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Background Cardiac electroporation is a promising novel non-thermal ablation method, gaining significant interest with recent first-in-man data suggesting effective cardiac lesion generation with no collateral damage. Nevertheless, significant knowledge gaps exist regarding its electrophysiological consequences in cardiomyocytes, including; cell specificity, protocol optimization, irreversibility threshold, recovery time-constants, and the mechanistic nature of its cytolytic and anti-arrhythmic properties. Purpose Establishing an innovative in-vitro model for the study of cardiac electroporation-ablation, utilizing human induced pluripotent stem cells (hiPSCs). Methods and results Healthy-control hiPSC-derived cardiomyocytes were enzymatically dissociated and seeded as circular cell sheets (hiPSC-CCSs). Electroporation-ablation experiments were performed using a custom designed high-frequency electroporation (HF-EP) generator. Two needle-shaped electrodes were used for HF-EP delivery (Figure 1). Subsequently, detailed voltage- and Ca2+-mapping studies of the hiPSC-CCSs were conducted (Figure 2). HF-EP application resulted in the generation of electrically isolated lesions within the hiPSC-CCSs (Figure 3). Further characterization of the temporal changes and electrophysiological properties following electroporation revealed that; (1) lesions persisted over prolonged periods of time (days), indicating irreversible electroporation, (2) a temporal decrease in lesion dimensions was noted, consistent with a significant reversible electroporation component (Figures 3–5), (3) most tissue recovery had occurred within the first 15 minutes following electroporation, with little recovery beyond that time-frame, (4) increasing pulse-number augmented lesion area as well as the proportion of irreversible damage, and (5) electroporation sensitization was achieved by increasing extracellular Ca2+, indicating its crucial role in electroporation cytolysis, potentially via direct cellular toxicity and apoptosis facilitation (Figures 5–6). Finally, evaluating for HF-EP anti-arrhythmic properties, we targeted multiple rotors or focal triggered-activity generated in the hiPSC-CCSs. HF-EP application generated sustained line-blocks, isolating arrhythmogenic substrates within the hiPSC-CCSs while blocking the propagation of arrhythmic wavefronts (Figure 7). Conclusion Our results demonstrate the ability to study cardiac electroporation utilizing hiPSC-derived cardiomyocytes, provide novel insights into its temporal and electrophysiological characteristics, facilitate electroporation protocol optimization, screen for potential electroporation sensitizers, and to study its mechanistic nature and anti-arrhythmic properties. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Division of Cardiology, and Tamman Cardiovascular Research Institute, Leviev Heart Center, Sheba Medical Center - Tel Hashomer, Ramat-Gan, Israel Figures 1–4Figures 5–7

Background One of the major barriers to an improved mechanistic understanding of atrial fibrillation (AF), and thus in the pipeline of drug development, has been a lack of appropriate tissue models, especially in small animals. Aim We propose an advanced anatomical ex-vivo model based on rat atria for acute assessment of AF susceptibility. This novel model could yield a better understanding of arrhythmia mechanisms as well as the development of potential therapeutic strategies for the prevention or termination of atrial arrhythmias. Methods Wistar rats atria (N=25) were isolated, flattened and pinned to a custom-made silicon plate. Atria were superfused with an oxygenized Tyrode's solution. Tissues were then loaded with a voltage-sensitive dye and mapped using a high-resolution optical mapping system. AF was induced with 1uM carbamylcholine (N=23) coupled with pacing maneuvers and treated with 30uM Vernakalant (N=10) or 10uM Flecainide (N=10). Finally, the feasibility of a new ablation technique (electroporation) was evaluated. Results Optical mapping results suggested that the superfusion procedure led to a fast atrial recovery. Sinus activity was conserved for all atria for a long period. All the anatomical landmarks were clearly visualized. The acquired optical signals were analyzed during sinus rhythm and pacing, which allowed the creation of detailed activation maps and measurements of action potential duration (APD) and conduction velocity (CV) at different pacing rates. The resulting APD restitution curves revealed electrical excitation at high pacing rates (cycle length between 50ms and 300ms) with a relatively flattened curve. AF was successfully induced and optically mapping confirmed the presence of reentrant activity. AF was successfully treated using Vernacalant and Flecainide. Finally, we demonstrated the feasibility of a new ablation approach (electroporation) for creation of a continuous linear lesion serving as a functional block. Conclusion The isolated superfused atria model, coupled with voltage-sensitive dyes, can be utilized for long-term high-resolution functional imaging of the atria during sinus rhythm, pacing and arrhythmogenic activity. This allows the study of the atrial electrophysiological properties, the mechanisms involved in AF initiation, perpetuation, and termination as well as the study of drug and new ablation modalities. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council (ERC) Spontaneous activation of isolated atria

Background and aim Epicardial fat (eFat) has been linked to atrial remodeling and fibrillation (AF). Small extracellular vesicles (sEVs) are heterogeneous membrane vesicles released by all cell types. They can both protect and damage tissues by the delivery of multiple different messengers. Surprisingly, the role of sEVs in the pathogenesis of AF has not been studied. Thus, we aimed to determine whether sEVs derived from eFat play a role in the pathogenesis of AF. Methods and results We collected eFat specimens from patients with and without chronic or paroxysmal AF undergoing open-heart surgery. Isolated eFat specimens were cut into small pieces and incubated as organ cultures. We isolated sEVs from the medium of the explant by differential ultra-centrifugation, high-density gradient or size exclusion chromatography (SEC), and characterized vesicle size distribution, morphology, specific markers, histology and molecular cargo. Immunostaining for macrophage accumulation, fibrosis and apoptosis confirmed the pro-inflammatory and pro-fibrotic properties of eFat sEVs from patients with AF (Fig. 1). eFat sEVs labeled with PKH26 were massively up taken by endothelial cells (Fig. 2). Real-time PCR showed an increased level of oxidative stress genes in endothelial cells. eFat sEVs from patients with AF caused more fibrosis after injection into rat hearts than those without AF. (Fig. 3). Finally, while eFat sEVs from patients with and without AF induced shorter action potential duration, only eFat sEVs from patients with AF induced sustained re-entry (rotor) in human induced pluripotent stem cell (iPS)-derived cardiomyocytes (Fig. 4) Conclusion We show, for the first time, that sEVs from eFat of patients with AF demonstrate unique pro-inflammatory, pro-fibrotic and pro-arrhythmic properties. Our findings suggest that eFat sEVs can induce cellular, molecular and electrophysiological remodeling that can subsequently lead to the development of AF. Funding Acknowledgement Type of funding source: None

Background The advent of human pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) provided exciting tools for cardiovascular physiological studies, disease modeling and drug testing applications. Current platforms for studying the mechanical properties of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as single-cells do not measure forces directly, require numerous assumptions, and cannot study cell mechanics at different loading conditions. Objective To establish a novel platform to assess the active and passive mechanical properties of single-cell hPSC-CMs at different loading conditions and to demonstrate the potential of this approach for drug testing and disease modeling applications. Methods and results To allow morphological maturation, hPSC-CMs were treated with Tri-iodo-thyronine hormone, dexamethasone and Insulin-like growth factor-1. The hPSC-CM were then lifted and attached to a highly sensitive optical-force transducer and a piezoelectric length controller and electrically-stimulated. The attached hPSC-CM remained intact and contractile allowing evaluation of their passive and active mechanical properties. Utilizing this technique, single-cell hPSC-CMs exhibited positive length-tension (Frank-Starling) relationships, and appropriate inotropic, klinotropic, and lusitropic changes in response to treatment with isoproterenol. The unique potential of the approach for drug testing and disease modeling was exemplified by treating the cells with doxorubicin (a potential cardiotoxic anti-cancer agent) and omecamtiv mecarbil (a positive ionotropic drug currently in stage 3 clinical trial). The results of these studies recapitulated the drugs' known actions to suppress (doxorubicin) and augment (omecamtiv mecarbil at low dose) cardiomyocyte contractility. Finally, novel insights were gained regarding the cellular effects of these drugs as doxorubicin treatment led to cellular mechanical alternans and high doses of omecamtiv mecarbil suppressed contractility and worsened the cellular diastolic properties. Conclusion A novel method that allows direct active and passive force measurements from single hPSC-CMs at different loading conditions for the first time was established and validated. Our results highlight the potential implications of this novel approach for pharmacological studies and disease modeling studies. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): European Research Council

The emerging technology of optogenetics uses optical and genetic means to monitor and modulate the electrophysiological properties of excitable tissues. While transforming the field of neuroscience, the technology has recently gained popularity also in the cardiac arena. Here, we describe the basic principles of optogenetics, the available and evolving optogenetic tools, and the unique potential of this technology for basic and translational cardiac electrophysiology. Specifically, we discuss the ability to control (augment or suppress) the cardiac tissue's excitable properties using optogenetic actuators (microbial opsins), which are light-gated ion channels and pumps that can cause light-triggered membrane depolarization or hyperpolarization. We then focus on the potential clinical implications of this technology for the treatment of cardiac arrhythmias by describing recent efforts for developing optogenetic-based cardiac pacing, resynchronization, and defibrillation experimental strategies. Finally, the significant obstacles and challenges that need to be overcome before any future clinical translation can be expected are discussed.

The objective of the current study was to characterize calcium handling in developing human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To this end, real-time polymerase chain reaction (PCR), immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal calcium imaging and surface membrane labeling with di-8-aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor-2, and inositol-1,4,5-trisphosphate (IP3) receptors. Store calcium function was manifested as action-potential-induced calcium transients. Time-to-target plots showed that these action-potential-initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC-CMs also exhibited local calcium events (“sparks”) that were localized to the surface membrane. The presence of caffeine-sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early-stage (with the initiation of beating), intermediate-stage (10 days post-beating [dpb]), and late-stage (30–40 dpb) hESC-CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3-releasable calcium pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2 μM 2-Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early-stage hESC-CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC-CMs both for developmental studies and for the development of future myocardial cell replacement strategies. Disclosure of potential conflicts of interest is found at the end of this article.

The adult human heart has limited regenerative capacity and therefore any significant myocardial damage results in progressive deterioration in its function. Cell-based cardiac repair represents an exciting approach for rebuilding the injured heart. Consequentially, during the last decade, significant efforts have been made to assess the potential role of skeletal myoblasts, foetal cardiomyocytes, bone marrow stem cells, endothelial progenitors, resident cardiac progenitor cells, and embryonic stem cells (ESC) to restore the myocardial performance. Clinical translation of these basic studies has progressed rapidly with several Phase I and II clinical trials, utilizing autologous bone marrow cells and skeletal myoblasts, being well underway. In this review, we will describe these basic and clinical efforts. A specific emphasis will be placed on the potential role of human ESC for myocardial repair. These unique pluripotent cell lines can be propagated in the undifferentiated state in culture and coaxed to differentiate into cell derivatives of all three germ layers, including cardiomyocytes. The potential applications of this unique differentiating system in the emerging field of cardiovascular regenerative medicine will be discussed with special emphasis on the steps required to fully harness their unique potential.

We report a case of a patient with a left posteroseptal accessory pathway associated with a coronary sinus (CS) aneurysm. The patient had undergone two previous failed ablation attempts at other institutions despite multiple radiofrequency applications delivered within and outside the CS aneurysm. Electroanatomical mapping was performed and allowed delineation of the three-dimensional anatomy of the aneurysm, so as to identify the ventricular insertion site, and to permit successful ablation of the pathway without any complications.