Search

Several super-resolution techniques have been developed over the recent years, offering sub-nanometre resolution of biological structures. DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography) utilises DNA binding kinetics to achieve non-diffraction limited images. The transient binding of fluorescently labelled oligonucleotides (imagers) to complementary strands (docking strands), anchored to the structure of interest, is detected as a single molecule event with high localisation precision. A super-resolution image is formed by piecing all the localisations acquired over several frames together. This approach offers great advantages over competing techniques; mainly, unlimited photon budget and straightforward implementation on a simple fluorescence microscope. In this thesis, the performance of DNA-PAINT in biological samples, especially on thick tissue sections, was explored. Limitations of the technique were investigated, in particular background signal in the form of non-specific binding events which compromises data interpretation and can prevent sparse targets from being detected. We established protocols that minimised non-specific binding events in post-processing. Control co localisation measures showed an increase in the overlap after removing the non-specific binding events, however the results varied depending on the size of the protein clusters; this finding suggested that the stochasticity of DNA-PAINT was the main factor in the non-consistent detection of the smaller protein clusters. Alternative co-localisation methods might be more suitable to analyse DNA-PAINT data; for example, Euclidean Distance Transform (EDT) based co-localisation. In addition to minimising non-specific binding events in post-processing, we explored DNA nanotechnology to ensure less background signal detection in data acquisition. Repeat DNA-PAINT suppressed background signal attributed to excess imagers diffusing in solution, as well as non-specific binding events. This strategy offers a way to mitigate critical limitations of DNA-PAINT, without compromising spatial resolution. DNA-PAINT based imaging techniques gain more and more prominence, as they show versatile biological applicability and easy implementation. Moreover, multiplex DNA-PAINT imaging was implemented to investigate the protein distribution of two cardiac receptors, namely RyR (Ryanodine Receptor) and InsP3R (Inositol Trisphosphate Receptor), located in the sarcoplasmic reticulum (SR). They are involved in Ca2+ signalling, a process essential to cardiac contractility. Efficient coupling between the sarcolemmal Ca2+ channels and SR is essential for synchronised Ca2+ release and hence synchronised cardiomyocyte contraction. In disease, reduced coupling increases spontaneous Ca2+ release, resulting in arrhythmogenic activity. The contribution to Ca2+ release via the InsP3Rs is elevated, suggesting a potential influence in the RyR Ca2+ release mechanism. We conducted co localisation analysis to determine the proximity of the two calcium channels in physiological conditions and compared the results with an ischaemic heart disease porcine model. Our study further supports InsP3R-RyR channel crosstalk as a strong factor in influencing Ca2+ signalling and contributing to arrhythmias in myocardial infarction (MI).

Cardiac contractility is often defined as the ability of the cardiac muscle to generate power and results from a number of mechanisms. Contractile work is also the trigger that causes an increase or decrease in ATP generation, via one of two main processes: fatty acid (FA) oxidation or carbon substrate metabolism. These two mechanisms are crucial in contraction and any changes can lead to cardiac dysfunction. To this effect, both age and obesity have been previously shown to play a significant role in cardiac dysfunction, primarily via changes to contractility and ATP generation and in pathways involving the citric acid cycle and uncoupling proteins. The primary aim of this thesis was to investigate cardiac contractility in rodent models of aging and high fat diet-induced obesity (HFD) in the presence and absence of inotropic drugs. The first part of this project was a physiological investigation into the effects of ageing (3, 6 and 18-month murine models) and high fat diet-induced obesity (6 and 18-month lean and obese murine models) on cardiac function. The second part of the project was a pharmacological investigation into the effects of inotrope-induced changes on cardiac contractility in ageing and high-fat diet-induced obesity (using the same models). To achieve this, three techniques were used: the Langendorff isolated heart model, the papillary muscle work-loop assay used to assess power output and western blotting protein analysis of Pyruvate dehydrogenase E1-α subunit (PDH) and Uncoupling protein 3 (UCP3). Cardiac function of the whole heart diminished with age, showing significant differences for the Rare pressure product (RPP) on the 18-month models when compared to the 3 and 6-month models (p<0.05), but the cardiac muscles also suffer time and age-dependant decreases in performance, when comparing the 6-month models to the 3-month models. This translated to reductions in power output (p<0.05) and total net-work done. In addition to this, and in agreement with previous literature, the results from the western blots on PDH have shown decreased phosphorylation in an age-dependant fashion and indicate that there is, in fact, a decrease in FA oxidation (p<0.0001 when comparing between the 6 and 18-month models with the 3-month model). The UCP3 results corroborate these findings and suggest that there is a decrease in the rate of FA oxidation, in an age-dependant fashion (p<0.0001 when comparing between the 6 and 18-month models with the 3-month model). HFD functional changes were also recorded, as the 6-month lean heart showed significant decreases in the absolute values for the haemodynamic parameters when compared to the 6-month HFD model (p<0.0001); the 18-month model showed a significant increase compared to the 6-month lean model, but not when compared to the 6-month HFD model, which aligns with previous literature which associated these changes with hypertrophic changes to the heart. On the muscle assay, significant decreases in power output, fatigue and total net-work done were recorded when comparing between lean and HFD muscles (p<0.01). Finally, the pPDH and UCP3 were shown to be effective biomarkers of obesity, as the formers levels decreased in the presence of the high-fat diet (p<0.0001), while the latter increased significantly. The measured increase in UCP3, specifically, is indicative of increased FA availability and its consequent usage. On the ageing pharmacological chapter, a phenomenon known as β-receptor desensitization was present, as the LVDP, HR and RPP for the 18-month dobutamine-treated hearts were shown to have age-dependant impairments, when compared to the 3 and 6-month models (p<0.0001). As for the western blot assay, it was found that PDH was reduced in an age-dependant fashion for all three drug-treated hearts, with seemingly no changes in expression between them (p<0.0001 when comparing between the 6 and 18-month models with the 3-month model). This seems to indicate that FA oxidation and ATP production are being affected, but not detected in the isolated heart models (other than on the Dobutamine-treated hearts). As for UCP3, interestingly no change was recorded for atenolol or itraconazole across different ages, but a significant decrease was recorded for the 6 and 18-month dobutamine treated hearts when compared to the 3-month heart (p<0.0001). It is therefore possible that there is a correlation between the observed β-receptor desensitization and the UCP3 levels, even though the exact link between the two is unclear. On the isolated heart model, between the lean and HFD atenolol-treated hearts, but both the Dobutamine and Itraconazole data showed severe impairment and exacerbation (respectively) in their inotropic effects on LVDP, HR and RPP, when treating the HFD models and comparing them to the lean models (p<0.01 for each parameter for Dobutamine and p<0.0001 for each parameter for Itraconazole). PDH was significantly changed for the HFD hearts treated with Dobutamine (p<0.0001) and Itraconazole (p<0.0001), with little to no change in our atenolol treated hearts. UCP3 on the other hand showed no significance between the dobutamine and atenolol treated HFD and lean hearts but showed a significant decrease for the itraconazole HFD treated hearts when compared to the lean hearts (p<0.001). In conclusion, these results showed that both age and HFD cause functional changes in the heart in both absence and presence of drug treatments and their combined synergistic effect can cause further detrimental changes on different haemodynamic parameters. In addition, PDH activity, specifically, seems to be heavily linked to the inotropic effect of both Dobutamine and Itraconazole in the presence of obesity, but further work will still be necessary in order to confirm this observation.

Extracellular matrix (ECM) remodelling is a hallmark of many cardiovascular diseases. Importantly, haemodynamic environments and disease propensity differ across vessels. A comprehensive ECM characterisation across non-diseased vascular territories, however, has not been performed. Here, I used human aortas, internal mammary arteries, radial arteries and saphenous veins (n=10 each) collected during surgery to characterise the vascular ECM by untargeted proteomics followed by targeted proteomics and glycoproteomics to quantify and investigate vascular ECM and glycosylation changes. 454 ECM and ECM-associated proteins were identified in the human vasculature, of which 184 were core ECM proteins. Arteries were rich in network-forming collagens IV and VIII which confer flexibility. In contrast, fibrillar collagens, conferring stiffness were reduced in aortas compared to the other vessels. Hyalectan proteoglycans (aggrecan and versican) providing viscoelasticity related to blood pressure, were abundant in aortas. IMAs, renowned for their resistance to atherosclerosis, showed reduced levels of proatherogenic small leucine rich proteoglycans (SLRPs) and increased levels of collagen XVIII, including its antiangiogenic and anti-atherosclerotic proteolytic fragments, endostatin and neostatin. Veins contained high levels of basement membrane (BM) glycoproteins (i.e. laminins and nidogen-2) and collagen VI, critical for BM stability, compared to arteries. For quantification, I selected proteotypic peptides to create a targeted panel of 125 ECM proteins, and applied it to aortas with (n=7) and without (n=6) type 2 diabetes (T2DM). These changes were validated in carotid endarterectomy samples from patients with (n=34) and without (n=70) T2DM. Targeted proteomics revealed higher levels of laminins β2, α4 and α5, and collagen α3(VI), in aortas of patients with T2DM compared to controls. This increase in BM proteins was also evident in carotid endarterectomy specimen from T2DM patients. Additionally, an increase in the SLRPs, biglycan, lumican, mimecan and prolargin was found, further corroborating the finding that the reduction in SLRPs in IMAs may relate to their decreased susceptibility for plaque formation. Differences in glycan composition on ECM proteins were detected in T2DM, which was supported by an increase in glycosylation enzymes. I provide the first comprehensive characterisation of the vascular ECM, revealing distinct ECM components of different vascular territories, which may influence disease propensity. Our targeted proteomics method allowed detection of changes in ECM proteins even before manifestation of overt atherosclerotic lesions in patients with T2DM, supporting the role of BM proteins, not only in diabetic micro-, but also macrovascular pathology. Additionally, I performed the first glycoproteomic characterisation of the ECM in the human diabetic aortas, demonstrating that T2DM alters the glycosylation status of vascular ECM proteins.

The transcription factor, Mesoderm posterior 1 (Mesp1), is essential for mesoderm formation in the early embryo. Overexpression in an embryonic stem cell (ESC) model leads to cardiac differentiation. However dependent upon conditions, Mesp1 overexpression can also induce skeletal muscle and haematopoietic lineages. The mechanism by which Mesp1 induces these different phenotypes is unknown but could be due to different interacting partners. Identifying if such interactions play a role in the differentiation of stem cells into cardiac progenitor cells (CPCs), as well as if we can use these factors to reprogram somatic cells into CPCs is the pivotal question to be answered in order to provide a novel CPC production pathway. The ability to produce a pure CPC population would allow us to make a breakthrough in their use in regenerative medicine, opening up new avenues of treatment for heart failure patients. To investigate this, we identified MESP1 and its binding partners through mass spectrometry of mouse embryonic stem cells (mESC) isolated at mesoderm stage of cardiac differentiation. Our analysis has identified 7 putative binding partners that have similar expression patterns to Mesp1 in mESCs undergoing cardiac differentiation. Furthermore, we have verified that 2 of these putative binding partners, Eomesodermin and Methyl- CpGbinding domain protein 3, interact with MESP1 when co-expressed in human embryonic kidney cells (HEK). Furthermore, using literature-based evidence, we have identified 1 other potential binding partner, WD Repeat Domain 5. Utilising mESCs, we have identified that these binding partners have an effect on cardiac differentiation, and other binding partner expression patterns, that previously observed in Mesp1 knockout mESCs. We further ascertained the effects of these binding partners in the reprogramming of cells. Overexpression in 3T3 fibroblasts has identified morphological and genetic changes, including inducing the expression of cardiac genes.

Primary Raynaud's phenomenon (PRP) is characterised by the periodic vasospasm of the digits precipitated by exposure to cold/emotional stimuli. PRP may involve vascular endothelium dysfunction, abnormalities in neural control of vascular tone and/or increases in circulating mediators which promote vasoconstriction. Cocoa products have been shown to promote vasodilation, and therefore could mitigate PRP symptoms. The work described in this thesis investigates the effects of cocoa flavanols on peripheral blood flow, thermoregulation and symptoms in people with PRP. The acute effects of high flavanol cocoa (HFC) consumption on cardiovascular parameters and peripheral thermoregulation was compared between people with PRP and control participants using a double blind, cross over design. The chronic effects of 3 months daily consumption of HFC was then investigated using a double blind, placebo-controlled, parallel group design in people with PRP. The experiencing of Raynaud's symptoms, their duration and level of pain, and an assessment of Raynaud's Condition Score were documented by participants in a symptoms diary throughout the intervention. Dietary macronutrient composition as well as total flavanol and epicatechin intake, were assessed monthly using diet diaries and Food Frequency Questionnaires, respectively, with Physical and Mental Health status being determined at these time points using the SF-36TM. A Finometer was used to measure cardiovascular variables and Laser Doppler Flowmetry used to assess skin blood flow before and after the supplementation period. In pilot studies, FMD was used to determine endothelial function following HFC consumption. Data from the pilot studies found that acute consumption of a HFC drink induces a greater vasodilatory response to a shear stress stimulus at 1hr compared to a LFC drink. In acute supplementation studies, it was found that consumption of a HFC drink did not impact peripheral thermoregulation in either PRP participants or controls. Meanwhile, consumption of HFC capsules in a chronic supplementation study reduced total peripheral resistance and increased cardiac output, but these beneficial effects were not accompanied by an increase in skin blood flow, or improvements in Raynaud's symptoms among PRP participants. It was also observed that in this cohort, having PRP did not appear to negatively impact mental or physical wellbeing, compared to a general population. The thesis proposes that it is feasible to recruit, retain, and provide follow-up with the participants for a full randomised control trial involving individuals with PRP in the future. The findings from acute supplementation studies demonstrated no differential effect of HFC drink on cardiovascular measures and skin blood flow in either PRP participants or controls, while data from chronic supplementation study found no differential effect of HFC capsules on skin blood flow and Raynaud's symptoms among PRP participants. Therefore, it is important that people with PRP are aware that cocoa flavanols probably will not help to reduce their symptoms, despite studies that suggest their consumption improves the compliance of resistance vessels and microvasculature in healthy individuals.

Exercise tolerance strongly predicts morbidity and mortality, and correlates with health-related quality of life. The mechanisms underpinning exercise intolerance were investigated in Chapter 3. Peripheral vascular dysfunction occurs when there is an imbalance between endothelial damage and endogenous repair mechanisms, and can contribute to exercise intolerance. Vascular dysfunction typically precedes a diagnosis of cardiovascular disease and is related to disease severity in cardiovascular disease patients (e.g. chronic heart failure; CHF). Exercise training increases vascular function and exercise tolerance in health and CHF, with high-intensity interval training (HIIT) often inducing greater improvements than continuous 'control' protocols. However, it is unknown whether the effectiveness of HIIT can be improved. Therefore, Chapter 4 investigated the acute and chronic effect of HIIT with and without blood flow restriction (BFR) on vascular endogenous repair capacity, ramp-incremental exercise tolerance and V̇O2peak in CHF. Four weeks of HIIT did not induce acute or chronic vascular adaptations; however, chronic HIIT increased ramp-incremental exercise tolerance and V̇O2peak. BFR did not increase training efficacy. Chapter 5 determined whether 6 weeks of HIIT at a high work rate with short work:recovery bouts (intended to maximise the volume of shear rate accumulated) increased vascular endogenous repair capacity, endothelial function, exercise tolerance and V̇O2peak more than intensity-matched continuous exercise training in healthy adults. No acute or chronic vascular adaptations were seen with HIIT. This type of HIIT increased post-training exercise tolerance and V̇O2peak but was no more effective than intensity-matched constant work rate exercise training. Overall, increases in exercise tolerance and V̇O2peak in both training studies were not underpinned by improvements in peripheral vascular endothelial function and/or increases in vascular endogenous repair capacity. Therefore, despite the efficacy of training to increase exercise tolerance, further work is needed to develop exercise strategies that effectively and specifically target improvements in vascular endogenous repair capacity and peripheral vascular function.

The maintenance of haematopoietic stem cell (HSC) self-renewal and differentiation throughout life is essential for ongoing haematopoiesis and is highly dependent upon cytokine-cytokine receptor interactions and direct cell-cell contact between the HSC and components of the perivascular bone marrow (BM) microenvironment. Thrombopoietin (TPO) is one of two such cytokines essential for HSC self-renewal. Although the majority of TPO is produced distally by the liver, lower amounts of TPO are thought to be produced locally in the BM, directly at the site of utilisation. However, the exact cellular sources of BM derived TPO are unclear and remains an active area of research. Contrary to previous studies, the results in this thesis indicate that megakaryocytes do not express Thpo, and instead LepR+/Cxcl12-DsRedhigh BM stromal cells (BMSCs) are major sources of Thpo in mice. Immune thrombocytopenia (ITP) is an acquired autoimmune condition characterised by reduced platelet production and increased platelet destruction by sustained immune attack. In this thesis, a novel mouse model of sustained ITP was generated and the effect on the immune and haematopoietic system was assessed. Platelet destruction was antibody dependent and appeared to be primarily driven by splenic macrophages. Additionally, ITP progression was associated with considerable progenitor expansion and BM remodelling. Single cell assays using Lin-Sca1+c-Kit+CD48-CD150+ long-term HSCs (LT-HSCs) revealed elevated LT-HSC activation and proliferation in vitro. However, LT-HSC functionality was maintained as measured by in vivo serial transplantations. ITP progression was associated with considerable BM vasodilation and angiogenesis, as well as a 2-fold increase in local production of CXCL12; a cytokine essential for LT-HSC function and BM homing expressed at high levels by LepR+ BMSCs. This was associated with a 1.5-fold increase in LepR+ BMSCs and a 5.5-fold improvement in progenitor homing to the BM. Whereas the increase in BMSCs was transient and reverted back to baseline after platelet count returned to normal, vasculature changes in the BM persisted. Together, these studies demonstrate that LT-HSCs expand in response to ITP, and that LT-HSC functionality during sustained haematopoietic stress is maintained through an adapting BM microenvironment.

For several years, the inability to replace the lost myocardium has been studied intensively to define the mechanisms that restrict the regenerative capability of the adult heart. Multiple evidence have pointed out that the turnover of postnatal mammalian cardiomyocytes is affected by different crucial pathways. However, a thorough understanding of the intrinsic molecular mechanisms that regulate this process is still far from being complete. It has been more than a decade since various laboratories initialised the study of ubiquitination processes in the heart, reaching the unanimous conclusion that it plays a critical role in manipulating virtually all heart functions. However, there is fragmentary information about the molecular function of this system on the regulation of cardiomyocyte proliferation and even less on how this could be exploited for therapeutic purposes. Hence, expanding our knowledge on the ubiquitination might devise new strategies for cardiac regeneration. In this work, we present the identification of ubiquitination factors that are essential for cardiomyocyte replication, which was identified by harnessing the High Throughput Screening approach. An RNAi-based screening was performed, in which approximately 600 ubiquitination factors were silenced individually in primary neonatal cardiomyocytes. After that, the top siRNAs inhibiting proliferation were selected and investigated further to validate their functions in vitro. The validation identified UBE2G1, an E2 conjugation enzyme, as the most effective factor induced cardiomyocyte proliferation. The depletion of UBE2G1 not only suppressed cell cycle progression but also stimulated hypertrophy and counteracted the effects of pro-proliferative miRNAs. Administration of AAV9-UBE2G1 in neonatal mice promoted the cycling of cardiomyocytes while it preserved the heart function in adult mice at the early time points after myocardial infarction. Activation of GSK-3β, ERK1/2, and STAT3 signalling pathways correlated with UBE2G1 activity, whereas its interacting partners remained to be identified. In summary, we have revealed the implicit potential of ubiquitination factors and highlighted their promising therapeutic use in a heart regeneration scenario.

Endothelial cells, which form the inner lining of all blood vessels, maintain various critical functions to enable the efficient transport of oxygen and nutrients to all metabolising cells of the body. Finely tuned regulation of endothelial function is important for the establishment and maintenance of an efficient and functional vascular transport system. As such, endothelial dysfunction can result in the development of tissue ischaemia and has been highlighted as a major step in the development of various degenerative diseases that affect the micro and macro vasculature including a variety of retinopathies such as Retinopathy of Prematurity (ROP). A vast repertoire of signalling pathways have been identified in regulating the functional maintenance of endothelial cells. Two such pathways known to have crucial regulatory roles governing endothelial function are the Notch and endothelial nitric oxide synthase (eNOS) signalling pathways: currently, the interaction between these pathways is poorly described. In part 1 we developed a heterologous system to investigate the effect of eNOS on Notch activation by overexpressing eNOS in HEK 293 cells; cells which are free of endogenous eNOS. eNOS overexpression resulted in increased cell-cell aggregation as well as increased N-Cadherin and Notch1 expression, the latter of which correlated with increased availability of the Notch intracellular domain (NICD). The results from chapter1 implicate eNOS in promoting an epithelial-like phenotype with increased cell-cell adhesion which was associated with enhanced Notch1-Dll4 signalling. In the presence of induced Dll4 expression the epithelial-like phenotype was further enhanced culminating in excessive cell-cell aggregation and the lack of any observable effect with γ-secretase inhibition indicated that augmented Dll4/Notch1 binding enhances cell aggregation. This provides evidence of the presence of Dll4 and Notch1 expression within adjacent cells leading to heterotypic cell-cell binding/adhesion. In part 2 of the study we investigated the functional role of constitutively expressed Dll4 on Notch1 activation in mature primary retinal microvascular endothelial cells (RMECs). We demonstrated using lentiviral-mediated shRNA knockdown that Dll4 has important roles in mediating cell-cell junction and that removal of Dll4 induces an enlarged senescent-like phenotype. These findings were linked to decreased Notch1 activation evidenced by reduced NICD levels indicating Notch 1 as the receptor mediating these effects. In retinal pericytes, the Notch1 receptor also showed evidence of regulating smooth muscle properties through increased Notch 3 signalling. Overall, these findings suggest a previously unknown role for the Notch1 isoform in retinal pericytes (RP) and endothelial cells (EC) in facilitating EC-P interaction. In part 3 using hyperoxia to model the early pathology of ROP we investigated the effect of hyperoxia on EC and on Notch signalling in particular. We demonstrated hyperoxia to have detrimental effects upon endothelial functions such as decreased proliferation with increased senescence and apoptosis while inducing a phenotypic switch to an elongated, spindle-shaped morphology with evidential decreased cell-cell adhesion as shown from epifluorescent and phase contrast images. We also presented evidence of increased Notch signalling under hyperoxia which included increased Notch1 cleavage, upregulation of target genes and activation of a fluorescent Notch1 reporter. Such target genes included the arterial markers Dll4 and Hey2, thus suggesting an arterial phenotype switch under hyperoxia. Collectively, the results generated in this thesis provide new insights into the complexity of the Dll4 and Notch1 signalling pathway in particular the differential signalling dynamics observed in the presence of eNOS and identified functional roles of Dll4 in mature retinal EC. The current study also identified detrimental effects of hyperoxia on endothelial function including decreased proliferation coinciding with increased apoptosis and senescence, all of which correlated with increased Notch signalling. The results provide insight into the pathology of ROP on a molecular and cellular level in particular the role of Notch1.

Background: Small conductance Ca2+-activated K+ current (ISK) may change cardiac atrial action potentials (AP) in response to altered [Ca2+]i; a potential therapeutic target for treating atrial fibrillation (AF). However, the contribution of ISK to atrial APs under physiological conditions is unclear. Furthermore, ISK may be enhanced in ventricles by heart failure, but whether by [Ca2+]i elevation in the non-failing ventricle is unknown. Aims: To test whether ISK flows under normal or increased global Ca2+]i, or with sub-sarcolemmal [Ca2+]i increase from APs in human and rabbit atrial cells. Also, to test an ISK blocker, ICAGEN (ICA), on rabbit left ventricular (LV) ion currents under [Ca2+]i elevation from Na+/Ca2+-exchanger (INa/Ca) stimulation. Methods: Myocytes were isolated enzymatically from hearts removed from anaesthetised rabbits, and from atrial tissues from consenting patients undergoing cardiac surgery. Whole-cell patch clamp (37°C) was used to record ion currents and APs (at 1, 2 or 3Hz), with [Ca2+]i measured using Fura-2. Results: A positive control tested stability/timing of K+ current (IK1) block: Ba2+(0.5 mM) significantly and reversibly decreased inward IK1 (at-115 mV) in 94% of LV cells, from -38.9±5.9 to -12.9±4.5 pA/pF (by 67%), and in 92% of atrial cells, by 43% (P<0.05, mean±SE, t-test, n=16-25 cells, 11-16 rabbits). Atrial ISK was investigated, under increasing [Ca2+]i (100-500 nM; with 5 mM BAPTA), with (100 nM) and ICA (1 μM). Neither drug affected inward or outward current (P > 0.05) at any [Ca2+]i, in rabbit or human (5-26 cells, 7-11 rabbits, 3-4 patients). APs recorded at 1 Hz (rabbit) were prolonged by 4-AP (ITO blocker; positive control): action potential depolarization at 30% repolarization (APD30) by 72%, at 70% repolarization (APD70) by 31%. By contrast, ICA (1 μM) had no effect on APD30-90, maximum diastolic potential (MDP), or Vmax, in human or rabbit. ICA at 10 μM (non-specific) increased APD70-90 vs time-matched controls. At 2 or 3 Hz, 1 μM ICA again had no effect on APs. In rabbit LV cells, stimulating INa/Ca increased [Ca2+]i (up to 2.8 μM) and inward /outward currents. ICA (1 μM) had no effect on [Ca2+]i or currents, whereas subsequent NiCl2 (10 mM; INa/Ca blocker) decreased them. By contrast, ICA 10 μM decreased outward (by 35%) and inward (49%) current, and [Ca2+]i (77%), with no effect of subsequent NiCl2. Conclusions: In rabbit and human atrial isolated myocytes, ISK may not flow under physiological conditions, nor during short bursts of supra-physiological stimulation, so atrial ISK activation (and thus its potential pharmacological inhibition during AF) may require changes to cellular electrophysiology or cell signalling systems to develop a sensitivity to ISK iii block. Furthermore, in non-failing LV myocytes, 1 μM ICA-sensitive ISK may not be activated by [Ca2+]i-elevation, and high ICA conc. may inhibit INa/Ca.

Cardiovascular disease is the primary cause of morbidity and mortality in patients with diabetes. Endothelial dysfunction is recognised to be a key early event in the atherogenic process that underpins vascular disease, and endothelial hyperglycaemic exposure or excursion could underpin this process. In addition, increased fructose consumption has been implicated in critical aspects of atherosclerosis. Although the adverse effects of high fructose consumption on hepatic metabolism have been extensively described, the direct effect of circulating fructose exposure on endothelial cell function is not fully understood. Furthermore, alteration of intracellular insulin signalling, described as vascular insulin resistance, could arise from the continuous hyperglycaemic exposure and consequent high insulin concentrations. In this study, the effect of chronic, acute and oscillating glucose exposure on a human-derived endothelial cell line, EA.Hy926, was examined. EA.hy926 cells were preconditioned with two distinctive pre-treatment glucose conditions during the cell growth phase: ("normal glucose", NG; NGPC cells) or 25 mM glucose ("high glucose", HG; HGPC cells). Both of these cell groups were exposed to three test carbohydrate concentrations (5.5, 12.5 and 25 mM) for 48 hours and the measurement of bioenergetics, cell function and key endothelial cell mediators compared for HGPC and NGPC cells. Further complexity was incorporated into the model by partial substitution of glucose with fructose and by the inclusion of insulin. The impact of the various test conditions was measured in terms of bioenergetics, using a metabolic analyser, and cell function by measurement of key endothelial cell mediators (nitric oxide (NO), endothelin-1 and thrombolytic factors). Chronic HG exposure modulated cell bioenergetics and decreased mitochondrial respiration, production of mediators of vessel tone (NO, endothelin-1) and fibrinolysis (tissue plasminogen activator, plasminogen activator inhibitor-1). These effects were not reversed after acute exposure to NG. Addition of fructose increased markers of metabolic activity, but reduced NO generation. The effect of insulin was complex, impacting both bioenergetics and NO generation and dependent upon the presence of fructose, as well as the glucose concentration of the medium. In conclusion, this study showed that chronic glucose exposure induces long-lasting changes in bioenergetics, antioxidant defence and production of mediators of vascular tone and fibrinolysis in EA.hy926 cells. The results highlighted deleterious and long-lasting changes to endothelial cell function induced by chronic hyperglycaemia which could not be reversed in an improved glycaemic environment. Moreover, fructose introduces an additional adverse effect on cell function, which is further complicated by the presence of insulin. All taken together, this study reinforces the importance of the link between carbohydrate load, diabetes, endothelial function and cardiovascular disease.

The sinoatrial node (SAN) is the primary cardiac pacemaker, which generates spontaneous action potentials (APs) and regulates the rhythms of the heart. The function of the SAN declines with age development, and this results in marked differences in the morphology and characteristics of APs and affects the pacemaking activities, consequently increases the incidence of sinus node dysfunction (SND) in older adults. These changes may be attributable to different sets of ion-channel interactions at different ages. The mechanism underlying the pathogenesis for cardiac pacemaker dysfunctions associated with ageing remain uncertain. In this project, using a computer modelling approach, we investigated the role of age-related remodelling of ion channels on pacemaking activites of the SAN at single-cell and tissue level. The research explained in this thesis utilised electrophysiological data from experiments in different species at the ion-channel level to develop novel mathematical models of neonatal, aged and diabetic conditions. A mathematical model of neonatal rabbit SAN cells was developed by modifying the current densities and/or kinetics of ion channels (INa, ICa,L, If, INaCa, IKr and IKs) in a SAN adult-cell model. At single cell level, simulation results showed that the ion channels altered during maturation play a functional role in slowing down the pacemaking APs. At the tissue level, these integral effects increase the activation time across the intact SAN-atrium, leading to decreased AP conduction velocity and heart rate. Moreover, vagal nerve activity elucidated a high sensitivity of neonatal SAN cells to acetylcholine. The effect of ACh amplified in neonates, under high concentration, leading to possible sinus arrest or conduction exit. Similarly, the ionic mechanisms underlying SND associated with ageing were assessed. A mathematical model of adult rat SAN cells was modified to investigate two case studies of cardiac pacemaker dysfunction, identified experimentally, which arose from different pathways of electrical remodelling in ion channels (If, ICaL, INaCa) and Ca2+ handling proteins proteins (RyR2, and SERCA2a) in the ageing rat heart. Our results suggest that the integral action of all remodelled ion channels and Ca2+ handling can be accounted for producing bradycardic effects as manifested by heart rate reduction, the remodelled ICa,L, either via a gain or loss of function, contributes primarily to ageing-related bradycardia. Therefore, ageing-related bradycardia can be linked to different remodelling 'pathways'. A one-dimensional string model of SAN-atrium was constructed, through incorporating the regional heterogeneity of SAN tissue, and coupled to atrial tissue. The new model was utilised to simulate the spontaneous activity and AP conduction across the SAN-atrium under normal conditions and SND associated with ion channel deficiency in diabetes. The model successfully produced similar 12 propagation and conduction activities to experimental observations in normal SAN.The functional alternation of ion channel; ICa,L, ICa,T, If and INaCa in diabetic rat closely resemble those observed in experimental SND. Finally, a mathematical model of mouse SAN cells was utilised to investigate the effect of changes in abundance of hyperpolarisation-activated cyclic nucleotide-gated channel (HCN) on the APs and pacemaker rate, during over-expression of gene target TBX18, which is used to enhance pacemaker function in a rat subsidiary atrial pacemaker (SAP), a model of sick sinus syndrome (SSS). Simulation results showed that the changes of the HCN abundance could explain the observed changes in beating rate. This suggests that TBX18 may have the potential to restore pacemaker function in human SSS.

Long non-coding RNAs (lncRNAs) are emerging as important regulators in many biological processes. However, despite over thousands of lncRNAs have been identified, only a few have been functionally characterised, especially in cardiac diseases and development. The use of anthracyclines such as doxorubicin (DOX) has improved mortality and morbidity in cancer patients, yet associated risks of cardiomyopathy have limited their clinical application. DOX-induced cardiomyopathy (DIC) is frequently irreversible and typically progresses to heart failure. However, little is known about the role of lncRNAs in the underlying mechanisms of DOX-induced cardiotoxicity. RNA-sequencing analysis of a DIC rat model revealed that the highly conserved lncRNA Cyrano was significantly down-regulated by DOX. To evaluate the functions and mechanisms of Cyrano, the highly conserved region and the promoter site were deleted separately by genome editing. Transcriptomic profiling of the transgenic lines showed that gene regulation by Cyrano does not solely depend on its conserved site. Capture hybridisation analysis of RNA targets revealed that Cyrano interacts with 3'UTRs of mRNAs and PABPN1, a crucial component of the mRNA 3'-end processing machinery. The depletion of Cyrano led to widespread 3'UTR lengthening of mRNAs. We propose that Cyrano may contribute to the dysregulation of alternative polyadenylation (APA) induced by DOX through its interaction with PABPN1 in cardiomyocytes. As genes and pathways important in cardiac development are often re-activated in heart failure, we hypothesised that Cyrano also regulates APA in cardiomyocyte differentiation. During the differentiation of cardiomyocytes from human induced pluripotent stem cells (hiPSCs), Cyrano showed a dynamic expression pattern that corresponded to the global APA changes. Together with the Cyrano-PABPN1 interaction in hiPSCs, these suggested that Cyrano may play a role in regulating APA in the early stages of cardiomyocyte differentiation. Further investigations are needed to elucidate the function and mechanisms of Cyrano in cardiomyocyte differentiation and cardiac development.

This thesis introduces and develops a number of mathematical models to investigate the propagation of waves in a stented artery. Generally, healthy arteries have smooth inner walls and blood flows through them easily. However, vessels may become occluded due to the accumulation of plaque on the inner walls, which can reduce blood flow or, in some instances, block it altogether. Intraluminal stenting is one technique that can be performed to restore adequate flow and avoid ischaemia (restriction of blood flow to tissues) occurring. Arterial walls are elastic and subjected to pulse waves originating from the left ventricle of the heart, the frequency of which vary with physical activity and possible disease states. It is important to identify how the reinforcement of arteries with stents may affect the propagation of these waves through the arterial network. New models are proposed here to analyse how elastic waves, induced by a pulsating flow in a stenotic artery containing several stents, are reflected and transmitted, both in the frequency and time domains. The reflection of waves in blood vessels is well documented in the literature, but it has generally been linked to a strong variation in geometry, such as the branching of vessels. The aim of this work is to detect the possibility of wave reflection in a stented artery due to the repetitive pattern of the stents, using the Bloch-Floquet approach and the analysis of reflection-transmission problems. The investigation of time-harmonic wave propagation and possible blockages is complemented with numerical simulations in the transient regime. Dispersion properties of the waves depend on the stent structure and are addressed throughout this thesis. Several vascular stenting procedures include overlapping stents; this configuration is included within the models, as well as the presence of atherosclerotic plaque. An additional analytical one-dimensional model is developed as an approximation to the full three-dimensional methods. The analytical derivations are accompanied by numerous illustrative examples and simulations. This thesis introduces a complete set of coupled fluid-solid models to analyse wave propagation in stented arteries. An appealing aspect of the work is that the onedimensional model is able to identify ranges of interest rapidly, which can then be investigated in more detail using the more precise, but time-consuming, full threedimensional models.

Cardiac computational modelling has offered an alternative method to experimental cardiology for heart function studies. Although different electrophysiological models in several species have been developed, no such models exist for the fish heart. Fish are vertebrate ectothermic animals, and their body temperature is affected by temperature changes in their aquatic environment. Given the prominence of global warming effects, temperature changes and the rise of water temperature influence different organs in the fish body, including the heart. A better understanding of the process of thermal acclimation of the fish heart at single cell and tissue levels where cardiac ion channels and pumps remodel after prolonged exposure to a different temperature, is of scientific interest. Also, a 3D model based on an accurate anatomy model is a valuable tool for the study of ventricular electrical activities. Furthermore, investigating the effects of heart diseases on cardiac mechanical dynamics and the contracting dynamics of the human ventricular cells is also valuable to study. Thus, the objective of this thesis consists of three parts: In part I, a novel biophysically computer model for the teleost fish ventricular myocyte has been developed at the physiological body temperature of 4°C. First, novel formulations of the major ionic currents including I_Na, I_CaL, I_Kr and I_K1, were developed at 4°C and then incorporated into the basal model developed by Luo and Rudy for the guinea-pig ventricle myocytes. The developed model was then used to assess the effects of changing temperatures on ventricle electrical dynamics. The developed models matched experimental findings quantitatively. In Part II, a novel 3D biophysically and anatomically accurate model for the teleost ventricle was developed. First, a 3D geometry of the heart was reconstructed and segmented into the heart's major chambers using a micro-CT scan with sustaining iodine technique. Then, the 3D geometry of the ventricle was incorporated into a 3D computational model to investigate the electrical excitation propagation in the teleost heart. In Part III, a multi-scale electromechanical model of human ventricle cells was modified to assess the impacts of hypertension (HP) on the contractile function of the human left ventricle myocytes. A single cell electromechanical model was modified by integrating experimental data from rat ventricular cells under Sham (control) and HP conditions. Simulations showed that HP prolonged APD₉₀ and increased [Ca²⁺]ᵢ with no marked change in the sarcomere length or the contractile force at cellular level, but in 2D tissue simulations, there was an increase in the tissue's vulnerability for initiation and maintenance of re-entrant excitation waves indicating a pro-arrhythmic effect of hypertension.

Transverse (t)-tubules are invaginations of the cell membrane vital for the synchronous rise of systolic calcium. In heart failure, t-tubules are lost leading to dys-synchronous calcium release. Nothing is known however, about the mechanisms that control atrial t-tubule formation or whether t-tubules can recover. Thus, this study aimed to investigate atrial t-tubule recovery following loss in heart failure, with emphasis on t-tubule density, calcium handling and proteins that contribute to t-tubule recovery. This study also aimed to gain understanding of how t-tubules form in the heart. Heart failure, and subsequent t-tubule loss, was induced in sheep by rapid ventricular pacing for ~ 36 days. Following 5 weeks of recovery, prompted by cessation of pacing, cardiac function, cellular hypertrophy and atrial tubule density all recovered. The organization of recovered tubules was altered however, with recovered tubules being predominately longitudinally orientated. Despite changes in orientation, recovered tubules were functional, in that, calcium was initially released along the tubules, followed by propagation to the rest of the cell. Recovery of atrial t-tubules was associated with increased expression of BIN1, Tcap and MTM1. Expressing these proteins in neonatal rat ventricular myocytes (NRVMs) demonstrated that BIN1 was sufficient to drive tubule formation, the structure of which was altered by coexpression with MTM1 and Tcap. Furthermore, NRVMs expressing variants 5, 8 and 9 of BIN1 exhibited increased systolic calcium transients and more synchronised calcium release. This is in contrast to skeletal muscle where tubule formation was dependent on the muscle specific variant 8 of BIN1. This study has shown that functional atrial t-tubules are restored following recovery from heart failure. The same proteins that were associated with t-tubule recovery were responsible for the formation of new tubule structures in the developing heart. This research identifies several targets, most notably BIN1, to facilitate t-tubule restoration and subsequent changes in calcium handling following loss in disease.

Transient dedifferentiation of cardiomyocytes has been established as a key mechanism behind the enhanced proliferation and cardiac regenerative capacity observed in lower vertebrates. In contrast, mature mammalian cardiomyocytes exist in a terminally differentiated state such that regeneration is significantly impaired. It has been previously demonstrated that somatic cells can be reprogrammed temporarily to a proliferative, dedifferentiated state in vitro and in vivo through transient overexpression of Oct3/4, Sox2, Klf4 and c-Myc (OSKM). However, the response of cardiomyocytes to transient OSKM expression has not previously been explored. In this thesis, the effects of adenoviral vector mediated OSKM delivery to cardiac cells in vitro and in vivo were investigated. Forced expression of OSKM in cultures of neonatal rat and mouse cardiomyocytes induced rapid dedifferentiation as evidenced by sarcomere dis-assembly and downregulation of cardiomyocyte-specific genes (Myh6, Myh7). This induced dedifferentiation was associated with an increase in cell cycle activity consistent with an enhanced proliferative capacity. The initiation of reprogramming was further confirmed by the expression of early stage reprogramming markers (ECad) however, late-stage reprogramming markers remained silenced and the generation of pluripotent stem cells was not observed. Furthermore, parallel to decreases in exogenous OSKM expression, partially reprogrammed cells regained a contractile cardiomyocyte-like morphology and a gene expression profile consistent with their redifferentiation towards functional cardiomyocytes. The effect of ectopic OSKM expression was also investigated in vivo through direct intramyocardial injection of the same adenoviral vector. Within 6 days after injection, upregulation of endogenous pluripotency genes (endogenous Oct3/4 and Gdf3) was observed indicating the activation of a reprogramming-like response within the adult mouse myocardium. The increased expression of such genes was maintained only temporarily consistent with a transient reprogramming response which was further evidenced by the absence of teratomas. Finally, the capacity of an OSKM expressing adenoviral vector to induce this transient reprogramming response in a mouse model of myocardial infarction was confirmed. In conclusion, this work reports the first evidence of adenoviral mediated transient reprogramming of cardiac cells both in vitro and in the healthy and injured myocardium in vivo. Furthermore, this study was the first to explore adenoviral vectors for in vivo OSKM delivery and highlighted challenges associated with this approach. Further work is necessary to understand the source and fate of partially reprogrammed cells as well as determine whether this strategy can provide any therapeutic benefit in the heart. However, it is hoped that the results presented here can help inform future research into the development and applications of transient reprogramming-based strategies.

The renin angiotensin system (RAS) is highly conserved across vertebrates. However, until recently it has not been extensively explored in zebrafish, an important model organism suitable for developmental studies, high-resolution in vivo imaging, and genetic and chemical screens. In common with mammals, the adult zebrafish kidney contains specialised renin-expressing mural cells that contain granules indicative of the processing and regulated secretion of active renin. Due to the translucent nature of zebrafish larvae, the functional pronephros is easily accessible for real-time imaging and in vivo studies of the RAS. The primary aim of this project was to develop novel tools to help elucidate the function of renin and the role of the RAS in zebrafish. I designed a zebrafish renin-luciferase reporter transgene using a previously published promoter sequence to investigate the transcriptional regulation of renin in real-time. The expression vector was injected into fertilised WIK zebrafish eggs and successful integration of the plasmid into the zebrafish genome was demonstrated. An in vivo assay was designed to select for highest luciferase expressers, but bioluminescent imaging revealed that the high signal stemmed from the yolk sac of zebrafish. Yolk sac expression appeared to originate from episomal transcription of the injected plasmid and, in the F0 fish, masked potential bona fide expression from the renin-expressing cells of the pronephros. A new transgene was generated using two reporters, driven by the same promoter sequence and the two reporters were separated by a '2A' sequence which codes for a self-cleaving peptide. This zebrafish line was generated to permit selection of tg(ren:LUC-2A-mCherry) fish for the strongest mCherry signal originating from renin expressing cells. However, no expression was observed at the anterior mesenteric artery (AMA). Further investigation revealed that when generating novel transgenic zebrafish, the reporter proteins are transiently expressed in the yolk sac of F0 founder fish and the ectopic expression can influence studies relying on quantitative reporters. Current studies largely rely on the transcriptional activity of RAS components and are restricted to timepoint-specific observations. The lack of antibodies prevents the measurement of RAS proteins in zebrafish. The amino acid sequence for zebrafish angiotensinogen is known and by comparison to mammalian sequences, I identified the amino acid sequences for angiotensin (Ang) I and II. The production of zebrafish Ang I and II peptides by solid phase peptide synthesis provided standards for the development of assays for zebrafish angiotensins. I used these assays to demonstrate that Captopril, which in mammals prevents the conversion of Ang I to Ang II by inhibiting the Angiotensin Converting Enzyme (ACE), is active in zebrafish, and that its administration leads to a dramatic decrease in Ang II. In parallel I designed and synthesised a fluorescent resonant energy transfer (FRET) probe to enable zebrafish renin activity to be measured. Using various zebrafish models I was able to demonstrate dynamic changes in renin activity and measure these using the first renin zebrafish FRET probe. I characterised a renin knockout zebrafish, generated by CrispRCas9 gene editing. Knockouts were identified by DNA sequencing which identified an 8bp deletion in exon 2 of the renin gene, causing a frameshift mutation and early termination of renin translation. Renin knockout fish proved to be viable and were screened for a phenotype using the high throughput automated screening system (VAST) which permits the precise imaging of a large number of live fish. Imaging revealed delayed development of the swim bladder and reduced fish length compared to age-matched controls. This was indicative of an overall developmental delay. The knockout fish were intercrossed with existing renal reporter lines to investigate phenotypes at a cellular level. Ren−/− tg(wt1b:EGFP) fish showed a delay in glomerular fusion of the pronephric kidney and a cross of the ren−/− with tg(ren:RFP-LifeAct) fish indicated a dramatic increase in renin-expressing cell along the renal mesonephric vasculature. Moreover, morphological examination revealed vacuolation of proximal tubules in the mesonephric kidney. The intercrossing of the ren−/− zebrafish to the tg(ren:RFP-LifeAct) and tg(acta2:EGFP) fluorescent reporters lead to the optimisation of a FAC sorting protocol and resulted in the first zebrafish ren- and acta2-expressing cells to be cultured and imaged using high-resolution microscopy imaging. In summary, my work has led to the first successful measurement of zebrafish AngI and AngII in mesonephric zebrafish tissue to demonstrate the effectiveness of Captopril in zebrafish as well as the first development of a zebrafish renin FRET probe to allow the measurement and quantification of renin activity in zebrafish, enabling more accurate studies of the zebrafish RAS. Lastly, successful phenotypic characterisation of the ren−/− fish will further our understanding of the role of renin and the RAS in zebrafish.

In both thrombosis and haemostasis, two distinct populations of activated platelets form, pro-aggregatory and pro-coagulant platelets. Pro-aggregatory platelets have active integrins and aggregate together via fibrinogen to form the haemostatic plug which prevents bleeding. Pro-coagulant platelets expose phosphatidylserine (PS) which acts as a surface for binding of the tenase (FIXa/VIIIa) and prothrombinase (FXa/Va) complexes that generate a burst of thrombin production responsible for occlusive clot formation. All current anti-platelet drugs are associated with bleeding side effects since they inhibit pro-aggregatory platelet activity. No drug exists which selectively targets the pro-coagulant platelet population, though this could be an alternative anti-thrombotic approach associated with fewer bleeding side effects if pro-aggregatory activity remains. In order to test such an approach, a selective inhibitor of pro-coagulant platelet activity must be developed. Critical to platelet PS exposure is activity of the main platelet scramblase, TMEM16F. This thesis aimed to develop inhibitors of TMEM16F and platelet scramblase activity. R5421 was the only previously described inhibitor of platelet scramblase activity. R5421, however, is shown to be a non-selective inhibitor of platelet scramblase activity, also affecting platelet pro-aggregatory activity by inhibiting αIIbβ3 integrin activation, α-granule release and cytosolic Ca2+ signalling in response to a range of agonists. Ligand-based in silico methods were applied to identify new inhibitors of platelet scramblase activity. The pesticide thiodicarb was identified as a novel inhibitor of platelet scramblase activity but also acts non-selectively. Since R5421 has wide ranging off-target effects and attempts to improve its selectivity were unsuccessful, other lead compounds were pursued. The polyphenolic compounds tannic acid and epigallocatechin gallate (EGCG') inhibit TMEM16F activity when over-expressed in cell lines. Both tannic acid and EGCG' are shown to inhibit platelet scramblase activity. An empirical screen of polyphenolic compounds defined an emerging pharmacophore for inhibition of platelet scramblase activity. All catechin gallate compounds inhibited platelet scramblase activity non-selectively. Structure-based in silico methods were applied to identify new inhibitors of platelet scramblase activity, first using the fungal nhTMEM16 structure and later the mTMEM16F structure. These screens identified novel inhibitors of platelet scramblase activity which do not affect platelet αIIbβ3 integrin activation, α-granule release or cytosolic Ca2+ signalling. Further work is required to confirm whether these compounds act directly on TMEM16F or inhibit platelet scramblase activity through a different mechanism.

Research in the past 15 years has shown that the adult heart, including human, contains cardiac fibroblasts exhibiting regenerative capacity. Little is known about the precise characteristics, properties, and differentiation potential of endogenous cardiac fibroblasts (CF) in the four cardiac chambers of the human heart, and how these are affected by the disease process. The fibroblast population in the heart is being redefined from an inert scar-forming cell type into one, which has an active role in terms of paracrine effects and cardiomyocyte coupling. Fibroblast characterisation shows overlap with the variously described mesenchymal stem cell populations, and these cells can be reprogrammed directly to cardiomyocytes. 3D engineered heart tissue (EHT) is made using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSC-CM). The addition of human foetal cardiac fibroblasts to the EHTs provided structural support, enhanced force output, improved calcium handling and induced expression of more mature sarcomeric proteins. In this thesis, for the first time the human CFs from all four chambers in patients with preserved biventricular function are investigated. A comparison is made between all four chambers and between left and right side of the heart. Fibroblasts identified by a range of markers, are characterised by growth pattern, rate of proliferative, secreted factors and expression of cardiogenic transcription factors. Moreover, the effect of chamber-specific fibroblasts on the function and gene expression of the iPSC-CM in EHT is studied. Finally, for the first time the secretory profile of EHT constructs is elucidated by the proteomic analysis of the medium bathing EHTs. This project aims to investigate the mechanism of this effect regarding cell coupling and paracrine effectors. The in-vitro gorwth of a truely defined human all four-chamber cardiac fibroblast population depicted that atrial fibroblasts have a better rate of proliferation, growth pattern and lesser predilection for myofriboblastic transformation as compasred to ventricular fibroblasts. The trancription factor expression of atrial fibroblasts is different from ventricular fibroblasts hence pointing towards their different embryological origin. The co-expression of chamber-specific fibroblasts in EHTs with cardiomyocytes of same origin showed that rigth ventricular fibroblasts based EHTs had the best kinetics. Finally the proteomic expression of EHTs is mainly dependent on presence of cardiomyocytes. The right ventricular fibroblasts based EHTs showed least amount of proteins expression in conjuction with best kinetics eluding to the fact that in-vivo many proteins expressed by cardiomyocytes and non-cardiomyocytes might have an inhibitory effect. This thesis provides an insight into phenotypic variablity, transcription factor expression, impact on kinetics and protein co-epxression in EHTs of chamber-specific fibroblasts with cardiomyocytes. Further investigations into the role of chamber-specific fibroblasts in EHTs in patients with impaired ventricular function can be explored subsequently.

Central (aortic) haemodynamics change markedly with age, as aortic stiffness and systolic pressure augmentation increase thereby elevating aortic blood pressure. Furthermore, the autonomic regulation of vascular sympathetic activity also changes; whereby the level of sympathetic nerve activity to the skeletal muscle vasculature, termed muscle sympathetic nerve activity (MSNA), increases, which can elevate peripheral blood pressure. The increase in blood pressure with age increases cardiovascular risk. Notably, exercise training is one recommended strategy to reduce this elevated risk. Although habitual endurance exercise is well known to elicit a range of physiological adaptations, the effects on aortic haemodynamics and peripheral blood pressure regulation are not well described. Therefore, the primary aim of this thesis was to investigate the effects of chronic habitual endurance exercise on aortic (central) haemodynamics and the autonomic regulation and neural control of brachial (peripheral) blood pressure in healthy ageing. To achieve this aim, normotensive middle-aged and young men were recruited into separate groups of chronically endurance-trained runners or recreationally-active nonrunners. Young men were studied to determine the effects of habitual exercise independently of age. Collectively, this thesis shows that in middle-aged men habitual exercise has no significant effects on aortic haemodynamics or the ability of MSNA to effect vascular responses during sympathoexcitation. However, the main finding from this thesis is that habitual endurance exercise, until middle-age, alters the autonomic regulation of MSNA at rest. Specifically, for a similar blood pressure, the arterial baroreflex is set higher in runners, which is associated with a greater likelihood of MSNA burst occurrence. However, there were no between-group differences in young men. Therefore, this greater likelihood of MSNA burst occurrence in middle-aged male runners may represent a fundamental physiological adaptation to chronic (~30 years) habitual endurance exercise to support resting peripheral blood pressure in an expanded cardiovascular system with greater reserve.

A 7 amino acid (7A, MHSPGAD) peptide was found alternatively translated from one of the transcript variants of mouse histone deacetylase 7 (Hdac7) mRNA. In this study, phosphorylated 7A (7Ap, MH[pS]PGAD) was able to increase Sca1+ vascular progenitor cells (VPCs) self-renewal with upregulation of E2F2 and cyclin D1 proteins and also improve angiogenesis in ischemic tissues in in vivo studies. A mutated 7Ap (7Am) was then designed to resist multiple proteases digestion in in vivo studies, retaining 7Ap’s cellular functions on cell migration and cell self-renewal. 7Am was also found to be able to stabilize glucose level in db/db mice model, together with evidence in in vitro studies that 7Ap could increase insulin secretion and glucose transporter 4 expression. Furthermore, 7A/7Ap could interact with HK1 and PKM2 increasing HK1’s activity in glycolysis as well as increasing both HK1 and PKM2 nuclear translocation related to their functions on gene transcription regulation. 7A was also found to possibly involved in protein glycosylation. In order to further understand the function of 7A in vivo, an Hd7-7sFLAG transgenic mice was generated, which could act as 7A knockout mice model, and it was confirmed that endogenous 7A existed. To further validate 7A’s role in vascular remodelling, a femoral artery injury model was introduced in the transgenic mice. Compared to wild type mice, the transgenic mice showed severe retardation of vascular injury repair, which was partially rescued by exogenous 7A supply. The 7A deficiency also abolished angiogenesis in ischemic tissues in a hindlimb ischemia model, leading to complete limb loss and death, which could also be rescued by exogenous 7A supply. Interestingly, 7A deficiency in those transgenic mice resulted in increased body weight gain under normal diet and increased amount of fat tissue, and this was ablated by administration of 7Am in male transgenic mice. We also discovered increased CD45+ cells from Sca1+ VPC population in transgenic mice, and those CD45+/Sca1+ cells may contribute to the body weight gain.

The aphorism “fat burns in the flame of carbohydrate” leads to the question of whether the oxidation of the ketone body, D-β-hydroxybutyrate (βHB), is facilitated by anaplerosis via glycogen and/or glucogenic amino acids. The work in this thesis aimed to investigate the importance of anaplerosis in ketone body metabolism in the isolated heart. The first study (Chapter 3) determined whether glycolysis from glycogen affected the heart’s ability to oxidise βHB. The glycogen content of isolated rat hearts was altered by perfusion with or without glucose, before perfusing with Krebs-Henseleit (KH) buffer containing [14C]-βHB or [5-3H]-glucose to measure βHB oxidation and glycolytic rates, respectively. βHB oxidation in hearts with low glycogen (LG) was 2-fold lower than those with high glycogen (HG), and βHB oxidation rates directly correlated with glycogen content. βHB oxidation decreased glycolytic rates to ~0.2 μmol. gww-1.min-1 in all hearts, redirecting glucose into glycogen in LG heart, but not in HG hearts. βHB alone or with glucose increased the Krebs cycle intermediates, citrate, 2-oxoglutarate and succinate, and the total nicotinamide adenine dinucleotide phosphate (NADP/H) pool measured using metabolomics. Thus, glycogen is required for βHB oxidation. The second study (Chapter 4) determined the effect of βHB oxidation on cardiac energetics using 31P-NMR spectroscopy. In HG hearts, βHB oxidation increased the free energy of ATP hydrolysis (∆GATP) by increasing the concentration of phosphocreatine ([PCr]) and the phosphorylation potential, calculated from the [ATP]/[ADP][Pi] ratio. βHB oxidation was unable to increase the low ∆GATP in LG hearts due to low glycolytic flux through pyruvate. The third study (Chapter 5) defined the effect of the glucogenic amino acids, asparagine, valine and glutamine, on βHB oxidation in hearts with decreased flux through pyruvate to oxaloacetate (glycogen-depleted and perfused with low glucose buffer). Asparagine increased βHB oxidation 2-fold by increasing the metabolites of the malate-aspartate shuttle and purine nucleotide cycle, aspartate, fumarate and guanosine triphosphate. Neither valine nor glutamine increased βHB oxidation rates. The final study (Chapter 6) determined the effect of substrates and loading conditions on cardiac efficiency (the ratio between hydraulic work and oxygen consumption) in working rat hearts. Fatty acids were found to decrease cardiac efficiency, via increased oxygen consumption and decreased hydraulic work, at both low and high pre- and afterloads. At high, but not at low, pre- and after-loads, βHB plus acetoacetate (AcAc) increased efficiency by increasing hydraulic work. Thus, βHB plus AcAc increased cardiac efficiency more than glucose alone or with fatty acids at high pre- and after-loads. In conclusion, βHB oxidation and glycolysis from glycogen have a synergistic relationship in heart, which serves to increase the ∆GATP and thereby cardiac efficiency.

Cyclooxygenase (COX), which can be expressed as COX-1 or COX-2 in endothelial cells has the unique ability to regulate microvascular tone through balanced production of dilator/constrictor prostanoids. This study investigated the roles of these isoforms in microvascular endothelial dilator function and how these are affected by supplement-derived ecdysteroids. Acetylcholine or 20-hydroxyecdysone relaxation were recorded in Skeletal muscle (SKM) and mesenteric (ME) arteries from healthy sheep and omental (OM) and subcutaneous (SC) fat arteries from obese humans by wire myography in the absence and presence of inhibitors of nitric oxide synthase, cyclooxygenase (COX) isoforms 1 and 2 and endothelium-derived hyperpolarizing factors. Gene and protein expression analysis were also carried out to fully characterize the roles of COX in these arteries. Non-selective COX inhibition attenuated acetylcholine relaxation in SKM arteries but enhanced it in ME arteries. Selective inhibition of COX-1 in both SKM and ME arteries also attenuated acetylcholine relaxation. In contrast, selective inhibition of COX-2 enhanced acetylcholine relaxation in ME arteries and had no effect in SKM arteries. In OM arteries from obese patients, selective inhibition of COX-1 but not COX-2 significantly improved acetylcholine relaxation. The OM arteries also displayed enhanced responsiveness to thromboxane A2 mimetic (U46619) compared with SC arteries. 20-hydroxyecdysone caused relaxation which was attenuated by NOS inhibition compared with COX inhibition in SKM and ME arteries. COX roles in microvascular endothelial dilator function are isoform-specific and dependent on type and health of the vasculature. In healthy arteries, COX-1 promotes but COX-2 opposes vasodilation. In human obesity, COX-1 opposes while COX plays no part in OM endothelial dilator function. Although 20-hydroxyecdysone alters COX expression, its vasodilatory effect is more eNOS-dependent than COX-dependent.

Optimal aortic valve function, limitation of blood damage, and frequency of thromboembolic events are all dependent upon the haemodynamics within the aortic root. Improved understanding of the young healthy physiological state via investigation of the fluid dynamics around and through the aortic valve is essential to identify detrimental changes leading to pathologies and develop novel therapeutic procedures. The aim of this study is to develop a numerical model that can support a better comprehension of the valve function and serve as a reference to identify the changes produced by specific pathologies and treatments. A Fluid-structure interaction (FSI) numerical model was developed and adapted to accurately replicate the conditions of a previous in vitro investigation into aortic valve dynamics, performed by means of particle image velocimetry (PIV). The model was validated on equivalent physical settings, in a pulse duplicator replicating the physiological healthy flow and pressure experienced in the left heart chambers. The resulting velocity fields and hydrodynamic valve performance indicators of the two analyses were qualitatively and quantitatively compared to validate the numerical model. The validated FSI model was then used to describe realistic young healthy, ageing and post treatment conditions, by eliminating the experimental and methodological limitations and approximations. In detail, in terms of treatments, both surgical and transcatheter valve replacement procedures were investigated. In terms of pathologies, typical alterations frequently due to ageing, namely thickening of the valve leaflets and progressive dilation of the aortic chamber, were studied. The analysis was performed by comparing the data obtained for the ageing and post treatment configurations with those of the young healthy root environment. The results were analysed in terms of leaflets kinematics, flow dynamics, pressure and valve performance parameters. The study suggests a new operating mechanism for the young healthy aortic valve leaflets considerably different from what reported in the literature to date and largely more efficient in terms of hydrodynamic performance.

Integrins are one of the major proteins that cells utilise to adhere to their extracellular matrix (ECM). Although integrins do not possess any catalytic capacity themselves, they facilitate and orchestrate the assembly of mechanically sensitive signalling hubs which provide essential cues to guide and inform cell behaviour. Within the heart, integrins are found in specialised adhesion structures called costameres which provide a physical link between the contractile sarcomere and the ECM. Via proteins such as vinculin and talin, integrin signalling has been reported to play a key role in developmental and diseased processes such as rigidity sensing and hypertrophy. Importantly, cardiomyocyte integrin expression is tightly regulated during development and diseased states where fibronectin binding integrin subtypes become dominant, whereas laminin binding subtypes are predominant in the healthy adult heart. Currently, the implications of this dynamic integrin expression profile for integrin signalling and cardiomyocyte behaviour is not well understood. Thus the aim of this thesis was to conduct a comparative investigation of cardiomyocyte spreading, morphology and contractility when cultured on fibronectin or laminin ligands. Initial investigations utilising PDMS substrates, nanopillar arrays and ligand micropatterning revealed that cardiomyocyte cultured on fibronectin exhibit greater spreading, contractility and vinculin enrichment than cells cultured on laminin. From these results, it was hypothesised that changes in integrin subtype expression may result in differential integrin signalling pathways. To understand the underpinning molecular mechanisms of these observations, we sought to investigate integrin clustering via the implementation of a DNA origami ligand nanopatterning platform. The results revealed key differences in the integrin clustering dynamics of fibronectin and laminin binding subtypes. This data provide new insights into the molecular mechanisms of integrin signalling in cardiomyocytes and advances our understanding of their role in development and disease.

Platelets are essential for the physiological process of haemostasis, but also drive pathological thrombosis. Platelet lifespan is a tightly controlled process through which platelets exist for approximately 10 days within the circulation of healthy individuals. However, in a number of disease states this process is dysregulated leading to an accelerated platelet turnover. Indeed, there are a number of reports suggesting that newly formed platelets are hyper-reactive and their presence has been associated with a higher risk of thrombosis. Whilst there are these indications of hyper-reactivity in young platelets, there are few systematic studies. Here I have used proteomics coupled with functional studies and immunofluorescence to show that there is a progressive decline in mitochondrial and cytoskeletal proteins as platelets age and an increase in apoptotic pathways. Given the apparent importance of mitochondria in supporting the predetermined platelet lifespan, it raised the question as to whether mitochondria are important for other platelet functional processes. Therefore, I sought to elucidate the impact of platelet activation on mitochondrial function and dynamics. Physiological stimulation causes an increase in mitochondrial respiration, consistent with an increase in energy demand. Interestingly, P2Y12 receptor inhibition causes a reduction in basal oxygen consumption, suggesting a dysregulation in mitochondrial function. Furthermore, this work highlights a role for mitochondria beyond energy production, with indications that stimulation causes platelets to package and release their mitochondria into microvesicles. Interestingly, these mitochondria-containing microvesicles have high P-selectin expression suggesting they may be more likely to interact with neutrophils than the rest of the microvesicle population. Indeed, incubation of neutrophils with mitochondria-positive microvesicles but not mitochondria-negative microvesicles causes alterations in the expression of surface markers; CD11b, CD66b and CXCR2, indicative of neutrophil activation potentially as a result of phagocytosis. This work highlights an important role of mitochondria in both platelet ageing and activation.

Nanoparticles are emerging drug delivery platforms for improved stability and bioavailability of drugs and vasoprotective nutraceutical compounds, such as resveratrol (RV) for the treatment of cardiovascular disease (CVD). Hypertension is a significant contributor to CVD, and while its pathophysiology is unclear, oxidative stress is thought to be a key contributor. It is evident that there is a growing need for treatment strategies to prevent future cardiovascular events. This project aimed to develop a potential therapeutic tool for the treatment of CVDs associated with oxidative stress. Nanoparticles (inorganic and organic) were synthesised and characterised using a range of chemianalytical techniques and their potential for the delivery of nutraceutical compounds assessed. The effects of the drug-loaded nanoparticles were assessed using human coronary artery endothelial cells (HCAECs) in vitro and using a developed model of acute hypertension in isolated coronary and cerebral vessels to replicate an oxidative environment. Silica nanoparticles (SiNPs) functionalised with cerium oxide (CeO2) reduced SiNP surface reactivity and demonstrated antioxidant capacity, improving biocompatibility in vitro. Uptake of novel RV-loaded NLCs (RV-NLCs) by HCAECs maintained their viability and reduced both mitochondrial and cytosolic superoxide levels; vessel incubation in RV-NLCs restored the magnitude of dilation via NO following acute pressure elevation, mediated via AMPK in the coronary artery. In contrast, organic nanoparticles (RV-NLCs and TMSliposomes) were incapable of restoring elevated pressure induced attenuated dilation in cerebral arteries, suggesting alternate mechanisms of impairment. Findings from the present study support the use of nanoparticles for the treatment of CVD, whereby they offer improved biocompatibility, potency and sustained drug release into the vasculature; whilst also highlighting the tissue-specific variability in treatment responses, hence a need for in-depth, comparative studies in the development of novel clinical solutions.

Introduction Hyperosmolarity is a concomitant factor to the hyperglycaemia observed in diabetes mellitus, a disease known to affect the function of endothelial cells and cause vascular damage. Previous studies have found that hyperglycaemia is capable of reducing the viability of endothelial cells and altering the activity of store-operated Ca2+ entry (SOCE). This study aims to determine the role of hyperosmolarity on the endothelial function and the activity of SOCE and Orai channels. Methods In this study, hyperosmolarity was simulated (in vitro) via the addition of mannitol at clinically relevant concentrations (19.5, 30 & 60 mM). Physical & biochemical assays were employed to determine the effect this stimulus had on endothelial function. Ca2+ imaging was employed to measure the effect of hyperosmolarity on Orai channel activity and the role of Reactive Oxygen Species was investigated with the use of biochemical dyes (H2DCFDA. Mitosox & Amplex red). Results Hyperosmolarity reduced endothelial cell migration (41% fewer migrated cells compared to control P=<0.0001) and increased the number of dead cells per field (160% increase from control P=<0.0001). SOCE was reduced under hyperosmolar conditions in human aortic endothelial cells and endothelial cell line (EA.hy926). SOCE in primary endothelial cells was found to be mediated by Orai channels, which was demonstrated by transfection of Orai siRNAs. All three types of Orai channels (Orai1-3) were sensitive to hyperosmolarity in the HEK293 cells overexpressing STIM1/Orai with all three subtypes showing a >50% reduction in peak influx compared with untreated cells (P=<0.05 for all groups). Cytosolic reactive oxygen species (ROS) and mitochondrial ROS were increased by hyperosmolarity. Hydrogen peroxide was capable of reducing SOCE in a concentration- dependent manner. Conclusion These results suggest that the reduced SOCE by hyperosmolarity may be mediated by an increase in intracellular ROS concentration. The findings in this study suggest that store- operated Orai channels regulated by hyperosmolarity may act as a new underlying mechanism for causing diabetic vascular injury, which allows further therapeutic exploration for diabetic complications.

Von Willebrand Factor and Factor XII have well established roles in haemostasis and coagulation. While VWF is a mediator of platelet capture to sites of vascular injury under high shear stress, FXII initiates the intrinsic pathway of coagulation. Interestingly, multiple studies have shown that absence or inhibition of FXII does not impair haemostatic function but has a thrombo-protective effect, rendering it a potentially safe anticoagulant target. In this study, I investigated a novel binding interaction between VWF and FXII. Using substrate based activation assays, I showed that VWF is a potent and previously unrecognised activator of FXII. Additionally, I demonstrated that both inactive FXII and activated FXII bind to VWF but not to VWF lacking the A1 domain suggesting that the FXII binding site lies within the A1 domain. By carrying out further binding studies using static binding assays and surface plasmon resonance with isolated and full-length VWF A1 domain mutants, I identified amino acids E1452 and D1459 on the α-6 helix of the A1 domain as the FXII binding site in VWF. The physiological relevance of the VWF/FXII interaction was examined using plate-based thrombolysis as well as perfusion assays investigating the formation of platelet rich blood clots under conditions of flow. Using adapted thrombolysis assays, I showed that clot lysis times were significantly reduced when either VWF, FXIIa or activity of both proteins was blocked. Furthermore, I demonstrated that the interaction between VWF and FXIIa facilitates VWF mediated platelet capture under conditions of high shear and that interference with FXII/VWF binding has a prohibitive effect on overall thrombus formation. Based on these results, I hypothesise binding of VWF to FXII is an important component of thrombus formation and inhibition of this interaction might be a novel and safe way to prevent thrombosis without increasing the risk of bleeding side effects.

The majority of data available for drug predictive safety and disease/polymorphism modelling are based on results from genetic association studies or from models other than humans, which can lead to conflicting mechanistic explanations. This thesis considers whether human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) could complement existing assays to improve drug safety screening and disease/polymorphism modelling by assessing the impact of drug- and gene-induced changes on cell function. To evaluate the suitability of hPSC-CMs for drug safety screening, changes in contractility and electrophysiology were quantitated. Protocols for the CellOPTIQ:hiPSC-CMs combination were established and unified via a drug ‘training’ set, allowing for the blinded evaluation of up to 27 drugs with known positive, negative or neutral inotropic effects. This demonstrated that the accuracy of CellOPTIQ:hiPSC-CMs configuration ranged from 50 to 60%, which was lower than might have been expected from a human cell-based system. Nevertheless, contraction and relaxation time appeared to be informative for positive inotropes, while contraction amplitude was informative for negative inotropes. Moreover, accuracy was further improved and increased to 75% (for CellOPTIQ) by refining the conditions that would reduce beat rate, increasing the signal to noise ration and enabling chronic long-term drug testing. The results show that hiPSC-CMs could be used to reduce socioeconomics cost and assist regulatory guidelines governing cardiac safety assessments. To evaluate whether hPSC-CMs could be used to understand the gene-induced changes and influence of polymorphic variation in the genome on cell behaviour, the ADRB2 locus (encodes βeta2-adrenergic receptor; β2AR) was used as an exemplar. This is because the action of beta-blockers may be influenced by polymorphisms within the β2AR, particularly at amino acid positions 16 and 27, and there is a potential association with cardiovascular disorders such as heart failure. To study β2AR polymorphism differences against a constant genetic background, CRISPR/Cas9 technology was used to genome-edit the HUES7 hESC line to produce 4 isogenic β2AR variants (single amino acid codes: GE, GQ, RE, RQ). The isogenic variants were able to retain their pluripotency and differentiation capabilities into hPSC-CMs whilst retaining responsiveness to β2AR pharmacology. For the functional assessment, an initial phenotypic analysis was carried out by luciferase-based real-time cAMP (cyclic adenosine monophosphates) assay to estimate the differential response during acute and chronic agonist exposure to individual β2AR variants. It was found that RQ has the lower initial β2AR density, GQ has the highest receptor desensitisation, Arg16 was resistant to desensitisation and GQ tended towards being the most downregulated. Since β2AR is regulated by multiple complex processes and their regulation has essential effects on signal transduction, it is expected that these isogenic models will help to understand the mechanisms that underlie ‘disease’ and drug- and gene-induced states. Moreover, the work shows how coupling hPSC-CMs with CRISPR/Cas9 technologies can be used to personalise diagnosis and identify new pathogenic variants for cardiovascular disease. Thus, this thesis shows there is value in exploring the use of hPSC-CMs in assessing the impact of drug-induced changes on cell function, and provide a tool to gain mechanistic insights into how polymorphisms in the genome alter signalling cascades.

The cost of developing new drugs has increased by ~100 fold in recent decades, largely due to high attrition rate in clinical development caused by cardiotoxicity. Since the recent discovery of human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs), it has been developed as a novel, promising in vitro research tool in preclinical testing of drug-induced cardiotoxicity. However, major limitation of currently available hiPSC-CMs is their immature phenotype, which can hinder evaluation of contractile dysfunction. Existing contractility monitoring on hiPSC-CMs is currently being done at low throughput or by using surrogate markers (e.g. impedance). In addition, there has been no detailed cross-site validation study to evaluate the effect of drugs on hiPSC-CMs contractility. A multinational consortium was established with the University of Nottingham as the academic lead, in order to address the CRACK-IT InPulse Challenge. The Challenge consisted of two phases that lasted for 3.5 years. This project was part of the consortium aimed to develop medium throughput technology platform that could measure contractility in hiPSC-CMs as a physiologically-relevant functional output for use in preclinical drug safety evaluation. The University of Nottingham utilised CellOPTIQ® platform, a medium-high throughput screening platform to measure contractility and electrophysiology. Protocols were unified across all academic partners via a drug training set in phase 1, allowing subsequent blinded multi-centre evaluation of drugs with known positive, negative, or neutral inotropic effects in phase 2. Accuracy ranged from 44% to 85% across the platform-cell configurations. Refinement to test conditions was addressed after blinded testing, which resulted in increased accuracy to 85% for 2D monolayers and 93% for 3D EHT system. Through this study, hiPSC-CMs cultured in 2D and 3D platform have been shown to considerably have a value in predictive safety pharmacology despite their immaturity status and current technology evolution.

Calcitonin gene-related peptide (CGRP) is primarily localised and released from sensory nerves. It is a potent vasodilator neuropeptide with cardioprotective effects. The most common cardiovascular-relevant form is αCGRP. It signals via the CGRP receptor which is composed of the G-protein linked calcitonin like receptor (CLR) and an associated receptor associated modified protein (RAMP1); both of which are essential for function. There remains an unmet need in the treatment of cardiovascular disease and we hypothesised that CGRP acts via the CGRP receptor in an endothelial-independent manner to mediate its vasodilator and protective effects in vivo. The aim was to investigate the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME)-induced hypertension model in adult C57BL/6 wild-type (WT) and global αCGRP knock-out (KO) mice, which have similar baseline haemodynamics. The results showed that: i) αCGRP KO mice develop exacerbated hypertension in the presence of L-NAME, compared with WT mice and this was associated with adverse cardiovascular remodelling. This indicates that CGRP acts, at least in part, independently of endothelial-derived nitric oxide. ii) use of BIBN 4096 BS, a selective CGRP receptor antagonist supported these results, demonstrating the essential role of the CGRP receptor. No evidence was found for the involvement of a second proposed CGRP receptor (CTR/RAMP1). iii) RAMP1, but not CLR protein, from the CGRP receptor complex, was upregulated with L-NAME treatment. iv) αCGRP infusion reversed L-NAME-induced hypertension in αCGRP KO mice to the normotensive range. v) studies into the vascular site of CGRP’s protective action indicated that the mesenteric vessels, rather than the skin, play an important role in regulating the protective effect of CGRP; specifically, in first order mesenteric arterioles. This study in mice reveals that the therapeutic benefits of αCGRP extends to situations where endothelial dysfunction associated with reduced nitric oxide production exists.

The main aim of this thesis is to characterise sympathetic neural activity and autonomic regulation of BP at high altitude, in Lowlanders during acclimatisation, and in highland native populations, who have adapted to high altitude hypoxia over generations. To address this aim, resting sympathetic neural activity to the skeletal muscle vasculature (MSNA) was assessed, via microneurography, and baroreflex control of MSNA (vascular sympathetic baroreflex) and baroreflex control of the heart (cardiovagal baroreflex) was assessed using the modified Oxford test. Experimental study 1 examines sympathetic neural activity and arterial baroreflex function in Lowlanders following 10–20 days at 5050 m and compares them to Nepalese Sherpa. Experimental study 2 examines sympathetic neural activity and arterial baroreflex function in Andean Quechua who have developed the maladaptation syndrome chronic mountain sickness (CMS), and compares them to healthy Andean Quechua. Experimental study 1 and 2 also examine the mechanistic contribution of the peripheral chemoreflex to sympathetic neural activity and arterial baroreflex function in both Lowlanders and highland native populations. Experimental study 3 aims to extend these findings by investigating the previously unexplored mechanistic role of pressure-sensitive receptors in the pulmonary arteries in Lowlanders at high altitude. The major findings of this thesis are 1) heightened sympathetic neural activity is a feature of high altitude exposure in both acclimatising Lowlanders and highland natives, compared to Lowlanders at low altitude. Nepalese Sherpa, however, appear to have adapted to favour lower basal sympathetic neural activity, compared to Lowlanders and Andean Quechua at high altitude. Thus, divergent pathways of physiological adaptation between highland populations extend to autonomic regulation of resting arterial pressure. Despite heightened sympathetic neural activity, the responsiveness of the sympathetic nervous system to acute fluctuations in BP (i.e. vascular sympathetic baroreflex gain) is well preserved at high altitude. The vascular sympathetic baroreflex is reset to a higher MSNA set-point in all populations, compared to Lowlanders at low altitude. Such resetting permits elevated basal sympathetic vasomotor activity that allows normal resting arterial pressure to be maintained in the face of hypoxic local vasodilation, and potentially altered vascular sensitivity. Despite maintained vascular sympathetic baroreflex gain, the responsiveness of the cardiovagal baroreflex is depressed in both Lowlanders and healthy highlanders at high altitude. This thesis also demonstrates that the peripheral chemoreflex does not play a major role in sympathoexcitation and vascular sympathetic baroreflex resetting at high altitude in either acclimatising Lowlanders or highland natives. Moreover, this thesis demonstrates, for the first time, that hypoxia induced elevations in pulmonary arterial pressure contribute to the sympathoexcitation and baroreflex resetting during acclimatisation to high altitude in Lowlanders.

The mouse is commonly used in cardiovascular disease research. Therefore, accurate and reliable measurement of blood pressure in conscious mice is essential. Blood pressure can be measured directly from a major systemic blood vessel using implantable devices (telemetry probes) or noninvasively from a peripheral site such as the tail. Telemetry allows continuous collection of data in freely moving mice, whereas the mice are restrained during the tail-cuff procedure. However major surgery and higher cost associated with telemetry result in the fact that the tail-cuff technique remains widely used. The tail-cuff technique is known to cause stress in the animals and therefore its reliability and validity are questioned. The hypothesis is that the handling technique may reduce stress associated with the tail cuff technique. The aim of this thesis was to perform a detailed analysis of different handling techniques and the stages of the tail-cuff technique in their impact on central blood pressure, heart rate and core body temperature using telemetry. I have revealed that all interventions caused significant increases in these parameters, with the restraint step inducing the largest change, which is maintained throughout the restraint period. These were not further exacerbated by increasing temperature or tail-cuff inflations. I saw no differences due to different handling techniques or the operator’s gender. Having compared the simultaneous recordings obtained by telemetry and the tail-cuff in the same mouse, I found that the tail-cuff system consistently produced lower readings than telemetry. However, the readings obtained by the tail-cuff were similar to those obtained by telemetry in non-disturbed mice on the same day in normal and hypertensive mice. The results show that the tail-cuff technique induces a stress response in mice, which is not alleviated by repeated exposure. Although these results support the use of the tail-cuff for monitoring blood pressure in the hypertension model, they also highlight the fact that measuring the blood pressure from the tail may not be representative of the systemic blood pressure. This is important new knowledge for researchers who use murine models of hypertension.

Vascular endothelial growth factor receptors (VEGFRs) regulate fundamental cellular responses such as cell proliferation, survival, and migration, and thus play key roles in angiogenesis and vascular physiology in higher eukaryotes such as vertebrates. The intracellular signaling pathways associated with VEGFRs have been well studied and characterised. However, there is a lack of understanding of their mechanism of activation. In this study, mammalian cell lines for tetracycline-inducible expression of VEGFR1, VEGFR2 and VEGFR3 were developed. A strategy was devised for expression, solubilisation and purification of VEGFR2. The first negative stain electron microscopy (neg stain-EM) observation of full-length VEGFR2 produced various negative stain class averages of VEGFR2 dimer bound to VEGF-A ligand. By biochemical and structural means, we also unraveled the process of differential activation of VEGFR2, its modulation on the cell membrane in multiple states against the previously reported ‘one structure’ model. Also implicated in cardiovascular disease, lectin-like low-density lipoprotein receptor-1 (LOX-1) is a member of the scavenger receptor family that binds to oxidized low-density lipoprotein (OxLDL). It is also proposed to activate downstream signal transduction that causes pro-atherogenic processes such as endothelial dysfunction, foam cell formation and apoptosis. LOX-1 does not recognize the native form of OxLDL, and the mechanism of ligand recognition by LOX-1 is unknown. Moreover, the structural changes brought about by LDL oxidation are also unknown. This study revealed that LOX-1 forms higher order multimers with OxLDL. Using cryo-electron microscopy (cryo-EM), the first 3D de novo model of the OxLDL particle was proposed. These studies also investigated the structural differences between native LDL (nLDL) and OxLDL, and identified that oxidation of LDL causes tighter packing of lipids and unravels hithero inaccessible binding domains of ApoB-100 (protein component) of the lipoprotein, which is recognized by LOX-1 receptor. This starts to provide some insight into a crucial event in the progression of atherosclerosis.

Background: Zebrafish are an excellent model to study cardiovascular development and disease. Transgenic reporter lines and state-of-the-art microscopy allow 3D visualization of the vasculature in vivo. Previous studies relied on subjective visual interpretation of vascular topology without objective quantification. Thus, there is the need to develop analysis approaches that model and quantify the zebrafish vasculature to understand the effect of development, genetic manipulation or drug treatment. Aim: To establish an image analysis pipeline to extract quantitative 3D parameters describing the shape and topology of the zebrafish vasculature, and examine how these are impacted during development, disease, and by chemicals. Methods: Experiments were performed in zebrafish embryos, conforming with UK Home Office regulations. Image acquisition of transgenic zebrafish was performed using a Z.1 Zeiss light-sheet fluorescence microscope. Pre-processing, enhancement, registration, segmentation, and quantification methods were developed and optimised using open-source software, Fiji (Fiji 1.51p; National Institutes of Health, Bethesda, USA). Results: Motion correction was successfully applied using Scale Invariant Feature Transform (SIFT), and vascular enhancement based on vessel tubularity (Sato filter) exceeded general filter outcomes. Following evaluation and optimisation of a variety of segmentation methods, intensity-based segmentation (Otsu thresholding) was found to deliver the most reliable segmentation, allowing 3D vascular volume measurement. Following successful segmentation of the cerebral vasculature, a workflow to quantify left-right intra-sample symmetry was developed, finding no difference from 2-to-5dpf. Next, the first vascular inter-sample registration using a manual landmark-based approach was developed and it was found that conjugate direction search allowed automatic inter-sample registration. This enabled extraction of age-specific regions of similarity and variability between different individual embryos from 2-to-5dpf. A workflow was developed to quantify vascular network length, branching points, diameter, and complexity, showing reductions in zebrafish without blood flow. Also, I discovered and characterised a previously undescribed endothelial cell membrane behaviour termed kugeln. Conclusion: A workflow that successfully extracts the zebrafish vasculature and enables detailed quantification of a wide variety of vascular parameters was developed.

Introduction: far infrared (FIR) is an invisible electromagnetic wave and a source of heat. It is involved in a range of biological effects which includes improvement in clinical outcomes in many pathological diseases especially in patients with chronic heart failure. FIR was also employed in animal and cellular models to further explore their effects and to conclude an understanding of its mechanistic pathways. However, the mechanisms behind the beneficial effects induced by FIR is still unclear. Therefore, this pilot study aims to investigate the effects of FIR on the vascular function in healthy subjects. This study will also explore some of the mechanisms including anti-oxidation and an endothelium-related gene that might be explained by FIR effects. Methods: in this thesis, 70 healthy, non-smoking volunteers (31.47 ± 11, mean ± SD) participated in this study. They received 4 sessions (40 minutes each) over 2 weeks of either FIR, heat or placebo. Vascular assessments were performed following two testing protocols. Protocol 1: at baseline, immediately after single session and 24-hour after the last session. Protocol 2: at baseline, 4-hours after single session and 4-hours after the last session. Participants were assigned blindly into 6 groups (n=10, except for the first two groups where n=15). Two groups received FIR to the back with two different testing protocols (protocol 1 or protocol 2), two groups received FIR to the forearm following testing protocol 1 and protocol 2, one group received heat and one placebo-receiving group. The latter two interventions were delivered to the back only following protocol 1. FIR was delivered using a FIR ray device, a heating pad set at 38°C was used for the heat group and for the placebo group a switched-off FIR device was used. Endothelial function was assessed using Laser Doppler imaging with the iontophoresis of an endothelium-dependent agent, acetylcholine (ACh) and endothelium-independent agent, sodium nitroprusside (SNP). Arterial stiffness was measured using pulse wave analysis to determine the augmentation index (AI@75%) using a Sphygmocor device. Blood was obtained to look at circulatory biomarkers including cytokines, adhesion molecules, and oxidative stress biomarkers. Furthermore, the expression of 9 genes regulated by the nuclear factor erythroid 2–related factor 2 (NRF2), NFE2L2 (gene name for NRF2), Kelch-like ECH-associated protein 1 (KEAP1) and endothelial nitric oxide synthase (NOS3) in peripheral blood mononuclear cells (PBMCs) was examined. Results: in comparison with baseline, the ACh response was significantly improved with FIR treated back following both protocols 1 and 2. There was no change seen after a single session; however, greater responses were seen after repeated FIR sessions to the back in protocols 1 and 2 relative to baseline (mean (SD), 374064(11086) to 458390(129347) p-value= 0.017, and 328951(105075) to 409808(101317) p-value=0.023, respectively). Responses to SNP remained unchanged in both protocols following FIR to the back. AI@75% significantly improved after one session of FIR compared to baseline following protocols 1 and 2 (mean (SD), 4.8(16.1) to 2.7(16.3), p-value= 0.025, and -2.3(13.2) to -7.13(13.5), p-value = 0.00, respectively) and further improvements were seen after the last session in protocols 1 and 2 (mean (SD) 4.8(16.1) to 1(17.4), p-value < 0.001 and -2.3(13.2) to -9.6(12.9), p-value < 0.001, respectively). FIR treated forearm (protocol 2) showed a significant reduction in AI@75% after the last FIR session compared to baseline (mean (SD), 3.2(19.8) to -0.8(19.0), p=0.02). For gene expression, NFE2LE did not change following FIR treatment to the back; KEAP1, however, was significantly increased after the last session of FIR to the back (protocol 1) compared to the baseline level. Significant expression of NOS3, as well as some NRF2 related genes including GCLM, TXNRD1, HSP70 and PRDX1 following FIR, treated back (protocol 2). Forearm group showed a significant increase in KEAP1 4 hours after the last session (protocol2). Apart from that, forearm group, heat and placebo groups showed no change in endothelial function, arterial stiffness, or gene expression. Circulatory biomarkers showed no change in all the assigned groups. Conclusion: FIR non-thermally improves vascular function in healthy subjects when repeatedly given possibly through the upregulation of NOS3 and NRF2, which might be a promising modality to promote vascular health.

Transverse axial tubules (TAT) and caveolae are essential structural microdomains in cardiomyocytes that recruit components of various signalling pathways. Of particular interest, β- adrenergic receptors (β-ARs) are localised to these structures and in response to catecholamines elicit compartmentalised cyclic adenosine 3′,5′-monophosphate (cAMP) signals. Both Popeye domain-containing (POPDC) and Caveolin-3 (CAV3) proteins are localised to these membrane compartments. POPDC1 is a CAV3-interacting protein, and the Popeye domain acts as a highaffinity cAMP binding site. The function of these proteins in atrial myocytes (AMs) remains elusive. This study has investigated whether AMs isolated from CAV3 and POPDC null mutants display an altered TAT structure and aberrant cAMP response. The TAT structure was investigated in AMs isolated from the left and right atria of CAV3-/- and Popdc1-/- mice. In both mutants, the TAT structure of AMs originating from the right atria were stronger affected than from the left. cAMP compartmentation was studied with the help of a transgenic FRET sensor. In response to β2-AR-stimulation, phosphodiesterase (PDE) 4 is critical compared to PDE3 for cAMP compartmentation. To understand how changes in TAT structure and cAMP signalling might alter atrial function, sinoatrial pacemaking and atrial conduction were studied in Popdc1-/- and Popdc2-/- isolated atrial tissues. Both mutants demonstrated abnormal pacemaker activity, associated with depressed sinoatrial pacemaking, enhanced ectopy and tachycardia-bradycardia arrhythmias increasing HR lability in response to β-AR stimulation. Additionally, Popdc1-/- displayed slower atrial conduction, increased fibrosis and downregulated connexin-43 expression. Along with the altered fast sodium current and the elevated late sodium current, these facilitated the development of atrial tachyarrhythmias in Popdc1-/- mutants. These data suggest that Cav3-/- and Popdc1-/- mutants are associated with structural changes resulting in aberrant cAMP compartmentation, which may result in an increased risk of developing atrial arrhythmogenesis.

Hydrogen sulphide (H2S), a toxic gas, has recently been shown to be generated endogenously by biological tissue and it has emerged as a novel and important biological mediator in the vascular system. In blood vessels, H2S has been shown to produce complex actions, including both vasorelaxation and vasoconstriction, through different mechanisms, depending on vessel origin and oxygen levels. This thesis examined the effects of H2S on blood vessels and examined the influence of oxygen concentration on vascular responsiveness and the mechanisms involved. This study also investigated whether H2S contributed to the hypoxic vascular response. Porcine splenic and pulmonary vessels and human chorionic blood vessels were set up in an isometric tension recording system, blood vessels were pre-contracted and responses to the H2S donor, NaHS, were produced in the presence and absence inhibitors, under 95% O2:5% CO2, 95% Air: 5% CO2 and 95% N2:5% CO2 gassing conditions. Western blot analysis was carried out to determine Cystathionine β synthase (CBS) and Cystathionine γ lyase (CSE) expression in porcine splenic and pulmonary arteries. Changing oxygen concentrations altered the nature of the response observed with NaHS. Generally, in both porcine splenic arteries (PSA) and porcine pulmonary veins (PPV), gassing with physiological levels of O2 (95% air: 5% CO2), was associated with NaHS causing a contractile response, while, gassing with sub-physiological levels (95% N2: 5% CO2), NaHS caused a relaxant response in PSA. The nitric oxide (NO) synthase inhibitor L-NAME, significantly abolished contractile response in PSA, indicating an interaction with NO. In porcine splenic veins (PSV) and porcine pulmonary arteries (PPA) under physiological gassing condition (95% N2: 5% CO2), NaHS-induced vasorelaxation was observed. The vasorelaxatory responses were abolished by L-NAME, showing that NaHS is activating NOS to generate NO and cause vasorelaxation. In human chorionic blood vessels, NaHS caused vasoconstriction under physiological gassing condition (95% N2: 5% CO2). L-NAME was without effect on the contraction, and so NO is not involved in this human blood vessel. Both CBS and CSE are expressed in porcine splenic and pulmonary arteries. L-cysteine induced a contractile response in PSA and PPA but this was not blocked by the H2S synthesis- enzymes inhibitors, indicating that L-cysteine-elicited contraction was not through a H2S-dependent mechanism. Hypoxia caused a biphasic response in the PSA; a transient contraction followed by prolonged relaxation. The relaxation response was attenuated by AOAA, suggesting that H2S participates in vascular responses to hypoxia with CBS the likely source of H2S production. Hypoxia-induced vasoconstriction was observed in both PSA and PPA. The vasocontractile responses were abolished by L-NAME, showing that hypoxia decreases the sustained release of NO from the vascular endothelium. In summary, these data show that O2 influenced H2S responses. At more physiological levels of O2 the predominant response is vasoconstriction, often mediated by H2S ‘mopping up’ endogenous NO. The predominant response in PSV and PPA was vasorelaxation, evoked by activating NOS to generate NO. Responses to hypoxia, in part, involve endogenous H2S generation, suggesting that H2S can act as an oxygen sensor in PSA.

The research conducted for this thesis was aimed at providing a comprehensive description of the dynamic and conformational landscape of the minimally thermostabilised avian β1-adrenergic receptor (tβ1AR) using solution state nuclear magnetic resonance (NMR) spectroscopy. The tβ1AR is a class A G-protein-coupled receptor (GPCR) and the human counterpart is an important drug target in diseases of the heart. In chapter 2, a wide range of constructs, labelling reagents and conditions were tested to make the tβ1AR accessible to 19F NMR spectroscopy. The conditions optimised in chapter 2 were used in chapter 3 to conduct comprehensive 19F NMR studies investigating the conformational plasticity of the cytoplasmic ends of transmembrane domains 6 and 7. Both domains have previously been proposed to be important structural hotspots for conformational changes during the activation of the receptor. The work described in chapter 3 tests the conformational response of both domains in response to a range of ligands and a Gs protein mimicking nanobody. The data obtained suggested that the cytoplasmic ends of the receptor sample through several inactive, pre-active and signalling active states and the populations of these states are tightly controlled by the Gs efficacies of ligands and the binding of intracellular binding partners. The results will help to explain how extracellular signals are conformationally transmitted through the receptor to the cytoplasmic face of the receptor. In chapter 4, I complemented the results obtained in the NMR spectroscopic studies of chapter 3 with structural data obtained through second harmonic generation (SHG) spectroscopy. In a final step in chapter 5, the knowledge gained from working with the tβ1AR has been applied to initiate an investigation of the conformational plasticity of a second GPCR target, the human Protease activated receptor 1 (PAR1). The PAR1 is mostly associated with regulation of haemostasis and as such a high potential target for clinical research. I established the construct, expression, purification and 19F labelling of the PAR1 and obtained initial 19F NMR spectra. The work is an important step towards understanding the PAR1 conformational landscape.

Neutrophils are major effectors of acute inflammation and microbial defence. Their infiltration and migration in injured tissue are critical for the inflammatory response. They are often observed navigating in a highly co-ordinated and directed manner leading to their aggregation at the target site of infection. This self-organised cell gathering is referred to as swarming. It is known that neutrophil swarming is driven by autocrine attractant production, notably of the lipid leukotriene B4 (LTB4). The decision to release attractants at the single-cell level is important and impacts the magnitude of the entire immune response. However, the precise mechanisms triggering this decision remain unclear. In this study, I employed in vivo imaging of zebrafish larvae to reveal the molecular processes that trigger the release of LTB4 and initiation of swarms. I developed a 2-Photon laser wound assay to elicit and visualise neutrophil swarming. A major limitation in previous studies of neutrophil swarms was the lack of tools to understand cell signalling dynamics during the response. To overcome this, I generated a new biosensor to probe for calcium levels in neutrophils as it correlates with the production of chemoattractants. Using this new tool, I revealed that neutrophils clustering at the target inflammatory site are experiencing sustained high calcium elevation. Using a new probe to follow the production dynamics of LTB4, I demonstrated that the rise of intracellular calcium promotes the biosynthesis of this key attractant. I further demonstrated that these calcium fluxes are triggered upon contact with necrotic tissue. This prompted me to interrogate the damage molecules driving these calcium signals in neutrophils. I discovered that the calcium fluxes were mediated by ATP that binds gated ion channels (P2X1) leading to rapid intracellular calcium uptake. Surprisingly, I found that live neutrophils can also trigger this calcium flux in other neutrophils upon mutual contact. Using chemical and genetic inhibition, I found that connexin-43 (Cx43) hemichannels, through their ability to release ATP, enable amplification of the calcium signal leading to chemoattractant production and subsequent neutrophil recruitment. I concluded that activation of LTB4 synthesis is a group decision reached via Cx43-dependent communication in pioneer clustering neutrophils. As Cx43 inhibition significantly reduced neutrophil aggregation at the target site, I investigated if this inhibition could have consequences for wound defence. For this, I developed a wound colonisation assay with Pseudomonas aeruginosa. Using this approach, I showed that Cx43 was crucial for the protection of wounds from opportunistic bacteria. Finally, I designed and generated an optogenetic tool to manipulate LTB4 biosynthesis dynamics in vivo. I demonstrated the effectiveness of this tool in immortalised cells and zebrafish larvae. The unique features of this tool make it very useful for a wide range of research applications on signalling dynamics. In conclusion, I have shown that by reinforcing damage signalling, Cx43 channels coordinate attractant biosynthesis in pioneer neutrophils. This generates an effective chemoattractant gradient source and promotes targeted aggregation and defence. This study, therefore, reveals a new mechanistic principle of collective behaviour that could be exploited in future pathological research.

The epicardium is a transcriptionally heterogeneous cell layer covering the heart, crucial to correct cardiovascular development. Following epithelial-to-mesenchymal transition (EMT), epicardial cells migrate into myocardium, form coronary smooth muscle cells and cardiac fibroblasts, and instruct cardiomyocytes to proliferate and mature. Adult mammalian epicardium is quiescent, but reactivates post-injury with limited effect. However, in zebrafish and in neonatal mouse, epicardial signalling enables robust cardiac regeneration after myocardial infarction. We hypothesise that manipulating human epicardial function could facilitate heart regeneration, via reactivation of embryonic processes. However, epicardial regulation remains incompletely understood; although understanding epicardial mechanisms could be key to potentially manipulating epicardium for therapeutic benefit. This PhD investigates a candidate transcription factor, Basonuclin 1 (BNC1), in functional regulation of human epicardial models, and identifies this gene as a potential key human epicardial regulator. Epicardial-like cells derived from human pluripotent stem cells (hPSC-epi) were previously used for single-cell RNA sequencing (scRNA-seq) in order to investigate possible human epicardial heterogeneity. This identified two distinct hPSC-epi subpopulations: one high in WT1 expression, the other high in TCF21. Bioinformatic analyses identified BNC1 as a potential key node in the hPSC-epi signalling network, via network inference modelling. BNC1 is a transcription factor known to regulate migration and proliferation in other epithelia. Given our network inference analyses and the literature evidence, I hypothesised that BNC1 would have functional relevance in human epicardium, so aimed to investigate its function in hPSC-epi differentiation and epicardial cell migration, as well as identify its putative epicardial targets. Firstly, scRNA-seq data describing hPSC-epi heterogeneity were validated in primary human foetal epicardium and BNC1 expression was confirmed in human epicardial models. BNC1 was subsequently investigated, both by siRNA-knockdown in hPSC-epi and foetal epicardial explants and inducible knockdown cell lines (siKD). siKD hPSC-epi had over 90% BNC1 reduction and displayed significantly altered expression of canonical epicardial genes WT1 and TCF21: hPSC-epi heterogeneity was thereby lost. Altered hPSC-epi proliferation and viability were also observed. siKD hPSC-epi was subsequently used in a simple epicardial EMT model (epi-EMT). siKD epi-EMT displayed impaired migration and pronounced cortical actin localisation. ChIP sequencing and bulk RNA sequencing identified potentially promising BNC1 targets, such as actin-binding protein supervillin, for future investigation. We conclude that BNC1 is a key functional epicardial regulator in vitro, paving the way for in vivo characterisation. The knowledge that manipulating BNC1 regulates epicardial heterogeneity and function may instruct efforts to harness epicardial potential for future therapeutic benefit.

Blood cells are crucial to human physiology, with functions in oxygen transport, infection control, and wound healing. Molecular mechanisms endogenous to blood cells have been implicated in the aetiologies of cancer, infection and inflammatory and immune disorders. The genetic determinants of blood cell function have not been comprehensively characterised, because it is too difficult to perform direct assays of cell function in large population samples. High-throughput flow cytometry can be used to measure functionally relevant phenotypes such as cell granulation, nucleic acid content, and cell size. Many of these phenotypes are important for the diagnosis of diseases such as sepsis, Szary disease, toxic granulation, and myelodysplastic syndromes, or correlate with assessments of cell morphology from blood smear images. Here, I report the results of my genome- wide association study of 63 previously genetically unstudied blood cell flow cytometry phenotypes. I have identified associated variants in loci containing genes coding for established drug targets with known roles in white cell function and immunity. I have colocalised the association signals with blood cell transcriptomic, blood proteomic, and disease risk, identifying possible causal roles for molecular mechanisms endogenous to white cells in the aetiology of a range of immune disorders, including atopic dermatitis, multiple sclerosis and celiac disease. My results have utility in drug design and therapeutic target selection, demonstrated by examples including the replication of the mechanism of action of Daclizumab, a treatment for multiple sclerosis, and evidence for the role of IL-18R1 in aetiology of celiac disease. Furthermore, mendelian randomisation analyses suggest a causal role for blood cell flow cytometry phenotypes in the aetiology of coronary artery disease, lung cancer, and asthma. In addition to my work on flow cytometry traits, I report a major contribution to the largest ever GWAS meta-analysis of routine clinical haematological phenotypes, including 563,085 individuals. I performed primary and conditional analyses, identifying parsimonious sets of independently associated variants. This is the largest genome-wide association study study of clinical haematological phenotypes to date and identifies 7,122 association signals.

Mitral valve (MV), together with left atrium (LA) form an important coupled structure inside human heart. Mitral valve dysfunction including mitral stenosis, prolapse and regurgitation, is one of the most common valvular heart disease and hence has attracted significant research interest. Left atrium dysfunction such as atrial fibrillation, fibrosis and dilation, etc., also greatly affects the normal heart performance and leads to serious consequences. Computational modelling of human MV and LA function can improve our understanding of the biomechanics and flow details, which are important for improving surgical procedures and medical therapies. More importantly, it provides a tool for isolating the effects of different physiological and anatomical changes that occur under pathological conditions. However, because of the challenges of modelling the highly complex MV and LA structures and their interaction with the blood flow, only limited progress in multi-physics modelling of MV and LA has been made to date. Therefore, this thesis aims to develop computational models for MV and LA that include detailed geometry information and the fluid-structure interaction (FSI) behaviour as well as to investigate various factors that affect their normal function. First, we present a FSI model of the mitral valve that uses an anatomically and physiologically realistic description of the MV leaflets and chordae tendineae. Three different chordae models --- complex, `pseudo-fibre', and simplified chordae --- are compared to determine how different chordae representations affect the dynamics of the MV. Interesting flow patterns and vortex formulation are observed in all three cases. Additionally, results show that the complex and pseudo-fibre chordae models yield good valve closure during systole, but that the simplified chordae model leads to poorer leaflet coaptation and an unrealistic bulge in the anterior leaflet belly. An energy budget analysis shows that the MV models with complex and pseudo-fibre chordae have similar energy distribution patterns, but the MV model with the simplified chordae consumes more energy, especially during valve closing and opening. To conclude, we find that the complex chordae and pseudo-fibre chordae have similar impact on the overall MV function, but that the simplified chordae representation is less accurate. Following the MV modelling work, we further develop a coupled left atrium - mitral valve model based on coronary computed tomography angiography (CTA). The LA model incorporates patient-specific LA geometry and detailed fibre structure defined by an atlas-based method. It is equipped with a transversely isotropic material model. Effects of pathological conditions, e.g. mitral valve regurgitation and atrial fibrillation, and geometric and structural variations, namely, uniform vs non-uniform atrial wall thickness, atlas-based vs rule-based fibre architectures, on the system are investigated. We show that in the case of atrial fibrillation, the reversal pulmonary venous flow at late diastole disappear and also the filling waves at left atrial appendage orifice during systole have reduced magnitude. In the case of mitral regurgitation, a higher atrial pressure and disturbed flows are seen, especially during systole where a large regurgitant jet can be found with the suppressed pulmonary venous flow. We also show that both the rule-based and atlas-based fibre defining methods lead to similar flow fields and atrial wall deformations. However, the changes in wall thickness from non-uniform to uniform tend to underestimate the atrial deformation. Using a uniform but thickened wall also lowers the overall strain level. The flow velocity within the left atrial appendage, which is important in terms of appendage thrombosis, increases with the thickness of the left atrial wall. Energy analysis shows that the kinetic and dissipation energies of the flow within the left atrium are altered differently under conditions of atrial fibrillation and mitral valve regurgitation, providing a useful indication of the atrial performance in pathological situations. The future development of the coupled model involves applying patient-specific pressure and flow boundary conditions, introducing physiological LV models and modelling the process of blood clotting, etc.

Chemokines regulate the migration of leukocytes through binding to chemokine receptors. The inflammatory CC chemokine receptors CCR1, CCR2, CCR3 and CCR5 (iCCRs) are expressed by several different leukocytes and are important in orchestrating inflammatory responses. These receptors can bind to multiple CC chemokines and many of these chemokines share receptors, resulting in a highly complex system in which the exact role of each receptor is not understood. To aid understanding of these receptors, two unique mouse strains have been developed. These are mice that are deficient for all four iCCRs (iCCR-KO) and mice that express fluorescent reporter proteins for each of the iCCRs (REP mice). Hypertension is when blood pressure (BP) is elevated above 140/90 mmHg. There is evidence for a role of inflammation in the development of hypertension and the consequent end organ damage which increases the risk of cardiovascular disease. Several chemokines and chemokine receptors have been implicated in hypertension but due to the complexity of the chemokine system, the role they play in the pathogenesis of the disease is unclear. Therefore the role of iCCRs in hypertension was investigated using iCCR-KO and REP mice. WT, REP and iCCR-KO mice were subject to 7 or 14 days of Angiotensin (Ang) II induced hypertension. iCCR expression was characterised in REP and WT mice and the effect of iCCR deficiency on BP, vascular function, inflammation and cardiac and vascular remodelling was assessed using the iCCR-KO mice. Aortic CCR2 and CCR5 expression increased in Ang II treated WT mice compared to control WT mice. Further, iCCR-KO mice were protected from Ang II induced vascular dysfunction but iCCR deficiency did not influence BP or remodelling. iCCR-KO mice were also shown to have altered circulating leukocyte populations in Ang II induced hypertension. Control iCCR-KO mice tended to have a lower BP than WT mice so the effect of iCCR deficiency on regulators of BP in the kidneys and kidney leukocyte infiltration was investigated. iCCR-KO mice had fewer inflammatory monocytes and reduced mineralocorticoid receptor mRNA expression. This could influence BP but further studies are needed. Overall, novel mouse models have been used to identify how iCCRs are involved in hypertension. The results described here suggest that iCCRs, in particular CCR2 and CCR5, are involved in regulating vascular dysfunction in hypertension. Through improving understanding of these receptors in the disease, there is increased potential to target them as treatments that would ultimately reduce the risk of cardiovascular disease.

This thesis focuses on fabricating a perfused microbioreactor platform to generate functional human engineered heart tissue to be used for drug testing. To evaluate the feasibility of this system and make the conditions optimized for culturing cardiomyocytes in 3D constructs, rats' heart tissue models were firstly set up to test the efficacy for long-term culture. Myocytes from embryonic rats were cultured in 200mg/ml fibrinogen gel with 5 million/ml cell densities. The tissues were formed in 24-well culture dish and then transferred into the micro-bioreactors with carbon electrodes to provide electrical stimulations. Culture medium was perfused to maintain chemostat environment. To measure contractile force, the key function of cardiomyocytes in vivo, rod-shaped tissue constructs were generated and attached onto two flexible PDMS pillars (1mm diameter and 12mm length). They worked as a force transducer and their deformation was measured to estimate cellular contractile force with accurate mechanical model. The dynamic perfusion system was stimulated to determine optimal design and operation of microbioreactor. By comparing the functions of the cells cultured in static and perfused environment, it was concluded that the perfused system would be necessary to generate more representative 3D in vitro models Electrical stimulation is an important tool to induce functions of cardiomyocytes cultured in vitro. To investigate the electrophysiological activities of engineered heart tissue, different levels of electrical stimulations were applied onto the engineered heart tissue to mimic those in native heart. All the tissues were cultured 5 days before electrical stimulations were initialled to ensure tissues are sufficiently stable. This can avoid the influence onto protein assembly due to early electrical stimulation. In the experiments, monophasic electrical signals were applied onto the samples with amplitudes ranging from 1V/cm to 5V/cm and frequencies varying between 1Hz and 5Hz. While comparing the stimulated and non-stimulated samples, the optimal electrical stimulation condition is 3V/cm, 2Hz on the basis of performance of rEHT in terms of contractile force, beating rate and microstructures. It is then used to induce higher maturation of iPSC-CMs for human EHT generation. After 20 days of long-term culture, the human engineered heart tissues are mature and functional and can be used for testing the effect of drugs. Five drugs, including Ouabain, Bay k8644, Milrinone, Amlodipine and Ryanodine, all clinically approved to treat various cardiovascular diseases, were used for the evaluation tests. The tested drugs at different dosages were added into the culture medium and the latter was perfused into the microbioreactor hosting the human engineered heart tissues. The dynamic responses of the hEHTs were recorded over 24 hours experiments. The responses were shown to be clinically relevant. Although further tests are required to validate the efficiency and efficacy of the developed microbioreactor platform, it showed potentials for high throughput testing and refining, reducing and replacing the use of animal tests.

Understanding the molecular processes that govern cardiovascular development has the potential to inform regenerative strategies for disease states. Due to their versatile roles, epicardial cells are an attractive therapeutic target. However, using epicardial cells for therapy is hindered by our limited understanding of epicardial biology. The embryonic epicardium is one of the main regulators of cardiovascular development, functioning to secrete essential growth factors and produce epicardium-derived cells (EPDCs) that contribute most coronary vascular smooth muscle cells and cardiac fibroblasts. Due to its complex biology, the molecular mechanisms that control epicardial formation and differentiation have not been fully elucidated. SRSF3 is an RNA-binding protein that has essential roles in RNA processing, and it is highly expressed in the proepicardium and later in the epicardial layer during heart development. Deletion of Srsf3 from the murine proepicardium using the Tg(Gata5-Cre) or embryonic day (E) 8.5 induction of Wt1CreERT2 led to proliferative arrest and impaired epithelial-to-mesenchymal transition (EMT), which prevented proper formation and function of the epicardial layer. The epicardial requirement for SRSF3 was further assessed in a mouse epicardial cell line using RNA interference, which revealed that SRSF3 is required for production of Cyclin D1 and for EMT in response to TGFβ. Induction of Srsf3 deletion with the Wt1CreERT2 after the proepicardial stage resulted in disrupted sprouting from the sinus venosus and elevated hypoxia at E13.5. Single-cell RNA-sequencing showed SRSF3-depleted epicardial cells were removed by E15.5. The remaining non-targeted cells became hyperproliferative and compensated for the loss, with normal epicardial contribution being detected at E17.5. Due to the compensation mechanism, the existence of epicardial subpopulations was explored through assessment of published heterogeneous marker genes: Sema3d, Scx, Tcf21, Tbx18 and Wt1, using RNAscope, PrimeFlow and Single-cell RNA-Sequencing, in both wild-type and SRSF3 mutants. Complete overlap of the markers was detected, with no evidence of epicardial subpopulations, raising questions regarding the specificity of previously reported Cre lines. This research supports a role for SRSF3 as a master regulator of RNA metabolism in epicardial cells, however, further studies are required to dissect the exact molecular mechanisms involved.

Cardiovascular diseases (CVD) are major causes of morbidity and mortality worldwide. Complications associated with CVD encompass the entire vascular system, including blood vessels that supply brain. Impairments to cerebral blood flow (CBF) and cerebrovascular function result in increased risk of cognitive impairment, vascular dementia and stroke. Identifying interventions that can attenuate the age-related decline in CBF and enhance cerebrovascular function are essential, especially in individuals at high risk of CVD. Remote ischaemic preconditioning (rIPC), which involves cyclical periods of ischaemia-reperfusion applied noninvasively to a limb, has been shown to improve measures of peripheral vascular function and cardiovascular endpoints when applied acutely (one episode) or repeatedly (e.g. daily, 3 times per week). There is also emerging evidence that repeated rIPC elicits beneficial effects within the cerebral circulation in stroke patients. However, the mechanisms of how rIPC can improve cerebrovascular function are unknown. The overreaching aim of this thesis was to investigate whether acute and repeated rIPC could enhance cerebral and peripheral vascular function in cohorts with increased risk of CVD. In a crossover study design, Study 1 aimed to assess the impact of a single acute bout of rIPC on cerebrovascular function. Eleven young healthy (28±4 years) and nine individuals at risk of CVD (53±7 years) underwent assessment of cerebrovascular function. Using Transcranial Doppler (TCD), markers of cerebrovascular function were assessed following either bilateral arm rIPC or sham condition. There was no change in middle cerebral artery velocity (MCAv) or blood pressure (BP) during rIPC. Application of rIPC did not alter cerebrovascular reactivity (CVR) compared to sham (0.002 MCACVC/mmHg, 95%CI= -0.001, 0.005, P=0.24), nor did it affect any parameter of dynamic cerebral autoregulation (dCA) (0.028 normalised gain%/mmHg-1, 95%CI= -0.080, 0.137, P=0.59). This study suggested that an acute bout of rIPC does not influence cerebrovascular function in healthy young individuals and older subjects with increased risk for CVD. In a randomised control-pilot design, Study 2 aimed to obtain estimates for the change in peripheral conduit and cerebrovascular function following a 7-day rIPC intervention. Twenty-one type 2 diabetes mellitus (T2DM) patients performed either 7-day daily rIPC or control (no rIPC). Peripheral conduit artery function was assessed using flow mediated dilation (FMD) before and after an endothelial ischemia-reperfusion (IR) injury. Cerebrovascular function was assessed using TCD to examine dCA (0.10Hz squat stand manoeuvres). All measurements were performed at three time points; pre, immediately post intervention, and 8 days post intervention. Using pre-intervention data as a covariate, the change from pre-post in FMD was 1.3% (95%CI= 0.69, 3.80; P=0.09) and 0.23 %/mmHg-1 (95%CI -0.12, 0.59; P=0.18) in dCA normalised gain with rIPC versus control. The directional changes outline FMD can be enhanced by daily rIPC in patients with T2DM, whilst cerebrovascular function is unaltered. In Study 3, in a randomised design, nineteen participants at risk of CVD were allocated into either 8 weeks of aerobic exercise training and rIPC (rIPC + Ex) or 8 weeks of rIPC only performed 3 times per week. Assessment of cerebrovascular function was performed using TCD and FMD was used to examine peripheral vascular function before and after an IR injury. Measurements were performed before (week 0) and immediately after (week 8) each intervention. Neither intervention changed resting CBFv, dCA (spontaneous or forced BP oscillations) or CVR. FMD increased by 1.6% (95% CI= 0.4, 2.8) in the rIPC + Ex intervention and by 0.3% (95% CI= -1.1, 1.5) in the rIPC only intervention, whilst no statistical difference was found between interventions (P=0.65). Data from this study suggests that combining exercise with rIPC does not result in greater changes in cerebral or peripheral vascular function. In Study 4, dCA and baroreflex sensitivity (BRS) data collected during 0.10Hz squat stands manoeuvres was obtained from 206 individuals and analysed using transfer function analysis. Cross-sectional associations between ages were examined using linear regression adjusting for sex. Multivariable linear regression was used to adjust for sex, health status and VO2max. Age, sex, CVD risk and VO2max do not impact on dCA parameters normalised gain, phase or coherence (P > 0.05). dCA (absolute) gain reduced with age when adjusting for sex, and CVD risk. The data from this study suggest that dCA parameters, when adjusted for BP, does not decline with age in either sex. Collectively, the data contained within this thesis suggests rIPC interventions appear to be effective in improving peripheral endothelial function, but have little effect on cerebrovascular function. Additionally dCA, a frequently measured marker of cerebrovascular function seems to be unaffected by aging, CVD risk factors or cardio-respiratory fitness.