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Brette, F., S. C. Calaghan, S. Lappin, E. White, J. Colyer, and J.-Y. Le Guennec. Biphasic effects of hyposmotic challenge on excitation-contraction coupling in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 279: H1963-H1971, 2000.--The effects of short (1 min) and long (7-10 min) exposure to hyposmotic solution on excitation-contraction coupling in rat ventricular myocytes were studied. After short exposure, the action potential duration at 90% repolarization ([APD.sup.90]), the intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]][sub.i]) transient amplitude, and contraction increased, whereas the L-type [Ca.sup.2+] current ([I.sub.Ca,L]) amplitude decreased. Fractional sarcoplasmic reticulum (SR) [Ca.sup.2+] release increased but SR [Ca.sup.2+] load did not. After a long exposure, [I.sub.Ca,L], [APD.sub.90], [[Ca.sup.2+]][sub.i] transient amplitude, and contraction decreased. The abbreviation of [APD.sub.90] was partially reversed by 50 [micro]M DIDS, which is consistent with the participation of [Cl.sup.-] current activated by swelling. After 10-min exposure to hyposmotic solution in cells labeled with di-8-aminonaphthylethenylpyridinium, t-tubule patterning remained intact, suggesting the loss of de-t-tubulation was not responsible for the fall in [I.sub.Ca,L]. After long exposure, [Ca.sub.2+] load of the SR was not increased, and swelling had no effect on the site-specific phosphorylation of phospholamban, but fractional SR [Ca.sup.2+] release was depressed. The initial positive inotropic response to hyposmotic challenge may be accounted for by enhanced coupling between [Ca.sup.2+] entry and release. The negative inotropic effect of prolonged exposure can be accounted for by shortening of the action potential duration and a fall in the [I.sub.Ca,L] amplitude. cardiac myocyte; hyposmotic swelling; action potential; sarcoplasmic reticulum; L-type calcium current

MXenes are prototypes of surface tunable 2D materials with vast potential for properties tuning. Accurately characterizing their surface functionalization and its role in electronic structure is crucial, X-ray photoelectron spectroscopy (XPS) being among the go-to methods to do so. Despite extensive use, XPS analysis remains however intricate. Focusing on the benchmark MXene Ti 3 C 2 T z , Density Functional Theory (DFT) calculations of core-level binding energy shifts (BE.s.) are combined with experiments in order to provide a quantitative interpretation of XPS spectra. This approach demonstrates that BE.s. are driven by the complex interplay between chemical, structural, and subtle electronic structure effects preventing analysis from intuitive arguments or comparison with reference materials. In particular, it is shown that O terminations induce the largest BE.s. at Ti 2p levels despite lower electronegativity than F. Additionally, F 1s levels show weak sensitivity to the F local environment, explaining the single contribution in the spectrum, whereas O 1s states are significantly affected by the local surface chemistry. Finally, clear indicators of surface group vacancies are given at Ti 2p and O 1s levels. These results demonstrate the combination of calculations with experiments as a method of the highest value for MXenes XPS spectra analysis, providing guidelines for otherwise complex interpretations., (© 2024 Wiley‐VCH GmbH.)

The exchange protein directly activated by cAMP (EPAC) has been implicated in cardiac proarrhythmic signaling pathways including spontaneous diastolic Ca 2+ leak from sarcoplasmic reticulum and increased action potential duration (APD) in isolated ventricular cardiomyocytes. The action potential (AP) lengthening following acute EPAC activation is mainly due to a decrease of repolarizing steady-state K + current (IK SS ) but the mechanisms involved remain unknown. This study aimed to assess the role of EPAC1 and EPAC2 in the decrease of IK SS and to investigate the underlying signaling pathways. AP and K + currents were recorded with the whole cell configuration of the patch-clamp technique in freshly isolated rat ventricular myocytes. EPAC1 and EPAC2 were pharmacologically activated with 8-(4-chlorophenylthio)-2'- O -methyl-cAMP acetoxymethyl ester (8-CPTAM, 10 µmol/L) and inhibited with R-Ce3F4 and ESI-05, respectively. Inhibition of EPAC1 and EPAC2 significantly decreased the effect of 8-CPTAM on APD and IK SS showing that both EPAC isoforms are involved in these effects. Unexpectedly, calmodulin-dependent protein kinase II (CaMKII) inhibition by AIP or KN-93, and Ca 2+ chelation by intracellular BAPTA, did not impact the response to 8-CPTAM. However, inhibition of PLC/PKC and nitric oxide synthase (NOS)/PKG pathways partially prevents the 8-CPTAM-dependent decrease of IK SS . Finally, the cumulative inhibition of PKC and PKG blocked the 8-CPTAM effect, suggesting that these two actors work along parallel pathways to regulate IK SS upon EPAC activation. On the basis of such findings, we propose that EPAC1 and EPAC2 are involved in APD lengthening by inhibiting a K + current via both PLC/PKC and NOS/PKG pathways. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. NEW & NOTEWORHTY Exchange protein directly activated by cAMP (EPAC) proteins modulate ventricular electrophysiology at the cellular level. Both EPAC1 and EPAC2 isoforms participate in this effect. Mechanistically, PLC/PKC and nitric oxide synthase (NO)/PKG pathways are involved in regulating K + repolarizing current whereas the well-known downstream effector of EPAC, calmodulin-dependent protein kinase II (CaMKII), does not participate. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. Thus, EPAC inhibition may be a new approach to prevent arrhythmias under pathological conditions.

The 32nd Ion Channel Meetings were organized by the Ion Channels Association from September 17 to 20, 2023 in the Occitanie region (Sète). Researchers, post-docs and students from France, Europe and non-European countries came together to present and discuss their work on various themes covering the field of neuroscience, stem cells, hypoxia and pathophysiology cardiac. Through the plenary conference given by Professor Emilio Carbone and the 5 conferences organized by the scientific committee, attention was paid this year to autism, neuromotor and cardiac disorders and tumor aggressive processes. The scientific exchanges were enriched by two general conferences on the biometric analysis of publications related to ion channels and a retrospective presentation of proven cases of scientific fraud. These presentations are summarized in this meeting report., (Copyright 2024, Mary Ann Liebert, Inc., publishers.)

Aims: Pulmonary hypertension (PH) is characterised by an increase in pulmonary arterial pressure, ultimately leading to right ventricular failure and death. We have previously shown that nerve growth factor (NGF) plays a critical role in PH. Our objectives here were to determine whether NGF controls Connexin-43 (Cx43) expression and function in the pulmonary arterial smooth muscle, and whether this mechanism contributes to NGF-induced pulmonary artery hyperreactivity., Methods and Results: NGF activates its TrkA receptor to increase Cx43 expression, phosphorylation, and localization at the plasma membrane in human pulmonary arterial smooth muscle cells, thus leading to enhanced activity of Cx43-dependent GAP junctions as shown by Lucifer Yellow dye assay transfer and fluorescence recovery after photobleaching -FRAP- experiments. Using both in vitro pharmacological and in vivo SiRNA approaches, we demonstrate that NGF-dependent increase in Cx43 expression and activity in the rat pulmonary circulation causes pulmonary artery hyperreactivity. We also show that, in a rat model of PH induced by chronic hypoxia, in vivo blockade of NGF or of its TrkA receptor significantly reduces Cx43 increased pulmonary arterial expression induced by chronic hypoxia and displays preventive effects on pulmonary arterial pressure increase and right heart hypertrophy., Conclusions: Modulation of Cx43 by NGF in pulmonary arterial smooth muscle cells contributes to NGF-induced alterations of pulmonary artery reactivity. Since NGF and its TrkA receptor play a role in vivo in Cx43 increased expression in PH induced by chronic hypoxia, these NGF/Cx43-dependent mechanisms may therefore play a significant role in human PH pathophysiology., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Atrial cardiomyopathy is defined as any complex of structural, architectural, contractile or electrophysiological changes affecting atria, with the potential to produce clinically relevant manifestations. Most of our knowledge about the mechanistic aspects of atrial cardiomyopathy is derived from studies investigating animal models of atrial fibrillation and atrial tissue samples obtained from individuals who have a history of atrial fibrillation. Several noninvasive tools have been reported to characterize atrial cardiomyopathy in patients, which may be relevant for predicting the risk of incident atrial fibrillation and its related outcomes, such as stroke. Here, we provide an overview of the pathophysiological mechanisms involved in atrial cardiomyopathy, and discuss the complex interplay of these mechanisms, including aging, left atrial pressure overload, metabolic disorders and genetic factors. We discuss clinical tools currently available to characterize atrial cardiomyopathy, including electrocardiograms, cardiac imaging and serum biomarkers. Finally, we discuss the clinical impact of atrial cardiomyopathy, and its potential role for predicting atrial fibrillation, stroke, heart failure and dementia. Overall, this review aims to highlight the critical need for a clinically relevant definition of atrial cardiomyopathy to improve treatment strategies., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Background: Brugada syndrome is a significant cause of sudden cardiac death (SCD), but the underlying mechanisms remain hypothetical., Objectives: This study aimed to elucidate this knowledge gap through detailed ex vivo human heart studies., Methods: A heart was obtained from a 15-year-old adolescent boy with normal electrocardiogram who experienced SCD. Postmortem genotyping was performed, and clinical examinations were done on first-degree relatives. The right ventricle was optically mapped, followed by high-field magnetic resonance imaging and histology. Connexin-43 and Na V 1.5 were localized by immunofluorescence, and RNA and protein expression levels were studied. HEK-293 cell surface biotinylation assays were performed to examine Na V 1.5 trafficking., Results: A Brugada-related SCD diagnosis was established for the donor because of a SCN5A Brugada-related variant (p.D356N) inherited from his mother, together with a concomitant NKX2.5 variant of unknown significance. Optical mapping demonstrated a localized epicardial region of impaired conduction near the outflow tract, in the absence of repolarization alterations and microstructural defects, leading to conduction blocks and figure-of-8 patterns. Na V 1.5 and connexin-43 localizations were normal in this region, consistent with the finding that the p.D356N variant does not affect the trafficking, nor the expression of Na V 1.5. Trends of decreased Na V 1.5, connexin-43, and desmoglein-2 protein levels were noted; however, the RT-qPCR results suggested that the NKX2-5 variant was unlikely to be involved., Conclusions: This study demonstrates for the first time that SCD associated with a Brugada-SCN5A variant can be caused by localized functionally, not structurally, impaired conduction., Competing Interests: Funding Support and Author Disclosures This work received financial support from the French Government as part of the “Investments of the Future” program managed by the National Research Agency (ANR-10-IAHU04-LIRYC), the Leducq-Foundation (RHYTHM network, 16CVD02), and the Fondation Coeur et Artères (FC17T2). Dr Barc is supported by the ANR JCJC LEARN (R21006NN, RPV21014NNA). Dr Schott is supported by IRP-, an I-SITE NExT health and engineering initiative (Ecole Centrale & Nantes University) and by the IRP- GAINES funded by INSERM and CNRS. Dr Marionneau is supported by the ANR-16-CE92-0013-01 and the National Institutes of Health (R01-HL148803). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with increased mortality and morbidity. The Exchange Protein directly Activated by cAMP (EPAC), has been implicated in pro-arrhythmic signaling pathways in the atria, but the underlying mechanisms remain unknown. Methods: In this study, we investigated the involvement of EPAC1 and EPAC2 isoforms in the genesis of AF in wild type (WT) mice and knockout (KO) mice for EPAC1 or EPAC2. We also employed EPAC pharmacological modulators to selectively activate EPAC proteins (8-CPT-AM; 10 μM), or inhibit either EPAC1 (AM-001; 20 μM) or EPAC2 (ESI-05; 25 μM). Transesophageal stimulation was used to characterize the induction of AF in vivo in mice. Optical mapping experiments were performed on isolated mouse atria and cellular electrophysiology was examined by whole-cell patch-clamp technique. Results: In wild type mice, we found 8-CPT-AM slightly increased AF susceptibility and that this was blocked by the EPAC1 inhibitor AM-001 but not the EPAC2 inhibitor ESI-05. Consistent with this, in EPAC1 KO mice, occurrence of AF was observed in 3/12 (vs. 4/10 WT littermates) and 4/10 in EPAC2 KO (vs. 5/10 WT littermates). In wild type animals, optical mapping experiments revealed that 8-CPT-AM perfusion increased action potential duration even in the presence of AM-001 or ESI-05. Interestingly, 8-CPT-AM perfusion decreased conduction velocity, an effect blunted by AM-001 but not ESI-05. Patch-clamp experiments demonstrated action potential prolongation after 8-CPT-AM perfusion in both wild type and EPAC1 KO mice and this effect was partially prevented by AM-001 in WT. Conclusion: Together, these results indicate that EPAC1 and EPAC2 signaling pathways differentially alter atrial electrophysiology but only the EPAC1 isoform is involved in the genesis of AF. Selective blockade of EPAC1 with AM-001 prevents AF in mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Guillot, Boileve, Walton, Harfoush, Conte, Sainte-Marie, Charron, Bernus, Recalde, Sallé, Brette and Lezoualc’h.)

The surface functionalization of 2D transition metal carbides or nitrides, so-called MXenes, is one of the fundamental levers allowing to deeply modify their physicochemical properties. Beyond new approaches to control this pivotal parameter, the ability to unambiguously assess their surface chemistry is thus key to expand the application fields of this large class of 2D materials. Using a combination of experiments and state of the art density functional theory calculations, we show that the NMR signal of the carbon─the element common to all MXene carbides and corresponding MAX phase precursors─is extremely sensitive to the MXene functionalization, although carbon atoms are not directly bonded to the surface groups. The simulations include the orbital part to the NMR shielding and the contribution from the Knight shift, which is crucial to achieve good correlation with the experimental data, as demonstrated on a set of reference MXene precursors. Starting with the Ti 3 C 2 T x MXene benchmark system, we confirm the high sensitivity of the 13 C NMR shift to the exfoliation process. Developing a theoretical protocol to straightforwardly simulate different surface chemistries, we show that the 13 C NMR shift variations can be quantitatively related to different surface compositions and number of surface chemistry variants induced by the different etching agents. In addition, we propose that the etching agent affects not only the nature of the surface groups but also their spatial distribution. The direct correlation between surface chemistry and 13 C NMR shift is further confirmed on the V 2 CT x , Mo 2 CT x , and Nb 2 CT x MXenes.

Cardiac excitation-contraction coupling can be different between regions of the heart. Little is known at the atria level, specifically in different regions of the left atrium. This is important given the role of cardiac myocytes from the pulmonary vein sleeves, which are responsible for ectopic activity during atrial fibrillation. In this study, we present a new method to isolate atrial cardiac myocytes from four different regions of the left atrium of a large animal model, sheep, highly relevant to humans. Using collagenase/protease we obtained calcium-tolerant atrial cardiac myocytes from the epicardium, endocardium, free wall and pulmonary vein regions. Calcium transients were slower (time to peak and time to decay) in free wall and pulmonary vein myocytes compared to the epicardium and endocardium. This is associated with lower t-tubule density. Overall, these results suggest regional differences in calcium transient and t-tubule density across left atria, which may play a major role in the genesis of atrial fibrillation.

Background: Atrial fibrillation is the most sustained form of arrhythmia in the human population that leads to important electrophysiological and structural cardiac remodeling as it progresses into a chronic form. Calcium is an established key player of cellular electrophysiology in the heart, yet to date, there is no information that maps calcium signaling across the left atrium., Objective: The aim of this study is to determine whether calcium signaling is homogenous throughout the different regions of the left atrium. This work tests the hypothesis that differences across the healthy left atrium contribute to a unique, region-dependent calcium cycling and participates in the pro-arrhythmic activity during atrial fibrillation., Methods: An animal model relevant to human cardiac function (the sheep) was used to characterize both the electrical activity and the calcium signaling of three distinct left atrium regions (appendage, free wall and pulmonary veins) in control conditions and after acetylcholine perfusion (5 μM) to induce acute atrial fibrillation. High-resolution dual calcium-voltage optical mapping on the left atria of sheep was performed to explore the spatiotemporal dynamics of calcium signaling in relation to electrophysiological properties., Results: Action potential duration (at 80% repolarization) was not significantly different in the three regions of interest for the three pacing sites. In contrast, the time to 50% calcium transient decay was significantly different depending on the region paced and recorded. Acetylcholine perfusion and burst pacing-induced atrial fibrillation when pulmonary veins and appendage regions were paced but not when the free wall region was. Dantrolene (a ryanodine receptor blocker) did not reduce atrial fibrillation susceptibility., Conclusion: These data provide the first evidence of heterogenous calcium signaling across the healthy left atrium. Such basal regional differences may be exacerbated during the progression of atrial fibrillation and thus play a crucial role in focal arrhythmia initiation without ryanodine receptor gating modification.

The TREK-1 channel belongs to the TREK subfamily of two-pore domains channels that are activated by stretch and polyunsaturated fatty acids and inactivated by Protein Kinase A phosphorylation. The activation of this potassium channel must induce a hyperpolarization of the resting membrane potential and a shortening of the action potential duration in neurons and cardiac cells, two phenomena being beneficial for these tissues in pathological situations like ischemia-reperfusion. Surprisingly, the physiological role of TREK-1 in cardiac function has never been thoroughly investigated, very likely because of the lack of a specific inhibitor. However, possible roles have been unraveled in pathological situations such as atrial fibrillation worsened by heart failure, right ventricular outflow tract tachycardia or pulmonary arterial hypertension. The inhomogeneous distribution of TREK-1 channel within the heart reinforces the idea that this stretch-activated potassium channel might play a role in cardiac areas where the mechanical constraints are important and need a particular protection afforded by TREK-1. Consequently, the main purpose of this mini review is to discuss the possible role played by TREK -1 in physiological and pathophysiological conditions and its potential role in mechano-electrical feedback. Improved understanding of the role of TREK-1 in the heart may help the development of promising treatments for challenging cardiac diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bechard, Bride, Le Guennec, Brette and Demion.)

MRI is widely used in cardiology to characterize the structure and function of the heart. Currently, gadolinium-based contrast agents are widely used to improve sensitivity and specificity of diagnostic images. Recently, Manganese, a calcium analogue, has emerged as a complementary contrast agent with the potential to reveal remaining viable cells within altered tissue. Imaging applications may be limited by substantial toxicity of manganese. Indeed, cardiac safety of manganese is not yet comprehensively assessed. In this study we investigated the effect of MnCl 2 (1-100 µM) on cardiac function. Hemodynamic function was determined ex vivo using an isolated working rat heart preparation. HL-1 cardiac myocytes were used to investigate cell viability (calcein AM) and calcium cycling (Cal-520 a.m.). Rat ventricular cardiomyocytes were dissociated by enzymatic digestion. Action potentials and calcium currents were recorded using the patch clamp technique. MRI experiments were performed at 1.5T on formalin-fixed rat hearts, previously perfused with MnCl 2 . MnCl 2 perfusion from 1 up to 100 µM in isolated working hearts did not alter left ventricular hemodynamic parameters. Contractility and relaxation index were not altered up to 50 µM MnCl 2 . In HL-1 cardiac myocytes, incubation with increasing concentrations of MnCl 2 did not impact cell viability. The amplitude of the calcium transients were significantly reduced at 50 and 100 µM MnCl 2 . In freshly isolated ventricular myocytes, action potential duration at 20, 50 and 90% of repolarization were not modified up to 10 µM of MnCl 2 . L-type calcium current amplitude was significantly decreased by 50 and 100 µM of MnCl 2 . MRI on heart perfused with 25 and 100 µM of MnCl 2 showed a dose dependent decrease in the T1 relaxation time. In conclusion, our results show that low concentrations of MnCl 2 (up to 25 µM) can be used as a contrast agent in MRI, without significant impact on cardiac hemodynamic or electrophysiology parameters., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lamonzie, Vaillant, Abell, Charron, El Hamrani, Quesson and Brette.)

Air pollution is an environmental hazard that is associated with cardiovascular dysfunction. Phenanthrene is a three-ringed polyaromatic hydrocarbon that is a significant component of air pollution and crude oil and has been shown to cause cardiac dysfunction in marine fishes. We investigated the cardiotoxic effects of phenanthrene in zebrafish (Danio rerio), an animal model relevant to human cardiac electrophysiology, using whole-cell patch-clamp of ventricular cardiomyocytes. First, we show that phenanthrene significantly shortened action potential duration without altering resting membrane potential or upstroke velocity (dV/dt). L-type Ca2+ current was significantly decreased by phenanthrene, consistent with the decrease in action potential duration. Phenanthrene blocked the hERG orthologue (zfERG) native current, IKr, and accelerated IKr deactivation kinetics in a dose-dependent manner. Furthermore, we show that phenanthrene significantly inhibits the protective IKr current envelope, elicited by a paired ventricular AP-like command waveform protocol. Phenanthrene had no effect on other IK. These findings demonstrate that exposure to phenanthrene shortens action potential duration, which may reduce refractoriness and increase susceptibility to certain arrhythmia triggers, such as premature ventricular contractions. These data also reveal a previously unrecognized mechanism of polyaromatic hydrocarbon cardiotoxicity on zfERG by accelerating deactivation and decreasing IKr protective current., (© 2021 Kompella et al.)

Transient receptor potential (TRP) channels are nonselective cationic channels that are generally Ca 2+ permeable and have a heterogeneous expression in the heart. In the myocardium, TRP channels participate in several physiological functions, such as modulation of action potential waveform, pacemaking, conduction, inotropy, lusitropy, Ca 2+ and Mg 2+ handling, store-operated Ca 2+ entry, embryonic development, mitochondrial function and adaptive remodelling. Moreover, TRP channels are also involved in various pathological mechanisms, such as arrhythmias, ischaemia-reperfusion injuries, Ca 2+ -handling defects, fibrosis, maladaptive remodelling, inherited cardiopathies and cell death. In this Review, we present the current knowledge of the roles of TRP channels in different cardiac regions (sinus node, atria, ventricles and Purkinje fibres) and cells types (cardiomyocytes and fibroblasts) and discuss their contribution to pathophysiological mechanisms, which will help to identify the best candidates for new therapeutic targets among the cardiac TRP family.

Background Papillary muscles are an important source of ventricular tachycardia (VT). Yet little is known about the role of the right ventricular (RV) endocavity structure, the moderator band (MB). The aim of this study was to determine the characteristics of the MB that may predispose to arrhythmia substrates. Methods Ventricular wedge preparations with intact MBs were studied from humans (n=2) and sheep (n=15; 40-50 kg). RV endocardium was optically mapped, and electrical recordings were measured along the MB and septum. S1S2 pacing of the RV free wall, MB, or combined S1-RV S2-MB sites were assessed. Human (n=2) and sheep (n=4) MB tissue constituents were assessed histologically. Results The MB structure was remarkably organized as 2 excitable, yet uncoupled compartments of myocardium and Purkinje. In humans, action potential duration heterogeneity between MB and RV myocardium was found (324.6±12.0 versus 364.0±8.4 ms; P<0.0001). S1S2-MB pacing induced unidirectional propagation via MB myocardium, permitting sustained macroreentrant VT. In sheep, the incidence of VT for RV, MB, and S1-RV S2-MB pacing was 1.3%, 5.1%, and 10.3%. Severing the MB led to VT termination, confirming a primary arrhythmic role. Inducible preparations had shorter action potential duration in the MB than RV (259.3±45.2 versus 300.7±38.5 ms; P<0.05), whereas noninducible preparations showed no difference (312.0±30.3 versus 310.0±24.6 ms, respectively). Conclusions The MB presents anatomic and electrical compartmentalization between myocardium and Purkinje fibers, providing a substrate for macroreentry. The vulnerability to sustain VT via this mechanism is dependent on MB structure and action potential duration gradients between the RV free wall and MB.