Atrial Arrhythmogenicity Research Articles

Characterization of Calcium Release Events Evoked by InsP3R Activation in Intact and Permeabilized Atrial Myocytes

In cardiac myocytes Ca2+ release events based on the intracellular second messenger inositol-1,4,5-trisphosphate receptor (InsP3R) activation are not conclusively characterized. This Ca2+ release mechanism may play a significant role under pathophysiological conditions, e.g. atrial arrhythmogenicity. In this study we characterize InsP3-induced SR-Ca2+ release events (Ca2+ puffs) and spontaneous SR-Ca2+ release events (Ca2+ sparks) based on RyRs openings in atrial myocytes. Local Ca2+ release events were examined in intact or permeabilized atrial myocytes acutely isolated from transgenic mouse hearts overexpressing InsP3Rs by using rapid 2-dimensional confocal imaging (150 fps). InsP3Rs were activated by rapid superfusion with InsP3 (permeabilized cells) or by ET-1 (intact cells). Xestospongin C was used as antagonist. Ca2+ puffs were solely appearing in the presence of intracellular InsP3. However, Ca2+ puffs may trigger Ca2+ sparks and both likely coexist, making the event separation and analysis from confocal images challenging. We compare two analytical approaches for quantitative Ca2+ event analysis based on different mathematical formalisms. One procedure operates with a spark as the rejection of the null hypothesis that the fluorescence change is noise. The second performs pixel-by-pixel fitting of the fluorescence signal and reconstructs underlying Ca2+ events via a clustering algorithm. Although rapid confocal imaging of the entire cell cross section always suffers from low signal-to noise ratio, this approach gives a more detailed and representative view of local Ca2+ events. Our new formalism for Ca2+ event analysis allows us to separate and characterize micro Ca2+ events (Ca2+ puffs, Ca2+ sparks) by their absolute number, amplitude, signal mass, area, FWHMX, FWHMY and kinetics using high speed imaging. We found that Ca2+ puffs exist as specific events in atrial myocytes, which could not be seen previously. Supported by SNF, SciEx and Novartis Res. Foundation.

Abstract 088: JNK2 Impairs Atrial Conduction And Enhances Arrhythmogenicity In Mouse Atria

The c-Jun N-terminal kinase (JNK) is a stress-activated kinase that is important in the development of cardiovascular diseases. We have previously shown that enhanced JNK activation was associated with suppressed gap junction Cx43, slow conduction, and increased atrial fibrillation (AF) in aged rabbit left atrium (LA). In an atrial cellular model (HL-1), we further demonstrated that JNK activation downregulated Cx43 and enhanced arrhythmogenicity. However, direct evidence for JNK inducing atrial arrhythmia in vivo is lacking. In the present study, we found that JNK activator anisomycin (aniso) treatment in wild-type (WT) mouse in vivo resulted in a 30% decrease in Cx43 protein in LA vs that of vehicle-treated WT LA (p<0.001; n=4, 6; each sample contained mixed LAs from 5 mice). From optical mapping recordings in the intact LA, conduction velocity (CV) in aniso-treated WT mice slowed by 36% (30±2 vs. 47±1 cm/sec in vehicle-treated WT, pacing cycle length (CL) = 100ms, p<0.01; n=4, 3). Additional in vivo treatment with SP600125 (a specific JNK inhibitor) reversed aniso-induced Cx43 suppression (n=3) and CV slowing (50±2 cm/sec; n=6) as compared with WT LA treated with aniso alone (p<0.001). Moreover, we assessed atrial arrhythmia susceptibility in vivo using burst pacing delivered via an octapolar cardiac catheter. JNK activated hearts exhibited an increased incidence of pacing-induced AF (13%; n=7), while no AF was induced in WT-vehicle mice (n=4). To further confirm the specific role of JNK activation in atrial arrhythmogenicity, JNK2 (one of the cardiac JNK isoforms) knockout mice (JNK2KO) were treated with aniso in vivo. Similar to the effect of JNK inhibition by SP600125, JNK2 knockout prevented aniso-induced Cx43 suppression (n=3) and slowing of CV (47±1cm/sec, CL=100ms; n=4) compared with aniso-treated WT (p<0.05 & p<0.001, respectively). Furthermore, a dramatically decreased susceptibility to pacing-induced AF (1.7%; n=6) was found in aniso-treated JNK2KO vs aniso-treated WT mice. In conclusion, our results strongly suggest that JNK, especially JNK2, activation contributes to impaired cell-cell communication that in turn promotes AF development. Modulation of JNK2 could be a novel therapeutic approach to prevent and treat AF in the elderly.

Unprovoked atrial tachyarrhythmias in aging spontaneously hypertensive rats: the role of the autonomic nervous system

Experimental models of unprovoked atrial tachyarrhythmias (AT) in conscious, ambulatory animals are lacking. We hypothesized that the aging, spontaneously hypertensive rat (SHR) may provide such a model. Baseline ECG recordings were acquired with radiotelemetry in eight young (14-wk-old) and eight aging (55-wk-old) SHRs and in two groups of four age-matched Wistar-Kyoto (WKY) rats. Quantification of AT and heart rate variability (HRV) analysis were performed based on 24-h ECG recordings in unrestrained rats. All animals were submitted to an emotional stress protocol (air-jet). In SHRs, carbamylcholine injections were also performed. Spontaneous AT episodes were observed in all eight aging SHRs (median, 91.5; range, 4-444 episodes/24 h), but not in young SHRs or WKY rats. HRV analysis demonstrated significantly decreased low frequency components in aging SHRs compared with age-matched WKY rats (P < 0.01) and decreased low/high frequency ratios in both young (P < 0.01) and aging (P = 0.01) SHRs compared with normotensive controls. In aging SHRs, emotional stress significantly reduced the number of arrhythmic events, whereas carbamylcholine triggered AT and significantly increased atrial electrical instability. This study reports the occurrence of unprovoked episodes of atrial arrhythmia in hypertensive rats, and their increased incidence with aging. Our results suggest that autonomic imbalance with relative vagal hyperactivity may be responsible for the increased atrial arrhythmogenicity observed in this model. We also provide evidence that, in this model, the sympatho-vagal imbalance preceded the occurrence of arrhythmia. These results indicate that aging SHRs may provide valuable insight into the understanding of atrial arrhythmias.

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.

The Relationship Between Conduction Velocity and Atrial Arrhythmogenicity under Conditions of Altered Ca2+ Homeostasis in RyR2-P2328S Murine Hearts

Atrial fibrillation (AF) is a significant contributor to mortality and health care expenditure; it is associated with abnormal sarcoplasmic reticulum (SR) Ca2+ release through cardiac ryanodine receptors. The initiation of AF requires an ectopic action potential (AP) triggered within a substrate of atria conducive to the formation of re-entry circuits. The present experiments explored whether increased diastolic SR Ca2+ release might contribute to atrial arrhythmogenicity in homozygotic gain-of-function RyR2-P2328S mice (RyR2s/s).Electrocardiographs and intracellular APs were recorded from Langendorff-perfused hearts at regular 8 Hz (S1) stimulation. RyR2s/s hearts showed depolarised resting membrane potentials that were associated with an increased incidence of ectopic APs and sustained tachyarrhythmias. During regular stimulation, the RyR2s/s showed reduced epicardial conduction velocities (CV), measured using a 64-electrode array, that corresponded with reduced S1 AP upstroke velocities ((dV/dt)max).When ectopic (S2) APs were imposed during S1 stimulation at progressively shorter S1S2 intervals, the inter-atrial CV and dV/dt(max) of the S2 APs decreased with each successive interval. Moderate reductions in S1S2 interval provoked sustained tachyarrhythmias in RyR2s/s hearts, whereas significantly shorter S1S2 intervals were required to provoke arrhythmias in WT hearts. Interestingly, the mean S1S2 interval required to provoke an arrhythmia in each variant resulted in similar inter-atrial CVs and dV/dt(max).These findings suggest that triggered activity is likely to lead to a sustained tachyarrhythmia in murine atria only when the CV of an ectopic AP is slow (<∼1.1 m/s). We have shown this is more likely to occur in RyR2s/s atria where increased diastolic SR Ca2+ release is associated with both increased ectopic APs and an intrinsically slow CV. We therefore suggest that abnormal Ca2+ homeostasis contributes to both the trigger and the re-entry substrate in sustained atrial tachyarrhythmias.

Angiotensin II type 1 receptor blocker attenuates diabetes-induced atrial structural remodeling

Diabetes mellitus promotes atrial structural remodeling, thereby producing atrial arrhythmogenicity, where advanced glycation endproducts (AGEs) and their receptor (RAGE) are implicated to play a role in the pathogenesis. We investigated the effects of candesartan, an angiotensin type II receptor blocker, on the diabetes-induced atrial structural change. Diabetes was induced in 8-week-old female Sprague-Dawley rats by intraperitoneal injection of streptozotocin at 70 mg/kg. Osmotic pumps were simultaneously set to infuse candesartan at a subdepressor dose of 0.05 mg/kg/day. Twelve weeks after the induction of diabetes, the blood glucose and glycated hemoglobin A1c were significantly higher in streptozotocin-injected rats than those in control rats, and were not affected by candesartan treatment. The atria of diabetic rats showed remarkable diffuse interstitial fibrosis with more enhanced protein expressions of RAGE and connective tissue growth factor (CTGF) compared with control ones. The treatment with candesartan significantly reduced CTGF expression and effectively suppressed the development of fibrotic deposition in diabetic animals. Candesartan reduced CTGF expression and attenuated the fibrosis in diabetic rat atria. These results implied the protective effects of candesartan on diabetes-related atrial arrhythmias.

Acute atrial arrhythmogenicity and altered Ca2+ homeostasis in murine RyR2-P2328S hearts

AimsThe experiments explored for atrial arrhythmogenesis and its possible physiological background in recently developed hetero-(RyR2+/S) and homozygotic (RyR2S/S) RyR2-P2328S murine models for catecholaminergic polymorphic ventricular tachycardia (VT) for the first time. They complement previous clinical and experimental reports describing increased ventricular arrhythmic tendencies associated with physical activity, stress, or catecholamine infusion, potentially leading to VT and ventricular fibrillation.Methods and resultsAtrial arrhythmogenic properties were compared at the whole animal, Langendorff-perfused heart, and single, isolated atrial myocyte levels using electrophysiological and confocal fluorescence microscopy methods. This demonstrated that: (i) electrocardiographic parameters in intact anaesthetized wild-type (WT), RyR2+/S and RyR2S/S mice were statistically indistinguishable both before and after addition of isoproterenol apart from increases in heart rates. (ii) Bipolar electrogram and monophasic action potential recordings showed significantly higher incidences of arrhythmogenesis in isolated perfused RyR2S/S, but not RyR2+/S, relative to WT hearts during either regular pacing or programmed electrical stimulation. The addition of isoproterenol increased such incidences in all three groups. (iii) However, there were no accompanying differences in cardiac anatomy or action potential durations at 90% repolarization and refractory periods. (iv) In contrast, episodes of diastolic Ca2+ release were observed under confocal microscopy in isolated fluo-3-loaded RyR2S/S, but not RyR2+/S or WT, atrial myocytes. The introduction of isoproterenol resulted in significant diastolic Ca2+ release in all three groups.ConclusionsThese findings establish acute atrial arrhythmogenic properties in RyR2-P2328S hearts and correlate these with altered Ca2+ homeostasis in an absence of repolarization abnormalities for the first time.

Atrial arrhythmogenicity in aged Scn5a+/∆KPQ 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+/ΔKPQ 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+/ΔKPQ than young WT. These levels increased with age in WT but not Scn5a+/ΔKPQ. Both young and aged Scn5a+/ΔKPQ mice showed lower heart rates and longer PR intervals than their WT counterparts. Young Scn5a+/ΔKPQ mice showed longer QT and QTc intervals than young WT. Aged Scn5a+/ΔKPQ 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+/ΔKPQ. Aged murine Scn5a+/ΔKPQ hearts thus exhibit an increased atrial arrhythmogenicity. The differing Nav1.5 expression and electrocardiographic indicators of slowed cardiac conduction between Scn5a+/ΔKPQ and WT, which show further variations associated with aging, may contribute toward atrial arrhythmia in aged Scn5a+/ΔKPQ hearts.

Potential atrial arrhythmogenicity of adipocytes: Implications for the genesis of atrial fibrillation

Atrial fibrillation is the most common sustained cardiac arrhythmia. Obesity and metabolic syndrome are independent risk factors for atrial fibrillation, and epicardial adipose tissues are highly associated with the genesis of atrial fibrillation, compared to visceral fat. Adipocytes can produce inflammatory cytokines and adipocytokines, which may enhance inflammation and oxidative stress in patients with obesity or metabolic syndrome. Moreover, it is possible that local interactions between epicardial adipose tissue and the adjacent myocardium can directly produce electrical or structural remodelings in the atrium. Taken together, we hypothesized that epicardial adipocytes contain distinctive arrhythmogenicity through increases in inflammatory cytokines, adipocytokines, and adipocyte-cardiomyocyte interactions to induce atrial fibrillation.

Abnormal Atrial Repolarization and Depolarization Contribute to the Inducibility of Atrial Fibrillation in Brugada Syndrome

Brugada syndrome is often accompanied by atrial tachyarrhythmia, such as atrial fibrillation (AF). The aim of this study was to examine atrial vulnerability in patients with Brugada syndrome. Two groups of patients were compared: 18 patients with Brugada syndrome (Brugada syndrome group) and 11 age-matched patients with neither organic heart disease nor AF episodes (control group). Programmed electrical stimulation was performed from the right atrium (RA), and the effective refractory period of the right atrium (ERP-RA), interatrial conduction time (IACT), monophasic action potentials (MAPs) at the high RA, and the inducibility of AF lasting > 30 seconds were studied. MAP duration at 80% repolarization (MAPD(80)) was calculated. AF was induced with a single extrastimulus or double extrastimuli in all patients with Brugada syndrome but in none of the control patients. The ERP-RA did not differ between the groups. IACT at the shortest diastolic interval was significantly increased in the Brugada syndrome group compared to that in the control group. The maximum slope of the MAPD(80) restitution curve was significantly steeper in the Brugada syndrome group than in the control group (2.4 + or - 2.0 versus 0.82 + or - 0.36, P < 0.02). Ventricular fibrillation was induced with ventricular programmed stimulation in all Brugada syndrome patients. Both abnormal interatrial conduction and steep restitution of action potential duration may contribute to the atrial arrhythmogenicity in Brugada syndrome.

Atrial arrhythmogenesis in wild-type and Scn5a+/Δ murine hearts modelling LQT3 syndrome

Long QT(3) (LQT3) syndrome is associated with abnormal repolarisation kinetics, prolonged action potential durations (APD) and QT intervals and may lead to life-threatening ventricular arrhythmias. However, there have been few physiological studies of its effects on atrial electrophysiology. Programmed electrical stimulation and burst pacing induced atrial arrhythmic episodes in 16 out of 16 (16/16) wild-type (WT) and 7/16 genetically modified Scn5a+/Δ (KPQ) Langendorff-perfused murine hearts modelling LQT3 (P < 0.001 for both), and in 14/16 WT and 1/16 KPQ hearts (P < 0.001 for both; Fisher’s exact test), respectively. The arrhythmogenic WT hearts had significantly larger positive critical intervals (CI), given by the difference between atrial effective refractory periods (AERPs) and action potential durations at 90% recovery (APD90), compared to KPQ hearts (8.1 and 3.2 ms, respectively, P < 0.001). Flecainide prevented atrial arrhythmias in all arrhythmogenic WT (P < 0.001) and KPQ hearts (P < 0.05). It prolonged the AERP to a larger extent than it did the APD90 in both WT and KPQ groups, giving negative CIs. Quinidine similarly exerted anti-arrhythmic effects, prolonged AERP over corresponding APD90 in both WT and KPQ groups. These findings, thus, demonstrate, for the first time, inhibitory effects of the KPQ mutation on atrial arrhythmogenesis and its modification by flecainide and quinidine. They attribute these findings to differences in the CI between WT and mutant hearts, in the presence or absence of these drugs. Thus, prolongation of APD90 over AERP gave positive CI values and increased atrial arrhythmogenicity whereas lengthening of AERP over APD90 reduced such CI values and produced the opposite effect.

Effects of Aging and Ouabain on Left Atrial Arrhythmogenicity

Aging increases atrial fibrillation (AF) vulnerability. The left atrium (LA) is important for the generation of AF. However, the effect of aging on the electrophysiological properties of the LA in general, on the specific LA sites, and of possible accentuation of regional differences between the LA sites with aging is not clear. The purpose of this study was to evaluate the effects of aging on the LA electrophysiological heterogeneity and ouabain-induced arrhythmogenicity. We used conventional microelectrodes to record the action potentials (APs) in isolated young (age, 3 months) and aged (age, 3 years) rabbit LA posterior wall (LAPW) and LA appendage (LAA) tissue specimens before and after the administration of ouabain. Young LAPWs (n = 10) had larger AP amplitudes than young LAAs (n = 10, P < 0.05), and aged LAPWs (n = 9) had longer AP durations than aged LAAs (n = 9, P < 0.05). Ouabain (1 microM) induced a higher incidence (80% vs 30%, P < 0.05) of delayed afterdepolarizations (DADs) and spontaneous activity (60% vs 10%, P < 0.05) in the young LAPWs than in the young LAAs. Compared with the young group, the aged LAs had a higher incidence of DADs with a less negative resting membrane potential and smaller maximum upstroke velocity. After the ouabain (1 microM) administration, the aged LAPWs had a greater shortening of the AP duration. Ouabain-induced spontaneous activity was similar between the young and aged groups. Aging enhanced the LA regional electrical heterogeneity and LAPW arrhythmogenesis.

What are Arrhythmogenic Substrates in Diabetic Rat Atria?

Diabetes mellitus is one of the significant independent risk factors for the development of atrial fibrillation (AF). However, the pathophysiological mechanisms of the relationship have not been fully elucidated. The genetic type II diabetes (GK) rats and their original (Wistar) ones were subjected to electrophysiological (n = 8 per group) and histological (n = 7 per group) studies. At 40 weeks old, when GK rats had significantly (P < 0.01) more increased plasma glucose and HbA(1c) values than Wistar rats, atrial electrical stimuli in the isolated-perfused hearts induced significantly greater number of repetitive atrial responses in GK rats than in Wistar rats (47.9 +/- 17.5 vs 3.1 +/- 1.3 beats, respectively, P < 0.01). GK rats showed significantly longer intra-atrial activation time than Wistar rats (18.3 +/- 0.4 ms vs 15.9 +/- 0.5 ms, P < 0.01) without any significant difference in the atrial refractoriness. The histological examination revealed significantly increased diffuse fibrotic deposition in GK rats atria compared with Wistar ones (P < 0.01). The present diabetic GK rat showed increased atrial arrhythmogenicity with intra-atrial conduction disturbance, and thus indicated that the structural remodeling of atrium characterized by diffuse interstitial fibrosis would be a major substrate for diabetes-related AF.

P.1.14 Atrial conduction changes and pulmonary vein arrhythmogenicity following pulmonary vein ablation for atrial fibrillation

P.1.14 Atrial conduction changes and pulmonary vein arrhythmogenicity following pulmonary vein ablation for atrial fibrillation