Focus issue: Sudden cardiac arrestExperimentalCytosolic calcium accumulation and delayed repolarization associated with ventricular arrhythmias in a guinea pig model of Andersen-Tawil syndrome
Introduction
Anderson-Tawil syndrome (ATS1) is an inherited channelopathy that results from loss of function of the inward-rectifier K+ current (IK1) secondary to mutations in KCNJ2, the gene that encodes the Kir2.1 channel.1, 2 ATS1 is characterized electrocardiographically by a prolonged QT interval (hence its classification as long QT syndrome type 7) and nonsustained ventricular tachycardias (VTs) that often are foreshadowed by frequent triggered activity and occur more frequently during hypokalemia.2, 3 Therefore, it has been proposed that arrhythmias in ATS1 may be caused by electrical substrate remodeling4, 5 giving rise to the prolonged QT interval and increased triggered activity frequency. Although heterogeneous action potential duration (APD) prolongation and increased dispersion, both transmural and interventricular, have been reported in experimental models of ATS1,5, 6, 7 whether these gradients of repolarization are sufficient for reentry to occur remains unknown.
The high frequency and focal nature of bidirectional VTs in ATS1 suggest that triggered activity underlies, at least in part, the observed arrhythmias in ATS1.6 In general, focal arrhythmias have been linked to cytosolic Ca2+ ([Ca2+]i) accumulation.8, 9, 10 Indeed, in silico models of ATS1 support the hypothesis that [Ca2+]i accumulation underlies increased triggered activity during partial IK1 blockade.11, 12 Based on ex vivo studies in drug-induced Anderson-Tawil syndrome (DI-ATS1) models, Morita et al6 and Poelzing and Veeraraghavan7 proposed that arrhythmia propensity in ATS1 derives from [Ca2+]i accumulation leading to increased triggered activity. However, [Ca2+]i accumulation has yet to be demonstrated in an experimental model of ATS1, in part due to limitations in whole-heart [Ca2+]i measurement techniques.
Although the development of ratiometric (i.e., dual wavelength) fluorescent Ca2+ probes has helped minimize artifacts due to inhomogeneities in fluorescence and motion, whole-heart Ca2+ optical mapping has lacked a calibration procedure that would satisfactorily account for multiple excitation light exposures.13, 14 Therefore, we designed and validated a ratiometric Ca2+ optical mapping system capable of simultaneous, quantitative, multisite measurements and use the system here to test the hypothesis that elevated [Ca2+]i concomitant with APD prolongation, rather than APD dispersion, underlies arrhythmia propensity during DI-ATS1.
We demonstrate in guinea pig Langendorff-perfused ventricles that gradients of epicardial APD dispersion in DI-ATS1 were insufficient for arrhythmia induction by premature stimuli. However, APD prolongation was associated with increased incidence and severity of spontaneous and rapid pacing induced arrhythmias. Importantly, we demonstrate that this increased arrhythmia incidence is associated with significant diastolic [Ca2+]i accumulation. Furthermore, APD abbreviation with the ATP-sensitive potassium channel opener pinacidil alleviated both diastolic [Ca2+]i accumulation and the consequent increased arrhythmia burden.
Section snippets
Methods
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the Institutional Animal Care and Use Committee of the University of Utah (Protocol No. 05-07002).
Guinea pig Langendorff preparation
Guinea pig ventricles were perfused as Langendorff preparations as previously described.7 In brief, adult male guinea pig breeders (weight 800–1,000 g) were anesthetized with sodium pentobarbital (30 mg/kg intraperitoneally). Their hearts were rapidly excised and the atria removed and perfused as Langendorff preparations (perfusion pressure 55 mmHg) with oxygenated (100% O2) Tyrode solution at 36.5°C of the following composition (in mmol/L): CaCl2 2, NaCl 140, KCl 4.5, dextrose 10, MgCl2 1, and
Statistical analysis
Statistical analysis was performed with two-tailed Student's t-test for paired and unpaired data. Multiple regression analyses were used to characterize fluorescence Ca2+ signal drift both in vitro and in ex vivo preparations. Fisher exact test was used to test differences in nominal data. P <.05 was considered significant. All values are reported as mean ± SE unless otherwise noted.
Drug induced-ATS1
A representative volume-conducted ECG shown in Figure 1A demonstrates QT-interval prolongation by approximately 60 ms during DI-ATS1 relative to control. Additionally, the T wave, which was monophasic under control conditions, was biphasic during DI-ATS1. Over all experiments, QTc during DI-ATS1 (286.7 ± 15.2 ms) was significantly longer relative to control (210.7 ± 5.2 ms, Figure 1B). Underlying the observed QTc prolongation during DI-ATS1 was APD prolongation illustrated by representative
Discussion
Several studies hypothesized that [Ca2+]i accumulation concomitant with APD prolongation underlies arrhythmias in ATS111, 17 and DI-ATS1.6, 7 However, [Ca2+]i accumulation had not been demonstrated in whole-heart preparations in part because of methodologic difficulties in quantitative [Ca2+]i measurement using ratiometric Ca2+ optical mapping. In this study, we demonstrate that DI-ATS1 was associated with [Ca2+]i accumulation concomitant with APD prolongation. Attenuating [Ca2+]i accumulation
Study limitations
Although APD gradients in guinea pig (present study) or canine5 were not associated with increased arrhythmia propensity, APD distribution and heterogeneity are know to vary among animal models.33, 34 The nature of electrophysiologic remodeling induced by chronic functional IK1 down-regulation, as occurs in patients with ATS1, remains unclear.5, 6, 7, 11, 17 Furthermore, it is well appreciated that pharmacologic models of cardiac disease should be interpreted cautiously due to the acute nature
Conclusion
This study suggests that arrhythmias during DI-ATS1 may be the result of triggered activity secondary to prolonged APD and altered [Ca2+]i cycling and less likely dependent on large gradients of repolarization acting as a substrate for reentrant arrhythmias. Therefore, ameliorating myocyte [Ca2+]i load may prove a more effective therapeutic goal in ATS1 compared to decreasing APD gradients.
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Intercellular Sodium Regulates Repolarization in Cardiac Tissue with Sodium Channel Gain of Function
2020, Biophysical JournalCitation Excerpt :All animal study protocols were approved by the Institutional Animal Care and Use Committee at the Virginia Polytechnic Institute and State University. Optical mapping of Langendorff-perfused guinea pig hearts has been extensively described by the Poelzing group (10,36). Briefly, male retired breeder guinea pigs were anesthetized with isoflurane inhalation, and hearts were rapidly excised and retrogradely perfused through the aorta.
Natural History and Risk Stratification in Andersen-Tawil Syndrome Type 1
2020, Journal of the American College of CardiologyEfficacy and safety of flecainide for ventricular arrhythmias in patients with Andersen-Tawil syndrome with KCNJ2 mutations
2015, Heart RhythmCitation Excerpt :APD prolongation also elicits an increase in calcium influx, leading to intracellular calcium overload. In addition, subsequent spontaneous calcium release may depolarize the membrane potential to the threshold of L-type calcium channel via transient inward currents carried by the Na+-Ca2+exchanger.4,25,26 APD prolongation, instability of the resting membrane potential, and calcium overload result in the onset of delayed afterdepolarization.4,24
Andersen-Tawil syndrome: Clinical and molecular aspects
2013, International Journal of CardiologyStore-dependent deactivation: Cooling the chain-reaction of myocardial calcium signaling
2013, Journal of Molecular and Cellular CardiologyCitation Excerpt :CPVT has been attributed to “leaky” RyR2s resulting in spontaneous Ca2 + release during diastole [51,69,68]. Whereas the link between spontaneous Ca2 + release and the consequent membrane depolarization (delayed after depolarization, DAD) is well established and involves NCX stimulated by elevated cytosolic Ca2 + [62,70–72], the mechanism responsible for spontaneous Ca2 + release in CPVT has been an area of intense investigation. Based on studies using recombinant RyR2, Chen and colleagues suggested that spontaneous release associated with CPVT is due to direct activation of SR Ca2 + release by luminal Ca2 + [28,51].
Inhibition of Na<sup>+</sup> channels ameliorates arrhythmias in a drug-induced model of Andersen-Tawil syndrome
2013, Heart RhythmCitation Excerpt :ATS1 is characterized electrocardiographically by a mildly prolonged QT interval and ventricular arrhythmias that are initiated by frequent PVA that occur more frequently during hypokalemia.1 Previously, our group2 as well as others3 have linked PVA to abnormal Ca2+ regulation, where the regional Na+/Ca2+-exchange (NCX) dominance coupled to slower sarcoplasmic reticulum (SR) Ca2+ loading was associated with a greater arrhythmia incidence.4 Specifically, increasing global NCX dominance by SR Ca2+ ATPase inhibition increased arrhythmia propensity, while NCX blockade significantly reduced these events.4
Dr. Poelzing was supported by the Nora Eccles Treadwell Foundation and National Institutes of Health Grant R21 HL094828-01A1.