Iolanda Feola

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.

Localized Optogenetic Targeting of Rotors in Atrial Cardiomyocyte Monolayers

Background: Recently, a new ablation strategy for atrial fibrillation has emerged, which involves the identification of rotors (ie, local drivers) followed by the localized targeting of their core region by ablation. However, this concept has been subject to debate because the mode of arrhythmia termination remains poorly understood, as dedicated models and research tools are lacking. We took a unique optogenetic approach to induce and locally target a rotor in atrial monolayers. Methods and Results: Neonatal rat atrial cardiomyocyte monolayers expressing a depolarizing light-gated ion channel (Ca2+-translocating channelrhodopsin) were subjected to patterned illumination to induce single, stable, and centralized rotors by optical S1-S2 cross-field stimulation. Next, the core region of these rotors was specifically and precisely targeted by light to induce local conduction blocks of circular or linear shapes. Conduction blocks crossing the core region, but not reaching any unexcitable boundary, did not lead to termination. Instead, electric waves started to propagate along the circumference of block, thereby maintaining reentrant activity, although of lower frequency. If, however, core-spanning lines of block reached at least 1 unexcitable boundary, reentrant activity was consistently terminated by wave collision. Lines of block away from the core region resulted merely in rotor destabilization (ie, drifting). Conclusions: Localized optogenetic targeting of rotors in atrial monolayers could lead to both stabilization and destabilization of reentrant activity. For termination, however, a line of block is required reaching from the core region to at least 1 unexcitable boundary. These findings may improve our understanding of the mechanisms involved in rotor-guided ablation.

Optogenetic manipulation of anatomical re-entry by light-guided generation of a reversible local conduction block

Aims Anatomical re-entry is an important mechanism of ventricular tachycardia, characterized by circular electrical propagation in a fixed pathway. It’s current investigative and therapeutic approaches are non-biological, rather unspecific (drugs), traumatizing (electrical shocks), or irreversible (ablation). Optogenetics is a new biological technique that allows reversible modulation of electrical function with unmatched spatiotemporal precision using light-gated ion channels. We therefore investigated optogenetic manipulation of anatomical re-entry in ventricular cardiac tissue. Methods and results Transverse, 150-&mgr;m-thick ventricular slices, obtained from neonatal rat hearts, were genetically modified with lentiviral vectors encoding Ca2+-translocating channelrhodopsin (CatCh), a light-gated depolarizing ion channel, or enhanced yellow fluorescent protein (eYFP) as control. Stable anatomical re-entry was induced in both experimental groups. Activation of CatCh was precisely controlled by 470-nm patterned illumination, while the effects on anatomical re-entry were studied by optical voltage mapping. Regional illumination in the pathway of anatomical re-entry resulted in termination of arrhythmic activity only in CatCh-expressing slices by establishing a local and reversible, depolarization-induced conduction block in the illuminated area. Systematic adjustment of the size of the light-exposed area in the re-entrant pathway revealed that re-entry could be terminated by either wave collision or extinction, depending on the depth (transmurality) of illumination. In silico studies implicated source–sink mismatches at the site of subtransmural conduction block as an important factor in re-entry termination. Conclusions Anatomical re-entry in ventricular tissue can be manipulated by optogenetic induction of a local and reversible conduction block in the re-entrant pathway, allowing effective re-entry termination. These results provide distinctively new mechanistic insight into re-entry termination and a novel perspective for cardiac arrhythmia management.

Optogenetic Engineering of Atrial Cardiomyocytes

Optogenetics is emerging in the cardiology field as a new strategy to explore biological functions through the use of light-sensitive proteins and dedicated light sources. For example, this technology allows modification of the electrophysiological properties of cardiac muscle cells with superb spatiotemporal resolution and quantitative control. In this chapter, the optogenetic modification of atrial cardiomyocytes (aCMCs) from 2-day-old Wistar rats using lentiviral vector (LV) technology and the subsequent activation of the light-sensitive proteins (i.e., ion channels) through light-emitting diodes (LEDs) are described.

A Mathematical Model of Neonatal Rat Atrial Monolayers with Constitutively Active Acetylcholine-Mediated K+ Current.

Atrial fibrillation (AF) is the most frequent form of arrhythmia occurring in the industrialized world. Because of its complex nature, each identified form of AF requires specialized treatment. Thus, an in-depth understanding of the bases of these arrhythmias is essential for therapeutic development. A variety of experimental studies aimed at understanding the mechanisms of AF are performed using primary cultures of neonatal rat atrial cardiomyocytes (NRAMs). Previously, we have shown that the distinct advantage of NRAM cultures is that they allow standardized, systematic, robust re-entry induction in the presence of a constitutively-active acetylcholine-mediated K+ current (IKACh-c). Experimental studies dedicated to mechanistic explorations of AF, using these cultures, often use computer models for detailed electrophysiological investigations. However, currently, no mathematical model for NRAMs is available. Therefore, in the present study we propose the first model for the action potential (AP) of a NRAM with constitutively-active acetylcholine-mediated K+ current (IKACh-c). The descriptions of the ionic currents were based on patch-clamp data obtained from neonatal rats. Our monolayer model closely mimics the action potential duration (APD) restitution and conduction velocity (CV) restitution curves presented in our previous in vitro studies. In addition, the model reproduces the experimentally observed dynamics of spiral wave rotation, in the absence and in the presence of drug interventions, and in the presence of localized myofibroblast heterogeneities.

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.

Optogenetics enables real-time spatiotemporal control over spiral wave dynamics in an excitable cardiac system

Propagation of non-linear waves is key to the functioning of diverse biological systems. Such waves can organize into spirals, rotating around a core, whose properties determine the overall wave dynamics. Theoretically, manipulation of a spiral wave core should lead to full spatiotemporal control over its dynamics. However, this theory lacks supportive evidence (even at a conceptual level), making it thus a long-standing hypothesis. Here, we propose a new phenomenological concept that involves artificially dragging spiral waves by their cores, to prove the aforementioned hypothesis in silico, with subsequent in vitro validation in optogenetically modified monolayers of rat atrial cardiomyocytes. We thereby connect previously established, but unrelated concepts of spiral wave attraction, anchoring and unpinning to demonstrate that core manipulation, through controlled displacement of heterogeneities in excitable media, allows forced movement of spiral waves along pre-defined trajectories. Consequently, we impose real-time spatiotemporal control over spiral wave dynamics in a biological system.

Response by Feola et al to Letter Regarding Article, “Localized Optogenetic Targeting of Rotors in Atrial Cardiomyocyte Monolayers”

We thank Houston et al for their interest in our study. In their letter, they raise the question whether the rotors and accompanied spiral waves observed in our study represent microreentrant circuits anchored to lines of conduction block/slowing (ie, anatomical reentry), instead of reentrant activity around an unexcited, yet excitable core region (ie, functional reentry). Their comment is based on a movie published on their website, showing high-resolution mapping, in an HL-1 culture, of reentrant activity that seems anchored to microregions of conduction abnormalities. Although appraisal of these data is difficult without a detailed description of methods and results, we still would like to …