Download pdf - Effects of L-type Ca 2+ channel antagonism on ventricular arrhythmogenesis in murine hearts containing a modification in the Scn5a gene modelling human long QT syndrome 3

J Physiol 578.1 (2007) pp 85–97 85

Effects of L-type Ca2+ channel antagonism on ventriculararrhythmogenesis in murine hearts containing amodification in the Scn5a gene modelling human long QTsyndrome 3

Glyn Thomas1, Iman S. Gurung2, Matthew J. Killeen2, Parvez Hakim2, Catharine A. Goddard1,Martyn P. Mahaut-Smith2, William H. Colledge2, Andrew A. Grace1 and Christopher L.-H. Huang2

1Section of Cardiovascular Biology, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK2Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK

Ventricular arrhythmogenesis in long QT 3 syndrome (LQT3) involves both triggered activityand re-entrant excitation arising from delayed ventricular repolarization. Effects of specificL-type Ca2+ channel antagonism were explored in a gain-of-function murine LQT3 modelproduced by a ∆KPQ 1505–1507 deletion in the SCN5A gene. Monophasic action potentials(MAPs) were recorded from epicardial and endocardial surfaces of intact, Langendorff-perfusedScn5a+/∆ hearts. In untreated Scn5a+/∆ 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 msand 53.7 ± 1.6 ms in Scn5a+/∆ and WT hearts, respectively (P > 0.05; n = 5). Epicardial earlyafterdepolarizations (EADs), often accompanied by spontaneous ventricular tachycardia (VT),occurred in 100% of MAPs from Scn5a+/∆ 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 followingperfusion with 10 nM, 100 nM, 300 nM and 1 µM nifedipine, respectively (P < 0.05; n = 5), givingan effective IC50 concentration of 79.3 nM. Programmed electrical stimulation (PES) inducedVT in all five Scn5a+/∆ hearts (n = 5) but not in any WT hearts (n = 5). However, repeat PESinduced VT in 3, 2, 2 and 0 out of 5 Scn5a+/∆ hearts following perfusion with 10 nM, 100 nM,300 nM and 1 µM nifedipine, respectively. Patch clamp studies in isolated ventricular myocytesfrom Scn5a+/∆ and WT hearts confirmed that nifedipine (300 nM) completely suppressed theinward Ca2+ current but had no effect on inward Na+ currents. No significant effects were seenon epicardial APD90, endocardial APD90 or ventricular effective refractory period in Scn5a+/∆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 specificanti-arrhythmic effects in Scn5a+/∆ hearts through suppression of EADs.

(Resubmitted 1 October 2006; accepted after revision 13 November 2006; first published online 16 November 2006)Corresponding author C. L.-H. Huang: Physiological Laboratory, University of Cambridge, Downing Street, CambridgeCB2 3EG, UK. Email: [email protected]

Clinically, long QT3 syndrome (LQT3) is characterizedby 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 encodesthe α-(pore-forming) subunit of the cardiac Na+

channel, leading to an increased late Na+ current(INa), prolonged action potential plateau and delayedrepolarization. Both triggered activity through earlyafterdepolarizations (EADs) and re-entrant excitationvia 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 acorresponding, experimental, murine model, producedby the introduction of the gain-of-function knock-in�KPQ 1505–1507 deletion into the murine SCN5Agene (Nuyens et al. 2001; Head et al. 2005), suggestinga possible translational application for this and othergenetically modified murine systems. Microelectroderecordings from isolated ventricular myocytes fromsuch Scn5a+/� hearts thus revealed prolonged action

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society DOI: 10.1113/jphysiol.2006.121921

86 G. Thomas and others J Physiol 578.1

potential durations (APDs) and spontaneous EADs. Inaddition, an adaptation of the clinical technique ofpaced electrogram fractionation analysis recently usedto assess arrhythmogenicity in patients with LQTS(Roden, 2006; Saumarez et al. 2006) identified significantelectrogram duration and conduction curve dispersionin intact, isolated Langendorff-perfused Scn5a+/�hearts, strongly suggestive of a concomitant re-entrantsubstrate. Furthermore, reducing INa using the class IBNa+ channel antagonist mexiletine in Scn5a+/� exerts ananti-arrhythmogenic effect through a combined reductionin EAD frequency and interventricular dispersion ofAPD90 (Fabritz et al. 2003b), consistent with clinicalobservations (Schwartz et al. 1995).

Current pharmacological treatment of LQTS primarilyinvolves β-adrenoreceptor antagonism (Priori et al. 2001)but this appears to be of less clinical benefit in LQT3 thanin 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 successfularrhythmia suppression with the phenylalkylamine-typeCa2+ channel antagonist verapamil (Shimizu et al. 1995;Komiya et al. 2004) have prompted suggestions that suchdrugs might be appropriate as adjunctive therapy in LQT1,LQT2 and even LQT3 patients (Shimizu et al. 2005).Thus, experimental reports suggest that Ca2+ channelantagonism by verapamil suppresses EADs and reducesTDR in feline wedge preparations made to model acquired(as opposed to congenital) LQTS though suppressing therapid (IKr) and slow (IKs) components of the delayedrectifier K+ current by E-4031 and chromanol 293B,respectively (Aiba et al. 2005), and in a recent intact,pharmacological rabbit preparation made to model LQT3through augmenting INa by veratridine (Milberg et al.2005a).

However, verapamil is known also to exert effects onINa (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 itseffect on the L-type Ca2+ current (ICa,L) (Triggle, 2006),whereas the dihydropyridine Ca2+ antagonist nifedipineexerts no effects upon upon T-type Ca2+ current ICa,T,INa, IK or I f at concentrations as high as 5 µm (Verheijcket al. 1999). The present experiments, therefore, report andcharacterize for the first time, the anti-arrhythmic effectof the specific L-type Ca2+ channel antagonist nifedipinein mice with targeted disruption of the Scn5a gene.These findings complement previous reports of nifedipineupon 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 targeteddisruption 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-bufferedKrebs-Henseleit solution (mm: NaCl 119, NaHCO3 25, KCl4, KH2PO4 1.2, MgCl2 1, CaCl2 1.8, glucose 10 and sodiumpyruvate 2, pH 7.4) bubbled with 95% O2−5% CO2. Asmall (3–4 mm) section of aorta was cannulated underthe buffer surface and sutured to a 21-gauge tailor-madecannula, pre-filled with ice-cold buffer solution, thensecured with a metallic clip for retrograde perfusionusing the above solution at 2–2.5 ml min−1 using aperistaltic pump (Watson-Marlow Bredel model 505S,Falmouth, Cornwall, UK) after passing through 200 µmand 5 µm filters (Millipore, Watford, UK) and warmingto 37◦C via a water jacket and circulator (Technemodel C-85A, Cambridge, UK). Healthy, viable heartssuitable for experimentation regained a homogeneouspink colouration and spontaneous rhythmic contractionon warming. Hearts not demonstrating these featureswere immediately discarded, to avoid false positiveresults. Complete atrioventricular (AV) block was inducedin selected preparations by crush-ablation of the AVnode using surgical forceps as previously described andconfirmed by the recording of dissociated atrial andventricular waveforms (Fabritz et al. 2003b; Milberg et al.2005b) although this procedure reduced the longevityof both Scn5a+/� and WT preparations. Hearts wereperfused with physiological perfusion buffer for 20 minprior to experimentation, to avoid possible residual effectsof endogenous catecholamine release.

Monophasic action potential recordings

Monophasic action potentials (MAPs) were recordedfrom the epicardium using a spring-loaded, AgClcontact (2 mm tip diameter) MAP electrode (LintonInstruments, Harvard Apparatus, UK) which waspositioned manually. Endocardial recordings wereobtained using a custom-built electrode, constructed fromtwo twisted strands of Teflon-coated (0.25 mm diameter)silver wire (99.99% purity) (Advent Research MaterialsLtd, UK), galvanically chlorided and introduced into theleft ventricular cavity through a small access windowcreated in the interventricular septum and rotated suchthat the tip came to rest against the free wall. The endo-cardial electrode was initially placed by hand, and theposition maintained by custom-designed magnetic gripspositioned on a metallic platform. The entire apparatuswas earthed to reduce electrical interference. Signalswere amplified and low-pass filtered appropriately formurine recordings (0.1 Hz to 300 Hz) (Gould 2400S,

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society

J Physiol 578.1 Ventricular arrhythmogenesis in �KPQ Scn5a hearts 87

Gould-Nicolet Technologies, Ilford, UK) (Fabritz et al.2003a) then digitized using a 1401plus analog-to-digitalconverter (Cambridge Electronic Design, Cambridge,UK). Analysis of the MAP waveforms was performedused Spike II software (Cambridge Electronic Design).Results were expressed as means ± s.e.m. and differentexperimental groups compared using ANOVA (SPSSsoftware).

Programmed electrical stimulation

For programmed electrical stimulation (PES) of the heartpaired (1 mm interpole spacing) platinum stimulating andrecording electrodes were placed on the basal epicardialsurfaces of the right and left ventricles, respectively. Theperiod of initial pacing used 2 ms square-wave stimuliwith amplitudes of three times excitation threshold (GrassS48 stimulator, Grass-Telefactor, Slough, UK) for 20 minat 125 ms basic cycle length (BCL). All experimentalmice were bred from a 129 genetic background, which,along with C57 mice, are less susceptible to PES-inducedarrhythmias than FBV or Black Swiss mice (Maguire et al.2003). Nevertheless, complex pacing protocols involvingdouble/triple extra-stimuli and rapid burst pacing wereavoided to reduce the risk of false positive results (Maguireet al. 2003). PES pacing protocols comprised a drivetrain of 8 paced S1 beats at 125 ms BCL, followed by apremature S2 extrastimulus every ninth beat. S1–S2intervals first equalled the pacing interval and were thensuccessively reduced by 1 ms with each 9 beat cycle untilventricular refractoriness was reached, whereupon the S2stimuli elicited no electrograms.

Isolation of single ventricular myocytes

Following cannulation, the heart was perfused in aretrograde fashion with Krebs-Henseleit buffer, warmedto 37◦C by means of a water jacket and circulator (Technemodel C-85A, Cambridge, UK), at a rate of 2–2.5 ml min−1

for 5 min, until the heart regained a homogeneous pinkcolouration and began contracting spontaneously. Theheart was then perfused for 5 min with a nitrilotriaceticacid-based perfusion buffer containing (mm): 125 NaCl,4.75 KCl, 5 MgSO4, 10 Hepes, 5 sodium pyruvate, 20glucose, 20 taurine and 4.5 nitrilotriacetic acid. Followingthis, the heart was perfused with a digestion bufferfor 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 of90 deg curved forceps were used to remove a wedge-shapedsegment of myocardium from the left ventricular freewall. Ventricular tissue samples were placed in a tubecontaining digestion buffer in addition to 1 mg ml−1

bovine serum albumin (Sigma-Aldrich, Poole, UK) for5 min before gentle trituration for a further 5 min in thesame solution. Tissue samples were subsequently spundown in a centrifuge (1000 r.p.m. (1860 g) for 3 min)before the supernatant from the tissue tubes was discardedand replaced with a wash buffer containing (mm): 135NaCl, 1.1 MgCl2, 1.8 CaCl2, 5.4 KCl, 10 Hepes, 10 glucoseand pH was adjusted to 7.35 with NaOH. Ventricularmyocytes were stored in the above wash buffer andwere studied within 4–6 h. Following initial perfusion ofthe heart, all subsequent steps were performed at roomtemperature (22-24◦C).

Single cell electrophysiology

Conventional whole-cell patch clamp recording in voltageclamp mode was carried out using an Axopatch 200Bamplifier (Axon Instruments, CA, USA) coupled to aDigidata series computer interface and controlled bypCLAMP software (Axon Instruments). Pipettes withresistances of 1–4 M� were pulled from borosilicateglass capillaries (1.5 mm outer and 0.86 inner diameter,GC150-10; Harvard Apparatus Ltd). Extracellular buffercontained (mm): 135 NaCl, 1.1 MgCl2, 1.8 CaCl2, 5.4KCl, 10 Hepes, 10 glucose, and pH was adjusted to 7.35with NaOH. Intracellular pipette saline contained (mm):130 KCl, 1 MgCl2, 10 Hepes, 5 Mg-ATP, 5 Na2-creatinephosphate, and pH was adjusted to 7.2 with KOH. Afterformation of a gigaseal, the whole-cell configuration wasachieved by applying gentle suction through the pipetteand a brief voltage (ZAP) pulse. Up to 75% series resistancecompensation was achieved. Inward Ca2+ currentswere triggered by applying a series of 10 mV incrementalvoltage pulses from −40 to 10 mV from a holding potentialof −40 mV, and inward Na+ currents were triggered withsimilar pulses from −100 to −40 mV from a holdingvoltage of −100 mV.

Pharmacological agents

All drugs (Sigma-Aldrich, Poole, UK) were first preparedas 1 mm stock solutions. Nifedipine was dissolved in96% ethanol. Final drug concentrations were achieved bydilution with buffer solution. Nifedipine stock solutionswere refrigerated at 4◦C and were kept wrapped in foil toprevent light degradation.

Results

Nifedipine suppresses afterdepolarizations andspontaneous arrhythmias in Scn5a+/∆ hearts

Early afterdepolarizations (EADs) are generally believedto initiate ventricular tachycardia (VT) in a range ofconditions including LQTS, which are then maintained



via re-entrant excitation (Antzelevitch & Shimizu, 2002).In the present study, simultaneous recording of leftventricular endocardial and epicardial monophasic actionpotentials (MAPs) from Langendorff-perfused wholeheart preparations from Scn5a+/� (n = 5) and WT(n = 5) mice produced high quality signals that satisfiedpreviously documented criteria of a stable baseline andtriangular MAP morphology, rapid upstroke phase, and aconsistent amplitude (Knollmann et al. 2001).

Firstly, Fig. 1A shows a representative trace of anepicardial MAP recording from a spontaneously beatingScn5a+/� heart following the induction of bradycardiathrough mechanical atrioventricular (AV) blockade.Epicardial EADs occurred in 49 ± 10.5% of MAPsobserved in 20 s epochs, randomly selected from a totalsample duration time of 20 min (n = 10 epochs fromeach of 2 Scn5a+/� hearts). Furthermore, episodes ofspontaneous VT, lasting a mean duration of 0.59 ± 0.1 swere observed in 4 out of 5 Scn5a+/� similarly preparedhearts, in keeping with previous findings (Nuyens et al.2001; Fabritz et al. 2003b). However, we noted, for the firsttime to our knowledge, that no such features were recordedfrom the endocardial sites in Scn5a+/� hearts duringsimilar sampling periods, each of 20 min per heart (n = 5).However, perfusion with physiological buffer containing1 µm nifedipine suppressed epicardial EADs to 3.9 ± 1.5%of MAPs, in identically selected epochs from a total sampleduration time of 20 min in each heart studied (n = 5;

Figure 1. Monophasic action potential recordings from Scn5a+/∆ and WT heartsRecordings were obtained from the epicardial surface of spontaneously beating Scn5a+/� and WT hearts. Multipleearly 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 sucharrhythmias 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).

P < 0.05). Furthermore, no episodes of spontaneous VTwere observed in comparable sampling periods in all fiveof the previously mentioned Scn5a+/� hearts (P < 0.05)(Fig. 1B).

Conversely, in WT hearts, no EADs or spontaneous VTwere recorded from either the endocardial or epicardialsurfaces (n = 5) (Fig. 1C) subject to a similar samplingscheme. Similarly, no such features were observedfollowing perfusion of all hearts with physiological buffercontaining 1 µm nifedipine (Fig. 1D).

Secondly, these initial findings were extended bysubjecting a further five Scn5a+/� hearts to serialperfusion with buffer containing a range of nifedipineconcentrations (1 nm, 10 nm, 100 nm, 300 nm and1 µm). Mechanical induction of complete AV blockwas specifically avoided in subsequent experiments tominimize the risk of myocardial trauma and ischaemia.Therefore, a mild hypokalaemic buffer solution (4.0 mmK+) was used to facilitate bradycardia and EADs aspreviously 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+/�hearts alone (n = 10 epochs from each of 5 Scn5a+/�hearts). However, EAD occurrence was reduced to62 ± 7.1%, 44 ± 9.7% and 10 ± 10% of MAPs followingperfusion with 10 nm, 100 nm and 300 nm nifedipine,respectively (P < 0.05; n = 5). Furthermore, perfusionwith 1 µm nifedipine completely suppressed all EADs in all



MAPs recorded from Scn5a+/� hearts (P < 0.05; n = 5)(Fig. 2A). No EADs were observed in identically treatedWT control hearts (n = 5) with or without nifedipine.

Thus, for EAD suppression in Scn5a+/� hearts, wecalculated an effective IC50 for nifedipine to be 79.3 nm(Fig. 2B), in keeping with previous studies in isolatedguinea pig myocytes whereby nifedipine specificallyblocked the L-type Ca2+ current (ICa,L) with an IC50 of50 nm (Shen et al. 2000).

Anti-arrhythmic effects of nifedipine are unrelated toalterations in transmural dispersion of actionpotential duration in Scn5a+/∆ hearts

Murine repolarization is known to demonstratemarked regional heterogeneity, which may influencearrhythmogenic propensity (Anumonwo et al. 2001).However, left ventricular endocardial action potentialduration (APD) has been shown to be either comparable(Anumonwo et al. 2001; Knollmann et al. 2001) or longer

Figure 2. Percentage of monophasic action potentialsdisplaying early afterdepolarizations in Scn5a+/∆ heartsPercentage of monophasic action potentials (MAPs) displayingearly afterdepolarizations (EADs) in untreated Scn5a+/� heartsand following perfusion with physiological buffer containingincreasing concentrations of nifedipine (1 nM, 10 nM, 100 nM,300 nM and 1 µM) (A) and corresponding log concentration–response curve (B).

(Dilly et al. 2006) than the corresponding epicardialAPD in a number of previous murine studies. Debatecontinues regarding the most appropriate methodof assessing repolarization heterogeneity in isolatedhearts. Although we consider transmural dispersion ofrepolarization (TDR) to be the sum of local activationtime (AT) and APD (Opthof & Coronel, 2005), wefound only insignificant differences in AT betweenendocardium and epicardium in Scn5a+/� (13 ± 2.1 msversus 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 wasseen in the presence of nifedipine between endo-cardium and epicardium in Scn5a+/� (13.3 ± 1.2 msversus 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 �APD90,obtained from the absolute difference between mean leftventricular 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 wasperformed in all preparations during right ventricularepicardial pacing at 125 ms to standardize for intrinsicdifferences in heart rate. Individual APD90 values for eachheart studied under any given pharmacological conditionwere derived from the mean values obtained from foursets of 10 individual MAPs selected through each 20 minsampling period. No significant differences in APD90

were observed in MAPs sampled during these successivesets within this 20 min sampling period whether fromScn5a+/� (n = 5) or WT (n = 5) hearts.

In Scn5a+/� hearts alone, epicardial APD90 was60.0 ± 0.9 ms compared with 46.9 ± 1.6 ms in WThearts alone (P < 0.05; n = 5). The correspondingendocardial APD90 values were 52.0 ± 0.7 ms and53.7 ± 1.6 ms in Scn5a+/� and WT hearts, respectively(P > 0.05; n = 5). Thus, �APD90 in Scn5a+/� heartswas larger and negative, due to the prolongedepicardial APD90, whereas in WT hearts, �APD90 wassmaller and positive, in parallel with their respectivearrhythmogenic or non-arrhythmogenic phenotypes.Therefore, mean �APD90 was –8.0 ± 1.1 ms in Scn5a+/�hearts compared with 6.8 ± 2.3 ms in WT hearts(n = 5). Following perfusion of Scn5a+/� hearts withphysiological buffer containing increasing concentrationsof nifedipine (1 nm, 10 nm, 100 nm, 300 nm and1 µm), no significant differences were observed inepicardial 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, endocardialAPD90 at 45.1 ± 6.6, 48.0 ± 6.2, 50.6 ± 5.7, 51.7 ± 5.2and 47.6 ± 6.0 ms, respectively, and thus �APD90 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 withphysiological buffer containing identical concentrationsof nifedipine (1 nm, 10 nm, 100 nm, 300 nm and1 µm), no significant differences were observed inepicardial 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, endocardialAPD90 at 52.6 ± 1.4, 54.6 ± 0.1, 53.5 ± 2.2, 47.9 ± 2.3and 46.9 ± 2.7 ms, respectively, and thus �APD90 at7.9 ± 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).

Nifedipine suppresses ventricular arrhythmias inScn5a+/∆ hearts following programmed electricalstimulation

The effect of nifedipine on arrhythmogenicity wasfurther evaluated during provocation using programmedelectrical stimulation (PES), as with previous murinemodels of arrhythmia syndromes (Berul, 2003). Falsepositive results, as can also occur in clinical situations, were

minimized in the present study by the use of mice from ahomogenous, 129 genetic background and an avoidanceof closely coupled drive trains, complex extra-stimuliprotocols and further pharmacological enhancements(Berul et al. 2001; Maguire et al. 2003).

Following a drive train of 8 paced beats at a restingphysiological rate of 125 ms cycle length, the applicationof a single premature beat induced VT in all five Scn5a+/�hearts, mean duration 2.2 ± 1.0 s (Fig. 4A), whereas suchmanoeuvres failed to induce VT in all WT hearts (n = 5)(Fig. 4B). Following perfusion with physiological buffercontaining 1 nm nifedipine, VT remained inducible inall five Scn5a+/� hearts. However, following perfusionwith 10 nm nifedipine, VT was inducible in only 3 outof 5 Scn5a+/� hearts, and only 1 out of 5 Scn5a+/�hearts following perfusion with 100 nm and 300 nmnifedipine, respectively. Furthermore, perfusion with 1 µmnifedipine completely suppressed VT induction in all5 Scn5a+/� hearts (Fig. 4C). No effects were observedfollowing perfusion of WT preparations with identicalconcentrations of nifedipine (n = 5). These findings takentogether are consistent with a specific action of nifedipineon ventricular arrhythmogenesis.

Nifedipine conserves ventricular effective refractoryperiod in Scn5a+/∆ hearts

In addition to re-entrant substrate, tissue refractorinessis known to be closely associated with the inducibilityor otherwise of ventricular arrhythmogenesis in murinehearts (Maguire et al. 2003) and therefore the effectsof nifedipine on ventricular effective refractory periods(VERPs) were also investigated in the present study.Following standard baseline ventricular pacing, VERP wasmeasured using the PES decremental pacing protocol.Pacing stimuli were applied to the right ventricularepicardial surface and recordings taken from the leftventricular epicardial surface. VERP was taken to be thelongest S1–S2 interval which did not elicit a correspondingelectrogram.

In Scn5a+/� 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 wasobserved in either Scn5a+/� or WT hearts followingperfusion with physiological buffer containing 1 µmnifedipine (Fig. 5).

Nifedipine abolishes inward Ca2+ current inScn5a+/∆ and WT myocytes

The effect of nifedipine upon the inward Ca2+ current wasdirectly observed in ventricular myocytes, enzymaticallydissociated from the left ventricle from Scn5a+/� andWT 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 fromScn5a+/� hearts (n = 10) compared with a mean currentof 1003 ± 374 pA in corresponding ventricular myocytesfrom WT hearts (n = 10; P > 0.05). However, followingthe application of nifedipine (300 nm), complete

Figure 3. Effect of nifedipine on endocardial and epicardial APD90 values in Scn5a+/∆ and WT heartsComparison of the effect of nifedipine (1 nM, 10 nM, 100 nM, 300 nM and 1 µM) on mean ± S.E.M. endocardial andepicardial APD90 values and �APD90 in Scn5a+/� (A) and WT hearts (B).

suppression of this current was observed in all cells fromboth Scn5a+/� and WT hearts (n = 20) (Fig. 6A). Finally,the absence of any demonstrable effect of nifedipine uponinward Na+ currents was confirmed in ventricularmyocytes from Scn5a+/� hearts (n = 5) usingdepolarizing pulses from −100 to −40 mV (Fig. 6B).



Discussion

The characteristically increased late Na+ current (INa)in long QT 3 syndrome (LQT3) prolongs cardiacaction potential duration (APD) and is thought therebyto facilitate ventricular tachycardia (VT) through acombination of triggered and re-entrant mechanisms(Restivo et al. 2004). Similar arrhythmogenic increasesin late INa have also been implicated in a rangeof common pathophysiological states including tissue

Figure 4. Effect of nifedipine on ventricular arrhythmogenesis in Scn5a+/∆ and WT heartsBipolar electrograms (BEG) were recorded from an isolated Scn5a+/� and WT heart during programmed electricalstimulation. A premature stimulus (asterisk) can be seen to induce ventricular tachycardia (VT) (labelled) in theScn5a+/� heart (A), but not in the WT heart (B) which eventually fails to respond to stimulation as the ventriculareffective refractory period (VERP) is reached (labelled). The number of Scn5a+/� hearts in which VT was affectedby nifedipine (1 nM, 10 nM, 100 nM, 300 nM and 1 µM) is shown in C (out of 5 hearts).

hypoxia, cardiac failure and following myocardialinfarction (Belardinelli et al. 2006; Noble & Noble, 2006).Such arrhythmogenic features have been successfullyreproduced in a murine model following the introductionof the �KPQ 1505–1507 deletion into the SCN5A gene(Nuyens et al. 2001; Head et al. 2005). However, thehearts from such mice show an unfavourable responseto in vivo therapy with β-adrenoreceptor antagonism, asdo isolated perfused Scn5a+/� hearts in vitro (Fabritzet al. 2005; Head et al. 2005) which parallel clinical



reports of a generally poor response of LQT3 patientsto β-adrenoreceptor antagonists compared with otherLQTS subtypes, particularly LQT1 (Priori et al. 2004).Nonetheless, antagonism of the Ca2+ channel has beenproposed 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 indirectexperimental evidence from feline ventricular wedgemodels of acquired LQTS in which rapid (IKr) and slow(IKs) components of the delayed rectifier K+ current had tobe suppressed by E-4031 and chromanol 293B, respectively(Aiba et al. 2005), and recently from an intact LQT3 rabbitheart 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 wholeheart and at the cellular level, the anti-arrhythmic effects ofL-type Ca2+ channel antagonism in a LQT3 model directlyderived from �KPQ Scn5a mice.

It is generally accepted that action potentialprolongation predisposes to early afterdepolarizations(EADs) which in turn can trigger ventriculararrhythmogenesis in LQTS (Volders et al. 2000).Such EADs are believed to follow reactivation of theL-type Ca2+ channel within a voltage ‘window’ (January& Riddle, 1989) which may be generated by the effects oflate INa upon the resting membrane potential. However,the use of the phenylalkylamine verapamil in earlieranimal models (Milberg et al. 2005a; Aiba et al. 2005)precluded specific comment on the precise involvementof the L-type Ca2 current (ICa,L) due to its knownwide-ranging pharmacological effects involving INa

(Pidoplichko & Verkhratskii, 1989) and IKr (Zhang et al.1999). In contrast, the dihydropyridine nifedipine isa highly specific antagonist against the L-type Ca2+

channel (Verheijck et al. 1999; Zhang et al. 1999) andhas previously demonstrated anti-arrhythmic effects inseveral experimental animal models (Nattel & Quantz,1988; Anderson et al. 1998) including a murine model oflong QT 5 syndrome (LQT5) generated through targeteddisruption of KCNE1 (Balasubramaniam et al. 2003).These observations, supported by our previous reportthat nifedipine reduces electrically evoked Ca2+ responsesin fluo-3-loaded, isolated murine ventricular myocytes(Balasubramaniam et al. 2004), directly implicates ICa,L inthe generation of EADs in Scn5a+/� hearts, in apparentcontradiction to earlier studies (Patterson et al. 1997;Choi et al. 2002). These findings permit a scheme wherebyprolonged epicardial APD observed in Scn5a+/� hearts,possibly reflecting regional differences in late INa, iscapable 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 thedihydropyridine nifedipine are well described in the

literature (Verheijck et al. 1999), although there is littleconsistency regarding the concentrations used. In thepresent experiments, we used a range of nifedipineconcentrations up to a maximum of 1 µm, based upona careful review of previously published work. Specifically,studies in isolated rodent myocytes reported 90–99%reductions of ICa,L with nifedipine concentrations from10 to 32 µm (Levi & Issberner, 1996; Levi et al. 1996;Wasserstrom & Vites, 1996) whereas other studies describeincomplete blockade of ICa,L even at a concentration of20 µm (Sipido et al. 1995). However, specific antagonismof the L-type Ca2+ channel has been shown with 10 µmnifedipine (Yao et al. 1998) and alternative experimentsin isolated mouse myocytes reported EAD suppressionwith nifedipine concentrations between 5 and 8 µm(Liu et al. 1990). Importantly, nifedipine was shown toblock ICa,L with an IC50 of 50 nm at a holding potentialof −40 mV, which is within the voltage ‘window’ forL-type Ca2+ reactivation (Shen et al. 2000). Indeed,in the present experiments, a concentration-dependentsuppression of EADs and spontaneous paroxysmal VT(pVT) was observed in Scn5a+/� hearts with increasingconcentrations of nifedipine (10 nm to 1 µm) givinga calculated IC50 of 79.3 nm. Furthermore, nifedipine(10 nm to 1 µm) also prevented inducible VT inScn5a+/� hearts subjected to programmed electricalstimulation. Importantly, we can confirm that thiseffect was not mediated through increases in ventriculareffective refractory period in keeping with our previousobservations with nifedipine (1 µm) in mice with targeteddisruption of KCNE1 (Balasubramaniam et al. 2003). Atsuch concentrations, this effect is unlikely to be due to theaction of nifedipine on any other channels. Indeed, in iso-lated rabbit myocytes, whereas 2 µm has been associatedwith complete suppression of ICa,L (Hagiwara et al. 1988),nifedipine concentrations as high as 5 µm have no effects

Figure 5. Effect of nifedipine on ventricular effective refractoryperiod in Scn5a+/∆ and WT heartsComparison of the effect of 1 µM nifedipine on ventricular effectiverefractory period (VERP) between Scn5a+/� and WT hearts.



upon ICa,T, INa, IK and I f (Verheijck et al. 1999).Nonetheless, the exclusive effect of nifedipine (300 nm)upon the inward Ca2+ current was confirmed using patchclamp techniques in isolated ventricular myocytes fromScn5a+/� hearts. Furthermore, we confirmed that at thesame concentration, no effect is seen upon the inwardNa+ currents in similarly isolated ventricular myocytesin Scn5a+/� hearts. Certainly, in the present study, anynon-specific actions of nifedipine, particularly affectingthe repolarizing K+ currents, could potentially negate anybeneficial effects upon the L-type Ca2+ channel. Sucheffects are considered highly unlikely for several reasons.Firstly, it has already been shown that IKr and IKs inisolated rodent myocytes are unaffected by nifedipineconcentrations ≤ 10 µm, although individual currentswere affected by nifedipine concentrations of 275 µm and

Figure 6. Patch clamp studies in isolatedventricular myocytes from Scn5a+/∆ heartsPatch clamp studies in isolated ventricular myocytesfrom left ventricle of Scn5a+/� hearts revealed thatnifedipine (300 nM) completely suppressed the inwardCa2+ current following depolarizing pulses from aholding voltage of −40 mV to 10 mV (A) but had noeffect on inward Na+ currents following depolarizingpulses from −100 mV to −40 mV (B).

360 µm, respectively (Zhabyeyev et al. 2000). Indeed, evenin 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 inScn5a+/� hearts at baseline in the present study remainedunchanged following perfusion with 1 µm nifedipineand no action potential prolongation was observed inthe corresponding WT hearts, thereby excluding anysignificant deleterious effects upon outward repolarizingcurrents.

Dispersion of repolarization across the ventricular wallin LQTS can subsequently facilitate the maintenanceof EAD-mediated triggered activity in the form ofre-entrant wavefronts (El-Sherif et al. 1996). Furthermore,a marked dispersion of repolarization across themyocardial wall has been shown to generate areas of



new focal activation following decremental epicardialpremature stimulation in the canine ventricular wedgepreparation made to indirectly simulate LQT3 conditionsthrough augmenting INa using anemone toxin II (ATX-II)(Ueda et al. 2004). Conversely, reducing transmuraldispersion of repolarization has been shown to confer ananti-arrhythmogenic effect in several animal models ofLQTS, including LQT3 (Shimizu & Antzelevitch, 1997,2000a,b; Fabritz et al. 2003b; Milberg et al. 2005a). Inthe present study, the preferential prolongation of theepicardial APD90 in Scn5a+/� hearts generated a valuefor �APD90 in Scn5a+/� hearts that was the inverseof the pattern seen in the WT control hearts. However,unlike in previous studies where anti-arrhythmogeniceffects of drugs including verapamil and mexiletine parallelsuppression of EADs and modification of re-entrantsubstrate (Fabritz et al. 2003b; Milberg et al. 2005a),the anti-arrhythmogenic effect of nifedipine occurreddespite a neutral effect upon �APD90 in Scn5a+/� hearts.Therefore, we can confirm for the first time in Scn5a+/�hearts that suppression of EADs alone is sufficient tosuppress arrhythmogenesis without alteration to under-lying re-entrant substrate.

An important theoretical consideration regarding thepotential clinical role for nifedipine in LQT3 is the well-recognized anti-hypertensive action with correspondingactivation of the baroreceptor reflex and associated relativetachycardia (Boddeke et al. 1987). However, LQT3 patientstend to show less frequent but more lethal cardiac eventsthat typically take place during rest or sleep, in contrastto other LQT subtypes, notably LQT1, whereby eventsare more commonly associated with exercise or strongemotion (Schwartz et al. 1995, 2001). Indeed, prophylactictherapy against sudden cardiac death in LQTS is with theuse of β-adrenoreceptor blockers (Priori et al. 2001), yetpredictably, LQT3 patients appear to derive less benefitfrom treatment with β-adrenoreceptor-blocking agentsthan 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 withslowed atrial, atrioventricular and ventricular conductionin carriers of �KPQ LQT3 (Zareba et al. 2001) anda bradycardic mode of cardiac death in some LQT3patients (van den Berg et al. 2001). Such observationshave also been reproduced in the laboratory. In the canineventricular wedge preparation made to model LQT3with ATX-II, β-adrenergic stimulation with isoproterenol(100 nm) was associated with an anti-arrhythmogeniceffect, unlike that seen with corresponding models ofLQT1 and LQT2; furthermore, in the LQT3 model,β-adrenergic antagonism with propranolol was indirectlypro-arrhythmic as it reversed the beneficial effects thathad previously been induced by isoproterenol (Shimizu &Antzelevitch, 2000a). Furthermore, increased heart rateshave also been associated with anti-arrhythmogenic effects

in �KPQ Scn5a mice using isoproterenol (Nuyens et al.2001) and pacing (Fabritz et al. 2003b).

In the present study, we demonstrate that thesuppression of EADs through antagonism of the L-typeCa2+ channel is associated with an anti-arrhythmogeniceffect. Although there is little clinical data to supportthe use of calcium antagonists in the prevention ofsudden cardiac death in LQT3 syndrome, the increasingexperimental data supporting their value consideredalongside the poor efficacy of β-adrenoreceptorantagonists in LQT3, would justify further explorationsin this direction.

References

Aiba T, Shimizu W, Inagaki M, Noda T, Miyoshi S, Ding WG,Zankov DP, Toyoda F, Matsuura H, Horie M & Sunagawa K(2005). Cellular and ionic mechanism for drug-induced longQT syndrome and effectiveness of verapamil. J Am CollCardiol 45, 300–307.

Anderson ME, Braun AP, Wu Y, Lu T, Schulman H & Sung RJ(1998). KN-93, an inhibitor of multifunctionalCa++/calmodulin-dependent protein kinase, decreases earlyafterdepolarizations in rabbit heart. J Pharmacol Exp Ther287, 996–1006.

Antzelevitch C & Shimizu W (2002). Cellular mechanismsunderlying the long QT syndrome. Curr Opin Cardiol 17,43–51.

Anumonwo JM, Tallini YN, Vetter FJ & Jalife J (2001). Actionpotential characteristics and arrhythmogenic properties ofthe cardiac conduction system of the murine heart. Circ Res89, 329–335.

Balasubramaniam R, Chawla S, Mackenzie L, Schwiening CJ,Grace AA & Huang CL (2004). Nifedipine and diltiazemsuppress ventricular arrhythmogenesis and calcium releasein mouse hearts. Pflugers Arch 449, 150–158.

Balasubramaniam R, Grace AA, Saumarez RC, Vandenberg JI &Huang CL (2003). Electrogram prolongation and nifedipine-suppressible ventricular arrhythmias in mice followingtargeted disruption of KCNE1. J Physiol 552, 535–546.

Belardinelli L, Shryock JC & Fraser H (2006). Inhibition of thelate sodium current as a potential cardioprotective principle:effects of the late sodium current inhibitor ranolazine. Heart92 (Suppl. 4), iv6–iv14.

Berul CI (2003). Electrophysiological phenotyping ingenetically engineered mice. Physiol Genomics 13, 207–216.

Berul CI, McConnell BK, Wakimoto H, Moskowitz IP, MaguireCT, Semsarian C, Vargas MM, Gehrmann J, Seidman CE &Seidman JG (2001). Ventricular arrhythmia vulnerability incardiomyopathic mice with homozygous mutantmyosin-binding protein C gene. Circulation 104, 2734–2739.

Boddeke HW, Wilffert B, Heynis JB, van de Haar Keuken V,Jonkman FA & van Zwieten PA (1987). A comparison of thecardiac and vasodilatory effects of some calcium entryblockers in perfused isolated guinea-pig hearts. Arch IntPharmacodyn Ther 288, 175–185.

Choi BR, Burton F & Salama G (2002). Cytosolic Ca2+ triggersearly afterdepolarizations and Torsade de Pointes in rabbithearts with type 2 long QT syndrome. J Physiol 543, 615–631.



Chouabe C, Drici MD, Romey G, Barhanin J & Lazdunski M(1998). HERG and KvLQT1/IsK, the cardiac K+ channelsinvolved in long QT syndromes, are targets for calciumchannel blockers. Mol Pharmacol 54, 695–703.

Dilly KW, Rossow CF, Votaw VS, Meabon JS, Cabarrus JL &Santana LF (2006). Mechanisms underlying variations inexcitation–contraction coupling across the mouse leftventricular free wall. J Physiol 572, 227–241.

El-Sherif N, Caref EB, Yin H & Restivo M (1996). Theelectrophysiological mechanism of ventricular arrhythmiasin the long QT syndrome. Tridimensional mapping ofactivation and recovery patterns. Circ Res 79, 474–492.

Fabritz L, Emmerich M, Fortmueller L, Franz MR, Breithardt G,Carmeliet P, Carmeliet E & Kirchhof P (2005). Aggravationof bradycardia by propranolol in deltaKPQ SCN5A (LongQT3) mice. Eur Heart J 26, (Abstract Suppl.) S299.

Fabritz L, Kirchhof P, Franz MR, Eckardt L, Monnig G, MilbergP, Breithardt G & Haverkamp W (2003a). Prolonged actionpotential durations, increased dispersion of repolarization,and polymorphic ventricular tachycardia in a mouse modelof proarrhythmia. Basic Res Cardiol 98, 25–32.

Fabritz L, Kirchhof P, Franz MR, Nuyens D, Rossenbacker T,Ottenhof A, Haverkamp W, Breithardt G, Carmeliet E &Carmeliet P (2003b). Effect of pacing and mexiletine ondispersion of repolarisation and arrhythmias in �KPQSCN5A (long QT3) mice. Cardiovasc Res 57, 1085–1093.

Hagiwara N, Irisawa H & Kameyama M (1988). Contributionof two types of calcium currents to the pacemaker potentialsof rabbit sino-atrial node cells. J Physiol 395, 233–253.

Head CE, Balasubramaniam R, Thomas G, Goddard CA, LeiM, Colledge WH, Grace AA & Huang CL-H (2005). Pacedelectrogram fractionation analysis of arrhythmogenictendency in KPQ Scn5a mice. J Cardiovasc Electrophysiol 16,1329–1340.

Jahnel U, Klemm P & Nawrath H (1994). Differentmechanisms of the inhibition of the transient outwardcurrent in rat ventricular myocytes. Naunyn SchmiedebergsArch Pharmacol 349, 87–94.

January CT & Riddle JM (1989). Early afterdepolarizations:mechanism of induction and block. A role for L-type Ca2+current. Circ Res 64, 977–990.

Knollmann BC, Katchman AN & Franz MR (2001).Monophasic action potential recordings from intact mouseheart: validation, regional heterogeneity, and relation torefractoriness. J Cardiovasc Electrophysiol 12, 1286–1294.

Komiya N, Tanaka K, Doi Y, Fukae S, Nakao K, Isomoto S, SetoS & Yano K (2004). A patient with LQTS in whom verapamiladministration and permanent pacemaker implantationwere useful for preventing torsade de pointes. Pacing ClinElectrophysiol 27, 123–124.

Levi AJ & Issberner J (1996). Effect on the fura-2 transient ofrapidly blocking the Ca2+ channel in electrically stimulatedrabbit heart cells. J Physiol 493, 19–37.

Levi AJ, Li J, Spitzer KW & Bridge JH (1996). Effect on theindo-1 transient of applying Ca2+ channel blocker for asingle beat in voltage-clamped guinea-pig cardiac myocytes.J Physiol 494, 653–673.

Liu TF, Cheng YZ & Wei JH (1990). Early afterdepolarizationin mouse atrial fibers induced by 3.0 mM K+ and theinhibitory effects of tetrodotoxin and nifedipine. Meth FindExp Clin Pharmacol 12, 95–98.

Maguire CT, Wakimoto H, Patel VV, Hammer PE, Gauvreau K& Berul CI (2003). Implications of ventricular arrhythmiavulnerability during murine electrophysiology studies.Physiol Genomics 15, 84–91.

Milberg P & Eckardt L (2005). Transmural dispersion in LQT3and arrhythmogenesis. Cardiovasc Res 68, 338–339.

Milberg P, Reinsch N, Osada N, Wasmer K, Monnig G,Stypmann J, Breithardt G, Haverkamp W & Eckardt L(2005a). Verapamil prevents torsade de pointes by reductionof transmural dispersion of repolarization and suppressionof early afterdepolarizations in an intact heart model ofLQT3. Basic Res Cardiol 100, 365–371.

Milberg P, Reinsch N, Wasmer K, Monnig G, Stypmann J,Osada N, Breithardt G, Haverkamp W & Eckardt L (2005b).Transmural dispersion of repolarization as a key factor ofarrhythmogenicity in a novel intact heart model of LQT3.Cardiovasc Res 65, 397–404.

Ming Z, Nordin C & Aronson RS (1994). Role of L-type calciumchannel window current in generating current-induced earlyafterdepolarizations. J Cardiovasc Electrophysiol 5, 323–334.

Moss AJ (1998). Management of patients with the hereditarylong QT syndrome. J Cardiovasc Electrophysiol 9, 668–674.

Moss AJ, Zareba W, Hall WJ, Schwartz PJ, Crampton RS,Benhorin J, Vincent GM, Locati EH, Priori SG, NapolitanoC, Medina A, Zhang L, Robinson JL, Timothy K, Towbin JA& Andrews ML (2000). Effectiveness and limitations ofbeta-blocker therapy in congenital long-QT syndrome.Circulation 101, 616–623.

Nattel S & Quantz MA (1988). Pharmacological response ofquinidine induced early afterdepolarisations in caninecardiac Purkinje fibres: insights into underlying ionicmechanisms. Cardiovasc Res 22, 808–817.

Noble D & Noble PJ (2006). Late sodium current in thepathophysiology of cardiovascular disease: consequences ofsodium-calcium overload. Heart 92 (Suppl. 4), iv1–iv5.

Nuyens D, Stengl M, Dugarmaa S, Rossenbacker T,Compernolle V, Rudy Y, Smits JF, Flameng W, Clancy CE,Moons L, Vos MA, Dewerchin M, Benndorf K, Collen D,Carmeliet E & Carmeliet P (2001). Abrupt rate accelerationsor premature beats cause life-threatening arrhythmias inmice with long-QT3 syndrome. Nat Med 7, 1021–1027.

Opthof T & Coronel R (2005). Transmural dispersion in LQT3and arrhythmogenesis. Cardiovascular Res 68, 336–337.

Patterson E, Scherlag BJ & Lazzara R (1997). Earlyafterdepolarizations produced by d,1-sotalol and clofilium.J Cardiovasc Electrophysiol 8, 667–678.

Pidoplichko VI & Verkhratskii AN (1989). [Frequency-relatedblocking of sodium channels in the membrane of isolated ratcardiomyocytes by the calcium antagonist verapamil]. FiziolZh 35, 87–89.

Priori SG, Aliot E, Blomstrom-Lundqvist C, Bossaert L,Breithardt G, Brugada P, Camm AJ, Cappato R, Cobbe SM,Di Mario C, Maron BJ, McKenna WJ, Pedersen AK, RavensU, Schwartz PJ, Trusz-Gluza M, Vardas P, Wellens HJ &Zipes DP (2001). Task Force on Sudden Cardiac Death of theEuropean Society of Cardiology. Eur Heart J 22, 1374–1450.

Priori SG, Napolitano C, Schwartz PJ, Grillo M, Bloise R,Ronchetti E, Moncalvo C, Tulipani C, Veia A, Bottelli G &Nastoli J (2004). Association of long QT syndrome loci andcardiac events among patients treated with beta-blockers.JAMA 292, 1341–1344.



Restivo M, Caref EB, Kozhevnikov DO & El-Sherif N (2004).Spatial dispersion of repolarization is a key factor in thearrhythmogenicity of long QT syndrome. J CardiovascElectrophysiol 15, 323–331.

Roden DM (2006). Probing the arrhythmogenic substrate.Heart Rhythm 3, 779–780.

Saumarez RC, Pytkowski M, Sterlinski M, Hauer RN, DerksenR, Lowe MD, Szwed H, Huang CL, Ward DE, Camm AJ &Grace AA (2006). Delayed paced ventricular activation in thelong QT syndrome is associated with ventricular fibrillation.Heart Rhythm 3, 771–778.

Schwartz PJ & Priori SG (2004). Long QT syndrome:Genotype–phenotype correlations. In CardiacElectrophysiology from Cell to Bedside, 4th edn, ed. Zipes DP& Jalife J, pp. 651–659. Elsevier, Philadelphia.

Schwartz PJ, Priori SG, Locati EH, Napolitano C, Cantu F,Towbin JA, Keating MT, Hammoude H, Brown AM & ChenLS (1995). Long QT syndrome patients with mutations of theSCN5A and HERG genes have differential responses to Na+channel blockade and to increases in heart rate. Implicationsfor gene-specific therapy. Circulation 92, 3381–3386.

Schwartz PJ, Priori SG, Spazzolini C, Moss AJ, Vincent GM,Napolitano C, Denjoy I, Guicheney P, Breithardt G, KeatingMT, Towbin JA, Beggs AH, Brink P, Wilde AA, 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 (2001). Genotype–phenotype correlation in thelong-QT syndrome: gene-specific triggers forlife-threatening arrhythmias. Circulation 103, 89–95.

Shen JB, Jiang B & Pappano AJ (2000). Comparison of L-typecalcium channel blockade by nifedipine and/or cadmium inguinea pig ventricular myocytes. J Pharmacol Exp Ther 294,562–570.

Shimizu W, Aiba T & Antzelevitch C (2005). Specific therapybased on the genotype and cellular mechanism in inheritedcardiac arrhythmias. Long QT syndrome and Brugadasyndrome. Curr Pharm Des 11, 1561–1572.

Shimizu W & Antzelevitch C (1997). Sodium channel blockwith mexiletine is effective in reducing dispersion ofrepolarization and preventing torsade des pointes in LQT2and LQT3 models of the long-QT syndrome. Circulation 96,2038–2047.

Shimizu W & Antzelevitch C (2000a). Differential effects ofβ-adrenergic agonists and antagonists in LQT1, LQT2 andLQT3 models of the long QT syndrome. J Am Coll Cardiol35, 778–786.

Shimizu W & Antzelevitch C (2000b). Effects of a K+ channelopener to reduce transmural dispersion of repolarizationand prevent torsade de pointes in LQT1, LQT2, and LQT3models of the long-QT syndrome. Circulation 102, 706–712.

Shimizu W, Ohe T, Kurita T, Kawade M, Arakaki Y, Aihara N,Kamakura S, Kamiya T & Shimomura K (1995). Effects ofverapamil and propranolol on early afterdepolarizations andventricular arrhythmias induced by epinephrine incongenital long QT syndrome. J Am Coll Cardiol 26,1299–1309.

Sipido KR, Carmeliet E & Pappano A (1995). Na+ current andCa2+ release from the sarcoplasmic reticulum during actionpotentials in guinea-pig ventricular myocytes. J Physiol 489,1–17.

Triggle DJ (2006). L-type calcium channels. Curr Pharm Des12, 443–457.

Ueda N, Zipes DP & Wu J (2004). Epicardial but notendocardial premature stimulation initiates ventriculartachyarrhythmia in canine in vitro model of long QTsyndrome. Heart Rhythm 1, 684–694.

van den Berg MP, Wilde AA, Viersma TJW, Brouwer J,Haaksma J, van der Hout AH, Stolte-Dijkstra I, BezzinaTCR, Van Langen IM, Beaufort-Krol GC, Cornel JH 2nd &Crijns HJ (2001). Possible bradycardic mode of death andsuccessful pacemaker treatment in a large family withfeatures of long QT syndrome type 3 and Brugada syndrome.J Cardiovasc Electrophysiol 12, 630–636.

Verheijck EE, van Ginneken AC, Wilders R & Bouman LN(1999). Contribution of L-type Ca2+ current to electricalactivity in sinoatrial nodal myocytes of rabbits. Am J PhysiolHeart Circ Physiol 276, H1064–H1077.

Viswanathan PC & Rudy Y (2000). Cellular arrhythmogeniceffects of congenital and acquired long-QT syndrome in theheterogeneous myocardium. Circulation 101, 1192–1198.

Volders PG, Vos MA, Szabo B, Sipido KR, de Groot SH, GorgelsAP, Wellens HJ & Lazzara R (2000). Progress in theunderstanding of cardiac early afterdepolarizations andtorsades de pointes: time to revise current concepts.Cardiovasc Res 46, 376–392.

Wasserstrom JA & Vites AM (1996). The role of Na+–Ca2+exchange in activation of excitation–contraction coupling inrat ventricular myocytes. J Physiol 493, 529–542.

Yao A, Su Z, Nonaka A, Zubair I, Lu L, Philipson KD, Bridge JH& Barry WH (1998). Effects of overexpression of theNa+-Ca2+ exchanger on [Ca2+]i transients in murineventricular myocytes. Circ Res 82, 657–665.

Zareba W, Sattari MN, Rosero S, Couderc JP & Moss AJ (2001).Altered atrial, atrioventricular, and ventricular conduction inpatients with the long QT syndrome caused by the �KPQSCN5A sodium channel gene mutation. Am J Cardiol 88,1311–1314.

Zhabyeyev P, Missan S, Jones SE & McDonald TF (2000).Low-affinity block of cardiac K+ currents by nifedipine.Eur J Pharmacol 401, 137–143.

Zhang S, Zhou Z, Gong Q, Makielski JC & January CT (1999).Mechanism of block and identification of the verapamilbinding domain to HERG potassium channels. Circ Res 84,989–998.

Acknowledgements

Supported by grants from the British Heart Foundation, theMedical Research Council, the Wellcome Trust, the HelenKirkland Fund for Cardiac Research and the Raymond andBeverly Sacker Medical Research Centre.
