Hypertension afflicts 15-20% of the human population and exerts a significant impact on health care resources. Several studies both in humans and animal models suggested a genetic predisposition to hypertension and its complications including malignant hypertension (MH). In this study, two complementary approaches were employed to study the genetic factors and mechanisms of target organ damage in MH. In the first approach, the mouse <i>Ren2</i> transgene was used to induce hypertension in different strains of rats and the incidence of MH was found to be dependent on genetic background. To identify the loci affecting the MH phenotype in rats, an informative genetic cross was carried out with phenotypic assessment and high throughput genome screening. QTL analysis identified a polygenic inheritance of MH lethality involving two modifier loci on chromosome 10 and 17 from the Fischer and Lewis strains, which encompass <i>Ace </i>and <i>At1</i> genes, both logical candidates for involvement in the manifestation of the MH phenotype. The effects of the loci were consistent with multiplicative effects. Furthermore, a difference in plasma ACE activity was found in rats inheriting Fischer and Lewis loci and supported the association of ACE and MH. To facilitate the study of the etiology of vascular lesion in MH, rats with inducible hypertension were generated. The strategy utilized the cytochrome P450, <i>Cyp1a1</i> promoter to drive expression of <i>Ren2</i> in transgenic rats. Transgene expression was thereby rendered inducible by xenobiotics, such as indole-3 carbinol (13C) and 3-methylcholanthrene. The level and duration of hypertension can be regulated in these rats by adjusting the dosage and nature of inducer. Kinetics study of plasma and tissue renin suggested that tissue uptake and activation of prorenin was implicated in development of hypertension. The rats exhibited clinical and pathological features of malignant hypertension upon induction with continuous dietary 13C administration.

Background: Non-invasive cardiac imaging provides important diagnostic and prognostic information in cardiovascular disease. Assessment of ventricular function remains the fundamental imaging request in clinical practice. Cardiac magnetic resonance (CMR) is now the recognised reference standard for quantification of left and right ventricular systolic function, but not diastolic filling. Due to cost and limited availability of CMR, echocardiography remains the first line imaging modality for assessing ventricular function in most cases. Several echocardiographic methods are available for quantifying global ventricular function however despite significant advances in cardiac imaging techniques, visual assessment of ventricular systolic function remains the standard by which ventricular function is reported in many centres. This method is subjective and introduces inter-observer bias. In an era of multi-modal imaging, accurate, reproducible and widely available methods for quantifying ventricular function, which exhibit good inter-modal inter-technique concordance, are desirable. The overall aim of this thesis was to examine the accuracy and reproducibility of several new echocardiographic imaging techniques for quantifying left and right ventricular systolic function, indexed against CMR reference standards, and to examine a novel CMR technique for assessing diastolic function, indexed against current reference standards (invasive catheter recording of left ventricular end diastolic filling pressure (LVEDP)), in a heterogeneous cohort of patients as seen in clinical practice. Methods: All imaging modalities were performed within three hours of each other. Study 1 was designed to compare the accuracy of speckle tracking strain echocardiography for quantifying LV systolic function against biplane Simpson’s rule (SR) and 3D-echocardiography, using CMR LV ejection fraction (LVEF) as the reference standard. Study 2 was designed to investigate the accuracy of a novel modified regional wall motion scoring index (RWMSI) for calculating LVEF, and compare its accuracy against SR and CMR LVEF. Study 3 was designed to explore the clinical utility of velocity encoded (VEC) CMR for diagnosing LV diastolic dysfunction. VEC CMR E/Em velocity ratio was compared to LVEDP recorded during left heart catheterisation. Study 4 was a head-to-head comparison of 10 echocardiographic non-volumetric indices of right ventricular systolic function, based on current European Association of Echocardiography recommendations, indexed against CMR RVEF as the reference standard. Results: In study 1 we demonstrate that speckle tracking strain may be superior to SR for quantifying LV systolic function. In study 2 we suggest that, when specialist imaging software is unavailable, a modified RWMSI may be superior to SR for calculating LVEF. In study 3, we demonstrate a significant correlation between VEC-CMR E/Em ratios and LVEDP, and conclude that VEC-CMR may be a useful tool to diagnose diastolic dysfunction, especially in patients with preserved LVEF. In study 4, we demonstrate that RV free wall strain has a closer correlation to CMR-RVEF than nine alternative echocardiographic indices of RV function, and may be the method of choice for assessing RV systolic function by 2D-echocardiography in the future. Conclusions: This series of studies has confirmed that novel non-invasive cardiac imaging techniques may be used to accurately quantify cardiac ventricular function, and may confer significant advantage over current methods.

Objectives: The prevalence of cardiovascular diseases including heart failure (HF), atrial fibrillation (AF) and peripheral vascular disease (PVD) rises sharply among those aged 85 years and over, who now constitute the most rapidly increasing age group worldwide. Majority of this disease burden remains undiagnosed. Most previous community-based studies of left ventricular (LV) dysfunction and HF included only small numbers in this age group. We conducted a community-based study of 85+ year olds using domiciliary echocardiography, electrocardiography and ankle brachial index (ABI) assessments to estimate the prevalence of LV dysfunction, AF and PVD. We cross-referenced our findings to pre-existing HF, AF and PVD diagnoses present in general practice (GP) medical records to estimate the proportion of undiagnosed cardiovascular pathology. We also assessed to diagnostic performance of NT-proBNP to detect underlying LV dysfunction. Design: Cross-sectional analysis of data from Newcastle 85+ Cohort Study. Setting: Primary care, North-East England. Participants: 427 men and women (60.9% women) aged 85+ years and above, from Newcastle 85+ Study. Measurements: Assessment was conducted in home setting. 2-D and Doppler echocardiography was performed, with LV systolic and diastolic function graded according to American Society of Echocardiography guidelines. A dyspnoea questionnaire was used to assign New York Heart Association (NYHA) functional severity class. ABI measurement and other measures to assess arterial stiffness including pulse wave velocity and pulse wave analysis were carried out by portable sphygmoCor and vicorder devices. Bloods samples were taken for NT-proBNP levels. Previous diagnoses of HF, AF and PVD were abstracted from the GP medical records. Results: Normal LV function (ejection fraction greater than 55% and normal/mildly impaired diastolic function) was found in just 37.2% of participants. 48.4% had LV systolic dysfunction and 14.4% had isolated diastolic dysfunction. 66.1% people with underlying LV systolic or diastolic dysfunction, had symptoms of breathlessness (NYHA II or above). Overall 37.4% of participants had undiagnosed symptomatic significant LV dysfunction (29.5% systolic, 7.9% isolated diastolic). 23.8% of participants with pre existing diagnosis of HF, had no echocardiographic evidence of underling systolic or diastolic dysfunction. Markers of arterial stiffness were not significantly associated with LV dysfunction. Diagnostic performance of NT-proBNP to detect underlying symptomatic or asymptomatic dysfunction was not robust. Prevalence of peripheral vascular disease was 22.1%. 19.0% of participants who had no formal GP diagnosis of PVD had definite PVD on the basis of ABI assessment. Prevalence of atrial fibrillation was 25.5% in the entire cohort. Nearly half (53.2%) of these patients had had no existing GP diagnosis of AF. 87.2% participants with AF were CHA2DS2-VASc score 3 or above. Only 15.6% participants with AF were taking warfarin. In remaining 84.4% participants with AF, who were not on warfarin, only 42.4% participants were taking antiplatelet medications. Conclusions: Systolic and diastolic LV dysfunction was much commoner in 85+ year olds than most previous studies have suggested, affecting around half of a community-dwelling sample; the majority of cases were symptomatic. Despite a national initiative to improve heart failure management within primary care in England, over 80% of very old people with symptomatic significant LV dysfunction remained undetected. Prevalence of AF and PVD is also much common in that rapidly expanding fraction of population, majority of which remains undiagnosed. There is need to establish effective ways to detect this cardiovascular disease burden in very old population.

Electrocardiography (ECG) records a complex curve composed of several waves (P, QRS and T) representing the electrical activity in the heart. Long QT syndrome (LQT) refers to a group of heart conditions and derives its name from the ECG interval ranging from the beginning of the QRS to the end of the T wave. Several LQT subtypes have been identified; together, the first three subtypes account for approximately 80% of the reported disease cases. Genetic mutations in cardiac ion channels have been identified as a cause of LQT. The details of how genetic and other elements interact to modulate the translation of genetic defects to lethal arrhythmias is not fully understood. The aim of this thesis is to develop computational biology techniques that facilitate the investigation of the (I) molecular properties of cardiac channel proteins and their role in the electrical behavior of cardiac cells and tissue, and (II) interplay of genetic and other factors in LQT arrhythmogenesis. Adopting an in silica approach, the particular objectives of this thesis are (1) to investigate the structure-function relationship of cardiac channel proteins; (2) to develop computational models correlating the molecular processes of cardiac channel proteins with the electrical dynamics of cardiac cells and tissue; (3) to investigate the role of genetic mutations in LQT arrhythmogenesis; (4) to investigate the interplay of genetic predisposition and other modifiers in the modulation of LQT arrhythmogenesis; and (5) to develop and evaluate mathematical and computational techniques facilitating multiscale modeling and simulation in biology. In addressing the listed aims and objectives, we have developed and evaluated a number of computational biology techniques in the stated areas. Studying the LQT with our tools on several levels of biological organization (cardiac ion channels, cells and tissue), we have generated a number of plausible hypotheses on the mechanisms underlying LQT.

Myocardial infarction is the largest cause of morbidity and mortality worldwide. Despite optimal treatment, patients have a mortality which approaches 12% at six months. Reperfusion of the ischaemic myocardium is essential to salvage myocardium. However, reperfusion itself is harmful, with up to 40% of myocardial necrosis occurring at this time. This is known as “Lethal Reperfusion Injury”. Opening of the mitochondrial permeability transition pore (MPTP), a channel situated in the inner mitochondrial membrane is central to this process. In its quiescent state, the MPTP remains closed, but once open it becomes non-selectively permeable to solutes of up to 1.5 kDa, resulting in rapidly advancing necrotic cell death. The molecular structure of the MPTP has not yet been fully determined, although cyclophilin D (Cyp D) has been shown to be essential to its function. Genetic ablation of Cyp D has been shown to result in delayed opening of the MPTP and resistance to myocardial damage after acute ischaemia-reperfusion injury. MPTP inhibition is cardioprotective, and may be achieved by a variety of means including ischaemic pre- and post-conditioning, and by pharmacological agents. The aim of this thesis is to investigate the role of the MPTP (cyclophilin D) in cardioprotection from acute ischaemia-reperfusion injury. In chapter 4 we investigate whether it is possible to protect the heart in mice deficient of cyclophilin D by applying a stronger ischaemic preconditioning stimulus to that previously investigated in vivo. We found that there was a non-statistically significant trend towards a protective effect in cyclophilin D deficient mice which were subjected to a stronger IPC stimulus than that previously studied in vivo. Chapter 5 explores whether it is possible to protect the heart whilst avoiding the deleterious extra-cardiac effects of cyclosporin A by sub-cellular targeting of cyclosporin A (CsA) to mitochondria in the first in vivo testing of a mitochondrial-specific form of CsA (mtCsA). We demonstrated that mtCsA administered at reperfusion did not protect the heart of wild type mice from ischaemia- reperfusion. Chapter 6 examines the possibility of achieving cardioprotection independent of cyclophilin D. In this chapter, the effects of the matrix metalloproteinase inhibitor, Ilomastat, are studied. We confirmed that MMP inhibition was cardioprotective in wild type mice and also showed for the first time in vivo that administration of ilomastat can protect the heart from ischaemia- reperfusion in mice deficient in cyclophilin D. In summary our data supports the hypothesis that it is possible to protect the heart from ischaemia – reperfusion in cyclophilin D deficiency by administration of an MMP inhibitor at reperfusion. We also showed a trend towards cardioprotection in cyclophilin D deficiency by administration of an increased IPC stimulus. Together, these data may suggest that necrotic cell death may not all be mediated by the MPTP, or possibly, that the MPTP may exhibit a threshold effect which is surmountable by an increase in inhibitory action of combined pharmacology or increase in IPC stimulus.

Introduction: Mutations in cardiac myosin binding protein-C (MYBPC3), the most common genetic cause of hypertrophic cardiomyopathy (HCM), have been reported to cause a comparatively benign and late-onset form of the disease with incomplete penetrance. Based upon selected families with small numbers of mutations, these early reports may be misleading however. This study aimed to redefine the clinical characteristics of HCM related to MYBPC3 by evaluating a large cohort of unselected patients and their families, in whom an MYBPC3 mutation had been identified. Methods: Index cases and their families underwent history, physical examination, electrocardiogram (ECG), transthoracic echocardiography, ambulatory ECG monitoring, metabolic exercise testing and mutation analysis. Long-term follow up data was collected where available. Results: 44 MYBPC3 mutations were identified in 59 index cases. 26 of 59 (44%) were missense with 11 (19%) insertions/deletions, 11 (19%) intronic, and 5 (8%) nonsense mutations. A further 6 (10%) had complex genetic status with two different sequence variations identified. Nine families shared the R502W missense mutation and haplotype analysis confirmed a common founder, the first to be described in a UK cohort. A further 111 mutation carriers were identified, of which 39 were clinically affected - disease penetrance was therefore incomplete (58%) and related to age and gender but not mutation type. Mean age at diagnosis was 40.1 +/- 15.9 years with a wide range (5-76); 91.8% of affected mutation carriers were diagnosed over the age of 20 years. Most had asymmetric septal hypertrophy (86.4%) and mean maximal wall thickness was 20 +/- 5.8mm. Families sharing identical mutations showed significant variability in disease penetrance, age at diagnosis and risk of sudden death, suggesting that modifying factors play a significant role in disease development. No clinically useful markers of early disease expression were apparent from tissue Doppler studies in unaffected genotyped relatives. During long term follow up (mean 7.9 +/- 4.5 years) 1 individual developed hypertrophy as an adult, 5 individuals died (3 suddenly) and overall survival was 94%. Discussion: The broad spectrum of mutations, disease severity and natural history of disease suggests that earlier reports of late-onset, benign disease related to MYBPC3 mutations were premature. In this study disease expression is broadly similar to non-genotyped HCM cohorts with disease severity ranging from mild to severe, risk of sudden death ranging from low to high and clinical disease being diagnosed in all decades of life. Such variance is not adequately explained by the sarcomeric protein gene or specific mutation per se and other genetic and environmental factors influence disease penetrance, severity and prognosis. The next generation of genotype-phenotype studies require a shift in focus from single gene analysis to include other genetic and environmental variables and an international collaborative database is recommended.

Purpose: Coronary heart disease (CHD) is a major health problem in Jordan and the leading cause of death. It is a progressive condition but effective secondary prevention (SP) that includes behavioural change and medication reduces risk. Little is known about the current provision of SP for patients with CHD in Jordan. This study was designed to explore the current provision of SP from perspective of professionals and to evaluate patients' risk factors, SP interventions received and associated outcomes. Method: A mixed methods, parallel, repeated measures research design was used with a purposive sample from three interventional hospitals. A questionnaire was completed by 16 key staff, 20 Jordanian health care professionals (JHCP) were interviewed and 180 patients were evaluated in hospital and after 6 months. All patients had either had an acute myocardial infarction (AMI) treated medically, a Percutaneous Coronary Intervention (PCI) or a coronary artery bypass graft (CASG). The quantitative data was collected using self-reported questionnaires and a medical record review during hospitalisation and six months later. The qualitative data was collected using semi-structured interviews with JHCP. The European guidelines on CHD prevention (2012) were used to define recommended targets. Results: Provision of SP services in Jordan was poor compared to Guideline recommendations. There was no cardiac rehabilitation, smoking cessation or SP available post-discharge. Interviews established that while health professionals expressed the importance of SP, multiple barriers existed. They were generally dissatisfied with current SP provision and wanted to improve it, but identified training and other issues that needed to be addressed in order to achieve this. The prevalence of risk factors in patients was high during hospitalisation and also at follow-up, but some small improvements at 6 months were observed. Obesity and overweight reduced from 77% to 75%, smoking reduced from 59% to 47% and physical inactivity reduced from 59% to 41%. Quality of life was low and anxiety and depression were high at both baseline and 6 months, but significant improvements over time were observed. Risk factors such as hypertension and blood sugar control deteriorated over time and patients' knowledge regarding their condition remained deficient at 6 months. Conclusion: Despite an extremely high prevalence of risk factors in this population, the provision of SP is poor and obstacles to its development are widespread. There was much greater focus on medical treatment arid medication rather than on lifestyle modifications. Secondary prevention of CHD in Jordan requires urgent improvement and the potential role of nurses in SP should be enhanced.

Due to critical dependence of the heart on oxidative metabolism, cardiac involvement in mitochondrial disease is common and may occur as the principal clinical manifestation or part of multisystem disease. The basic features of cardiac mitochondrial disease expression remain uncertain and no effective treatment exists. Previous research has suggested that cardiac involvement in mitochondrial disease is an important cause of morbidity and early mortality in paediatric populations. In this thesis, a retrospective study confirms the frequent occurrence of cardiac involvement in adults, and demonstrates a significant impact on survival; the importance of specific mt-tRNA mutations and age of symptom onset as predictors of cardiac involvement is also highlighted. Conversely, in children with end-stage cardiomyopathy of unknown aetiology, a prospective study identifies respiratory chain disease as an important cause of disease, altering patient management in a high-risk population in whom mitochondrial disease was not suspected. Using histochemical and immunohistochemical analysis of cardiac tissue, profound complex I deficiency is demonstrated in all cardiomyocytes displaying any evidence of COX-deficiency but also in cells without COX deficiency, supporting the primacy of this factor in patients with well-characterised mt-tRNA mutations. Differences in cardiac complex I expression between patients harbouring m.3243A>G and m.8344A>G mutations may impact on cardiac phenotype; chamber-specific respiratory chain abnormalities are noted and, while tissue segregation may play a role in frequency and severity of cardiac involvement, skeletal muscle mitochondrial DNA mutation load is not a consistent marker of risk. Advanced imaging techniques are used to demonstrate early concentric hypertrophic remodelling, and specific changes in intramyocardial strains and torsion, in patients harbouring the m.3243A>G or m.8344A>G mutations without clinical evidence of cardiac involvement. However, an endurance exercise interventional study shows that patients experience comparable cardiac hypertrophic and haemodynamic adaptations to sedentary controls and confirms the safety and efficacy of 16 weeks’ training.

Coronary heart disease is still the leading cause of death in the UK, despite significant advances in clinical treatments. Stem cell transplantation has the potential to improve cardiac function and patient outcome, but optimal cell types, cell preparation methods and cell delivery routes are yet to be established. The heart contains a small population of progenitor cells that, in culture, contribute to spontaneously formed spheroids known as cardiospheres (Csphs). Following further culture, Csphs give rise to cardiosphere derived cells (CDCs). Both Csphs and CDCs show paracrine benefit including neovascularisation in myocardial ischaemia, leading to improvement in heart function. The aims of this project were to use mouse models to (i) investigate the effect of hypoxic preconditioning on the pro-angiogenic potential of CDCs and (ii) characterise the contribution of cardiac fibroblasts (CFs) to CDCs. I used Col1a2-CreERT;Rosa26-STOP-YFP mice to track YFP-expressing CFs in myocardial tissue and in CDC culture. Co-staining experiments showed only partial overlap of YFP with other CF markers (vimentin and Fsp1) in heart tissue, which may be due to the heterogeneity of CFs and/or incomplete activation of YFP in CFs. I showed that CF-derived cells exist in all stages of CDC culture, and a small subset of these cells also expressed the stem cell markers Sca-1 or cKit, suggesting CF derived cells may contribute to the progenitor cell population. My results showed that preconditioning CDCs with 3%O2 enhances cell outgrowth from heart explants and promotes expression of stem cell and pro-angiogenic markers. I then assessed the pro-angiogenic potential of CDCs in vivo using a sub-dermal matrigel plug assay and showed that CDCs alone have limited pro-angiogenic potential. However, 3%O2 preconditioning of CDCs significantly enhances this process. Further research will increase our understanding of CDC-mediated angiogenesis and improve clinical therapies for MI patients.

Evaluation of platelet dependent thrombosis and the role of dual antiplatelet therapy on blood thrombogenicity in patients with type 2 diabetes mellitus and coronary artery disease The prevalence of type 2 diabetes mellitus (T2DM) is rapidly increasing. Recurrent thrombotic events and cardiac mortality are higher in T2DM. Aims: My overall aim was to identify abnormalities in blood thrombogenicity in T2DM and assess the response to dual antiplatelet therapy. Hypotheses: Study 1 ACS study: Patients with T2DM have increased blood thrombogenicity following non ST-elevation acute coronary syndromes (NSTE-ACS). Study 2 CAD study (double blinded RCT): Addition of clopidogrel to standard treatment will reduce blood thrombogenicity in T2DM and stable coronary artery disease (CAD). Methods: Study 1 ACS study: Eighty patients (40 with T2DM) with troponin positive NSTE-ACS on aspirin and clopidogrel underwent thrombogenicity studies. Study 2 CAD study: Ninety patients with T2DM and proven but stable CAD were randomised to either clopidogrel 75 mg od or placebo and were studied at base line and one week after therapy. I performed i) Badimon chamber study, ii) thromboelastography, iii) VerifyNow® aggregometry, iv) scanning electron microscopy (SEM), v) Multiplate® aggregometry, vi) biomarkers of platelet reactivity assays and vii) serum pro-inflammatory cytokines assays. Results: In T2DM patients with NSTE-ACS, there was higher thrombus area compared to non-diabetic patients. Diabetic thrombus showed lower viscoelastic tensile ii strength and was more resistant to autolysis. On SEM, fibrin fibres in diabetic thrombus were thinner, with higher lateral interlinkage and mesh-like organisation. Thrombus correlated inversely with the rate of thrombus retraction. P selectin, CD40 ligand and inflammatory cytokines were higher in T2DM. Clopidogrel decreased thrombus area in stable CAD patients with T2DM, lowered platelet content of thrombus and increased fibrin diameter and density. Fibrin fibre diameter correlated negatively with shear elastic force of the thrombus. In a post hoc analysis, thrombus area of non diabetic ACS patients’ was similar to that of T2DM patients with stable CAD. Thrombus area and point of care tests showed no correlation. Conclusions: T2DM had higher blood thrombogenicity after NSTE-ACS and platelet dependent thrombus was reduced in stable CAD patients after clopidogrel therapy. Clopidogrel resulted in favourable ultra structural changes to thrombus. Patients with T2DM and stable CAD were “ACS equivalent” and addition of clopidogrel may improve their clinical outcomes. Novel pharmacotherapy to target fibrin and inflammation in T2DM may reduce blood thrombogenicity.