A. Introduction

The field of translational cardiac electrophysiology has flourished in the last two decades, leading to multiple new treatments and tools being translated into clinical practice. One of the main reasons why translational electrophysiology has thrived during this period is the advent and development of multiple novel translational research platforms. The use of these research platforms has enabled researchers to understand the pathophysiology of different arrhythmias in greater detail, culminating in the development of novel therapeutic strategies.

In this chapter, we will describe some of the most common and important translational research platforms currently used in the field of cardiac electrophysiology. The use of multi-electrode arrays to map complex arrhythmias and to study conduction properties will be described. We will then provide an overview of cardiac optical mapping, a technique increasing in popularity that allows for the non-invasive recording of optical action potentials from tens of thousands of sites simultaneously, thereby allowing for detailed mapping of arrhythmia circuits. The growing role of imaging in electrophysiology research will be discussed, followed by a description of the use of experimental models and computational modeling in translational electrophysiology research.

B. Multi-Electrode Arrays

The extracellular electrogram forms the basis of the majority of recordings in the clinical cardiac electrophysiology laboratory. Extracellular electrogram recordings have also been used to a great extent in translational research. However, unlike in the clinical setting where the number of electrogram recordings is often limited, electrograms are often recorded from a large number of sites using multi-electrode arrays in translational research, which provides greater spatial resolution for arrhythmia mapping. Here, we describe their use in a number of settings in the translational research laboratory.

Multi-electrode arrays in vivo

The use of multi-electrode recordings in vivo has provided important insights into the mechanisms of arrhythmogenesis. Here, we provide descriptions of two approaches to in vivo electrogram recordings, in models of atrial fibrillation (AF) and post-myocardial infarction ventricular tachycardia (VT).

In the seminal paper from the laboratory of Maurits Allessie (1), which coined the phrase “AF begets AF”, the authors chronically implanted electrodes in the atria in a goat model of atrial fibrillation. The implantation of electrodes was done surgically, via a thoracotomy and a total of 27 electrodes were sutured to the epicardial surface of the goat atria. Three silicon strips containing platinum electrodes were strategically placed on the epicardial surfaces of both the right and left atria, allowing electrograms to be recorded from important locations in both atria. The electrode leads were then tunneled subcutaneously, exteriorized and connected to data acquisition systems. This approach of implanting electrodes surgically and then recovering the animals allowed electrograms to be recorded over long durations in these chronically-instrumented goats. Using this research platform, the authors were able to show that atrial fibrillation can cause remodeling of the electrophysiological properties of atrial myocardium, thereby making the maintenance of atrial fibrillation more likely.

Multi-electrode arrays can also be used in open-chested studies, as was performed in the study by Peters et al (2). In that study, the authors sutured 9 x 13 cm flexible polymer sheets containing 292 bipolar electrodes onto the left ventricular surface of canine hearts that had previously been subjected to myocardial infarction. The electrodes were connected to a bioamplifier and a data acquisition system, allowing for recording from a large number of sites. Using this technique, they were able to map the circuit of post-myocardial ventricular tachycardia, identify lines of functional block and identify locations of the common central pathways of VT circuits.

Multi-electrode arrays ex-vivo

Multi-electrode arrays are also increasingly used on ex vivo perfused, whole hearts. The Langendorff isolated, perfused heart preparation is a popular experimental preparation in the translational research laboratory as it readily allows for the study of ischemia, infarction and reperfusion, as well as for testing new pharmacological agents (3,4).

In our laboratory, we have been using flexible multi-electrode arrays to record unipolar and bipolar electrograms from the epicardial surface of atria and ventricles of isolated, perfused rat hearts. These electrode arrays (Multi Channel Systems, Reutlingen, Germany) are known as FlexMEAs, and are made from flexible polyimide foil, with the biosensors only 12μm thick. These lightweight arrays contain 32 or 64 titanium nitride electrodes that can be custom designed to cover a specified area of interest. When applied to the wet surface of an isolated, perfused heart, these electrode arrays can record electrograms of excellent quality as shown in Fig. 1. The data can then be analyzed to calculate conduction velocities across the area covered by the array and also to calculate activation recovery intervals, as a surrogate for action potential duration.

Figure 1. Representative unipolar electrode recordings obtained using a flexible multi-electrode array applied to the epicardial surface of a rat left ventricle. The area covered by this 8×8 array measured 5mm x 5mm.

Electrode arrays on myocardial slices and cell monolayers

In the translational laboratory, electrode arrays are also often used to investigate electrophysiological properties in both myocardial slices and in cell culture monolayers. Although these are not whole heart preparations, there are potential advantages to recording electrograms from these specialized preparations.

Camelita et al demonstrated the feasibility and reproducibility of using multi-electrode arrays to study the electrophysiology of vibratome-cut myocardial slices (5). Using biopsy specimens obtained from patients with heart failure and also from experimental canine models, they were able to prepare multiple slices of approximately 300μm in thickness, which were superfused and placed on a multi-electrode array plate. Using these techniques, conduction velocities can be measured in the myocardial slices with preserved structural, biochemical and electrophysiological properties, and this approach can be used to study the electrophysiology of heart failure and also for high-throughput drug screening.

Multi-electrode plates are also useful for recording electrograms from cell culture monolayers. Experiments can be performed using neonatal rat ventricular myocytes (6), induced pluripotent stem-cells (iPSCs) (7) or cells of cardiomyocyte tumor lineage that retain differentiated cardiac morphological, biochemical, and electrophysiological properties, such as the HL-1 cell (8). The use of cell culture monolayers provides the benefit of being able to create monolayers with different patterns (9), which then allows the relationship between myocardial architecture and electrophysiological properties to be investigated.

C. Cardiac Optical Mapping

Although the extracellular electrogram recording systems described above can provide important information about the conduction properties of intact myocardium, the number of recordings that can be made simultaneously is often limited. Another limitation is that the extracellular recordings do not provide any information on the action potential and transmembrane voltage at the cellular level. These limitations have led to the development and increasing use of cardiac optical mapping.

Optical mapping is a technique that allows for the recording of biological data simultaneously over a large number (>10000) of sites using non-invasive optical methods. Its most important application in electrophysiology is the optical mapping of transmembrane voltage (Vm) transients using fluorescent voltage-sensitive dyes. Cardiac optical mapping of transmembrane voltage was established in the 1970s following on from pioneering work in the neurosciences (10). The first cardiac optical mapping experiments were performed by Salama and Morad in 1976 (11). Since then, this field has grown exponentially and optical mapping systems can now be found in most translational electrophysiology laboratories. In the following section, a brief description in this technique is provided, but the reader is directed to multiple excellent reviews on this technique for further details (12-14).

Principles of optical mapping of transmembrane voltage (Vm)

During a typical optical mapping experiment, an ex vivo heart is perfused and stained with a voltage-sensitive dye, which binds to the cell membrane. The potentiometric dye is then excited with a light source of an appropriate wavelength. When the excited dye molecules fall back to its baseline state from its excited state, they emits photons of a longer wavelength. The system of optics then filters the emitted photons and focuses the light onto a photodetector, which quantifies the amount of emitted light (Fig. 2).

Figure 2. Optical mapping hardware. A dual photodiode array (PDA) system is depicted. The heart is illuminated using an LED light source. The fluorescent light is then collected and split using two dichroic mirrors. The CCD camera collects light for plain images, whilst the two PDAs can be used to record transmembrane voltage (Vim) and calcium (Ca) transients.

Because of the unique property of potentiometric dyes, the quantity of filtered light detected by the photodetector is proportional to transmembrane voltage (Vm), and this allows for the non-invasive measurement of Vm changes and the recording of optical action potentials. One important difference to note between optical action potentials and those recorded with microelectrodes is that the optical action potential signal arises from a volume of cells and represents a multicellular spatial average of transmembrane potentials from cells within that volume of tissue.

Potentiometric dyes

Multiple potentiometric dyes have been synthesized to allow measurement of transmembrane voltage changes. Some common examples include di-4-ANNEPS, di-8-ANNEPS and RH237. These dyes all exhibit voltage-dependent shifts of their emission spectra, and they work by a mechanism known as electrochromism, whereby dye molecules within the cell membrane redistribute charge within the molecule when the cell depolarises. The charge shift within each molecule shifts the emission spectrum of the dye in response to changes in transmembrane voltage. Fig. 3 shows representative transmembrane voltage data acquired using the potentiometric dye RH237.

Figure 3. Representative optical action potentials recorded from an infarcted rat heart. The panel on the left shows the heart silhouette in blue and data from six pixels are shown on the right panel. Note that there is a progressive change in action potential morphology from top (recorded from non-infarcted region) to bottom (recorded from within the myocardial infarction zone).

Eliminating motion artifact during optical mapping

One of the main technical challenges of optical mapping studies is dealing with motion artefact. The vigorous contractions of heart muscle moves the heart within the optical mapping chamber and prevents the recording of optical action potential signals from the same site on the epicardial surface over the course of a cardiac cycle. As a result, strategies are required to reduce or eliminate motion artifact during optical mapping experiments. These include mechanical stabilisation, by gently wedging the heart within an optical mapping chamber, or using pharmacological agents such as blebbistatin, an inhibitor the adenosine triphosphatases (ATPases) associated with class II myosin isoforms, to uncouple cardiac excitation from contraction (15).

Detectors for cardiac optical mapping

There are three main classes of photodetectors commonly used for cardiac optical mapping experiments: Photodiode arrays (PDAs), Coupled-charge devices (CCDs), and Complementary metal–oxide–semiconductor (CMOS) detectors. Photodiode arrays consist of multiple photodiodes, with each diode detecting optical signals from a specific location on the heart. Each photodiode instantaneously converts photoexcited charge carriers into current flow, and the current is then converted to voltage using a preamplifier. Each photodiode is connected to its own preamplifier, and this parallel readout of the photodiodes provides a high temporal resolution, which is a major advantage over CCD systems, where pixels are read out sequentially. However, PDAs have poorer spatial resolution as typical systems consist of only several hundred photodiodes, whereas CCD arrays can contain up to several hundred thousand elements. Although photodiode arrays and CCD cameras have been the two main forms of detectors used for cardiac optical mapping, over recent years, CMOS cameras have been developed for optical mapping and these newer cameras have both excellent temporal and spatial resolutions.

Applications of cardiac optical mapping

Because of its excellent spatial resolution and the ability to record transmembrane voltage changes, cardiac optical mapping has been used to great effect in mapping complex arrhythmias and also in studying changes in activation and repolarization. Some of the major optical mapping studies include studies identifying and confirming the role of spiral waves and rotors in sustaining fibrillation (16,17). Optical mapping studies have also helped provide greater understanding of mechanisms of defibrillation (18), and also provided evidence for important electrophysiological concepts such as source-sink mismatch (9). Recently, optical mapping studies of human myocardium has given us insight into the electrophysiological remodeling changes that occur in human heart failure (19-21).

In addition to mapping transmembrane voltage, recent optical mapping studies have focused on multi-parametric imaging, combining the mapping of transmembrane voltage with calcium or metabolic imaging. Studies of dual voltage and calcium mapping have demonstrated the important relationship between voltage and calcium changes preceding the onset of arrhythmias (22). There has also been growing interest in metabolic imaging, for instance in measuring the intrinsic fluorescent signal of NADH to quantify local ischemia (23). Studies have also been carried out to investigate changes in the inner mitochondrial membrane potential during ischemia using the dye tetramethyl rhodamine methyl ester (TMRM) (24).

D. Imaging in Electrophysiology

Clinical cardiovascular magnetic resonance (CMR) imaging is a well established, validated technique, indicated to aid the diagnosis and treatment of a wide variety of cardiac pathologies (25). Of particular interest to the electrophysiologist is the ability of CMR to provide non-invasive information of the structural changes occurring in the myocardium that provide a substrate for arrhythmogenesis. The role of CMR continues to evolve as imaging modalities are developed; this section will focus on the application of delayed-enhanced and diffusion-weighted MR imaging to animal and human studies of cardiac arrhythmias.

Delayed-Enhancement MR Imaging

CMR with delayed-enhancement (DE-MRI) of Gadolinium is able to identify fibrosis and myocardial scar in animal and human subjects post-myocardial infarction. DE-MRI is able to distinguish ischemic and non-ischemic cardiomyopathies, and the presence and extent of DE is an independent predictor of the occurrence of ventricular arrhythmias (26) and sudden cardiac death (SCD) (27).

In patients with resuscitated SCD or sustained monomorphic ventricular tachycardia (VT) DE-MRI improved the diagnostic yield when compared against non-CMR imaging modalities (28). In patients undergoing cardioverter-defibrillator implantation for ischemic cardiomyopathy, DE-MRI quantification of infarct transmurality was able to identify a subset of patients at higher risk of SCD and ventricular arrhythmias (29). In combination with electro-anatomical mapping, DE-MRI can accurately predict the location of arrhythmia origin (30) and has been used to successfully guide radiofrequency ablation of the substrate in patients with ischemic VT (31).

Diffusion-weighted MR Imaging

Diffusion-weighted MR imaging utilizes the probabilistic diffusion of water molecules within the myocardium to generate 3-dimensional, non-destructive virtual histology (32). For a detailed explanation of the physics of diffusion imaging and a review of individual diffusion based imaging modalities (tensor, spectrum, tractography) the reader is directed to several excellent review articles available in the literature (33,34).

Diffusion tensor MR imaging (dtMRI) has been pivotal in understanding the complex geometry of myocardium with early work focusing on correlating traditional, destructive histology with virtual histology from dtMRI in normal animal hearts (35,36). dtMRI has been used to probe the structural changes that occur in animal models of infarction (37), including in our laboratory where we have used dtMRI to examine structural changes as shown in Fig. 4.

Figure 4. Top: Single slice through infarcted rat heart showing comparison between conventional (Masson’s Trichrome) histology and fiber orientation from the Primary Eigenvector derived from dtMRI. Bottom: Demonstration of reduced fractional anisotropy, increased mean diffusivity and increased fiber disarray derived from dtMRI in the infarct area compared to remote.

Diffusion spectrum MR imaging (DSI) and MR tractography, which offer greater spatial resolution compared to dtMRI have been applied in a rat model of infarction (38). 3-dimensional MR tractography allows for visualization of both whole heart structure as well as regional structure, demonstrating the altered orientation of fibers from epicardium to endocardium as shown in Fig. 5.

Figure 5. MR Tractography derived from dtMRI of a normal rat heart demonstrating (left) normal whole heart tract architecture and (center, right) altered tract orientation from epicardium to midmyocardium to endocardium.

Due to the complexity of the scanning sequence, human diffusion imaging studies in vivo are limited. dtMRI has been used alongside late-gadolinium and cine MRI in patients post-infarction to correlate fiber architecture with viability and wall motion (39) and to correlate the sequential changes in myocardial microstructure post-infarction (40).

E. Experimental Models of Arrhythmogenesis

Due to the low population incidence of sudden cardiac death and spontaneous VT/VF, the study of the mechanisms of arrhythmogenesis relies on experimental models of disease with a high incidence of arrhythmias. This section considers the role of a variety of models of disease, including animal models (both surgical and genetic), isolated human preparations and the emerging use of induced pluripotent stem cells.

Animal Models

Animal models of arrhythmia are well established and have been extensively reviewed(41-43). In this section we consider the main surgical and genetic models of disease, specifically relevant to human pathology.

Surgical Models

Surgical models of disease in vivo allow for the study and determination of the mechanisms of arrhythmogenesis in a variety of disease states including ischemia, heart failure and atrial fibrillation. The most widespread surgical model, occlusion or ligation of the left-anterior descending artery, is used to study the effects of myocardial infarction and ischemia-reperfusion. This model was first applied successfully by use of the Harris two-stage procedure in large animals in the 1950s (44) and has since been used in rabbit, rat and mouse studies (45) with the advantage of large scale reproducibility and lower cost. These models have proved vital in allowing the study of the mechanical, electrical and arrhythmic consequences of ischemia and infarction (46).

Surgical models of heart failure (HF) have been developed in large animals, where rapid pacing is usually employed to induce HF (47). In small animals, pressure overload (e.g. aortic banding) is normally used to induce HF or ligation of the left anterior descending coronary artery where animals are allowed to recover post-infarction surgery. The development of left ventricular dilatation and HF in rats post-MI surgery formed the basis for the heart failure model which led to the discovery of the advantageous effects of reverse remodeling (improved LV function and mortality) of ACE inhibitor treatment (48), a therapeutic intervention which has been directly translated to human patients with HF.

Atrial fibrillation (AF), the commonest arrhythmia in humans, has also been extensively studied in a variety of large and small animal models. Large animal models include AF induced by burst pacing (1), AF in the context of structural changes (such as HF) and AF induced by atrial stretch (49). There are few reliable small animal surgical models of AF although genetic models such as the spontaneously hypertensive rat (50) and knock-out mouse models have been employed.

Genetic Models

The use of small animal genetic models of disease for studying cardiomyopathies is well established and the reader is directed to the excellent recent review by McCauley and Wehrens (51). In this section, we describe examples of genetic models with significant arrhythmia phenotypes that are of interest to a translational researcher in cardiac electrophysiology.

Arrhythmogenic right ventricular cardiomyopathy (ARVC), a heritable cardiomyopathy characterized by preferential RV fibrotic remodeling and SCD has been characterized using mouse models (52) with desmosome protein mutations (mimicking the human phenotype) in desmoplakin, plakophilin-2 and junction plakoglobin. Similar mouse models have been developed for the study of hypertrophic and dilated cardiomyopathy with mutations in genes encoding the alpha-myosin heavy chain, binding protein C or troponin T proteins (53) and genes encoding the cytoskeleton, sarcomeric and nuclear envelope proteins (54) respectively.

In addition to cardiomyopathies, ion channelopathies have also been studied using genetic small animal models. One of the earliest models of arrhythmic disease was the LQT-3 mouse, generated by knock-in of the KPQ deletion on the SCN5A gene (55) resulting in prolongation of the action potential and episodes of spontaneous VT, consistent with the human phenotype. A variety of knock-in models (LQT1, LQT2 and LQT4) have improved the understanding of the mechanistic basis for arrhythmogenesis although their relevance to the human phenotype has been difficult to establish due to differences in murine electrophysiology, namely the intrinsically higher heart rate and altered ion channel expression resulting in lack of the plateau phase of the AP.

Brugada syndrome, a condition associated with idiopathic VT/VF and ST-segment elevation in the anterior chest leads is associated with a mutation in the SCN5A gene for which a SCN5A knockout mouse has provided a genetic model for translational research (56). Knockout models with mutations in the ryanodine receptor or calsequestrin have been fundamental in improving the understanding of the mechanisms underlying abnormalities in sarcoplasmic reticulum calcium handling resulting in arrhythmogenesis in CPVT(57). In addition to the described models of human arrhythmia syndromes, several knock-out murine models have also been developed that allow for a broader understanding of the cellular mechanism of arrhythmogenesis such as the potassium-channel knock-out, the myotonic dystrophy knock-out and the Cx-43 knock-out mouse (58).

Zebrafish

Over the last 15 years, the Zebrafish, a tropical freshwater teleost, has been increasingly used as an experimental model for studying cardiac development, arrhythmogenesis and human cardiac disease (59). The Zebrafish model offers several advantages over conventional small rodent models of cardiac electrophysiology. The Zebrafish embryo is transparent, allowing for assessment of development, heart rate, and, using fluorescent-labeled proteins optical mapping of voltage and calcium transients. In addition, Zebrafish embryos are able to survive in the absence of a developed circulatory system allowing for the study of severe defects, which would lead to early mortality in other animal models of disease. The use of high throughput forward genetic screens in developing Zebrafish embryos has allowed for the identification of novel genes and targets responsible for cardiogenesis, differentiation and early cardiac function (60).

However, there are limitations with the Zebrafish model; Zebrafish have a cold-blooded, two-chamber circulation, absent t-tubuli, limited sarcoplasmic reticulum function and absence of several ion channels (Ito1, Ikur) found in humans. Despite these limitations, the well-characterized Zebrafish model still has a role as a model of human disease, most notably long QT syndrome (61) and pharmacological studies (62).

Human Models

Although animal models of disease have provided a wealth of information, the differences between human and animal electrophysiology and the ability to translate findings to clinical relevant models require the study of human tissue.

Optical mapping with voltage-sensitive dyes of isolated wedges of explanted human hearts has significantly improved our understanding of the electrophysiology of the human sino-atrial node, AV node and the dual-pathway dependent nature of re-entrant tachycardias. Optical mapping studies in failing human hearts has demonstrated heterogeneous remodeling of excitation-contraction coupling and calcium handling (21) in addition to increased action potential (AP) duration, reduced transmural AP dispersion and transmural conduction slowing (19).

In addition to isolated wedges, vibratome-cut slices from human left ventricle biopsies have proved a metabolically and electrically stable preparation for the study of heart failure and drug screening(5).

Induced Pluripotent Stem Cell Models

Human somatic cells can be reprogrammed to generate human induced pluripotent stem cells (iPSC), which can be both patient- and disease- specific. Human iPSC are able to differentiate to form functional cardiomyocytes and provide a new experimental model for the study of human arrhythmias (63). Human iPSC models of arrhythmogenesis have been described for LQT1, LQT2, LQT3/Brugada syndrome and CPVT.

Limitations exist with the use of human iPSC models; derived cardiomyocytes tend to express an immature electrophysiological phenotype due to altered ion channel expression compared to adult cardiomyocytes with cell populations often expressing a mixed phenotype (64). However, the ability to generate disease-specific human cardiomyocytes produces an experimental model with significant advantages compared to animal models and has significant potential in both enhancing our understanding of arrhythmogenesis and allowing for testing of potential therapeutic agents.

F. Computational Modeling of Cardiac Electrophysiology

Computational modeling has become an increasingly important research platform for the translational researcher in the past decade, as the efficiency of computer hardware has improved. Computational models of cardiac electrophysiology are composed of models of membrane excitability that act within an environment that describes the cardiac structure. Membrane excitability is defined by ion channel currents derived from experimental studies, whereas cardiac structure incorporates different cellular (e.g. myocytes, fibroblasts) and spatial (e.g. gap junctions, extracellular matrix) variables (65). Computer modeling allows the description of action potential (AP) characteristics and propagation within tissue as well as allowing for hypothesis testing on preparations that are otherwise experimentally difficult or impossible.

Biophysically Detailed Electrophysiology Models

The Nobel Prize winning work of Hodgkin and Huxley, published in 1952, was the first of its kind to attempt to understand and model the flow of excitable current in biological tissue (66). The formulation of a mathematical model, incorporating voltage and time-dependent currents forms the underpinning of current models of the cardiac action potential. These biophysically detailed models are formulated and validated using experimental data from a range of animal and human tissues, recombinant ion channels and/or whole-cell (patch clamp) recordings. Mathematical models in widespread use today incorporate the major human ion currents and can accurately reproduce the normal human AP at rest and the AP changes that occur during acute ischemia (67,68)

3-Dimensional Electrophysiology Models

In addition to modeling the single-cell AP, mathematical models can now be applied at a whole-heart level (69). These models rely on representative histological-anatomical models (70), commonly derived by extracting and fitting fiber orientation information from diffusion MR scans to a segmented, 3-dimensional mesh (71). By incorporating fiber orientation, anatomically accurate 3D models of AP propagation in both normal and infarcted porcine hearts have been developed and validated by in-vivo electrophysiological testing and ex-vivo optical mapping (72). In addition to animal studies, anatomically detailed 3D computer modeling has been applied in studies of human sinus and AV node physiology, atrial structure and function and vulnerability of the right and left ventricle to electrical shock (73).

G. Conclusions

In this chapter, we have provided a description of some of the most important research platforms in translational cardiac electrophysiology. This is by no means an exhaustive list, but instead serves to provide the translational researcher with an impression of the vast array of techniques currently available in cardiac electrophysiology. Multiple new research platforms are also currently on the horizon, including electrocardiographic imaging which allows for non-invasive arrhythmia mapping in humans (74), and new stretchable electronics for in vivo electrophysiological research (75). The wide range of research platforms currently available should provide any translational researcher with sufficient tools to probe and address the important research questions in cardiac electrophysiology.

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