Spontaneous Atrial Fibrillation Research Articles

Quantitative 3D electron microscopy characterization of mitochondrial structure, mitophagy, and organelle interactions in murine atrial fibrillation

Atrial fibrillation (AF) is the most common clinical arrhythmia, however there is limited understanding of its pathophysiology including the cellular and ultrastructural changes rendered by the irregular rhythm, which limits pharmacological therapy development. Prior work has demonstrated the importance of reactive oxygen species (ROS) and mitochondrial dysfunction in the development of AF. Mitochondrial structure, interactions with other organelles such as sarcoplasmic reticulum (SR) and T-tubules (TT), and degradation of dysfunctional mitochondria via mitophagy are important processes to understand ultrastructural changes due to AF. However, most analysis of mitochondrial structure and interactome in AF has been limited to two-dimensional (2D) modalities such as transmission electron microscopy (EM), which does not fully visualize the morphological evolution of the mitochondria during mitophagy. Herein, we utilize focused ion beam-scanning electron microscopy (FIB-SEM) and perform reconstruction of three-dimensional (3D) EM from murine left atrial samples and measure the interactions of mitochondria with SR and TT. We developed a novel 3D quantitative analysis of FIB-SEM in a murine model of AF to quantify mitophagy stage, mitophagosome size in cardiomyocytes, and mitochondrial structural remodeling when compared with control mice. We show that in our murine model of spontaneous and continuous AF due to persistent late sodium current, left atrial cardiomyocytes have heterogenous mitochondria, with a significant number which are enlarged with increased elongation and structural complexity. Mitophagosomes in AF cardiomyocytes are located at Z-lines where they neighbor large, elongated mitochondria. Mitochondria in AF cardiomyocytes show increased organelle interaction, with 5X greater contact area with SR and are 4X as likely to interact with TT when compared to control. We show that mitophagy in AF cardiomyocytes involves 2.5X larger mitophagosomes that carry increased organelle contents. In conclusion, when oxidative stress overcomes compensatory mechanisms, mitophagy in AF faces a challenge of degrading bulky complex mitochondria, which may result in increased SR and TT contacts, perhaps allowing for mitochondrial Ca2+ maintenance and antioxidant production.

Spontaneous atrial fibrillation occurrence following transesophageal atrial burst pacing in rats

Abstract Purpose Animal models play a crucial role in elucidating the complex pathophysiology of atrial fibrillation (AF) and in identifying and testing novel antiarrhythmic strategies. We aimed to develop and characterize a new experimental rat model of AF, induced by long-term transesophageal atrial electrical stimulation, and to investigate whether the "AF begets AF" concept applies to small rodent hearts. Method Twelve adult male Wistar rats were randomized into two groups: Sham (n = 5) and AF (n = 7). All rats were implanted at the beginning of the study with ECG radiotelemetry devices and underwent continuous ECG monitoring throughout the study. To induce AF, the rats in the AF group underwent atrial pacing for 10 consecutive days (15 successive cycles of stimulation/day, 20 s each, with a free interval of 5 min between cycles; 4,000 stimuli/min, stimulus duration 6 ms, voltage 3 V above the diastolic threshold). A similar procedure was applied to the Sham group, but without performing the actual electrical stimulation. The number and duration of spontaneous AF episodes were evaluated based on continuous ECG monitoring prior to, during, and for one week after transesophageal atrial pacing. The inducibility of AF and the occurrence and duration of spontaneous AF episodes were evaluated and compared between the two groups. Results Atrial fibrillation was induced in 85.7% of the rats subjected to atrial electrical pacing (Figure, A), with an average global inducibility of 44.0% ± 16.9% and a mean AF duration of 123.2 ± 96.3 s. No induced or spontaneous AF episodes were recorded in any of the Sham animals. In the AF group, none of the animals presented spontaneous AF before transesophageal atrial pacing. However, in the AF rats, after atrial electrical stimulation initiation, the number of atrial premature beats significantly increased (21.3 ± 6.8 / 24-h post-stimulation vs. 7.3 ± 6.5 / 24-h at baseline; p< 0.01; Figure, B), and all rats presented spontaneous AF episodes (Figure, C). At the end of the study, AF rats presented a total number of 23.9 ± 2.3 AF episodes / 24-h (Figure, D), with a mean AF burden of 337.7 ± 25.06 s / 24-h. Conclusions Our data demonstrate that, despite the small size of the atria, the "AF begets AF" concept is applicable in small rodents. The study presents a novel rat model of AF, in which spontaneous AF occurs following long-term atrial electrical stimulation. Given that the model is relatively rapid and easy to implement, it provides a setup for further investigation of AF pathophysiology, and for identifying and testing novel therapeutic strategies for AF.Figure 1

Therapeutic advances in atrial fibrillation based on animal models.

Atrial fibrillation (AF) is the most prevalent sustained cardiac arrhythmia among humans, with its incidence increasing significantly with age. Despite the high frequency of AF in clinical practice, its etiology and management remain elusive. To develop effective treatment strategies, it is imperative to comprehend the underlying mechanisms of AF; therefore, the establishment of animal models of AF is vital to explore its pathogenesis. While spontaneous AF is rare in most animal species, several large animal models, particularly those of pigs, dogs, and horses, have proven as invaluable in recent years in advancing our knowledge of AF pathogenesis and developing novel therapeutic options. This review aims to provide a comprehensive discussion of various animal models of AF, with an emphasis on the unique features of each model and its utility in AF research and treatment. The data summarized in this review provide valuable insights into the mechanisms of AF and can be used to evaluate the efficacy and safety of novel therapeutic interventions.

Predictors of nonpulmonary vein triggers for atrial fibrillation: A clinical risk score

BackgroundTargeting non-pulmonary vein triggers (NPVTs) after pulmonary vein isolation may reduce atrial fibrillation (AF) recurrence. Isoproterenol infusion and cardioversion of spontaneous or induced AF can provoke NPVTs but typically require vasopressor support and increased procedural time. ObjectiveThe purpose of this study was to identify risk factors for the presence of NPVTs and create a risk score to identify higher-risk subgroups. MethodsUsing the AF ablation registry at the Hospital of the University of Pennsylvania, we included consecutive patients who underwent AF ablation between January 2021 and December 2022. We excluded patients who did not receive NPVT provocation testing after failing to demonstrate spontaneous NPVTs. NPVTs were defined as non-pulmonary vein ectopic beats triggering AF or focal atrial tachycardia. We used risk factors associated with NPVTs with P <.1 in multivariable logistic regression model to create a risk score in a randomly split derivation set (80%) and tested its predictive accuracy in the validation set (20%). ResultsIn 1530 AF ablations included, NPVTs were observed in 235 (15.4%). In the derivation set, female sex (odds ratio [OR] 1.40; 95% confidence interval [CI] 0.96–2.03; P = .080), sinus node dysfunction (OR 1.67; 95% CI 0.98–2.87; P = .060), previous AF ablation (OR 2.50; 95% CI 1.70–3.65; P <.001), and left atrial scar (OR 2.90; 95% CI 1.94–4.36; P <.001) were risk factors associated with NPVTs. The risk score created from these risk factors (PRE2SSS2 score; [PRE]vious ablation: 2 points, female [S]ex: 1 point, [S]inus node dysfunction: 1 point, left atrial [S]car: 2 points) had good predictive accuracy in the validation cohort (area under the receiver operating characteristic curve 0.728; 95% CI 0.648–0.807). ConclusionA risk score incorporating predictors for NPVTs may allow provocation of triggers to be performed in patients with greatest expected yield.

Cardiac-specific overexpression of CREM-IbΔC-X via CRISPR/Cas9 in mice presents a new model of atrial cardiomyopathy with spontaneous atrial fibrillation

Atrial cardiomyopathy (ACM) forms the substrate for atrial fibrillation (AF) and underlies the potential for atrial thrombus formation and subsequent stroke. However, generating stable animal models that accurately replicate the entire progression of atrial lesions, particularly the onset of AF, presents significant challenges. In the present study, we found that the isoform of CRE-binding protein modulator (CREM-IbΔC-X), which is involved in the regulation of cardiac development and atrial rhythm, was highly expressed in atrial biopsies from patients with AF. Building upon this finding, we employed CRISPR/Cas9 technology to create a mouse model with cardiac-specific overexpression of CREM-IbΔC-X (referred to as CS-CREM mice). This animal model effectively illustrated the development of ACM through electrophysiological and structural remodelings over time. Proteomics and Chip-qPCR analysis of atrial samples revealed significant upregulation of cell-matrix adhesion and extracellular matrix structural components, alongside significant downregulation of genes related to atrial functions in the CS-CREM mice. Furthermore, the corresponding responses to anti-arrhythmia drugs, i.e., amiodarone and propafenone, suggested that CS-CREM mice could serve as an ideal in vivo model for drug testing. Our study introduced a novel ACM model with spontaneous AF by cardiac-specifically overexpressing CREM-IbΔC-X in mice, providing valuable insights into the mechanisms and therapeutic targets of ACM.

A Genomic Link From Heart Failure to Atrial Fibrillation Risk: FOG2 Modulates a TBX5/GATA4-Dependent Atrial Gene Regulatory Network.

The relationship between heart failure (HF) and atrial fibrillation (AF) is clear, with up to half of patients with HF progressing to AF. The pathophysiological basis of AF in the context of HF is presumed to result from atrial remodeling. Upregulation of the transcription factor FOG2 (friend of GATA2; encoded by ZFPM2) is observed in human ventricles during HF and causes HF in mice. FOG2 expression was assessed in human atria. The effect of adult-specific FOG2 overexpression in the mouse heart was evaluated by whole animal electrophysiology, in vivo organ electrophysiology, cellular electrophysiology, calcium flux, mouse genetic interactions, gene expression, and genomic function, including a novel approach for defining functional transcription factor interactions based on overlapping effects on enhancer noncoding transcription. FOG2 is significantly upregulated in the human atria during HF. Adult cardiomyocyte-specific FOG2 overexpression in mice caused primary spontaneous AF before the development of HF or atrial remodeling. FOG2 overexpression generated arrhythmia substrate and trigger in cardiomyocytes, including calcium cycling defects. We found that FOG2 repressed atrial gene expression promoted by TBX5. FOG2 bound a subset of GATA4 and TBX5 co-bound genomic locations, defining a shared atrial gene regulatory network. FOG2 repressed TBX5-dependent transcription from a subset of co-bound enhancers, including a conserved enhancer at the Atp2a2 locus. Atrial rhythm abnormalities in mice caused by Tbx5 haploinsufficiency were rescued by Zfpm2 haploinsufficiency. Transcriptional changes in the atria observed in human HF directly antagonize the atrial rhythm gene regulatory network, providing a genomic link between HF and AF risk independent of atrial remodeling.

Posterior Wall Isolation Improves Outcomes for Persistent AF With Rapid Posterior Wall Activity: CAPLA Substudy

BackgroundPulmonary vein isolation (PVI) is less effective in persistent atrial fibrillation (PerAF) than in paroxysmal atrial fibrillation (AF). However, the CAPLA (Effect of Catheter Ablation Using Pulmonary Vein Isolation With vs Without Posterior Left Atrial Wall Isolation on Atrial Arrhythmia Recurrence in Patients With Persistent Atrial Fibrillation: The CAPLA randomized clinical trial) of PVI vs posterior wall isolation (PWI) did not support empiric PWI in PerAF. We examined pulmonary vein (PV) and posterior wall (PW) electrical characteristics to determine if select patients may benefit from additional PWI. ObjectivesThis study sought to determine the impact of PV and PW electrical characteristics on AF ablation outcomes in the CAPLA randomized study. MethodsParticipants in spontaneous AF at the time of ablation were included from the CAPLA study. The mean, shortest, and longest PV, PW, and left atrial (LA) appendage cycle length measurements were annotated preablation using a multipolar catheter for 100 consecutive cycles. Next, cardioversion was performed with a high-density LA voltage map completed. Cox proportional hazards regression was utilized to determine clinical and electroanatomic predictors of AF recurrence overall and according to ablation strategy. Follow-up included twice daily single-lead electrocardiograms or continuous monitoring for 12 months. ResultsA total of 151 patients (27% female, age 65 ± 9 years, 18% long-standing PerAF, LA volume index 52 ± 16 mL/m2, median AF duration 5 months [IQR: 2-10 months]) were in AF on the day of procedure and were randomized to PVI alone (50%) or PVI+PWI (50%) according to the CAPLA randomized clinical trial protocol. Baseline clinical, echocardiographic, and electroanatomic parameters were comparable between groups (all P > 0.05) including PV and PW characteristics. After 12 months, freedom from AF off antiarrhythmic drug therapy was 51.7% in PVI and 49.7% in PVI+PWI (log-rank P = 0.564). Rapid PW activity was defined as less than the median of the shortest PW cycle length (140 ms) and rapid PV activity was defined as less than the median of the shortest PV cycle length (126 ms). In those with rapid PW activity, the addition of PWI was associated with greater arrhythmia-free survival (56.4%) vs PVI alone (38.6%) (HR: 0.78; 95% CI: 0.67-0.94; log-rank P = 0.030). Moreover, in those undergoing PVI only, the risk of AF recurrence was higher in those with rapid PW activity (55.3% vs 46.5% in slower PW activity; HR: 1.50, 95%CI 1.11-2.26; log-rank P = 0.036). Rapid PV activity and PV cycle length (individual PVs or average of all 4 PVs) were not associated with outcome (all P > 0.05) regardless of ablation strategy. There was no correlation between PW cycle length and posterior low voltage (r = −0.06, P = 0.496). The addition of PWI did not improve arrhythmia-free survival in subgroups with LA enlargement (LA volume index >34 mL/m2) (HR: 0.69; 95% CI: 0.39-1.25; P = 0.301), posterior low-voltage zone (HR: 1.06; 95% CI: 0.68-1.66; P = 0.807), or long-standing PerAF (HR: 1.10; 95% CI: 0.71-1.72; P = 0.669). ConclusionsRapid PW activity is associated with an increased risk of AF recurrence post–catheter ablation. The addition of PWI in this subgroup was associated with a significant improvement in freedom from AF compared with PVI alone. The presence of rapid PW activity may identify patients with PerAF likely to benefit from PWI.

Percolation theory as a conceptual framework to explain spontaneous atrial fibrillation termination: a pilot study.

Atrial fibrillation (AF) strikingly possesses the ability to abruptly transition into more organized electrical activity and spontaneously terminate, even after persisting for long periods. Despite being central to the clinical behavior and treatment of AF, these phenomena remain incompletely understood. In this paper, we hypothesized that the spontaneous termination of AF may represent a type of percolation phase transition, which is more likely to occur when a domain spanning cluster of refractory sites in the atrium are connected (called a 'percolation cluster'). This was assessed in n=50 computational simulations of AF developed using the Aliev-Panfilov (APV) 2-dimensional cell model. In self-terminating simulations of AF, it was found that the average refractory cluster size, χ(p) (median: 647.7), and the largest refractory cluster size, M1 (median: 1702.3), abruptly increased just prior to AF spontaneously terminating, indicating the onset of the formation of a domain spanning percolation cluster. In contrast, simulations of sustained AF did not demonstrate an increase in χ(p) (median: 463.0) and M1 (median: 1448.2). Self-terminating AF simulations also demonstrated hallmark properties characteristic of a percolation phase transition, such as an abrupt increase in χ(p) at the critical occupation probability pc. The cluster size distribution was also shown to obey a power law for various occupation probabilities p, also indicative of a percolation phase transition. Collectively, these properties suggests that the spontaneous termination of AF could be a form of percolation phase transition, which could provide new insights for AF treatment.

Artificial intelligence-enabled spatio-temporal dispersion mapping for persistent AF: Similarities and differences between pacing-induced or spontaneous AF

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Volta Medical Saint Joseph Hospital Marseille, France Background Spatiotemporal dispersion is an electrical footprint of atrial fibrillation (AF) drivers that has been successfully implemented to target extra-pulmonary veins (PVs) regions during persistent AF ablation. Purpose The aim of the study is to characterize spatiotemporal dispersion extent and location and to compare dispersion atrial localization and extent between pacing-induced AF and spontaneous AF. Methods Spatiotemporal dispersion maps were built with an artificial intelligence software (VX1, Volta Medical) and analyzed in 71 consecutives persistent (66%) and long-standing persistent (34%) AF patients admitted for a first ablation procedure. Fifty-two patients were in spontaneous AF (73%) at the outset of the procedure while AF was induced in 19 patients (27%) by burst pacing ± isoproterenol infusion. A semi-quantitative visual quantification of dispersion extent was conducted by implementing an atrial segmentation into 22 regions and a region-centered score from 0 (no dispersion) to 3 (high dispersion). Also, an automated quantification was performed as follows: (i) a gradient filter designed to extract atrial shapes from background was applied, (ii) dispersion areas were segmented using a color thresholding method, and (iii) a structuring element was used to connect segmented dispersion areas. Results The regional characterization of dispersion shows that dispersion distribution follows a similar pattern and is present in similar atrial regions, regardless of whether AF is spontaneous or induced (Figure 1). Global dispersion extent, however, is significantly higher in the left atrium of patients in spontaneous AF compared to patients with induced AF (15.43% ± 9.04 versus 9.86% ± 6.41, P=0.0025, Figure 2). Accordingly, the regional dispersion score tends to be lower when AF is induced. Dispersion hotspots are: LSPV anterior antrum -ridge (R1), RSPV anterior antrum (R5), anterior wall (R9), roof (R10), posterior wall (R11), and low left atrial septum (R15) in the left atrium. In the right atrium: low right atrial septum (R15) and posterior right atrium (R20) (Figure 1). Conclusions Artificial intelligence-enabled dispersion persistent AF mapping indicates that dispersion is reduced in induced vs. spontaneous-AF but that its distribution follows a similar pattern.

PO-02-207 PREDICTION OF CORRELATION LENGTH FROM THE SURFACE ELECTROCARDIOGRAM IN ATRIAL FIBRILLATION

Correlation length in atrial fibrillation (AF) can be calculated from intracardiac electrograms (EGMs) and conceptualized as the distance over which electrical signals remain synchronized. Emerging data shows correlation length predicts AF termination and is associated with outcomes after AF ablation. The capacity to estimate correlation length non-invasively would be a major advance for its applicability to clinical care. Association with non-invasive electrocardiographic (ECG) measurements is currently unknown. We sought to determine if correlation length, measured from EGMs at the time of AF ablation, can be predicted non-invasively from the surface ECG in AF. We conducted a prospective, multicentre, observational study, with a published and registered protocol (ACTRN12619001172190p) of participants undergoing clinically indicated AF ablation. Data collected included (1) detailed clinical phenotyping of participants pre-procedurally, (2) intraprocedural mapping of spontaneous or induced AF with a HD Grid catheter, recording 1-minute EGM segments in 6 right atrial and 10 left atrial anatomic locations, sampled at 2000Hz, with simultaneously acquired ECG. Correlation length is a measure of the distance over which EGMs remain synchronized, measured by calculating the cross-correlation of nearby EGMs as a function of distance. Predictors of correlation length were modelled with a multilevel mixed-effects gamma generalised linear regression. The study recruited n=42 participants. Correlation length could be predicted by surface ECG metrics, including ECG dominant frequency (DF) (coefficient -0.0509, 95% CI -0.0881- -0.0136, p=0.007), Hursts exponent (coefficient -11.66, 95% CI -20.90 - -2.42, p=0.013), Kurtosis (coefficient -0.0034, 95%CI -0.0059 - -0.0009, p=0.007), and Shannon entropy (coefficient -0.1701, 95% CI -0.3319- -0.0083, p=0.039) (see figure 1). The directionality of these findings is biologically plausible. ECG features which did not predict the invasive correlation length included ECG standard deviation (p=0.466), ECG skewness (p=0.332), ECG period (p=0.314) and ECG mean (p=0.261). Intracardiac correlation length is associated with surface ECG features. Importantly, ECG features are measurable non-invasively, meaning this approach could be expanded to patients not undergoing AF ablation, or prior to AF ablation. Given correlation length associates with clinical parameters and outcomes, this metric may help to personalize AF therapy in the future.

Overview of programmed electrical stimulation to assess atrial fibrillation susceptibility in mice.

Atrial fibrillation (AF) is the most common human arrhythmia and is associated with increased risk of stroke, dementia, heart failure, and death. Among several animal models that have been used to investigate the molecular determinants of AF, mouse models have become the most prevalent due to low cost, ease of genetic manipulation, and similarity to human disease. Programmed electrical stimulation (PES) using intracardiac or transesophageal atrial pacing is used to induce AF as most mouse models do not develop spontaneous AF. However, there is a lack of standardized methodology resulting in numerous PES protocols in the literature that differ with respect to multiple parameters, including pacing protocol and duration, stimulus amplitude, pulse width, and even the definition of AF. Given this complexity, the selection of the appropriate atrial pacing protocol for a specific model has been arbitrary. Herein we review the development of intracardiac and transesophageal PES, including commonly used protocols, selected experimental models, and advantages and disadvantages of both techniques. We also emphasize detection of artifactual AF induction due to unintended parasympathetic stimulation, which should be excluded from results. We recommend that the optimal pacing protocol to elicit an AF phenotype should be individualized to the specific model of genetic or acquired risk factors, with an analysis using several definitions of AF as an endpoint.

Renal denervation alleviates chronic obstructive sleep apnea-induced atrial fibrillation via inhibition of atrial fibrosis and sympathetic hyperactivity.

Previous studies have reported that renal denervation (RDN) prevents the occurrence of atrial fibrillation (AF) related to obstructive sleep apnea (OSA). However, the effect of RDN on chronic OSA (COSA)-induced AF is still unclear. Healthy beagle dogs were randomized into the OSA group (sham RDN + OSA), OSA-RDN group (RDN + OSA), and CON group (sham RDN + sham OSA). The COSA model was built via repeated apnea and ventilation rounds for 4h each day lasting 12weeks, and RDN was employed after 8weeks of modeling. All dogs were implanted Reveal LINQ™ to detect spontaneous AF and AF burden. Circulating levels of norepinephrine, angiotensin II, and interleukin-6 were determined at baseline and end of the study. In addition, measurements of the left stellate ganglion, AF inducibility, and effective refractory period were conducted. The bilateral renal artery and cortex, left stellate ganglion, and left atrial tissues were collected for molecular analysis. Of 18 beagles, 6 were randomized to each ofthe groups described above.RDN remarkably attenuated ERP prolongation and AF episodes and duration. RDN markedly suppressed the LSG hyperactivity and atrial sympathetic innervation, decreased the serum concentrations of Ang II and IL-6, further inhibited fibroblast-to-myofibroblast transformation via the TGF-β1/Smad2/3/α-SMA pathway, and reduced the expression of MMP-9, thus decreasing OSA-induced AF. RDN may reduce AF by inhibiting sympathetic hyperactivity and AF in a COSA model.

Abstract 12847: Atrial Fibrillation and Atrial Flutter: What Are the Substrate Differences Seen in 3D Panoramic Optical Mapping?

Background: Panoramic optical mapping (POM) is an effective tool for 3D visualization and analysis of action potential duration (APD) heterogeneity, a key driver for initiation and persistence of reentry. POM may elucidate cardiac electrophysiological predisposition to develop chaotic arrhythmia such as atrial fibrillation (AF) or organized arrhythmia such as atrial flutter (AFL). Objective: To determine the APD differences in AF and AFL in a transgenic (TG) murine model. Methods: We performed high resolution POM with 4 simultaneous CMOS cameras on 14 Langendorff perfused TG murine hearts (SCN5A mutation at lidocaine binding site F1759A) with spontaneous AF and AFL. We generated serial 2D activation and APD maps in normal sinus rhythm (NSR), while phase movies were created in AF/AFL. All optical maps were then reconstructed in 3D. We averaged the APD80 and APD dispersion values over each atrial chamber and compared the mean differences: i) within AF and AFL across right atrium (RA) and left atrium (LA); and ii) within RA and LA across AF and AFL. Results: AF (n=7) and AFL (n=7) were present at baseline (8/14) or induced by 10Hz burst pacing (6/14). Mean APD80 in RA/LA was: 57.03 ± 4.66 / 50.93± 3.38ms (p&lt;0.05) in AF vs 77.18 ± 4.30 / 58.25 ± 3.88ms (p&lt; 0.01) in AFL, and mean dispersion: 6.23 ± 1.04/ 7.25± 1.71ms in AF vs 5.49 ± 0.80/ 8.42 ± 1.15ms (p&lt;0.01) in AFL. High mean APD80 in the RA with low intra-atrial dispersion in the RA lead to AFL rotational reentry cores, while lower overall APD80 values with high intra-atrial dispersion in the LA resulted in the presence of AF phase singularity points. Conclusion: Development of AF was associated with high intra-atrial APD dispersion in NSR, whereas AFL was associated with high inter-atrial APD dispersion in NSR (1.4-fold higher mean APD80 in the RA). Certain atrial APD dispersion patterns observed in NSR are associated with development of chaotic vs. organized atrial arrhythmias.

Abstract 14868: Identification of Novel Small Molecule Inhibitors of Calcium/Calmodulin-Dependent Protein Kinase II Delta for Treatment of Beta-Blocker Resistant Arrhythmia

Introduction. CaMKIId is a validated drug target in atrial fibrillation (AF), ventricular arrhythmia, and HF. As a key mediator of Ca 2+ action, it is hyperactivated by dysregulated Ca 2+ homeostasis with pathological consequences, which can be normalized by a variety of genetic and pharmacological manipulations. Objectives. Develop an ATP competitive CaMKIId inhibitor with nanomolar potency for initial efficacy in an AF mouse model and further optimization of selectivity, PK and low CNS penetration. Methods and Results. Molecular modeling using the crystal structure of human CaMKIId with several scaffolds as a starting point, led to development of a consistent structure activity relationship model and design of AS283. The molecule is an ATP competitive inhibitor with an IC 50 of 6nM and Ki of 1nM. Unlike KN-93, a common tool inhibitor of CaMKII in preclinical studies, AS283 inhibits the autophosphorylated, Ca 2+ -independent form of the kinase. AS283 shows preclinical proof of concept in both ventricular arrhythmia and AF models, with the latter described here. Mice with a gain of function mutation in the RyR2 receptor (R 178Q/+ ) do not undergo spontaneous AF but have an increased vulnerability to AF in response to atrial pacing. Intracardial electrophysiology with recording of atrial and ventricular electrograms (Na Li and Xander Wehrens, Baylor College) were used to select mutant and wild-type mice that exhibited AF in pacing trials. AF-positive animals were randomly assigned to treatment with placebo or AS283, with KN-93 as control. AS283 completely suppressed incidence of AF in comparison to placebo and exhibited a dose response. Autophosphorylation of CaMKIId at Thr287 was used as a biomarker of target engagement. As anticipated, atrial pacing increases intracellular Ca 2+ leading to activation of CaMKII and increased P-Thr287. AS283 blocked the increase in P-Thr287. Conclusion. AS283 is a novel, potent CaMKIId inhibitor with robust preclinical efficacy, supporting its further optimization for PK and low CNS penetration. Consistent with studies using genetic and pharmacological tools, an optimized, small molecule inhibitor of CaMKII has a promise in treating arrhythmias that are sub-optimally managed with or contraindicated to beta blockers.

Mouse models of spontaneous atrial fibrillation.

Atrial fibrillation (AF) is the most common arrhythmia in adults, with a prevalence increasing with age. Current clinical management of AF is focused on tertiary prevention (i.e., treating the symptoms and sequelae) rather than addressing the underlying molecular pathophysiology. Robust animal models of AF, particularly those that do not require supraphysiologic stimuli to induce AF (i.e., showing spontaneous AF), enable studies that can uncover the underlying mechanisms of AF. Several mouse models of AF have been described to exhibit spontaneous AF, but pathophysiologic drivers of AF differ among models. Here, we describe relevant AF mechanisms and provide an overview of large and small animal models of AF. We then provide an in-depth review of the spontaneous mouse models of AF, highlighting the relevant AF mechanisms for each model.

PO-685-04 DIFFERING DISTRIBUTION OF PREMATURE ATRIAL COMPLEXES THAT DO AND DO NOT PRECIPITATE SPONTANEOUS ATRIAL FIBRILLATION BY NON-INVASIVE IMAGING

It remains unclear which premature atrial complexes (PACs) will or will not induce spontaneous atrial fibrillation (AF), and their implication in the success of ablation therapy. Identify the anatomical location and relative timing of PACs at spontaneous AF onset and their relation with the acute success of pulmonary vein isolation (PVI). ECG imaging (ECGI) was reconstructed from 62 electrodes using the zero-order Tikhonov method in 10 patients referred for AF ablation (8 male, 69±6 years) and N=17 Premature Atrial Complexes (PAC) that did and did not initiate AF were identified (Fig. A-B). Focal and re-entrant sites were identified by phase analysis on ECGI maps (fig C). PACs inducing AF were more premature compared to non-inducing AF PACs: 45±15% vs. 70±18% of previous atrial cycle (p=0.03). PACs that induced AF were more likely to arise from the PVs than non-AF inducing PACs (p=0.04), and PVs harboured more focal (6±9 vs 2±9, fig. D) and re-entrant sources (5±6 vs 1±1, fig. E). Moreover, focal PACs were more likely to arise from the PVs in patients in whom PVI did versus did not acutely terminate AF: 19±1% vs 8±12% (fig. F). The anatomical distribution of PACs supports the role of PV in spontaneous AF onset, and may predicts patients in whom PVI is acutely successful. Non-invasive imaging of PACs may help to stratify AF patients as well as to plan ablation procedures.

Atrial AMP-activated protein kinase is critical for prevention of dysregulation of electrical excitability and atrial fibrillation.

Metabolic stress is an important cause of pathological atrial remodeling and atrial fibrillation. AMPK is a ubiquitous master metabolic regulator, yet its biological function in the atria is poorly understood in both health and disease. We investigated the impact of atrium-selective cardiac AMPK deletion on electrophysiological and structural remodeling in mice. Loss of atrial AMPK expression caused atrial changes in electrophysiological properties and atrial ectopic activity prior to the onset of spontaneous atrial fibrillation. Concomitant transcriptional downregulation of connexins and atrial ion channel subunits manifested with delayed left atrial activation and repolarization. The early molecular and electrophysiological abnormalities preceded left atrial structural remodeling and interstitial fibrosis. AMPK inactivation induced downregulation of transcription factors (Mef2c and Pitx2c) linked to connexin and ion channel transcriptional reprogramming. Thus, AMPK plays an essential homeostatic role in atria, protecting against adverse remodeling potentially by regulating key transcription factors that control the expression of atrial ion channels and gap junction proteins.

Transesophageal Atrial Burst Pacing for Atrial Fibrillation Induction in Rats.

Animal studies have brought important insights into our understanding regarding atrial fibrillation (AF) pathophysiology and therapeutic management. Reentry, one of the main mechanisms involved in AF pathogenesis, requires a certain mass of myocardial tissue in order to occur. Due to the small size of the atria, rodents have long been considered 'resistant' to AF. Although spontaneous AF has been shown to occur in rats, long-term follow-up (up to 50 weeks) is required for the arrhythmia to occur in those models. The present work describes an experimental protocol of transesophageal atrial burst pacing for rapid and efficient induction of AF in rats. The protocol can be successfully used in rats with healthy or remodeled hearts, in the presence of a wide variety of risk factors, allowing the study of AF pathophysiology, identification of novel therapeutic targets, and evaluation of novel prophylactic and/or therapeutic strategies.

Attenuating persistent sodium current-induced atrial myopathy and fibrillation by preventing mitochondrial oxidative stress.

Mechanistically driven therapies for atrial fibrillation (AF), the most common cardiac arrhythmia, are urgently needed, the development of which requires improved understanding of the cellular signaling pathways that facilitate the structural and electrophysiological remodeling that occurs in the atria. Similar to humans, increased persistent Na+ current leads to the development of an atrial myopathy and spontaneous and long-lasting episodes of AF in mice. How increased persistent Na+ current causes both structural and electrophysiological remodeling in the atria is unknown. We crossbred mice expressing human F1759A-NaV1.5 channels with mice expressing human mitochondrial catalase (mCAT). Increased expression of mCAT attenuated mitochondrial and cellular reactive oxygen species (ROS) and the structural remodeling that was induced by persistent F1759A-Na+ current. Despite the heterogeneously prolonged atrial action potential, which was unaffected by the reduction in ROS, the incidences of spontaneous AF, pacing-induced after-depolarizations, and AF were substantially reduced. Expression of mCAT markedly reduced persistent Na+ current–induced ryanodine receptor oxidation and dysfunction. In summary, increased persistent Na+ current in atrial cardiomyocytes, which is observed in patients with AF, induced atrial enlargement, fibrosis, mitochondrial dysmorphology, early after-depolarizations, and AF, all of which can be attenuated by resolving mitochondrial oxidative stress.

Genetic inhibition of nuclear factor of activated T-cell c2 prevents atrial fibrillation in CREM transgenic mice.

Abnormal intracellular calcium (Ca2+) handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signalling pathway contributes to atrial remodelling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models. Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF (cAF). NFATc1 and NFATc2 mRNA levels were elevated in cAF patients compared with those in normal sinus rhythm (NSR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM:NFATc2-/- mice. Programmed electrical stimulation revealed that CREM:NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with wild-type controls, which was reversed in CREM:NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM:NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on ryanodine receptor type 2 (RyR2) in CREM:RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras and EF-hand domain-containing protein (Rasef) as a direct target of NFATc2-mediated transcription. Our findings reveal activation of the NFAT signalling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model.