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.

Shaping surface acoustic waves for cardiac tissue engineering

The heart is a non-regenerating organ that gradually suffers a loss of cardiac cells and functionality. Given the scarcity of organ donors and complications in existing medical implantation solutions, it is desired to engineer a three-dimensional architecture to successfully control the cardiac cells in vitro and yield true myocardial structures similar to native heart. This thesis investigates the synthesis of a biocompatible gelatin methacrylate hydrogel to promote growth of cardiac cells using biotechnology methodology: surface acoustic waves, to create cell sheets. Firstly, the synthesis of a photo-crosslinkable gelatin methacrylate (GelMA) hydrogel was investigated with different degree of methacrylation concentration. The porous matrix of the hydrogel should be biocompatible, allow cell-cell interaction and promote cell adhesion for growth through the porous network of matrix. The rheological properties, such as polymer concentration, ultraviolet exposure time, viscosity, elasticity and swelling characteristics of the hydrogel were investigated. In tissue engineering hydrogels have been used for embedding cells to mimic native microenvironments while controlling the mechanical properties. Gelatin methacrylate hydrogels have the advantage of allowing such control of mechanical properties in addition to easy compatibility with Lab-on-a-chip methodologies. Secondly in this thesis, standing surface acoustic waves were used to control the degree of movement of cells in the hydrogel and produce three-dimensional engineered scaffolds to investigate in-vitro studies of cardiac muscle electrophysiology and cardiac tissue engineering therapies for myocardial infarction. The acoustic waves were characterized on a piezoelectric substrate, lithium niobate that was micro-fabricated with slanted-finger interdigitated transducers for to generate waves at multiple wavelengths. This characterization successfully created three-dimensional micro-patterning of cells in the constructs through means of one- and two-dimensional non-invasive forces. The micro-patterning was controlled by tuning different input frequencies that allowed manipulation of the cells spatially without any pre- treatment of cells, hydrogel or substrate. This resulted in a synchronous heartbeat being produced in the hydrogel construct. To complement these mechanical forces, work in dielectrophoresis was conducted centred on a method to pattern micro-particles. Although manipulation of particles were shown, difficulties were encountered concerning the close proximity of particles and hydrogel to the microfabricated electrode arrays, dependence on conductivity of hydrogel and difficult manoeuvrability of scaffold from the surface of electrodes precluded measurements on cardiac cells. In addition, COMSOL Multiphysics software was used to investigate the mechanical and electrical forces theoretically acting on the cells. Thirdly, in this thesis the cardiac electrophysiology was investigated using immunostaining techniques to visualize the growth of sarcomeres and gap junctions that promote cell-cell interaction and excitation-contraction of heart muscles. The physiological response of beating of co-cultured cardiomyocytes and cardiac fibroblasts was observed in a synchronous and simultaneous manner closely mimicking the native cardiac impulses. Further investigations were carried out by mechanically stimulating the cells in the three-dimensional hydrogel using standing surface acoustic waves and comparing with traditional two-dimensional flat surface coated with fibronectin. The electrophysiological responses of the cells under the effect of the mechanical stimulations yielded a higher magnitude of contractility, action potential and calcium transient.

An investigation of the possible health-promoting modes of action of regular- and super- doses of phytase in the broiler chicken

The overall objective of this thesis was to study the effects of regular and high (super-) doses of phytase in the gut of broilers, with the aim of documenting the mechanism of their action leading to improvements in animal health. Phytase is often supplemented to commercial broiler diets to facilitate the hydrolysis of plant phytate and release of phosphorus for utilisation. Although not the original intention of its addition, phytase supplementation leads to improvements in growth performance parameters and enhanced nutrient utilisation. Further benefits have also been observed following the addition of super-doses of phytase which are not explained by an increase in phosphorus release, and thus have been termed ‘extra-phosphoric effects’. Using diets formulated to be adequate or marginally deficient in available phosphorus (aP; forming the negative control, NC), phytase was supplemented at 1,500 and 3,000 FTU/kg phytase in the first study (both super-doses) and the partitioning of nutrients within the body was investigated. It appeared that there were some metabolic changes between 1,500 and 3,000 FTU/kg, switching between protein and fat accretion, potentially as a consequence of nutrient availability, although these changes were not reflected by changes in growth performance parameters. However, the loss of the NC treatment without phytase on day 12 limits the comparison of the phytase within the NC treatment, but does allow for comparison of each dose at adequate or low dietary aP levels. As expected, a greater degree of phytate hydrolysis was achieved with 3,000 than with 1,500 FTU/kg phytase, but changes in carcass accretion characteristics were greater with 1,500 than 3,000 FTU/kg. Using these findings and the observation that there were no further changes in the parameters measured by increasing phytase from 1,500 to 3,000 FTU/kg (aside from phytate hydrolysis), 1,500 FTU/kg phytase was selected as the super-dose to be used in subsequent studies. The next study considered the influence of regular (500 FTU/kg) and super doses (1,500 FTU/kg) of phytase from within the gut. Overall, it was observed that changes were occurring to the gut environment, which ultimately would influence the absorptive capacity and conditions for further phytate hydrolysis. Dietary treatment influenced gut conditions such as pH, intestinal morphology and bacterial populations which can subsequently influence nutrient utilisation and potential for growth. The subsequent study was designed to investigate the effects within the gut in more detail. The release of nutrients from phytate hydrolysis and their bioavailability within the digesta can influence conditions within intestine, facilitating enhanced absorption. One of the parameters investigated was the expression of genes involved in the transport of nutrients in the intestine. Overall, there were few significant dietary treatment influences on gene expression in the intestine, however there was a dose-dependent response of phytase on the expression of the jejunual divalent mineral transporter. This indicates a change in divalent mineral bioavailability in the intestine, with correlations with inositol phosphate esters (IPs) being identified. This is likely explained by the IPs produced by phytase hydrolysis and accumulating in the digesta, differing between regular and high doses of phytase. It became apparent that interactions between the products of phytate hydrolysis (IP3, IP4) and minerals in the digesta had the potential to influence the gut environment and subsequent nutrient bioavailability and overall phytase action. The final study was designed to increase the content of the IPs, and investigate the influence of phytase under these conditions. As the complete hydrolysis of phytate to myo-inositol has been reported to be beneficial due to its proposed insulin mimetic effects, myo-inositol was also supplemented to one of the diets to see if any further benefits would be observed when supplemented alongside super-doses of phytase. Neither increased concentrations of the higher IP esters (IP6, IP5 and IP4) nor myo-inositol (myo-) had any effect on broiler growth performance, however there were still apparent beneficial influences of phytase supplementation. The results suggest considerable and important interactions between minerals and IP esters within the digesta, which ultimately have the potential to influence gut conditions and thus nutrient utilisation and growth performance. Reduced concentrations of blood glucose in the high IP ester diet with additional phytase supplementation suggest some insulin-like effects of myo- production. Additionally, the lack of effect of myo- supplementation on blood glucose and insulin concentrations suggests a difference between the structure of phytase-produced myo- and supplemented myo-. Although there were no improvements in growth performance by increasing phytase from 500 to 1,500 FTU/kg, there were changes occurring at the level of the gut and expression of genes in the intestine, influencing nutrient utilisation and the partitioning of nutrients within the body. There are many factors to be considered when supplementing phytase, with dietary nutrient content and nutrient release and IP production during phytate hydrolysis having an influence on phytase action, nutrient absorption and conditions within the gut. Super-doses of phytase may be beneficial for maintaining optimal gut conditions, clearing IP esters from the digesta, reducing their potential to form complexes with minerals and other nutrients, ultimately influencing the efficiency of production.