig. 2A), whereas quinidine (10 µM) appeared to increase the VERP and EGD ratio of the non-arrhythmogenic Scn5a+/ heart (Fig. 2B). Figure 3 illustrates these findings with typical BEG traces recorded following S2 extra-stimuli imposed during PES at 8 Hz; electrogram spreading in the arrhythmogenic Scn5a+/ heart (Fig. 3A) is reduced in the presence of flecainide (Fig. 3B). Conversely, electrogram spreading in the non-arrhythmogenic Scn5a+/ heart (Fig. 3C) is increased in the presence of quinidine (Fig. 3D).
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hearts subjected to PES at 8 Hz and 10 Hz, respectively (mean ±
S.E.M.). First, Table 3A demonstrates that interventions that reduced arrhythmic tendency in Scn5a+/ hearts correlated with a reduced EGD ratio, whereas interventions that increased arrhythmic tendency correlated with an increased EGD ratio. Thus, flecainide significantly reduced EGD ratios in the arrhythmogenic Scn5a+/ hearts whether paced at 8 Hz or 10 Hz, which correlates with the absence of VT in Table 1. In contrast, quinidine markedly increased EGD ratios, to a considerably greater extent than flecainide, in the non-arrhythmogenic Scn5a+/ hearts paced at 8 Hz, which correlates with the arrhythmogenic effects of quinidine shown in Table 1.
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hearts. Thus, flecainide markedly prolonged VERPs in the arrhythmogenic Scn5a+/ hearts paced at 8 Hz and 10 Hz, whereas quinidine also prolonged VERPs in the non-arrhythmogenic Scn5a+/ hearts paced at 8 Hz. In contrast, Table 1 had shown that flecainide produced antiarrhythmic effects in arrhythmogenic Scn5a+/ hearts, while quinidine produced proarrhythmic effects in non-arrhythmogenic Scn5a+/ hearts. Thirdly, Table 3C demonstrates that changes in conduction latency following pharmacological intervention similarly failed to correlate with arrhythmic tendency in Scn5a+/ hearts. Thus, neither flecainide nor quinidine elicited marked effects on conduction latency in the non-arrhythmogenic Scn5a+/ hearts, whereas flecainide significantly increased conduction latency in the arrhythmogenic Scn5a+/ hearts paced at 8 Hz.
Finally, flecainide (10 µM) exerted an arrhythmogenic effect in 11 out of 16 WT hearts; subsequent experiments used a reduced concentration of (5.0 µM) flecainide to permit their direct comparison with Scn5a+/ hearts in which VT was abolished during PES (Table 1). Flecainide markedly increased EGD ratios (Table 3A), VERPs (Table 3B) and conduction latencies (Table 3C) in the WT hearts. Quinidine exerted arrhythmogenic effects in only one out of the five WT hearts (Table 1) and markedly reduced EGD ratios (Table 3A) in contrast to flecainide, but significantly prolonged VERPs (Table 3B) and conduction latencies (Table 3C).
Effects of flecainide and quinidine on monophasic action potential (MAP) waveforms
The remaining experiments explored changes of action potential (AP) waveforms in WT and Scn5a+/ hearts under comparable conditions in the presence and absence of flecainide and quinidine. Both epicardial and endocardial MAPs were recorded; pacing was attempted at 8 Hz and 10 Hz, but the higher 10 Hz rate prevented full recovery of the MAP in Scn5a+/ hearts between stimuli. Arrhythmic tendency was determined using pulse protocols identical to those applied when obtaining BEGs. Figures 4 and 5 illustrate examples of MAPs recorded from the epicardium and endocardium of typical WT (Fig. 4), arrhythmogenic (Fig. 5A–C) and non-arrhythmogenic (Fig. 5D–F) Scn5a+/ hearts paced at 8 Hz prior to (Figs. 4 and 5, A and D) and during treatment with flecainide (10 µM: Fig. 4B and C; 5.0 µM: Fig. 5B and C) and quinidine (10 µM: Figs 4 and 5, E and F). Such MAPs were derived from recordings that satisfied previously established criteria including a rapid upstroke phase with consistent amplitude, a smooth contoured repolarization phase and a stable baseline (Fabritz et al. 2003). The top panels in both Figs 4 and 5 overlay epicardial and endocardial MAPs to emphasize AP waveform differences in untreated hearts. The lower two panels illustrate the effects of flecainide and quinidine on AP waveform by superimposing records obtained from the epicardium (‘epi’: Figs 4B and E and 5B and E) and endocardium (‘endo’: Figs 4C and F and 5C and F) before (designated ‘epi’ or ‘endo’) and during treatment (‘+ flecainide’ or ‘+ quinidine’). AP abnormalities in the form of early afterdepolarizations were nowhere observed under any of the genetic or experimental conditions examined here.
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hearts had shorter repolarization phases than their corresponding epicardial MAPs. Furthermore, the epicardial MAPs obtained from both arrhythmogenic and non-arrhythmogenic Scn5a+/ hearts had longer repolarization phases than the epicardial MAPs obtained from WT hearts. Likewise, the endocardial MAPs obtained from both arrhythmogenic and non-arrhythmogenic Scn5a+/ hearts had shorter repolarization phases than the endocardial MAPs obtained from WT hearts. The presence of flecainide resulted in a more rapid return to baseline of the epicardial MAPs obtained from arrhythmogenic Scn5a+/ hearts (Fig. 5B); endocardial MAPs had longer repolarization phases than their respective untreated controls (Fig. 5C). However, in the presence of quinidine, the epicardial MAPs obtained from non-arrhythmogenic Scn5a+/ hearts were similar in waveform to their respective untreated controls (Fig. 5E), whereas the endocardial MAPs had longer repolarization phases than their respective untreated controls (Fig. 5F). Quantitative representation of epicardial and endocardial action potential (AP) time courses
Figures 6–8 quantify the above effects of flecainide and quinidine on epicardial and endocardial AP waveforms. The point of maximum positive deflection for each MAP was considered the point of 0% repolarization, and the point of full return to baseline that of 100% repolarization. The intervening waveform was described in terms of APDx measurements that were made at x
= 90% (APD90), 70% (APD70) and 50% (APD50) repolarization (mean ±
S.E.M.). The effect of heterogeneous repolarization on the difference between epicardial and endocardial AP recovery was expressed empirically as the difference (APDx) between endocardial APDx and epicardial APDx values. Figure 6A–C illustrates epicardial (Fig. 6A) and endocardial (Fig. 6B) APDx and APDx (Fig. 6C) at 90%, 70% and 50% repolarization (ms) (mean ±
S.E.M.) in WT hearts paced at 8 Hz in the absence (open bars) and presence of flecainide (10 µM: crossed bars) and quinidine (10 µM: striped bars). In untreated WT hearts, AP recovery was more gradual in the endocardium than in the epicardium, and gave positive APDx values that differed significantly from zero. Flecainide (10 µM) markedly reduced both epicardial and endocardial APD90 values, but left APD90 unchanged despite the persistence of VT in three out of six WT hearts. A lower (5.0 µM) concentration neither exerted arrhythmogenic effects nor significantly altered either epicardial or endocardial APDx or APDx (n
= 14) (P > 0.05), in agreement with the BEG studies summarized in Table 1. Quinidine significantly prolonged epicardial APD90, but did not significantly alter APD90, and no VT was observed in any of the 14 hearts studied during PES.
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hearts in the absence (open bars) and presence of flecainide (5.0 µM: crossed bars) and quinidine (10 µM: striped bars). They demonstrate that AP recovery was significantly slower in the epicardium than in the endocardium, giving negative APDx values that differ significantly from zero and from WT hearts (all P < 0.05). Flecainide reduced epicardial but increased endocardial APD90 to give positive APD90 values that differed significantly from corresponding untreated controls; VT was abolished in 19 out of 22 arrhythmogenic Scn5a+/ hearts. In contrast, quinidine prolonged endocardial APD90, but exerted no significant effect on APD90; VT was induced in 10 out of 14 non-arrhythmogenic Scn5a+/ hearts during PES.
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hearts, whereas VERPs were prolonged in non-arrhythmogenic Scn5a+/ hearts. Nevertheless, APD90 values were negative in all Scn5a+/ hearts in contrast to positive values in WT hearts. Flecainide and quinidine exerted sharply differing effects in Scn5a+/ and WT hearts. Thus, flecainide reduced EGD ratios in arrhythmogenic Scn5a+/ hearts, restored APD90 and abolished VT. Quinidine increased EGD ratios in non-arrhythmogenic Scn5a+/ hearts, conserved APD90 and increased the incidence of VT. In contrast, flecainide increased EGD ratios and was pro-arrhythmic in WT hearts, whereas quinidine reduced EGD ratios. Neither flecainide nor quinidine affected APD90 in WT hearts. |
Discussion |
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-subunit of the cardiac (Nav1.5) voltage-gated Na+ channel, causing incomplete Na+ channel inactivation (Wang et al. 1995). This results in a persistent, ‘late’
INa, which prolongs the plateau phase of the cardiac action potential (AP) (Bennett et al. 1995; Kass, 1997). Such abnormal prolongation of repolarization may increase the incidence of arrhythmogenic early afterdepolarizations (January & Riddle, 1989). In addition, altered spatiotemporal dispersion of repolarization and gradients of refractoriness resulting from altered ionic currents may ultimately generate re-entrant substrates, in turn predisposing to torsades de pointes and ventricular fibrillation (Zareba et al. 1998; Baker et al. 2000). LQT3 contrasts with the Brugada syndrome (BrS) in which peak INa is reduced due to ‘loss-of-function’ mutations of SCN5A (Brugada & Roberts, 2001; Grant et al. 2002; Brugada et al. 2003). LQT3 and BrS respond differently to pharmacological agents. In LQT3, flecainide exerts antiarrhythmic effects (Windle et al. 2001; Moss et al. 2005), inhibiting inward INa in preference to outward Ito (Benhorin et al. 2000) and correcting the associated repolarization abnormalities (Wang et al. 1997; Antzelevitch & Fish, 2001; Restivo et al. 2004). These heterogeneous effects of flecainide on AP duration (APD) have been demonstrated experimentally in canine ventricular myocardium (Krishnan & Antzelevitch, 1991). However, these favour the development of phase 2 re-entry and extra-systolic activity (Krishnan & Antzelevitch, 1993), and can accentuate arrhythmic tendency in the BrS, particularly in asymptomatic patients (Brugada et al. 1999; Priori et al. 2000; Gasparini et al. 2003). In contrast, quinidine can induce or exacerbate LQT syndrome (Roden et al. 1986; Clancy et al. 2003); it prolongs APD at 90% repolarization in both Langendorff-perfused rat heart preparations (Farkas & Curtis, 2003) and canine purkinje fibres (Roden & Hoffman, 1985), causing torsades de pointes and ventricular fibrillation in whole canine hearts (Inoue et al. 1986; Inoue & Sugimoto, 1992). Quinidine treatment has also been associated with QT interval prolongation and pro-arrhythmic effects following its administration for clinical AF (Selzer & Wray, 1964; Bauman et al. 1984; Ometto et al. 1990; Lin & Quasny, 1997). However, in BrS, quinidine restores the balance between outward (Ito) and inward (INa) currents early in the AP, reverses the associated electrocardiographic ST-segment elevation and exerts antiarrhythmic effects (Alings et al. 2001; Belhassen et al. 2004; Hermida et al. 2004).
This study compared for the first time the effects of flecainide and quinidine on the electrophysiological properties of wild-type (WT) and genetically modified (Scn5a+/) mice lacking the same KPQ residues as LQT3 patients (Nuyens et al. 2001). These studies had previously demonstrated that the Scn5a+/ system is markedly susceptible to arrhythmia, in agreement with clinical findings, thus proving its usefulness as a model for the investigation of arrhythmogenesis in LQT3 (Head et al. 2005). The present experiments first determined arrhythmic tendency using a programmed electrical stimulation (PES) technique (Saumarez & Grace, 2000; Saumarez et al. 2003, 2006; Turner et al. 2005) in the presence and absence of pharmacological intervention. A significant proportion of Scn5a+/ hearts were arrhythmogenic in response to extra-stimuli (S2) compared to WT hearts, confirming previous studies (Fabritz et al. 2003; Nuyens et al. 2001), despite there being no sex or age differences. However, flecainide reduced whereas quinidine increased the incidence of ventricular tachycardia (VT) in the arrhythmogenic and initially non-arrhythmogenic Scn5a+/ hearts, respectively, during PES.
Secondly, these PES findings were quantified by application of a technique derived from the paced electrogram fractionation analysis (PEFA) procedure used clinically to stratify risks of SCD (Saumarez & Grace, 2000; Saumarez et al. 2003, 2006; Turner et al. 2005). PEFA was originally developed to identify arrhythmic substrates by timing the activation of particular ventricular myocardial sites in response to increasingly premature extra-stimuli applied to the ventricular free wall as opposed to the conduction system. The resulting conduction curves permitted detection of abnormal patterns of myocardial activation through changes of electrogram duration (EGD), ventricular effective refractory period (VERP) and conduction latency. This approach had identified re-entrant as opposed to focal substrates with increases in EGD when first applied to murine models of human LQT3 and LQT5 (Balasubramaniam et al. 2003; Head et al. 2005). The present results went on similarly to implicate EGDs, expressed as EGD ratios normalized to their corresponding values obtained at long S1–S2 intervals, in arrhythmogenesis. Thus, arrhythmogenic Scn5a+/ hearts had prolonged EGD ratios, whereas non-arrhythmogenic Scn5a+/ hearts had prolonged VERPs relative to WT hearts. Furthermore, flecainide reduced EGD ratios and arrhythmic tendency in arrhythmogenic Scn5a+/ hearts, whereas quinidine increased EGD ratios and arrhythmic tendency in non-arrhythmogenic Scn5a+/ hearts. In contrast, flecainide increased EGD ratios and arrhythmic tendency in WT hearts, whereas quinidine reduced EGD ratios and exerted minor arrhythmogenic effects.
Thirdly, epicardial and endocardial monophasic AP waveforms were compared in order to assess for alterations in the differences between their recovery kinetics. The greater density of Ito in the epicardium compared to endocardium establishes transmural repolarization gradients that normally protect against arrhythmia (Vos & Jungschleger, 2001; Costantini et al. 2005). However, the abnormal gradients implicated in LQT3, caused by incomplete Na+ channel inactivation, result in a lengthening of the electrocardiographic QT interval (Antzelevitch et al. 1998). In contrast to the effect of a homogeneous prolongation or shortening of APD throughout the ventricular wall, non-uniform repolarization may support re-entrant events following premature excitation, in turn generating torsades de pointes (Baker et al. 2000). These spatial gradients have been observed in pharmacological rabbit, guinea pig and canine wedge models of LQT3 (Shimizu & Antzelevitch, 1997; Restivo et al. 2004; Milberg et al. 2005) and actually associated with arrhythmogenesis in LQT3 mouse models (Nuyens et al. 2001; Fabritz et al. 2003). However, epicardial APDs are more sensitive to factors influencing INa than endocardial APDs, owing to the significantly greater density of Ito in the epicardium compared to the endocardium, and the right rather than the left ventricle (Antzelevitch & Dumaine, 2001). Thus, mexiletine shortened APD and reduced such transmural differences in arterially perfused ventricular wedge preparations treated with ATX-II to mimic LQT3 (Sicouri et al. 1997). Accordingly, Na+ channel blockade as might result from flecainide treatment prevents re-entrant excitation.
The present experiments attribute the tachyarrhythmias observed in the genetically modified Scn5a+/ hearts studied to a lengthening of the epicardial AP in combination with a shortening of the endocardial AP. This resulted in a negative difference, expressed as APDx, particularly in the arrhythmogenic Scn5a+/ hearts compared to WT. These results complement previous observations from systems made to model LQT3 by pharmacological manipulation (Shimizu & Antzelevitch, 1997) in which re-entrant tendencies correlated with an increased spatial dispersion of repolarization (Restivo et al. 2004). Flecainide then shortened epicardial APs and lengthened endocardial APs in arrhythmogenic Scn5a+/ hearts, and restored APDx at 90% repolarization (APD90). In contrast, quinidine selectively lengthened endocardial APs in non-arrhythmogenic Scn5a+/ hearts, consistent with the clinical observations of QT interval prolongation, yet did not significantly alter APD90. In WT hearts, flecainide shortened both epicardial and endocardial APs, and exerted contrasting proarrhythmic effects despite not altering APD90. Quinidine lengthened epicardial APs to an extent that might correlate with its minor arrhythmogenic effects (Balser, 2002), and likewise conserved APD90. In contrast, AP abnormalities in the form of early afterdepolarizations previously associated with focal arrhythmias (Hoffman & Dangman, 1987) were nowhere observed under any of the genetic or experimental conditions examined here.
In summary, the arrhythmogenic features of genetically modified Scn5a+/ murine systems closely resemble those of the human phenotype and demonstrate that pharmacological interventions succeed in altering arrhythmic tendency and associated electrophysiological parameters. Flecainide reduces arrhythmic tendency, restores EGD ratio and APD90 in arrhythmogenic Scn5a+/ hearts. Quinidine increases arrhythmic tendency and EGD ratio, but does not significantly alter APD90 in non-arrhythmogenic Scn5a+/ hearts. In contrast, in WT hearts, flecainide increases arrhythmic tendency and EGD ratio, whereas quinidine exerts minor arrhythmogenic effects and reduces EGD ratios; neither intervention altered APD90. The findings in this paper therefore support the hypothesis that arrhythmogenesis can result from an increased distribution of conduction velocities associated with re-entrant substrates, but that arrhythmogenesis in Scn5a+/ hearts also depends on differences in repolarization kinetics through the ventricular myocardial wall. Thus, incomplete Na+ channel inactivation generates functional substrates dependent on altered refractoriness that cause abnormalities in activation and conduction of subsequent cardiac impulses. Any spatial heterogeneities between the epicardial and endocardial layers would thus cause fragmentation of the activation wavefront (Turner et al. 2005). Electrograms recorded from abnormal tissue are accordingly fractionated owing to variable delays created by these abnormal activation paths through functionally altered tissue (Saumarez et al. 2006). Furthermore, flecainide is antiarrhythmic in Scn5a+/ hearts yet proarrhythmic in WT hearts; quinidine is proarrhythmic in Scn5a+/ hearts and much less so in WT hearts. Thus, those pharmacological manoeuvres that increase arrhythmic tendency, increase EGD ratio but leave APD90 intact. In contrast, those pharmacological manoeuvres that reduce arrhythmic tendency also reduce EGD ratio, and restore APD90 in Scn5a+/ but not WT hearts.
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