Functional-Molecular Mechanisms of Sympathetic-Parasympathetic Dysfunction in PVC-Induced Cardiomyopathy Revealed by Dual Stressor PVC-Exercise Challenge.

BACKGROUND: The significance of autonomic dysfunction in premature ventricular contraction-induced cardiomyopathy (PVC-CM) remain unknown. OBJECTIVE: Utilizing a novel "dual stressor" provocative challenge combining exercise with premature ventricular contraction (PVCs), the authors characterized the functional and molecular mechanisms of cardiac autonomic (cardiac autonomic nervous system) remodeling in a PVC-CM animal model. METHODS: In 15 canines (8 experimental, 7 sham), we implanted pacemakers and neurotelemetry devices and subjected animals to 12 weeks of bigeminal PVCs to induce PVC-CM. Sympathetic nerve activity (SNA), vagal nerve activity (VNA), and heart rate were continuously recorded before, during, and after treadmill exercise challenge with and without PVCs, at baseline and after development of PVC-CM. Western blot and enzyme-linked immunosorbent assay were used to evaluate molecular markers of neural remodeling. RESULTS: Exercise triggered an increase in both SNA and VNA followed by late VNA withdrawal. With PVCs, the degree of exercise-induced SNA augmentation was magnified, whereas late VNA withdrawal became blunted. After PVC-CM development, SNA was increased at rest but failed to adequately augment during exercise, especially with PVCs, coupled with impaired VNA and heart rate recovery after exercise. In the remodeled cardiac autonomic nervous system, there was widespread sympathetic hyperinnervation and elevated transcardiac norepinephrine levels but unchanged parasympathetic innervation, indicating sympathetic overload. However, cardiac nerve growth factor was paradoxically downregulated, suggesting an antineurotrophic counteradaptive response to PVC-triggered sympathetic overload. CONCLUSIONS: Sympathetic overload, sympathetic dysfunction, and parasympathetic dysfunction in PVC-CM are unmasked by combined exercise and PVC challenge. Reduced cardiac neurotrophic factor might underlie the mechanisms of this dysfunction. Neuromodulation therapies to restore autonomic function could constitute a novel therapeutic approach for PVC-CM.

A cross species thermoelectric and spatiotemporal analysis of alternans in live explanted hearts using dual voltage-calcium fluorescence optical mapping.

Objective.Temperature plays a crucial role in influencing the spatiotemporal dynamics of the heart. Electrical instabilities due to specific thermal conditions typically lead to early period-doubling bifurcations and beat-to-beat alternans. These pro-arrhythmic phenomena manifest in voltage and calcium traces, resulting in compromised contractile behaviors. In such intricate scenario, dual optical mapping technique was used to uncover unexplored multi-scale and nonlinear couplings, essential for early detection and understanding of cardiac arrhythmia.Approach.We propose a methodological analysis of synchronized voltage-calcium signals for detecting alternans, restitution curves, and spatiotemporal alternans patterns under different thermal conditions, based on integral features calculation. To validate our approach, we conducted a cross-species investigation involving rabbit and guinea pig epicardial ventricular surfaces and human endocardial tissue under pacing-down protocols.Main results.We show that the proposed integral feature, as the area under the curve, could be an easily applicable indicator that may enhance the predictability of the onset and progression of cardiac alternans. Insights into spatiotemporal correlation analysis of characteristic spatial lengths across different heart species were further provided.Significance.Exploring cross-species thermoelectric features contributes to understanding temperature-dependent proarrhythmic regimes and their implications on coupled spatiotemporal voltage-calcium dynamics. The findings provide preliminary insights and potential strategies for enhancing arrhythmia detection and treatment.

Innovative Characterization of Alternans Onset and Development in Dual Voltage-Calcium Whole-Heart Optical Mapping Signals at Multiple Thermal States.

Cardiac electrical dynamics show complex space-time instabilities, like period-doubling bifurcation and beat-to-beat alternans, known to occur as pro-arrhythmic phenomena and linked to membrane voltage and intracellular calcium kinetics. Besides, cellular ionic dynamics are critically affected by temperature oscillations, further enhancing the complexity of such arrhythmias precursors that lead to irregular cardiac contraction. In this complex scenario, fluorescence dual optical mapping techniques allow the unveiling of nonlinear and multi-scale couplings. In this contribution, we propose a novel methodological analysis of synchronous dual voltage-calcium traces obtained from whole rabbit hearts for (i) detecting alternans onset and evolution, (ii) characterizing novel restitution curves, and (iii) defining spatio-temporal alternans patterns at four thermal states. We validate our approach against well-accepted analyses considering complete pacing-down restitution protocols. The proposed methodology computes integral features, e.g., area under the curve, suggesting that a novel, easy-to-use indicator, may advance predictability on alternans onset and evolution, further providing insights into spatio-temporal cardiac analyses.Clinical Relevance- This work introduces new methods for the early detection of cardiac alternans onset and development as precursors of arrhythmias and fibrillation at different temperatures.

Complex repolarization dynamics in ex vivo human ventricles are independent of the restitution properties.

AIMS: The mechanisms of transition from regular rhythms to ventricular fibrillation (VF) are poorly understood. The concordant to discordant repolarization alternans pathway is extensively studied; however, despite its theoretical centrality, cannot guide ablation. We hypothesize that complex repolarization dynamics, i.e. oscillations in the repolarization phase of action potentials with periods over two of classic alternans, is a marker of electrically unstable substrate, and ablation of these areas has a stabilizing effect and may reduce the risk of VF. To prove the existence of higher-order periodicities in human hearts. METHODS AND RESULTS: We performed optical mapping of explanted human hearts obtained from recipients of heart transplantation at the time of surgery. Signals recorded from the right ventricle endocardial surface were processed to detect global and local repolarization dynamics during rapid pacing. A statistically significant global 1:4 peak was seen in three of six 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. CONCLUSION: We present evidence of complex higher-order periodicities and the co-existence of such regions with stable non-chaotic areas in ex vivo human hearts. We infer that the oscillation of the calcium cycling machinery is the primary mechanism of higher-order dynamics. These higher-order regions may act as niduses of instability and may provide targets for substrate-based ablation of VF.

Higher-Order Dynamics Beyond Repolarization Alternans in Ex-Vivo Human Ventricles are Independent of the Restitution Properties.

Background: Repolarization 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. Methods: We 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. Results: A 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. Discussion: We 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.

Beyond Alternans: Detection of Higher-Order Periodicity in Ex-Vivo Human Ventricles Before Induction of Ventricular Fibrillation.

Background: Repolarization alternans, defined as period-2 oscillation in the repolarization phase of the action potentials, is one of the cornerstones of cardiac electrophysiology as it provides a mechanistic link between cellular dynamics and ventricular fibrillation (VF). Theoretically, higher-order periodicities (e.g., period-4, period-8,...) are expected but have very limited experimental evidence. Methods: We studied explanted human hearts, obtained from the recipients of heart transplantation at the time of surgery, using optical mapping technique with transmembrane voltage-sensitive fluorescent dyes. The hearts were stimulated at an increasing rate until VF was induced. The signals recorded from the right ventricle endocardial surface just before the 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: A prominent and statistically significant 1:4 peak (corresponding to period-4 dynamics) was seen in three of the six studied hearts. Local analysis revealed the spatiotemporal distribution of higher-order periods. Period-4 was localized to temporally stable islands. Higher-order oscillations (period-5, 6, and 8) were transient and primarily occurred in arcs parallel to the activation isochrones. Discussion: We present evidence of higher-order periodicities and the co-existence of such regions with stable non-chaotic areas in ex-vivo human hearts before VF induction. This result is consistent with the period-doubling route to chaos as a possible mechanism of VF initiation, which complements the concordant to discordant alternans mechanism. The presence of higher-order regions may act as niduses of instability that can degenerate into chaotic fibrillation.

Optical mapping of contracting hearts.

Optical mapping is a widely used tool to record and visualize the electrophysiological properties in a variety of myocardial preparations such as Langendorff-perfused isolated hearts, coronary-perfused wedge preparations, and cell culture monolayers. Motion artifact originating from the mechanical contraction of the myocardium creates a significant challenge to performing optical mapping of contracting hearts. Hence, to minimize the motion artifact, cardiac optical mapping studies are mostly performed on non-contracting hearts, where the mechanical contraction is removed using pharmacological excitation-contraction uncouplers. However, such experimental preparations eliminate the possibility of electromechanical interaction, and effects such as mechano-electric feedback cannot be studied. Recent developments in computer vision algorithms and ratiometric techniques have opened the possibility of performing optical mapping studies on isolated contracting hearts. In this review, we discuss the existing techniques and challenges of optical mapping of contracting hearts.

Circle Method for Robust Estimation of Local Conduction Velocity High-Density Maps From Optical Mapping Data: Characterization of Radiofrequency Ablation Sites.

Conduction velocity (CV) slowing is associated with atrial fibrillation (AF) and reentrant ventricular tachycardia (VT). Clinical electroanatomical mapping systems used to localize AF or VT sources as ablation targets remain limited by the number of measuring electrodes and signal processing methods to generate high-density local activation time (LAT) and CV maps of heterogeneous atrial or trabeculated ventricular endocardium. The morphology and amplitude of bipolar electrograms depend on the direction of propagating electrical wavefront, making identification of low-amplitude signal sources commonly associated with fibrotic area difficulty. In comparison, unipolar electrograms are not sensitive to wavefront direction, but measurements are susceptible to distal activity. This study proposes a method for local CV calculation from optical mapping measurements, termed the circle method (CM). The local CV is obtained as a weighted sum of CV values calculated along different chords spanning a circle of predefined radius centered at a CV measurement location. As a distinct maximum in LAT differences is along the chord normal to the propagating wavefront, the method is adaptive to the propagating wavefront direction changes, suitable for electrical conductivity characterization of heterogeneous myocardium. In numerical simulations, CM was validated characterizing modeled ablated areas as zones of distinct CV slowing. Experimentally, CM was used to characterize lesions created by radiofrequency ablation (RFA) on isolated hearts of rats, guinea pig, and explanted human hearts. To infer the depth of RFA-created lesions, excitation light bands of different penetration depths were used, and a beat-to-beat CV difference analysis was performed to identify CV alternans. Despite being limited to laboratory research, studies based on CM with optical mapping may lead to new translational insights into better-guided ablation therapies.

Methodology for Cross-Talk Elimination in Simultaneous Voltage and Calcium Optical Mapping Measurements With Semasbestic Wavelengths.

Most cardiac arrhythmias at the whole heart level result from alteration of cell membrane ionic channels and intracellular calcium concentration ([Ca2+] i ) cycling with emerging spatiotemporal behavior through tissue-level coupling. For example, dynamically induced spatial dispersion of action potential duration, QT prolongation, and alternans are clinical markers for arrhythmia susceptibility in regular and heart-failure patients that originate due to changes of the transmembrane voltage (V m) and [Ca2+] i . We present an optical-mapping methodology that permits simultaneous measurements of the V m - [Ca2+] i signals using a single-camera without cross-talk, allowing quantitative characterization of favorable/adverse cell and tissue dynamical effects occurring from remodeling and/or drugs in heart failure. We demonstrate theoretically and experimentally in six different species the existence of a family of excitation wavelengths, we termed semasbestic, that give no change in signal for one dye, and thus can be used to record signals from another dye, guaranteeing zero cross-talk.

Quantifying arrhythmic long QT effects of hydroxychloroquine and azithromycin with whole-heart optical mapping and simulations.

BACKGROUND: In March 2020, hydroxychloroquine (HCQ) alone or combined with azithromycin (AZM) was authorized as a treatment for COVID-19 in many countries. The therapy proved ineffective with long QT and deadly cardiac arrhythmia risks, illustrating challenges to determine the new safety profile of repurposed drugs. OBJECTIVE: To investigate proarrhythmic effects and mechanism of HCQ and AZM (combined and alone) with high doses of HCQ as in the COVID-19 clinical trials. METHODS: Proarrhythmic effects of HCQ and AZM are quantified using optical mapping with voltage-sensitive dyes in ex vivo Langendorff-perfused guinea pig (GP) hearts and with numerical simulations of a GP Luo-Rudy and a human O'Hara-Virag-Varro-Rudy models, for Epi, Endo, and M cells, in cell and tissue, incorporating the drug's effect on cell membrane ionic currents. RESULTS: Experimentally, HCQ alone and combined with AZM leads to long QT intervals by prolonging the action potential duration and increased spatial dispersion of action potential (AP) repolarization across the heart, leading to proarrhythmic discordant alternans. AZM alone had a lesser arrhythmic effect with less triangulation of the AP shape. Mathematical cardiac models fail to reproduce most of the arrhythmic effects observed experimentally. CONCLUSIONS: During public health crises, the risks and benefits of new and repurposed drugs could be better assessed with alternative experimental and computational approaches to identify proarrhythmic mechanisms. Optical mapping is an effective framework suitable to investigate the drug's adverse effects on cardiac cell membrane ionic channels at the cellular level and arrhythmia mechanisms at the tissue and whole-organ level.

A Network-based Cardiac Electrophysiology Simulator with Realistic Signal Generation and Response to Pacing Maneuvers.

Diagnosis and localization of cardiac arrhythmias, especially supraventricular tachycardia (SVT), by inspecting intracardiac signals and performing pacing maneuvers is the core of electrophysiology studies. Acquiring and maintaining complex skill sets can be facilitated by using simulators, allowing the operator to practice in a safe and controlled setting. An electrophysiology simulator should not only display arrhythmias but it has to respond to the user's arbitrary inputs. While, in principle, it is possible to model the heart using a detailed anatomical and cellular model, such a system would be unduly complex and computationally intensive. In this paper, we describe a freely available web-based electrophysiology simulator (http://svtsim.com), which is composed of a visualization/interface unit and a heart model based on a dynamical network. In the network, nodes represent the points of interest, such as the sinus and the atrioventricular nodes, and links model the conduction system and pathways. The dynamics are encoded explicitly in the state machines attached to the nodes and links. Simulated intracardiac signals and surface ECGs are generated from the internal state of the heart model. Reentrant tachycardias, especially various forms of SVT, can emerge in this system in response to the user's actions in the form of pacing maneuvers. Additionally, the resulting arrhythmias respond realistically to various inputs, such as overdrive pacing and delivery of extra stimuli, cardioversion, ablation, and infusion of medications. For nearly a decade, svtsim.com has been used successfully to train electrophysiology practitioners in many institutions. We will present our experience regarding best practices in designing and using electrophysiology simulators for training and testing. We will also discuss the current trends in clinical cardiac electrophysiology and the anticipated next generation electrophysiology simulators.

Not all Long-QTs Are The Same, Proarrhytmic Quantification with Action Potential Triangulation and Alternans.

Long-QT is commonly associated with an increased risk of polymorphic ventricular tachycardia from drug therapy. However, not all drugs prolonging QT interval are proarrhythmic. This study aimed to characterize cellular and tissue mechanisms under which QT-interval prolonging drugs and their combination are proarrhythmic, examining arrhythmia susceptibility due to action potential (AP) triangulation and spatial dispersion of action potential duration (APD). Additionally, we aimed to elucidate that Torsades de Pointe (TdP) associated with long-QT are not necessarily caused by early-after-depolarization (EADs) but are related to the presence of AP alternans in both time and space. Isolated Guinea Pig hearts were Langendorff perfused, and optical mapping was done with a voltage dye-sensitive dye. Two commonly used drugs at the beginning of the COVID-19 pandemic, hydroxychloroquine (HCQ) and Azithromycin (AZM), were added to study the effects of QT interval prolongation. Alternans in time and space were characterized by performing restitution pacing protocols. Comparing APs, HCQ prolongs APD during phase-III repolarization, resulting in a higher triangulation ratio than AZM alone or AZM combined with HCQ. Lower triangulation ratios with AZM are associated with phase-II prolongation, lower arrhythmia, and lower incidence of spatially discordant alternans.

Interactive Simulation of the ECG: Effects of Cell Types, Distributions, Shapes and Duration.

The shape of the ECG depends on the lead positions but also on the distribution and dispersion of different cell types and their action potential (AP) durations and shapes. We present an interactive JavaScript program that allows fast simulations of the ECG by solving and displaying the dynamics of cardiac cells in tissue using a web browser. We use physiologically accurate ODE models of cardiac cells of different types including SA node, right and left atria, AV node, Purkinje, and right and left ventricular cells with dispersion that accounts for apex-to-base and epi-to-endo variations. The software allows for real-time variations for each cell type and their spatial range so as to identify how the shape of the ECG varies as a function of cell type, distribution, excitation duration and AP shape. The propagation of the wave is visualized in real time through all the regions as parameters are kept fixed or varied to modify ECG morphology. The code solves thousands of simulated cells in real time and is independent of operating system, so it can run on PCs, laptops, tablets and cellphones. This program can be used to teach students, fellows and the general public how and why lead positions and the different cell physiology in the heart affect the various features of the ECG.

Unimapper: An Online Interactive Analyzer/Visualizer of Optical Mapping Experimental Data.

Time series of spatially-extended two-dimensional recordings are the cornerstone of basic and clinical cardiac electrophysiology. The data source may be either multipolar catheters, multi-electrode arrays, optical mapping with the help of voltage and calcium-sensitive fluorescent dyes, or the output of simulation studies. The resulting data cubes (usually two spatial and one temporal dimension) are shared either as movie files or, after additional processing, various graphs and tables. However, such data products can only convey a limited view of the data. It will be beneficial if the data consumers can interactively process the data, explore different processing options and change its visualization. This paper presents the Unified Electrophysiology Mapping Framework (Unimapper) to facilitate the exchange of electrophysiology data. Its pedigree includes a Java-based optical mapping application. The core of Unimapper is a website and a collection of JavaScript utility functions for data import and visualization (including multi-channel visualization for simultaneous voltage/calcium mapping), basic image processing (e.g., smoothing), basic signal processing (e.g., signal detrending), and advanced processing (e.g., phase calculation using the Hilbert transform). Additionally, Unimapper can optionally use graphics processing units (GPUs) for computationally intensive operations. The Unimapper ecosystem also includes utility libraries for commonly used scientific programming languages (MATLAB, Python, and Julia) that allow the data producers to convert images and recorded signals into a standard format readable by Unimapper. Unimapper can act as a nexus to share electrophysiology data - whether recorded experimentally, clinically or generated by simulation - and enhance communication and collaboration among researchers.

Interactive 3D Human Heart Simulations on Segmented Human MRI Hearts.

Understanding cardiac arrhythmic mechanisms and developing new strategies to control and terminate them using computer simulations requires realistic physiological cell models with anatomically accurate heart structures. Furthermore, numerical simulations must be fast enough to study and validate model and structure parameters. Here, we present an interactive parallel approach for solving detailed cell dynamics in high-resolution human heart structures with a local PC's GPU. In vitro human heart MRI scans were manually segmented to produce 3D structures with anatomically realistic electrophysiology. The Abubu.js library was used to create an interactive code to solve the OVVR human ventricular cell model and the FDA extension of the model in the human MRI heart structures, allowing the simulation of reentrant waves and investigation of their dynamics in real time. Interactive simulations of a physiological cell model in a detailed anatomical human heart reveals propagation of waves through the fine structures of the trabeculae and pectinate muscle that can perpetuate arrhythmias, thereby giving new insights into effects that may need to be considered when planning ablation and other defibrillation methods.

Generation of Monophasic Action Potentials and Intermediate Forms.

The monophasic action potential (MAP) is a near replica of the transmembrane potential recorded when an electrode is pushed firmly against cardiac tissue. Despite its many practical uses, the mechanism of MAP signal generation and the reason it is so different from unipolar recordings are not completely known and are a matter of controversy. In this work, we describe a method to simulate realistic MAP and intermediate forms, which are multiphasic electrograms different from an ideal MAP. The key ideas of our method are the formation of compressed zones and junctional spaces-regions of the extracellular and bath or blood pool directly in contact with electrodes that exhibit a pressure-induced reduction in electrical conductivity-and the presence of a complex network of passive components that acts as a high-pass filter to distort and attenuate the signal that reaches the recording amplifier. The network is formed by the interaction between the passive tissue properties and the double-layer capacitance of electrodes. The MAP and intermediate forms reside on a continuum of signals, which can be generated by the change of the model parameters. Our model helps to decipher the mechanisms of signal generation and can lead to a better design for electrodes, recording amplifiers, and experimental setups.

Fatal arrhythmias: Another reason why doctors remain cautious about chloroquine/hydroxychloroquine for treating COVID-19.

BACKGROUND: Early during the current coronavirus disease 19 (COVID-19) pandemic, hydroxychloroquine (HCQ) received a significant amount of attention as a potential antiviral treatment, such that it became one of the most commonly prescribed medications for COVID-19 patients. However, not only has the effectiveness of HCQ remained questionable, but mainly based on preclinical and a few small clinical studies, HCQ is known to be potentially arrhythmogenic, especially as a result of QT prolongation. OBJECTIVE: The purpose of this study was to investigate the arrhythmic effects of HCQ, as the heightened risk is especially relevant to COVID-19 patients, who are at higher risk for cardiac complications and arrhythmias at baseline. METHODS: An optical mapping technique utilizing voltage-sensitive fluorescent dyes was used to determine the arrhythmic effects of HCQ in ex vivo guinea pig and rabbit hearts perfused with the upper therapeutic serum dose of HCQ (1000 ng/mL). RESULTS: HCQ markedly increased action potential dispersion, resulted in development of repolarization alternans, and initiated polymorphic ventricular tachycardia. CONCLUSION: The study results further highlight the proarrhythmic effects of HCQ.

High-Resolution Optical Measurement of Cardiac Restitution, Contraction, and Fibrillation Dynamics in Beating vs. Blebbistatin-Uncoupled Isolated Rabbit Hearts.

Optical mapping is a high-resolution fluorescence imaging technique, that uses voltage- or calcium-sensitive dyes to visualize electrical excitation waves on the heart surface. However, optical mapping is very susceptible to the motion of cardiac tissue, which results in so-called motion artifacts in the fluorescence signal. To avoid motion artifacts, contractions of the heart muscle are typically suppressed using pharmacological excitation-contraction uncoupling agents, such as Blebbistatin. The use of pharmacological agents, however, may influence cardiac electrophysiology. Recently, it has been shown that numerical motion tracking can significantly reduce motion-related artifacts in optical mapping, enabling the simultaneous optical measurement of cardiac electrophysiology and mechanics. Here, we combine ratiometric optical mapping with numerical motion tracking to further enhance the robustness and accuracy of these measurements. We evaluate the method's performance by imaging and comparing cardiac restitution and ventricular fibrillation (VF) dynamics in contracting, non-working vs. Blebbistatin-arrested Langendorff-perfused rabbit hearts (N = 10). We found action potential durations (APD) to be, on average, 25 ± 5% shorter in contracting hearts compared to hearts uncoupled with Blebbistatin. The relative shortening of the APD was found to be larger at higher frequencies. VF was found to be significantly accelerated in contracting hearts, i.e., 9 ± 2Hz with Blebbistatin and 15 ± 4Hz without Blebbistatin, and maintained a broader frequency spectrum. In contracting hearts, the average number of phase singularities was N PS = 11 ± 4 compared to N PS = 6 ± 3 with Blebbistatin during VF on the anterior ventricular surface. VF inducibility was reduced with Blebbistatin. We found the effect of Blebbistatin to be concentration-dependent and reversible by washout. Aside from the electrophysiological characterization, we also measured and analyzed cardiac motion. Our findings may have implications for the interpretation of optical mapping data, and highlight that physiological conditions, such as oxygenation and metabolic demand, must be carefully considered in ex vivo imaging experiments.