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RELATED PAPERS |
1 Section of Cardiovascular Biology, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK
2 Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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APD90: endocardial minus epicardial APD90) (–2.5 ± 2.4 ms, compared with 12.4 ± 1.1 ms in WT, P < 0.001; n
= 5). (3) Frequent epicardial early afterdepolarizations (EADs) and spontaneous ventricular tachycardia (VT) in 4 out of 5 KCNE1–/– hearts but not WT (n
= 5). EADs were especially frequent following temporary cessations of ventricular pacing. (4) Monomorphic VT lasting 1.36 ± 0.2 s in 5 out of 5 KCNE1–/– hearts, following premature stimuli but not WT (n
= 5). (5) Epicardial APD alternans. Perfusion of KCNE1–/– hearts with 1 µM nifedipine induced potentially anti-arrhythmic changes including: (1) restored epicardial APD90 (from 57.1 ± 0.5 ms to 42.3 ± 0.4 ms, P < 0.001; n
= 5); (2) altered APD90 to values (11.2 ± 2.6) close to WT (P > 0.05; n
= 5); (3) EAD suppression during both spontaneous activity and following cessation of ventricular pacing (n
= 5) to give similar features to WT controls (n
= 5); (4) suppression of programmed electrical stimulation-induced VT; and (5) suppression of APD alternans. These findings suggest arrhythmic effects of reduced outward currents expected in KCNE1–/– hearts and their abolition by antagonism of inward L-type Ca2+ current.
(Received 28 July 2006;
accepted after revision 2 November 2006;
first published online 9 November 2006)
Corresponding author C. L.-H. Huang: Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK. Email: clh11{at}cam.ac.uk
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Introduction |
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- and 
-subunits of a potassium channel which conducts the slow component, IKs, of the delayed rectifier current (Sanguinetti et al. 1996). Reduced IKs, which may accompany mutations within KCNE1, is implicated in the prolongation of the cardiac action potential, and associated ventricular arrhythmogenesis characteristic of LQT5 (Splawski et al. 1997; Duggal et al. 1998). The phenotypic consequences of reduced IKs have previously been investigated in several murine models. Homozygote mice with targeted disruption of KCNE1 or KCNQ1 have bilateral sensorineural deafness and exhibit Shaker/Waltzer movements, due to severe structural and functional abnormalities (Vetter et al. 1996; Drici et al. 1998; Kupershmidt et al. 1999; Lee et al. 2000; Casimiro et al. 2001). However, the corresponding cardiac phenotype has been less well defined. Indeed, QT interval abnormalities have been described in surface ECG recordings from KCNE1–/– mice in response to changes in heart rate (Drici et al. 1998) whereas other groups describe no such abnormalities in either surface ECG recordings (Kupershmidt et al. 1999) nor differences in action potential duration (APD) in microelectrode recordings from isolated right ventricular endocardial preparations from KCNE1–/– hearts (Charpentier et al. 1998). Furthermore, widespread abnormalities were recorded from surface ECG in sedated mice with targeted disruption of KCNQ1, yet no significant differences were observed in monophasic action potentials (MAPs) recorded from the endocardial and epicardial surfaces of such hearts when isolated (Casimiro et al. 2001).
Nonetheless, despite such contrasting reports, at the whole-heart level, an arrhythmogenic phenotype has been reported in the atria of KCNE1–/– hearts (Temple et al. 2005), and in the ventricles of both KCNQ1–/– (Tosaka et al. 2003) and KCNE1–/– hearts (Balasubramaniam et al. 2003). Furthermore, based upon observations in isolated, Langendorff-perfused KCNE1–/– hearts, using an adapted version of the clinical tool of paced electrogram fractionation analysis (PEFA) which identifies features indicative of a re-entrant excitation, it was speculated (Balasubramaniam et al. 2003; Roden, 2006; Saumarez et al. 2006) that a triggered mechanism may actually underlie such arrhythmogenicity, which was further supported by the anti-arrhythmogenic effects observed following perfusion with the dihydropyridine, L-type Ca2+ antagonist nifedipine (Balasubramaniam et al. 2003).
In the present study, we accordingly extend the work of Balasubramaniam et al. (2003) by simultaneously recording endocardial and epicardial MAPs for the first time in whole, isolated perfused KCNE1–/– mice subjected to programmed electrical stimulation (PES) under various pharmacological conditions. Firstly, we demonstrate electrical features potentially associated with an arrhythmogenic substrate including: (1) prolonged epicardial and endocardial action potential duration (APD) which occurs in association with (2) an altered repolarization gradient between epicardium and endo-cardium, and (3) action potential alternans. Secondly, we report early afterdepolarizations (EADs) and triggered arrhythmogenesis for the first time in isolated, whole KCNE1–/– hearts. Thirdly, we extend previous work describing the anti-arrhythmogenic effects of the dihydropyridine L-type Ca2+ antagonist nifedipine in KCNE1–/– hearts (Balasubramaniam et al. 2003) by reporting for the first time the effects of nifedipine on all these characteristics that would be expected to exert anti-arrhythmic effects through actions on inward Ca2+ currents in contrast to the reduced outward currents expected in KCNE1–/– hearts.
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Methods |
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The KCNE1–/– mice were supplied by Vetter et al. (1996), inbred on the 129/sv strain. In our laboratory, breeding pairs of homozygote animals were established to provide both KCNE1–/– and WT lines. KCNE1–/– mice display Shaker/Waltzer behaviour and have sensorineural deafness, in common with the human phenotype, but exhibit no increased mortality compared to corresponding WT mice, a feature of such genetically modified systems that has already been discussed in detail (Nerbonne et al. 2001; Warth & Barhanin, 2002; London, 2004; Napolitano, 2004). All mice were maintained in an animal house facility at room temperature (21 ± 1°C) and subjected to 12 h light–dark cycles. Sterile rodent chow and drinking water were freely available. Experiments were performed on male and female mice aged approximately 6 months.
Preparation of Langendorff-perfused hearts
Whole hearts from mice killed by rapid cervical dislocation without anaesthesia, in accordance with Schedule 1 of the 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 (Balasubramaniam et al. 2003). A small (3–4 mm) section of aorta was cannulated under the buffer surface and secured to a 21-gauge tailor-made cannula, pre-filled with ice-cold buffer solution, then used for retrograde perfusion with 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 during PES (1 heart out of a total of 11 was discarded due to inadequate cannulation and subsequent regional hypoperfusion). Hearts were perfused with physiological perfusion buffer for 20 min prior to experimentation.
Monophasic action potential recordings
MAPs were recorded from the epicardium using a commercially available, 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 positioned by hand, and the contact maintained by custom-designed magnetic grips positioned on a metallic platform. Signals were amplified and low-pass filtered appropriately for murine recordings (0.1 Hz to 1.0 k Hz) (Gould 2400S, Gould-Nicolet Technologies, Ilford, Essex, UK) then digitized using a 1401plus analog-to-digital converter (Cambridge Electronic Design, Cambridge, UK). Analysis of the MAP waveforms was performed using 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
Programmed electrical stimulation (PES) of the heart was performed using paired (1 mm interpole spacing) platinum stimulating and recording electrodes, manually positioned 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. All experimental mice were bred from a 129 genetic background, which, along with C57 mice, are less susceptible to PES-induced arrhythmias following extra-systolic (S2) stimuli than FBV or Black Swiss animals (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).
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.
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Results |
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All simultaneously recorded epicardial and endocardial MAPs recorded from KCNE1–/– (n = 5) and WT hearts (n = 5) in the present study satisfied the established criteria of baseline stability and typically triangular murine morphology, consisting of a rapid upstroke and minimal plateau phase in the WT (Knollmann et al. 2001). Epicardial MAPs recorded from intact KCNE1–/– hearts were markedly prolonged, often with evidence of a plateau phase, compared to those of WT hearts, particularly in the latter stages of recovery, in agreement with action potential recordings at the cellular level in ventricular myocytes from other genetically modified, IK-depleted mice (Xu et al. 1999b; Brunner et al. 2001) (Fig. 1A). No such features were observed in relation to the time course of the corresponding endocardial APD from KCNE1–/– as compared with WT hearts (Fig. 1B).
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The kinetic differences in Fig. 1A and B were confirmed by this quantification of APD values, which demonstrated significantly shorter values of APD30 and APD50, whereas APD70 and APD90 by contrast were more prolonged in the KCNE1–/– hearts compared with WT controls. Epicardial APD30 and APD50 were significantly shorter in KCNE1–/– hearts at 2.3 ± 0.06 and 6.1 ± 0.2 ms, respectively, compared with 6.6 ± 0.01 ms and 14.0 ± 0.02 ms, respectively, in WT hearts (P < 0.001; n = 5). Conversely, corresponding APD70 and APD90 were significantly prolonged in KCNE1–/– hearts at 31.5 ± 0.6 ms and 57.1 ± 0.5 ms, compared with 25.1 ± 0.05 ms and 36.1 ± 0.07 ms, respectively, in WT hearts (P < 0.001; n = 5) (Fig. 1C). Endocardial APD30 and APD50 were also significantly shorter in KCNE1–/– hearts at 5.1 ± 0.03 ms and 12.1 ± 0.07 ms, respectively, compared with 7.9 ± 0.01 ms and 14.8 ± 0.03 ms in WT hearts (P < 0.001; n = 5). APD70 was no different between KCNE1–/– and WT hearts at 26.8 ± 0.2 ms and 26.4 ± 0.08 ms, respectively (P > 0.05) although APD90 was significantly prolonged in KCNE1–/– hearts at 54.4 ± 2.4 ms compared with 48.5 ± 0.3 ms in WT hearts (P < 0.05; n = 5) (Fig. 1D).
KCNE1–/– hearts demonstrate altered spatial repolarization gradients
Heterogeneity of repolarization, known to influence arrhythmogenic propensity in clinical situations (Restivo et al. 2004), has also been described in previous murine models of arrhythmic syndromes (Baker et al. 2000; Fabritz et al. 2003b). However, under normal circumstances, murine repolarization is known to demonstrate marked regional heterogeneity (Anumonwo et al. 2001; Knollmann et al. 2001; Dilly et al. 2006); recent work has demonstrated that this may actually prevent arrhythmias (Costantini et al. 2005). The spatial (transmural) gradient was accordingly explored in KCNE1–/– hearts by comparing the difference in APD90 (APD90) plus local activation time (AT) between simultaneously recorded left ventricular endocardial free wall, and left ventricular epicardial MAPs (Opthof & Coronel, 2005) during steady-state, right ventricular epicardial pacing at 125 ms CL in order to suppress EADs, known to directly influence local electrical heterogeneity (Volders et al. 2000). No significant differences in AT were found between endocardium and epicardium in KCNE1–/– (14.8 ± 0.5 ms versus 15.3 ± 0.9 ms, respectively, P > 0.05) and WT 14.6 ± 1.4 ms versus 13.6 ± 0.7 ms, respectively (P > 0.05) hearts. Consequently, the transmural gradient of repolarization was simply calculated as endocardial APD90 minus epicardial APD90. Thus, in the arrhythmogenic KCNE1–/– hearts (n
= 5), 54.4 ± 2.4 ms minus 57.1 ± 0.5 ms gave a mean APD90 of –2.5 ± 2.4 ms, compared with 48.5 ± 0.3 ms minus 36.1 ± 0.07 ms to give a APD90 of 12.4 ± 1.1 ms in WT hearts (P < 0.001; n
= 5) (Fig. 2).
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Premature ventricular contractions, non-sustained ventricular tachycardia and atrial fibrillation have been previously described during radiotelemetry recordings in ambulant KCNE1–/– and KCNQ1–/– mice (Tosaka et al. 2003; Temple et al. 2005), along with ventricular tachycardia in whole, Langendorff-perfused KCNE1–/– hearts in response to the autonomic stimulant nicotine (100 µM) (Tosaka et al. 2003) or programmed electrical stimulation (PES) (Balasubramaniam et al. 2003). However, the mechanisms underlying these ventricular arrhythmias remain unexplored.
For the first time to our knowledge, our simultaneous epicardial and endocardial MAP recording of intrinsic activity demonstrated frequent epicardial EADs and spontaneous VT in 4 out of 5 KCNE1–/– hearts (Fig. 3A and B) but not in any WT controls (n = 5) (Fig. 3C). Furthermore, EADs observed in KCNE1–/– hearts demonstrated marked kinetic differences from those described in previous murine models of LQTS (Fabritz et al. 2003a). The EADs thus occurred much later during the course of repolarization (phase 3–4 of the action potential), but well before full repolarization was achieved, thereby differentiating them from delayed afterdepolarizations (Fig. 3B). Such epicardial EADs were observed to follow MAPs in 23.9 ± 9.7% of n = 10 sampling periods. Each sampling period lasted 10 s and two of these were each randomly selected from a total recording time of 20 min in each heart, from each of five KCNE1–/– hearts. These phenomena were never observed anywhere during the inspection of entire experimental traces obtained from WT hearts (P < 0.05; n = 5). EADs were especially frequent following temporary cessations of steady-state ventricular pacing, occurring within the first five intrinsic beats in 80% of KCNE1–/– hearts (n = 16 out of 20 pauses in 5 individual hearts) following such interruptions after a mean pause of 2.1 ± 0.3 s (Fig. 3D). In contrast, no EADs were observed (n = 0 out of 20 pauses in 5 individual hearts) following resumption of intrinsic activity, which occurred after a significantly shorter mean pause of only 0.4 ± 0.07 s after the cessation of ventricular pacing (P < 0.05) in WT controls (Fig. 3E). These EADs and spontaneous VT were suppressed by commencement of right ventricular pacing at 125 ms cycle length (CL) in 4 out of 5 KCNE1–/– hearts, although spontaneous VT lasting a mean of 2.48 ± 0.7 s persisted in 1 out of 5 KCNE1–/– hearts. In contrast to these observations from epicardial recordings, no EADs were recorded from the endocardial surfaces of KCNE1–/– or WT hearts either during recording of intrinsic activity (n = 5) or steady-state pacing (n = 5).
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Previous optical mapping experiments in transgenic mice demonstrating reduced slowly inactivating K+ current, IK,slow had indicated that VT induced during programmed electrical stimulation (PES) was the result of functional block of the premature stimulus due to spatial dispersion of repolarization and subsequent re-entrant excitation (Baker et al. 2000). Likewise, an adapted version of the clinical tool paced electrogram fractionation analysis (PEFA) applied to KCNE1–/– hearts during PES had revealed features indicative of unidirectional block and re-entrant excitation (Balasubramaniam et al. 2003). However, a marked anti-arrhythmogenic effect had followed perfusion with the dihydropyridine, L-type Ca2+ antagonist nifedipine, without apparent alteration to the underlying re-entrant substrate, thus leading to speculation of a concomitant triggered mechanism of arrhythmogenesis (Balasubramaniam et al. 2003). Such EAD-related triggered activities under long QT conditions are currently thought to initiate VT, which is thereafter maintained by re-entrant excitation (Yan et al. 2001).
In the present study, in 5 out of 5 KCNE1–/– hearts, following a drive train of 8 paced beats at 125 ms CL, a single right ventricular epicardial premature stimulus applied within 26–40 ms of the previous paced beat induced episodes of monomorphic, non-sustained VT of mean duration 1.36 ± 0.2 s (n = 5) (Fig. 4A). VT kinetics in KCNE1–/– hearts were uniform, with mean CL of 50.7 ± 3.8 ms (n = 5) (Fig. 4B, left), the waveform of each deflection closely resembling individual EADs frequently observed in spontaneously beating preparations which did not progress to VT (Fig. 4B, right). Importantly, each deflection during VT failed to return to baseline prior to the next depolarization until spontaneous termination occurred (Fig. 4A). These observations differ from VT induced in other genetically modified, arrhythmogenic murine systems, in which monomorphic initiating sequences eventually give way to polymorphic VT, more typical of re-entrant excitation (Baker et al. 2000; Fabritz et al. 2003a). Indeed, polymorphic VT was seen in 0 out of 5 KCNE1–/– or WT hearts.
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Spatially discordant APD alternans induces increases in dispersion of refractoriness, which can present a favourable substrate for re-entrant excitation (Weiss et al. 2006) seen in LQTS (Zareba et al. 1994), and corresponding animal models (Chinushi et al. 1998; Shimizu & Antzelevitch, 1999; Fabritz et al. 2003b). Drici et al. reported T wave alternans in surface ECG recordings from 1 out of 2 KCNE1–/– mice and 2 out of 3 WT mice following intraperitoneal injections of isoproterenol (isoprenaline), but not under control conditions (Drici et al. 1998). However, in the present study, APD alternans was observed for the first time in epicardial, but not endocardial, records in KCNE1–/– (n = 6) but not WT hearts (n = 6) by measuring the differences in APD90 between consecutive MAPs recorded from the left ventricular epicardium and endocardium following commencement of steady-state right ventricular pacing at 125 ms CL. Figure 5A (left panel) shows a representative trace of epicardial MAPs recorded continuously from a KCNE1–/– heart, alongside a plot of the APD90 of each consecutive MAP during a typical 10 s recording (right panel). There was a clear alternation in individual APD90 values with each successive beat in KCNE1–/– hearts, whereas in WT hearts, variations in APD90 did not show this alternating pattern (Fig. 5B). Spontaneous VT was observed in 1 out of 6 KCNE1–/– during such steady-state pacing, but not in any WT hearts (n = 6).
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Previous studies had reported effects of the L-type Ca2+ antagonist nifedipine upon KCNE1–/– hearts without alteration to any underlying re-entrant substrate as detected by PEFA, which implied a triggered mechanism of arrhythmia (Balasubramaniam et al. 2003), especially as Ca2+, whether transferred through the L-type Ca2+ channel (ICa,L) or the Na+–Ca2+ exchanger (INa–Caxc) is considered the principal inward charge carrier responsible for initiating EADs (Zeng & Rudy, 1995). Previously, L-type Ca2+ channel antagonism with the phenylalkylamine verapamil, has suppressed EADs and reduced dispersion of repolarization and VT in indirect animal models of LQTS (Aiba et al. 2005; Milberg et al. 2005), which supports similar clinical observations (Cosio et al. 1991; Shimizu et al. 1995; Komiya et al. 2004), although verapamil exerts effects on K+ and Na+ as well as Ca2+ channels (Pidoplichko & Verkhratskii, 1989; Chouabe et al. 1998; Zhang et al. 1999). Thus, in the present study, the precise electrophysiological effects of the specific dihydropyridine nifedipine (Zhang et al. 1999) were investigated in KCNE1–/– hearts.
Firstly, perfusion with physiological buffer containing 1 µM nifedipine, significantly reduced epicardial APD90 in KCNE1–/– hearts from 57.1 ± 0.5 ms to 42.3 ± 0.4 ms (P < 0.001; n = 5) (Fig. 6A). In contrast, no significant differences were seen in endocardial APD90 values (54.4 ± 2.4 ms and 53.4 ± 1.6 ms, respectively, P > 0.05; n = 5) (Fig. 6B). Following perfusion of WT controls with physiological buffer containing 1 µM nifedipine, no significant differences were observed in either epicardial APD90 (36.1 ± 0.07 ms to 40.8 ± 0.1 ms, P > 0.05; n = 5) or endocardial APD90 (48.5 ± 0.3 ms to 49.1 ± 0.4 ms, P > 0.05; n = 5). Such selective shortening of epicardial APD by an L-type Ca2+ channel antagonist in the intact KCNE1–/– hearts mirrors previous observations in arterially perfused feline ventricular preparations with combined block of the rapidly activating delayed rectifier current, IKr and IKs (using E-4031 and chromanol 293B, respectively) (Aiba et al. 2005).
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APD90 in KCNE1–/– hearts (n
= 5). No significant difference in AT was seen in the presence of nifedipine between endocardium and epicardium in KCNE1–/– (12.6 ± 1.2 ms versus 13.7 ± 0.9 ms, respectively, P > 0.05) and WT 13.8 ± 0.8 ms versus 12.2 ± 1.2 ms, respectively (P > 0.05). Thus, endocardial APD90 minus epicardial APD90 (53.4 ± 1.6 ms minus 42.3 ± 0.4 ms) restored mean APD90 to 11.2 ± 2.6 ms, which did not differ significantly from the APD90 value seen in the non-arrhythmogenic WT hearts (endocardial APD90 to 49.1 ± 0.4 ms, minus APD90 40.8 ± 0.1 ms, to give APD90 of 8.2 ± 0.4 ms, respectively, P > 0.05; n
= 5) (Fig. 6C). Nifedipine suppresses arrhythmic tendency
Perfusion with physiological buffer containing 1 µM nifedipine, completely suppressed all EADs in all KCNE1–/– hearts (P < 0.05; n = 10) during both spontaneous activity (Fig. 6D) and following sudden cessation of ventricular pacing, paralleling similar features in WT controls (n = 5). Furthermore, the combined effect of these alterations upon inducibility of VT was assessed in KCNE1–/– hearts using PES procedures identical to those reported previously, whereupon premature stimuli failed to induce VT in all preparations (0 out of 5) (Fig. 6E).
Ventricular refractoriness is known to affect arrhythmogenicity in murine hearts (Maguire et al. 2003) and therefore the effect of nifedipine on ventricular effective refractory period (VERP) was also investigated. VERP was measured at the left ventricular epicardial surface and considered to be the longest S1–S2 interval which did not elicit a corresponding electrogram. In KCNE1–/– hearts, mean VERP was 50 ± 5.4 ms (n = 5) compared with 52.8 ± 7.8 in WT controls (n = 5; P > 0.05). Following perfusion with physiological buffer containing 1 µM nifedipine, mean VERP was 47.3 ± 8.1 ms and 63.8 ± 11.3 ms in KCNE1–/– and WT hearts, respectively (n = 5), although ultimately no statistical difference was observed (P > 0.05) between the groups (Fig. 7).
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APD alternans (calculated as the difference in APD90 between successive pairs of MAPs obtained from three 10 s sampling windows, randomly selected from a total recording time of 20 min, in each of six KCNE1–/– and six WT hearts) was reduced from 7.42 ± 1.2 ms to 2.67 ± 0.9 ms in KCNE1–/– hearts (P < 0.05; n
= 6), compared with 1.5 ± 0.3 ms and 1.6 ± 0.4 ms, respectively (P > 0.05; n
= 6) in identical epicardial traces from WT hearts (Fig. 9).
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Discussion |
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Firstly, we report for the first time significant prolongation of epicardial and endocardial MAPs, recorded from intact KCNE1–/– hearts, an anticipated consequence of IKs reduction, as seen in other genetically modified, mice lacking H+ currents (Xu et al. 1999a; Brunner et al. 2001). Such prolongation can be reconciled with previous reports of QT interval abnormalities observed in surface ECG recordings from KCNE1–/– mice (Drici et al. 1998). Nonetheless, known regional differences in APD within the WT murine heart (Anumonwo et al. 2001; Knollmann et al. 2001; Liu et al. 2004; Dilly et al. 2006) may result from regional heterogeneity of K+ channel (Kv4.2, Kv4.3, Kv1.5, Kv2.1) expression within the left ventricular endocardium and epicardium of adult WT (C57BL6, FVB and Sv129) mice (Xu et al. 1999b; Brunet et al. 2004). Indeed, greater densities of IK along with expression of the fast component of the transient outward current, Ito,f are known to occur in epicardial cells within the murine LV wall as compared with those from the endocardial surface (Brunet et al. 2004), which may account for the preferential prolongation of epicardial APD observed in KCNE1–/– hearts.
Other groups have reported no such abnormalities in surface ECG recordings (Kupershmidt et al. 1999), nor differences in action potential prolongation in microelectrode recordings from isolated right ventricular endocardial preparations from KCNE1–/– hearts (Charpentier et al. 1998). Likewise, widespread abnormalities recorded from surface ECG in KCNQ1–/– hearts were not reflected in abnormalities of MAPs recorded from the endocardial and epicardial surfaces of such hearts when isolated (Casimiro et al. 2001). However, such marked regional differences in APD seen in the whole heart may not be reflected in recordings from isolated tissue preparations (Charpentier et al. 1998) or single cell studies (Casimiro et al. 2001).
Secondly, we report for the first time contrasting left ventricular repolarization gradients between KCNE1–/– and WT hearts brought about through the preferential prolongation of the epicardial APD in KCNE1–/–. It is recognized that heterogeneously distributed K+ current densities are implicated in the generation of functional repolarization gradients that are necessary to permit synchronized repolarization and protect against re-entrant (Nerbonne & Guo, 2002) and triggered (Burashnikov & Antzelevitch, 2002) arrhythmias. Indeed, previous murine studies have demonstrated increased vulnerability to ventricular arrhythmogenesis through alterations in K+ currents which result in both increases (London et al. 1998; Baker et al. 2000) and decreases in repolarization gradients (Costantini et al. 2005).
Thirdly, we report for the first time frequent early afterdepolarizations (EADs) and spontaneous VT in KCNE1–/– mice, confirming previous predictions (Balasubramaniam et al. 2003). EADs were observed only in epicardial and not in endocardial sites and as such represent local events in specific areas of myocardium, rather than propagated events initiated from the atrioventricular node or Purkinje tissue and conducting to the myocardial cells from which the recordings were taken.
Furthermore, PES-induced episodes of repetitive triggered activity, giving rise to monomorphic, non-sustained VT in all KCNE1–/– hearts, unlike the polymorphic VT, more typical of re-entrant excitation mechanisms described in alternative murine models (Baker et al. 2000; Fabritz et al. 2003b). The involvement of EAD-mediated triggered activity in the initiation of ventricular arrhythmias has been proven experimentally under conditions of prolonged repolarization associated with LQT2 (reduced IKr) and LQT3 (augmented INa), although their role in LQT1 and LQT5 (reduced IKs) remains uncertain (Volders et al. 2000). Previously, in experimental preparations using isolated canine ventricular tissue (Burashnikov & Antzelevitch, 2002) supported by simulation studies using Markov models (Silva & Rudy, 2005), IKs appears to provide a ‘repolarization reserve’, protecting against action potential prolongation and EADs in the event of concomitant reductions in other repolarizing currents, specifically IKr. Indeed, selective IKs reduction in several previous genetically modified murine models was associated with frequent premature ventricular beats and episodes of non-sustained VT in ambulant mice (Tosaka et al. 2003) and triggered activity in isolated hearts (Bale et al. 2003).
Fourthly, APD alternans was observed for the first time in MAPs recorded from epicardium but not endocardium in KCNE1–/– but not WT hearts. Furthermore, we demonstrated that alternans in KCNE1–/– hearts was not associated with abnormalities of APD restitution, thereby supporting the involvement of abnormal Ca2+ handling, in keeping with previous studies (Pruvot et al. 2004; Wan et al. 2005). However, although the association between APD alternans and arrhythmogenicity in a range of cardiac conditions including LQTS is clear (Rosenbaum et al. 1994), the precise underlying mechanisms (electrical restitution or abnormalities of Ca2+ handling) remain contentious (Pruvot et al. 2004).
Finally, we describe for the first time electrophysiological effects consistent with a reduction in arrhythmogenic propensity in KCNE1–/– hearts following perfusion with physiological perfusion buffer containing 1 µM nifedipine. Nifedipine is known 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) although specific antagonism of the L-type Ca2+ channel has also been shown with 10 µM nifedipine (Yao et al. 1998) and alternative experiments in isolated mouse myocytes reported early afterdepolarization (EAD) suppression with nifedipine concentrations between 5 and 8 µM (Liu et al. 1990). In addition, 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 been shown to have no effect upon ICa,T, INa, IK and If (Verheijck et al. 1999). In the present study, effects of nifedipine included (1) attenuation of prolonged epicardial APD90, thereby (2), exerting a marked alteration in APD90 in KCNE1–/– hearts to mirror that seen in non-arrhythmogenic, untreated WT hearts, (3) suppression of all epicardial EADs during both spontaneous activity and following sudden cessation of ventricular pacing, (4) reduction in VT inducibility during PES procedures, and (5) suppression of APD alternans previously observed in records from epicardial, but not endocardial KCNE1–/– or WT hearts. Whilst not excluding other effects on altered Ca2+ homeostasis generally following changes in ICa, these findings support the previous speculation that (1) an imbalance between ICa and IK in the terminal phases of repolarization, brought about by a reduction in IK through targeted disruption of KCNE1, may promote arrhythmogenicity in KCNE1–/– hearts, (2) such imbalance may lead to EADs through re-activation of the L-type Ca2+ channel, and (3) that restoration of this ICa–IK balance may underlie the anti-arrhythmic effects observed with antagonism of the L-type Ca2+ channel using the dihydropyridine nifedipine in KCNE1–/– hearts (Balasubramaniam et al. 2003). Such a scheme is certainly compatible with other observations with the phenylalkylamine verapamil in both clinical case reports (Cosio et al. 1991; Shimizu et al. 1995; Komiya et al. 2004) and in animal models of LQTS (Aiba et al. 2005; Milberg et al. 2005) despite the known high affinity block of potassium (IKr) currents (Chouabe et al. 1998; Zhang et al. 1999) in addition to antagonism of L-type calcium channels (ICa,L) (Triggle, 2006). Unfortunately, there is currently little consistent data to support the clinical use of Ca2+ channel antagonists for suppression of ventricular arrhythmogenesis. Indeed, the significant reduction in sudden death in non-ischaemic heart failure reported in the Prospective Randomized Amlodipine Survival Evaluation (PRAISE) trial (Packer et al. 1996) was not replicated in the subsequent study, PRAISE-2 (Thackray et al. 2000). Nonetheless, such experimental findings may merit further testing at the clinical level.
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Anumonwo JM, Tallini YN, Vetter FJ & Jalife J (2001). Action potential characteristics and arrhythmogenic properties of the cardiac conduction system of the murine heart. Circ Res 89, 329–335.
Babij P, Askew GR, Nieuwenhuijsen B, Su CM, Bridal TR, Jow B, Argentieri TM, Kulik J, DeGennaro LJ, Spinelli W & Colatsky TJ (1998). Inhibition of cardiac delayed rectifier K+ current by overexpression of the long-QT syndrome HERG G628S mutation in transgenic mice. Circ Res 83, 668–678.
Baker LC, London B, Choi BR, Koren G & Salama G (2000). Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia. Circ Res 86, 396–407.
Balasubramaniam R, Chawla S, Mackenzie L, Schwiening CJ, Grace AA & Huang CL (2004). Nifedipine and diltiazem suppress ventricular arrhythmogenesis and calcium release in mouse hearts. Pflugers Arch 449, 150–158.[CrossRef][Medline]
Balasubramaniam R, Grace AA, Saumarez RC, Vandenberg JI & Huang CL (2003). Electrogram prolongation and nifedipine-suppressible ventricular arrhythmias in mice following targeted disruption of KCNE1. J Physiol 552, 535–546.
Brunet S, Aimond F, Li H, Guo W, Eldstrom J, Fedida D, Yamada KA & Nerbonne JM (2004). Heterogeneous expression of repolarizing, voltage-gated K+ currents in adult mouse ventricles. J Physiol 559, 103–120.
Brunner M, Guo W, Mitchell GF, Buckett PD, Nerbonne JM & Koren G (2001). Characterization of mice with a combined suppression of Ito and IK,slow. Am J Physiol Heart Circ Physiol 281, H1201–H1209.
Burashnikov A & Antzelevitch C (2002). Prominent IKs in epicardium and endocardium contributes to development of transmural dispersion of repolarization but protects against development of early afterdepolarizations. J Cardiovasc Electrophysiol 13, 172–177.[CrossRef][Medline]
Camm AJ, Janse MJ, Roden DM, Rosen MR, Cinca J & Cobbe SM (2000). Congenital and acquired long QT syndrome. Eur Heart J 21, 1232–1237.
Casimiro MC, Knollmann BC, Ebert SN, Vary JC Jr, Greene AE, Franz MR, Grinberg A, Huang SP & Pfeifer K (2001). Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange-Nielsen Syndrome. Proc Natl Acad Sci U S A 98, 2526–2531.
Charpentier F, Merot J, Riochet D, Le Marec H & Escande D (1998). Adult KCNE1-knockout mice exhibit a mild cardiac cellular phenotype. Biochem Biophys Res Commun 251, 806–810.[CrossRef][Medline]
Chinushi M, Restivo M, Caref EB & El-Sherif N (1998). Electrophysiological basis of arrhythmogenicity of QT/T alternans in the long-QT syndrome: tridimensional analysis of the kinetics of cardiac repolarization. Circ Res 83, 614–628.
Chouabe C, Drici MD, Romey G, Barhanin J & Lazdunski M (1998). HERG and KvLQT1/IsK, the cardiac K+ channels involved in long QT syndromes, are targets for calcium channel blockers. Mol Pharmacol 54, 695–703.
Cosio FG, Goicolea A, Lopez Gil M, Kallmeyer C & Barroso JL (1991). Suppression of Torsades de Pointes with verapamil in patients with atrio-ventricular block. Eur Heart J 12, 635–638.
Costantini DL, Arruda EP, Agarwal P, Kim KH, Zhu Y, Zhu W, Lebel M, Cheng CW, Park CY, Pierce SA, Guerchicoff A, Pollevick GD, Chan TY, Kabir MG, Cheng SH, Husain M, Antzelevitch C, Srivastava D, Gross GJ, Hui CC, Backx PH & Bruneau BG (2005). The homeodomain transcription factor Irx5 establishes the mouse cardiac ventricular repolarization gradient. Cell 123, 347–358.[CrossRef][Medline]
Dilly KW, Rossow CF, Votaw VS, Meabon JS, Cabarrus JL & Santana LF (2006). Mechanisms underlying variations in excitation–contraction coupling across the mouse left ventricular free wall. J Physiol 572, 227–241.
Drici MD, Arrighi I, Chouabe C, Mann JR, Lazdunski M, Romey G & Barhanin J (1998). Involvement of IsK-associated K+ channel in heart rate control of repolarization in a murine engineered model of Jervell and Lange-Nielsen syndrome. Circ Res 83, 95–102.
Duggal P, Vesely MR, Wattanasirichaigoon D, Villafane J, Kaushik V & Beggs AH (1998). Mutation of the gene for IsK associated with both Jervell and Lange-Nielsen and Romano-Ward forms of Long-QT syndrome. Circulation 97, 142–146.
El-Sherif N, Caref EB, Yin H & Restivo M (1996). The electrophysiological mechanism of ventricular arrhythmias in the long QT syndrome. Tridimensional mapping of activation and recovery patterns. Circ Res 79, 474–492.
Fabritz L, Kirchhof P, Franz MR, Eckardt L, Monnig G, Milberg P, Breithardt G & Haverkamp W (2003a). Prolonged action potential durations, increased dispersion of repolarization, and polymorphic ventricular tachycardia in a mouse model of proarrhythmia. Basic Res Cardiol 98, 25–32.[CrossRef][Medline]
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 on dispersion of repolarisation and arrhythmias in DeltaKPQ SCN5A (long QT3) mice. Cardiovasc Res 57, 1085–1093.
Felipe A, Knittle TJ, Doyle KL, Snyders DJ & Tamkun MM (1994). Differential expression of Isk mRNAs in mouse tissue during development and pregnancy. Am J Physiol Cell Physiol 267, C700–C705.
Garfinkel A, Kim YH, Voroshilovsky O, Qu Z, Kil JR, Lee MH, Karagueuzian HS, Weiss JN & Chen PS (2000). Preventing ventricular fibrillation by flattening cardiac restitution. Proc Natl Acad Sci U S A 97, 6061–6066.
Hagiwara N, Irisawa H & Kameyama M (1988). Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol 395, 233–253.
Knollmann BC, Katchman AN & Franz MR (2001). Monophasic action potential recordings from intact mouse heart: validation, regional heterogeneity, and relation to refractoriness. J Cardiovasc Electrophysiol 12, 1286–1294.[CrossRef][Medline]
Komiya N, Tanaka K, Doi Y, Fukae S, Nakao K, Isomoto S, Seto S & Yano K (2004). A patient with LQTS in whom verapamil administration and permanent pacemaker implantation were useful for preventing torsade de pointes. Pacing Clin Electrophysiol 27, 123–124.[CrossRef][Medline]
Kupershmidt S, Yang T, Anderson ME, Wessels A, Niswender KD, Magnuson MA & Roden DM (1999). Replacement by homologous recombination of the minK gene with lacZ reveals restriction of minK expression to the mouse cardiac conduction system. Circ Res 84, 146–152.
Lee MP, Ravenel JD, Hu RJ, Lustig LR, Tomaselli G, Berger RD, Brandenburg SA, Litzi TJ, Bunton TE, Limb C, Francis H, Gorelikow M, Gu H, Washington K, Argani P, Goldenring JR, Coffey RJ & Feinberg AP (2000). Targeted disruption of the Kvlqt1 gene causes deafness and gastric hyperplasia in mice. J Clin Invest 106, 1447–1455.[Medline]
Liu TF, Cheng YZ & Wei JH (1990). Early afterdepolarization in mouse atrial fibers induced by 3.0 mM K+ and the inhibitory effects of tetrodotoxin and nifedipine. Methods Find Exp Clin Pharmacol 12, 95–98.[Medline]
Liu G, Iden JB, Kovithavongs K, Gulamhusein R, Duff HJ & Kavanagh KM (2004). In vivo temporal and spatial distribution of depolarization and repolarization and the illusive murine T wave. J Physiol 555, 267–279.
London B (2004). Mouse models of cardiac arrhythmias. In Cardiac Electrophysiology from Cell to Bedside, ed. Zipes DP & Jalife J, pp. 433–443. Elsevier, Philadelphia.
London B, Pan X-H, Lewarchik C & Lee C (1998). QT interval prolongation and arrhythmias in heterozygous Merg1-targeted mice. Circulation 98, I-56. (Abstract)
Lu CW, Lin JH, Rajawat YS, Jerng H, Rami TG, Sanchez XV, Defreitas G, Carabello B, Demayo F, Kearney DL, Miller G, Li H, Pfaffinger PF, Bowles NE, Khoury DS & Towbin JA (2006). Functional and clinical characterization of a mutation in KCNJ2 associated with Andersen-Tawil syndrome. J Med Genet 43, 653–659.
Maguire CT, Wakimoto H, Patel VV, Hammer PE, Gauvreau K & Berul CI (2003). Implications of ventricular arrhythmia vulnerability during murine electrophysiology studies. Physiol Genomics 15, 84–91.
Milberg P, Reinsch N, Osada N, Wasmer K, Monnig G, Stypmann J, Breithardt G, Haverkamp W & Eckardt L (2005). Verapamil prevents torsade de pointes by reduction of transmural dispersion of repolarization and suppression of early afterdepolarizations in an intact heart model of LQT3. Basic Res Cardiol 100, 365–371.[CrossRef][Medline]
Mohler PJ, Schott JJ, Gramolini AO, Dilly KW, Guatimosim S, duBell WH, Song LS, Haurogne K, Kyndt F, Ali ME, Rogers TB, Lederer WJ, Escande D, Le Marec H & Bennett V (2003). Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature 421, 634–639.[CrossRef][Medline]
Napolitano C (2004). Transgenic models in cardiac arrhythmias: how close can we get to the bedside? Cardiovasc Res 61, 206–207.
Nerbonne JM & Guo W (2002). Heterogeneous expression of voltage-gated potassium channels in the heart: roles in normal excitation and arrhythmias. J Cardiovasc Electrophysiol 13, 406–409.[CrossRef][Medline]
Nerbonne JM, Nichols CG, Schwarz TL & Escande D (2001). Genetic manipulation of cardiac K+ channel function in mice: what have we learned, and where do we go from here? Circ Res 89, 944–956.
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 accelerations or premature beats cause life-threatening arrhythmias in mice with long-QT3 syndrome. Nat Med 7, 1021–1027.[CrossRef][Medline]
Opthof T & Coronel R (2005). Transmural dispersion in LQT3 and arrhythmogenesis. Cardiovascular Res 68, 336–337.
Packer M, O'Connor CM, Ghali JK, Pressler ML, Carson PE, Belkin RN, Miller AB, Neuberg GW, Frid D, Wertheimer JH, Cropp AB & DeMets DL (1996). Effect of amlodipine on morbidity and mortality in severe chronic heart failure. Prospective Randomized Amlodipine Survival Evaluation Study Group. N Engl J Med 335, 1107–1114.
Pidoplichko VI & Verkhratskii AN (1989). [Frequency-related blocking of sodium channels in the membrane of isolated rat cardiomyocytes by the calcium antagonist verapamil]. Fiziol Zh 35, 87–89.
Pruvot EJ, Katra RP, Rosenbaum DS & Laurita KR (2004). Role of calcium cycling versus restitution in the mechanism of repolarization alternans. Circ Res 94, 1083–1090.
Restivo M, Caref EB, Kozhevnikov DO & El-Sherif N (2004). Spatial dispersion of repolarization is a key factor in the arrhythmogenicity of long QT syndrome. J Cardiovasc Electrophysiol 15, 323–331.[Medline]
Roden DM (2006). Probing the arrhythmogenic substrate. Heart Rhythm 3, 779–780.[CrossRef][Medline]
Roepke TK, Anantharam A, Kirchhoff P, Busque SM, Young JB, Geibel JP, Lerner DJ & Abbott GW (2006). The KCNE2 potassium channel ancillary subunit is essential for gastric acid secretion. J Biol Chem 281, 23740–23747.
Rosenbaum DS, Jackson LE, Smith JM, Garan H, Ruskin JN & Cohen RJ (1994). Electrical alternans and vulnerability to ventricular arrhythmias. N Engl J Med 330, 235–241.
