This thesis examined the effect of heat stress, dehydration and exercise on global left ventricular (LV) function and LV twist, untwisting and strain (LV mechanics) in healthy individuals. The primary aim was to identify whether the different haemodynamics induced by heat stress, dehydration and exercise would be associated with alterations in systolic and diastolic LV mechanics as assessed by two-dimensional speckle tracking echocardiography. Study one showed that enhanced systolic and diastolic LV mechanics during progressively increasing heat stress at rest likely compensate in part for a lower venous return, resulting in a maintained stroke volume (SV). In contrast, heat stress during knee-extensor exercise did not significantly increase LV twist, suggesting that exercise attenuates the increase in LV mechanics seen during passive heat stress. Study two revealed that dehydration enhances systolic LV mechanics whilst diastolic mechanics remain unaltered at rest, despite pronounced reductions in preload. The maintenance of systolic and diastolic LV mechanics with dehydration during knee-extensor exercise further suggests that the large decline in SV with dehydration and hyperthermia is caused by peripheral cardiovascular factors and not impaired LV mechanics. During both, heat stress and dehydration, enhanced systolic mechanics were achieved solely by increases in basal rotation. In contrast, the third study demonstrated that when individuals are normothermic and euhydrated, systolic and diastolic basal and apical mechanics increase significantly during incremental exercise to approximately 50% peak power. The subsequent plateau suggests that LV mechanics reach their peak at sub-maximal exercise intensities. Together, the present findings emphasise the importance of acute adjustments in both, basal and apical LV mechanics, during periods of increased cardiovascular demand.

Death due to lethal cardiac arrhythmias is the leading cause of mortality in Western society. Many of the fundamental mechanisms underlying the onset of arrthythmias, their maintenance and termination, still remain poorly understood. The specialized conduction (or His-Purkinje) system is fundamental to ventricular electrophysiological function and is a key player in various cardiac diseases. In recent years, computational simulation has become an important tool in im- proving our understanding ofthese mechanisms. Current state-of-the-art computational ventric- ular electrophysiology models often do not feature a detailed representation of the specialized conduction system. Ventricular models that do incorporate the specialized conduction system often use a simplified anatomical description and are commonly based on the monodomain equations, rather than the more general bidomain equations. Thus, using computational simula- tion to investigate both normal physiological function of the specialized conduction system and pathologies in which it is involved presents difficulties. This thesis develops the techniques and tools required to model the specialized conduction sys- tem at the ventricular scale. We derive one-dimensional bidomain equations that model elec- trical propagation in the system by reducing the equations associated with a three-dimensional fibre. To complement the derived equations, we develop a numerical solution scheme for the model that is efficient enough to allow ventricular simulations. The one-dimensional bido- main model allows defibrillation studies to be performed with the specialized conduction sys- tem. Secondly, we investigate the imaging and mesh generation tools required to integrate an anatomically detailed mesh of the specialized conduction system into a current state-of-the-art ventricular mesh. Using these tools, a highly detailed rabbit-specific specialized conduction system anatomical model is developed. Simulations are performed that dem~strate the re- sponse of the specialized conduction system to defibrillation strength shocks and we compare activation sequences generated using the model to experimental recordings. Finally, we investi- gate variability in the anatomy of the system. The tools and ventricular model presented in this thesis fulfil an important role in allowing the study of the e1ectrophysiological function of the specialized conduction system at the ventricular scale.

Every year 400,000 - 600,000 people in the United States die from sudden cardiac death. Sudden cardiac death is often caused by irregular electrical impulses, or arrhythmias, in the heart. Arrhythmias can be corrected through pharmacological therapies, device therapies, or both. One type of device therapy, pacemakers, are inserted in the heart to correct arrhythmias. After a period of ventricular pacing, cardiac memory is defined by changes in the T-wave that are persistent upon return to normal activation pathways. During ventricular pacing, regional stroke work in areas closest to the pacing electrode is significantly decreased. We hypothesize that the mechanical function in the region around the pacing site will continue to have altered mechanical function after cession of pacing, in effect showing a mechanical cardiac memory. To test the hypothesis, nine canine models were implanted with pacing electrodes in both the atrium and ventricle. After a forty- minute stabilization period, baseline data were obtained during atrial pacing. Cardiac memory was induced in five canine models through a two-hour period of ventricular pacing followed immediately by atrial pacing. The remaining canine models served as controls, undergoing atrial pacing for two hours. High- density mapper (HDM) was used to determine mechanical function in a region centered approximately 1 cm away from the pacing electrode. No differences in global function (tau, developed pressure, dP/dtmax, dP/dtmin) were found after two hours of ventricular pacing upon return to normal activation pathways. There was a significant decrease in regional stroke work in an area close to the electrode between baseline (5.7 ± 2.6 %), during ventricular pacing (-3.8 ± 0.9 %)(p<0.05) and after two hours of ventricular pacing upon return to normal activation pathways (2.4 ± 1.6 %)(p<0.05). Further, systolic area contraction was also significantly different between baseline (5.0 ± 6.6 %) and after two hours of ventricular pacing upon return to normal activation pathways (0.2 ± 7.4 %)(p<0.05). Diastolic twist and diastolic twist rates showed no significant differences. Finally, contractile principal strain increased by inducing cardiac memory (-2.6 ± 0.3 %) as compared to baseline (-1.1 ± 0.5 %)(p<0.05). These findings suggest there is a mechanical correlation to electrical cardiac memory.

The severity of cardiac dysfunction predicts mortality in septic patients. In this thesis, I have investigated the pathophysiology and the novel therapeutic strategy to attenuate cardiac dysfunction in experimental sepsis. I have developed a model of cardiac dysfunction caused by lipopolysaccharide (LPS)/peptidoglycan (PepG) co-administration or polymicrobial sepsis in young and old, male and female mice. There is good evidence that females tolerate sepsis better than males. Here, I have demonstrated for the first time that the cardiac dysfunction caused by sepsis was less pronounced in female than in male mice; this protection was associated with cardiac activation of a pro-survival pathway [Akt and endothelial nitric oxide synthase], and the decreased activation of a pro-inflammatory signalling pathway [nuclear factor (NF)-κB]. Patients with chronic kidney disease (CKD) requiring dialysis have a higher risk of sepsis and a 100-fold higher mortality. Activation of NF-κB is associated with sepsis-induced cardiac dysfunction and NF-κB is activated by IκB kinase (IKK). Here, I have shown that 5/6th nephrectomy for 8 weeks caused a small, but significant, cardiomyopathy, cardiac activation of NF-κB and expression of inducible nitric oxide synthase (iNOS). When subjected to LPS or polymicrobial sepsis, CKD mice exhibited exacerbation of cardiac dysfunction and cardiac activation of NF-κB and iNOS expression, which were attenuated by a specific IKK inhibitor (IKK 16). Thus, selective inhibition of IKK may represent a novel therapeutic approach for the sepsis-induced cardiac dysfunction in CKD patients. Activation of transient receptor potential vanilloid receptor type 1 (TRPV1) improves outcome in sepsis/endotoxaemia. The identity of the endogenous activators of TRPV1 and the role of the channel in the cardiac dysfunction caused by sepsis/endotoxaemia is unknown. Here, I have shown that activation of TRPV1 by 12-(S)-HpETE and 20-HETE (potent ligands of TRPV1) leads to the release of calcitonin gene-related peptide (downstream mediator of TRPV1 activation), which protects the heart against the cardiac dysfunction caused by LPS.

The IKs current constitutes part of the repolarisation reserve in the human myocardium, and whilst it does not play a major role at resting heart rates, it becomes a crucial component of repolarisation in the setting of increased sympathetic tone and high heart rates. The formation of the IKs current requires the KCNQ1 α-subunit and the KCNE1 β-subunit. Mutations in either of these subunits can lead to long QT syndrome types 1 and 5, respectively. Loss-of-function mutations in the IKs channel can reduce the repolarisation reserve and lead to action potential prolongation, predisposing to lethal cardiac arrhythmias such as torsades de pointes and ventricular fibrillation. It is widely recognised that the IKs channel requires the minor membrane phospholipid PIP2 for its function, and previous work in this laboratory found that mutations in a PIP2-binding region in KCNQ1 led to retention of the channel in the endoplasmic reticulum, suggesting that PIP2 may play a role in anterograde trafficking. Here, the rapamycin-inducible dimerisation system was used to manipulate levels of PIP2 and/or PI4P at the plasma membrane or Golgi, and the effect of this on IKs channel trafficking and function was investigated using molecular biology, confocal microscopy and electrophysiology. Despite difficulties with optimising the rapamycin-induced dimerisation system, it was observed that the IKs channel does not require PIP2 for anterograde trafficking, but is heavily reliant on PIP2 for channel opening. In addition, activation of the β1-adrenergic receptor (β 1-AR) led to an increase in the IKs current amplitude. The potential interplay between β1-AR and PIP2 signalling was also explored by depleting PIP2 during β1-AR stimulation. PIP2 depletion was less effective at inhibiting the IKs current during β1-AR stimulation, but this requires further investigation. In conclusion, the results suggest that the IKs channel is reliant on PIP2 for function, but not anterograde trafficking.

Trauma-induced secondary cardiac injury (TISCI) represents an under recognised complication of severe injury with haemorrhage. A limited number of clinical studies have supported the development of adverse cardiac events, such as arrhythmia, in association with biomarker proven TISCI. Pre-clinical studies using small animal models have provided insights into potential mechanisms and key effector molecules involved in the development of TISCI, but there remains a general lack of understanding regarding the in vivo functional implications of this indirect cardiac injury resulting from trauma-haemorrhage. This project aimed to investigate the implications of cardiac injury on myocardial systolic function. A robust, translatable model of TISCI was developed, which reflected the cardiac biomarker profile identified in clinical studies and, for the first time, demonstrated a significant, dose-dependent rise in Heart-type Fatty Acid Binding Protein (H-FABP) in response to trauma-haemorrhage. Non-invasive echocardiography was used to determine the acute myocardial response to injury and haemorrhage and also to assess the response of the left ventricle to resuscitation after an antecedent 60-minute period of trauma-haemorrhage. The functional studies presented here have enabled real time visualisation of the impact of trauma-haemorrhage upon systolic left ventricular function over 1 to 6 hours, both with and without resuscitation. Having established the trends in in vivo systolic function over time, further studies were then conducted to test the combination of adenosine, lidocaine and magnesium (ALM) as a cardiovascular rescue agent in TISCI. ALM, as an adjunct to fluid resuscitation, has shown great promise as a therapeutic agent which improves haemodynamic outcomes, reduces the volume of resuscitation fluid required and favours survival in the murine model of TISCI.

The glucocorticoid receptor (GR) is expressed throughout the cardiovascular system and glucocorticoids (GC) are known to influence cardiovascular processes ranging from angiogenesis and vascular tone to cardiomyocyte hypertrophy and inflammation. Genetic variation in the human GR gene that associates with relative glucocorticoid resistance is also linked to hypertension and increased risk of cardiovascular disease. Mice with global GR haploinsufficiency (GR+/-) are similarly glucocorticoid resistant, with increased hypothalamic-pituitary-adrenal (HPA) axis activity and elevated blood pressure in adulthood. Previous work from the laboratory has demonstrated that the GR is essential for normal growth and maturation of the foetal heart in late gestation and in vitro studies show that GC can alter cardiomyocyte function and induce cardiomyocyte hypertrophy. I hypothesised that reduced GR density during development would have consequences for cardiovascular function and disease risk in adulthood and that cardiovascular GR signalling is important for postnatal growth of the heart, as well as physiological and pathological cardiac remodeling in adulthood. I tested this hypothesis in GR+/- mice with global alteration in GR density as well as in SMGRKO mice, with deletion of GR in cardiomyocytes and vascular smooth muscle. To investigate the association between GC resistance and cardiovascular disease risk, I have characterised the cardiac phenotype of GR+/- mice, basally and following physiological and pathological cardiac remodeling induced by a swim training programme and Angiotensin II treatment, respectively. Survival to weaning was reduced by 35% in GR+/- mice compared with wild-type (WT) littermates. Ultrasound analysis revealed impairment of systolic cardiac function in utero (E17.5) and at postnatal day (P) 2. However, by P7 cardiac function had normalised in surviving GR+/- mice and remained equivalent to WT littermates in adulthood. Heart weight and morphology were normal in GR+/- mice in adulthood but cardiomyocyte cross sectional area was reduced, in combination with an increase in nuclei per unit area implying an increased number of cardiomyocytes. This could arise from a delay in the developmental transition from hyperplasic to hypertrophic growth of cardiomyocytes and suggests that GR+/- mice may have a reduced ability to respond to the increased cardiac workload at birth and during the early postnatal period. Further cardiac challenge may be posed by the elevated blood pressure, compensatory increase in HPA axis activity and aldosterone levels previously reported in GR+/- mice. Adaptation to pathological cardiac challenge was assessed in adult GR+/- mice and WT littermates in response to AngII treatment, which has a direct hypertrophic effect on cardiomyocytes and, at higher doses, elevates blood pressure. GR+/- and WT mice showed an equivalent, dose-dependent increase in cardiomyocyte hypertrophy and cardiac fibrosis in response to AngII, as well as similar alterations in expression of Ca2+ handling genes. Functionally, these changes to the myocardium resulted in matched reductions in ejection fraction in GR+/- and WT mice. In contrast, when cardiac hypertrophy was induced by the physiological challenge of swim training, normal cardiac function was maintained in both GR+/- mice and WT controls. The physiological cardiac hypertrophy induced by swim training was not associated with cardiac fibrosis or pathological changes to left ventricle (LV) gene expression profiles. GR+/- mice have elevated HPA axis activity at baseline and swim training increased adrenal gland weight to a greater extent in GR+/- mice suggesting that raised GC levels due to compensatory HPA activation in GR+/- mice, may mask the role of GR in cardiac remodeling. To remove the effects of compensatory HPA axis activation and to achieve a greater degree of GR deficiency in the cardiovascular system, homozygous SMGRKO mice were investigated. Similar to GR+/- mice, survival to weaning compared with control littermates was impaired, by 46% and 65% in males and females respectively. Doppler measurements of transmitral inflow and transaortic outflow of blood showed a detrimental increase in the myocardial performance index (MPI), a load-independent measure of combined systolic and diastolic function. This was due to prolongation of the isovolumetric contraction time, indicating impairment of the initial LV contractile phase. Heart/body weight ratio was increased in both and male and female SMGRKO mice. Interestingly, cross sectional area was reduced in adult female SMGRKO mice cardiomyocyte, as was found in the GR+/- mice. In contrast, in male SMGRKO mice, cardiomyocyte cross sectional area and nuclei per unit area were equivalent to control littermates at 6 weeks of age, when heart/body weight ratio was already elevated. By 12 weeks of age, cardiomyocyte cross sectional area was greater in male SMGRKO mice than control littermates. In addition, levels of mRNA encoding myosin heavy chain-β, a marker of pathological cardiac hypertrophy, were greater in the LV of male but not female SMGRKO mice at 12 weeks. These findings suggest that cardiomyocyte hyperplasia in early neonatal life, possibly in combination with physiological elongation of cardiomyocytes, may underlie the elevated heart weight in female SMGRKO mice, whereas in male SMGRKO mice there is a transition to potentially pathological hypertrophy of cardiomyocytes. This may occur at puberty, in response to increased androgens, when marked LV growth occurs in males. Histopathology showed LV fibrosis in SMGRKO mice in both sexes, accompanied by elevated levels of mRNA encoding pro-fibrotic and matrix-remodeling genes in the LV. Intriguingly, levels of mRNA encoding the mineralocorticoid receptor (MR) were elevated in both sexes, which may be causal in the development of fibrosis. Indeed, in the LV, levels of mRNA encoding MR were already elevated in 6 week old SMGRKO males, at a time when cardiac collagen levels were only modestly increased. Levels of mRNA encoding the ryanodine receptor, which is essential for cardiac excitation contraction coupling, were reduced in the LV of female, but not male SMGRKO mice. The mechanisms underlying gender differences could be further investigated by comparing neonatal cardiac development in male and female SMGRKO mice. In conclusion, deletion of GR in cardiomyocytes and vascular smooth muscle causes gender specific pathological cardiac remodeling, demonstrating the essential role of cardiovascular GR signalling in cardiac maturation and function. Global GR deficiency alters the trajectory of cardiac development and increases risk of mortality. In surviving GR+/- mice, compensatory adaptations occur in response to the functional impairment seen in utero but subtle cardiac abnormalities remain in adulthood which, together with the elevated blood pressure and GC levels, may contribute to cardiovascular disease risk.

Myocardial infarction is the most common cause of heart failure and remains one of the leading causes of morbidity and mortality in humans. Stem cells are important in the maintenance and repair of adult tissues. Hoechst effluxing cells, termed side population cells are a rare subset of cells found in adult tissues that are highly enriched for stem and progenitor cell activity. Recent studies have suggested that Sca-1+/CD31- cardiac side population cells are capable of differentiation into cardiomyocytes in vitro. However, the response of cardiac side population cells to myocardial injury remains unknown in vivo. In this study, we directly transplanted Sca-1+/CD31- cardiac side population cells into an acutely infarcted mouse heart. After two weeks, the transplanted cells were found to express cardiomyocyte or endothelial cell markers. Importantly, when these cells were transplanted into a remote nonischemic part of the heart after MI, they were able to migrate to the damaged myocardium. Consistent with these cells homing property, we found that SDF-1α, a chemotactic chemokine and its receptor, CXCR4 were up-regulated in the damaged myocardium and on Sca-1+/CD31- cardiac SP cells respectively following an acute myocardial infarction. We further showed that SDF-1α was able to induce migration of Sca-1+/CD31- cardiac side population cells in vitro. Our results have therefore suggested that Sca-1+/CD31- cardiac side population cells are able to migrate to damaged myocardium from non-ischemic myocardium and differentiate into cardiomyocytes as well as endothelial cells in the acutely infarcted mouse heart. We postulate that the SDF-1α/CXCR4 interaction may play an important role in the migration of these cells. Understanding and enhancing these processes may hold enormous potential possibilities for therapeutic myocardial regeneration for the treatment of cardiovascular disease.

The newborn heart possesses a higher tolerance to ischemia in comparison to adult hearts. Post-ischemic interventions that increase energy production are beneficial for recovery. These data suggest that the newborn heart holds on a very tight energetic plasticity and may not be capable to effectively respond to increases in energetic demand. Congenital heart defects can lead to the development of cardiac hypertrophy and often require surgical intervention. Using an animal model of newborn hypertrophy and biventricular isolated working heart we confirm the metabolic profile of the newborn rabbit heart, in which fatty acid oxidation provides the vast majority of energy to the heart. Our findings show that when right ventricle load is added, the increasing energy requirements are met by increasing glucose oxidation rates. Our data generated by the isolated biventricular working heart model further supports the concept of the newborn heart in a state of deficient energy reserve.

Mindin (Spondin 2) is a highly conserved extracellular matrix protein of the Mindin-F-spondin family and a regulator of host innate immunity. Despite its expression in the heart, its role in cardiac stress response is unknown. The objective of this study was to determine the role of mindin following myocardial infarction (MI). C57/BL6 wild-type (mindin+/+) or mindin knockout (mindin-/-) mice were randomized to permanent left anterior descending (LAD) coronary artery ligation or sham operation. Mindin expression level increased maximally on day 7 post MI, but returned to baseline by day 28. Mindin-/- mice showed reduced mortality, rupture rate, cardiac MMP-9/-2 activities, NF-kB activation, cytokines and macrophage recruitment. We concluded that mindin is a significant contributor to mortality and acute adverse remodeling post MI, partly through its unique attributes of innate immune regulator and inhibitor of angiogenesis. Mindin may function as a potential biomarker or therapeutic target post MI.