Background Low aerobic fitness and obesity are associated with atherosclerosis, and thereforegreatly increase the risk of cardiovascular disease (CVD) and early death. It has long been known that atherosclerosis my begin early in life. Despite this fact, it remains unknown how obesity and aerobic fitness early in life influence the risks of atherosclerosis, CVD and death. Furthermore, it is unknown whether high aerobic fitness can compensate for the risks associated with obesity, and how genetic confounding affects the relationshipsof aerobic fitness with CVD and all-cause mortality. Thus, the main aims of this thesis were to investigate the associations of aerobic fitness in late adolescence with myocardial infarction (Study I), stroke (Study II) and all-cause mortality (Study III), and how genetic confounding influences the relationshipsof aerobic fitness with CVD, diabetes and death (Study IV). Methods The study population comprised up to1.3 million men who participated in mandatory Swedish military conscription. During conscription, all conscripts underwent highly standardized tests to assess aerobic fitness, body mass index, blood pressure and cognitive function. A physician also examined all conscripts. Data on subjects’ diagnoses, death and socioeconomic status during follow-up were retrieved using record linkage. Subjects were subsequently followed until the study endpoint, date of death or date of any outcome of interest. Associations between baseline variables and the risks of adverse outcomes were assessed using Cox’s proportional hazard models. Genetic confounding of the relationships between aerobic fitness and diabetes, CVD and death was assessed using a twin population and a paired logistic regression model. Results In Study I, low aerobic fitness at conscription was associated with an increased risk of myocardial infarction (MI) during follow-up (hazard ratio [HR] 0.82 per standard deviation increase). Similarly, in Study II, high aerobic fitness reduced the risk of stroke (HR 0.84 for ischemic stroke, HR 0.82 for hemorrhagic stroke; P < 0.001 for all), and obesity was associated with an increased risk of stroke (HR 1.15 for ischemic stroke, HR 1.18 for hemorrhagic stroke; P < 0.001 for all). In Study III, high aerobic fitness was also associated with reduced all-cause mortality later in life (HR 0.49, P < 0.001). High aerobic fitness exerted the strongest protection against death from substance and alcohol abuse, suicide and trauma (HRs 0.20, 0.41 and 0.52, respectively; P < 0.001 for all). Obese individuals with aerobic fitness were at higher risk of MI and all-cause mortality than were normal-weight individuals with low fitness (Studies I and III). In Study IV, fit twins had no reduced risk of CVD or death during follow-up compared with their unfit twin siblings (odds ratio 1.11, 95% confidence interval 0.88–1.40), regardless of how large the difference in fitness was. However, the fitter twins were protected against diabetes during follow-up. Conclusions Already early in life, aerobic fitness is a strong predictor of CVD and all-cause mortality later in life. In contrast to the “fat but fit” hypothesis, it seems that high aerobic fitness cannot fully compensate for the risks associated with obesity. The associationsof aerobic fitness with CVD and all-cause mortality appear to be mediated by genetic factors. Together, these findings have implications for the view of aerobic fitness as a causal risk factor for CVD and early death.

Cardiovascular risk scores predict an individual’s risk of developing cardiovascular disease. Many were developed and validated in study cohorts on risk-factor lowering treatment – a cause of inaccuracy. In addition, risk scores are criticised as being biased towards the elderly due to the prominence of age as a risk predictor. Although present guidelines advocate the use of short-term (5-10 year) absolute risk scores, other approaches to redress this perceived imbalance such as lifetime risk scores are being considered. The overall objective of this thesis is to identify the most appropriate cardiovascular risk score for use in general practice, taking account of the impact of treatment and time on assessed risk. This objective was met by three different methods. First, a systematic review of cardiovascular risk scores was conducted. This explored the derivation of each score, including the extent of treatment. Next, doctors were interviewed in depth to understand their perception and use of risk scores. Finally, mathematical models were devised to determine whether a true difference in life expectancy exists at different ages but the same short-term cardiovascular risk. The models incorporated age-specific case fatality rates, competing risks and time preference to estimate the potential years of life lost due to a five-year treatment delay in different age groups with the same short-term coronary heart disease risk. The findings demonstrate that cardiovascular risk scores do not take account of treatment effects. This significantly affects their application in clinical practice. In addition, there is little difference in potential life years lost between ages at the same risk level because of higher case-fatalities in older people. When time preference is considered, any residual case for treating the same level of short-term risk differently at different ages is abolished. The overall conclusion is that the five to ten-year absolute cardiovascular risk score is the most appropriate approach to primary cardiovascular disease prevention. By overestimating risk in the young, other approaches benefit the few at the expense of the many.

Radiotherapy has been shown to increase the subsequent risk of heart disease among survivors of breast cancer, but little is known about factors, other than the dose of radiation delivered to the heart, which determine the magnitude of the risk. In addition, survivors of teenage and young adult cancer are internationally acknowledged as an understudied population, and limited information is available on their late health risks. This thesis sought to utilise the largest observational datasets available to date for these populations: the Collaborative Group on Observational Studies of Breast Cancer Survivors and the Teenage and Young Adult Cancer Survivor Study. These were used to firstly characterise the radiation-related risks of heart disease and lung cancer, and secondly to provide an overview of the long-term risk of heart disease for the entire spectrum of cancers diagnosed in teenagers and young adults aged 15 to 39. Initially, a methodology study and systematic review demonstrated that selection effects and other biases can be very problematic during analyses of observational cohorts, particularly when using a radiotherapy comparison. However, in the case of heart disease and lung cancer, one can take advantage of the breast being a paired organ and use a laterality comparison, particularly when laterality played little effect in treatment selection. This comparison was used throughout the analyses of breast cancer patients. This thesis demonstrated that adjuvant radiotherapy for breast cancer significantly increased the risk of heart disease among women with left-sided breast cancer and those patients with ipsilateral lung cancer. Interestingly, younger women were at the highest risk of heart disease, and a progressive proportional decrease in risk with increasing age at diagnosis was found, which has not been shown before. It also suggested that radiotherapy and chemotherapy combined may further increase the risk of heart disease among breast cancer patients. Survivors of teenage and young adult cancer, particularly Hodgkin lymphoma, were at a significantly raised cardiac mortality risk compared to the matched general population. The findings of this thesis provide evidence to support continued follow-up for cancer patients, as survivors were found to be at a substantial risk into the second or third decade after treatment. It has permitted the detection of groups of individuals at particularly increased risks, for example younger patients and survivors of Hodgkin lymphoma diagnosed in teenagers and young adults, for whom closer monitoring for late effects or measures to reduce the risk, such as adaptations to treatment, may be appropriate. Finally, evidence was also presented to support the development of clinical follow-up guidelines specifically for survivors of teenage and young adult cancer.

Modulation of cardiac rate and contraction through calcium-dependent and independent means are of central import to the ability of an organism to adapt to its environment. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent calcium-releasing second messenger across a broad range of tissues and organisms. In cardiac myocytes, NAADP is thought to stimulate calcium release from acidic stores which then bolsters filling and release during CICR. Questions remain: as to the potential need for amplification to generate the size of responses observed and the physiological role of the NAADP pathway. In contractile myocytes, photorelease of NAADP caused significant increase in calcium transient amplitude and velocity of transient upstroke and decay. Effects were absent during NAADP photorelease in the presence of Ned-19 or CaMKII inhibitors. Cellular calcium transient responses to β-adrenergic stimulation were significantly reduced in the presence of inhibitors of the NAADP pathway. These data support the hypothesis that NAADP-induced calcium release is relevant during adrenergic stimulation and requires amplification through CaMKII. Rate modulation at the sino-atrial node can occur through the hyperpolarisation-activated current I_(f). Basal cardiac rate is a major determinant in cardiac mortality and compounds which specifically affect rate have clinical utility. A compound currently used to treat inflammatory conditions was found to have a significant rate-reducing effect in sino-atrial node preparations mediated by inhibition of I_(f). Apelin, an endogenous peptide, has been reported to potently generate improved contractility without development of hypertrophy. Study of its effects in single cells have provided conflicting information, at least in part because of the difficulty in working with the compound. A method for the consistent observation of apelin-mediated contractile responses is presented, focusing on the timecourse of cell contraction. These observations suggest a role for apelin in both inotropy and lusitropy and will enable further research.

Cardiac stem cells (CSCs), isolated from heart tissue explants and expanded via the formation of cardiospheres (Csp), are a promising candidate for cell therapy to prevent heart failure following myocardial infarction. To allow early administration to patients, isolation and expansion of CSCs must be performed in the shortest time possible. Hence, this project aimed to optimize culture conditions and characterize the cardiac explant-derived cells (EDCs), Csp and Csp-derived cells (CDCs) produced. Rat neonatal EDCs contained 4-7% c-kit⁺ cells, measured using flow cytometry. Optimal Csp growth conditions were determined, such that plating 3 x 10^4 EDCs per well of a 24-well plate coated with 16.7 µg/ml poly-D-lysine, in CGM containing 7% serum, improved Csp production and generated 1.5 x 10^7 CDCs in 16 days, a sufficient number for cell therapy. The CDCs expressed the stemness markers; c-kit, Oct3/4, SOX2, and Klf-4, and the cardiac differentiation markers; GATA4 and Nkx2.5. The therapeutic effect of CDCs may be limited by the low, 3 ± 0.1%, c-kit⁺ cell numbers. To increase c-kit⁺ cells in CDCs, an alternate culture method for Csp and different extracellular matrices (ECM) for cell expansion were tested. The hanging drop culture method produced Csp with higher levels of c-kit⁺ cells (9 ± 2%) than poly-D-lysine-coated and low-bind culture dishes. Of five ECM tested, collagen IV was found to enhance EDC migration and CDC proliferation, and produced 11 ± 0.4% c-kit⁺ cells, with Csp cultured in hanging drops. Intramyocardial injection of CDCs improved left ventricular ejection fractions of infarcted rat hearts by 9% and prevented the peri-infarct wall from thinning, measured in vivo using MRI over 16 weeks. To improve cell tracking using MRI, two MR positive contrast agents, gadolinium-DTPA and gadonanotubes were tested. Gd-DTPA had low sensitivity after labelling (1.4 x 10^5 cells/mm2); whereas gadonanotubes did not provide positive contrast at 11.7 T. Thus, neither contrast agent could be used for cell tracking using high magnetic field. In conclusion, CDCs were an effective source of stem cells that could be used for heart repair, although cells could not be tracked using positive MR contrast.

Myocardial and skeletal muscle high energy phosphate metabolism is abnormal in heart failure, but the pathophysiology is not understood. Plasma non-esterified fatty acids (NEFA) increase in heart failure due to increased sympathetic drive, and regulate the transcription of mitochondrial uncoupling protein-3 (UCP3), through peroxisome proliferator-activated receptor-α. The aim of the work in this thesis was to determine whether cardiac PCr/ATP ratios and skeletal muscle PCr kinetics during exercise were related to cardiac and skeletal muscle UCP3 levels respectively, thus providing a mechanism for the apparent mitochondrial dysfunction observed in heart failure. Patients having cardiac surgery underwent pre-operative testing, including cardiac and gastrocnemius 31P magnetic resonance spectroscopy. Intra-operatively, ventricular, atrial and skeletal muscle biopsies were taken for measurement of mitochondrial protein levels by immunoblotting, along with mitochondrial function by tissue respiration rates. Fasting plasma NEFA concentrations increased in patients with ventricular dysfunction and with New York Heart Association (NYHA) class. Ventricular UCP3 levels increased and cardiac PCr/ATP decreased with NYHA class, however, demonstrated no relationship to each other. In skeletal muscle, maximal rates of oxidative ATP synthesis (Qmax) related to functional capacity. Skeletal muscle UCP3 levels increased with NYHA class but were unrelated to skeletal muscle Qmax. Tissue respiration experiments revealed no relationship between ventricular function and indices of mitochondrial coupling, furthermore, indices of mitochondrial coupling were unrelated to tissue UCP3 levels. No evidence was found to support mitochondrial uncoupling, mediated through UCP3, as a cause of the abnormalities in cardiac and skeletal muscle high energy phosphate metabolism.

Stroke is a major cause of premature death and disability throughout the world and atrial fibrillation (AF) is one of the most common preventable causes of stroke. AF affects about 10% of individuals aged ≥80 years, but warfarin is substantially under-used in this age group despite being effective in preventing AF-related thromboembolic events. AF-related ischaemic strokes tend to be severe and incur high care costs, and non-cerebral systemic embolism secondary to AF is also a major clinical burden. Despite that, there are few population-based studies on AF-related ischaemic stroke, and no recent study of the burden of AF-related thromboembolism and the population impact of under-treatment. I have used data from the Oxford Vascular Study (OXVASC), a prospective, population-based incidence study of vascular disease in all territories, which was started in April 2002 and is on-going. The study population comprises of 92,728 individuals registered with 100 family physicians in nine general practices and uses multiple overlapping methods of “hot” and “cold” pursuit to achieve near-complete ascertainment of all patients with acute vascular events. There are several findings described by the research in this thesis which have important implications for public health and can be utilised to improve secondary prevention in stroke. First, I have shown that one-third of all incident embolic events were related to AF and 60% of AF-related embolic events occurred at ≥80 years. Second, I have shown that only 9% of patients aged ≥80 years with incident embolic event related to known prior AF were on premorbid warfarin, and consequently three quarters of those previously independent were dead or disabled six months post event. Third, I have shown that there has been no reduction in age-specific incidence of AF-related ischaemic stroke in Oxfordshire over the last 25 years. Fourth, I have shown that assuming age-specific incidence does not continue to rise, if prevention is not improved, the number of embolic events at age ≥80 years would be expected to treble by 2050 (72,975 AF-related embolic events), with 84% of events at all ages occurring at age ≥80. Fifth, I have shown through a meta-analysis that one in five incident strokes had a history of prior AF of which only 19% were on premorbid warfarin, and AF was related to one in three incident ischaemic strokes. Sixth, I have shown that 1 in 5 stroke patients with known prior AF subsequently became institutionalised and incurred high acute and long-term care costs. Seventh, I have shown that one in five patients with undetermined cerebral ischaemic event subsequently had AF-related late recurrent stroke. Eighth, I have shown that even though TIA or ischaemic stroke patients who subsequently turned out to have new AF at follow-up had significantly higher baseline NT-proBNP compared to non-AF group, its utility is limited by low sensitivity and specificity. Ninth, I have shown in another meta-analysis that the duration of cardiac monitoring after cerebral ischaemic events was the main determinant of the observed rate of pAF, and that 5-7 days of monitoring may be adequate in unselected patient populations. Finally, I have shown that using 5-day event loop recording in clinic patients with TIA and minor ischaemic stroke could detect 12% new AF and the delay in monitoring did not reduce the sensitivity of pAF detection.

The endothelial nitric oxide synthase (eNOS) co-factor tetrahydrobiopterin (BH4) has been shown to play a pivotal role in maintaining endothelial function in experimental vascular disease models. In BH4-deficient states, eNOS becomes enzymatically ‘uncoupled’, generating reactive oxygen species instead of nitric oxide, thus promoting endothelial dysfunction. In humans with coronary artery disease (CAD), higher vascular BH4 levels have been shown to be associated with improved endothelial function, and genetic variation in endogenous BH4 synthesis has implicated a causal role. Accordingly, BH4 has been proposed as a potential therapeutic target in vascular disease states. The work in this thesis aims to further elucidate the roles of exogenous and endogenous BH4 in humans. In a randomised, placebo-controlled clinical trial of oral BH4 therapy in patients with CAD, exogenous BH4 had no effect on endothelial function or vascular oxidative stress. Subsequent pharmacokinetic and pharmacodynamic analysis revealed that oral BH4 significantly augmented BH4 levels in plasma and in venous tissue (but not in arterial tissue), but also increased levels of the oxidation product dihydrobiopterin (BH2), which lacks eNOS cofactor activity. Thus, there was a null effect on overall biopterin redox status. To further understand the mechanics of exogenous BH4 oxidation, ex vivo studies of human blood and vascular tissue demonstrated that exogenous BH4 is very rapidly oxidised to BH2; co-administration with an antioxidant had only a modest effect on preventing BH4 oxidation in blood, with no beneficial effect on biopterin redox state in the vasculature. Finally, using a “Mendelian randomisation” approach, I studied the effects of a haplotype of GCH1 (the gene encoding the rate limiting enzyme in BH4 synthesis) on endogenous BH4 bioavailability and vascular function in healthy individuals. In patients with CAD, this haplotype has been associated with decreased BH4 bioavailability and eNOS uncoupling, however in healthy individuals the haplotype exerted no significant effect, likely due to reduced inflammatory stimulation of GCH1.

Background: Acute myocardial infarction (AMI) is a major public health concern. There are limited recent national-level population-based epidemiological data on AMI in England. As a result, the current burden of disease is difficult to quantify. Aim: This thesis addresses gaps in knowledge on AMI in England. It aims to provide a comprehensive analysis of AMI epidemiology over the last decade. Methods: This is a population-based study using person-linked routine hospital and mortality data for England for the period from 1 April 1998 to 31 March 2008. Main outcome measures include: trends in event rate, case fatality, and mortality for AMI, as well as trends in characteristics of, and hospital care for, the AMI patient population between 1999 and 2007; rates of occurrence and case fatality for first and recurrent AMI in 2007; and five-year survival and risk of a second AMI for 2003 to 2007. Results: Total age-standardised AMI mortality rate fell by around half, while the age-standardised event rate and case fatality rate each declined by around one third between 1999 and 2007. Approximately half of the decline in AMI mortality was attributed to a decline in event rate and half to improved survival. During the 2000s, the hospitalised AMI patient population became increasingly elderly, presented with more comorbidities, underwent more revascularisation procedures, and spent less time in hospital. In 2007, approximately 90,000 AMIs occurred in England, of which around one third were fatal, one in seven were reinfarctions, and three quarters were AMIs in those aged 65 years and older. Among 30-day survivors of a first AMI, around one in three men and one in four women died within five years, and about one in eight men and one in six women experienced a second AMI in the same time period. Conclusions: There have been substantial improvements in AMI occurrence, survival, and mortality over the last decade in England. This was driven by improvements in prevention and acute medical treatment. The results in this thesis emphasise the importance of both.

Background: Excess adiposity, diabetes mellitus, and ischaemic heart disease are common important causes of morbidity and premature mortality in postmenopausal women in the UK. A large amount of data exists on known risk factors for these conditions, and for risk factors men and women share there is little evidence to suggest sex-based differences. It has been suggested that factors unique to women (such as parity and breastfeeding) may also influence risk. The nature of the relationship between childbearing and these conditions remains to be clarified. In this thesis I explore the association between women’s childbearing histories and their adiposity, and risk of diabetes or ischaemic heart disease after the menopause, to provide evidence on the character, repeatability and public health relevance of the associations. Aim: To explore the hypothesis that childbearing (specifically parity and breastfeeding) is associated with women’s body weight and risk of excess adiposity, and also with women’s risk of diabetes mellitus, and ischaemic heart disease after the menopause. Methods: Data are analysed from a large population-based cohort of middle-aged UK women recruited in 1996 to 2001 (the Million Women Study) with complete childbearing information, and who had baseline anthropometry, and were followed for incident diabetes or ischaemic heart disease through repeat survey questionnaires, hospital admission records, and central registry databases. Results: In a large ethnically homogeneous population of postmenopausal UK women increasing parity was associated with an increase in BMI, however this increase was offset in women who breastfed. The associations between parity, breastfeeding and BMI were of a similar order of magnitude to established risk factors known to be associated with BMI, for example smoking, and physical activity. The associations between childbearing and women’s risk of diabetes mellitus after the menopause appear to be largely due to the effects of childbearing on maternal BMI. There is only limited evidence to suggest a direct effect of childbearing on women’s risk of diabetes after the menopause. There is statistically significant evidence of an association between childbearing and women’s risk of ischaemic heart disease after the menopause. Parity was associated with a modest increase in risk whereas breastfeeding was associated with a small decrease in risk, however the effects were small in comparison to known important risk factors. Conclusions In a large population of UK women childbearing was found to have a persistent influence on women’s mean BMI after the menopause, and through this postmenopausal risk of diabetes mellitus. Childbearing was also found to be mod-estly associated with women’s risk of ischaemic heart disease after the menopause.