High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate lectrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology

Optical mapping and optogenetics in cardiac electrophysiology research and therapy:a state-of-the-art review

State-of-the-art innovations in optical cardiac electrophysiology are significantly enhancing cardiac research. A potential leap into patient care is now on the horizon. Optical mapping, using fluorescent probes and high-speed cameras, offers detailed insights into cardiac activity and arrhythmias by analysing electrical signals, calcium dynamics, and metabolism. Optogenetics utilizes light-sensitive ion channels and pumps to realize contactless, cell-selective cardiac actuation for modelling arrhythmia, restoring sinus rhythm, and probing complex cell–cell interactions. The merging of optogenetics and optical mapping techniques for ‘all-optical’ electrophysiology marks a significant step forward. This combination allows for the contactless actuation and sensing of cardiac electrophysiology, offering unprecedented spatial–temporal resolution and control. Recent studies have performed all-optical imaging ex vivo and achieved reliable optogenetic pacing in vivo, narrowing the gap for clinical use. Progress in optical electrophysiology continues at pace. Advances in motion tracking methods are removing the necessity of motion uncoupling, a key limitation of optical mapping. Innovations in optoelectronics, including miniaturized, biocompatible illumination and circuitry, are enabling the creation of implantable cardiac pacemakers and defibrillators with optoelectrical closed-loop systems. Computational modelling and machine learning are emerging as pivotal tools in enhancing optical techniques, offering new avenues for analysing complex data and optimizing therapeutic strategies. However, key challenges remain including opsin delivery, real-time data processing, longevity, and chronic effects of optoelectronic devices. This review provides a comprehensive overview of recent advances in optical mapping and optogenetics and outlines the promising future of optics in reshaping cardiac electrophysiology and therapeutic strategies

Temporal irregularity quantification and mapping of optical action potentials using wave morphology similarity

Background Cardiac optical mapping enables direct and high spatio-temporal resolution recording of action potential (AP) morphology. Temporal alterations in AP morphology are both predictive and consequent of arrhythmia. Here we sought to test if methods that quantify regularity of recorded waveforms could be applied to detect and quantify periods of temporal instability in optical mapping datasets in a semi-automated, user-unbiased manner. Methods and results We developed, tested and applied algorithms to quantify optical wave similarity (OWS) to study morphological temporal similarity of optically recorded APs. Unlike other measures (e.g. alternans ratio, beat-to-beat variability, arrhythmia scoring), the quantification of OWS is achieved without a restrictive definition of specific signal points/features and is instead derived by analysing the complete morphology from the entire AP waveform. Using model datasets, we validated the ability of OWS to measure changes in AP morphology, and tested OWS mapping in guinea pig hearts and mouse atria. OWS successfully detected and measured alterations in temporal regularity in response to several proarrhythmic stimuli, including alterations in pacing frequency, premature contractions, alternans and ventricular fibrillation. Conclusion OWS mapping provides an effective measure of temporal regularity that can be applied to optical datasets to detect and quantify temporal alterations in action potential morphology. This methodology provides a new metric for arrhythmia inducibility and scoring in optical mapping datasets

Transcriptional Regulation of Arrhythmia: from Mouse to Human

In the last two decades, our understanding of cardiac arrhythmias has been accelerated immensely by the development of genetically engineered animals. Transgenic and knockout mice have been the “gold standard” platforms for delineating disease mechanisms. Much of our understanding of the pathogenesis of atrial and ventricular arrhythmias is gained from mouse models that alter the expression of specific ion channels or other proteins. However, cardiac arrhythmias such as atrial fibrillation are heterogeneous diseases with numerous distinct conditions that could not be explained exclusively by the disruption of ionic currents. Increasing evidence suggests disruption of signaling pathways in the pathogenesis of cardiac arrhythmias. Although crucial for studying disease mechanisms, animal models often fail to predict human response to treatments due to inter-species genetic and physiological differences. Cardiac slices obtained from human hearts have been demonstrated as an accurate model that more faithfully recapitulates human cardiac physiology. However, the use of the human cardiac slices for evaluating the transcriptional regulation of arrhythmia is hampered by tissue remodeling and dedifferentiation in long-term culture of the slices. The first part of this dissertation aims to elucidate one of the potential mechanisms of sick sinus syndrome and atrial fibrillation induced by transient reactivation of Notch, a critical transcription factor during cardiac development and has been shown to be reactivated in the adult heart following cardiac injury. When Notch is transiently reactivated in the adult mice to mimic the injury response, the animals exhibits slowed heart rate, increased heart rate variability, frequent sinus pauses, and slowed atrial conduction. The electrical remodeling of the atrial myocardium results in increased susceptibility to atrial fibrillation. The transient reactivation of Notch also significantly altered the atrial gene expression profile, with many of the disrupted genes associated with cardiac arrhythmias by genome-wide association study. The second part of this dissertation aims to address the lack the translation from animal research to human therapies by extending the human cardiac slice viability in culture. With the optimized culture parameters, human cardiac slices obtained from the left ventricular free wall remained electrically viable for up to 21 days in vitro and routinely maintained normal electrophysiology for up to 4 days. To genetically alter the human cardiac slices, a localized gene delivery technique was evaluated and optimized. The third part of the dissertation aims to further improve long-term culture of human cardiac slices and to increase the availability of human tissue for research by developing a self-contained heart-on-a-chip system for automated culture of human cardiac slices. The system maintains optimal culture conditions and provides electrical stimulation and mechanical anchoring to minimize tissue dedifferentiation. The work allows for accelerated optimization of long-term culturing of human cardiac slice, which will enable study of arrhythmia mechanisms on human cardiac tissue via targeted control of transcription factors

A multi-channel system for use in cardiac electrophysiologic studies

The location of accessory pathways in Wolff-Parkinson-White syndrome patients is performed manually during open heart surgery at Groote Schuur Hospital, using a hand-held roving electrode. This manual procedure is slow and tedious, prolonging the operation and the time for which the patient remains on cardiac bypass. A multichannel electrogram acquisition and display system with a storage facility would significantly reduce the time taken and improve the reliability of locating the accessory pathways. Having considered a number of currently available cardiac mapping systems it was decided that a new system be developed for specific application within Groote Schuur Hospital. The main design goals of this system are to improve accuracy, increase reliability and enhance the speed of the entire mapping procedure with direct benefit to staff and patients. The system is based on an IBM compatible computer and allows for the acquisition of a maximum of thirty-two electrogram inputs. A typical configuration would acquire twenty epicardial, two references (one each from atrium and ventricle), one roving electrode and two surface lead signals. The epicardial signals are obtained from a custom-built electrode belt which is placed around the heart over the atrioventricular groove. The project includes the development of front-end hardware and software for processing, display and storage of electrogram signals. The relative activation times of the signals are displayed under software control in order to facilitate the location of any accessory pathway(s)

Endocardial activation mapping of human atrial fibrillation

Successful ablation of arrhythmias depends upon interpretation of the mechanism. However, in persistent atrial fibrillation (AF) ablation is currently directed towards the mechanism that initiates paroxysmal AF. We sought to address the hypothesis that atrial activation patterns during persistent AF may help determine the underlying mechanism. Activation mapping of AF wavefronts is labor intensive and often restricted to short time segments in limited atrial locations. RETRO-Mapping was developed to identify uniform wavefronts that occur during AF, and summate all wavefront vectors on to an orbital plot. Uniform wavefronts were mapped using RETRO-Mapping during sinus rhythm, atrial tachycardia, and atrial fibrillation, and validated against detailed manual analysis of the same wavefronts with conventional isochronal mapping. RETRO-Mapping was found to have comparable accuracy to isochronal mapping. RETRO-Mapping was then used to investigate atrial activation patterns during persistent AF. Atrial activation patterns demonstrated evidence of spatiotemporal stability over long time periods. Orbital plots created at different time points in the same location remained unchanged. Together with this important discovery, both fractionation and bipolar voltage were also demonstrated to express stability over time. Spatiotemporal stability during persistent AF enables sequential mapping as an acceptable technique. This property also allowed the development of a method for displaying sequentially mapped locations on a single map – RETRO-Choropleth Map. These findings go against the multiple wavelet hypothesis with random activation. Having gained insights in to these stable activation patterns, extensive analysis was undertaken to identify the presence of focal activation. Focal activations were identified during persistent AF. RETRO-Mapping was used to show that adjacent activation patterns were not related to focal activations. Lastly, the effect of pulmonary vein isolation (PVI) was studied by mapping atrial activation patterns before and after PVI. RETRO-Mapping showed that PVI leads to increased organisation of AF in most patients, supporting a mechanistic role of the pulmonary veins in persistent AF. In conclusion, a new technique has been developed and validated for automated activation mapping of persistent AF. These techniques could be used to guide additional ablation strategies beyond PVI for patients with persistent AF.Open Acces

High Resolution Multi-parametric Diagnostics and Therapy of Atrial Fibrillation: Chasing Arrhythmia Vulnerabilities in the Spatial Domain

After a century of research, atrial fibrillation (AF) remains a challenging disease to study and exceptionally resilient to treatment. Unfortunately, AF is becoming a massive burden on the health care system with an increasing population of susceptible elderly patients and expensive unreliable treatment options. Pharmacological therapies continue to be disappointingly ineffective or are hampered by side effects due to the ubiquitous nature of ion channel targets throughout the body. Ablative therapy for atrial tachyarrhythmias is growing in acceptance. However, ablation procedures can be complex, leading to varying levels of recurrence, and have a number of serious risks. The high recurrence rate could be due to the difficulty of accurately predicting where to draw the ablation lines in order to target the pathophysiology that initiates and maintains the arrhythmia or an inability to distinguish sub-populations of patients who would respond well to such treatments. There are electrical cardioversion options but there is not a practical implanted deployment of this strategy. Under the current bioelectric therapy paradigm there is a trade-off between efficacy and the pain and risk of myocardial damage, all of which are positively correlated with shock strength. Contrary to ventricular fibrillation, pain becomes a significant concern for electrical defibrillation of AF due to the fact that a patient is conscious when experiencing the arrhythmia. Limiting the risk of myocardial injury is key for both forms of fibrillation. In this project we aim to address the limitations of current electrotherapy by diverging from traditional single shock protocols. We seek to further clarify the dynamics of arrhythmia drivers in space and to target therapy in both the temporal and spatial domain; ultimately culminating in the design of physiologically guided applied energy protocols. In an effort to provide further characterization of the organization of AF, we used transillumination optical mapping to evaluate the presence of three-dimensional electrical substrate variations within the transmural wall during acutely induced episodes of AF. The results of this study suggest that transmural propagation may play a role in AF maintenance mechanisms, with a demonstrated range of discordance between the epicardial and endocardial dynamic propagation patterns. After confirming the presence of epi-endo dyssynchrony in multiple animal models, we further investigated the anatomical structure to look for regional trends in transmural fiber orientation that could help explain the spectrum of observed patterns. Simultaneously, we designed and optimized a multi-stage, multi-path defibrillation paradigm that can be tailored to individual AF frequency content in the spatial and temporal domain. These studies continue to drive down the defibrillation threshold of electrotherapies in an attempt to achieve a pain-free AF defibrillation solution. Finally, we designed and characterized a novel platform of stretchable electronics that provide instrumented membranes across the epicardial surface or implanted within the transmural wall to provide physiological feedback during electrotherapy beyond just the electrical state of the tissue. By combining a spatial analysis of the arrhythmia drivers, the energy delivered and the resulting damage, we hope to enhance the biophysical understanding of AF electrical cardioversion and xiii design an ideal targeted energy delivery protocol to improve upon all limitations of current electrotherapy