JACC: Clinical Electrophysiology

BackgroundWe performed high-density mapping of persistent atrial fibrillation (AF) in animals and patients (1) to test that AF is due to [&ge;]1 reentries, and (2) to characterize activation delay and reentries pre/ post pulmonary vein isolation (PVI). We determined electrophysiological characteristics that may predispose to the induction, maintenance, and reduction of AF. Methods and ResultsThis study includes 48 dogs and nine patients. 43 AF- and five sinus/ paced rhythm dogs (3-14 weeks rapid atrial pacing) were studied at open chest surgery with 117 epicardial electrograms (EGMs) (2.5mm dist.) in 6 bi-atrial regions. Rotational activity automatically detected with a new algorithm tracking the earliest and latest activation in all regions (5{+/-}2 per region) were stable over 424{+/-}505ms [120- 4940ms]. Reentry stability was highest in the right atrial appendage (RAA) (405{+/-}219ms) and the posterior left atrium (PLA) (267{+/-}115ms) and anchored between >=3 zones of activation delay (15{+/-}5ms, median 13ms) defined as >10ms per 2.5mm. Cycle length (CL) and degree of focal fibrosis were highest in the PLA and left atrial free wall (LAFW) with 94{+/-}7ms, 96{+/-}5ms, and 49{+/-}14%, 47{+/-}19%. Fiber crossing density correlated with the stability of rotational activity (R=0.6, P<0.05). Activation delay was 2x higher in AF compared to sinus rhythm/paced rhythm (interval 200-500ms). Activation delay zones > 10ms were at the same locations, but increased 4x during AF vs. SR and were located at fiber crossings, fibrosis/ fat zones. Stability of rotational activity correlated with Organization Index (OI), Fraction Index (FI), Shannons Entropy (ShEn), and CL (R>0.5, p< 0.0001). PVI in five hearts increased CL [2-14%] and reduced stability of rotational activity in nearly all regions remote to the pulmonary veins (PVs). Also in the clinical evaluation in nine patients using the HD-catheter (16 electrodes, 3mm dist.) activation delay at the reentrant trajectory was 2x higher at edges with maximal delay (20.5{+/-}8.1ms, median 19.6ms) vs (9.3{+/-}8.8ms, median 9.2ms) and 1.4 x higher during AF (13.0{+/-}18.7ms, median 7.2ms) compared to SR/ CS-pacing (18.0{+/-}11.6ms, median 17.7ms). ConclusionRotational activities in all bi-atrial regions anchored between small frequency-dependent activation delay zones in AF. PVI led to beneficial remodeling in bi-atrial regions remote to the PVs. These data may identify a new paradigm for persistent AF. Subject TermsArrhythmias, Atrial Fibrillation, Cardiac Electrophysiology, High-Density Mapping, Catheter Ablation, Pulmonary Vein Isolation, Fibrosis Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIRotational activity trajectories based on high-resolution mapping follow propagation line patterns. C_LIO_LIRotational activities anchor frequently between small frequency-dependent slow conduction zones in all bi-atrial regions. C_LIO_LISlow conduction zones are fiber crossings zones and develop into fibrosis and fat regions over time. C_LIO_LIPVI reduces slow conduction zones and AF drivers in regions remote to the PVs in both atria. C_LI What Are the Clinical Implications?O_LIThe new method for the robust detection of rotational activity based on the earliest and latest activation may be useful for an improved AF treatment. C_LIO_LIStability of rotational activity may be predicted with the correlated substrate characteristic fiber crossings density, with slow conduction zones, and with established electrogram measures in the different atrial regions. C_LIO_LIPVI leads to beneficial remodeling in all regions remote to the PVs in the left atrium and right atrium. C_LI

Background: Despite being central to atrial fibrillation (AF) and ventricular fibrillation (VF) mechanisms and therapy, the factors governing AF and VF termination are poorly understood. It has been noted that ratio of system size (L) and the two-point spatial correlation length ({xi}2) are associated with time until termination in transient spatiotemporally chaotic systems, but the relationship between these characteristics and termination has not been systematically studied in human AF and VF. Objective: We aimed assess whether the time to cardiac fibrillation termination can be predicted using a novel estimator, the spatial autocorrelation dimension (Di), defined as the ratio of L and {xi}2, in human AF and VF. Methods: Di was computed and compared in a multi-centre, multi-system study with data for sustained versus spontaneously terminating human AF/VF. VF data was collected during coronary-bypass surgery; and AF data during clinically indicated AF ablation. We analyzed: i) VF mapped using a 256-electrode epicardial sock (n=12pts); ii) AF mapped using a 64-electrode constellation basket-catheter (n=15pts); iii) AF mapped using a 16-electrode HD-grid catheter (n=42pts). To investigate temporal fibrillation persistence, the response of AF-episodes to flecainide (n=7pts) was also studied. Results: Spontaneously terminating fibrillation demonstrated a lower Di (P<0.001 all systems). Lower Di was also seen in paroxysmal compared to persistent AF (P=0.002). Post-flecainide, Di decreased over time (P<0.001). Lower Di was also associated with longer-lasting episodes of AF/VF (R2>0.90, P<0.05 in all cases). Using k-means clustering, two distinct clusters and their centroids were identified i) a cluster of spontaneously terminating episodes, and ii) a cluster of sustained epochs. Conclusion: Di predicts the temporal persistence of cardiac fibrillation. This finding provides potentially important insights into a possible common pathway to termination and therapeutic approaches.

BackgroundDuring radiofrequency (RF) ablation, lesion size increases with increasing contact force (CF), RF power and application time. The effects of CF and RF power on lesion size during high-power and short-duration (HP-SD) ablation have not been well-determined. This study aimed to, during HP-SD ablation: 1) examine the relationship between lesion size and CF, RF power and time, and 2) prospectively validate the ability of a novel logarithmic formula, incorporating CF, RF power and time (Force-Power-Time-Index, FPTI, gram x Watt x sec) to predict lesion size using a swine beating heart model. MethodsEight closed-chest swine were studied. A 7.5Fr CF ablation catheter with a 3.5mm irrigated-tip electrode containing 6 surface thermocouples (Qdot-Micro) was positioned in the right and left ventricles. In five swine (Phase1-Study), RF was delivered at [&le;]90Watts (modulated to maintain the surface electrode temperature<65{degrees}C) for 4sec to 103 ventricular sites with various CF (range 5-54g). Swine were sacrificed and lesion size was measured. A new logarithmic FPTI-Formula was created based on the relationship between lesion depth and CF, power and time. In the prospective validation study using the remaining three swine (Phase2-Study), RF(90W) was delivered for 4 sec at 72 sites with FPTI-Formula predicted lesion depths of 2-6mm. Actual lesion depth was compared to the predicted lesion depth. ResultsIn the Phase1-study, there was a close relationship between lesion depth and the product of Force x Power x Time (R=0.711, p<0.0001), creating a novel logarithmic FPTI-Formula to predict lesion depth. In the Phase2-study, lesion depth predicted by the FPTI-Formula correlated highly with actual lesion depth (1.9-6.1mm), with {+/-}1mm accuracy in 68/72(94%) lesions (R=0.867, p<0.0001). No steam pop or thrombus formation occurred. ConclusionDuring HP-SD ablation, the new FPTI-Formula prospectively predicted lesion depth with high accuracy while the surface electrode temperature control prevented steam pop and thrombus formation.

Background and AimsSubstantial sex-based differences have been reported in atrial fibrillation (AF), with female patients experiencing worse symptoms, increased complications from drug side effects or ablation, and elevated risk of AF-related stroke and mortality. Recent studies revealed sex-specific alterations in AF-associated Ca2+ dysregulation, whereby female cardiomyocytes more frequently exhibit potentially proarrhythmic Ca2+-driven instabilities compared to male cardiomyocytes. In this study, we aim to gain a mechanistic understanding of the Ca2+-handling disturbances and Ca2+-driven arrhythmogenic events in males vs females and establish their responses to Ca2+-targeted interventions. Methods and ResultsWe incorporated known sex differences and AF-associated changes in the expression and phosphorylation of key Ca2+-handling proteins and in ultrastructural properties and dimensions of atrial cardiomyocytes into our recently developed 3D atrial cardiomyocyte model that couples electrophysiology with spatially detailed Ca2+-handling processes. Our simulations of quiescent cardiomyocytes show increased incidence of Ca2+ sparks in female vs male myocytes in AF, in agreement with previous experimental reports. Additionally, our female model exhibited elevated propensity to develop pacing-induced spontaneous Ca2+ releases (SCRs) and augmented beat-to-beat variability in action potential (AP)-elicited Ca2+ transients compared with the male model. Parameter sensitivity analysis uncovered precise arrhythmogenic contributions of each component that was implicated in sex and/or AF alterations. Specifically, increased ryanodine receptor phosphorylation in female AF cardiomyocytes emerged as the major SCR contributor, while reduced L-type Ca2+ current was protective against SCRs for male AF cardiomyocytes. Furthermore, simulations of tentative Ca2+-targeted interventions identified potential strategies to attenuate Ca2+-driven arrhythmogenic events in female atria (e.g., t-tubule restoration, and inhibition of ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca{superscript 2}-ATPase), and revealed enhanced efficacy when applied in combination. ConclusionsOur sex-specific computational models of human atrial cardiomyocytes uncover increased propensity to Ca2+-driven arrhythmogenic events in female compared to male atrial cardiomyocytes in AF, and point to combined Ca2+-targeted interventions as promising approaches to treat AF in female patients. Our study establishes that AF treatment may benefit from sex-dependent strategies informed by sex-specific mechanisms. Translational perspectiveAccumulating evidence demonstrates substantial sex-related differences in atrial fibrillation (AF), which is the most common arrhythmia, with female patients faring worse with the condition. By integrating known sex-differential components into our computational atrial cardiomyocyte model we found that female atrial cardiomyocytes in AF exhibit greater propensity to develop Ca2+-driven arrhythmia than male cardiomyocytes. Model analyses provided novel mechanistic insights and suggested strategies such as t-tubule restoration, correction of Ca2+-handling disturbances, and the combination of both, as promising approaches to treat AF in female patients. Our study uncovers and validate sex-specific AF mechanisms and inform the development of targeted anti-AF strategies. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/583217v2_figa1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@d3ec3borg.highwire.dtl.DTLVardef@13a2c7borg.highwire.dtl.DTLVardef@3e4605org.highwire.dtl.DTLVardef@6726b4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Sex-specific 3D spatiotemporal models of human atrial cardiomyocyte Ca2+ signaling reveal a greater propensity to develop Ca2+-driven arrhythmic events in female vs male atrial cardiomyocytes in AF. Model analysis links sex-specific AF remodeling to arrhythmogenic mechanisms. AF, atrial fibrillation; SCR, spontaneous Ca2+ release; CaT, cytosolic Ca2+ transient; RyR2-P, phosphorylated ryanodine receptor type 2 (RyR2); CSQ, calsequestrin; LTCC, L-type Ca2+ channel; PLB, phospholamban; SERCA, sarcoendoplasmic reticulum Ca2+ ATPase; SR, sarcoplasmic reticulum. C_FIG

Resident cardiac macrophages are understood to facilitate atrioventricular (AV) node conduction because they purportedly couple to AV node myocytes via connexin43 (Cx43) containing gap junctions. We tested this mechanism using biophysical modelling, high-resolution imaging of mouse and human AV conduction tissue, and pharmacological macrophage depletion. In silico, coupling macrophage membrane phenotypes to HCN4+ AV node myocytes imposed an electrotonic load that suppressed pacemaking and promoted conduction slowing, including stable 2:1 block in strand simulations. Anatomically, HCN4-defined components of the mouse AV conduction axis were essentially devoid of Cx43 and overlap of CD68+ macrophages and Cx43 was negligible in both mouse AV node and human penetrating bundle. Finally, near-complete macrophage depletion with CSF1R inhibition (PLX5622) did not alter AV electrical activity in vivo or ex vivo. Together, these data argue against a physiologically relevant role for Cx43-mediated macrophage-myocyte electrical coupling in normal AV node function. HIGHLIGHTSO_LIModelling predicts that AV node automaticity and conduction would be suppressed if macrophages coupled to AV node myocytes C_LIO_LIThe mouse AV conduction axis is essentially devoid of Cx43, currently considered responsible for macrophage-AV node myocyte coupling C_LIO_LIOverlap of macrophages and Cx43 expression is not discernible in the Cx43-expressing human distal AV node C_LIO_LIMacrophage depletion by CSF1R inhibition does not impact AV electrical activity in vivo or ex vivo C_LI

BackgroundElectrographic flow (EGF) mapping algorithms employing Horn-Schunck flow estimations can create temporospatial visualizations of atrial electrical wavefront propagations during atrial fibrillation (AF). Reproducible patterns of centrifugal EGF activation from discrete sites may represent sites of AF origin or sources. Our objectives were to assess the patterns and prevalence of AF sources using EGF mapping. MethodsUnipolar electrograms were recorded for 1-minute with 64-pole basket catheters. Flow estimates were constructed by passing consecutive frames through an algorithm to learn and then compare typical wave direction patterns to describe flow-field evolution. During each 2-second segment, sites initiating centrifugal activation patterns were defined as AF sources. Maps of source location/activity duration were generated. ResultsThe EGF method was applied to 405 prospective and retrospective patients with persistent or long-standing persistent AF. Mean age 62.5 years; mean LA size 54 mm; mean AF duration 4.6 years. EGF mapping found 6.6 {+/-} 2.4 AF sources/patient (range 1 to 17). Distribution was 55% LA and 45% RA. Dominant sources (prevalence [&ge;]20%) were demonstrated in 185 (45.7%) patients, but only 10.7% of all sources were dominant. While AF cycle length (CL) was not affected by source prevalence, CL variance significantly decreased as source prevalence increased. ConclusionsComplex AF conduction patterns make ablation challenging, but EGF mapping enables detection and organization of time-dependent AF behaviors. Although many low prevalence sources are detected, they may not be clinically relevant, while higher prevalence sources seem to modulate AF. Recording durations of 1 minute facilitate source discrimination.

Atrial fibrillation (AF) is a progressive disease involving both structural and functional remodeling. To investigate the contribution of cell-scale functional remodeling to AF pathogenesis, we combined personalized 3D anatomical models with pathology-specific ionic models. The latter were developed using recordings in myocytes isolated from patients in sinus rhythm, paroxysmal, postoperative, and persistent AF. To quantify AF dynamics, we developed a novel algorithm for locating RDs by backtracking the conduction velocity field from the wavebreak regions. We demonstrate that our novel algorithm is at least 700 times faster than the traditional phase singularity analysis. The inducibility of simulated AF was not pathology-dependent, but pathological models demonstrate a more extensive arrhythmogenic substrate compared to the sinus rhythm. AF driver locations depend on electrophysiological remodeling; differences between pathology-specific models are explained by differences in wavebreak patterns. Specifically, RDs tend to dwell in the regions with the highest wavebreak probability.

BackgroundThoracic epidural anesthesia (TEA) has been shown to reduce the burden of ventricular tachyarrhythmias (VT) in small case-series of patients with refractory VT and cardiomyopathy. However, its electrophysiological and autonomic effects in diseased hearts remain unclear and its use after myocardial infarction (MI) is limited by concerns for potential RV dysfunction. MethodsMI was created in Yorkshire pigs (N=22) by LAD occlusion. Six weeks post-MI, an epidural catheter was placed at the C7-T1 vertebral level for injection of 2% lidocaine. RV and LV hemodynamics were recorded using Millar pressure-conductance catheters, and ventricular activation-recovery intervals (ARIs), a surrogate of action potential durations, by a 56-electrode sock and 64-electrode basket catheter. Hemodynamics and ARIs, baroreflex sensitivity (BRS) and intrinsic cardiac neural activity, and ventricular effective refractory periods (ERP) and slope of restitution (Smax) were assessed before and after TEA. VT/VF inducibility was assessed by programmed electrical stimulation. ResultsTEA reduced inducibility of VT/VF by 70%. TEA did not affect RV-systolic pressure or contractility, although LV-systolic pressure and contractility decreased modestly. Global and regional ventricular ARIs increased, including in scar and border zone regions post-TEA. TEA reduced ARI dispersion specifically in border zone regions. Ventricular ERPs prolonged significantly at critical sites of arrhythmogenesis, and Smax was reduced. Interestingly, TEA significantly improved cardiac vagal function, as measured by both BRS and intrinsic cardiac neural activity. ConclusionTEA does not compromise RV function in infarcted hearts. Its anti-arrhythmic mechanisms are mediated by increases in ventricular ERP and ARIs, decreases in Smax, and reductions in border zone heterogeneity. TEA improves parasympathetic function, which may independently underlie some of its observed anti-arrhythmic mechanisms. This study provides novel insights into the anti-arrhythmic mechanisms of TEA, while highlighting its applicability to the clinical setting. Abstract IllustrationMyocardial infarction is known to cause cardiac autonomic dysfunction characterized by sympathoexcitation coupled with reduced vagal tone. This pathological remodeling collectively predisposes to ventricular arrhythmia. Thoracic epidural anesthesia not only blocks central efferent sympathetic outflow, but by also blocking ascending projections of sympathetic afferents, relieving central inhibition of vagal function. These complementary autonomic effects of thoracic epidural anesthesia may thus restore autonomic balance, thereby improving ventricular electrical stability and suppressing arrhythmogenesis. DRG=dorsal root ganglion, SG=stellate ganglion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/585127v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1150b29org.highwire.dtl.DTLVardef@8fd741org.highwire.dtl.DTLVardef@17d16bforg.highwire.dtl.DTLVardef@1608799_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundRepolarization alternans, defined as period-2 oscillation in the repolarization phase of the action potentials, provides a mechanistic link between cellular dynamics and ventricular fibrillation (VF). Theoretically, higher-order periodicities (e.g., periods 4, 6, 8,...) are expected but have minimal experimental evidence. MethodsWe studied explanted human hearts obtained from recipients of heart transplantation at the time of surgery. Optical mapping of the transmembrane potential was performed after staining the hearts with voltage-sensitive fluorescent dyes. Hearts were stimulated at an increasing rate until VF was induced. Signals recorded from the right ventricle endocardial surface prior to induction of VF and in the presence of 1:1 conduction were processed using the Principal Component Analysis and a combinatorial algorithm to detect and quantify higher-order dynamics. Results were correlated to the underlying electrophysiological characteristics as quantified by restitution curves and conduction velocity. ResultsA prominent and statistically significant global 1:4 peak (corresponding to period-4 dynamics) was seen in three of the six studied hearts. Local (pixel-wise) analysis revealed the spatially heterogeneous distribution of periods 4, 6, and 8, with the regional presence of periods greater than two in all the hearts. There was no significant correlation between the underlying restitution properties and the period of each pixel. DiscussionWe present evidence of higher-order periodicities and the co-existence of such regions with stable non-chaotic areas in ex-vivo human hearts. We infer from the independence of the period to the underlying restitution properties that the oscillation of the excitation-contraction coupling and calcium cycling mechanisms is the primary mechanism of higher-order dynamics. These higher-order regions may act as niduses of instability that can degenerate into chaotic fibrillation and may provide targets for substrate-based ablation of VF.

BACKGROUNDAtrial fibrillation (AF) susceptibility is heightened in endurance athletes but the underlying mechanisms are incompletely understood. Because pulmonary vein (PV) myocyte triggers are critical determinants of AF, we investigated PV electrophysiological remodelling in animal models of the athletes heart. METHODSThe following experiments were performed in canines and mice after 16 or 6 weeks, respectively, of daily exercise training (ExT), and compared to sedentary (Sed) controls: ECG recording, echocardiography, pharmacological autonomic block, extrastimulus pacing, multielectrode array mapping, monophasic and intracellular action potential (AP) recording with custom-designed pattern recognition analysis, histology, RNAseq and spatial in situ transcriptomics. RESULTSAF propensity was significantly increased in ExT animals. Mapping studies identified heightened rotational activity in the PV-left atrial (LA) junction of ExT vs. Sed canines in vivo, and enhanced automaticity, triggered activity and AP duration variability ex vivo in ExT canines and mice. Intracellular recordings in mouse PV cardiomyocytes determined at least six AP subtypes with increased frequency of pacemaker-like APs in ExT PV, concomitant with increased expression of pacemaking HCN4, Cav1.3 and Cav3.1 channels. PV spontaneous excitability was also significantly enhanced. Subcellular resolution spatial transcriptomics in mouse PV-LA identified diffuse ion channel remodelling and activation of established AF-promoting pro-inflammatory and pro-fibrotic cytokines and chemokines in ExT PV cardiomyocytes. Conduction slowing in the ExT PV-LA junction was attributable to: gap junction remodelling, reduced Na+ channel expression and increased extracellular matrix deposition with enhanced myofibroblast number and proximity to PV cardiomyocytes. CONCLUSIONSEndurance exercise elicits proarrhythmic electro-anatomical remodelling of the PV-LA junction with enhanced pacemaking ion channel expression and immune-inflammatory pathway activation in PV myocytes as prominent contributors. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABSO_LIThis work is the first demonstration that endurance training results in proarrhythmic electrophysiological remodelling of PV sleeve myocytes and extracellular matrix deposition in the PV-LA junction. C_LIO_LIWe register electrical and molecular heterogeneity of the PV-LA junction at single cell and subcellular resolution, and for the first time identify the molecular events that underlie increased proarrhythmic activity of the trained PV. These include enhanced pacemaking ion channel expression (e.g., HCN4, Cav1.3, and Cav3.1), pro-inflammatory cytokine activation (e.g., TNF, IL-6), increased myofibroblasts and extracellular matrix deposition. C_LI What are the clinical implications?O_LIWe identify the molecular determinants of PV proarrhythmic activity in the trained heart and present new therapeutic targets for AF prevention in athletes. C_LIO_LIOur findings provide mechanistic rationale for the efficacy of pulmonary vein isolation for AF in athletes. C_LI

BackgroundAlthough atrial electrograms (EGMs) are thought to reflect pathophysiological substrate for atrial fibrillation (AF), it is not known which electrograms are suitable targets during AF ablation. We hypothesized that electrogram morphology recurrence (EMR) better reflects arrhythmogenic AF substrate than traditional frequency and complexity measures of AF. In a canine rapid atrial pacing (RAP) model of AF, we assessed the relationship between EMR and traditional AF electrogram measures, rotational activity in the atria, fibrosis, myofiber orientation and parasympathetic innervation. MethodsPersistent AF was induced in 13 dogs by RAP for 6-8 weeks. High-density epicardial mapping (117 electrodes) was performed in six atrial sub-regions. EMR measures Recurrence percentage (Rec%) and cycle length of the most frequent electrogram morphology (CLR), Fractionated Interval (FI), Organization Index (OI), Dominant Frequency (DF) and Shannons Entropy (ShEn) were analyzed before and after atropine administration. Myocyte fiber orientation, amount of fibrosis and spatial distribution of parasympathetic nerve fibers were quantified. ResultsRec% was greatest in the appendages, and CLR was lowest in the posterior left atrium. Rec%/CLR correlated with FI, OI and the complexity measure ShEn, but not with DF. All electrogram measures were poorly correlated with fibrosis and myofiber anisotropy. Rec% correlated closely with stability of rotational activity. Unlike other measures, Rec% correlated closely with spatial heterogeneity of parasympathetic nerve fibers; this was reflected in CLR response to atropine. ConclusionEMR correlates closely with stability of rotational activity and with the pattern of atrial parasympathetic innervation. CLR may therefore be a viable therapeutic target in persistent AF.

Background: Pulsed-field ablation (PFA) systems increasingly provide impedance-based contact indicators, such as tissue proximity indication (TPI), derived from local impedance changes relative to a blood-pool baseline. These indicators are largely binary and do not quantify post-application catheter?tissue energy coupling. We evaluated Bipolar Local Impedance Delta (BiLID), the peri-application bipolar local impedance drop, as a complementary impedance-based index of delivered energy coupling. Methods: We retrospectively analyzed 1,556 VARIPULSE applications in 23 patients undergoing pulmonary vein isolation. BiLID was derived from 29,822 paired pre-/post-ablation impedance measurements obtained from numeric local impedance readouts displayed by the mapping system, without proprietary data export or waveform estimation. Reproducibility was assessed by intraclass correlation. Associations with TPI status, vein anatomy, application order, and peak creatine kinase-MB (CK-MB) were examined using linear mixed-effects models with within-patient clustering and exploratory patient-level analyses. Results: BiLID showed excellent interobserver reproducibility and increased stepwise with the number of TPI-positive electrodes per pair (0?2; P < 0.0001), while varying widely among TPI-positive signals. BiLID differed by electrode position and was lower during right than left pulmonary vein ablation (both P < 0.0001). Total BiLID correlated with peak CK-MB (r = 0.71; 95% CI, 0.42?0.87; P = 0.0001), whereas application count (r = 0.16, P = 0.4711) and TPI-positive signals (r = 0.26, P = 0.2334) did not. Lower CK-MB elevation was associated with larger left atrial volume index, female sex, and heart failure, suggesting substrate-modulated biomarker release. Conclusions: BiLID is a reproducible, continuous index of catheter?tissue energy coupling that complements pre-delivery binary contact indicators by quantifying the response after PFA delivery. It captures graded contact quality and anatomical heterogeneity and may inform individualized, coupling-guided PFA titration.

Background: Three pulsed-field ablation (PFA) systems are FDA-approved for atrial fibrillation (AF), yet whether their safety profiles differ from each other and from radiofrequency (RF) ablation has not been systematically characterized using real-world adverse event data. We compared platform-specific complication profiles across three FDA-approved PFA systems and contemporary RF catheters in MAUDE. Methods: We analyzed 2,262 manually adjudicated MAUDE adverse event reports (760 PFA, 1,502 RF) through July 2025. Neurologic events underwent independent adjudication into five tiers by three auditors. Disproportionality was assessed using Reporting Odds Ratios (ROR) with Benjamini-Hochberg (BH) correction. Results: Pooled PFA had significantly lower BH-adjusted ROR for tamponade (0.52, 95% CI 0.41-0.67) and esophageal injury (0.09, 0.01-0.66), consistent with a tissue-selective reporting profile across platforms. Platform-level analysis, however, revealed substantial heterogeneity: the stroke signal was driven by Varipulse (ROR 16.41, 8.61-31.28) and was not observed with Farapulse (ROR 1.26, NS). Pooled PFA had higher ROR for imaging-confirmed stroke (3.84, 2.27-6.49) and arrhythmia (2.57, 1.91-3.45). Coronary vasospasm (24 vs. 0 events) and hemolysis (15 vs. 1 events) were PFA-specific. Composite serious adverse events were similar. In a pre-specified extension period analysis (August-December 2025), the pooled PFA stroke signal attenuated to non-significance (ROR 1.60, 0.89-2.85), consistent with notoriety bias following the FDA Safety Communication. Conclusions: PFA adverse-event reporting shows substantial platform heterogeneity across approved systems. Varipulse was associated with a disproportionate neurologic reporting signal, while all PFA platforms show tissue-selective reporting patterns relative to RF. These findings support platform-aware clinical decision-making and post-market surveillance.

AimsThe behavior of pacemaker cardiomyocytes (PCs) in the sinoatrial node (SAN) is modulated by neurohormonal and paracrine factors, many of which signal through G-protein coupled receptors (GPCRs). The aims of the present study are to catalog GPCRs that are differentially expressed in the mammalian SAN and to define the acute physiological consequences of activating the cholecystokinin-A signaling system in isolated PCs. Methods and ResultsUsing bulk and single cell RNA sequencing datasets, we identify a set of GPCRs that are differentially expressed between SAN and right atrial tissue, including several whose roles in PCs and in the SAN have not been thoroughly characterized. Focusing on one such GPCR, Cholecystokinin-A receptor (CCKAR), we demonstrate expression of Cckar mRNA specifically in mouse PCs, and further demonstrate that subsets of SAN fibroblasts and neurons within the cardiac intrinsic nervous system express cholecystokinin, the ligand for CCKAR. Using mouse models, we find that while baseline SAN function is not dramatically affected by loss of CCKAR, the firing rate of individual PCs is slowed by exposure to sulfated cholecystokinin-8 (sCCK-8), the high affinity ligand for CCKAR. The effect of sCCK-8 on firing rate is mediated by reduction in the rate of spontaneous phase 4 depolarization of PCs and is mitigated by activation of beta-adrenergic signaling. Conclusions(1) PCs express many GPCRs whose specific roles in SAN function have not been characterized, (2) Activation of the the cholecystokinin-A signaling pathway regulates PC automaticity.

BackgroundSpontaneous Ca2+ release events and waves are frequent in isolated ventricular cardiomyocytes from failing hearts (HF) and are proposed to initiate arrhythmias in the intact heart. However, evidence supporting whether single-cell Ca2+ waves trigger tissue-wide depolarization in the intact heart is scarce, particularly in human HF. We characterized Ca2+ waves at single-cell resolution within the multicellular network of the intact heart and identified propagating dynamics and mechanisms facilitating arrhythmogenesis at tissue level. MethodsLiving myocardial slices (LMS) from HF and non-HF human hearts were prepared from left ventricular tissue and paced at 2 Hz under adrenergic stimulation. Ca2+ transients and waves were recorded by wide-field imaging of Fluo-8. Ca2+ waves in relation to single-cell structures within each LMS were identified using custom algorithms. Computational modelling assessed whether experimentally observed HF Ca2+ waves dynamics can lead to focal excitation in tissue models. ResultsFollowing pacing, early onset Ca2+ waves, initiating within the first 2 seconds, were more frequent in HF compared to non-HF, and HF cardiomyocytes had more foci, where Ca2+ waves originate, than non-HF. Spatial mapping showed that early onset waves in HF occurred frequently in clusters of neighboring cells. Although early onset Ca2+ waves propagated similar distances in HF and non-HF cardiomyocytes, they more frequently crossed cell boundaries in HF. Particularly, HF LMS exhibited more side-to-side Ca2+ propagation, correlating with increased connexin 43 distribution to lateral membranes. Furthermore, HF LMS exhibited more local and global triggered Ca2+ activities compared to non-HF LMS, correlating with local tissue depolarization. Simulations of HF Ca2+ wave dynamics in remodeled tissue demonstrated a greater capacity to elicit focal excitation. ConclusionsIn human HF, a higher incidence of early onset Ca2+ waves combines with altered intercellular connectivity to create synchrony in clusters of nearby cells that can overcome the current sink, thereby increasing arrhythmia susceptibility. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/651991v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@12582forg.highwire.dtl.DTLVardef@5b9893org.highwire.dtl.DTLVardef@17de348org.highwire.dtl.DTLVardef@1d4c420_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundHeart rhythm relies on complex interactions between electrogenic membrane proteins and intracellular Ca2+ signaling in sinoatrial node (SAN) myocytes; however, mechanisms underlying the functional organization of proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, but the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND). MethodsBiochemical co-purification, in vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca2+ imaging, and immunofluorescent and electron microscopy analyses were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking. ResultsIn both mouse and human SANs, caveolae compartmentalized HCN4, Cav1.2, Cav1.3, Cav3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local SR Ca2+ release events (LCRs), which diffuse across [~]15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca2+ current, a negative shift of the HCN current (If) activation curve, and a 40% reduction of Na+/Ca2+-exchanger function, along with [~]2.3-times widening of the sarcolemma-SR distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extra-caveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by LCRs and trigger action potentials. HF remodeling mirrored caveolae disruption leading to NCX1-LCR uncoupling and SND. ConclusionsSAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, potentially demonstrating a new dimension of SAN remodeling and providing a newly recognized target for therapy.

The hearts ability to beat with high precision, with a regular and steady rhythm relies on the synchronised activity of pacemaker cells in the sinoatrial node (SAN), which communicate with one another through gap junctions. This process ensures that electrical impulses are organised and reach a critical mass to ignite the electrical activity of the surrounding atrial tissue and trigger the regular heartbeat. HypothesisWe hypothesise that hyperpolarisation-activated, cyclic nucleotide-gated (HCN) channels play a pivotal role in maintaining this synchronisation process. This idea aligns with the well-established role of HCN channels in stabilising the membrane potential in the voltage range of the slow diastolic depolarisation, counteracting voltage fluctuations and effectively filtering out variations in the beating rate from neighbouring cells. AimWe focus on two specific HCN channel subtypes--HCN1 and HCN4--and their contributions to the rapid synchronisation of pacemaker cells, a phenomenon known as phasic entrainment. Using two HCN channel-mutant mouse models, we dissect the distinct roles of HCN1 and HCN4 in this process. MethodsWe employed patch-clamp electrophysiology to determine phase response curves (PRCs) to predict the ability of single cells to interact in the SAN. Using computer simulations, the behaviour of the SAN at the network level was determined. ResultsWe found that HCN1, but not HCN4, is essential for the fast synchronisation of pacemaker cells in the SAN and propose a mechanism by which HCN1 channels regulate this process. ConclusionThese findings highlight HCN1 as a critical component for ensuring the precise and rapid coordination needed for synchronisation of the SAN, for a regular and consistent heartbeat. Translational perspectiveOur work provides essential insights into the cellular and molecular mechanisms governing SAN function and lays the groundwork for several clinically relevant applications. Understanding how If blockers may affect heart rate and rhythm stability is crucial for assessing potential side effects of current and future subtype-specific HCN channel inhibitors. Moreover, our findings could support new diagnostic strategies for detecting patients at risk of SAN dysfunction. Ultimately, these findings pave the way for innovative therapeutic approaches, including targeted channel modulation and future cell or gene therapies to restore pacemaker stability.

BackgroundAblation strategies for patients with symptomatic atrial fibrillation (AF) and isolated pulmonary veins (PV) vary and their impact on arrhythmia recurrence remains unclear. This prospective randomized German multi--center trial sought to compare two ablation strategies in this patient cohort. MethodsPatients with AF despite durable PV isolation were randomly assigned at seven centers to undergo low-voltage area (LVA) ablation using 3D mapping and irrigated radiofrequency current ablation (group A) or empirical left atrial appendage isolation (LAAI) utilizing the cryoballoon (CB) followed by staged interventional LAA closure (group B). The primary endpoint was freedom from atrial tachyarrhythmias between 91 and 365 days after index ablation. The study was powered for superiority of LAAI compared to LVA. ResultsPatients (40% female, mean age 68.8{+/-}8 years) with paroxysmal (32%) or persistent AF (68%) were randomized to undergo LVA ablation (n=79) or CB guided LAAI (n=82). After a planned interim analysis enrollment was halted on January 10th 2023. In the LAAI group 77/82 LAAs were successfully isolated with subsequent LAAC in 57 patients. Procedure related complications occurred in 4 (5%) and 11 (13.5%) patients in group A and B, respectively (P=0.10). The median follow-up was 367 (IQR 359-378) days. The Kaplan Meier point estimate for the freedom from a primary endpoint event was 51.7% (CI 40.9-65.4%) for group A and 55.5% (CI 44.4-69.2%; p=0.8069). ConclusionsThe present study did not detect superiority of CB guided LAAI over LVA ablation in patients with AF despite durable PVI. It was registered at https://clinicaltrials.gov/study/NCT04056390 Clinical PerspectivesO_ST_ABSWhat is new?C_ST_ABSO_LIThis is the first randomized multi-center study to compare two different ablation strategies in AF patients with durable PVI. C_LIO_LIEmpirical LAAI was not associated with better outcome in comparison to low-voltage area ablation. C_LI What are the clinical implications?O_LILAAI should not be advocated as a stand-alone ablation strategy for patients with AF recurrences after prior ablation. C_LIO_LIThe patient with AF recurrence after prior catheter ablation should be informed that if all PVs are found durably isolated the optimal ablation strategy remains uncertain. C_LI

Voltage-gated sodium (NaV) channels drive cardiac excitability. While NaV1.5 is the primary cardiac isoform, the composition and functional contributions of non-NaV1.5 isoforms in the heart remain unclear. Here, we developed a chemical-genetic mouse model (NaV1.5-GX) in which NaV1.5 can be selectively and reversibly inhibited by acyl- and aryl-sulfonamide compounds (GX drugs). NaV1.5-GX mice exhibited normal cardiac function at baseline, but acute GX drug administration caused profound conduction defects and arrhythmias. Whole-heart optical mapping revealed dose-dependent chamber-specific sensitivity to NaV1.5 inhibition, with the right ventricle (RV) being the most sensitive, followed by the left ventricle (LV), left atrium (LA), and right atrium (RA). Patch-clamp recordings of isolated cardiomyocytes with application of NaV isoform-selective inhibitors showed that NaV1.5 contributed 93% of sodium current in the LV, 81% in the RV and 78% in the LA. Non-NaV1.5 isoforms were differentially enriched across chambers: NaV1.8 in the LV, NaV1.1/1.3 in the RV, and NaV1.2/1.6/1.7 in the atria. These results reveal a surprising chamber-specific isoform landscape of cardiac sodium currents which may underlie the right ventricular predominant phenotype of Brugada syndrome and highlight non-NaV1.5 isoforms as potential mediators of chamber-specific cardiac pathologies and as pharmacological targets.

BackgroundFractionated potential (FP) ablation during atrioventricular nodal reentrant tachycardia (AVNRT), is an effective strategy that minimizes redundant radiofrequency (RF) applications. This study aimed to evaluate the utility of cryoablation targeting FPs to effectively terminate AVNRT while further minimizing redundant cryoapplications. Moreover, we observed what appeared to be compact AVN (cAVN) or proximal His potentials--tiny, dull potentials (TDPs) with continuity to the His potential during sinus rhythm (SR) and AVNRT--in the anteroseptal area. The second aim of this study was to explore the significance of those potentials. MethodsAnalyzed were 53 slow-fast AVNRT patients who underwent ablation procedures. Ultra-high resolution activation maps in the triangle of Koch were obtained during SR (n=34) and AVNRT (n=46). TDPs during SR and AVNRT in the anteroseptal area were identified and annotated using the LUMIPOINT Activation Search tool. ResultsFP areas were observed in 19 patients (56%) during SR and in 46 (100%) during AVNRT. This area corresponded to the AVNRT termination and/or successful ablation site in all, with peak numbers of 8.8{+/-}1.4 during AVNRT and 5.3{+/-}1.3 during SR. The number of ablation points was 3.6{+/-}1.5 for the FP-guided cryoablation (n=32) (Bonferroni corrected P<0.05 vs. anatomical RF; and P<0.05 vs. FP-guided RF), 5.4{+/-}2.1 for the FP-guided RF ablation (n=11) (P=0.0825 vs. anatomical RF), and 8.2{+/-}3.2 for the conventional RF ablation (n=10). Transient AV block occurred in 11 patients (21%). All AV block sites overlapped with the TDP area in the phase just before the His potential during AVNRT and SR, with a confidence setting of [&ge;]24% (35[24-60]%). Conversely, in 42 patients without AV block, no ablation was performed in this area. ConclusionThe FP-guided cryoablation strategy targeting AVNRT termination required fewer cryoapplications than RF ablation. The RF/cryo application in the TDP area during SR and AVNRT posed a risk of AV block.