In vivo ratiometric optical mapping enables high-resolution cardiac electrophysiology in pig models

AIMS: Cardiac optical mapping is the gold standard for measuring complex electrophysiology in ex vivo heart preparations. However, new methods for optical mapping in vivo have been elusive. We aimed at developing and validating an experimental method for performing in vivo cardiac optical mapping in pig models. METHODS AND RESULTS: First, we characterized ex vivo the excitation-ratiometric properties during pacing and ventricular fibrillation (VF) of two near-infrared voltage-sensitive dyes (di-4-ANBDQBS/di-4-ANEQ(F)PTEA) optimized for imaging blood-perfused tissue (n = 7). Then, optical-fibre recordings in Langendorff-perfused hearts demonstrated that ratiometry permits the recording of optical action potentials (APs) with minimal motion artefacts during contraction (n = 7). Ratiometric optical mapping ex vivo also showed that optical AP duration (APD) and conduction velocity (CV) measurements can be accurately obtained to test drug effects. Secondly, we developed a percutaneous dye-loading protocol in vivo to perform high-resolution ratiometric optical mapping of VF dynamics (motion minimal) using a high-speed camera system positioned above the epicardial surface of the exposed heart (n = 11). During pacing (motion substantial) we recorded ratiometric optical signals and activation via a 2D fibre array in contact with the epicardial surface (n = 7). Optical APs in vivo under general anaesthesia showed significantly faster CV [120 (63-138) cm/s vs. 51 (41-64) cm/s; P = 0.032] and a statistical trend to longer APD90 [242 (217-254) ms vs. 192 (182-233) ms; P = 0.095] compared with ex vivo measurements in the contracting heart. The average rate of signal-to-noise ratio (SNR) decay of di-4-ANEQ(F)PTEA in vivo was 0.0671 ± 0.0090 min-1. However, reloading with di-4-ANEQ(F)PTEA fully recovered the initial SNR. Finally, toxicity studies (n = 12) showed that coronary dye injection did not generate systemic nor cardiac damage, although di-4-ANBDQBS injection induced transient hypotension, which was not observed with di-4-ANEQ(F)PTEA. CONCLUSIONS: In vivo optical mapping using voltage ratiometry of near-infrared dyes enables high-resolution cardiac electrophysiology in translational pig models.The CNIC is supported by the Ministry of Science, Innovation and Universities and the Pro CNIC Foundation. The CNIC is a Severo Ochoa Center of Excellence (SEV-2015-0505). This study was supported by grants from Fondo Europeo de Desarrollo Regional (CB16/11/00458), the Spanish Ministry of Science, Innovation and Universities (SAF2016-80324-R, PI16/02110, and DTS17/00136), and by the European Commission (ERA-CVD Joint Call [JTC2016/APCIN-ISCIII-2016], grant#AC16/00021). The study was also partially supported by the Fundacio´n Interhospitalaria para la Investigacio´n Cardiovascular (FIC) and the Heart Rhythm section of the Spanish Society of Cardiology. The work at the University of Connecticut was supported by grant EB001963 from the National Institutes of Health.S

Two-photon excitation of FluoVolt allows improved interrogation of transmural electrophysiological function in the intact mouse heart

Background and aims: Two-photon excitation of voltage sensitive dyes (VSDs) can measure rapidly changing electrophysiological signals deep within intact cardiac tissue with improved three-dimensional resolution along with reduced photobleaching and photo-toxicity compared to conventional confocal microscopy. Recently, a category of VSDs has emerged which records membrane potentials by photo-induced electron transfer. FluoVolt is a novel VSD in this category which promises fast response and a 25% fractional change in fluorescence per 100 mV, making it an attractive optical probe for action potential (AP) recordings within intact cardiac tissue. The purpose of this study was to characterize the fluorescent properties of FluoVolt as well as its utility for deep tissue imaging. Methods: Discrete tissue layers throughout the left ventricular wall of isolated perfused murine hearts loaded with FluoVolt or di-4-ANEPPS were sequentially excited with two-photon microscopy. Results: FluoVolt loaded hearts suffered significantly fewer episodes of atrio-ventricular block compared to di-4-ANEPPS loaded hearts, indicating comparatively low toxicity of FluoVolt in the intact heart. APs recorded with FluoVolt were characterized by a lower signal-to-noise ratio and a higher dynamic range compared to APs recorded with di-4-ANEPPS. Although both depolarization and repolarization parameters were similar in APs recorded with either dye, FluoVolt allowed deeper tissue excitation with improved three-dimensional resolution due to reduced out-of-focus fluorescence generation under two-photon excitation. Conclusion: Our results demonstrate several advantages of two-photon excitation of FluoVolt in functional studies in intact heart preparations, including reduced toxicity and improved fluorescent properties

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

A study on the effect of β-adrenergic stimulation on the electrophysiology of the isolated heart

Background: A coordinated heart beat relies on the propagation of a rapid depolarising event throughout the atria and ventricles and the subsequent coupling of this electrical signal to a transient contraction in every atrial and ventricular cardiomyocyte. The rate of propagation, known as conduction velocity (CV) is mainly determined by cellular expression of Na channels and gap junctional proteins (connexins), however there is emerging evidence that both proteins may be functionally regulated by intrinsic signaling processes. Previous studies indicate that stimulation of the β-adrenergic pathway increases CV, but little consistent data exists on the magnitude, associated adrenoreceptor pharmacology or time course of the effect. This study investigates the effect of β-AR stimulation – using either the β-agonist isoproterenol (ISO) or by directly raising cAMP via addition of Forskolin (Fsk) and/or 3-Isobutyl-1-methylxanthine (IBMX) - on ventricular CV in the intact rat heart. The aim was to measure the response of CV to β-AR stimulation and investigate the mechanisms behind this response. Action potential (AP) and intracellular Ca2+ measurements were also made to determine the effect of β-AR stimulation on cellular electrophysiology over the same time-course as the CV response to β-AR stimulation. Methods: Adult male Wistar rats (250-350g) were euthanized by cervical dislocation and excised hearts retrogradely perfused with modified Tyrode's solution. CV measurements were taken using a custom-built probe, consisting of bipolar stimulating and recording electrode pairs placed flat against the epicardium of the left ventricle (LV). The CV probe also incorporated a fibre-optic light guide, allowing ratiometric measurements of voltage and intracellular Ca2+ from the LV epicardium. Results and Conclusions: β-AR stimulation increased LV longitudinal CV by approximately 10%. This increase in CV was found to be cAMP mediated. This effect was not due to changes in Ca2+ handling alone and although an increase in AP amplitude (APA) suggested that INa was increased, the magnitude was thought insufficient to explain the change in CV. This suggested a potential role for gap junction conductance (GJC) in mediating CV changes. This view was supported by preliminary data indicating the magnitude of the response was larger when measuring transverse CV: transverse conduction involves proportionally more GJC than longitudinal conduction. β-AR stimulation was confirmed to increase CV, a response mediated via β1AR subtype, and which required an increase in cAMP: cAMP was increased by activation of adenylyl cyclase (AC) with forskolin (Fsk) or through inhibition of phosphodiesterases (PDEs) by IBMX. The increase in CV was shown to be mediated through the cAMP sensitive kinase, PKA; another cAMP target, Epac, appeared not have a role in this pathway. Understanding the regulation of CV by β-AR stimulation is crucial to understanding sympathetic regulation of the heart and may lead to further understanding of the interplay between downregulated β-AR signaling and arrhythmia generation in the diseased heart

Examination of myocardial electrophysiology using novel panoramic optical mapping techniques

Optical mapping of voltage signals has revolutionised the field and study of cardiac electrophysiology by providing the means to visualise changes in electrical activity at a high temporal and spatial resolution from the cellular to the whole heart level under both normal and disease conditions. The aim of this thesis was to develop a novel method of panoramic optical mapping using a single camera and to study myocardial electrophysiology in isolated Langendorff-perfused rabbit hearts. First, proper procedures for selection, filtering and analysis of the optical data recorded from the panoramic optical mapping system were established. This work was followed by extensive characterisation of the electrical activity across the epicardial surface of the preparation investigating time and heart dependent effects. In an initial study, features of epicardial electrophysiology were examined as the temperature of the heart was reduced below physiological values. This manoeuvre was chosen to mimic the temperatures experienced during various levels of hypothermia in vivo, a condition known to promote arrhythmias. The facility for panoramic optical mapping allowed the extent of changes in conduction timing and pattern of ventricular activation and repolarisation to be assessed. In the main experimental section, changes in epicardial electrical activity were assessed under various pacing conditions in both normal hearts and in a rabbit model of chronic MI. In these experiments, there was significant changes in the pattern of electrical activation corresponding with the changes in pacing regime. These experiments demonstrated a negative correlation between activation time and APD, which was not maintained during ventricular pacing. This suggests that activation pattern is not the sole determinant of action potential duration in intact hearts. Lastly, a realistic 3D computational model of the rabbit left ventricle was developed to simulate the passive and active mechanical properties of the heart. The aim of this model was to infer further information from the experimental optical mapping studies. In future, it would be feasible to gain insight into the electrical and mechanical performance of the heart by simulating experimental pacing conditions in the model

The effect of hypothermia and rewarming on cardiac electrophysiology and mechanical function

Hypothermia is defined as a core body temperature of 35°C or less and can be induced (i.e. therapeutic) or accidental. It is well established that hypothermia leads to a positive inotropic response which causes an increase in the magnitude of cardiac contraction, however rewarming from hypothermia is associated with a negative inotropic response, and the underlying mechanisms of this remain unclear. Accidental hypothermia is further complicated by risk of ventricular arrhythmias and cardiac arrest. This contributes to high mortality rates among these patients. Although hypothermia is used extensively as a therapeutic intervention and survival is possible after extreme exposure, treatment of arrhythmias during rewarming is still challenging. In order to develop targeted anti-arrhythmic strategies in this very specific situation, we first need to understand the basis for pro-arrhythmia during cooling and rewarming. This study aimed to examine the effect of hypothermia and rewarming on aspects of cardiac inotropy and excitability. An in vitro model of hypothermia and rewarming using isolated rat ventricular cardiomyocytes showed that following 3 hours of hypothermia there was a significant reduction in shortening upon rewarming. This was not accompanied by a change in intracellular Ca2+, suggesting a rewarming induced decrease in myofilament sensitivity to Ca2+. In separate experiments, animals underwent an in vivo hypothermia/rewarming procedure and displayed evidence of rewarming induced contractile dysfunction. Epicardial action potential (AP) measurements on these hearts showed a shortened AP duration (APD) when compared to normothermic control animals, which suggests that a sustained electrophysiological effect that could manifest as a shortened QT interval. In contrast to this, a period of transient hypothermia had alternative detrimental effects on the cardiac APD when compared to prolonged hypothermia, an effect that could predispose to the induction of long QT related arrhythmias and ventricular tachycardia. Separate experiments assessed the effect of moderate (31˚C) and severe (17˚C) hypothermia on cardiac excitability in Langendorff perfused rabbit hearts. Moderate hypothermia prolonged PR and QT intervals whilst in severe hypothermia all ECG parameters were prolonged. Ventricular activation times were unaffected at 31°C whilst action potential duration (APD90) was significantly prolonged. At 17°C there were significant and proportionally similar delays in both activation and repolarisation. Ventricular fibrillation (VF) threshold was significantly reduced at 31°C (pro-arrhythmic), but at 17°C VF threshold was >2x baseline (37°C) (anti-arrhythmic). At 31°C, transverse conduction (CVt) was relatively insensitive to cooling versus longitudinal conduction (CVl) but at 17°C both CVt and CVl were proportionately reduced to a similar extent. The gap junction uncoupler heptanol had a larger relative effect on CVt than CVl, and at 31°C was able to restore the CVt/CVl ratio, returning VF threshold to baseline values. This suggests that moderate hypothermia creates repolarisation abnormalities and is pro-arrhythmic. These divergent effects appear to be linked to a lower temperature sensitivity of gap junctions, a conclusion supported by the anti-arrhythmic effect of heptanol at 31°C

Development of a medium-high throughput electrophysiology method to study cellular heterogeneity in the rabbit heart

Sudden cardiac death (SCD) is a prominent cause of death worldwide today, mainly occurring as a result of coronary heart disease, cardiomyopathies, and inherited or induced arrhythmia syndromes. Survival following sudden cardiac arrest (SCA) has improved in the past decades, but the majority of cases of SCD remain unwitnessed. Although advances have been made towards the investigation of the mechanisms behind SCD, it remains a poorly understood phenomenon. Environmental factors have been identified and associated with increased arrhythmic risk, and most prominently, drug-induced arrhythmias constitute a serious hurdle to both cardiac and non-cardiac drug development. The past decade has seen pro-arrhythmic screening of new compounds become routine, and develop into a major point of interest for drug development. Specifically, the onset of drug-induced polymorphic ventricular tachycardia, such as torsade de pointes (TdP), is of particular interest to cardiac research. The concept of electrophysiological heterogeneity in cardiac muscle holds exciting potential for explaining the pathophysiology of TdP, but quantifying cellular heterogeneity using conventional methods is a challenge. This work developed and refined a fluorescence-based, medium/high-throughput electrophysiological assay to process large cell populations (~50-500 cells) from single hearts. Using this novel approach, transmural electrophysiological differences were found between regions of individual hearts, replicating published work with a 3 to 4-fold reduction in hearts sampled, and additionally providing a previously unknown quantification of cellular heterogeneity in isolated cardiomyocyte populations, in both healthy and failing rabbit hearts. Further classification of electrophysiological differences within smaller regions of the ventricle yielded evidence of repolarisation gradients across the myocardium, with vast overlap in repolarisation duration, challenging the dogma of region-specific repolarisation duration. Lastly, by specifically blocking hERG channels and L-type calcium channels in cardiac subregions (sub-epicardial apex and base) strong evidence was found for heterogeneous electrophysiology response amongst isolated cell populations. Specifically, sub-epicardial action potential shortening using nifedipine was strongly APD dependent, whereby baseline AP duration determined the extent of APD shortening via drug-induced ICa-L blockade. Sub-epicardial AP prolongation mediated via IKr block using dofetilide also produced non-homogeneous cell response in the form of two distinct population responses: (i) The majority (~85%) was made up of normal responding cells, experiencing ~20-30ms AP prolongation not dependent on baseline APD (P100ms AP prolongation, beyond the pacing cycle length (>500ms) without any evidence of early-afterdepolarisations. Large experimental samples of AP parameters gathered in this study can provide real-world data parameter space ranges for mathematical model development, showing that ion channel conductance ranges used today to predict drug responses at the organ level may be too restrictive, or inaccurate. Iterative model adjustment using large experimental datasets can help constrain models and improve their predictive power, saving time by reducing computational power required

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