Saïd F.A. Askar

Epicardial substrate mapping for ventricular tachycardia ablation in patients with non-ischaemic cardiomyopathy: a new algorithm to differentiate between scar and viable myocardium developed by simultaneous integration of computed tomography and contrast-enhanced magnetic resonance imaging.

AIMS During epicardial electroanatomical mapping (EAM), it is difficult to differentiate between fibrosis and fat, as both exhibit attenuated bipolar voltage (BV). The purpose of this study was to assess whether unipolar voltage (UV), BV, and electrogram characteristics (EC) can distinguish fibrosis from viable myocardium and fat during epicardial EAM for ventricular tachycardia (VT) ablation in non-ischaemic cardiomyopathy (NICM). METHODS AND RESULTS Ten NICM patients (7 males, 56 ± 13 years) with VT underwent epicardial EAM with real-time integration of computed tomography-derived epicardial fat and contrast-enhanced MRI-derived scar. Bipolar voltage (filtered 30-400 Hz), UV (filtered 1-240 Hz), and EC (duration and morphology) were correlated with the presence of fat and scar. At sites devoid of fat, the optimal cutoff values to differentiate between scar and myocardium were 1.81 mV for BV and 7.95 mV for UV. Bipolar voltage, UV, and electrogram duration >50 ms distinguished scar from myocardium in areas covered with <2.8 mm fat (all P < 0.001), but not ≥ 2.8 mm fat. In contrast, electrogram morphology-characteristics could also detect scar covered with ≥ 2.8 mm fat (P = 0.001). A newly developed three-step algorithm combining electrogram morphology, duration, and UV could correctly identify scar with a sensitivity of 75%. Unipolar voltage but not BV could detect intramural scar in the absence of fat. CONCLUSIONS Both BV ≤ 1.81 mV and UV ≤ 7.95 mV are useful for detection of scar during epicardial EAM, in the absence of ≥ 2.8 mm fat. However, EC can be used to detect scar covered with fat. A newly developed algorithm combining UV and EC can differentiate between scar and viable myocardium. Unipolar voltage but not BV could detect intramural scar.

Light-induced termination of spiral wave arrhythmias by optogenetic engineering of atrial cardiomyocytes

AIMS Atrial fibrillation (AF) is the most common cardiac arrhythmia and often involves reentrant electrical activation (e.g. spiral waves). Drug therapy for AF can have serious side effects including proarrhythmia, while electrical shock therapy is associated with discomfort and tissue damage. Hypothetically, forced expression and subsequent activation of light-gated cation channels in cardiomyocytes might deliver a depolarizing force sufficient for defibrillation, thereby circumventing the aforementioned drawbacks. We therefore investigated the feasibility of light-induced spiral wave termination through cardiac optogenetics. METHODS AND RESULTS Neonatal rat atrial cardiomyocyte monolayers were transduced with lentiviral vectors encoding light-activated Ca(2+)-translocating channelrhodopsin (CatCh; LV.CatCh∼eYFP↑) or eYFP (LV.eYFP↑) as control, and burst-paced to induce spiral waves rotating around functional cores. Effects of CatCh activation on reentry were investigated by optical and multi-electrode array (MEA) mapping. Western blot analyses and immunocytology confirmed transgene expression. Brief blue light pulses (10 ms/470 nm) triggered action potentials only in LV.CatCh∼eYFP↑-transduced cultures, confirming functional CatCh-mediated current. Prolonged light pulses (500 ms) resulted in reentry termination in 100% of LV.CatCh∼eYFP↑-transduced cultures (n = 31) vs. 0% of LV.eYFP↑-transduced cultures (n = 11). Here, CatCh activation caused uniform depolarization, thereby decreasing overall excitability (MEA peak-to-peak amplitude decreased 251.3 ± 217.1 vs. 9.2 ± 9.5 μV in controls). Consequently, functional coresize increased and phase singularities (PSs) drifted, leading to reentry termination by PS-PS or PS-boundary collisions. CONCLUSION This study shows that spiral waves in atrial cardiomyocyte monolayers can be terminated effectively by a light-induced depolarizing current, produced by the arrhythmogenic substrate itself, upon optogenetic engineering. These results provide proof-of-concept for shockless defibrillation.

Electrical activation of sinus venosus myocardium and expression patterns of RhoA and Isl-1 in the chick embryo

Electrical Activity and RhoA in the Embryo. Introduction: Myocardium at the venous pole (sinus venosus) of the heart has gained clinical interest as arrhythmias can be initiated from this area. During development, sinus venosus myocardium is incorporated to the primary heart tube and expresses different markers than primary myocardium. We aimed to elucidate the development of sinus venosus myocardium, including the sinoatrial node (SAN), by studying expression patterns of RhoA in relation to other markers, and by studying electrical activation patterns of the developing sinus venosus myocardium.

Atrium-Specific Kir3.x Determines Inducibility, Dynamics and Termination of Fibrillation by Regulating Restitution-Driven Alternans

Background— Atrial fibrillation is the most common cardiac arrhythmia. Ventricular proarrhythmia hinders pharmacological atrial fibrillation treatment. Modulation of atrium-specific Kir3.x channels, which generate a constitutively active current (IK,ACh-c) after atrial remodeling, might circumvent this problem. However, it is unknown whether and how IK,ACh-c contributes to atrial fibrillation induction, dynamics, and termination. Therefore, we investigated the effects of IK,ACh-c blockade and Kir3.x downregulation on atrial fibrillation. Methods and Results— Neonatal rat atrial cardiomyocyte cultures and intact atria were burst paced to induce reentry. To study the effects of Kir3.x on action potential characteristics and propagation patterns, cultures were treated with tertiapin or transduced with lentiviral vectors encoding Kcnj3- or Kcnj5-specific shRNAs. Kir3.1 and Kir3.4 were expressed in atrial but not in ventricular cardiomyocyte cultures. Tertiapin prolonged action potential duration (APD; 54.7±24.0 to 128.8±16.9 milliseconds; P<0.0001) in atrial cultures during reentry, indicating the presence of IK,ACh-c. Furthermore, tertiapin decreased rotor frequency (14.4±7.4 to 6.6±2.0 Hz; P<0.05) and complexity (6.6±7.7 to 0.6±0.8 phase singularities; P<0.0001). Knockdown of Kcnj3 or Kcnj5 gave similar results. Blockade of IK,ACh-c prevented/terminated reentry by prolonging APD and changing APD and conduction velocity restitution slopes, thereby altering the probability of APD alternans and rotor destabilization. Whole-heart mapping experiments confirmed key findings (eg, >50% reduction in atrial fibrillation inducibility after IK,ACh-c blockade). Conclusions— Atrium-specific Kir3.x controls the induction, dynamics, and termination of fibrillation by modulating APD and APD/conduction velocity restitution slopes in atrial tissue with IK,ACh-c. This study provides new molecular and mechanistic insights into atrial tachyarrhythmias and identifies Kir3.x as a promising atrium-specific target for antiarrhythmic strategies.

Engraftment Patterns of Human Adult Mesenchymal Stem Cells Expose Electrotonic and Paracrine Proarrhythmic Mechanisms in Myocardial Cell Cultures

Background—After intramyocardial injection, mesenchymal stem cells (MSCs) may engraft and influence host myocardium. However, engraftment rate and pattern of distribution are difficult to control in vivo, hampering assessment of potential adverse effects. In this study, the role of the engraftment patterns of MSCs on arrhythmicity in controllable in vitro models is investigated. Methods and Results—Cocultures of 4×105 neonatal rat cardiomyocytes and 7% or 28% adult human MSCs (hMSCs) in diffuse or clustered distribution patterns were prepared. Electrophysiological effects were studied by optical mapping and patch-clamping. In diffuse cocultures, hMSCs dose-dependently decreased neonatal rat cardiomyocyte excitability, slowed conduction, and prolonged action potential duration until 90% repolarization (APD90). Triggered activity (14% versus 0% in controls) and increased inducibility of re-entry (53% versus 6% in controls) were observed in 28% hMSC cocultures. MSC clusters increased APD90, slowed conduction locally, and increased re-entry inducibility (23%), without increasing triggered activity. Pharmacological heterocellular electric uncoupling increased excitability and conduction velocity to 133% in 28% hMSC cocultures, but did not alter APD90. Transwell experiments showed that hMSCs dose-dependently increased APD90, APD dispersion, inducibility of re-entry and affected specific ion channel protein levels, whereas excitability was unaltered. Incubation with hMSC–derived exosomes did not increase APD in neonatal rat cardiomyocyte cultures. Conclusions—Adult hMSCs affect arrhythmicity of neonatal rat cardiomyocyte cultures by heterocellular coupling leading to depolarization–induced conduction slowing and by direct release of paracrine factors that negatively affect repolarization rate. The extent of these detrimental effects depends on the number and distribution pattern of hMSCs. These results suggest that caution should be urged against potential adverse effects of myocardial hMSC engraftment.

Similar arrhythmicity in hypertrophic and fibrotic cardiac cultures caused by distinct substrate-specific mechanisms

AIMS Cardiac hypertrophy and fibrosis are associated with potentially lethal arrhythmias. As these substrates often occur simultaneously in one patient, distinguishing between pro-arrhythmic mechanisms is difficult. This hampers understanding of underlying pro-arrhythmic mechanisms and optimal treatment. This study investigates and compares arrhythmogeneity and underlying pro-arrhythmic mechanisms of either cardiac hypertrophy or fibrosis in in vitro models. METHODS AND RESULTS Fibrosis was mimicked by free myofibroblast (MFB) proliferation in neonatal rat ventricular monolayers. Cultures with inhibited MFB proliferation were used as control or exposed to phenylephrine to induce hypertrophy. At Day 9, cultures were studied with patch-clamp and optical-mapping techniques and assessed for protein expression. In hypertrophic (n = 111) and fibrotic cultures (n = 107), conduction and repolarization were slowed. Triggered activity was commonly found in these substrates and led to high incidences of spontaneous re-entrant arrhythmias [67.5% hypertrophic, 78.5% fibrotic vs. 2.9% in controls (n = 102)] or focal arrhythmias (39.1, 51.7 vs. 8.8%, respectively). Kv4.3 and Cx43 protein expression levels were decreased in hypertrophy but unaffected in fibrosis. Depolarization of cardiomyocytes (CMCs) was only found in fibrotic cultures (-48 ± 7 vs. -66 ± 7 mV in control, P < 0.001). L-type calcium-channel blockade prevented arrhythmias in hypertrophy, but caused conduction block in fibrosis. Targeting heterocellular coupling by low doses of gap-junction uncouplers prevented arrhythmias by accelerating repolarization only in fibrotic cultures. CONCLUSION Cultured hypertrophic or fibrotic myocardial tissues generated similar focal and re-entrant arrhythmias. These models revealed electrical remodelling of CMCs as a pro-arrhythmic mechanism of hypertrophy and MFB-induced depolarization of CMCs as a pro-arrhythmic mechanism of fibrosis. These findings provide novel mechanistic insight into substrate-specific arrhythmicity.

Constitutively Active Acetylcholine-Dependent Potassium Current Increases Atrial Defibrillation Threshold by Favoring Post-Shock Re-Initiation

Electrical cardioversion (ECV), a mainstay in atrial fibrillation (AF) treatment, is unsuccessful in up to 10–20% of patients. An important aspect of the remodeling process caused by AF is the constitutive activition of the atrium-specific acetylcholine-dependent potassium current (IK,ACh → IK,ACh-c), which is associated with ECV failure. This study investigated the role of IK,ACh-c in ECV failure and setting the atrial defibrillation threshold (aDFT) in optically mapped neonatal rat cardiomyocyte monolayers. AF was induced by burst pacing followed by application of biphasic shocks of 25–100 V to determine aDFT. Blocking IK,ACh-c by tertiapin significantly decreased DFT, which correlated with a significant increase in wavelength during reentry. Genetic knockdown experiments, using lentiviral vectors encoding a Kcnj5-specific shRNA to modulate IK,ACh-c, yielded similar results. Mechanistically, failed ECV was attributed to incomplete phase singularity (PS) removal or reemergence of PSs (i.e. re-initiation) through unidirectional propagation of shock-induced action potentials. Re-initiation occurred at significantly higher voltages than incomplete PS-removal and was inhibited by IK,ACh-c blockade. Whole-heart mapping confirmed our findings showing a 60% increase in ECV success rate after IK,ACh-c blockade. This study provides new mechanistic insight into failing ECV of AF and identifies IK,ACh-c as possible atrium-specific target to increase ECV effectiveness, while decreasing its harmfulness.

Forced fusion of human ventricular scar cells with cardiomyocytes suppresses arrhythmogenicity in a co-culture model

AIMS Fibrosis increases arrhythmogenicity in myocardial tissue by causing structural and functional disruptions in the cardiac syncytium. Forced fusion of fibroblastic cells with adjacent cardiomyocytes may theoretically resolve these disruptions. Therefore, the electrophysiological effects of such electrical and structural integration of fibroblastic cells into a cardiac syncytium were studied. METHODS AND RESULTS Human ventricular scar cells (hVSCs) were transduced with lentiviral vectors encoding enhanced green fluorescent protein alone (eGFP↑-hVSCs) or together with the fusogenic vesicular stomatitis virus G protein (VSV-G/eGFP↑-hVSCs) and subsequently co-cultured (1:4 ratio) with neonatal rat ventricular cardiomyocytes (NRVMs) in confluent monolayers yielding eGFP↑- and VSV-G/eGFP↑-co-cultures, respectively. Cellular fusion was induced by brief exposure to pH = 6.0 medium. Optical mapping experiments showed eGFP↑-co-cultures to be highly arrhythmogenic [43.3% early afterdepolarization (EAD) incidence vs. 7.7% in control NRVM cultures, P < 0.0001], with heterogeneous prolongation of action potential (AP) duration (APD). Fused VSV-G/eGFP↑-co-cultures displayed markedly lower EAD incidence (4.6%, P < 0.001) than unfused co-cultures, associated with decreases in APD, APD dispersion, and decay time of cytosolic Ca(2+) waves. Heterokaryons strongly expressed connexin43 (Cx43). Also, maximum diastolic potential in co-cultures was more negative after fusion, while heterokaryons exhibited diverse mixed NRVM/hVSC whole-cell current profiles, but consistently showed increased outward Kv currents compared with NRVMs or hVSCs. Inhibition of Kv channels by tetraethylammonium chloride abrogated the anti-arrhythmic effects of fusion in VSV-G/eGFP↑-co-cultures raising EAD incidence from 7.9 to 34.2% (P < 0.001). CONCLUSION Forced fusion of cultured hVSCs with NRVMs yields electrically functional heterokaryons and reduces arrhythmogenicity by preventing EADs, which is, at least partly, attributable to increased repolarization force.

QRS prolongation after premature stimulation is associated with polymorphic ventricular tachycardia in nonischemic cardiomyopathy: Results from the Leiden Nonischemic Cardiomyopathy Study

BACKGROUND Progressive activation delay after premature stimulation has been associated with ventricular fibrillation in nonischemic cardiomyopathy (NICM). OBJECTIVES The objectives of this study were (1) to investigate prolongation of the paced QRS duration (QRSd) after premature stimulation as a marker of activation delay in NICM, (2) to assess its relation to induced ventricular arrhythmias, and (3) to analyze its underlying substrate by late gadolinium enhancement cardiac magnetic resonance imaging (LGE-CMR) and endomyocardial biopsy. METHODS Patients with NICM were prospectively enrolled in the Leiden Nonischemic Cardiomyopathy Study and underwent a comprehensive evaluation including LGE-CMR, electrophysiology study, and endomyocardial biopsy. Patients without structural heart disease served as controls for electrophysiology study. RESULTS Forty patients with NICM were included (mean age 57 ± 14 years; 33 men [83%]; left ventricular ejection fraction 30% ± 13%). After the 400-ms drive train and progressively premature stimulation, the maximum increase in QRSd was larger in patients with NICM than in controls (35 ± 18 ms vs. 23 ± 12 ms; P = .005) and the coupling interval window with QRSd prolongation was wider (47 ± 23 ms vs. 31 ± 14 ms; P = .005). The maximum paced QRSd exceeded the ventricular effective refractory period, allowing for pacing before the offset of the QRS complex in 20 of 39 patients with NICM vs. 1 of 20 controls (P < .001). In patients with NICM, QRSd prolongation was associated with the inducibility of polymorphic ventricular tachycardia (16 of 39 patients) and was related to long, thick strands of fibrosis in biopsies, but not to focal enhancement on LGE-CMR. CONCLUSION QRSd is a simple parameter used to quantify activation delay after premature stimulation, and its prolongation is associated with the inducibility of polymorphic ventricular tachycardia and with the pattern of myocardial fibrosis in biopsies.

Response to the letter by Wolkowicz et al.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prolongation of minimal action potential duration in sustained fibrillation decreases complexity by transient destabilization