The RyR2-P2328S mutation downregulates Nav1.5 producing arrhythmic substrate in murine ventricles.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) predisposes to ventricular arrhythmia due to altered Ca(2+) homeostasis and can arise from ryanodine receptor (RyR2) mutations including RyR2-P2328S. Previous reports established that homozygotic murine RyR2-P2328S (RyR2 (S/S)) hearts show an atrial arrhythmic phenotype associated with reduced action potential (AP) conduction velocity and sodium channel (Nav1.5) expression. We now relate ventricular arrhythmogenicity and slowed AP conduction in RyR2 (S/S) hearts to connexin-43 (Cx43) and Nav1.5 expression and Na(+) current (I Na). Stimulation protocols applying extrasystolic S2 stimulation following 8 Hz S1 pacing at progressively decremented S1S2 intervals confirmed an arrhythmic tendency despite unchanged ventricular effective refractory periods (VERPs) in Langendorff-perfused RyR2 (S/S) hearts. Dynamic pacing imposing S1 stimuli then demonstrated that progressive reductions of basic cycle lengths (BCLs) produced greater reductions in conduction velocity at equivalent BCLs and diastolic intervals in RyR2 (S/S) than WT, but comparable changes in AP durations (APD90) and their alternans. Western blot analyses demonstrated that Cx43 protein expression in whole ventricles was similar, but Nav1.5 expression in both whole tissue and membrane fractions were significantly reduced in RyR2 (S/S) compared to wild-type (WT). Loose patch-clamp studies similarly demonstrated reduced I Na in RyR2 (S/S) ventricles. We thus attribute arrhythmogenesis in RyR2 (S/S) ventricles resulting from arrhythmic substrate produced by reduced conduction velocity to downregulated Nav1.5 reducing I Na, despite normal determinants of repolarization and passive conduction. The measured changes were quantitatively compatible with earlier predictions of linear relationships between conduction velocity and the peak I Na of the AP but nonlinear relationships between peak I Na and maximum Na(+) permeability.

Action potential wavelength restitution predicts alternans and arrhythmia in murine Scn5a(+/-) hearts.

Reductions in cardiac action potential wavelength, and the consequent wavebreak, have been implicated in arrhythmogenesis. Tachyarrhythmias are more common in the Brugada syndrome, particularly following pharmacological challenge, previously modelled using Scn5a(+/-) murine hearts. Propagation latencies and action potential durations (APDs) from monophasic action potential recordings were used to assess wavelength changes with heart rate in Langendorff-perfused wild-type (WT) and Scn5a(+/-) hearts. Recordings were obtained from right (RV) and left (LV) ventricular, epicardial and endocardial surfaces during incremental pacing, before and following flecainide or quinidine challenge. Conduction velocities (θ'), action potential wavelengths (λ' = APD × Î¸'), and their corresponding alternans depended non-linearly upon diastolic interval (DI). Maximum θ' was lower in Scn5a(+/-) RV epicardium than endocardium. Flecainide further reduced θ', accentuating this RV conduction block. Quinidine reduced maximum θ' in WT and caused earlier conduction failure in the RV of both Scn5a(+/-) and WT. Use of recovery wavelengths (λ'0 = DI × Î¸') rather than DI, provided novel λ restitution plots of λ' against λ'0, which sum to a basic cycle distance permitting feedback analysis. λ' restitution gradient better correlated with alternans magnitude than either APD or θ restitution gradient. The large differences in θ' and APD restitution contrasted with minor differences in maximum λ' between epi- and endocardia of untreated hearts, and quinidine-treated WT hearts. Strikingly, all regions and conditions converged to a common instability point, implying a conserved relationship. Flecainide or quinidine decreased the pacing rates at which this occurred, through reducing basic cycle distance, in the Scn5a(+/-) RV epicardium, directly predictive of its arrhythmic phenotype.

Conduction slowing contributes to spontaneous ventricular arrhythmias in intrinsically active murine RyR2-P2328S hearts.

INTRODUCTION: The familial condition catecholaminergic polymorphic ventricular tachycardia (CPVT) is characterized by episodic bidirectional ventricular tachycardia (BVT), polymorphic ventricular tachycardia (PVT), and ventricular fibrillation following adrenergic challenge. It is associated with mutations involving the cardiac ryanodine receptor (RyR2). METHODS AND RESULTS: We explored for a slowing of myocardial conduction that could potentially result in a substrate for the spontaneous arrhythmogenesis that was observed following introduction of isoproterenol and caffeine in intrinsically beating murine RyR2-P2328S hearts. Such pharmacological challenge increased the number of arrhythmic episodes in electrocardiographic recordings from intact anesthetized mice, with the greatest effects in the homozygote RyR2(S/S). Arrhythmias took the form of bigeminy, BVT, monomorphic ventricular tachycardia, and PVT, as found in human CPVT. Ventricular epicardial conduction velocities (CVs) measured using multielectrode array recordings and maximum action potential upstroke rates, (dV/dt)(max), measured using intracellular microelectrodes were indistinguishable in untreated wild-type (WT) and RyR2(S/S). Pharmacological challenge of RyR2(S/S), but not WT hearts, then reduced CV and (dV/dt)(max) and also revealed a strongly arrhythmic phenotype. There was no evidence of gross structural or fibrotic changes in either RyR2(+/S) or RyR2(S/S) hearts on light microscopy. CONCLUSIONS: We associate altered ventricular myocardial CV potentially resulting in arrhythmogenic substrate with arrhythmic properties associated with genetic RyR2 alterations for the first time.

The age-dependence of atrial arrhythmogenicity in Scn5a+/- murine hearts reflects alterations in action potential propagation and recovery.

1. In the present study, we investigated the effect of age on atrial electrophysiological properties in Scn5a(+/-) hearts used to model corresponding increases in atrial arrhythmic tendency in human Brugada syndrome. 2. Atrial action potential initiation, propagation and recovery were compared in young (3 month old) and aged (12 month old), wild-type (WT) and Scn5a(+/-) hearts. Multielectrode array recordings assessed the spatial propagation of intrinsic electrical activity in superfused atrial preparations, whereas bipolar electrogram recordings measured basic cycle lengths (BCL) in Langendorff preparations. The duration of electrogram activity (EGD) during regular and extrasystolic stimulation with programmed electrical stimulation provided EGD ratios and atrial effective refractory periods (AERP). Monophasic recordings measured action potential durations (APD). 3. Systematic statistical explorations for independent and interacting effects of age and the Scn5a(+/-) condition demonstrated that both young and aged Scn5a(+/-) mice exhibited slowed propagation of atrial excitation relative to corresponding WT mice, with the greatest effects in aged Scn5a(+/-) mice, which additionally exhibited increased intrinsic BCL. 4. Young Scn5a(+/-) mice exhibited greater EGD and EGD ratios, as well as APD/AERP ratios, suggesting increased arrhythmic tendency compared with WT mice. 5. Aged Scn5a(+/-) mice exhibited normal EGD, EGD ratios and APD compared to aged WT and young Scn5a(+/-), and increased AERP and smaller APD/AERP ratios compared with young Scn5a(+/-). 6. These electrophysiological findings indicate increased atrial arrhythmogenicity with maximal effects on both conduction and repolarization characteristics in young compared with aged Scn5a(+/-) mice.

Delayed conduction and its implications in murine Scn5a(+/-) hearts: independent and interacting effects of genotype, age, and sex.

We explored for relationships between SCN5A haploinsufficiency, implicated in clinical arrhythmogenicity, and right ventricular (RV) conduction disorders in Langendorff-perfused, male and female, and young (3 months) and old (>12 month old) Scn5a ( +/-) and wild type (WT) hearts. The investigated conditions of genotype, age, and sex affected latencies but not repolarization time courses of RV monophasic action potentials. This prompted examination of the patterns of RV epicardial activation, its dispersion, and their interrelationships as possible arrhythmic mechanisms using a 64-channel, multi-electrode array. Mean ventricular activation times (T*(MEAN)), spatial dispersions (D* (S)) between recording channels/cardiac cycle, and maximum activation times (T* (MAX)) representing the slowest possible conduction in any given heart were all higher in old male Scn5a ( +/-) compared with young male and old female Scn5a ( +/-) and old male WT. Temporal dispersions (D*(T)) of recording channels were similarly higher in old male Scn5a (+/-) compared with old male WT. All groupings of D*(T), D*(S), and T*(MAX) nevertheless linearly correlated with T*(MEAN), with indistinguishable slopes. The variates explored thus influence D*(T), D*(S), and T*(MAX) through actions on T*(MEAN). These findings in turn correlated with increased levels of fibrosis in young male, young female, and old male Scn5a ( +/-) compared with the corresponding WTs. We thus demonstrate for the first time independent and interacting effects of genotype, age, and sex on epicardial conduction and its dispersions at least partially attributable to fibrotic change, resulting in the greatest effects in old male Scn5a ( +/-) in an absence of alterations in repolarization time courses. This directly implicates altered depolarization in the clinical arrhythmogenicity associated with Scn5a ( +/-).

Mapping of reentrant spontaneous polymorphic ventricular tachycardia in a Scn5a+/- mouse model.

Two major mechanisms have been postulated for the arrhythmogenic tendency observed in Brugada Syndrome (BrS): delays in conduction or increased heterogeneities in repolarization. We use a contact mapping system to directly investigate the interacting roles of these two mechanisms in arrhythmogenesis using a genetic murine model for BrS for the first time. Electrograms were obtained from a multielectrode recording array placed against the left ventricle and right ventricle (RV) of spontaneously beating Langendorff-perfused wild type (WT) and Scn5a+/- mouse hearts. Scn5a+/- hearts showed activation waves arriving at the epicardial surface consistent with slowed conduction, which was exacerbated in the presence of flecainide. Lines of conduction block across the RV resulting from premature ventricular beats led to the formation of reentrant circuits and polymorphic ventricular tachycardia. WT hearts showed an inverse relationship between activation times and activation recovery intervals measured at the epicardial surface, which resulted in synchronicity of repolarization times. In contrast, Scn5a+/- hearts, despite having smaller mean activation recovery intervals, demonstrated a greater heterogeneity compared with WT. Isochronal maps showed that their normal activation recovery interval gradients at the epicardial surface were disrupted, leading to heterogeneity in repolarization times. We thus directly demonstrate the initiation of arrhythmia in the RV of Scn5a+/- hearts. This occurs as a result of the combination of repolarization heterogeneities leading to lines of conduction block and unidirectional conduction, with conduction slowing allowing the formation of reentrant circuits. The repolarization heterogeneities may also be responsible for the changing pattern of block, leading to the polymorphic character of the resulting ventricular tachycardia.

Atrial arrhythmogenicity in aged Scn5a+/DeltaKPQ mice modeling long QT type 3 syndrome and its relationship to Na+ channel expression and cardiac conduction.

Recent studies have reported that human mutations in Nav1.5 predispose to early age onset atrial arrhythmia. The present experiments accordingly assess atrial arrhythmogenicity in aging Scn5a+/KPQ mice modeling long QT3 syndrome in relationship to cardiac Na(+) channel, Nav1.5, expression. Atrial electrophysiological properties in isolated Langendorff-perfused hearts from 3- and 12-month-old wild type (WT), and Scn5a+/KPQ mice were assessed using programmed electrical stimulation and their Nav1.5 expression assessed by Western blot. Cardiac conduction properties were assessed electrocardiographically in intact anesthetized animals. Monophasic action potential recordings demonstrated increased atrial arrhythmogenicity specifically in aged Scn5a+/DeltaKPQ hearts. These showed greater action potential duration/refractory period ratios but lower atrial Nav1.5 expression levels than aged WT mice. Atrial Nav1.5 levels were higher in young Scn5a+/DeltaKPQ than young WT. These levels increased with age in WT but not Scn5a+/DeltaKPQ. Both young and aged Scn5a+/DeltaKPQ mice showed lower heart rates and longer PR intervals than their WT counterparts. Young Scn5a+/DeltaKPQ mice showed longer QT and QTc intervals than young WT. Aged Scn5a+/DeltaKPQ showed longer QRS durations than aged WT. PR intervals were prolonged and QT intervals were shortened in young relative to aged WT. In contrast, ECG parameters were similar between young and aged Scn5a+/DeltaKPQ. Aged murine Scn5a+/DeltaKPQ hearts thus exhibit an increased atrial arrhythmogenicity. The differing Nav1.5 expression and electrocardiographic indicators of slowed cardiac conduction between Scn5a+/DeltaKPQ and WT, which show further variations associated with aging, may contribute toward atrial arrhythmia in aged Scn5a+/DeltaKPQ hearts.

Nonlinearity between action potential alternans and restitution, which both predict ventricular arrhythmic properties in Scn5a+/- and wild-type murine hearts.

Electrocardiographic QT- and T-wave alternans, presaging ventricular arrhythmia, reflects compromised adaptation of action potential (AP) duration (APD) to altered heart rate, classically attributed to incomplete Na(v)1.5 channel recovery prior to subsequent stimulation. The restitution hypothesis suggests a function whose slope directly relates to APD alternans magnitude, predicting a critical instability condition, potentially generating arrhythmia. The present experiments directly test for such correlations among arrhythmia, APD alternans and restitution. Mice haploinsufficient in the Scn5a, cardiac Na(+) channel gene (Scn5a(+/-)), previously used to replicate Brugada syndrome, were used, owing to their established arrhythmic properties increased by flecainide and decreased by quinidine, particularly in right ventricular (RV) epicardium. Monophasic APs, obtained during pacing with progressively decrementing cycle lengths, were systematically compared at RV and left ventricular epicardial and endocardial recording sites in Langendorff-perfused Scn5a(+/-) and wild-type hearts before and following flecainide (10 µM) or quinidine (5 µM) application. The extent of alternans was assessed using a novel algorithm. Scn5a(+/-) hearts showed greater frequencies of arrhythmic endpoints with increased incidences of ventricular tachycardia, diminished by quinidine, and earlier onsets of ventricular fibrillation, particularly following flecainide challenge. These features correlated directly with increased refractory periods, specifically in the RV, and abnormal restitution and alternans properties in the RV epicardium. The latter variables were related by a unique, continuous higher-order function, rather than a linear relationship with an unstable threshold. These findings demonstrate a specific relationship between alternans and restitution, as well as confirming their capacity to predict arrhythmia, but implicate mechanisms additional to the voltage feedback suggested in the restitution hypothesis.

Frequency distribution analysis of activation times and regional fibrosis in murine Scn5a+/- hearts: the effects of ageing and sex.

Both Brugada Syndrome (BrS) and progressive cardiac conduction defect (PCCD) are associated respectively with diffuse and discrete alterations in conduction pathways affected by ageing and sex. This study assessed for contributions of such processes to the mechanism of conduction changes in Scn5a(+/-) and WT hearts stratified by age (3 and 12 months) and sex. In vivo electrocardiographic chest-lead assessment demonstrated greater incidences of bundle branch block in all Scn5a(+/-) mice compared to WT. Frequency analysis of right ventricular (RV) epicardial activation obtained from a 64-channel multi-electrode array demonstrated greater prominence of late conducting components in Scn5a(+/-) compared to WT male, and in male compared to female Scn5a(+/-) following stratification by genotype and sex. Similar differences were observed between old male Scn5a(+/-) and young male Scn5a(+/-), old female Scn5a(+/-), and old male WT, following stratification by genotype, age and sex. These findings directly correlated with histomorphometric assessment of regional fibrosis in both septa and free walls preferentially involving the RV. We demonstrate complex alterations in conduction distributions suggesting a conversion of normal to slow-conducting tissue, modulated by ageing and sex, coupled with fibrosis in Scn5a(+/-) hearts. These features suggest an overlap between pathophysiological processes related to BrS and PCCD in Scn5a(+/-) hearts.

Reduced Na(+) and higher K(+) channel expression and function contribute to right ventricular origin of arrhythmias in Scn5a+/- mice.

Brugada syndrome (BrS) is associated with ventricular tachycardia originating particularly in the right ventricle (RV). We explore electrophysiological features predisposing to such arrhythmic tendency and their possible RV localization in a heterozygotic Scn5a+/- murine model. Na(v)1.5 mRNA and protein expression were lower in Scn5a+/- than wild-type (WT), with a further reduction in the RV compared with the left ventricle (LV). RVs showed higher expression levels of K(v)4.2, K(v)4.3 and KChIP2 in both Scn5a+/- and WT. Action potential upstroke velocity and maximum Na(+) current (I(Na)) density were correspondingly decreased in Scn5a+/-, with a further reduction in the RV. The voltage dependence of inactivation was shifted to more negative values in Scn5a+/-. These findings are predictive of a localized depolarization abnormality leading to slowed conduction. Persistent Na(+) current (I(pNa)) density was decreased in a similar pattern to I(Na). RV transient outward current (I(to)) density was greater than LV in both WT and Scn5a+/-, and had larger time constants of inactivation. These findings were also consistent with the observation that AP durations were smallest in the RV of Scn5a+/-, fulfilling predictions of an increased heterogeneity of repolarization as an additional possible electrophysiological mechanism for arrhythmogenesis in BrS.