Cardiac arrhythmias arise from excitable-media dynamics that are often challenging to teach and visualize in real time. To address this, we developed iMyocyte , an interactive web-based platform for real-time demonstration of excitation wave propagation, conduction block, and reentry dynamics using a simplified cellular automaton model (i.e., a network of discrete cells that change state based on neighbouring cells). In iMyocyte , a network is built from student devices, each representing a model cell coupled to its neighbours to propagate excitation. Unlike traditional teaching materials, iMyocyte lets students manipulate key model parameters and immediately observe how these shape wave dynamics and reentry conditions. Pilot classroom deployments with undergraduate life science students at McGill University indicate high engagement and suggest improved conceptual clarity, motivating future work to improve the platform’s usability, robustness, and visualization features, and to conduct formal learning assessment. ### Competing Interest Statement The authors have declared no competing interest.

OBJECTIVE:Frequent premature ventricular complexes (PVCs) can lead to adverse health conditions such as cardiomyopathy. The linear correlation between PVC frequency and heart rate (as positive, negative, or neutral) on a 24-hour Holter recording has been proposed as a way to classify patients and guide treatment with beta-blockers. Our objective was to evaluate the robustness of this classification to measurement methodology, different 24-hour periods, and nonlinear dependencies of PVCs on heart rate. MATERIALS AND METHODS:We analyzed 82 multi-day Holter recordings (1-7 days) collected from 48 patients with frequent PVCs (burden 1%-44%). For each record, linear correlation between PVC frequency and heart rate was computed for different 24-hour periods and using different length intervals to determine PVC frequency. RESULTS:Using a 1-hour interval, the correlation between PVC frequency and heart rate was consistently positive, negative, or neutral on different days in only 36.6% of patients. Using shorter time intervals, the correlation was consistent in 56.1% of patients. Shorter time intervals revealed nonlinear and piecewise linear relationships between PVC frequency and heart rate in many patients. DISCUSSION:The variability of the correlation between PVC frequency and heart rate across different 24-hour periods and interval durations suggests that the relationship is neither strictly linear nor stationary. A better understanding of the mechanism driving the PVCs, combined with computational and biological models that represent these mechanisms, may provide insight into the observed nonlinear behavior and guide more robust classification strategies. CONCLUSION:Linear correlation as a tool to classify patients with frequent PVCs should be used with caution. It is sensitive to the specific 24-hour period analyzed and the methodology used to segment the data. More sophisticated classification approaches that can capture nonlinear and time-varying dependencies should be developed and considered in clinical practice.

OBJECTIVE:Reentrant arrhythmias are life-threatening cardiac events that are difficult to study due to limited experimental control over the complex circuit dynamics. We present a real-time coupled cardiac system that allows in real-time dynamic manipulation of reentrant pathways in vitro using physiologically relevant simulations. METHODS:We designed a closed-feedback loop system that couples a cultured cardiac monolayer with a two-dimensional computational simulation of cardiac tissue. The simulation, based on GPU-accelerated models (e.g., cellular automata), predicts wave propagation in real-time using the Abubu.js library. Optical mapping captures monolayer activation patterns, and simulation outputs are converted into light-based stimulation via optogenetics, using LEDs and microcontrollers to depolarize cardiac tissue. RESULTS:Our platform is capable of accurately detecting and responding to electrical waves in real-time, enabling interactive modulation of reentrant circuits. The system replaces traditional fixed-delay stimulation protocols with computationally guided interventions, better mimicking physiological conduction dynamics. CONCLUSION:This coupled system provides a novel and responsive method to study reentrant arrhythmias. Its integration of optical stimulation, real-time modeling, and tissue feedback enables the construction of user-defined reentry pathways and dynamic interaction with reentrant circuit behavior. SIGNIFICANCE:By merging computational and biological systems, this work introduces a versatile experimental framework for investigating arrhythmias. Built from inexpensive and accessible components, it lowers technical and financial barriers, increasing accessibility across a broad range of researchers and research environments. The platform may inform future control and anti-arrhythmic strategies and pave the way for personalized cardiac electrophysiology studies.

Phase resetting of cardiac oscillators underlies some complex arrhythmias. Here we use optogenetic stimulation to construct phase response curves (PRC) for spheroids of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) and a computational cardiomyocyte model to identify ionic mechanisms shaping the PRC. The clinical utility of the human PRCs is demonstrated by adding a patient-based conduction delay to the same equations to explain complex multi-day Holter ECG dynamics and cardiac arrhythmias. Periodic stimulation of these patient-based models and the computational model of human iPSC-CM reveal similar bifurcation patterns and entrainment zones. Cell therapy by injecting iPSC-CM into diseased hearts can induce ectopic foci-based engraftment arrhythmias. The PRC analysis offers a potential strategy to entrain these foci in a parameter space that avoids such arrhythmias.

Abstract: Reentrant arrhythmias are life-threatening cardiac events that are difficult to study due to limited experimental control over the complex circuit dynamics. We aimed to develop a real-time digital heart twin that allows in real-time dynamic manipulation of reentrant pathways in vitro using physiologically relevant simulations. We designed a closed-feedback loop system that couples a cultured cardiac monolayer with a two-dimensional computational simulation of cardiac tissue. The simulation, based on GPU-accelerated models (e.g., cellular automata), predicts wave propagation in real-time using the Abubu.js library. Optical mapping captures monolayer activation patterns, and simulation outputs are converted into light-based stimulation via optogenetics, using LEDs and microcontrollers to depolarize cardiac tissue. Our platform is capable of accurately detecting and responding to electrical waves in real-time, enabling interactive control of reentrant circuits. The system replaces traditional fixed-delay stimulation protocols with computationally guided interventions, better mimicking physiological conduction dynamics. This digital twin provides a novel and responsive method to study reentrant arrhythmias. Its integration of optical stimulation, real-time modeling, and tissue feedback enables the construction of user-defined reentry pathways under dynamic control. By merging computational and biological systems, this work introduces a versatile experimental framework for investigating arrhythmias. The platform may inform future control and anti-arrhythmic strategies and pave the way for personalized cardiac electrophysiology studies. ### Competing Interest Statement The authors have declared no competing interest.

Previously, we introduced a microscope design that enabled rapid, random-access well plate imaging [ eLife, 10, e56426 (2021)10.7554/eLife.56426]. Here, we implement this design in a low-cost, compact, and portable prototype (the Exeter Multiscope) and apply it to the problem of capturing the contraction of cardiomyocyte monolayers, which have been plated into nine wells within a 96-well plate. Using a transmissive rather than reflective geometry, each well is sampled using 500 × 500 pixels across a 1.4 × 1.4 mm field of view, acquired in three colours at 3.7 Hz per well. The use of multiple illumination wavelengths provides post-hoc focus selection, further increasing the level of automation. The performance of the Exeter Multiscope is benchmarked against industry standard methods using a commercial microscope with a motorised stage and demonstrates that the Multiscope can acquire data almost 40 times faster. The data from both Multiscope and the commercial systems are processed by a 'pixel variance' algorithm that uses information from the pixel value variability over time to determine the timing and amplitude of tissue contraction. This algorithm is also benchmarked against an existing algorithm that employs an absolute difference measure of tissue contraction.

Cardiovascular toxicity remains a primary concern in drug development, accounting for a significant portion of post-market drug withdrawals due to adverse reactions such as arrhythmias. Traditional preclinical models, predominantly based on animal cells, often fail to replicate human cardiac physiology accurately, complicating the prediction of drug-induced effects. Although human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a more genetically relevant system, their use in 2D, static cultures does not sufficiently mimic the dynamic, 3D environment of the human heart. 3D cardiac organoids made from human iPSC-CMs can potentially bridge this gap. However, most traditional electrophysiology assays, developed for single cells or 2D monolayers, are not readily adaptable to 3D organoids. This study uses optical calcium analysis of human organoids combined with miniaturized fluorescence microscopy (miniscope) and heart-on-a-chip technology. This simple, inexpensive, and efficient platform provides robust on-chip calcium imaging of human cardiac organoids. The versatility of the system is demonstrated through cardiotoxicity assay of drugs known to impact cardiac electrophysiology, including dofetilide, quinidine, and thapsigargin. The platform promises to advance drug testing by providing a more reliable and physiologically relevant assessment of cardiovascular toxicity, potentially reducing drug-related adverse effects in clinical settings.

We apply a novel microscope architecture, the Exeter Multiscope, to the problem of acquiring image data in rapid succession from nine wells of a 96 well plate. We demonstrate that the new microscope can detect contraction in cardiomyocyte monolayers which have been plated into these wells. Each well is sampled using 500 x 500 pixels across a 1.4 x 1.4 mm field of view, acquired in three colours at 3.7 Hz per well. The use of multiple illumination wavelengths provides post-hoc focus selection, further increasing the level of automation. The performance of the Exeter Multiscope is benchmarked against industry standard methods using a commercial microscope with a motorised stage and demonstrates that the Multiscope can acquire data almost 40 times faster. The data from both Multiscope and the commercial systems are processed by a ‘pixel variance’ algorithm that uses information from the pixel value variability over time to determine the timing and amplitude of tissue contraction. This algorithm is also benchmarked against an existing algorithm that employs an absolute difference measure of tissue contraction. ### Competing Interest Statement The authors have declared no competing interest.

Many natural and man-made systems are prone to critical transitions-abrupt and potentially devastating changes in dynamics. Deep learning classifiers can provide an early warning signal for critical transitions by learning generic features of bifurcations from large simulated training data sets. So far, classifiers have only been trained to predict continuous-time bifurcations, ignoring rich dynamics unique to discrete-time bifurcations. Here, we train a deep learning classifier to provide an early warning signal for the five local discrete-time bifurcations of codimension-one. We test the classifier on simulation data from discrete-time models used in physiology, economics and ecology, as well as experimental data of spontaneously beating chick-heart aggregates that undergo a period-doubling bifurcation. The classifier shows higher sensitivity and specificity than commonly used early warning signals under a wide range of noise intensities and rates of approach to the bifurcation. It also predicts the correct bifurcation in most cases, with particularly high accuracy for the period-doubling, Neimark-Sacker and fold bifurcations. Deep learning as a tool for bifurcation prediction is still in its nascence and has the potential to transform the way we monitor systems for critical transitions.

A cardiac arrhythmia is an abnormality in the rate or rhythm of the heart beat. We study a type of arrhythmia called a premature ventricular complex (PVC), which is typically benign, but in rare cases can lead to more serious arrhythmias or heart failure. There are three known mechanisms for PVCs: reentry, an ectopic focus, and triggered activity. We develop minimal models for each mechanism and attempt the inverse problem of determining which model (and therefore which mechanism) best describes the beat dynamics observed in an ambulatory electrocardiogram. We demonstrate our approach on a patient who exhibits frequent PVCs and find that their PVC dynamics are best described by a model of triggered activity. Better identification of the PVC mechanism from wearable device data could improve risk stratification for the development of more serious arrhythmias.

In an excitable medium, a stimulus generates a wave that propagates in space until it reaches the boundary or collides with another wave and annihilates. We study the dynamics generated by two periodic sources with different frequencies in excitable cardiac tissue culture using optogenetic techniques. The observed rhythms, which can be modeled using cellular automata and studied analytically, show unexpected regularities related to classic results in number theory. We apply the results to identify cardiac arrhythmias in people that are due to a putative mechanism of two competing pacemakers.

Resolving the relationships between cellular mechanics and ion signalling has remained a core focus of biophysics since the discovery of excitable tissues. Many attempts to understand the material properties of excitable cells have been challenged by their active nature, whereby it has been necessary to probe with a known force and measure the resulting displacement. Here we present Passive Periodic Rheology (PPR), a new approach which uses the contractile forces exerted by the excitable cell as the periodic driving force, instead of an external stimulus. Internal cellular displacements can be quantified using traction force microscopy, allowing us to recover a spatio-temporal map of the cellular mechanics. We demonstrate cardiomyocytes as a model system for performing PPR by revealing the phase delay between calcium signalling, contractility, and intracellular displacements to provide a spatial map of the active energy of the cell. Diverse changes in these signals and their relationships are known to lead to cardiac dysfunction. Quantifying the active properties of cardiomyocytes could therefore give insight into future diagnostics and treatments for cardiovascular disease, as well as offer a new tool for more accessible and comprehensive insights into dynamic cellular systems.

Coupled electromechanical waves define a heart's function in health and diseases. Optical mapping of electrical waves using fluorescent labels offers mechanistic insights into cardiac conduction abnormalities. Dye-free/label-free mapping of mechanical waves presents an attractive non-invasive alternative. In this study, we developed a simultaneous widefield voltage and interferometric dye-free optical imaging methodology that was used as follows: (1) to validate dye-free optical mapping for quantification of cardiac wave properties in human iPSC-cardiomyocytes (CMs); (2) to demonstrate low-cost optical mapping of electromechanical waves in hiPSC-CMs using recent near-infrared (NIR) voltage sensors and orders of magnitude cheaper miniature industrial CMOS cameras; (3) to uncover previously underexplored frequency- and space-varying parameters of cardiac electromechanical waves in hiPSC-CMs. We find similarity in the frequency-dependent responses of electrical (NIR fluorescence-imaged) and mechanical (dye-free-imaged) waves, with the latter being more sensitive to faster rates and showing steeper restitution and earlier appearance of wavefront tortuosity. During regular pacing, the dye-free-imaged conduction velocity and electrical wave velocity are correlated; both modalities are sensitive to pharmacological uncoupling and dependent on gap-junctional protein (connexins) determinants of wave propagation. We uncover the strong frequency dependence of the electromechanical delay (EMD) locally and globally in hiPSC-CMs on a rigid substrate. The presented framework and results offer new means to track the functional responses of hiPSC-CMs inexpensively and non-invasively for counteracting heart disease and aiding cardiotoxicity testing and drug development.

The mechanical function or failure of the heart is defined by the adaptability of its contractility under different conditions. This, coupled with dynamic changes of heart mechanics, via hemodynamics or pathological stiffening, have driven decades of research to understand how external mechanical cues affect function of cardiac muscle cells (CMs). Despite a growing body of research on the effect of microenvironment mechanics (often stiffness) on contractility of CMs, there has been little appreciation of how this adaptation evolves over time. In vivo, CMs experience a gradual increase in the stiffness of their microenvironment, from ∼1 kPa in neonatal heart, to ∼10 kPa for adult heart to ∼50 kPa in fibrotic myocardium (e.g post myocardial infarction). While such mechanical changes have shown to affect CMs behaviour, the role of chronic adaptation over time is often ignored. Here we quantified the evolution of contractile work of CMs over six days. Primary CMs were cultured on substrates of neonatal, adult, and fibrotic myocardium and we measured their contractility using Traction Force Microsocpy (TFM) on days 6, 8, 10, and 12. We observed a gradual increase in contractility in all conditions, suggesting an adaptation mechanism controlling the acute response over longer time scales. To study their electrical response, we used dye-free diffraction imaging. We observed that monolayers on 1 kPa substrates show higher autonomous beating rates, and while substrates with stiffnesses of 10 and 50 kPa show similar beating rates, the monolayers on the 50 kPa substrate have a smaller contraction magnitude. We verified these results with fluorescent imaging of a calcium indicator (FlouO-4). CMs on neonatal stiffness showed significantly faster action potentials (smaller duration) than their counterparts on stiffer substrates. These results indicate the importance of the time-dependent response of CMs to the mechanics of their microenvironment.

Fast volumetric imaging is essential for understanding the function of excitable tissues such as those found in the brain and heart. Measuring cardiac voltage transients in tissue volumes is challenging, especially at the high spatial and temporal resolutions needed to give insight to cardiac function. We introduce a new imaging modality based on simultaneous illumination of multiple planes in the tissue and parallel detection with multiple cameras, avoiding compromises inherent in any scanning approach. The system enables imaging of voltage transients in situ, allowing us, for the first time to our knowledge, to map voltage activity in the whole heart volume at KHz rates. The high spatiotemporal resolution of our method enabled the observation of novel dynamics of electrical propagation through the zebrafish atrioventricular canal.

Cardiac action potential (AP) shape and propagation are regulated by several key dynamic factors such as ion channel recovery and intracellular Ca2+ cycling. Experimental methods for manipulating AP electrical dynamics commonly use ion channel inhibitors that lack spatial and temporal specificity. In this work, we propose an approach based on optogenetics to manipulate cardiac electrical activity employing a light-modulated depolarizing current with intensities that are too low to elicit APs (sub-threshold illumination), but are sufficient to fine-tune AP electrical dynamics. We investigated the effects of sub-threshold illumination in isolated cardiomyocytes and whole hearts by using transgenic mice constitutively expressing a light-gated ion channel (channelrhodopsin-2, ChR2). We find that ChR2-mediated depolarizing current prolongs APs and reduces conduction velocity (CV) in a space-selective and reversible manner. Sub-threshold manipulation also affects the dynamics of cardiac electrical activity, increasing the magnitude of cardiac alternans. We used an optical system that uses real-time feedback control to generate re-entrant circuits with user-defined cycle lengths to explore the role of cardiac alternans in spontaneous termination of ventricular tachycardias (VTs). We demonstrate that VT stability significantly decreases during sub-threshold illumination primarily due to an increase in the amplitude of electrical oscillations, which implies that cardiac alternans may be beneficial in the context of self-termination of VT.

Parasystole in an arrhythmia caused by the emergence of a second pacemaker, in addition to the sinoatrial (SA) node. This ectopic pacemaker can trigger early cardiac contractions in the atria or in the ventricles and if frequent, they can lead to cardiomyopathies. Published models of parasystole suggest that the wave dynamics can be predicted from P, the ratio of the period of the ectopic pacemaker to that of the SA node, and the refractory period θ. These models, despite providing valuable insight into the arrhythmia, do not model the excitable media which carries electronic impulses between the two pacemakers. In this project, we modelled two independently beating oscillators embedded in excitable media, using computational and experimental methods. The theoretical models are based on a one-dimensional line of discretized cells representing a strip of excitable media. We simulated wave propagation using a discrete cellular automaton as well as a system of continuous Fitzhugh-Nagumo differential equations. The experimental model is based on an all-optical non-contact system that allowed us to stimulate and record from bioengineered cardiac monolayers for long periods of time in a physiologically stable environment. To study parasystolic dynamics, we track the sequence N of numbers of normal SA node waves between consecutive ectopic beats. Results from these three models are consistent: given the ratio P, the refractory period θ and a point X in space, there are only three unique values in N and they're identical across the models. These results add another degree of complexity to parasystolic dynamics earlier described: the spatial structure also impacts the observable wave dynamics. These results enable the development of models of parasystole in two dimensions based on experimental data.

Abstract Aims Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility. Methods and results NOX2-Tg mice showed a 2- to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1β, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype. Conclusion Together, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.

Aims Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility. .................................................................................................................................................................................................... Methods and results NOX2-Tg mice showed a 2to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1b, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2þ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype. .................................................................................................................................................................................................... Conclusion Together, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.