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

Electrophysiology

The outstanding evolution of recording techniques paved the way for better understanding of electrophysiological phenomena within the human organs, including the cardiovascular, ophthalmologic and neural systems. In the field of cardiac electrophysiology, the development of more and more sophisticated recording and mapping techniques made it possible to elucidate the mechanism of various cardiac arrhythmias. This has even led to the evolution of techniques to ablate and cure most complex cardiac arrhythmias. Nevertheless, there is still a long way ahead and this book can be considered a valuable addition to the current knowledge in subjects related to bioelectricity from plants to the human heart

Nonlinear physics of electrical wave propagation in the heart: a review

The beating of the heart is a synchronized contraction of muscle cells (myocytes) that are triggered by a periodic sequence of electrical waves (action potentials) originating in the sino-atrial node and propagating over the atria and the ventricles. Cardiac arrhythmias like atrial and ventricular fibrillation (AF,VF) or ventricular tachycardia (VT) are caused by disruptions and instabilities of these electrical excitations, that lead to the emergence of rotating waves (VT) and turbulent wave patterns (AF,VF). Numerous simulation and experimental studies during the last 20 years have addressed these topics. In this review we focus on the nonlinear dynamics of wave propagation in the heart with an emphasis on the theory of pulses, spirals and scroll waves and their instabilities in excitable media and their application to cardiac modeling. After an introduction into electrophysiological models for action potential propagation, the modeling and analysis of spatiotemporal alternans, spiral and scroll meandering, spiral breakup and scroll wave instabilities like negative line tension and sproing are reviewed in depth and discussed with emphasis on their impact in cardiac arrhythmias.Peer ReviewedPreprin

Interactions Between Activation And Repolarisation In Predisposition Towards Cardiac Arrhythmia

The lethal cardiac arrhythmias ventricular fibrillation (VF) and ventricular tachycardia (VT) are a leading cause of death in heart disease. We hypothesised that dynamic activation and repolarisation interactions will vary according to autonomic tone and the nature of the myocardial substrate as affected by disease states. This hypothesis was tested in a series of human and murine experiments. Incorporation of data from human electrophysiological studies into a linear computer model was able to predict activation dynamics of sequential extrastimuli. This served as a validation of the concept of dynamic interactions between activation and repolarisation in man. A human model of mental stress demonstrated that activation and repolarisation dynamics are altered by intrinsic autonomic stimulation. Specifically, a reduction in activation potential duration and an increase in dispersion of repolarisation occurred at short coupling intervals during stress. A weak increase in conduction velocity and excitability was also observed. Patients with early-stage arrhythmogenic right ventricular cardiomyopathy (ARVC) were seen to exhibit conduction changes prior to the onset of structural disease. This was used to determine potential diagnostic criteria based on surface ECG correlates of intracardiac observations. These criteria are able to distinguish early ARVC from benign right ventricular outflow tract tachycardia. Finally, the mechanism of modulation of tissue level activation dynamics were further studied using a novel thin-tissue slice murine model. Conduction velocity and excitability were modulated by both sympathetic and parasympathetic stimuli, parasympathetic modulation is demonstrated to be dependent on the Gαi2 regulatory pathway at the tissue level. The tissue slice method provides a novel tissue-level platform for the study of cardiac electrophysiology in genetically modified mice. In conclusion, this work demonstrates that modulations of activation and repolarisation dynamics are seen in pro-arrhythmic states, specifically in sympathetically active states and in arrhythmogenic right ventricular cardiomyopathy

Ursodeoxycholic acid: a potential anti-arrhythmic and anti-fibrotic agent in adult hearts

Acute myocardial ischaemia and reperfusion (I-R) are major causes of ventricular arrhythmias. In the chronic post-ischaemic heart, the presence of a healed fibrotic scar contributes to the occurrence of malignant arrhythmias, and development of post-myocardial infarction (MI) left ventricular (LV) remodelling and heart failure (HF). The aim of the work in this thesis was to investigate if ursodeoxycholic acid (UDCA) protects against acute I-R-induced arrhythmias, and if it plays cardioprotective and anti-arrhythmic roles in the chronic post-MI adult myocardium. An ex vivo rat model of acute I-R was used to study the effect of UDCA on arrhythmia incidence. UDCA administration reduced acute ischaemia-induced arrhythmias, with no effect on reperfusion arrhythmias. The antiarrhythmic effect of UDCA is partially mediated by an increase in cardiac wavelength, due to the attenuation of conduction velocity (CV) slowing, and the preservation of Connexin43 phosphorylation during acute ischaemia. Multiple in vitro models of cardiac fibrosis were used to study the potential of UDCA as treatment of cardiac fibrosis. UDCA was proven to reduce cardiac fibrosis and preserve the associated changes in contractile functions and electrophysiology. The antifibrotic mechanism of action of UDCA is partially mediated by TGR5 modulation via dephosphorylation of ERK protein. A sixteen-week post-MI model was generated to explore the effects of UDCA on late post-MI arrhythmias and LV remodeling. UDCA prevented the adverse LV remodeling associated with the progression of MI and reduced fibrosis and the healed ischaemic border zone (IBZ) sizes. This resulted in reduced late susceptibility to ventricular arrhythmias and improved CV across the IBZ in UDCA-treated hearts at 16 weeks post MI. We generated robust novel data highlighting the potential application of UDCA in the prevention of ventricular arrhythmias during acute MI in the adult myocardium as well as against cardiac arrhythmias that are associated with cardiac fibrosis, due to its cardioprotective effect in the post-MI heart.Open Acces

The Effects of Gap Junction Modulation on Myocardial Structure and Function

The aim of the work in this thesis was to investigate the effects of gap junction modulation during acute myocardial infarction (MI) on ventricular arrhythmogenesis in the settings of acute reperfusion and chronic post-myocardial infarction. Ventricular arrhythmias can occur during reperfusion because of abrupt changes in electrophysiology, whilst arrhythmias occur post-MI because the healed infarct scar forms a substrate for re-entry, with increased inhomogeneity of scarring being associated with greater arrhythmia susceptibility. The effects gap junction modulation on reperfusion arrhythmogenesis were studied in an ex vivo rat model of acute ischaemia-reperfusion. Gap junction modulators were administered to hearts subjected to left anterior descending artery occlusion followed by reperfusion. The electrophysiological changes that accompany ischaemia-reperfusion were studied using optical mapping. Gap junction modulators, AAP10 and carbenoxolone, reduced the incidence of reperfusion arrhythmias. This was associated with the attenuation of the abrupt recovery of conduction during reperfusion, which may underlie their antiarrhythmic effects. A four-week rat chronic myocardial infarction model was developed to study the effects of acute gap junction modulation on late post-MI arrhythmias. Gap junction modulators, rotigaptide and carbenoxolone, were administered acutely for 7 days from the time of surgical MI, and rats were studied at 4 weeks post-MI with ECG-telemetry, programmed electrical stimulation, optical mapping, histomorphometry and connexin43 immunohistochemistry. Enhancing gap junction coupling with rotigaptide acutely during MI reduced heterogeneities in infarct border zone scarring and reduced susceptibility to ventricular arrhythmias on programmed electrical stimulation. Histomorphometric studies support a possible mechanism whereby homogenisation of the acute ischaemic insult and the cell death process may result in more homogeneous scarring and a less arrhythmic healed substrate. Gap junction modulation was anti-arrhythmic in the acute reperfusion setting, and the enhancement of coupling during acute MI may represent a novel therapeutic strategy to modify the morphology of the healed infarct and alter post-infarction arrhythmia susceptibility

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

Multiscale Modeling of the Ventricles: From Cellular Electrophysiology to Body Surface Electrocardiograms

This work is focused on different aspects within the loop of multiscale modeling: On the cellular level, effects of adrenergic regulation and the Long-QT syndrome have been investigated. On the organ level, a model for the excitation conduction system was developed and the role of electrophysiological heterogeneities was analyzed. On the torso level a dynamic model of a deforming heart was created and the effects of tissue conductivities on the solution of the forward problem were evaluated

Calcium Remodeling through Different Signaling Pathways in Heart Failure: Arrhythmogenesis Studies of Pyk2, Dystrophin, and β-adrenergic Receptor Signaling

Heart failure is a common clinical syndrome that ensues when the heart is no longer able to generate sufficient cardiac output to meet the demands of the body. It is one of the leading causes of death worldwide but with limited and non-ideal therapies at the moment. One reason behind this may be the complexity of significant alterations in multiple signaling pathways and concomitant structural and functional remodeling, especially Ca handling. Ca is critical in both the electrical and mechanical properties of cardiac myoctyes, and much is known about ionic currents and the normal excitation-contraction coupling process. In heart failure, distinct impaired signaling pathways induce significant alterations in how cardiac Ca handling is regulated. These alterations either directly cause certain arrhythmias or facilitate arrhythmias by association with electrical remodeling. The goal of this dissertation was to investigate the mechanisms of calcium remodeling through different signaling pathways in heart failure, and mechanisms on how the intricate and dynamic interactions between Ca handling and signaling pathways impairment facilitate arrhythmias in heart failure. To achieve this goal, a dual optical mapping system was designed to investigate electrical activity and Ca transient simultaneously. High spatio-temporal resolution mapping allows for quantifying conduction, repolarization and Ca cycling, especially on the interactions between action potential and Ca handling. In this dissertation, I investigated Ca remodeling in three different signaling pathways: stress activated signaling, cytoskeletal signaling and β adrenergic receptor signaling pathway. Proline-rich tyrosine kinase 2: Pyk2) is a non-receptor protein kinase regulated by intracellular Ca. It mediates a typical stress activated signaling pathways along with c-Src, P38 MAPK and regulates a broad range of key biological responses. By optically mapping the genetically engineered mouse model: Pyk2 knockout, I detected a protective role of Pyk2 with respect to ventricular tachyarrhythmia during parasympathetic stimulation by regulation of gene expression related to calcium handling. The mdx mouse model was introduced in the investigation of cytoskeletal signaling pathway. mdx mice is a common model for Duchenne muscular dystrophy, which is a clinical syndrome resulted from recessive of dystrophin and eventually develops into heart failure. The project suggested the association of mechanical stimulation and deficiency of dystrophin account for the cardiac mechanical defects and resulting Ca mishandling, but not either of the two above-mentioned entities alone. Ca mishandling leads to Ca cycling dispersion, which facilitates generation of arrhythmias. β Adrenergic receptor signaling pathway was investigated on explanted donor and failing human hearts. Distinct β adrenergic receptor subtypes were found to regulate remodeling differently. The association between remodeling of action potential and Ca transient provides crucial arrhythmic drivers and substrate in heart failure