Spontaneous Atrial Fibrillation Research Articles

A missense mutation in the RSRSP stretch of Rbm20 causes dilated cardiomyopathy and atrial fibrillation in mice

Dilated cardiomyopathy (DCM) is a fatal heart disease characterized by left ventricular dilatation and cardiac dysfunction. Recent genetic studies on DCM have identified causative mutations in over 60 genes, including RBM20, which encodes a regulator of heart-specific splicing. DCM patients with RBM20 mutations have been reported to present with more severe cardiac phenotypes, including impaired cardiac function, atrial fibrillation (AF), and ventricular arrhythmias leading to sudden cardiac death, compared to those with mutations in the other genes. An RSRSP stretch of RBM20, a hotspot of missense mutations found in patients with idiopathic DCM, functions as a crucial part of its nuclear localization signals. However, the relationship between mutations in the RSRSP stretch and cardiac phenotypes has never been assessed in an animal model. Here, we show that Rbm20 mutant mice harboring a missense mutation S637A in the RSRSP stretch, mimicking that in a DCM patient, demonstrated severe cardiac dysfunction and spontaneous AF and ventricular arrhythmias mimicking the clinical state in patients. In contrast, Rbm20 mutant mice with frame-shifting deletion demonstrated less severe phenotypes, although loss of RBM20-dependent alternative splicing was indistinguishable. RBM20S637A protein cannot be localized to the nuclear speckles, but accumulated in cytoplasmic, perinuclear granule-like structures in cardiomyocytes, which might contribute to the more severe cardiac phenotypes.

Late Sodium Current in Atrial Cardiomyocytes Contributes to the Induced and Spontaneous Atrial Fibrillation in Rabbit Hearts.

Increased late sodium current (INa) induces long QT syndrome 3 with increased risk of atrial fibrillation (AF). The role of atrial late INa in the induction of AF and in the treatment of AF was determined in this study. AF parameters were measured in isolated rabbit hearts exposed to late INa enhancer and inhibitors. Late INa from isolated atrial and ventricular myocytes were measured using whole-cell patch-clamp techniques. We found that induced-AF by programmed S1S2 stimulation and spontaneous episodes of AF were recorded in hearts exposed to either low (0.1-3 nM) or high (3-10 nM) concentrations of ATX-II (n = 10). Prolongations in atrial monophasic action potential duration at 90% completion of repolarization and effective refractory period by ATX-II (0.1-15 nM) were greater in hearts paced at slow than at fast rates (n = 5-10, P < 0.05). Both endogenous and ATX-II-enhanced late INa density were greater in atrial than that in ventricular myocytes (n = 9 and 8, P < 0.05). Eleclazine and ranolazine reduced AF window and AF burden in association with the inhibition of both endogenous and enhanced atrial late INa with half maximal inhibitory concentrations (IC50) of 1.14 and 9.78, and 0.94 and 8.31 μM, respectively. The IC50s for eleclazine and ranolazine to inhibit peak INa were 20.67 and 101.79 μM, respectively, in atrial myocytes. In conclusion, enhanced late INa in atrial myocytes increases the susceptibility for AF. Inhibition of either endogenous or enhanced late INa, with increased atrial potency of drugs is feasible for the treatment of AF.

Activation of AMP-Activated Protein Kinases Prevents Atrial Fibrillation.

Atrial fibrillation (AF) is common, yet there is no preventive therapy for AF. We tested the efficacy of AMP-activated protein kinase (AMPK) activators, metformin, and aspirin, in primary prevention of AF in cardiac-specific liver kinase B1 (LKB1) knockout (KO) mouse model of AF. Incidence of spontaneous AF was significantly reduced in treated KO mice with metformin (10mg/kg/day) (8.3% in male and 10.3% in female) and aspirin (20mg/kg/day) (29.4% in male and 21.4% in female) compared with untreated littermates (81% in male and 67% in female) at 8weeks (p < 0.05). Prevention of AF was associated with activation of AMPK in treated mice and thereby improvement of mitochondrial function, gap junction proteins (connexin 40/43), and intra- and inter-cellular ultrastructure in atrial myocardium. Fibrosis was significantly less in treated mice atria. Pharmacological activation of AMPK is an effective upstream therapy for the primary prevention of AF in susceptible heart. Graphical abstract.

Abstract 550: FOG2 Modulates a TBX5-Dependent Gene Regulatory Network for Cardiac Rhythm Homeostasis

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Recent work has implicated numerous transcription factors in AF risk, including ZFPM2 (FOG2), GATA4, and TBX5. FOG2 is overexpressed in human heart failure, a major AF risk factor. We found that genetic inducible physiologic overexpression of FOG2 in the adult mouse heart caused spontaneous atrial fibrillation. Single cell electrophysiology revealed action potential prolongation and inappropriate depolarizations in FOG2-OE atrial cardiomyocytes. RNA-seq of the FOG2-OE left atrium prior to AF onset demonstrated that FOG2 suppresses a network of calcium cycling genes, providing a possible mechanism for disrupted cardiomyocyte electrophysiology. FOG2 ChIP-seq demonstrated genomic localization only at locations co-occupied by GATA4. Unexpectedly, we observed little overlap between genes activated by GATA4 and repressed by FOG2. Instead, we observed highly significant overlap between genes repressed by FOG2 and activated by TBX5. To identify the FOG2-dependent atrial gene regulatory network, we performed differential deep sequencing of ncRNAs. Comparison of atrial FOG2-dependent non-coding transcripts with previously performed TBX5-dependent ncRNA profiling indicated that TBX5 activated and FOG2 repressed a shared atrial rhythm gene regulatory network. Integration of FOG2, GATA4, and TBX5 ChIP-seq revealed that FOG2 only affected ncRNA transcription, indicative of enhancer activity changes, at locations co-occupied by TBX5. TBX5-dependent activation of specific regulatory elements for calcium handling genes was abolished by FOG2 in vitro . The genomic TBX5/FOG2 interaction predicted a genetic interaction in-vivo , in which cardiac conduction and arrhythmia abnormalities caused by Tbx5 haploinsufficiency were rescued by FOG2 haploinsufficiency. Non-coding RNA profiling thereby predicted a novel functional TF interaction between FOG2 and TBX5. This work reveals a specific genomic model of atrial rhythm control in which FOG2 is recruited to GATA4 and TBX5 bound locations to modulate a TBX5-dependent atrial gene regulatory network for calcium handling and cardiac rhythm homeostasis.

Unsupervised Classification of Atrial Fibrillation Triggers Using Heart Rate Variability Features Extracted from Implantable Cardiac Monitor Data.

Catheter ablation is a common treatment of atrial fibrillation (AF), but its success rate is around 60%. It is believed that the success rate can be improved if the procedure were to be guided by the specific AF triggers found in the "Flashback", i.e. the trend of around 500 ventricular beats preceding the AF onset stored in an implantable cardiac monitor (ICM). The need to automatically classify these different triggers: atrial tachycardia (AT), atrial flutter, premature atrial contractions (PAC) or Spontaneous AF has motivated the design in this paper of an unsupervised classification method evaluating statistical and geometrical Heart Rate Variability (HRV) features extracted from the Flashback. From a cohort of 132 patients (57± 12 years, male 67%), 528 Flashbacks were extracted and classified into 5 different clusters after the Principal Component Analysis (PCA) was computed on the HRV features. 2 principal components explained more than 95% of the variance and were a combination of the mean R-R interval, Square root of the mean squared differences of successive R-R intervals (RMSSD), Standard deviation of the R-R intervals (SDNN) and Poincare descriptors, SD1 and SD2. RMSSD and SD1 were significantly different among all clusters (p-value < 0.05, with Holm's correction) showing that distinct patterns can be found using this method.Clinical Relevance-Preliminary step towards ablation strategy guidance using the AF trigger patterns to improve catheter ablation success rates.

Detection of atrial fibrillation with implantable loop recorders in horses.

BackgroundCardiac arrhythmias in horses are diagnosed by auscultation or electrocardiogram (ECG), which results in a low sensitivity for detecting arrhythmias that occur sporadically. Implantable loop recorders (ILRs) are small ECG devices placed subcutaneously, to automatically detect arrhythmias in human patients.ObjectivesTo test ILRs ability to detect atrial fibrillation (AF) in horses. Furthermore, we hypothesised that anatomical location of the implant site might influence signal quality. Signal quality was evaluated both during exercise and over time.Study designExperimental study.MethodsIn five Standardbred mares, eleven ILRs were implanted subcutaneously in up to three different positions (Front: pectoral region, Left‐6: sixth left intercostal space and Ventral: xiphoid region) and AF induced. The R‐ and T‐wave amplitudes were measured in all positions over time during AF. AF burden automatically registered by the ILRs over a 2‐month period was compared with selected Holter ECG recordings.ResultsAll three positions had stable R‐ and T‐wave amplitudes during the study period and were of sufficient quality to allow AF detection at rest. The position Left‐6 showed significantly higher R‐ and T‐wave amplitudes compared with the other positions. During submaximal exercise only the Left‐6 position was able to record ECG signals of diagnostic quality. No position yielded diagnostic signals at maximum exercise due to artefacts.Main limitationsFew horses and ILRs included and no spontaneous AF episodes were studied.ConclusionsThis preliminary study indicates that ILRs can be used for AF detection in horses, but the anatomical location is important for optimal ECG quality. Despite insufficient quality during exercise, ILRs were suitable for AF detection at rest. Therefore, the ILR may be a valuable diagnostic tool for detecting paroxysmal AF in horses.

Loss of SORBS2 in Cardiomyocytes Leads to Dilated Left Ventricle Cardiomyopathy in Mice

Through rigorous bioinformatic and wet‐lab investigations, we identified SORBS2 as a cardiac‐enriched gene that is consistently upregulated at mRNA and protein levels across diverse mouse models of heart failure (HF; genetic, ischemic, pressure‐overload) and in failing human hearts (ischemic, idiopathic). Significant reduction in SORBS2 protein levels are also found in aged heart. SORBS2 is among a family of adaptor proteins (SORBS1‐3) involved in cell adhesion and cytoskeletal organization; SORBS proteins localize to myocyte z‐discs and aid in aligning sarcomere and mitochondrial networks (Loveless et al. 2017 MBC). Global SORBS2 knockout mice display early death (100% at 4–6 months), potentially due to cardiac arrhythmia and right ventricle (RV) cardiomyopathy (Ding et al. 2019 BioRxiv). Beyond this, the role of SORBS2 in cardiomyocyte biology remains largely untested. We created a cardiomyocyte‐specific knockout of SORBS2 (SORBS2‐cKO) via MYH6‐CRE transgenic mice bred to floxed‐SORBS2 mice. To delete SORBS2 postnatally and separate the role of SORBS2 in adult cardiac function from development, floxed‐SORBS2 mice were also injected at 3 weeks of age with AAV2/9‐CRE virus. Heart function was measured from 3 months thru one year of age. Contrary to the work of Ding, et al., we report here that neither constitutive nor inducible cardiomyocyte‐restricted SORBS2 knockout leads to severe arrhythmias, RV dysfunction, or early death in mice. Instead, SORBS2‐cKO mice develop aging‐related left ventricle (LV) cardiomyopathy starting with LV wall motion abnormalities at ~ 8 months‐old, progressing to ~50% reduced LVEF and 30% increased HW/BW by 10 months‐old, and dilating with heart failure by 12 months (~400% increase LVEDV), with no detectable change to RV structure/function. Surface EKG recordings in SORBS2‐cKO mice show obvious conduction deficiencies including bifid P‐wave, prolonged QRS, and bundle branch block, starting at 3 months‐age and persisting through life. Notably, no mice showed irregular R‐R intervals, spontaneous atrial fibrillation or ventricular tachycardia. While exploring the mechanism of SORBS2 loss leading to conduction defects, we did not find reduced gap junction protein GJA1 levels in SORBS2‐cKO mouse hearts. This is interesting given that Ding, et al., reported a 90% decrease in GJA1 protein levels in global SORBS2‐KO mouse hearts. Consistent with prior literature, our bioinformatic analyses show a strong association for SORBS2 with cytoskeletal proteins and also point to new potential connections to cardiac signaling receptors/channels including SCN5A. Electrophysiology studies were conducted in isolated neonatal rat cardiomyocytes with siRNA‐mediated knockdown of SORBS2; however, no changes in SCN5A current properties were found. Altogether, these results highlight the importance of SORBS2 to cardiomyocyte biology likely through its primary role as a cytoskeletal adapter. Necessary, ongoing work will continue to precisely define how loss of SORBS2 in cardiomyocytes leads to dilated cardiomyopathy, assessing sarcomere organization, excitation‐contraction coupling, and cardiomyocyte force mechanics.Support or Funding InformationThis work was funded by 19POST34380640 to JMM, HL137272 to DSM, HL147545 to BL, and HL144717 and HL148796 to RLB.

Spontaneous Atrial Fibrillation in Transgenic Goats With TGF (Transforming Growth Factor)-β1 Induced Atrial Myopathy With Endurance Exercise.

There is increasing evidence that endurance exercise is associated with increased risk of atrial fibrillation (AF). However, it is unknown if the relationship between endurance exercise and AF is dependent on an atrial myopathy. Six cardiac-specific TGF (transforming growth factor)-β1 transgenic and 6 wild-type (WT) goats were utilized for these studies. Pacemakers were implanted in all animals for continuous arrhythmia monitoring and AF inducibility. AF inducibility was evaluated using 5 separate 10 s bursts of atrial pacing (160-200 ms). Three months of progressive endurance exercise (up to 90 minutes at 4.5 mph) was performed. Quantitative assessment of circulating microRNAs and inflammatory biomarkers was performed. Sustained AF (≥30 s) was induced with 10 s of atrial pacing in 4 out of 6 transgenic goats compared with 0 out of 6 WT controls at baseline (P<0.05). No spontaneous AF was observed at baseline. Interestingly, between 2 and 3 months of exercise 3 out of 6 transgenic animals developed self-terminating spontaneous AF compared with 0 out of 6 WT animals (P<0.05). There was an increase in AF inducibility in both transgenic and WT animals during the first 2 months of exercise with partial normalization at 3 months (transgenic 67%; 100%; 83% versus WT 0%; 67%; 17%). These changes in AF susceptibility were associated with a decrease in circulating microRNA-21 and microRNA-29 during the first 2 months of exercise with partial normalization at 3 months in both transgenic and WT animals. Finally, MMP9 (matrix metallopeptidase 9) was increased during the second and third months of exercise training. This study demonstrates a novel transgenic goat model of cardiac fibrosis (TGF-β1 overexpression) to demonstrate that endurance exercise in the setting of an underlying atrial myopathy increases the incidence of spontaneous AF. Furthermore, endurance exercise seems to increase inducible AF secondary to altered expression of key profibrotic biomarkers that is independent of the presence of an atrial myopathy.

Atriale en ventriculaire elektrische en contractiele “remodelling” en “reverse remodelling” van chronisch “pacing”-geïnduceerde atriale fibrillatie bij paarden: eerste resultaten

Na een langdurige periode van atriumfibrillatie neemt de elektrische en contractiele herstelperiode bij de mens meerdere weken in beslag. Momenteel is er maar weinig bekend over dit fenomeen bij het paard. In de voorliggende studie werden bij zes paarden een neurostimulator en een pacemaker ingepland om atriumfibrillatie (AF) te kunnen induceren en te onderhouden via een “burst-pacing”-mechanisme; en om de atriale en ventriculaire elektrofysiologie te kunnen bestuderen. Bij alle paarden ontstond persisterende AF na “burst pacing” gedurende twee tot vier weken. Parameters, zoals de atriale fibrillatiecyclus, de rechter atriale en ventriculaire refractaire periode en de gevoeligheid van het hart voor ritmestoornissen, zoals atriale aritmieën, atriale tachyaritmieën of AF, werden zowel voor en tijdens een AF-periode van vier maanden en gedurende een herstelperiode van twee maanden opgevolgd. De atriale en ventriculaire contractiliteit uitgedrukt in fractionele “shortening” en de grootte van de hartcompartimenten werden opgevolgd via 2D-echocardiografie. Bij twee van de zes paarden kon de procedure niet volledig opgevolgd worden ten gevolge van te hoge drempelwaarden (1 paard) of door een infectie ter hoogte van de “pocket” (1 paard). Bij de andere vier paarden konden er reeds na 48 uur van AF significante verschillen gemeten worden ten opzichte van de basale waarden. Tijdens de herstelperiode keerden alle waarden na één tot twee maanden naar de basale waarden terug. In dit onderzoek wordt aangetoond dat geïnduceerde chronische AF, elektrische en contractiele veranderingen teweegbrengt ter hoogte van het hart bij het paard maar dat deze veranderingen omkeerbaar zijn eens het sinusritme hersteld is. Deze resultaten suggereren dat er geen permanente schade veroorzaakt wordt door vier maanden pacing-geïnduceerde AF en dat een rustperiode van zes tot acht weken gunstig zou zijn alvorens het werk te hervatten.

P1010Electrophysiological characteristics of paroxysmal tachycardia in children with short PQ interval

Abstract Introduction It is well known that children with short PQ interval can have different types paroxysmal tachycardia (PT). Combination of short PQ interval with PT is called Lown-Ganong-Levine (LGL) syndrome. We have hypothesized that in children with short PQ interval electrophysiological types of PT do not correlate with shortening of PQ interval. Purpose The aim of the study was to assess the PT types based on results of the electrophysiological studies and radiofrequencycatheter ablation (RFA), and to estimate the AV conduction parameters and their dynamic changes before and after the procedure. Materials and methods of research: 55 children with supraventricular tachycardia (SVT) and short PQ interval were examined. The examination included:ECG, 24-hour Holter monitor, transesophageal pacing study (TEPS), intracardiac electrophysiological study (EPS). 22 children underwent RFA procedure. During the period of the study 55 children had 111 TEPS and 28 EPS studies. Results 21 (38,2%) children had orthodromic AV reentrant tachycardia with accessory pathway (AVRT), 27 (49,1%) had typical (slow-fast) AV nodal reentrant tachycardia (AVNRT), 2 (3,6%) had atypical AVNRT, 1 (1,8%) had combination of typical and atypical AVNRT, 1 (1,8%) had atrial ectopic tachycardia combined with atypical AVNRT, 4 (7,3%) had spontaneous atrial fibrillation or atrial flutter, 3 (5,4%) had combination with typical AVNRT and 1 child (1,8%) had AVRT. PQ interval measured 107,2 ms (83–120ms). 22 (40%) children underwent RFA-13 children with typical AVNRT, 9 with AVRT. In children with AVNRT tachycardia cycle length was 229–425 ms (309,8±67,6ms). Primary RFA was effective in 10 (76,9%) children. The AVNRT recurrence was found in 3 (13,6%) cases after 7,2±4,3 month from the time of procedure. After slow pathway ablation PQ interval length didn't change (107,1±9,1ms compared to 108,2±7,1ms; p=0,2). No change occurred with the maximal rate of 1:1 conduction through AV node (187,8±14,8 imp/min compared to187±12,5 imp/min; p=0,2). Effective refractory period of AV node (AVNERP) increased by a small amount (288,9±14ms, compared to 266±42,7ms; p=0,06). 9 (40,9%) children underwent RFA of the accessory pathway (AP). In all of the cases there were concealed AP with the left-sided localization. AVRT cycle length was 259–382 ms (304,2±41,2ms). After RFA of concealed AP maximal rate of 1:1 conduction through AV node increased by a small amount (220±26,4 imp/min compared to 191,2±23,6 imp/min) and AVNERP decreased (256,7±25,2ms compared to 268,6±38,9ms; p=0,09).After RFA of AP PQ interval length didn't change (110±5,9ms compared to 110±5ms; p=0,1). Conclusion In children with short PQ interval PT was associated with dual physiology of AV connection or with concealed AP. After the successful RFA in children with AVNRT and AVRT there was no PQ interval normalization, which remained short in all examinations. Thus, short PQ- interval is a co-existing phenomenon in children with AVNRT and AVRT.

P343Comparison of Focal Impulse and Rotor Modulation Ablation (FIRM) only versus second-generation cyroballoon ablation in patients with paroxysmal atrial fibrillation

Abstract Background Rotors have been postulated to be a major driver of atrial fibrillation (AF). Initial studies demonstrated, that focal impulse and rotor modulation (FIRM) might be an effective therapy for the treatment of paroxysmal AF (PAF). However, data about FIRM-guided ablation strategies without PVI is sparse. Objective To compare the safety and efficacy of FIRM-guided catheter ablation (without PVI; FIRM arm) and second generation cryoballoon (CB2, CB2 arm) based PVI in patients with paroxysmal atrial fibrillation (PAF) and de-novo catheter ablation of AF. Methods In this retrospective single-center study patients with PAF undergoing de-novo ablation of PAF between February 2016 and January 2017 were enrolled. Patients treated with FIRM-guided AF ablation as a standalone therapy without PVI were included and compared with patients undergoing CB2 based PVI. All patients in the FIRM arm were part of the randomized multicenter FIRMAP AF trial (results of this trial will be presented at this meeting). In patients undergoing FIRM-guided ablation, 3D electroanatomical mapping of both atria was performed. Rotor mapping using FIRM technology was conducted in spontaneous or induced AF. The procedural endpoint was the elimination of all rotors and focal impulses; no PVI was performed in those patients. In the CB2 arm, CB based PVI with the procedural endpoint of isolation of all veins was performed. Procedural data and arrhythmia-free survival after 12 months were compared. Results FIRM-guided and CB2 based AF ablation was performed in 22 and 86 patients, respectively. Follow up was completed in 20 and 79 patients LA diameter differed between groups. Otherwise, baseline characteristics did not differ between the FIRM group (mean age 60±11 years, 59.1% males) and the CB2 group (mean age 62±13, 62.4% male). Arrhythmia-free survival including a 90-day blanking period was 25.0% (15/20) in the FIRM group and 86.1% (11/79) in the CB2 PVI group (p=0.000; Figure 1). Procedure duration was significantly longer in the FIRM group (152 [120; 176] minutes) compared to the CB2 PVI group (122 [110; 145] minutes) (p=0.031), whereas radiation dose was lower in the FIRM group (1266 [1027; 2281] cGy·cm2 vs. 3020 [1677; 4215] cGy·cm2). Adverse events (groin complications) occurred in 1 patient (1.2%) in the CB2 PVI group and 5 patients (22.7%) in the FIRM group. Figure 1. Kaplan-Meier-survival curve dem Conclusion De novo ablation of PAF using a FIRM-guided AF ablation only (without PVI) is associated with poor arrhythmia-free survival after 12 months compared to CB2 PVI. These results underline the importance of PVI as the first-line approach in catheter ablation of AF.

P6290Enhanced IL-6/pSTAT3/fibroblast signaling pathway contributes to spontaneous postoperative atrial fibrillation in a novel simulated cardiac surgery mouse model

Abstract Background Cumulative evidences have shown that IL-6 in atrium might play an important role in the pathogenesis of postoperative AF (POAF) via activation of atrial fibrosis in patients undergoing CABG. However, whether atria produces IL-6 after the stimulation of CABG and its causal relationship with spontaneous POAF (sPOAF) and its specific pathways is still unclear. Purpose To test the hypothesis that atrium will produce IL-6 after CABG and causes sustained sPOAF (ssPOAF) through activating pSTAT3-mediated fibroblast proliferation. Methods To determine the causal relationship between IL-6 and sPOAF, IL-6−/− and wild type (WT) mice were both divided into three groups (10 mice/group): CABG group (NAI, mimic CABG), anti-inflammatory group (AI, mimic CABG with pericardial administration of methylprednisolone for 3 days via chest tube), and control group (anesthesia only). Mice were monitored for ssPOAF for 7 days using implanted telemetry device. Another two sets of mice, using the same models mentioned above, were euthanatized at 48th hours postoperatively. The atria of one set animals were excised and separated into pericardium (PC), pulmonary vein (PV), left atrium (LA), and right atrium (RA) and cultured for 4 hours. IL-6 levels in the supernatant were measured at 10 min and 4 hours of culture using ELISA test. The region producing the largest amount of IL-6 in the other set of animals was harvested for analyzing expressions of IL-6, pSTAT3/STAT3, connexin 43 and 40, fibroblast deposition, and collagen I and III. Path analysis was performed to determine the causal relationship of CABG induced IL-6 release, pSTAT3/fibroblast signaling, and the onset of ssPOAF. Results 40% NAI-WT mice developed ssPOAF (Figure 1A) which was completely protected in IL-6−/− and AI groups. IL-6 was produced by all 4 atrial regions at 4hrs after CABG stimulation with the LA producing the highest amount. Western blotting (Figure 1B), RT-CPR, Masson staining, and immunofluorescence all showed a significantly upregulation of IL-6, pSTAT3/STAT3, fibroblasts, collagen I and III, and downregulation of Cx40 an 43 in NAI-WT mice, but not in IL-6−/− and AI mice. IL-6 was colocalized with vimentin to a large extent in cytoplasm (Figure 1C). IL-6 had strong positive correlation with pSTAT3/STAT3, collagen I and III (all r&gt;0.700, P&lt;0.001), moderate and weak negative correlation with Cx40 and 43 (r=−0.505, P&lt;0.001; r=−0.307, P=0.048, respectively). Path analysis (Figure 1D) revealed that every 1 unit increase in IL-6 upregulated a 0.589 unit increase in ssPOAF, which was mediated by pSTAT3/collagen indirectly and collagen I/ collagen III directly. Conclusion Our study, for the first time, to the best of our knowledge, established a novel pathophysiological role of IL-6/pSTAT3/fibroblast signaling in the pathogenesis of ssPOAF and demonstrated that inhibition of atrial IL-6 might be a potential novel sPOAF prevention strategy. Acknowledgement/Funding The National Natural Science Foundation (No.81170170)

Heterogeneity of the action potential duration is required for sustained atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia and accounts for substantial morbidity and mortality. Recently, we created a mouse model with spontaneous and sustained AF caused by a mutation in the NaV1.5 channel (F1759A) that enhances persistent Na+ current, thereby enabling the investigation of molecular mechanisms that cause AF and the identification of novel treatment strategies. The mice have regional heterogeneity of action potential duration of the atria similar to observations in patients with AF. In these mice, we found that the initiation and persistence of the rotational reentrant AF arrhythmias, known as spiral waves or rotors, were dependent upon action potential duration heterogeneity. The centers of the rotors were localized to regions of greatest heterogeneity of the action potential duration. Pharmacologically attenuating the action potential duration heterogeneity reduced both spontaneous and pacing-induced AF. Computer-based simulations also demonstrated that the action potential duration heterogeneity is sufficient to generate rotors that manifest as AF. Taken together, these findings suggest that action potential duration heterogeneity in mice and humans is one mechanism by which AF is initiated and that reducing action potential duration heterogeneity can lessen the burden of AF.

A calcium transport mechanism for atrial fibrillation in Tbx5-mutant mice.

Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific Tbx5-mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5-deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca2+ sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca2+-dependent pathway for AF risk.

Atrial fibrillation and electrophysiology in transgenic mice with cardiac-restricted overexpression of FKBP12.

Cardiomyocyte-restricted overexpression of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice develop spontaneous atrial fibrillation (AF). The aim of the present study is to explore the mechanisms underlying the occurrence of AF in αMyHC-FKBP12 mice. Spontaneous AF was documented by telemetry in vivo and Langendorff-perfused hearts of αMyHC-FKBP12 and littermate control mice in vitro. Atrial conduction velocity was evaluated by optical mapping. The patch-clamp technique was applied to determine the potentially altered electrophysiology in atrial myocytes. Channel protein expression levels were evaluated by Western blot analyses. Spontaneous AF was recorded in four of seven αMyHC-FKBP12 mice but in none of eight nontransgenic (NTG) controls. Atrial conduction velocity was significantly reduced in αMyHC-FKBP12 hearts compared with NTG hearts. Interestingly, the mean action potential duration at 50% but not 90% was significantly prolonged in αMyHC-FKBP12 atrial myocytes compared with their NTG counterparts. Consistent with decreased conduction velocity, average peak Na+ current ( INa) density was dramatically reduced and the INa inactivation curve was shifted by approximately +7 mV in αMyHC-FKBP12 atrial myocytes, whereas the activation and recovery curves were unaltered. The Nav1.5 expression level was significantly reduced in αMyHC-FKBP12 atria. Furthermore, we found increases in atrial Cav1.2 protein levels and peak L-type Ca2+ current density and increased levels of fibrosis in αMyHC-FKBP12 atria. In summary, cardiomyocyte-restricted overexpression of FKBP12 reduces the atrial Nav1.5 expression level and mean peak INa, which is associated with increased peak L-type Ca2+ current and interstitial fibrosis in atria. The combined electrophysiological and structural changes facilitated the development of local conduction block and altered action potential duration and spontaneous AF. NEW & NOTEWORTHY This study addresses a long-standing riddle regarding the role of FK506-binding protein 12 in cardiac physiology. The work provides further evidence that FK506-binding protein 12 is a critical component for regulating voltage-gated sodium current and in so doing has an important role in arrhythmogenic physiology, such as atrial fibrillation.

Enhanced Cardiomyocyte NLRP3 Inflammasome Signaling Promotes Atrial Fibrillation.

Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The NLRP3 (NACHT, LRR, and PYD domain containing protein 3) inflammasome mediates caspase-1 activation and interleukin-1β release in immune cells but is not known to play a role in cardiomyocytes (CMs). Here, we assessed the role of CM NLRP3 inflammasome in AF. NLRP3 inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal AF or long-standing persistent (chronic) AF. To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knockin mouse model expressing constitutively active NLRP3 (CM-KI) was established. In vivo electrophysiology was used to assess atrial arrhythmia vulnerability. To evaluate the mechanism of AF, electric activation pattern, Ca2+ spark frequency, atrial effective refractory period, and morphology of atria were evaluated in CM-KI mice and wild-type littermates. NLRP3 inflammasome activity was increased in the atrial CMs of patients with paroxysmal AF and chronic AF. CM-KI mice developed spontaneous premature atrial contractions and inducible AF, which was attenuated by a specific NLRP3 inflammasome inhibitor, MCC950. CM-KI mice exhibited ectopic activity, abnormal sarcoplasmic reticulum Ca2+ release, atrial effective refractory period shortening, and atrial hypertrophy. Adeno-associated virus subtype-9-mediated CM-specific knockdown of Nlrp3 suppressed AF development in CM-KI mice. Finally, genetic inhibition of Nlrp3 prevented AF development in CREM transgenic mice, a well-characterized mouse model of spontaneous AF. Our study establishes a novel pathophysiological role for CM NLRP3 inflammasome signaling, with a mechanistic link to the pathogenesis of AF, and establishes the inhibition of NLRP3 as a potential novel AF therapy approach.

Abstract 16713: Defining the Tbx5-Dependent miRNA Regulatory Networks of Atrial Fibrillation

Atrial fibrillation (AF) is the most commonly diagnosed arrhythmic disorder, characterized by random electrical activity in the heart. Complications of AF include stroke and heart failure, which makes understanding the molecular mechanism underlying this conduction defect imperative. The Tbx5 locus has been implicated by genome wide association studies (GWAS) to have an increased risk for AF. Our lab has previously demonstrated the absence of this transcription factor leads to spontaneous and sustained AF, which corroborates the importance of Tbx5 in the maintenance of cardiac rhythm. While Tbx5 is known to drive the network for cardiac rhythm, the downstream regulatory components need to be further characterized, including the downstream miRNAs. The components involved in cardiac rhythm are dose sensitive, therefore small perturbations in gene expression can lead to arrhythmias. Maintaining stable levels of gene expression can be regulated with miRNAs, which provide a negative feedback in dose sensitive pathways. Tbx5 -deficient mice developed spontaneous, sustained AF; in order to further interrogate the TBX5-dependent pathway that led to AF, we transcriptionally profiled small RNAs from the left atrium of Tbx5 -deficient mice. The most downregulated TBX5-dependent miRNA was miR10b. In concordance with previous publications, our miR10b mimic and inhibitor experiments in HL-1 cardiomyocytes demonstrate that miR10b negatively regulates Tbx5 expression. Therefore, we hypothesized that miR10b was crucial in the maintenance of atrial rhythm. Cellular electrophysiology studies with the overexpression of miR10b in HL-1 cardiomyocytes led to the elongation of atrial action potentials, similar to the phenotype we saw in the Tbx5 -deficient atrial tissue. Using whole mouse electrophysiology, we found that the mir10b removal in the hearts leads to increased AF susceptibility. Thus, providing evidence that mirR10b is crucial for the maintenance of normal cardiac conduction. In order to identify other miRNAs essential for cardiac conduction, we examined the other TBX5-dependent miRNAs in a high-throughput calcium screen. Further defining the miRNAs can provide a better understanding of the regulatory mechanisms crucial for cardiac conduction.

Homozygous CREM-IbΔC-X Overexpressing Mice Are a Reliable and Effective Disease Model for Atrial Fibrillation.

Background: Atrial fibrillation (AF) is a significant cause of morbidity and mortality with foreseeably increasing prevalence. While large animal models of the disease are well established but resource intensive, transgenic AF mouse models are not yet widely used to develop or validate novel therapeutics for AF. Hemizygous mice with a cardiomyocyte-specific overexpression of the human cAMP response element modulator (CREM) isoform IbΔC-X spontaneously develop AF on grounds of an arrhythmogenic substrate consisting of alterations in structure, conduction, and calcium handling.Objective: We investigated if homozygous expression of the CREM-IbΔC-X transgene in mice alters the time course of AF development, and if homozygous CREM-IbΔC-X transgenics could be suitable as a disease model of AF.Methods: Southern Blot, quantitative real-time PCR, and immunoblotting were used to identify and verify homozygous transgenics. Cardiac gravimetry, quantitative real-time RT-PCR, histology, survival analysis, and repeated ECG recordings allowed assessment of phenotypic development and effects of antiarrhythmic drugs.Results: Homozygous animals could be identified by Southern blot and quantitative PCR, showing a strong trend to increased transgenic protein expression. In homozygous animals, atrial hypertrophy appeared earlier and more pronounced than in hemizygous animals, going along with an earlier onset of spontaneous AF, while no increased early mortality was observed. Application of a rate-controlling drug (esmolol) led to the expected result of a decreased heart rate. Application of a rhythm-controlling drug (flecainide) showed effects on heart rate variability, but did not lead to a definitive conversion to sinus rhythm.Conclusion: We suggest homozygous CREM-IbΔC-X overexpressing mice as a reliable model of early onset, rapidly progressive AF.

Atrial Infarction-Induced Spontaneous Focal Discharges and Atrial Fibrillation in Sheep: Role of Dantrolene-Sensitive Aberrant Ryanodine Receptor Calcium Release.

The mechanisms underlying spontaneous atrial fibrillation (AF) associated with atrial ischemia/infarction are incompletely elucidated. Here, we investigate the mechanisms underlying spontaneous AF in an ovine model of left atrial myocardial infarction (LAMI). LAMI was created by ligating the atrial branch of the left anterior descending coronary artery. ECG loop recorders were implanted to monitor AF episodes. In 7 sheep, dantrolene-a ryanodine receptor blocker-was administered in vivo during the 8-day observation period (LAMI-D, 2.5 mg/kg, IV, BID). LAMI animals experienced numerous spontaneous AF episodes during the 8-day monitoring period that were suppressed by dantrolene (LAMI, 26.1±5.1; sham, 4.3±1.1; LAMI-D, 2.8±0.8; mean±SEM episodes per sheep, P<0.01). Optical mapping showed spontaneous focal discharges (SFDs) originating from the ischemic/normal-zone border. SFDs were calcium driven, rate dependent, and enhanced by isoproterenol (0.03 µmol/L, from 210±87 to 3816±1450, SFDs per sheep) but suppressed by dantrolene (to 55.8±32.8, SFDs per sheep, mean±SEM). SFDs initiated AF-maintaining reentrant rotors anchored by marked conduction delays at the ischemic/normal-zone border. NOS1 (NO synthase-1) protein expression decreased in ischemic zone myocytes, whereas NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase and xanthine oxidase enzyme activities and reactive oxygen species (DCF [6-carboxy-2',7'-dichlorodihydrofluorescein diacetate]-fluorescence) increased. CaM (calmodulin) aberrantly increased [3H]ryanodine binding to cardiac RyR2 (ryanodine receptors) in the ischemic zone. Dantrolene restored the physiological binding of CaM to RyR2. Atrial ischemia causes spontaneous AF episodes in sheep, caused by SFDs that initiate reentry. Nitroso-redox imbalance in the ischemic zone is associated with intense reactive oxygen species production and altered RyR2 responses to CaM. Dantrolene administration normalizes the CaM response, prevents LAMI-related SFDs, and AF initiation. These findings provide novel insights into the mechanisms underlying ischemia-related atrial arrhythmias.

Atrial Fibrillation Initiated by Early Afterdepolarization-Mediated Triggered Activity during Acute Oxidative Stress: Efficacy of Late Sodium Current Blockade.

The mechanism of Atrial Fibrillation (AF) that emerges spontaneously during acute oxidative stress is poorly defined and its drug therapy remains suboptimal. We hypothesized that oxidative activation of Ca-calmodulin dependent protein kinase (CaMKII) promotes Early Afterdepolarization-(EAD)-mediated triggered AF in aged fibrotic atria that is sensitive to late Na current (INa-L) blockade. High-resolution voltage optical mapping of the Left and Right Atrial (LA & RA) epicardial surfaces along with microelectrode recordings were performed in isolated-perfused male Fisher 344 rat hearts in Langendorff setting. Aged atria (23-24 months) manifested 10-fold increase in atrial tissue fibrosis compared to young/adult (2-4 months) atria (P<0001. Spontaneous AF arose in 39 out of 41 of the aged atria but in 0 out of 12 young/adult hearts (P<001) during arterial perfusion of with 0.1 mm of hydrogen peroxide (H2O2). Optical Action Potential (AP) activation maps showed that the AF was initiated by a focal mechanism in the LA suggestive of EAD-mediated triggered activity. Cellular AP recordings with glass microelectrodes from the LA epicardial sites showing focal activity confirmed optical AP recordings that the spontaneous AF was initiated by late phase 3 EAD-mediated triggered activity. Inhibition of CaMKII activity with KN-93 (1 μM) (N=6) or its downstream target, the enhanced INa-L with GS-967 (1 μM), a specific blocker of INa-L (N=6), potently suppressed the AF and prevented its initiation when perfused 15 min prior to H2O2 (n=6). Increased atrial tissue fibrosis combined with acute oxidative activation of CaMK II Initiate AF by EAD-mediated triggered activity. Specific block of the INa-L with GS-967 effectively suppresses the AF. Drug therapy of oxidative AF in humans with traditional antiarrhythmic drugs remains suboptimal; suppressing INa-L offers a potential new strategy for effective suppression of oxidative human AF that remains suboptimal.