When modelling tissue-level cardiac electrophysiology, a continuum approximation to the discrete cell-level equations, known as the bidomain equations, is often used to maintain computational tractability. The bidomain equations are derived from the discrete equations using a mathematical technique known as homogenisation. As part of this derivation conductivity tensors are specified for use in the continuum model. Analysing the derivation of the bidomain equations allows us to investigate how microstructure, in particular gap junctions that electrically connect cells, affect tissue-level conductivity properties and model solutions. We perform two distinct but related strands of investigation in this thesis. In the first, we consider the effect of including gap junctions on the results of both discrete and continuum simulations, and identify when the continuum model fails to be a good approximation to the discrete model. Secondly, we perform a comprehensive study into how cell-level microstructure properties, such as cell shape, impact the homogenised conductivities to be used in a tissue-level continuum model. This will allow us to predict how the onset of a disease or a change in cellular microstructure will affect the propagation of action potentials. To do this, we first derive a modified version of the bidomain equations that explicitly takes gap junctions into account. We then derive analytic solutions for the homogenised conductivity tensors on a simplified two-dimensional geometry and find that diseased gap junctions have a large impact on the results of homogenisation. On this same geometry we compare the results of discrete and continuum simulations and find a significant discrepancy between model conduction velocities when we introduce gap junctions with lower coupling strength, or when we consider elongated cells. From this, we conclude that the bidomain equations are less likely to give an accurate representation of the underlying discrete system when modelling diseased states whose symptoms include reduced gap junction coupling or an increase in myocyte length. We then use a more realistic two-dimensional geometry and numerically approximate the homogenised conductivity tensors on this geometry. We discover that the packing of cells has a substantial effect on conduction, with a brick-wall geometry particularly beneficial for fast propagation, and that gap junctions also have a large effect on conduction. Finally, we consider a three-dimensional cellular geometry and show that the effect of changing gap junction properties is different when compared to two dimensions, and discover that the anisotropy ratios of the tissue are highly sensitive to changes in gap junction parameters. Overall, we conclude that gap junctions and cell structure have a large effect on discrete and continuum model results, and on homogenised conductivity calculations in tissue-level cardiac electrophysiology.

Reproducibility of results in biomedical research is an area of concern that should be paramount for all researchers. Importantly, this issue has been examined for experiments concerning cardiac electrophysiology. Specifically, multiple labs have found differences in results when comparing cardiac conduction velocity (CV) between healthy mice and mice that were heterozygous null for the gap junction (GJ) forming protein, Connexin 43. While the results of the comparison study showed differing extracellular ionic concentrations of the perfusates, specifically sodium, potassium, and calcium ([Na+]o, [K+]o, and [Ca2+]o), there was a lack of understanding why certain combinations of the aforementioned ions led to specific CV changes. However, more research from our lab indicates that these changes can predict modifications to a secondary form of cardiac coupling known as ephaptic coupling (EpC). Therefore the work in this dissertation was twofold, 1) to examine the effects of modulating EpC through perfusate ionic concentrations while also modulating GJC and 2) to investigate the effects of modulating all three of the main ions contributed with cardiac conduction (Na+, K+, Ca2+) and the interplay between them. Firstly I designed and tested changes from the use of 3D printed bath for optical mapping procedures. After verification that the bath did not modify electrophysiological or contrile parameters, I studied the effects of physiologic changes to EpC determinants ([Na+]o and [K+]o) on CV during various states of GJ inhibition using the non-specific GJ uncoupler carbenoxolone (CBX). Multiple pacing rates were used to further modify EpC, as an increased pacing rate leads to a decrease in sodium channel availability through modification of the resting membrane potential. with no to low (0 and 15 µM CBX) GJ inhibition, physiologic changes in [Na+]o and [K+]o did not affect CV, however increasing pacing rate decreased CV as expected. When CBX was increased to 30 µM, a combination of decreasing [Na+]o and increasing [K+]o significantly decreased cardiac CV, specifically when pacing rate was increased. Next, the combinatory effects of cations associated with EpC (Na+, K+, and Ca2+) were tested in to examine how cardiac CV reacts to changes in perfusate solution and how this may explain differences in experimental outcomes between laboratories. Briefly, experiments were run where [K+]o was varied throughout an experiment and the values for [Na+]o and [Ca2+]o were at one of two specific values during an experiment. 30 µM CBX was added to half of the experiments to see the changes in the CV-[K+]o relationship with GJ inhibition. With unaltered GJ coupling, elevated [Na+]o maintains CV during hyperkalemia. Interestingly, both [Na+]o and [Ca2+]o must be increased to maintain normal CV during hyperkalemia with reduced GJ coupling. These data suggest that optimized fluids can sustain normal conduction under pathophysiologic conditions like hyperkalemia and GJ uncoupling. Taken as a whole, this dissertation attempts to shed light on the importance of ionic concentration balance in perfusate solutions on cardiac conduction. / Ph. D.

In recent years there has been more focus on investigating sex differences across all medical fields, including cardiology. There are sex differences in disease presentation, treatment and baseline function. These differences are critical to understand in order to properly treat both men and women. Even with an increased focus on this field, research has a male bias and there is more work to be done. Cardiac conduction is a highly synchronized process. Electrical signals are passed cell to cell through two mechanisms, ephaptic coupling and gap junctional coupling. These methods of electrical communication rely on gap junctions, sodium channels and the perinexus. When conduction is disrupted it causes arrhythmias. When investigating these three critical determinants of cardiac conduction in guinea pig hearts, we determined that there are sex differences in two of three investigated determinants. It appears that females are more susceptible to sodium channel modulation while males are more susceptible to gap junction modulation. Understanding these differences is critical to clinical care. It has been shown that females have higher mortality following cardiothoracic surgery and the reason for this is unknown. During cardiothoracic surgery the heart is arrested and maintained by a fluid, cardioplegia solution. Cardioplegia solutions contain components that are known to modulate conduction. We investigated the sex differences in cardiac electrophysiology with a focus on cardiac conduction and components of a common cardioplegia solution; we determined that there are electrophysiologic sex differences in response to both magnesium and mannitol. The sex substrates in three of the major determinants of conduction (sodium channels, gap junctions and perinexal width) and the differences in the effects of cardioplegia components on males and females may help to explain the higher mortality of females post cardiothoracic surgery. / Master of Science / In recent years there has been more focus on investigating sex differences across all medical fields, including cardiology. There are sex differences in disease presentation, treatment and baseline function. These differences are critical to understand in order to properly treat both men and women. Even with an increased focus on this field, research has a male bias and there is more work to be done. Cardiac conduction is a highly synchronized process. Electrical signals are passed cell to cell through two mechanisms, ephaptic coupling and gap junctional coupling. These methods of electrical communication rely on gap junctions, sodium channels and the perinexus. When conduction is disrupted it causes arrhythmias. When investigating these three critical determinants of cardiac conduction in guinea pig hearts, we determined that there are sex differences in two of three investigated determinants. It appears that females are more susceptible to sodium channel modulation while males are more susceptible to gap junction modulation. Understanding these differences is critical to clinical care. It has been shown that females have higher mortality following cardiothoracic surgery and the reason for this is unknown. During cardiothoracic surgery the heart is arrested and maintained by a fluid, cardioplegia solution. Cardioplegia solutions contain components that are known to modulate conduction. We investigated the sex differences in cardiac electrophysiology with a focus on cardiac conduction and components of a common cardioplegia solution; we determined that there are electrophysiologic sex differences in response to both magnesium and mannitol. The sex substrates in three of the major determinants of conduction (sodium channels, gap junctions and perinexal width) and the differences in the effects of cardioplegia components on males and females may help to explain the higher mortality of females post cardiothoracic surgery.

Stimulation of cardiac β-adrenoceptors is the primary mechanism by which cardiac output is increased to meet metabolic demands of the body. Recently, nicotinic acid adenine dinucleotide phosphate (NAADP), has been implicated as a novel component of the β-adrenoceptor signalling pathway. NAADP is thought to mobilise Ca²⁺ from acidic endolysosomal stores which then supplements sarcoplasmic reticulum Ca²⁺ load, leading to a positive inotropic effect. Recent progress in the field has been made with the identification of two-pore channel 2 (TPC2) as a candidate NAADP receptor. Isolated ventricular myocytes from mice lacking TPC2 proteins (Tpcn2^-/-) displayed blunted maximal responses to the β-adrenoceptor agonist isoprenaline. This blunted response was also evident in Langendorff-perfused Tpcn2^-/- hearts. Furthermore, a blunted response was observed in guinea pig ventricular myocytes which had been treated with bafilomycin A1, which disrupts the integrity of acidic endolysosomal stores. These data add to the body of evidence that NAADP signalling forms an important additional component of the β-adrenoceptor signalling pathway. Chronic activation of the β-adrenoceptor pathway is associated with certain disease states including heart failure and arrhythmias. Anandamide is an endogenous cannabinoid, ('endocannabinoid'), with similar properties to δ⁹-tetrahydrocannabinol (δ⁹-THC), the primary active constituent of Cannabis sativa. These compounds have widespread physiological effects through actions at cannabinoid CB₁ and CB₂ receptors, which are negatively coupled to adenylyl cyclases and are expressed in cardiac muscle. Exposure of guinea pig ventricular myocytes to anandamide resulted in a reduction in the amplitude of contractions and Ca²⁺ transients. This was associated with a reduction in action potential duration and amplitude of I_CaL. These effects of anandamide could not be prevented by cannabinoid receptor antagonists, and could not be mimicked by cannabinoid receptor agonists. An inhibition of IKs was also observed. Given the reported Gi-protein coupling of cannabinoid receptors, it may be expected that additional negative inotropic actions of anandamide might be observed when adenylyl cyclases are stimulated. However, the effects of anandamide to reduce amplitude of contraction and I_CaL were no greater in the presence of isoprenaline. Furthermore, the effects of anandamide were not prevented by pre-treatment of myocytes with pertussis toxin (PTX). This is in contrast to the actions of adenosine, which displayed clear PTX sensitive actions in the presence of isoprenaline. These data suggest that cannabinoid receptors are not involved in mediating the negative inotropic actions of anandamide. Another endocannabinoid, 2-arachidonoylglycerol, was without significant effect on action potentials or contractions in the absence or presence of isoprenaline. δ⁹-THC shared many of the actions of anandamide. It appears that anandamide and δ⁹-THC exert significant effects on cardiac muscle through direct modulation of ion channel function, although additional actions, for example, on the sarcoplasmic reticulum or myofilaments, cannot be ruled out.

No description available.

Iroquois homeobox (Irx) family members, a group of highly conserved homeodomain containing transcription factors, are involved in the patterning and the proper functions of vertebrate organs. They can act as transcriptional activators or repressors in a context-dependent manner. Preliminary data indicated that Irx3 and Irx5 are functionally redundant during cardiac morphogenesis, and they physically interact with other cardiac transcription factors. At E14.5, outflow tract septation failure and ventricular septation failure were observed in Irx3/5DKO mouse hearts. Loss of Irx3/5 in neural crest and endothelial cell lineages led to outflow tract septation failure and ventricular septal defect. In adult mice, Irx3 is expressed in the atrioventricular conduction system, and loss of Irx3 leads to slower ventricular conduction velocity. qRT-PCR analysis and immunofluorescence staining revealed that the expression of gap junction proteins, Cx40 and Cx43, are affected by the loss of Irx3. Over-expression of Irx3 and a dominant repressor form of Irx3, Irx3-EnR, resulted in Cx40 upregulation, indicating that Irx3 acts as an indirect positive regulator of Cx40. Irx3-EnR over-expression in vivo resulted in postnatal onset of atrial enlargement, ventricular hypertrophy, and conduction failure. Taken together, this study demonstrates the significance of Irx3/5 in both cardiovascular development and cardiac electrophysiology.

Iroquois homeobox (Irx) family members, a group of highly conserved homeodomain containing transcription factors, are involved in the patterning and the proper functions of vertebrate organs. They can act as transcriptional activators or repressors in a context-dependent manner. Preliminary data indicated that Irx3 and Irx5 are functionally redundant during cardiac morphogenesis, and they physically interact with other cardiac transcription factors. At E14.5, outflow tract septation failure and ventricular septation failure were observed in Irx3/5DKO mouse hearts. Loss of Irx3/5 in neural crest and endothelial cell lineages led to outflow tract septation failure and ventricular septal defect. In adult mice, Irx3 is expressed in the atrioventricular conduction system, and loss of Irx3 leads to slower ventricular conduction velocity. qRT-PCR analysis and immunofluorescence staining revealed that the expression of gap junction proteins, Cx40 and Cx43, are affected by the loss of Irx3. Over-expression of Irx3 and a dominant repressor form of Irx3, Irx3-EnR, resulted in Cx40 upregulation, indicating that Irx3 acts as an indirect positive regulator of Cx40. Irx3-EnR over-expression in vivo resulted in postnatal onset of atrial enlargement, ventricular hypertrophy, and conduction failure. Taken together, this study demonstrates the significance of Irx3/5 in both cardiovascular development and cardiac electrophysiology.

<p>Instability in the propagation of nonlinear electro-chemical waves in the heart is responsible for life-threatening disease. This thesis describes an investigation of the effects of boundaries on cardiac wave propagation that arises from a site where an electrical stimulus is applied or from boundaries beyond which current does not flow. It is generally believed that the spatial scale for boundary effects is approximately equal to the passive length constant, lambda, of the tissue, the distance over which a a voltage pulse decays when it is below the threshold for wave generation. From the results of <em>in vitro</em> experiments with bullfrog cardiac tissue and through numerical simulations, I find that boundaries affect wave propagation over a much larger spatial scale and that the spatial variation in some cardiac restitution properties is correlated statistically with the onset of alternans, a possible precursor to fibrillation in the human heart.</p><p>An optical imaging system using novel illumination based on LEDs is used to determine the spatial dependence of action potential duration (APD) and the slope of the dynamic restitution curve S_DRC, which describes the relationship between steady-state APD and diastolic interval. For tissue with nearly identical cells, I find that APD is longest near the stimulus and shortest near the physical boundary with significant changes (~100 ms) over a distance of ~10lambda. S_DRC decreases with distance from the stimulus at a constant rate (~0.1-1.5 /mm) over the surface of the tissue. Simulations using a two-variable cardiac model confirm that spatial patterns of APD and S_DRC can be induced by boundaries.</p><p>Additional measurements with the simultaneous impalement of two microelectrodes are used to determine the spatial differences of other restitution properties. These studies indicate that APD and S_DRC, as well as the slopes of the constant-BCL and S1S2 restitution curves, vary in space and that the spatial differences and onset of alternans at rapid pacing are correlated. If similar correlations are evident in humans, such measurements may identify patients who are susceptible to arrhythmias and allow for early treatment.</p> / Dissertation

<p>The role of cardiac tissue structure in both normal and abnormal impulse conduction has been extensively studied by researchers in cardiac electrophysiology. However, much is left unknown on how specific micro- and macroscopic structural features affect conduction and conduction block. Progress in this field is constrained by the inability to simultaneously assess intramural cardiac structure and function, as well as the intrinsic complexity and variability of intact tissue preparations. Cultured monolayers of cardiac cells, on the other hand, present a well-controlled in vitro model system that provides the necessary structural and functional simplifications to enable well-defined studies of electrical phenomena. In this thesis, I developed a novel, reproducible cell culture system that accurately replicates the realistic microstructure of cardiac tissues. This system was then applied to systematically explore the influence of natural structure (e.g. tissue boundaries, expansions, local fiber directions) on normal and arrhythmogenic electrical conduction.</p><p>Specifically, soft lithography techniques were used to design cell cultures based on microscopic DTMRI (diffusion tensor magnetic resonance imaging) measurements of fiber directions in murine ventricles. Protein micropatterns comprised of mosaics of square pixels with angled lines that followed in-plane cardiac fiber directions were created to control the adhesion and alignment of cardiac cells on a two-dimensional substrate. The high accuracy of cell alignment in the resulting micropatterned monolayers relative to the original DTMRI-measured fiber directions was validated using immunofluorescence and image processing techniques.</p><p>Using this novel model system, I first examined how specific structural features of murine ventricles influence basic electrical conduction. (1) Realistic ventricular tissue boundaries, either alone or with (2) microscopic fiber directions were micropatterned to distinguish their individual functional roles in action potential propagation. By optically mapping membrane potentials and applying low-rate pacing from multiple sites in culture, I found that ventricular tissue boundaries and fiber directions each shaped unique spatial patterns of impulse propagation and additively increased the spatial dispersion of conduction velocity.</p><p>To elucidate the roles that natural tissue structure play in arrhythmogenesis, I applied rapid-rate pacing from multiple sites in culture in an attempt to induce unidirectional conduction block remote from the pacing site--a precursor to reentry. The incidence of remote block was found to be highly dependent on the direction of wave propagation relative to the underlying tissue structure, and with a susceptibility that was synergistically increased by both realistic tissue boundaries and fiber directions. Furthermore, all instances of remote block in these micropatterned cultures occurred at the anterior and posterior junctions of the septum and right ventricular free wall. At these sites, rapid excitation yielded more abrupt conduction slowing and promoted wavefront-waveback interactions that ultimately evolved into transmural lines of conduction block. The location and shape of these lines of block was found to strongly correlate with the spatial distribution of the electrotonic source-load mismatches introduced by ventricular structures, such as tissue expansions and sharp turns in fiber direction.</p><p>In summary, the overall objective of the work described in this thesis was to reveal the distinct influences of realistic cardiac tissue structure on action potential conduction and conduction block by engineering neonatal rat cardiomyocyte monolayers that reproducibly replicated the anatomical details of murine ventricular cross-sections. In the future, this novel model system is expected to further our understanding of structure-function relationships in normal and structurally diseased hearts, and possibly enable the development of novel gene, cell, and ablation therapies for cardiac arrhythmias.</p> / Dissertation

Binary transgenic (BT) mice with doxycycline (DOX)-suppressible cardiac-specific over-expression of endothelin 1 (ET 1) exhibit progressive heart failure, QRS prolongation, and death following DOX withdrawal. However, the molecular basis and reversibility of the electrophysiological abnormalities in this model were not known. Here we assess the mechanisms underlying ET 1 mediated electrical remodelling, and its role in heart failure. Prior attempts to prevent this model of ET-1 induced cardiomyopathy with ET receptor antagonism were not beneficial. We now propose to evaluate the effectiveness of blocking the synthesis of ET-1 with CGS 26303, a dual inhibitor of endothelin converting enzyme (ECE) and neutral endopeptidase. BT vs. littermate control mice were withdrawn from DOX and serially studied with ultrasound biomicroscopy, octapolar catheters, multi-electrode epicardial mapping, histopathology, Western blot, immunohistochemistry and qRT-PCR. Prolonged ventricular activation and depressed rate of ventricular activation were detected as early as 4 wks after transgene activation, when structure and function of the heart remained unaffected. By 8 wks of ET-1 over-expression, biventricular systolic and diastolic dysfunction, myocardial fibrosis, cardiomyocyte hypertrophy, prolonged ventricular activation and repolarization, depressed rate of ventricular activation, and abnormal atrioventricular nodal function were observed. Within 4 wks of ET-1 induction, reduction were observed in connexin-43 mRNA, protein, and phosphorylation, Nav1.5 mRNA and protein, Na+ conductance, K+ channel interacting protein-2 mRNA and Kv4.2 mRNA. Chromatin immunoprecipitation revealed that nuclear factor κB preferentially binds to Cx43 and Nav1.5 promoters. Importantly, the associated electrophysiological abnormalities at this time point were reversible upon suppression of ET 1 over-expression and completely prevented the development of structural and functional remodelling. Treatment with CGS-26303 (5 mg/kg/day) failed to improve survival, or hemodynamic and contractile decline. ET-1-mediated ventricular conduction delays correlates with gap junction and ion channel remodelling, and precedes heart failure. The sequence and reversibility of this phenotype suggest that a primary abnormality in electrical remodelling may contribute to the pathogenesis of heart failure. CGS 26303 failed to prevent this cardiomyopathic phenotype. These data suggest that chronically high levels of bigET-1, as seen in heart failure, may induce increased ECE activity and/or non-ECE ET-1 synthesis, thus circumventing the efficacy of ECE blockade in this model.