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. 2006 Nov 16;578(Pt 1):85–97. doi: 10.1113/jphysiol.2006.121921

Effects of L-type Ca2+ channel antagonism on ventricular arrhythmogenesis in murine hearts containing a modification in the Scn5a gene modelling human long QT syndrome 3

Glyn Thomas 1, Iman S Gurung 2, Matthew J Killeen 2, Parvez Hakim 2, Catharine A Goddard 1, Martyn P Mahaut-Smith 2, William H Colledge 2, Andrew A Grace 1, Christopher L-H Huang 2
PMCID: PMC2075124  PMID: 17110414

Abstract

Ventricular arrhythmogenesis in long QT 3 syndrome (LQT3) involves both triggered activity and re-entrant excitation arising from delayed ventricular repolarization. Effects of specific L-type Ca2+ channel antagonism were explored in a gain-of-function murine LQT3 model produced by a ΔKPQ 1505–1507 deletion in the SCN5A gene. Monophasic action potentials (MAPs) were recorded from epicardial and endocardial surfaces of intact, Langendorff-perfused Scn5a+/Δ hearts. In untreated Scn5a+/Δ hearts, epicardial action potential duration at 90% repolarization (APD90) was 60.0 ± 0.9 ms compared with 46.9 ± 1.6 ms in untreated wild-type (WT) hearts (P < 0.05; n = 5). The corresponding endocardial APD90 values were 52.0 ± 0.7 ms and 53.7 ± 1.6 ms in Scn5a+/Δ and WT hearts, respectively (P > 0.05; n = 5). Epicardial early afterdepolarizations (EADs), often accompanied by spontaneous ventricular tachycardia (VT), occurred in 100% of MAPs from Scn5a+/Δ but not in any WT hearts (n = 10). However, EAD occurrence was reduced to 62 ± 7.1%, 44 ± 9.7%, 10 ± 10% and 0% of MAPs following perfusion with 10 nm, 100 nm, 300 nm and 1 μm nifedipine, respectively (P < 0.05; n = 5), giving an effective IC50 concentration of 79.3 nm. Programmed electrical stimulation (PES) induced VT in all five Scn5a+/Δ hearts (n = 5) but not in any WT hearts (n = 5). However, repeat PES induced VT in 3, 2, 2 and 0 out of 5 Scn5a+/Δ hearts following perfusion with 10 nm, 100 nm, 300 nm and 1 μm nifedipine, respectively. Patch clamp studies in isolated ventricular myocytes from Scn5a+/Δ and WT hearts confirmed that nifedipine (300 nm) completely suppressed the inward Ca2+ current but had no effect on inward Na+ currents. No significant effects were seen on epicardial APD90, endocardial APD90 or ventricular effective refractory period in Scn5a+/Δ and WT hearts following perfusion with nifedipine at 1 nm, 10 nm, 100 nm, 300 nm and 1 μm nifedipine concentrations. We conclude that L-type Ca2+ channel antagonism thus exerts specific anti-arrhythmic effects in Scn5a+/Δ hearts through suppression of EADs.

Clinically, long QT3 syndrome (LQT3) is characterized by QT-interval prolongation and ventricular tachycardia (VT), which typically occurs at rest or during sleep (Schwartz & Priori, 2004) and is associated with gain-of-function mutations in the Scn5a gene which encodes the α-(pore-forming) subunit of the cardiac Na+ channel, leading to an increased late Na+ current (INa), prolonged action potential plateau and delayed repolarization. Both triggered activity through early afterdepolarizations (EADs) and re-entrant excitation via transmural dispersion of repolarization (TDR) are implicated in the genesis of VT in long QT syndromes (LQTS) (Volders et al. 2000; Restivo et al. 2004).

These features have recently been paralleled in a corresponding, experimental, murine model, produced by the introduction of the gain-of-function knock-in ΔKPQ 1505–1507 deletion into the murine SCN5A gene (Nuyens et al. 2001; Head et al. 2005), suggesting a possible translational application for this and other genetically modified murine systems. Microelectrode recordings from isolated ventricular myocytes from such Scn5a+/Δ hearts thus revealed prolonged action potential durations (APDs) and spontaneous EADs. In addition, an adaptation of the clinical technique of paced electrogram fractionation analysis recently used to assess arrhythmogenicity in patients with LQTS (Roden, 2006; Saumarez et al. 2006) identified significant electrogram duration and conduction curve dispersion in intact, isolated Langendorff-perfused Scn5a+/Δ hearts, strongly suggestive of a concomitant re-entrant substrate. Furthermore, reducing INa using the class IB Na+ channel antagonist mexiletine in Scn5a+/Δ exerts an anti-arrhythmogenic effect through a combined reduction in EAD frequency and interventricular dispersion of APD90 (Fabritz et al. 2003b), consistent with clinical observations (Schwartz et al. 1995).

Current pharmacological treatment of LQTS primarily involves β-adrenoreceptor antagonism (Priori et al. 2001) but this appears to be of less clinical benefit in LQT3 than in the other LQTS subtypes, particularly LQT1 (Moss, 1998; Moss et al. 2000; Schwartz et al. 2001; Priori et al. 2004). On the other hand, clinical reports of successful arrhythmia suppression with the phenylalkylamine-type Ca2+ channel antagonist verapamil (Shimizu et al. 1995; Komiya et al. 2004) have prompted suggestions that such drugs might be appropriate as adjunctive therapy in LQT1, LQT2 and even LQT3 patients (Shimizu et al. 2005). Thus, experimental reports suggest that Ca2+ channel antagonism by verapamil suppresses EADs and reduces TDR in feline wedge preparations made to model acquired (as opposed to congenital) LQTS though suppressing the rapid (IKr) and slow (IKs) components of the delayed rectifier K+ current by E-4031 and chromanol 293B, respectively (Aiba et al. 2005), and in a recent intact, pharmacological rabbit preparation made to model LQT3 through augmenting INa by veratridine (Milberg et al. 2005a).

However, verapamil is known also to exert effects on INa (IC50 of 5–50 μm) (Pidoplichko & Verkhratskii, 1989) and exert high affinity block of IKr (IC50 of 143 nm) (Chouabe et al. 1998; Zhang et al. 1999) in addition to its effect on the L-type Ca2+ current (ICa,L) (Triggle, 2006), whereas the dihydropyridine Ca2+ antagonist nifedipine exerts no effects upon upon T-type Ca2+ current ICa,T, INa, IK or If at concentrations as high as 5 μm (Verheijck et al. 1999). The present experiments, therefore, report and characterize for the first time, the anti-arrhythmic effect of the specific L-type Ca2+ channel antagonist nifedipine in mice with targeted disruption of the Scn5a gene. These findings complement previous reports of nifedipine upon electrically evoked Ca2+ transients in isolated, fluo-3-loaded ventricular myocytes from isoproterenol (isoprenaline)-treated WT hearts (Balasubramaniam et al. 2004) as well as intact hearts from mice with targeted disruption of KCNE1 modelling long QT syndrome 5 (LQT5) (Balasubramaniam et al. 2003).

Methods

Preparation of Langendorff-perfused hearts

Whole hearts from mice killed by cervical dislocation (Schedule 1: UK Animals (Scientific Procedures) Act 1986) were excised and placed in ice-cold bicarbonate-buffered Krebs-Henseleit solution (mm: NaCl 119, NaHCO3 25, KCl 4, KH2PO4 1.2, MgCl2 1, CaCl2 1.8, glucose 10 and sodium pyruvate 2, pH 7.4) bubbled with 95% O2−5% CO2. A small (3–4 mm) section of aorta was cannulated under the buffer surface and sutured to a 21-gauge tailor-made cannula, pre-filled with ice-cold buffer solution, then secured with a metallic clip for retrograde perfusion using the above solution at 2–2.5 ml min−1 using a peristaltic pump (Watson-Marlow Bredel model 505S, Falmouth, Cornwall, UK) after passing through 200 μm and 5 μm filters (Millipore, Watford, UK) and warming to 37°C via a water jacket and circulator (Techne model C-85A, Cambridge, UK). Healthy, viable hearts suitable for experimentation regained a homogeneous pink colouration and spontaneous rhythmic contraction on warming. Hearts not demonstrating these features were immediately discarded, to avoid false positive results. Complete atrioventricular (AV) block was induced in selected preparations by crush-ablation of the AV node using surgical forceps as previously described and confirmed by the recording of dissociated atrial and ventricular waveforms (Fabritz et al. 2003b; Milberg et al. 2005b) although this procedure reduced the longevity of both Scn5a+/Δ and WT preparations. Hearts were perfused with physiological perfusion buffer for 20 min prior to experimentation, to avoid possible residual effects of endogenous catecholamine release.

Monophasic action potential recordings

Monophasic action potentials (MAPs) were recorded from the epicardium using a spring-loaded, AgCl contact (2 mm tip diameter) MAP electrode (Linton Instruments, Harvard Apparatus, UK) which was positioned manually. Endocardial recordings were obtained using a custom-built electrode, constructed from two twisted strands of Teflon-coated (0.25 mm diameter) silver wire (99.99% purity) (Advent Research Materials Ltd, UK), galvanically chlorided and introduced into the left ventricular cavity through a small access window created in the interventricular septum and rotated such that the tip came to rest against the free wall. The endocardial electrode was initially placed by hand, and the position maintained by custom-designed magnetic grips positioned on a metallic platform. The entire apparatus was earthed to reduce electrical interference. Signals were amplified and low-pass filtered appropriately for murine recordings (0.1 Hz to 300 Hz) (Gould 2400S, Gould-Nicolet Technologies, Ilford, UK) (Fabritz et al. 2003a) then digitized using a 1401plus analog-to-digital converter (Cambridge Electronic Design, Cambridge, UK). Analysis of the MAP waveforms was performed used Spike II software (Cambridge Electronic Design). Results were expressed as means ± s.e.m. and different experimental groups compared using ANOVA (SPSS software).

Programmed electrical stimulation

For programmed electrical stimulation (PES) of the heart paired (1 mm interpole spacing) platinum stimulating and recording electrodes were placed on the basal epicardial surfaces of the right and left ventricles, respectively. The period of initial pacing used 2 ms square-wave stimuli with amplitudes of three times excitation threshold (Grass S48 stimulator, Grass-Telefactor, Slough, UK) for 20 min at 125 ms basic cycle length (BCL). All experimental mice were bred from a 129 genetic background, which, along with C57 mice, are less susceptible to PES-induced arrhythmias than FBV or Black Swiss mice (Maguire et al. 2003). Nevertheless, complex pacing protocols involving double/triple extra-stimuli and rapid burst pacing were avoided to reduce the risk of false positive results (Maguire et al. 2003). PES pacing protocols comprised a drive train of 8 paced S1 beats at 125 ms BCL, followed by a premature S2 extrastimulus every ninth beat. S1–S2 intervals first equalled the pacing interval and were then successively reduced by 1 ms with each 9 beat cycle until ventricular refractoriness was reached, whereupon the S2 stimuli elicited no electrograms.

Isolation of single ventricular myocytes

Following cannulation, the heart was perfused in a retrograde fashion with Krebs-Henseleit buffer, warmed to 37°C by means of a water jacket and circulator (Techne model C-85A, Cambridge, UK), at a rate of 2–2.5 ml min−1 for 5 min, until the heart regained a homogeneous pink colouration and began contracting spontaneously. The heart was then perfused for 5 min with a nitrilotriacetic acid-based perfusion buffer containing (mm): 125 NaCl, 4.75 KCl, 5 MgSO4, 10 Hepes, 5 sodium pyruvate, 20 glucose, 20 taurine and 4.5 nitrilotriacetic acid. Following this, the heart was perfused with a digestion buffer for 12–15 min containing (mm): 125 NaCl, 4.75 KCl, 5 MgSO4, 10 Hepes, 5 sodium pyruvate, 20 glucose, 20 taurine, 0.6 CaCl2 and 1 mg ml−1 collagenase type 2 (Worthington, UK), 1 mg ml−1 hyaluronidase (Sigma, -Aldrich, Poole, UK). After this period, a small pair of 90 deg curved forceps were used to remove a wedge-shaped segment of myocardium from the left ventricular free wall. Ventricular tissue samples were placed in a tube containing digestion buffer in addition to 1 mg ml−1 bovine serum albumin (Sigma-Aldrich, Poole, UK) for 5 min before gentle trituration for a further 5 min in the same solution. Tissue samples were subsequently spun down in a centrifuge (1000 r.p.m. (1860 g) for 3 min) before the supernatant from the tissue tubes was discarded and replaced with a wash buffer containing (mm): 135 NaCl, 1.1 MgCl2, 1.8 CaCl2, 5.4 KCl, 10 Hepes, 10 glucose and pH was adjusted to 7.35 with NaOH. Ventricular myocytes were stored in the above wash buffer and were studied within 4–6 h. Following initial perfusion of the heart, all subsequent steps were performed at room temperature (22-24°C).

Single cell electrophysiology

Conventional whole-cell patch clamp recording in voltage clamp mode was carried out using an Axopatch 200B amplifier (Axon Instruments, CA, USA) coupled to a Digidata series computer interface and controlled by pCLAMP software (Axon Instruments). Pipettes with resistances of 1–4 MΩ were pulled from borosilicate glass capillaries (1.5 mm outer and 0.86 inner diameter, GC150-10; Harvard Apparatus Ltd). Extracellular buffer contained (mm): 135 NaCl, 1.1 MgCl2, 1.8 CaCl2, 5.4 KCl, 10 Hepes, 10 glucose, and pH was adjusted to 7.35 with NaOH. Intracellular pipette saline contained (mm): 130 KCl, 1 MgCl2, 10 Hepes, 5 Mg-ATP, 5 Na2-creatine phosphate, and pH was adjusted to 7.2 with KOH. After formation of a gigaseal, the whole-cell configuration was achieved by applying gentle suction through the pipette and a brief voltage (ZAP) pulse. Up to 75% series resistance compensation was achieved. Inward Ca2+ currents were triggered by applying a series of 10 mV incremental voltage pulses from −40 to 10 mV from a holding potential of −40 mV, and inward Na+ currents were triggered with similar pulses from −100 to −40 mV from a holding voltage of −100 mV.

Pharmacological agents

All drugs (Sigma-Aldrich, Poole, UK) were first prepared as 1 mm stock solutions. Nifedipine was dissolved in 96% ethanol. Final drug concentrations were achieved by dilution with buffer solution. Nifedipine stock solutions were refrigerated at 4°C and were kept wrapped in foil to prevent light degradation.

Results

Nifedipine suppresses afterdepolarizations and spontaneous arrhythmias in Scn5a+/Δ hearts

Early afterdepolarizations (EADs) are generally believed to initiate ventricular tachycardia (VT) in a range of conditions including LQTS, which are then maintained via re-entrant excitation (Antzelevitch & Shimizu, 2002). In the present study, simultaneous recording of left ventricular endocardial and epicardial monophasic action potentials (MAPs) from Langendorff-perfused whole heart preparations from Scn5a+/Δ (n = 5) and WT (n = 5) mice produced high quality signals that satisfied previously documented criteria of a stable baseline and triangular MAP morphology, rapid upstroke phase, and a consistent amplitude (Knollmann et al. 2001).

Firstly, Fig. 1A shows a representative trace of an epicardial MAP recording from a spontaneously beating Scn5a+/Δ heart following the induction of bradycardia through mechanical atrioventricular (AV) blockade. Epicardial EADs occurred in 49 ± 10.5% of MAPs observed in 20 s epochs, randomly selected from a total sample duration time of 20 min (n = 10 epochs from each of 2 Scn5a+/Δ hearts). Furthermore, episodes of spontaneous VT, lasting a mean duration of 0.59 ± 0.1 s were observed in 4 out of 5 Scn5a+/Δ similarly prepared hearts, in keeping with previous findings (Nuyens et al. 2001; Fabritz et al. 2003b). However, we noted, for the first time to our knowledge, that no such features were recorded from the endocardial sites in Scn5a+/Δ hearts during similar sampling periods, each of 20 min per heart (n = 5). However, perfusion with physiological buffer containing 1 μm nifedipine suppressed epicardial EADs to 3.9 ± 1.5% of MAPs, in identically selected epochs from a total sample duration time of 20 min in each heart studied (n = 5; P < 0.05). Furthermore, no episodes of spontaneous VT were observed in comparable sampling periods in all five of the previously mentioned Scn5a+/Δ hearts (P < 0.05) (Fig. 1B).

Figure 1. Monophasic action potential recordings from Scn5a+/Δ and WT hearts.

Figure 1

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Recordings were obtained from the epicardial surface of spontaneously beating Scn5a+/Δ and WT hearts. Multiple early afterdepolarizations (EADs) (*) are seen, along with an episode of non-sustained ventricular tachycardia (VT) (A). Increased motion of the preparation during tachycardia is reflected in the baseline variability. All such arrhythmias however, were suppressed following perfusion with physiological buffer solution containing 1 μm nifedipine (B). No such EADs or VT were observed in WT hearts before (C) or after perfusion with 1 μm nifedipine (D).

Conversely, in WT hearts, no EADs or spontaneous VT were recorded from either the endocardial or epicardial surfaces (n = 5) (Fig. 1C) subject to a similar sampling scheme. Similarly, no such features were observed following perfusion of all hearts with physiological buffer containing 1 μm nifedipine (Fig. 1D).

Secondly, these initial findings were extended by subjecting a further five Scn5a+/Δ hearts to serial perfusion with buffer containing a range of nifedipine concentrations (1 nm, 10 nm, 100 nm, 300 nm and 1 μm). Mechanical induction of complete AV block was specifically avoided in subsequent experiments to minimize the risk of myocardial trauma and ischaemia. Therefore, a mild hypokalaemic buffer solution (4.0 mm K+) was used to facilitate bradycardia and EADs as previously described (Fabritz et al. 2003a; Milberg et al. 2005a). Consequently, EADs were now observed in 100% of MAPs during identical 20 s epochs in Scn5a+/Δ hearts alone (n = 10 epochs from each of 5 Scn5a+/Δ hearts). However, EAD occurrence was reduced to 62 ± 7.1%, 44 ± 9.7% and 10 ± 10% of MAPs following perfusion with 10 nm, 100 nm and 300 nm nifedipine, respectively (P < 0.05; n = 5). Furthermore, perfusion with 1 μm nifedipine completely suppressed all EADs in all MAPs recorded from Scn5a+/Δ hearts (P < 0.05; n = 5) (Fig. 2A). No EADs were observed in identically treated WT control hearts (n = 5) with or without nifedipine.

Figure 2. Percentage of monophasic action potentials displaying early afterdepolarizations in Scn5a+/Δ hearts.

Figure 2

Figure 2

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Percentage of monophasic action potentials (MAPs) displaying early afterdepolarizations (EADs) in untreated Scn5a+/Δ hearts and following perfusion with physiological buffer containing increasing concentrations of nifedipine (1 nm, 10 nm, 100 nm, 300 nm and 1 μm) (A) and corresponding log concentration–response curve (B).

Thus, for EAD suppression in Scn5a+/Δ hearts, we calculated an effective IC50 for nifedipine to be 79.3 nm (Fig. 2B), in keeping with previous studies in isolated guinea pig myocytes whereby nifedipine specifically blocked the L-type Ca2+ current (ICa,L) with an IC50 of 50 nm (Shen et al. 2000).

Anti-arrhythmic effects of nifedipine are unrelated to alterations in transmural dispersion of action potential duration in Scn5a+/Δ hearts

Murine repolarization is known to demonstrate marked regional heterogeneity, which may influence arrhythmogenic propensity (Anumonwo et al. 2001). However, left ventricular endocardial action potential duration (APD) has been shown to be either comparable (Anumonwo et al. 2001; Knollmann et al. 2001) or longer (Dilly et al. 2006) than the corresponding epicardial APD in a number of previous murine studies. Debate continues regarding the most appropriate method of assessing repolarization heterogeneity in isolated hearts. Although we consider transmural dispersion of repolarization (TDR) to be the sum of local activation time (AT) and APD (Opthof & Coronel, 2005), we found only insignificant differences in AT between endocardium and epicardium in Scn5a+/Δ (13 ± 2.1 ms versus 11.3 ± 0.7 ms, respectively; P > 0.05) and WT (13.7 ± 1.5 ms versus 11 ± 0.6 ms, respectively; P > 0.05) hearts. Similarly, no significant difference in AT was seen in the presence of nifedipine between endocardium and epicardium in Scn5a+/Δ (13.3 ± 1.2 ms versus 13.3 ± 1.3 ms, respectively; P > 0.05) and WT (12 ± 1.2 ms versus 11 ± 0.6 ms, respectively; P > 0.05) as previously reported (Milberg & Eckardt, 2005). Thus, in the present study, TDR is expressed simply as ΔAPD90, obtained from the absolute difference between mean left ventricular endocardial APD90 and mean left ventricular epicardial APD90 as used in previous models of LQT3 (Milberg et al. 2005b).

Measurement of endocardial and epicardial APD90 was performed in all preparations during right ventricular epicardial pacing at 125 ms to standardize for intrinsic differences in heart rate. Individual APD90 values for each heart studied under any given pharmacological condition were derived from the mean values obtained from four sets of 10 individual MAPs selected through each 20 min sampling period. No significant differences in APD90 were observed in MAPs sampled during these successive sets within this 20 min sampling period whether from Scn5a+/Δ (n = 5) or WT (n = 5) hearts.

In Scn5a+/Δ hearts alone, epicardial APD90 was 60.0 ± 0.9 ms compared with 46.9 ± 1.6 ms in WT hearts alone (P < 0.05; n = 5). The corresponding endocardial APD90 values were 52.0 ± 0.7 ms and 53.7 ± 1.6 ms in Scn5a+/Δ and WT hearts, respectively (P > 0.05; n = 5). Thus, ΔAPD90 in Scn5a+/Δ hearts was larger and negative, due to the prolonged epicardial APD90, whereas in WT hearts, ΔAPD90 was smaller and positive, in parallel with their respective arrhythmogenic or non-arrhythmogenic phenotypes. Therefore, mean ΔAPD90 was –8.0 ± 1.1 ms in Scn5a+/Δ hearts compared with 6.8 ± 2.3 ms in WT hearts (n = 5). Following perfusion of Scn5a+/Δ hearts with physiological buffer containing increasing concentrations of nifedipine (1 nm, 10 nm, 100 nm, 300 nm and 1 μm), no significant differences were observed in epicardial APD90 at 62.6 ± 2.4, 69.3 ± 1.9, 68.4 ± 1.4, 69.4 ± 1.0 and 66.0 ± 1.1 ms, respectively, endocardial APD90 at 45.1 ± 6.6, 48.0 ± 6.2, 50.6 ± 5.7, 51.7 ± 5.2 and 47.6 ± 6.0 ms, respectively, and thus ΔAPD90 at −17.5 ± 7.0, −21.3 ± 6.5, −17.8 ± 5.9, −17.7 ± 5.3 and −18.4 ± 6.1 ms, respectively (P > 0.05; n = 5) (Fig. 3A). Similarly, following perfusion of WT hearts with physiological buffer containing identical concentrations of nifedipine (1 nm, 10 nm, 100 nm, 300 nm and 1 μm), no significant differences were observed in epicardial APD90 at 44.7 ± 1.1, 45.0 ± 1.6, 44.9 ± 2.3, 43.2 ± 1.3 and 43.1 ± 3.0 ms, respectively, endocardial APD90 at 52.6 ± 1.4, 54.6 ± 0.1, 53.5 ± 2.2, 47.9 ± 2.3 and 46.9 ± 2.7 ms, respectively, and thus ΔAPD90 at 7.9 ± 1.8, 9.6 ± 1.6, 8.6 ± 3.2, 4.7 ± 2.6 and 3.8 ± 4.0 ms, respectively (P > 0.05; n = 5) (Fig. 3B).

Figure 3. Effect of nifedipine on endocardial and epicardial APD90 values in Scn5a+/Δ and WT hearts.

Figure 3

Figure 3

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Comparison of the effect of nifedipine (1 nm, 10 nm, 100 nm, 300 nm and 1 μm) on mean ± s.e.m. endocardial and epicardial APD90 values and ΔAPD90 in Scn5a+/Δ (A) and WT hearts (B).

Nifedipine suppresses ventricular arrhythmias in Scn5a+/Δ hearts following programmed electrical stimulation

The effect of nifedipine on arrhythmogenicity was further evaluated during provocation using programmed electrical stimulation (PES), as with previous murine models of arrhythmia syndromes (Berul, 2003). False positive results, as can also occur in clinical situations, were minimized in the present study by the use of mice from a homogenous, 129 genetic background and an avoidance of closely coupled drive trains, complex extra-stimuli protocols and further pharmacological enhancements (Berul et al. 2001; Maguire et al. 2003).

Following a drive train of 8 paced beats at a resting physiological rate of 125 ms cycle length, the application of a single premature beat induced VT in all five Scn5a+/Δ hearts, mean duration 2.2 ± 1.0 s (Fig. 4A), whereas such manoeuvres failed to induce VT in all WT hearts (n = 5) (Fig. 4B). Following perfusion with physiological buffer containing 1 nm nifedipine, VT remained inducible in all five Scn5a+/Δ hearts. However, following perfusion with 10 nm nifedipine, VT was inducible in only 3 out of 5 Scn5a+/Δ hearts, and only 1 out of 5 Scn5a+/Δ hearts following perfusion with 100 nm and 300 nm nifedipine, respectively. Furthermore, perfusion with 1 μm nifedipine completely suppressed VT induction in all 5 Scn5a+/Δ hearts (Fig. 4C). No effects were observed following perfusion of WT preparations with identical concentrations of nifedipine (n = 5). These findings taken together are consistent with a specific action of nifedipine on ventricular arrhythmogenesis.

Figure 4. Effect of nifedipine on ventricular arrhythmogenesis in Scn5a+/Δ and WT hearts.

Figure 4

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Bipolar electrograms (BEG) were recorded from an isolated Scn5a+/Δ and WT heart during programmed electrical stimulation. A premature stimulus (asterisk) can be seen to induce ventricular tachycardia (VT) (labelled) in the Scn5a+/Δ heart (A), but not in the WT heart (B) which eventually fails to respond to stimulation as the ventricular effective refractory period (VERP) is reached (labelled). The number of Scn5a+/Δ hearts in which VT was affected by nifedipine (1 nm, 10 nm, 100 nm, 300 nm and 1 μm) is shown in C (out of 5 hearts).

Nifedipine conserves ventricular effective refractory period in Scn5a+/Δ hearts

In addition to re-entrant substrate, tissue refractoriness is known to be closely associated with the inducibility or otherwise of ventricular arrhythmogenesis in murine hearts (Maguire et al. 2003) and therefore the effects of nifedipine on ventricular effective refractory periods (VERPs) were also investigated in the present study. Following standard baseline ventricular pacing, VERP was measured using the PES decremental pacing protocol. Pacing stimuli were applied to the right ventricular epicardial surface and recordings taken from the left ventricular epicardial surface. VERP was taken to be the longest S1–S2 interval which did not elicit a corresponding electrogram.

In Scn5a+/Δ hearts, mean VERP was 45.5 ± 5.7 ms (n = 5) compared with 52.6 ± 13.8 ms in WT controls (n = 5; P > 0.05). No difference in mean VERP was observed in either Scn5a+/Δ or WT hearts following perfusion with physiological buffer containing 1 μm nifedipine (Fig. 5).

Figure 5. Effect of nifedipine on ventricular effective refractory period in Scn5a+/Δ and WT hearts.

Figure 5

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Comparison of the effect of 1 μm nifedipine on ventricular effective refractory period (VERP) between Scn5a+/Δ and WT hearts.

Nifedipine abolishes inward Ca2+ current in Scn5a+/Δ and WT myocytes

The effect of nifedipine upon the inward Ca2+ current was directly observed in ventricular myocytes, enzymatically dissociated from the left ventricle from Scn5a+/Δ and WT hearts. Depolarizing pulses from a holding voltage of −40 mV to 10 mV triggered a mean inward Ca2+ current of 824 ± 68 pA in ventricular myocytes from Scn5a+/Δ hearts (n = 10) compared with a mean current of 1003 ± 374 pA in corresponding ventricular myocytes from WT hearts (n = 10; P > 0.05). However, following the application of nifedipine (300 nm), complete suppression of this current was observed in all cells from both Scn5a+/Δ and WT hearts (n = 20) (Fig. 6A). Finally, the absence of any demonstrable effect of nifedipine upon inward Na+ currents was confirmed in ventricular myocytes from Scn5a+/Δ hearts (n = 5) using depolarizing pulses from −100 to −40 mV (Fig. 6B).

Figure 6. Patch clamp studies in isolated ventricular myocytes from Scn5a+/Δ hearts.

Figure 6

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Patch clamp studies in isolated ventricular myocytes from left ventricle of Scn5a+/Δ hearts revealed that nifedipine (300 nm) completely suppressed the inward Ca2+ current following depolarizing pulses from a holding voltage of −40 mV to 10 mV (A) but had no effect on inward Na+ currents following depolarizing pulses from −100 mV to −40 mV (B).

Discussion

The characteristically increased late Na+ current (INa) in long QT 3 syndrome (LQT3) prolongs cardiac action potential duration (APD) and is thought thereby to facilitate ventricular tachycardia (VT) through a combination of triggered and re-entrant mechanisms (Restivo et al. 2004). Similar arrhythmogenic increases in late INa have also been implicated in a range of common pathophysiological states including tissue hypoxia, cardiac failure and following myocardial infarction (Belardinelli et al. 2006; Noble & Noble, 2006). Such arrhythmogenic features have been successfully reproduced in a murine model following the introduction of the ΔKPQ 1505–1507 deletion into the SCN5A gene (Nuyens et al. 2001; Head et al. 2005). However, the hearts from such mice show an unfavourable response to in vivo therapy with β-adrenoreceptor antagonism, as do isolated perfused Scn5a+/Δ hearts in vitro (Fabritz et al. 2005; Head et al. 2005) which parallel clinical reports of a generally poor response of LQT3 patients to β-adrenoreceptor antagonists compared with other LQTS subtypes, particularly LQT1 (Priori et al. 2004). Nonetheless, antagonism of the Ca2+ channel has been proposed as an alternative therapy (Shimizu et al. 2005) on the basis of clinical observations (Shimizu et al. 1995; Komiya et al. 2004) which are supported by indirect experimental evidence from feline ventricular wedge models of acquired LQTS in which rapid (IKr) and slow (IKs) components of the delayed rectifier K+ current had to be suppressed by E-4031 and chromanol 293B, respectively (Aiba et al. 2005), and recently from an intact LQT3 rabbit heart model whereby INa was augmented by veratridrine (Milberg et al. 2005a). Prompted by these earlier studies, we report and characterize for the first time in the whole heart and at the cellular level, the anti-arrhythmic effects of L-type Ca2+ channel antagonism in a LQT3 model directly derived from ΔKPQ Scn5a mice.

It is generally accepted that action potential prolongation predisposes to early afterdepolarizations (EADs) which in turn can trigger ventricular arrhythmogenesis in LQTS (Volders et al. 2000). Such EADs are believed to follow reactivation of the L-type Ca2+ channel within a voltage ‘window’ (January & Riddle, 1989) which may be generated by the effects of late INa upon the resting membrane potential. However, the use of the phenylalkylamine verapamil in earlier animal models (Milberg et al. 2005a; Aiba et al. 2005) precluded specific comment on the precise involvement of the L-type Ca2 current (ICa,L) due to its known wide-ranging pharmacological effects involving INa (Pidoplichko & Verkhratskii, 1989) and IKr (Zhang et al. 1999). In contrast, the dihydropyridine nifedipine is a highly specific antagonist against the L-type Ca2+ channel (Verheijck et al. 1999; Zhang et al. 1999) and has previously demonstrated anti-arrhythmic effects in several experimental animal models (Nattel & Quantz, 1988; Anderson et al. 1998) including a murine model of long QT 5 syndrome (LQT5) generated through targeted disruption of KCNE1 (Balasubramaniam et al. 2003). These observations, supported by our previous report that nifedipine reduces electrically evoked Ca2+ responses in fluo-3-loaded, isolated murine ventricular myocytes (Balasubramaniam et al. 2004), directly implicates ICa,L in the generation of EADs in Scn5a+/Δ hearts, in apparent contradiction to earlier studies (Patterson et al. 1997; Choi et al. 2002). These findings permit a scheme whereby prolonged epicardial APD observed in Scn5a+/Δ hearts, possibly reflecting regional differences in late INa, is capable of generating the necessary critical voltage ‘window’ described for L-type Ca2+ channel reactivation (January & Riddle, 1989; Ming et al. 1994; Viswanathan & Rudy, 2000).

The specific L-type Ca2+-blocking properties of the dihydropyridine nifedipine are well described in the literature (Verheijck et al. 1999), although there is little consistency regarding the concentrations used. In the present experiments, we used a range of nifedipine concentrations up to a maximum of 1 μm, based upon a careful review of previously published work. Specifically, studies in isolated rodent myocytes reported 90–99% reductions of ICa,L with nifedipine concentrations from 10 to 32 μm (Levi & Issberner, 1996; Levi et al. 1996; Wasserstrom & Vites, 1996) whereas other studies describe incomplete blockade of ICa,L even at a concentration of 20 μm (Sipido et al. 1995). However, specific antagonism of the L-type Ca2+ channel has been shown with 10 μm nifedipine (Yao et al. 1998) and alternative experiments in isolated mouse myocytes reported EAD suppression with nifedipine concentrations between 5 and 8 μm (Liu et al. 1990). Importantly, nifedipine was shown to block ICa,L with an IC50 of 50 nm at a holding potential of −40 mV, which is within the voltage ‘window’ for L-type Ca2+ reactivation (Shen et al. 2000). Indeed, in the present experiments, a concentration-dependent suppression of EADs and spontaneous paroxysmal VT (pVT) was observed in Scn5a+/Δ hearts with increasing concentrations of nifedipine (10 nm to 1 μm) giving a calculated IC50 of 79.3 nm. Furthermore, nifedipine (10 nm to 1 μm) also prevented inducible VT in Scn5a+/Δ hearts subjected to programmed electrical stimulation. Importantly, we can confirm that this effect was not mediated through increases in ventricular effective refractory period in keeping with our previous observations with nifedipine (1 μm) in mice with targeted disruption of KCNE1 (Balasubramaniam et al. 2003). At such concentrations, this effect is unlikely to be due to the action of nifedipine on any other channels. Indeed, in isolated rabbit myocytes, whereas 2 μm has been associated with complete suppression of ICa,L (Hagiwara et al. 1988), nifedipine concentrations as high as 5 μm have no effects upon ICa,T, INa, IK and If (Verheijck et al. 1999). Nonetheless, the exclusive effect of nifedipine (300 nm) upon the inward Ca2+ current was confirmed using patch clamp techniques in isolated ventricular myocytes from Scn5a+/Δ hearts. Furthermore, we confirmed that at the same concentration, no effect is seen upon the inward Na+ currents in similarly isolated ventricular myocytes in Scn5a+/Δ hearts. Certainly, in the present study, any non-specific actions of nifedipine, particularly affecting the repolarizing K+ currents, could potentially negate any beneficial effects upon the L-type Ca2+ channel. Such effects are considered highly unlikely for several reasons. Firstly, it has already been shown that IKr and IKs in isolated rodent myocytes are unaffected by nifedipine concentrations ≤ 10 μm, although individual currents were affected by nifedipine concentrations of 275 μm and 360 μm, respectively (Zhabyeyev et al. 2000). Indeed, even in the most sensitive of all the native rodent K+ currents, the IC50 for nifedipine is 30 μm (Jahnel et al. 1994). Secondly, the action potential prolongation observed in Scn5a+/Δ hearts at baseline in the present study remained unchanged following perfusion with 1 μm nifedipine and no action potential prolongation was observed in the corresponding WT hearts, thereby excluding any significant deleterious effects upon outward repolarizing currents.

Dispersion of repolarization across the ventricular wall in LQTS can subsequently facilitate the maintenance of EAD-mediated triggered activity in the form of re-entrant wavefronts (El-Sherif et al. 1996). Furthermore, a marked dispersion of repolarization across the myocardial wall has been shown to generate areas of new focal activation following decremental epicardial premature stimulation in the canine ventricular wedge preparation made to indirectly simulate LQT3 conditions through augmenting INa using anemone toxin II (ATX-II) (Ueda et al. 2004). Conversely, reducing transmural dispersion of repolarization has been shown to confer an anti-arrhythmogenic effect in several animal models of LQTS, including LQT3 (Shimizu & Antzelevitch, 1997, 2000a,b; Fabritz et al. 2003b; Milberg et al. 2005a). In the present study, the preferential prolongation of the epicardial APD90 in Scn5a+/Δ hearts generated a value for ΔAPD90 in Scn5a+/Δ hearts that was the inverse of the pattern seen in the WT control hearts. However, unlike in previous studies where anti-arrhythmogenic effects of drugs including verapamil and mexiletine parallel suppression of EADs and modification of re-entrant substrate (Fabritz et al. 2003b; Milberg et al. 2005a), the anti-arrhythmogenic effect of nifedipine occurred despite a neutral effect upon ΔAPD90 in Scn5a+/Δ hearts. Therefore, we can confirm for the first time in Scn5a+/Δ hearts that suppression of EADs alone is sufficient to suppress arrhythmogenesis without alteration to underlying re-entrant substrate.

An important theoretical consideration regarding the potential clinical role for nifedipine in LQT3 is the well-recognized anti-hypertensive action with corresponding activation of the baroreceptor reflex and associated relative tachycardia (Boddeke et al. 1987). However, LQT3 patients tend to show less frequent but more lethal cardiac events that typically take place during rest or sleep, in contrast to other LQT subtypes, notably LQT1, whereby events are more commonly associated with exercise or strong emotion (Schwartz et al. 1995, 2001). Indeed, prophylactic therapy against sudden cardiac death in LQTS is with the use of β-adrenoreceptor blockers (Priori et al. 2001), yet predictably, LQT3 patients appear to derive less benefit from treatment with β-adrenoreceptor-blocking agents than the other LQTS subtypes (Moss, 1998; Moss et al. 2000; Schwartz et al. 2001; Priori et al. 2004). Furthermore, β-adrenoreceptor blockade appears to correlate with slowed atrial, atrioventricular and ventricular conduction in carriers of ΔKPQ LQT3 (Zareba et al. 2001) and a bradycardic mode of cardiac death in some LQT3 patients (van den Berg et al. 2001). Such observations have also been reproduced in the laboratory. In the canine ventricular wedge preparation made to model LQT3 with ATX-II, β-adrenergic stimulation with isoproterenol (100 nm) was associated with an anti-arrhythmogenic effect, unlike that seen with corresponding models of LQT1 and LQT2; furthermore, in the LQT3 model, β-adrenergic antagonism with propranolol was indirectly pro-arrhythmic as it reversed the beneficial effects that had previously been induced by isoproterenol (Shimizu & Antzelevitch, 2000a). Furthermore, increased heart rates have also been associated with anti-arrhythmogenic effects in ΔKPQ Scn5a mice using isoproterenol (Nuyens et al. 2001) and pacing (Fabritz et al. 2003b).

In the present study, we demonstrate that the suppression of EADs through antagonism of the L-type Ca2+ channel is associated with an anti-arrhythmogenic effect. Although there is little clinical data to support the use of calcium antagonists in the prevention of sudden cardiac death in LQT3 syndrome, the increasing experimental data supporting their value considered alongside the poor efficacy of β-adrenoreceptor antagonists in LQT3, would justify further explorations in this direction.

Acknowledgments

Supported by grants from the British Heart Foundation, the Medical Research Council, the Wellcome Trust, the Helen Kirkland Fund for Cardiac Research and the Raymond and Beverly Sacker Medical Research Centre.

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