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

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

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

A Regional Reduction in Ito and IKACh in the Murine Posterior Left Atrial Myocardium Is Associated with Action Potential Prolongation and Increased Ectopic Activity.

BACKGROUND: The left atrial posterior wall (LAPW) is potentially an important area for the development and maintenance of atrial fibrillation. We assessed whether there are regional electrical differences throughout the murine left atrial myocardium that could underlie regional differences in arrhythmia susceptibility. METHODS: We used high-resolution optical mapping and sharp microelectrode recordings to quantify regional differences in electrical activation and repolarisation within the intact, superfused murine left atrium and quantified regional ion channel mRNA expression by Taqman Low Density Array. We also performed selected cellular electrophysiology experiments to validate regional differences in ion channel function. RESULTS: Spontaneous ectopic activity was observed during sustained 1Hz pacing in 10/19 intact LA and this was abolished following resection of LAPW (0/19 resected LA, P<0.001). The source of the ectopic activity was the LAPW myocardium, distinct from the pulmonary vein sleeve and LAA, determined by optical mapping. Overall, LAPW action potentials (APs) were ca. 40% longer than the LAA and this region displayed more APD heterogeneity. mRNA expression of Kcna4, Kcnj3 and Kcnj5 was lower in the LAPW myocardium than in the LAA. Cardiomyocytes isolated from the LAPW had decreased Ito and a reduced IKACh current density at both positive and negative test potentials. CONCLUSIONS: The murine LAPW myocardium has a different electrical phenotype and ion channel mRNA expression profile compared with other regions of the LA, and this is associated with increased ectopic activity. If similar regional electrical differences are present in the human LA, then the LAPW may be a potential future target for treatment of atrial fibrillation

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

Familial atrial fibrillation mutation M1875T-SCN5A increases early sodium current and dampens the effect of flecainide

Aims Atrial fibrillation (AF) is the most common cardiac arrhythmia. Pathogenic variants in genes encoding ion channels are associated with familial AF. The point mutation M1875T in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel Nav1.5, has been associated with increased atrial excitability and familial AF in patients. Methods and results We designed a new murine model carrying the Scn5a-M1875T mutation enabling us to study the effects of the Nav1.5 mutation in detail in vivo and in vitro using patch clamp and microelectrode recording of atrial cardiomyocytes, optical mapping, electrocardiogram, echocardiography, gravimetry, histology, and biochemistry. Atrial cardiomyocytes from newly generated adult Scn5a-M1875T+/− mice showed a selective increase in the early (peak) cardiac sodium current, larger action potential amplitude, and a faster peak upstroke velocity. Conduction slowing caused by the sodium channel blocker flecainide was less pronounced in Scn5a-M1875T+/− compared to wildtype atria. Overt hypertrophy or heart failure in Scn5a-M1875T+/− mice could be excluded. Conclusion The Scn5a-M1875T point mutation causes gain-of-function of the cardiac sodium channel. Our results suggest increased atrial peak sodium current as a potential trigger for increased atrial excitability

Reduced plakoglobin increases the risk of sodium current defects and atrial conduction abnormalities in response to androgenic anabolic steroid abuse

Androgenic anabolic steroids (AAS) are commonly abused by young men. Male sex and increased AAS levels are associated with earlier and more severe manifestation of common cardiac conditions, such as atrial fibrillation, and rare ones, such as arrhythmogenic right ventricular cardiomyopathy (ARVC). Clinical observations suggest a potential atrial involvement in ARVC. Arrhythmogenic right ventricular cardiomyopathy is caused by desmosomal gene defects, including reduced plakoglobin expression. Here, we analysed clinical records from 146 ARVC patients to identify that ARVC is more common in males than females. Patients with ARVC also had an increased incidence of atrial arrhythmias and P wave changes. To study desmosomal vulnerability and the effects of AAS on the atria, young adult male mice, heterozygously deficient for plakoglobin (Plako+/−), and wild type (WT) littermates were chronically exposed to 5α-dihydrotestosterone (DHT) or placebo. The DHT increased atrial expression of pro-hypertrophic, fibrotic and inflammatory transcripts. In mice with reduced plakoglobin, DHT exaggerated P wave abnormalities, atrial conduction slowing, sodium current depletion, action potential amplitude reduction and the fall in action potential depolarization rate. Super-resolution microscopy revealed a decrease in NaV1.5 membrane clustering in Plako+/− atrial cardiomyocytes after DHT exposure. In summary, AAS combined with plakoglobin deficiency cause pathological atrial electrical remodelling in young male hearts. Male sex is likely to increase the risk of atrial arrhythmia, particularly in those with desmosomal gene variants. This risk is likely to be exaggerated further by AAS use

Development and application of novel processing tools and methods for cardiac optical mapping

Cardiac optical mapping provides unparalleled spatio-temporal resolution information of cardiac electrophysiology. It has hence emerged as an important technology in understanding cardiac electrical behaviour in physiological and pathophysiological states. There is a requirement for effective data analysis tools that are high-throughput, robustly characterised and flexible with regards to a growing array of experimental models. In this thesis a MATLAB based software, ElectroMap, was developed for analysis of diverse optical mapping datasets. ElectroMap incorporates existing and novel methods to allow quantification and mapping of action potential and calcium transient morphology and activation/repolarisation times. Automated pacing cycle length detection and segmentation were implemented, realising high-throughput analysis of beat-to-beat responses and transient behaviour. Standalone modules dedicated to calculation of conduction velocity and alternans were introduced, allowing thorough integration of key factors in arrhythmogenesis. Semi-automated analysis of temporal variations in wave morphology were developed from previous methodologies for electrogram analysis. Algorithms to use fractional rate of change of fluorescence as a measure of conduction were also introduced to the software. Algorithms were tested in silico datasets, mouse and guinea pig optical mapping datasets and preliminary experiments also showed use for in vivo human electrogram mapping of atrial fibrillation