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Cardiovascular disease is the commonest complication of kidney disease. The risk of cardiovascular disease increases as estimated glomerular filtration rate (eGFR) declines and is highest in patients with advanced chronic kidney disease (CKD) on dialysis or with a kidney transplant - termed 'kidney failure'. Despite this, patients with kidney disease have been excluded from most cardiovascular trials over the last three decades. Given this systematic disenfranchisement of patients with kidney disease, our understanding of the diagnosis, incidence, treatment, and outcomes of cardiovascular disease in these patients is limited. My aim was to use large-scale, contemporaneous regional and national routinely-collected, linked Scottish healthcare data to illustrate, update, and address CKD-related disparities in the diagnosis, treatment, outcomes, and temporal trends of cardiovascular disease in patients with kidney disease. First, I performed a pre-specified secondary analysis of a stepped-wedge, cluster-randomised controlled trial to evaluate the implementation of a high-sensitivity cardiac troponin I assay on the diagnosis, management, and outcomes of patients with and without kidney impairment identified as having myocardial injury or infarction. In 46,927 consecutive patients presenting to hospital with suspected acute coronary syndrome, 19% had kidney impairment. As kidney function declined, the frequency of elevated cardiac troponin concentrations increased six-fold, but the proportion diagnosed with type 1 myocardial infarction halved. Implementation of high-sensitivity cardiac troponin testing increased the diagnosis of type 1 myocardial infarction in both patients with and without kidney impairment. Following implementation, rates of coronary angiography, revascularisation and the prescription of evidencebased treatments increased in patients with normal kidney function, but not in patients with kidney impairment. Despite the identification of more patients at risk, rates of myocardial infarction or cardiovascular death at one year were unchanged in patients with kidney impairment following implementation of high-sensitivity cardiac troponin testing. Second, I performed a retrospective, national data-linkage study to assess temporal trends in the incidence, treatment and outcomes myocardial infarction and stroke in patients with kidney failure in Scotland over the past 20 years. Amongst 16,072 patients with kidney failure, there were 1,992 and 996 incident episodes of myocardial infarction and stroke between 1996 and 2016. During this period, the incidence of myocardial infarction and stroke decreased by ~1.5-fold in patients with kidney failure. When compared with the general population, patients with kidney failure had a ~5 times higher overall incidence of myocardial infarction and a ~3 times higher overall incidence of stroke. Since 2009, there has also been a ~3-fold increase in the prescription of antiplatelet therapy following incident stroke and a ~1.5-fold increase in the prescription of dual antiplatelet therapy following incident myocardial infarction. Finally, although 1-year cardiovascular case fatality has improved substantially following myocardial infarction and stroke in patients with kidney failure over the past 20 years in both sexes and across all kidney replacement therapy modalities, overall outcomes for these patients remain poor. Third, I performed a nationwide data-linkage study comparing the clinical characteristics, microbiology, and long-term outcomes of infective endocarditis in patients with and without kidney failure in Scotland over the past three decades. One-fifth of patients with kidney failure died within 30 days of their incident infective endocarditis hospitalisation, half died within one year, and two-thirds within three years. Compared to those without kidney failure, patients with kidney failure were twice as likely to die within one year of hospitalisation and more than three times as likely to die within three years. Fourth, I investigated how infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) modified existing short- and medium-term cardiovascular risk in ~37,000 patients with and without CKD. In patients with CKD, those with COVID-19 had a higher risk of cardiovascular and all-cause death than those without COVID-19 throughout follow-up, but especially within 30 days of SARS-CoV-2 testing. During this early period, patients with CKD and COVID-19 had a more than two-fold increased risk of cardiovascular death - and a more than four-fold increased risk of all-cause death - than patients with CKD who tested negative. Compared to patients without CKD, those with CKD were also more likely to test positive. Following a positive test, patients with CKD had higher rates of cardiovascular complications - including hospitalisations and cardiovascular death - than those without CKD. Moreover, the risks of cardiovascular, COVID-19-related, and all-cause death increased as kidney function declined. My findings highlight the power and potential of contemporaneous routinelycollected, linked healthcare data to define and address established CKDrelated disparities in cardiovascular disease and outcomes. Moreover, my results will provide an evidence base for public health policy and inform planning of healthcare resource allocation for all patients with kidney disease. My future work will focus on using high-fidelity routinely-collected data to improve the cardiovascular risk stratification of patients with kidney failure listed for kidney transplantation and to comprehensively define how age, sex, and socioeconomic status interact to influence the development, treatment, progression, and outcomes of cardiovascular and kidney disease.

Background: Environmental exposure to inorganic arsenic (iAs), one of the most abundant elements in the earth's crust, is widely recognised as a major global health risk - millions of people are estimated to be chronically exposed to arsenic worldwide. Yet, compared to arsenic-related cancer and skin lesions, research on arsenic-related cardiometabolic disease, particularly at lower exposure levels, has been limited. This PhD thesis aims to investigate the epidemiological relationship between chronic environmental arsenic exposure and several sub-types of cardiometabolic disease, with a primary focus on cardiovascular disease (CVD), specifically myocardial infarction (MI), as well as diabetes mellitus (DM) and hypertension. Data sources: Firstly, to explore the relationship between toenail arsenic (including arsenic metabolites and metabolism markers) with MI, the Bangladesh Risk of Acute Vascular Events (BRAVE) study, a case-control study of 1,532 MI cases and 1,334 controls, was used. Secondly, to explore the dose-response relationship between arsenic and several CVD endpoints, a systematic review and two-stage dose-response meta-analyses were performed on 35 studies. Thirdly, to assess the association of environmental arsenic exposure with two major CVD risk factors, diabetes mellitus and hypertension, a systematic review and two-stage dose-response meta-analyses were performed on 71 studies. Lastly, to explore whether genetic variation may be at least partially responsible for inter-individual variation in arsenic-related cardiometabolic disease susceptibility, a narrative systematic review on 14 unique candidate gene-environment (cGxE) studies reporting on 650 single nucleotide polymorphisms (SNPs) in 145 genes was used. Main results: In BRAVE, MI was positively associated with higher toenail monomethylated arsenic (MMA) in μg/g, higher MMA% and a higher primary methylation index (PMI) in both categorical and continuous logistic regression models, and negatively associated with higher iAs% and a higher secondary methylation index (SMI). No statistically significant associations were found for total arsenic (tAs), iAs and dimethyalated arsenic (DMA) in μg/g, nor for DMA%. Dose-response meta-analyses found consistent positive associations between water arsenic concentrations and coronary heart disease (CHD) [fatal, non-fatal, overall], non-fatal stroke, CVD (fatal, non-fatal, overall), hypertension, DM type 2, and gestational diabetes mellitus (GDM). Analyses on low water arsenic concentrations showed positive associations below 10 μg/l for several of the cardiometabolic outcomes studied. From the total of 35 studies on CVD endpoints, and 71 studies on hypertension or DM, only 4 (low quality) studies reported statistically significant negative associations. In the narrative systematic review, 29 SNPs in 21 genes as well as 3 haplotypes (involved in e.g. arsenic metabolism, DNA damage repair, endothelial function, inflammation) were indicative of SNP-arsenic interactions associated with cardiometabolic outcomes. Conclusion: This PhD thesis presents further supports evidence that people exposed to chronic environmental arsenic pollution may be at greater risk for cardiovascular disease, hypertension, diabetes mellitus type 2 and gestational diabetes. Evidence is most conclusive at moderate and high levels of arsenic exposure (i.e., water arsenic > 100 μg/l). However, results at lower arsenic levels suggest that a potential downward revision of the World Health Organization (WHO) guidelines (10 μg/l) may be beneficial for health protection. Whilst studies on individual arsenic species and arsenic metabolism markers are less consistent, results in this thesis suggest a potential role for arsenic metabolism in the development of CVD - potentially particularly at lower arsenic levels. Various SNPs and genes have been suggested to be involved in the susceptibility to cardiometabolic disease, however, these results should be approached with appropriate caution due to the lack of reproducible findings, and the cGxE nature of the included studies. Overall, further long-term prospective studies of individual person data that assess arsenic through different media and include speciation analyses, combined with experimental, mechanistic and genetic studies, are needed to improve understanding of the observed relationship between arsenic and cardiometabolic disease, and the ways in which arsenic may disrupt cell physiology and promote pathophysiology.

Electrocardiographic analysis has the capability to diagnose and locate abnormalities relating to the heart. There are several lead systems, such as the 12-lead ECG, that are commonly used in clinical settings. One aspect of this thesis is to derive additional posterior and right-sided chest leads from the 12-lead ECG, and to evaluate the performance of the derived leads in the detection of ECG changes associated with myocardial ischaemia. The 12-lead ECG is also not practicable for long term ambulatory monitoring, especially in the detection of paroxysmal cardiac abnormalities such as unstable angina. Therefore, the second study of this thesis introduces a novel patch-based short-spaced lead system sensitive to ST-segment changes associated with ischaemia. With the increasing numbers of electronically-stored patient data, it is imperative that clinicians can develop their own algorithms. In the third study, a framework for biomedical algorithm development is introduced, with a focus on its use by non-coders to pass data through multi-lingual scripts. Derived posterior (V7-V12) and right-sided chest leads (V3R-V6R) from the 12-lead ECG were closely correlated to those recorded. Myocardial infarction detection was improved as additional leads were added to the 12-lead ECG, however, this was not statistically significant. A patch-based short spaced lead system that was sensitive to ST-segment changes associated with ischaemia was suggested. It consisted of two bipolar leads. Coefficients were generated to derive this short spaced lead system from the 12-lead ECG. ST-segment changes associated with ischaemia were detected with the highest F1 score (86.7%). A web-based framework was introduced to reduce the barrier to entry in biomedical digital signal processing for non-coders. A Python framework was used with the MATLAB Engine to allow users to create algorithms consisting of multi-lingual scripts capable of processing patient data, without the need to write code. The framework was reproducable and scalable.

The human heart cannot regenerate following myocardial infarction and instead forms a fibrotic scar that impairs cardiac function and can lead to heart failure. Cardiac regeneration aims to treat the injured heart by stimulating the heart to repair itself. The Astyanax mexicanus is a uniquely suited model of cardiac regeneration as it comprises two closely related populations: (1) the 'regenerative' surface (SF) population and (2) the 'non-regenerative' Pachόn (PF) cave population. The immune response to injury is known to be a key regulator of successful regeneration. However, how this response differs between SF and PF populations is unknown. To fully characterise the A. mexicanus immune response, single cell RNA-sequencing, differential gene expression analysis, histological staining and in situ hybridisation were used to determine the immune cell populations present in the heart at 1-, 3-, 7-, 14- and 30 days post-cryoinjury (dpci). Pharmacological perturbations of the A. mexicanus immune response were then used to explore whether identified differences in the regenerative and scarring immune responses to cardiac injury were exerting control on regenerative capacity. Striking spatiotemporal differences were found in the dynamics of both myeloid and lymphoid populations in the PF and SF. Immediately after injury, the non-regenerative PF showed a stronger response to injury with a significantly greater influx of neutrophils into the wound (1dpci p=0.0156; 3dpci p=0.0075). By 7dpci, this inflammatory response was resolved, and differential gene expression analysis showed that PF neutrophils had returned to the uninjured state. In contrast, the regenerative SF showed a greater immune response at the late stages of cardiac healing (7-, 14- and 30dpci). Specifically, at 7- and 14dpci, a transcriptionally unique population of neutrophils was observed in the SF that remained activated and upregulated TNFα-NFκB signalling. Furthermore, a stark contrast in the B cell response was observed: SF displayed a significant influx of B cells at 14- and 30dpci that was absent in the PF (p<0.005). Inhibition of the SF immune response using 7-14dpci Dexamethasone treatment did not impair the regenerative capacity of the SF but did significantly disrupt scar organisation, resulting in scars that closely resembled time-matched PF scars. This research represents a significant leap forward in our understanding of how the immune response differs between successful regeneration (SF) and scarring (PF). However, further studies will be required to determine the role of late-stage SF neutrophils and B cells in successful regeneration before novel therapeutic targets can be identified for immunomodulation in heart attack patients.

Cardiovascular disease, including myocardial infarction, remains the number one cause of worldwide morbidity and mortality. The major cause of myocardial infarction is arterial thrombosis, driven by platelet aggregation. Adenosine diphosphate (ADP)-induced platelet aggregation is mediated by the Gi-protein-coupled receptor (GPCR), P2Y12. Therefore, P2Y12 antagonists are clinically used to prevent thrombotic events. However, current antiplatelet drugs have several drawbacks such as the increased risk of bleeding, difficulty in fine-tuning the antiplatelet effects of irreversible antagonists, and variability in patient response. Furthermore, the nucleoside-based, reversible drug ticagrelor has been reported to cause dyspnoea due to off-target effects. Additionally, the binding modes of the P2Y12 ligands are not fully known. Interestingly, the recently solved crystal structure of P2Y12 has revealed that the orthosteric site is composed of two sub-pockets. This thesis had two complementary aims: 1) to further understand the mechanism of action of cangrelor - the most recently approved, and only intravenously acting P2Y12 antagonist; and 2) to discover novel, competitively acting, non-nucleotide-based reversible inhibitor(s) of ADP-induced platelet aggregation. A plate-based aggregometry assay and platelet-rich plasma (PRP) isolated from the blood of human donors were used to show that cangrelor (in nM and µM concentrations) may act in a non-competitive manner to ADP (up to mM concentrations). This is in contrast with reports in the literature that cangrelor is a competitive antagonist of the P2Y12 receptor. Interestingly, it acted in a competitive manner when the P2Y12 receptor was stimulated with the synthetic and more potent agonist, 2-methylthio-ADP (2MeSADP). The cangrelor analogue, AR-C66096, acted in a competitive manner with both agonists. Subsequently, a multiplexed flow cytometric assay assessing phosphorylated platelet vasodilator-stimulating phosphoprotein (pVASP) levels in platelets was successfully optimised. For this assay, a technique called barcoding was used with a novel combination of dye and fluorophore-conjugated antibody, opening a new avenue for barcoding. This assay further showed that ADP (up to 1mM) + cangrelor (100nM) Emax did not reach that of ADP (1mM) + vehicle, whereas AR-C66096 did. Electrostatic field potential analysis of the two compounds revealed that AR-C66096 had a field of negative electrostatic potential that was missing in cangrelor. Additionally, these results suggested that there may be mechanistic differences in the activation of the receptor by ADP and 2MeSADP. To achieve the second aim, ligand-based in silico tools were used to virtually screen over 440,000 molecules to identify novel scaffolds possessing reasonable similarities in 3D shape and electrostatic properties in reference to the experimental P2Y12 antagonist, AZD1283. Docking of the best hits was performed against the recently solved crystal structure of P2Y12. Following the meticulous inspection of docked poses, as well as similarity indices with the query ligand, 33 compounds were purchased for in vitro validation. From these, two competitively acting, novel scaffolds (namely compound B6 and B11) were identified, which showed consistent inhibition of ADP-induced aggregation of platelets from human blood donors. These compounds were predicted to have comparable interactions with the receptor to the co-crystallised antagonist, AZD1283. Of these two best hits, compound B6, which is a 2-aryl benzoxazole derivative, was chosen for further investigation. To establish the structure-activity relationship (SAR) analysis around the B6 scaffold, nine analogues of this compound were purchased and experimentally tested using the assays described above. This led to the identification of another novel inhibitor of ADP-induced platelet aggregation, namely compound S8. However, despite good docking profiles of the compounds against the crystal structure of P2Y12, the latter could not be confirmed as their target upon analysis of pVASP levels. Further work is required to confirm the mechanism by which these compounds inhibit platelet aggregation. To summarise, this thesis has increased our understanding of cangrelor's mechanism of action, and several 2-aryl benzoxazole derivatives are described as competitive and reversibly acting inhibitors of ADP-induced platelet aggregation.

Background: Periodontitis is increasingly associated with atherosclerotic cardiovascular disease, particularly with coronary artery disease (CAD), stroke and essential hypertension. Although no studies have explored the association of the extent and severity of CAD outcomes with the severity of clinical and microbiological indicators of the periodontal disease. Periodontal bacterial species that colonise deep periodontal pockets and can become pathogenic under altered conditions, also known as "pathobionts". Five bacterial species are known to transition to pathobiont status in the periodontal environment, promoting oral dysbiosis and the chronic inflammation of periodontitis: Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans and Prevotella intermedia. Transcriptomic studies demonstrated that the expression of virulence factors of these bacterial species contribute to periodontitis progression and systemic bacterial dissemination. Previous case-control reports have indicated that patients with periodontitis are at higher risk of developing myocardial infarction (MI) compared to periodontally healthy individuals. Although a great variation is observed with reported risk estimates, possibly due to methodological differences and the confounding effect of risk factors common to periodontitis and MI. Methods: The present thesis is comprised of two different study designs, cross-sectional and case- control analyses. For cross-sectional study design, 160 acute type-1 myocardial infarction (MI) patients underwent periodontal examination to quantify periodontal inflamed surface area (PISA), probing pocket depths (PPD), clinical attachment loss (CAL) using a computerised periodontal probe. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans and Prevotella intermedia bacterial loads were quantified in subgingival plaques of all study participants using polymerase chain reaction (qPCR). Salivary and serum anti-P. gingivalis antibodies were measured using enzyme- linked immunosorbent assays (ELISA). In a MI subgroup (n=10), periodontal bacteria were quantified in aspirated intracoronary thrombi, venous blood and deployment balloons. In a separate MI subgroup (n=40), the expression of thirteen well-recognised periodontal bacterial virulence genes was quantified in subgingival plaques with reverse transcription PCR. In MI patients, myocardial injury extent was estimated by 12-hour troponin (TnI) and severity of CAD burden by SYNTAX-I scores. For a case-control analysis, 50 MI patients were matched 1:1 with controls for the common risk factors. The controls underwent a similar periodontal examination, along with quantification of the subgingival loads of the aforementioned bacterial species. In the cross-sectional study, hierarchical linear regression modelling was used to predict two outcome variables: myocardial injury [troponinI (TnI)] and CAD burden (SYNTAX-I scores). Improvement in the explained proportion of variation (?R2) was assessed after including each periodontal predictor of interest in the model. The correlation between subgingival and intra-coronary loads of periodontal bacteria was assessed using spearman's correlation. For a case-control study, a conditional logistic regression analysis was used to estimate the risk of MI in patients with periodontitis compared to periodontally healthy individuals, matched for the common risk factors. Results: The cross-sectional study analysis revealed that in MI patients, after CAD risk factors adjustment, increased PISA (ΔR²=5%), mean-PPD (ΔR²=3%), mean-CAL (ΔR²=2%), P. gingivalis load (ΔR²=4%) and upregulated expression of virulence genes bioF- 3, fimA, prtH, prtP, ltxA, cdtB (ΔR²=4-9%) predicted higher TnI release, all p < 0.05. Increased mean-PPD (ΔR²=5%), mean-CAL (ΔR²=4%), P. gingivalis load (ΔR²=5%) and upregulated expression of bioF-3, fimA, prtP and ltxA (ΔR²=4-9%) predicted higher SYNTAX-I score, all p < 0.05. The linear regression modelling showed that 19,000, 32,000, 38,000 and 10,000 ng/L increase in TnI release with every 200 mm² PISA, 1mm mean PPD, 1mm mean CAL and 10⁹ P. gingivalis copies/ng DNA, respectively. Similarly, SYNTAX-I scores worsened by 3, 4 and 1 unit with every 1mm mean PPD, 1mm mean CAL and 10⁹ P. gingivalis copies/ng DNA, respectively. The upregulated expression of bioF-3, fimA, prtH, prtP, ltxA, cdtB virulence genes were predictors of Tnl release (p < 0.05 for all), while upregulated expression of bioF-3, fimA, prtP and ltxA virulence genes predicted SYNTAX-I scores (p < 0.05 for all). Periodontal bacterial DNA was detected in intracoronary thrombi and subgingival plaques of the same patients, with a strong correlation for P. gingivalis and T. forsythia bacterial loads (Spearman's rho=0.6, p < 0.05 for both). The matched case-control analysis disclosed that the clinical, bacterial and immunological indicators of periodontitis were significantly elevated in MI patients than matched controls (p < 0.05 for all). The conditional logistic regression disclosed that periodontitis patients are 5.5 times more likely to suffer from MI than periodontally healthy individuals. Conclusions: The findings strengthen the association between the severity of periodontitis and the risk of suffering from the worst CAD-related outcomes. Periodontal pathobionts which underlie the process of periodontitis are novel targets as risk reduction strategies in coronary artery disease which need to be addressed in clinical cardiology and dental practices. Periodontitis should be considered as a modifiable risk factor for the CAD-related primary and secondary prevention strategies. Regular periodontal care for the reduction of periodontitis and pathobiont microbial burden may offer novel approaches for coronary artery disease risk reduction.

Ischaemic heart disease (IHD) is a leading cause of mortality worldwide. MI is associated with complications such as ventricular arrhythmias (VAs) and contractile dysfunction which have also been observed in HF and attributed to altered intracellular calcium (Ca2+) handling. Within the myocyte, the excitation-contraction (EC) coupling process facilitates the rise and fall of Ca2+ with resultant cardiac contraction and relaxation and relies on key structures such as the ryanodine receptor (RyR) and thus, ultrastructural alterations can affect Ca2+ handling. Whilst these changes have been extensively studied in HF, there is limited information on Ca2+ handling and RyR organizational changes following MI. This study has developed a large mammal MI model to elucidate the temporal evolution of Ca2+ handling and RyR organizational changes following MI, with a focus on the MI border zone (BZ) to determine the association with arrhythmogenesis and contractile dysfunction. A large mammal MI model was established demonstrating the typical ST-T segment electrocardiographic changes with VAs observed on MI induction along with the rise and fall of serum troponin levels and an established infarct on cardiac extraction. Following MI, contractile and electrical remodelling changes were observed during the acute, latent and chronic phases, with some contractile dysfunction present at 8 weeks (chronic phase). Compared to control, spontaneous Ca2+ sparks were increased in amplitude and size in 8 week MI BZ cells, with an increased incidence of potentially arrhythmogenic Ca2+ waves at all time points following MI. In contrast to HF, the Ca2+ transient amplitude, rate of decay and synchronicity of Ca2+ rise were unaltered in this model, potentially explaining the modest degree of LV dysfunction seen. At 8 weeks, there was increased RyR inter-cluster distances with a greater number of clusters separated by more than 1 µm and an increased number of single receptor clusters. Additionally, cluster size variation was also increased at 8 weeks. Taken together, this suggests cluster fragmentation and dispersion with increased cluster size heterogeneity which has been associated with altered Ca2+ handling and increased propensity for Ca2+ waves. In conclusion, a large mammal MI model has been established demonstrating changes in Ca2+ handling and RyR organization potentially explaining the increased arrhythmogenicity following MI. This model has the potential to be a highly translatable model of IHD which may allow the discovery of novel therapeutic targets.

Myocardial infarction (MI) triggers an immune response, whereby phagocytic cells remove dead tissue and assist with the subsequent remodelling and repair of the infarcted heart. In adult mice, MI activates cardiac lymphatics, which function to drain the build-up of interstitial fluid (oedema) and traffic macrophages to mediastinal lymph nodes (MLNs), reducing inflammatory/fibrotic cell content and improving cardiac output. Mice at postnatal day 1 (P1) fully regenerate their heart following MI in a pro-regenerative macrophage-dependent manner, whereas similar injury at P7 leads to scarring driven by pro-fibrotic macrophages. The role of cardiac lymphatics in the regenerative capacity of neonatal mice remains unexplored. Therefore, we hypothesised that lymphatics respond and function differently following MI during this regenerative window (P1 to P7), to clear macrophage specific subtypes depending upon their requirement for regeneration (P1) or fibrotic repair (P7). To understand the spatiotemporal changes that take place in the cardiac lymphatic vasculature following birth, we initially quantified the expansion of the vascular network along the sub-epicardium. This revealed lymphatic growth and sprouting until P16, and strain-dependent developmental differences. We then investigated the maturation status of lymphatic endothelial cell junctions, which suggested a potential transition from "zipper" (impermeable) to "button"-type (permeable) junctions during the first two weeks of life. In addition, we examined the lymphangiogenic response and the trafficking efficiency of cardiac lymphatics after surgically induced MI. Using 3D light-sheet and confocal imaging; we found that VEGFR3-expressing lymphatics have limited lymphangiogenic response in P1 compared to P7 hearts 7 days after MI. To assess trafficking of macrophages to MLNs, we performed adoptive transfer of adult splenic hCD68-eGFP labelled monocytes into the myocardium of P1 and P7 recipient mice undergoing coronary artery ligation. Imaging of MLNs from these animals indicated a less efficient clearance of GFP-labelled cells from P1 compared to P7 hearts 7 days after MI . To further investigate differences in immune cell trafficking in P1 versus P7 hearts and their association with regeneration/repair, we made use of mice lacking Lyve1 that exhibit impaired transmigration of interstitial macrophages to lymphatic vessels in adult mice. Unexpectedly, MRI analysis of Lyve1-/- mice revealed impaired cardiac regeneration after P1 MI, while no changes were observed in cardiac function after P7 MI compared to the respective intact controls. Lastly, to gain insight into the molecular underpinnings of lymphatic endothelium-macrophage interactions in P1 versus P7, we generated unbiased single cell RNA sequencing datasets from samples collected at different time-points after MI. A summary of the initial computational analyses, as well as future approaches are discussed here. The results of this project show that cardiac lymphatics continue to grow and mature postnatally and support the hypothesis that cardiac lymphatics respond and function less efficiently after P1 MI, compared to P7, in line with a need to retain pro-regenerative macrophages in the neonatal heart versus clearance of pro-inflammatory/fibrotic macrophages 7-days later. Further analysis is required to uncover the molecular mechanisms that lead to this differential response.

Background: Microvascular obstruction (MVO) is an important predictor of short- and longer-term outcome following primary percutaneous coronary intervention (PPCI) treatment of ST-elevation myocardial infarction (STEMI). The evidence base supporting the role of adenosine and sodium nitroprusside (SNP), as the most evaluated adjunctive therapies aimed at attenuating MVO and infarct size (IS), remains weak as the trials have had variable endpoints, differing drug doses and delivery. As such, the results regarding benefit are conflicting. Objectives: To determine whether intracoronary administration of adenosine or SNP following thrombus aspiration reduce IS and MVO measured by cardiac magnetic resonance (CMR) in patients undergoing PPCI within 6h of onset of STEMI. Design and Setting: Multi-centre, prospective, randomized, controlled and open-label trial with blinded end-point analysis undertaken at four high-volume PPCI centres in the UK. Participants: Patients presenting with STEMI undergoing planned PPCI with Thrombolysis in Myocardial Infarction (TIMI) 0/1 flow in the infarct-related artery (IRA) and no significant bystander coronary artery disease on angiography. Interventions: Participants were allocated to three groups: (1) PPCI with adjunctive intracoronary (IC) adenosine, (2) PPCI with adjunctive IC SNP, (3) control (standard PPCI), following Bivalirudin anticoagulation and thrombus aspiration in all patients. Main outcome measures: The primary outcome was IS (determined as a percentage of total left ventricular mass, LVM) measured by CMR undertaken at 48-72 hours post-PPCI. Secondary outcome measures included MVO (hypoenhancement within the infarct core) on CMR, electrocardiographic and angiographic markers of microvascular perfusion and MACE during median 6-month follow-up. The study aimed to recruit 240 patients (powered at 80% to detect a 5% absolute reduction in IS). Results: In total, 247 patients (79% male with mean age 59.3±12.3 years) were randomized and CMR was undertaken in 207 patients with 197 (80%) completing for the primary outcome. There was no significant difference in IS between groups. However, adjusting for infarct location, there was a borderline significant increase in mean IS in the adenosine group (HR 2.86, 95% CI: +0.01 to +5.72, p=0.05) compared to control. Furthermore, on per-protocol analysis, IS was increased in adenosine treated patients compared to control (12.0 vs 8.3, p=0.031). Increased LV volumes and reduced EF were also observed in the adenosine arm. There was a significant increase in MACE in patients undergoing adenosine-facilitated PPCI compared to control driven by heart failure HR 5.39 [1.18-24.60], LogRank P=0.04) at 30 days and 6 months (HR 6.53 [1.46-29.2], LogRank P=0.01) post-randomisation. Conclusions: The randomised REFLO-STEMI trial tested the potential impact of locally delivered Adenosine and SNP, using a sensitive marker (CMR), on MVO and infarct size. There was no demonstrated efficacy with either drug. However, an increase in MACE rate was observed with adenosine. Adenosine is not routinely used prophylactically and so this is unlikely to lead to any change in current guidelines. The results probably have no bearing on Adenosine used to relieve observed angiographic no-reflow although they must be noted. However, the use of high-dose intra-coronary adenosine may be associated with cardiac toxicity and should not be used routinely in the setting of PPCI to prevent reperfusion injury.

Background: Myocardial infarction (MI) is a major cause of death and disability worldwide with 20% and 45% mortality at 1- and at 5-year respectively. Following MI several detrimental changes are known to occur in the myocardium and a better understanding of these changes would be beneficial for the prevention and treatment of the disease. The aim of this research project was to develop and characterise a large animal model of closed-chest balloon MI with ischaemia reperfusion injury for diagnostic and therapeutic purposes. Methods: From November 2016 to March 2018, a closed-chest MI model using percutaneous coronary occlusion was performed in 65 Yorkshire swine. This thesis reports the clinical, haemodynamic, and biochemical parameters related to the model. In addition, this thesis reports the findings cardiac magnetic resonance (CMR) studies performed in the acute and chronic phase after MI. Results: In total, 65 experiments were performed. Overall experiment-related mortality was 6.1% with 40% of the animals demonstrating malignant arrhythmia during the induction of MI. Longer periods of coronary occlusion (90 minutes vs 60 minutes) resulted in a higher incidence of complications and mortality rate. The average scar size by CMR in the acute phase was 20.70±7.24 grams (as percentage of LV mass = 21.61±7.58 %) at early CMR, and at chronic time point was 8.38±3.5 grams (as percentage of LV mass = 7.93±3.36 %). The left ventricular function (LVEF) decreased significantly at early CMR with a mean value of 43.21±8.62 %. The mean value of cardiac troponin I four hours after MI was 87.47 ± 48.62 ng/ml. Conclusion: With this work we have been able to develop a reliable and highly reproducible model of acute MI in large animal. The model is affected by a very low failure rate, produces a consistent myocardial scar, and can be used for multiple therapeutic and diagnostic purposes in MI.

Recent studies have revealed that postnatal human hearts possess a marked ability for renewal, raising hope for a new therapeutic approach for cardiac repair through stimulating endogenous cardiomyocyte proliferation. Modulation of the Hippo pathway, a key regulator of cardiomyocyte proliferation and survival, has been shown to improve cardiac structure and function post-MI. However, targeting the core components of the pathway for therapeutic purpose is difficult due to the limited understanding on its upstream regulatory mechanism. MicroRNAs (miRNAs), small non-coding ribonucleic acids (RNAs), are considered potential therapeutic targets because of their ability to modulate the expression of genes, including those related to the Hippo pathway. The aim of this study is to identify novel miRNA(s) that can modulate the Hippo pathway and enhance cardiac regenerative capacity. First, we performed literature review, bioinformatics study, and Yes-associated protein (YAP) luciferase assay to screen candidate miRNAs for their ability to enhance the activity of YAP, the main effector of the Hippo pathway, and identified miR-411 as a potential Hippo regulator. Overexpression of miR-411 can significantly induce cardiomyocyte proliferation by reducing phosphorylation of Hippo component large tumour suppressor kinase 1 (LATS1) and YAP. To test the effects of miR-411 in vivo, we overexpress miR-411 by injecting miR-411 mimics resuspended with non-viral vector polyethilenimine directly into the myocardium of a normal heart or following myocardial infarction (MI). Overexpression of miR-411 can induce cardiomyocyte cell cycle re-entry in the adult heart and improve cardiac structures and functions at four weeks post-MI. To investigate miR-411 target genes in cardiomyocytes, the expression of 30 candidate target genes was examined following in vitro and in vivo overexpression of miR-411. The expression of the serotonin transporter gene (SERT) was found to be significantly downregulated and the interaction between miR-411 and the 3' untranslated region (3'UTR) of SERT messenger RNA (mRNA) was confirmed by luciferase assay, indicating that SERT is a direct target of miR-411. Interestingly, small interfering RNA (siRNA)-mediated knockdown of SERT resulted in a significant reduction in LATS1 phosphorylation and an increase in YAP activity, a phenotype similar to that of miR-411 treated neonatal rat cardiomyocytes (NRCMs). In addition, experiments using serotonin receptor antagonist supported the finding that miR-411 mediated Hippo modulation was linked with SERT-dependent signalling. However, the exact mechanism is yet to be investigated. In conclusion, miR-411 enhances cardiac regeneration and improves cardiac function post-MI by modulating the Hippo pathway. The mechanism is likely through the inhibition of SERT expression.

Inflammation plays a key role in the disease processes of both atherosclerosis and myocardial infarction (MI). Regulatory T (Treg) cells are a subset of the adaptive immune system involved in immune tolerance. Animal models show that increasing Treg cell numbers in atherosclerosis can limit disease progression. Several small clinical studies have used low dose interleukin-2 (ld-IL-2) to increase Treg cells in patients with various autoimmune but not cardiovascular diseases. Therefore, a phase 1/2a, randomised, placebo-controlled, double-blind, dose-escalation, two-part clinical trial was performed to test ld-IL-2 (aldesleukin) given once daily subcutaneously, for five consecutive days. In Part A, 25 patients with stable ischaemic heart disease were treated in 5 dose groups (0.3, 0.6, 1.2, 2.4 and 3 x106 IU/day); whilst in Part B, 16 patients with acute coronary syndrome were treated in two dose groups (1.5 and 2.5 x106 IU/day). Results showed that ld-IL-2 was safe and well tolerated across the dose range. The 1.5 x106 IU/day dose increased Treg cell levels by the pre-specified 75% and did not increase effector T cells numbers. There were additional novel findings in other immune cells and cytokine markers. B cell subsets in animal models worsen post-MI inflammation and remodelling. Antibody mediated depletion of B cells in mice limited myocardial injury and improved cardiac function. Rituximab is a monoclonal antibody targeted against human B cells and has been used in the treatment of certain autoimmune diseases and cancers. However, its use in cardiovascular disease is untested and is currently contraindicated. Therefore, a phase 1/2a, open-label, dose-escalation, single-arm, clinical trial was set up to test rituximab in patients with ST-elevation MI. In total, 24 patients were treated in 4 escalating dose groups (200, 500, 700 and 1000 mg). Rituximab was administrated intravenously as a single dose within 48hours of symptoms onset. Rituximab was well tolerated at all doses. Rituximab caused rapid and significant depletion of B cells within 30 minutes of starting the infusion which suggests its use in acute MI is biologically plausible. At 6 months B cell repopulation occurred in a dose-dependent manner. In addition, several new insights into the pharmacodynamics of rituximab were gained. These results have paved the way for future phase 2b trials, one of which is underway.

Autophagy is an important process to maintain cellular homeostasis in many cell types including cardiomyocytes. One type of selective autophagy which degrades defective mitochondria is called mitophagy. In the heart, defective autophagy and/or mitophagy in response to pathological stimuli may lead to the development of adverse cardiac remodelling and eventually heart failure. The microtubule-associated protein 1S (MAP1S) has previously been identified as an interacting partner of the major autophagy regulator LC3; however, its role in the heart is still unknown. In this study I hypothesised that MAP1S may play an essential role in regulating autophagy in the heart. I used mice with genetic knockout of the Map1s gene (MAP1S-/-) and neonatal rat cardiomyocytes (NRCM) with siRNA-mediated gene silencing to study the role of MAP1S in the heart and in cardiomyocytes. In response to autophagic stimulation using rapamycin and chloroquine treatment (Rap/Chl), MAP1S-deficient cardiomyocytes displayed reduction in autophagic flux with an indication of autophagososme-lysosome fusion impairment. This finding was supported by data from electron microscopy analysis of Rap/Chl- induced MAP1S-/- mice, which showed evidence of higher numbers of lysosomal structures as well as indications of altered autophagosome-lysosome fusion in MAP1S-/- mice. Furthermore, in vitro analyses using GFP-LC3 + MitoTracker co-staining and an mt-mKeima reporter system suggested that MAP1S-deficient cardiomyocytes were characterized by impairment of mitochondrial binding with autophagosomes. In addition, analysis of mitochondrial function using a Seahorse analyser showed that MAP1S depletion resulted in the reduction of mitochondrial function. Equally important, MAP1S-knockdown cardiomyocytes exhibited increased apoptosis. To study the role of MAP1S in pathological conditions in vivo, I subjected MAP1S-/- mice to myocardial infarction. Following MI, there was significantly higher mortality in MAP1S-/- mice than in WT controls, despite a comparable degree of infarction between groups as assessed by cTnI level and the fibrotic infarct area. Echocardiography analysis also suggested a reduction in ejection fraction in MAP1S-/- mice compared to WT after MI. Importantly, TUNEL assay indicated higher apoptosis in MAP1S-/- mice which might contribute to the low survival rate. This phenotype might be attributable to altered autophagy or mitophagy in the knockout animals. Taken together, my findings indicate that MAP1S plays an essential role in regulating autophagy and mitophagy in the heart. Ablation of MAP1S reduces survival and leads to the severe impairment of cardiac function after MI.

Reactive oxygen species (ROS) are known to play a central role in the pathology of acute myocardial infarction (AMI), where their production is driven by the metabolism of the citric acid cyclic intermediate succinate that accumulates during ischaemia. This is a major driver of damage upon reperfusion of ischaemic tissue. ROS are however increasingly understood to not only have negative effects, but also to act as biological signalling molecules that are able to activate protective pathways. In order to investigate how such antagonistic processes might interplay in the setting of ischaemia/reperfusion injury (IRI), it is necessary to have a tool that allows the titration of a precise amount of ROS, ideally occurring in a manner that closely mimics endogenous ROS production in both location and the exact reactive species. To this end we validate that the mitochondria-targeted compound MitoPQ produces ROS in this manner and also develop a bespoke control compound that matches the molecular properties of MitoPQ closely with the exception that it does not redox cycle at complex I in the mitochondrial respiratory chain. These compounds are then used in model systems in vitro and in the mouse in vivo to show that whilst high doses of exogenous ROS are damaging to the heart undergoing ischaemia/reperfusion, low doses protect the heart against this insult as shown by smaller infarct sizes. Such a biphasic relationship is also seen between mitochondrial ROS and a range of cellular functions, and further allow us to demonstrate clearly for the first time that a primary increase in mitochondrial ROS may result in these changes rather than the other way around. We also validate two further tools for use in vivo that enable the investigation of the effects of changes in mitochondrial redox status. MitoNeoD is an improved probe for the detection of mitochondrial superoxide using the exomarker approach, while MitoCDNB allows the selective disruption of mitochondrial thiol redox state. The metabolic changes occurring in IRI are also investigated, applying mass spectrometry and magnetic resonance spectroscopy to measure the accumulation of succinate during ischaemia and show that the existing cardioprotective strategies of ischaemic preconditioning and moderate cooling do not alter the extent to which succinate is accumulated during ischaemia. The use of disodium malonate to inhibit the metabolism of succinate at reperfusion is conversely found to protect the heart against IRI. A pilot experiment is also conducted to validate a protocol of volitional exercise post AMI that has been reported to have beneficial effects on heart function during the development of heart failure with a view to investigating a potential role of succinate, but no difference is observed between exercised and sedentary mice in our hands.

Background: Phase IV cardiac rehabilitation (CR) improves outcomes and facilitates behaviour change following myocardial infarction. Although one-third of myocardial infarction (MI) patients in Scotland are under 65 years old, research is lacking in this age group. Aim: To explore the experiences, illness perceptions (Leventhal) and self-identity (Charmaz) of working age (18-65 year olds) individuals attending Phase IV cardiac rehabilitation, including around initiation and maintenance of lifestyle change and the role of spouses. Methods: 16 participants (14 male, 2 female) and 12 spouses (10 female, 2 male) completed nearly 23 hours of semi-structured interviews at the start and end of Phase IV cardiac rehabilitation in Fife and Dundee. Participants completed health behaviour and illness perceptions questionnaires at these time points and after 3 months. Data were integrated and analysed using framework analysis. Results: The MI conflicted with participants' self-identity. Fear and perceived consequences led to restrictive and protective control behaviour from both participants and spouses. CR aided participants goal of 'getting back to normal' - providing safety, monitoring and knowledge reduced fear, allowing return to pre-MI activities and behaviour, both healthy and unhealthy. Perceived cause, communication from health professionals, social influences all impacted health behaviour outcomes, which were varied. Although there were improvements in health behaviour at end of CR, these were not sustained at 3-month follow-up. Identity disruption, mood changes and medication side-effects persisted at 3-month follow-up. Psychological and longer-term support was lacking. Discussion: Cardiac rehabilitation is a time of transition, with experiences and outcomes varying between individuals. Participants and health professionals desired behaviour may conflict. Further research should explore long-term outcomes and opportunities for intervention and support in this group, as well as female attendance and gender. Using longitudinal mixed-methods, and theoretical models provides insight into processes over time but require careful data management and analysis.

Approximately 18 million people die of cardiovascular disease (CVD) around the world each year, making CVDs the number one cause of death in the world1. The extracellular signal-regulated protein kinase 5 (ERK5) has been found to play an essential role in cell growth and survival in heart disease, however, its role in other cell functions is still under investigation. Therefore, the objective of this study was to uncover the potential participation of ERK5 in cardiac metabolism, particularly under pathological conditions. In the setting of high-fat diet (HFD)-induced obesity, it was demonstrated that ERK5 regulates peroxisome proliferator-activated receptor gamma co-activator alpha (PGC1alpha) expression through myocyte enhancer factor 2 a (MEF2A) and MEF2D, this way maintaining mitochondrial biogenesis, fatty acid oxidation (FAO), and oxidative stress control. Mimicking HFD stress, saturated fatty acid treatment in adult rat cardiomyocytes (ARCMs) showed that the oxidative stress produced by GP91PHOX activates calpain1, which degrades ERK5. Preventing ERK5 loss by Gp91phox or Calpain- 1 inhibition preserved mitochondrial function and FAO. Similarly, restoring ERK5 expression in ERK5-deficient hearts using adeno-associated virus 9 (AAV9) prevented the development of heart failure after HFD feeding. In the context of myocardial infarction (MI), it was established that ERK5 loss leads to a dampened cardiac insulin response, increased apoptosis and accelerated cardiac dysfunction through an increased inhibition of the insulin receptor substrate 1 (IRS1) by miR128-3p. Subjecting ARCMs and human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to hypoxia, it was discovered that ERK5 regulates miR128-3p by activation of the CAMP responsive element binding protein (CREB)-CCAAT/enhancer-binding protein beta (CEBPbeta) pathway. Consistent with these observations, ERK5 restoration, CEBPbeta overexpression, or miR128-3p inhibition in ERK5-deficient mice by AAV9 injection normalized IRS1 level and cardiac function after MI. These results indicate that the ERK5-CEBPbeta pathway is essential for the prevention of insulin resistance after an ischaemic insult by protecting IRS1. ERK5 was found to be a cardioprotective regulator of lipid and glucose metabolism under stress; therefore, indicating its potential as a treatment to prevent heart failure.

Vaccination of older adults is a key component of public health policy, but further evidence is required to understand its effectiveness in practice. Electronic health records (EHRs) present a potential alternative to the gold-standard evidence of clinical trials, particularly for populations, such as older adults, who may be under-represented in trials due to ethical and practical constraints in recruitment. Importantly, EHRs also allow the real-world study of an intervention as it is delivered in practice, and its effect in clinically important sub-groups. However, EHRs are not purposed to collect informaton on confounders, which may bias results from the analyisis of routinely-collected data. This motivated my review of quasi-experimental (QE) methods as a means of indirectly adjusting for confounding. My published methodological review found that the longitudinal information available in EHRs offer many opportunities for mitigating against confounding bias, but many methods may be under-utilised. The prior event rate ratio (PERR) and its alternative formulation, described under the Pairwise framework, is a recently developed method that utilises longitudinal information. This before-and-after approach can be applied to rate and survival data, allowing an easy comparison to many trial results. The data on vaccination in UK older adults was also the basis for further study of the performance and limitations of the method beyond exisiting simulation studies. Through comparison to weighted regression, I demonstrated how the source of confounding and robustness of the results could be explored. In a novel application of the PERR and Pairwise methods to interactions, I investigated the effectiveness of the pneumococcal vaccine in older patients, and found evidence for an increase in effectiveness with age across the years of policy implementation, 2003-2005. In my investigation of the influenza vaccine in annual cohorts from 1997 to 2011, I found consistent evidence of a moderately protective effect against myocardial infarction, but that this may decrease with age. The evidence also indicated a protective effect against influenza itself, but no age trend in its effectiveness was detected.

Ischaemic heart disease remains the single leading cause of death throughout the world. Rupture of an advanced atheromatous coronary plaque precipitates the majority of these clinical events, resulting in thrombosis and myocardial infarction. Post-mortem studies have identified thin-cap fibroatheroma (TCFA) as the plaque subtype most prone to rupture with prospective virtual-histology intravascular ultrasound (VH-IVUS) studies linking VH-TCFA to future adverse clinical events. VH-TCFA are however common along the coronary tree with the majority remaining clinically silent, suggesting that factors other than plaque phenotype play an important role in determining rupture and future plaque behaviour. Rupture is thought to occur when the structural stress within the plaque exceeds the material strength of the overlying fibrous cap. Previous histological work has demonstrated that ruptured plaques are associated with higher stress compared to non-ruptured controls, with in vivo VH-IVUS studies linking higher plaque structural stress (PSS) with the presentation of acute coronary syndrome. Wall shear stress (WSS) on the other hand has been implicated in early plaque development and plaque growth suggesting that both PSS and WSS can influence future plaque behaviour. The work presented in this thesis is associated with a number of novel findings. First, it is the only work to demonstrate that in vivo PSS is higher in coronary atherosclerotic plaques with rupture vs. no rupture across a range of plaque subtypes and irrespective of whether analysis of the entire plaque or of regions close to the minimal luminal area is performed. Second, it shows that the pattern and extent of plaque progression and regression defined as an increase and decrease in plaque area, respectively, are associated with specific biomechanical environments at baseline, in the only study that examines the role of both PSS and WSS in this process. More specifically, high PSS is associated with changes consistent with increased vulnerability both in areas of progression and regression. On the other hand, lower WSS at baseline is associated with greater increases in plaque area and burden in areas that progress and with smaller decreases in areas that regress largely due to changes in fibrous tissue. Although the role of WSS in determining future plaque behaviour has been previously examined, this is the first time that this is assessed specifically in areas of progression and regression, particularly important in view of the dynamic nature of atherosclerotic plaques. More importantly, the work presented in this thesis demonstrates that the interplay of these biomechanical forces is associated with specific patterns of plaque progression and regression despite the fact that PSS and WSS are independent of each other. This has never been previously demonstrated and further suggests that incorporation of biomechanical analysis can play role in the identification of plaques that lead to future clinical events. Finally, the ability of PSS to identify plaques that lead to adverse clinical events was assessed through a propensity core matched analysis of the PROSPECT (A Prospective Natural-History Study of Coronary Atherosclerosis) study. The analysis presented here is the largest, most extensive and thus most significant work to ever examine this with results suggesting that incorporation of PSS and associated parameters can improve the capability of VH-IVUS to identify plaques that lead to such events. In summary, the results of this thesis suggest that coronary PSS plays an important role in the pathophysiology of plaque rupture, and that its incorporation in routine plaque assessment may improve our current ability to identifying coronary plaques that lead to future adverse clinical events. The interplay between PSS and WSS may also affect future plaque behaviour and in particular progression and regression. Prospective studies are now required to fully evaluate the role of these biomechanical forces in plaque development, and whether their incorporation in plaque evaluation can be of clinical significance.

Acute myocardial infarction is the commonest cause of death in firefighters, accounting for 45% of all deaths on duty. Compared with an average life expectancy of 77 years in the general population, the average age of cardiovascular death in firefighters is 50 years suggesting that occupational hazards are responsible for premature disease. The risk of acute myocardial infarction is increased 12- to 136-fold during rescue and firefighting duties, and is likely to reflect a combination of factors including strenuous physical exertion, mental stress, heat and pollutant exposure. Previous studies have established that the duties of a firefighter, in particular fire suppression, put inordinate strain on the cardiovascular system yet the exact mechanisms underlying the increased risk of myocardial infarction remain poorly defined. In a series of studies, I assessed the effect of occupation-specific risk factors on cardiovascular health in a combination of controlled and real-life studies in order to better define these mechanisms, hypothesising that exposure to high temperatures, strenuous physical exertion, psychological stress and air pollution either alone or in combination caused vascular dysfunction and thrombosis. In order to assess if firefighters had a greater cumulative risk of cardiovascular disease due to their occupation at baseline, I assessed the cardiovascular function of group of healthy, off-duty firefighters and compared this to a group of healthy age- and sex-matched off-duty police officers; an occupational group with similar responsibilities but a much lower risk of on-duty cardiovascular events. I was able to demonstrate that traditional cardiovascular risk factors, vascular endothelial function and thrombogenicity were similar in the two groups concluding that the excess of cardiovascular events and deaths in on-duty firefighters are due to the acute and transient effects of strenuous physical exertion, psychological stress, heat and exposure to air pollutants. Having established that off-duty firefighters had no apparent increased risk of cardiovascular events, I then went on to clarify the effects of combustion derived air pollution in the form of wood smoke on the cardiovascular system. The suppression of wildland or forest fires is globally the single most important duty of the fire service. Previous work within our institution has demonstrated the adverse effects of combustion derived air pollution, in the form of diesel exhaust, on the cardiovascular system. In a similar fashion, I assessed the effect of a wood smoke inhalation in a group of healthy off-duty firefighters by performing controlled exposures to wood smoke utilising a unique and well characterised facility. Interestingly, unlike diesel-exhaust, the exposure to wood smoke had no adverse effect on vascular endothelial function or thrombogenicity in this group concluding that cardiovascular events during wildland fire suppression may not be directly related to wood smoke inhalation but instead precipitated by other mechanisms such as strenuous physical exertion or dehydration. Latterly, I proceeded to evaluate the effects of strenuous physical exertion and heat exposure by comprehensively assessing a number of cardiovascular end points following controlled exposure to a fire simulation activity in a group of healthy, off-duty firefighters. I was able to demonstrate that exposure to extreme heat and physical exertion impaired vasomotor function and increased thrombus formation. Moreover, I demonstrated cardiac troponin concentrations increased suggesting that fire suppression activity may cause myocardial injury. These important findings suggest pathogenic mechanisms to explain the association between fire suppression activity and acute myocardial infarction. In the final phase of work, I endeavoured to assess the effects of real-life firefighter activities on the cardiovascular system. In an ambitious study, I attempted to undertake a comprehensive assessment of cardiovascular function in healthy firefighters following three periods of duty: fire suppression, alarm response and non-emergency activity. I was unable to complete enough studies to adequately power an analysis and draw any firm conclusions about the effect of these duties on cardiovascular health. Further work is required in a real-world setting to more clearly define the occupational risk factors underlying the increased risk of cardiovascular events associated with specific firefighter duties Understanding the biological mechanisms and environmental factors that predispose firefighters to cardiovascular events is essential if we are to develop effective methods for the prevention of acute myocardial infarction on-duty. This body of work has greatly improved the understanding of the mechanisms underlying the increased risk of cardiovascular events on duty and calls for the immediate evaluation of current practice in order to minimise risk to firefighters in the future. Examples of where improvements should be made include strategies to ensure adequate hydration and cooling following exposure to heat and physical exertion, change to working patterns to limit the duration of extreme exposures, and education, training and screening programmes to reduce the impact of traditional and occupational cardiovascular risk factors.

Myocardial infarction is a leading cause of morbidity and mortality worldwide. The purpose of this thesis was to develop strategies for the assessment of patients with suspected myocardial infarction using a high-sensitivity cardiac troponin I assay, and to evaluate the relationship between the aetiology of myocardial infarction and long term clinical outcomes to identify opportunities to modify outcomes. In the United Kingdom, approximately 1 million patients present to hospital with chest pain each year and are assessed for suspected myocardial infarction, yet fewer than 20% of patients receive this diagnosis. Prior clinical standards mandated the admission of patients for serial cardiac troponin testing to identify myocardial necrosis and determine if myocardial infarction had occurred. However, new high-sensitivity assays offer a magnitude improvement in diagnostic precision, and as such provide a novel approach to diagnose or exclude myocardial infarction at an earlier stage. In our first study, I evaluate the performance of a high-sensitivity cardiac troponin I assay as a risk stratification tool in patients with suspected acute coronary syndrome. A systematic review and individual patient-level data meta-analysis was performed, including prospective studies measuring high-sensitivity cardiac troponin I in patients with suspected acute coronary syndrome, where the diagnosis was adjudicated according to the universal definition of myocardial infarction. The primary outcome was myocardial infarction or cardiac death during the index hospitalization or at 30 days. Meta-estimates for primary and secondary outcomes were derived using a binomial-normal random effects model. Performance was evaluated in subgroups and across a range of troponin concentrations (2-16 ng/L) using individual patient data. A total of 22,457 patients were included in the meta-analysis (age 62 [15.5] years; n=9,329 (41.5%) women), of whom 2,786 (12.4%) experienced myocardial infarction or cardiac death at 30 days. Cardiac troponin I concentrations were < 5 ng/L at presentation in 11,012 (49%) patients, with a negative predictive value of 99.5% (95% confidence interval [CI] 99.3-99.6) for myocardial infarction or cardiac death at 30 days. Lower thresholds did not improve safety, but did significantly reduce the proportion identified as low risk. This threshold of 5 ng/L formed the basis for the development of a diagnostic pathway for patients with suspected acute coronary syndrome. In a cohort study of 1,218 patients with suspected acute coronary syndrome who underwent high-sensitivity cardiac troponin I measurement at presentation, 3 and 6 or 12 hours, I derived and validated a novel pathway (rule out myocardial infarction if < 5 ng/L at presentation, or change < 3 ng/L and < 99th centile at 3 hours), and compared this with the established European Society of Cardiology 3-hour pathway (rule out myocardial infarction if < 99th centile at presentation, or at 3 hours if symptoms < 6 hours). The primary outcome was a comparison of the negative predictive value (NPV) of both pathways for myocardial infarction or cardiac death at 30 days. The primary outcome was evaluated in pre-specified subgroups stratified by age, gender, time of symptom onset and known ischaemic heart disease. In those < 99th centile at presentation, the ESC pathway ruled out myocardial infarction in 28.1% (342/1,218) and 78.9% (961/1,218) at presentation and 3 hours respectively, missing 18 index and two 30-day events (NPV 97.9%, 95% confidence intervals [CI] 96.9-98.7%). The novel pathway ruled out 40.7% (496/1,218) and 74.2% (904/1,218) at presentation and 3 hours, missing two index and two 30-day events (NPV 99.5%, 95% CI 99.0-99.9%; P < 0.001 for comparison). The NPV of the novel pathway was greater than the ESC pathway overall (P < 0.001), and in all subgroups including those presenting early or known to have ischaemic heart disease. There are a number of additional approaches for the rule out of myocardial infarction. Clinical risk scores apply conventional risk factors to estimate the probability of myocardial infarction. The most widely implemented scores, HEART, EDACS, GRACE and TIMI, have been extensively validated when used alongside contemporary troponin assays, however, their impact on pathways applying high-sensitivity cardiac troponin testing is less clear. In 1,935 patients with suspected acute coronary syndrome, I evaluated the safety and efficacy of our novel pathway or the European Society of Cardiology 3-hour pathway alone, or in conjunction with low-risk TIMI (0 or 1), GRACE (≤108), EDACS (< 16) or HEART (≤3) scores. Myocardial infarction or cardiac death at 30-days occurred in 14.3% (276/1,935). The ESC pathway ruled out 70% with 27 missed events giving a negative predictive value (NPV) of 97.9% (95% confidence interval [CI], 97.1 to 98.6%). Addition of a HEART score ≤3 reduced the proportion ruled out by the ESC pathway to 25%, but improved the NPV to 99.7% (95%CI 99.0 to 100%, P < 0.001). The novel pathway ruled out 65% with three missed events for a NPV of 99.7% (95%CI 99.4 to 99.9%). No risk score improved the NPV, but all reduced the proportion ruled out (24-47%, P < 0.001 for all). Whilst myocardial infarction due to atherosclerotic plaque rupture and thrombosis (type 1) is well described, the natural disease course of myocardial infarction due to oxygen supply-demand imbalance without atherothrombosis (type 2) is poorly understood. I aimed to define long-term outcomes and explore risk stratification in patients with type 2 myocardial infarction and myocardial injury. Consecutive patients (n=2,122) with elevated cardiac troponin I concentrations (≥0.05 μg/L) were identified at a tertiary cardiac centre. All diagnoses were adjudicated as per the Universal Definition of Myocardial Infarction. The primary outcome was all-cause death. Secondary outcomes included major adverse cardiovascular events (MACE; non-fatal myocardial infarction or cardiovascular death) and non-cardiovascular death. To explore competing risks, cause-specific hazard ratios were obtained using Cox regression models. The adjudicated index diagnosis was type 1 or type 2 myocardial infarction or myocardial injury in 1,171 (55.2%), 429 (20.2%) and 522 (24.6%) patients, respectively. At five years, all-cause death rates were higher in those with type 2 myocardial infarction (62.5%) or myocardial injury (72.4%) compared with type 1 myocardial infarction (36.7%). The majority of excess deaths in those with type 2 myocardial infarction or myocardial injury were due to non-cardiovascular causes (HR 2.32, 95%CI 1.92-2.81, versus type 1 myocardial infarction). Despite this, the observed crude MACE rates were similar between groups (30.6% versus 32.6%), with differences apparent after adjustment for co-variates (HR 0.82, 95%CI 0.69-0.96). Coronary heart disease was an independent predictor of MACE in those with type 2 myocardial infarction or myocardial injury (HR 1.71, 95%CI 1.31-2.24). Patients with type 2 myocardial infarction were less likely to receive secondary prevention therapy, suggesting a treatment gap may exist and there may be potential to modify clinical outcomes. A risk stratification threshold has been defined using high-sensitivity cardiac troponin I which identifies patients at very low risk of myocardial infarction or cardiac death. A diagnostic pathway incorporating this risk stratification threshold appears safer than established guidelines which apply the 99th centile alone. The use of clinical risk scores does not appear to improve the safety of this approach, however, does significantly reduce efficacy. Overall, these findings demonstrate the potential of high-sensitivity cardiac troponin testing to improve the efficiency of the assessment of patients with suspected acute coronary syndrome without compromising patient safety. The observations in those with myocardial injury and infarction have identified a phenotype of patients with type 2 myocardial infarction and coronary artery disease who are at increased cardiovascular risk, and who may benefit from targeted secondary prevention. The studies presented will inform the design of future clinical trials, and may inform international guidelines for the assessment of patients with suspected acute coronary syndrome.

Unlike other regenerative organs such as the liver, the adult mammalian heart does not regenerate tissue lost following injury such as myocardial infarction (MI). Instead a non-contractile fibrous scar develops that in the longer term leads to the development of heart failure (HF). In contrast to the adult, neonatal mammals, including mice and man, retain potent cardiac regenerative capacities and can replace myocardium lost following injury. Understanding the mechanisms underlying scar free repair in the neonate may help in development of new approaches to reduce the impact of myocardial injury in adults. In this thesis MI was induced by coronary artery ligation in mice at post-natal day 1 (P1). Novel electrocardiogram gated high resolution cardiac ultrasound was developed to permit non-invasive confirmation of injury 1 day later and regeneration 21 days later by loss, then restoration, of contractile function. Macrophages (MФ) play important roles in organ growth and homeostasis, and are required for scar-free regeneration of the neonatal mouse heart following MI. WNTs are secreted lipophilic proteins with multiple roles in development. MФ-derived WNTs are essential for scar free tissue regeneration following injury in the kidney, liver, and gut, but their role in the heart is unknown. The primary aim of this thesis was to investigate the role of MФ, and in particular MФ-derived WNTs in determining normal growth of the myocardium from neonate to adult and also in regeneration of the neonatal heart following injury. In wild-type neonatal mouse hearts, Csf1r-expressing cells density (mostly macrophages) was consistent across all time points studied. Three populations of resident cardiac mononuclear phagocytes were identified by flow cytometry: F4/80hi, CD11blo, Ly6C-ve - F4/80lo, CD11bhi, Ly6C-ve - F4/80lo, CD11bhi, Ly6C+ve. F4/80hi, CD11blo, Ly6C-ve cells were hypothesised to correspond to yolk-sac derived mononuclear phagocytes and F4/80lo, CD11bhi, Ly6C-ve - F4/80lo, CD11bhi, Ly6C+ve to foetal liver/bone marrow derived mononuclear phagocytes. Three phases of myocardial growth were identified by ultrasound and histological techniques: hyperplastic (P2-P8, with increased Ki67 and cardiac troponin immunopositive cells), hypertrophic/reorganisation (P8-P21, with increasing cardiomyocyte size and no change in left ventricle wall thickness), and finally hypertrophic solely (P21-P42, with increasing cardiomyocyte size and left ventricle wall thickness). Average coronary vessel size was shown to decrease between P2 and P8 whilst vessel density was increased. The number of α-smooth muscle actin (αSMA) coated vessels greatly increased between P8 and P42, indicating vessel maturation. Throughout all phases cardiac systolic function was maintained at steady state. Diastolic function was however shown to mature from a foetal to an adult pattern between P2 and P8, with reversal of the E:A wave ratio on Doppler ultrasound. In mice globally deficient in MФ due to a germline knock-out of the Csf1r gene (Csf1rnull mice), both body and heart weights were decreased from P7 onwards. The number of proliferating (Ki67+ve) cardiomyocytes at P1 and P7 was unchanged in Csf1r-null mice but there was a trend towards decreased cardiomyocyte size at P7, suggesting an influence on hypertrophic rather than hyperplastic growth of the myocardium. There was also a trend for slowed vascular network maturation, with a delay in the shift from large to smaller vessels in hearts from Csf1r-null mice. In mice with MФ-directed (Csf1r-icre mediated) depletion of Porcupine (Porcn), a gene encoding an enzyme required for WNT acylation and secretion cardiac growth, vascularisation, fibrosis and function were all similar in Cre-ve and Cre+ve animals until P41, when cardiomyocyte size and cardiac systolic function were both significantly increased in Cre+ve animals. However, the underlying mechanism is unknown. In the neonatal mice, Csf1r expressing cells, mostly MФ, were identified in association with regenerating myocardium after induction of MI at P1. Flow cytometry data showed that by P7 the putative resident yolk-sac derived population had mostly disappeared from the heart and was replaced by F4/80lo cells, similar to the pattern reported in the adult. In the regenerating myocardium, Axin2 expression was increased consistent with activation of canonical Wnt signalling. Expression of Wnt5b and Fzd2 receptor, both associated with fibrosis, was significantly increased relative to age matched uninjured hearts. MФ-directed depletion of Porcn did not influence either the functional decrease at day 1 or recovery at day 21 following induction of MI at P1. Coronary re-vascularisation was also unaffected by the genotype. However, retention of intra-myocardial fibrosis (picrosirius red staining) was significantly increased in hearts at day 21 post-MI from mice with MФ-directed depletion of Porcn. MФ-derived WNTs are therefore required for scar-free wound healing in the heart, as they are in the liver and the kidney where they regulate matrix metalloproteinase activity. In summary, novel ECG-gated high-resolution in vivo ultrasound developed in this project has allowed characterisation of cardiac structure and function during early post-natal growth and following injury and regeneration in neonatal mice. The resident MФ population of the heart is established pre-natally, and may play a role in determining maturation of the developing vascular network, although this does not involve MФ-derived Wnt signalling. Following MI, the MФ population may expand from bone marrow cells and MФ accumulate around the regenerating tissue. MФ derived WNTs are not required for regeneration of the neonatal myocardium but do have a role in ensuring scar free wound healing and this merits further investigation.

Efferocytosis, the clearance of apoptotic cells including apoptotic neutrophils by macrophage phagocytosis, is a key cellular mechanism for resolution of inflammation and tissue repair. Cyclin-dependent kinases (CDKs) 7 and 9 phosphorylate RNA polymerase II that is vital for neutrophil transcriptional capacity. CDK inhibitors such as R-roscovitine, and the more selective inhibitor AT7519, induce neutrophil apoptosis and promote resolution of several mouse models of inflammation including acute lung inflammation. The hypothesis investigated here was that AT7519 would promote neutrophil apoptosis (i) in the infarcted heart, leading to macrophage polarisation, angiogenesis, reduced infarct expansion and retention of cardiac function and (ii) in the peritoneum, enhancing resolution of sterile peritonitis. AT7519 (1μM) induced apoptosis of mouse unstimulated-bone marrow derived neutrophils and thioglycollate-stimulated neutrophils in vitro in a time- and caspase-dependent manner, but did not alter activation assessed by calcium flux in response to the synthetic formyl peptide (fMLF) or platelet-activating factor (PAF). Only high concentrations of AT7519 (10 μM) induced monocyte/macrophage apoptosis and this was likely due to saturated phagocytosis of apoptotic neutrophils induced by high concentration of AT7519. Myocardial infarction (MI) was induced by coronary artery ligation in adult male mice and infarct volume was assessed 7 or 21 days later by in vitro optical projection tomography (OPT). The novel use of OPT for this purpose was validated by demonstrating correlation with infarct volume obtained by late-gadolinium enhanced magnetic resonance imaging in vivo and with infarct area assessed by histological staining (Masson’s Trichrome) in tissue sections. AT7519 (30 mg/kg i.p.) increased the number of apoptotic neutrophils (cleaved caspase-3 and Ly6G +ve) in the heart when administered after MI, but this was not associated with any subsequent alteration in macrophage polarisation, vessel density, infarct expansion or structural and functional remodelling of the left ventricle. In contrast, induction of neutrophil apoptosis by AT7519 (30mg/kg i.p.) successfully promoted macrophage polarisation and the resolution of inflammation associated with peritonitis elicited by either 10% thioglycollate or by 1mg zymosan. AT7519 treatment also reduced the number of CD19+ B cells, Foxp3+CD4+ T cells and eosinophils in peritoneal lavage, and prolonged the phase of monocyte recruitment in zymosan-induced peritonitis. In conclusion, AT7519 successfully induced mouse neutrophil apoptosis in vitro, as well as in vivo in experimental MI and peritonitis. Subsequent promotion of inflammation resolution in peritonitis was not matched by improved outcome following MI. Unexpected effects of CDK inhibition on monocytes, T cells and eosinophils that are necessary for myocardial infarct repair may have compromised any beneficial effects resulting from promotion of in situ neutrophil apoptosis. CDK inhibition may therefore have therapeutic potential for the treatment of peritonitis, but not for prevention of infarct expansion and detrimental ventricular remodelling after MI.

Cardiac troponins are released into the circulation following myocardial injury with measurement of serum troponin concentration integral to the diagnosis of myocardial infarction. The next generation of high-sensitivity cardiac troponin assays have enhanced precision at very low concentrations and for the first time permit troponin concentrations to be reliably quantified outwith acute coronary syndromes. My aim was to evaluate the impact of more sensitive troponin assays on the diagnosis of myocardial infarction and to determine the potential for novel applications in stable cardiovascular diseases. In 2,929 consecutive hospitalised patients with myocardial injury I assessed the impact of implementing a contemporary sensitive troponin assay and a lower diagnostic threshold, on the incidence, management and outcome of patients with primary (type 1) and secondary (type 2) causes of myocardial infarction. Lowering the threshold improved outcomes in patients with type 1 myocardial infarction, but disproportionately increased the diagnosis of type 2 myocardial infarction, and identified a group of patients who remained at high-risk of death over the next 12 months. In 1,126 consecutive patients with suspected acute coronary syndrome I evaluated the impact of a high-sensitivity cardiac troponin I assay and sex-specific diagnostic thresholds on the diagnosis of myocardial infarction. Whilst having little effect in men, the use of sex-specific diagnostic thresholds would double the diagnosis of myocardial infarction in women, identifying those at increased risk of recurrent events. In a second cohort of 4,870 patients, I evaluated the optimal threshold to rule out myocardial infarction. The negative predictive value for myocardial infarction or cardiac death at 30 days was evaluated for a range of troponin concentrations. Troponin concentrations < 5 ng/L on presentation identified half of all patients with suspected acute myocardial infarction as low risk. Implementation of this assay and approach would markedly reduce hospital admissions and improve the diagnosis of myocardial infarction, particularly in women. High-sensitivity cardiac troponin assays may help us to manage patients with chronic cardiovascular diseases, such as aortic stenosis or stable angina pectoris. In patients with aortic stenosis, cardiac troponin was associated with an advanced hypertrophic response on electrocardiography and the presence of myocardial fibrosis on cardiac magnetic resonance imaging. Furthermore, cardiac troponin concentrations were a strong independent predictor of cardiovascular death or aortic valve replacement. In patients with stable angina, cardiac troponin was associated with transient ST-segment depression on ambulatory monitoring, suggesting that troponin concentrations may be modified by silent myocardial ischaemia. My findings suggest that high-sensitivity troponin assays have major potential to improve the assessment, treatment and outcome of patients with suspected acute coronary syndrome. The potential for cardiac troponins to improve risk stratification and monitoring of disease progression in patients with chronic cardiovascular disease now needs to be evaluated in prospective cohort studies.

Glucocorticoid production by the adrenal gland is regulated by hypothalamicpituitary- adrenal (HPA) axis activity. Within cells, glucocorticoid levels are modulated by 11β-hydroxysteroid dehydrogenase (11β-HSD), which interconverts active and intrinsically inert glucocorticoids. Glucocorticoids have widespread physiological effects and, in the cardiovascular system, they play a crucial role in heart development and maturation, blood pressure control, and myocardial calcium cycling. Mice which are unable to regenerate the physiological glucocorticoid, corticosterone, from 11-dehydrocorticosterone due to deletion of the type 1 11β-HSD isozyme (11β-HSD1) have previously been shown to have smaller, lighter hearts but unaltered systolic function. Moreover, a single nucleotide polymorphism (SNP) in the Hsd11b1 gene has been associated with reduced left ventricular mass in humans, suggesting a role for 11β-HSD1 in regulating cardiac size. After myocardial infarction (MI), 11β-HSD1 deficient mice have an augmented inflammatory response, increased numbers of pro-reparative alternatively-activated macrophages in the heart, enhanced peri-infarct angiogenesis and improved cardiac function compared to C57BL/6 controls. However, the role of ‘cardiovascular’ 11β-HSD1 in the development of these phenotypes, both basally and after MI, is unknown. It was hypothesised that ‘cardiovascular’ 11β-HSD1 deficiency would result in smaller hearts, and that this selective deletion would lead to altered calcium handling protein expression and diastolic abnormalities. Furthermore, it was hypothesised that ‘cardiovascular’ 11β-HSD1 deletion would reproduce the beneficial post-MI phenotype previously seen in global 11β-HSD1 deficient mice. The first aim was to characterise the cardiac phenotype of mice with global deletion of 11β-HSD1 (DelI mice), and mice in which deletion is restricted to cardiomyocytes and vascular smooth muscle cells (SMAC mice). SMAC mice have ‘floxed’ 11β- HSD1 alleles and a Cre recombinase transgene inserted into the Sm22α gene. Sm22α is expressed in vascular smooth muscle cells, and transiently in cardiomyocytes during development. Thus, Cre expression in these cells results in deletion of exon three of the Hsd11b1 gene and gives rise to a non-functional protein. Controls for DelI mice were C57BL/6 mice, and controls for SMAC mice were their Cre- littermates. DelI, but not SMAC, mice have smaller, lighter hearts, which may be explained by their shorter cardiomyocytes measured following isolation using a Langendorff preparation. Cardiomyocyte cross-sectional area is unchanged. In vivo measurement of cardiac function using ultrasound imaging showed systolic function is comparable between DelI mice and SMAC mice and their respective controls. However, there is mild diastolic dysfunction in both DelI and SMAC mice, characterised by reduced E wave deceleration and an increased mitral valve deceleration time. This phenotype occurred following pharmacological inhibition of 11β-HSD1, by administration of UE2316, a selective 11β-HSD1 inhibitor, to adult C57BL6/SJL mice. While ventricular collagen content is unaltered in DelI, SMAC and UE2316-treated mice compared to their respective controls, expression of sarcoplasmic reticulum Ca2+ ATPase (SERCA) is reduced, suggesting that altered calcium handling, rather than changes in stiffness, may underlie this phenotype. The second aim was to determine whether the beneficial acute outcomes seen previously in 11β-HSD1 deficient mice after MI could be reproduced by selective cardiovascular deletion of the enzyme. Seven days after MI, compared to Cre- littermate controls, SMAC mice have similar peri-infarct angiogenesis, total macrophage and alternatively-activated macrophage infiltration into the heart, infarct size, ventricular dilatation and systolic function. This suggests 11β-HSD1 deletion in another cell type, or types, is responsible for the phenotype seen in global 11β-HSD1 deficient mice. The final aim was to assess the impact of global 11β-HSD1 deficiency and ‘cardiovascular’ 11β-HSD1 deletion on the development of heart failure, using magnetic resonance imaging to determine structure and function. Eight weeks after MI, mice globally deficient in 11β-HSD1 have attenuated expression of ANP and β- MHC, RNA markers of heart failure, and show attenuated pulmonary oedema, reduced chamber dilatation, preserved systolic function and smaller infarcts compared to control. None of these parameters are altered in SMAC mice relative to control. In conclusion, the data presented in this thesis shows that cardiovascular 11β-HSD1 influences physiological cardiac function, potentially through regulation of calcium handling. 11β-HSD1 in other cells influences cardiomyocyte length, resulting in smaller hearts in its absence. Despite this, global 11β-HSD1 deficiency prevents heart failure development after MI, suggesting that pharmacological inhibition of 11β-HSD1 may be of benefit in treating this condition. Cardiovascular 11β-HSD1 does not, however, account for the changes in infarct healing or remodelling associated with this beneficial outcome, therefore these effects must be related to 11β-HSD1 deficiency elsewhere, such as fibroblasts or myeloid cells.

Based on data from the World Healthcare Organisation, cardiovascular diseases are the primary cause of disease-related death globally, with myocardial infarction (MI) being the most prevalent. If not treated effectively, MI can progress to heart failure (HF). With 70 million prescriptions for HF in 2014 and 515 people in the UK being hospitalised daily with MI, the British Heart Foundation calls for novel robust treatments. Even though cardiac stem cell (CSC) therapy for MI has been under investigation for more than a decade, there still has not been a consensus over the identity of the adult endogenous CSC. Recent clinical trials, using selected Ckit+ cells or the cardiosphere-derived cells (CDCs) have shown moderate results. The aim of this thesis was to develop a digestion-based method for isolation of cardiac progenitor cells (CPCs) from the mouse atria. The resulting "CTs" were isolated by collagenase/trypsin (where their name has resulted from) digestion with a prolonged period step for cell attachment. CTs were compared to isolated CDCs for their marker expression, using RT-PCR and Immunocytochemistry, showing cells with a mesenchymal phenotype which expressed SCA1 and CKIT. The CDCs had more of a fibroblast phenotype with higher Ddr2 and Wt1 expression. Using a TGF-β1 differentiation protocol, the CTs could be differentiated more effectively to a CM lineage than could the CDCs. In addition, Oleic acid (OA) supplementation stimulated the Peroxisome proliferator-activated receptor alpha pathway and led to maturation of the CT cells, both before and after differentiation. The differentiated CTs begin to express Tnnt2, while OA led to Myh7 increase and upregulated their oxidative metabolism. Finally, the CTs were more able to survive under serum-starvation than the CDCs, and transfection with miR-210 could enhance CT survival under these conditions and increased VEGF secretion. By digestion of the whole atria and allowing a prolonged time for attachment, we have developed a novel isolation protocol which generates a cell population containing a range of progenitors. Cells within this population can survive under serum starvation and can be differentiated to a CM lineage, making them a promising therapeutic population.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter that regulates vascular function and blood pressure, and also protects the heart from injury associated with myocardial infarction (MI). The mitochondrial enzyme thiosulfate sulfurtransferase (TST) has a putative role in the breakdown of H2S but its role in the cardiovascular system is unknown. I hypothesised that TST reduces cardiovascular H2S availability and that inhibiting TST activity may therefore ameliorate cardiovascular pathology. In the heart, TST was expressed by cardiomyocytes and vascular smooth muscle cells. Tst-/- mice all survived to adulthood and had normal cardiac structure and function. Cardiac and hepatic H2S breakdown rates were reduced and H2S levels were higher in the blood of Tst-/- mice. However, in heart tissue, protein levels for the H2S-activated Nrf2 downstream targets, thioredoxin (Trx1) and heme oxygenase-1 (HO-1) were comparable. In contrast, protein levels for the cardiac specific H2S-synthetic enzyme, cystathionine gamma lyase (CSE) was reduced, suggesting a homeostatic negative feedback mechanism to maintain H2S at non-toxic levels. Respiration, measured using an oxygen-sensing electrode was normal in isolated mitochondria from whole Tst-/- compared to control C57BL6 hearts. Endothelial nitric oxide synthase (eNOS) protein expression was lower in Tst-/- hearts, highlighting potential cross talk between H2S and nitric oxide (NO) signalling. TST was expressed in whole aorta homogenates and in isolated endothelial cells from aorta and small intramuscular vessels of the hindlimb from C57BL/6N control mice. Myography and western blotting revealed a greater influence of NO in aorta from Tst-/- mice that was associated with increased phosphorylation of the activating serine1177 residue of eNOS (PeNOSSer1177). NO plays a lesser role in resistance arteries, but in comparison to control vessels, small mesenteric vessels from Tst-/- mice was more reliant on small and intermediate calcium activated potassium channels for relaxation. Tst-/- mice were normotensive, despite this alteration in the regulation of vascular tone. However, metabolic cage experiments identified that Tst-/- mice presented with diuresis, polydipsia, and increased urinary electrolyte excretion of sodium, potassium and chloride, possibly to compensate for increased vascular tone in order to maintain stable blood pressure. To investigate the role of TST in regulating the response to pathological challenge, MI was induced by coronary artery ligation (CAL). In control mice, gene expression of CSE was downregulated by 2 days after CAL, but TST expression was 12-fold increased, suggesting regulation of H2S bioavailability during the acute MI-healing phase. Tst-/- male mice had a 40% greater incidence of cardiac rupture during infarct healing and surviving Tst-/- mice had greater left ventricular dilatation and impaired function compared to controls. Ex vivo, isolated perfused hearts from Tst-/- mice were more susceptible to ischaemia/ reperfusion injury, suggesting an additional role of TST in determining cardiomyocyte susceptibility to injury. In conclusion, these data indicate that cardiovascular H2S bioavailability is regulated through degradation by TST. The data presented here provide evidence for significant tissue specific crosstalk between H2S synthetic and degradative mechanisms and between H2S and other local regulatory mechanisms, including ion channels and NOS. We infer TST has a physiological role in the kidney where its loss leads to changes in renal electrolyte and water handling, although other compensatory mechanisms prevent a change in blood pressure. Under conditions of pathological challenge following MI, loss of TST is detrimental, illustrating its key role in removal of H2S. The data refute the original hypothesis that TST inhibition would be protective against cardiovascular pathology. Further studies in mice with tissue specific deletion of TST are now required to more fully reveal the cardiovascular role of TST.

One of the most prevalent pollutants in urban cities is diesel exhaust particulate (DEP). Air pollution has been linked with increased risk of recurrent myocardial infarction (MI) and MI related death (Brook, 2008). This may be due, in part, to effects on atherosclerotic plaque stability and blood clotting tendency. Whether exposure to DEP changes the response of the heart to ischaemia, resulting in increased damage after MI is less well documented. The work described in this thesis was designed to investigate the hypothesis that pulmonary instillation of DEP would increase vulnerability of the heart to subsequent myocardial reperfusion injury secondary to activation of a systemic inflammatory response, endothelial dysfunction and triggering of transient receptor potential vanilloid 1 (TRPV1) mediated autonomic reflexes in the lung. Examination of bronchoalveolar lavage (BAL) fluid revealed pulmonary inflammation 6 h after exposure to DEP, characterised by neutrophil infiltration, raised levels of the inflammatory mediator interleukin-6 (IL-6) and an increase in alveolar permeability demonstrated by increased levels of protein in the lavage fluid. Pulmonary inflammation was largely resolved 24 h after exposure. While there was no indication of systemic inflammation at 6 h after DEP instillation, the levels of two inflammatory mediators, IL-6 and tumour necrosis factor alpha (TNFα) were increased in the plasma by 24 h after exposure. DEP had no affect on blood flow responses to the endothelium dependent dilator acetylcholine (ACh) in rat hind-limb vasculature in vivo at 6 or 24 h. In summary, while exposure of rats to DEP can induce both pulmonary and systemic inflammation, it does not modify endothelium-dependent vasodilatation. Ischaemia-reperfusion (I/R) was induced in vivo in anaesthetised rats and ex vivo in buffer perfused hearts from rats that had received DEP in vivo 6 h earlier. In both in vivo and ex vivo I/R models, infarct size (unstained by triphenyltetrazolium choride) was significantly increased in hearts from DEP-instilled rats relative to hearts from saline-instilled or non-instilled rats. Baseline oxidant stress, determined by electron paramagnetic spin resonance (EPR) in heart perfusate, was also significantly higher in perfusate of hearts from DEP-instilled rats. In summary, a single exposure of the lung to DEP leads to priming of the myocardium for I/R injury. As the results cited above illustrated, priming of hearts appeared unlikely to be due to either coronary vascular endothelial dysfunction or systemic inflammation. At 6 h post exposure, DEP was associated with increased blood pressure and myocardial hypersensitivity to ischaemia-induced arrhythmias, both suggestive of sympathetic activation. The beta 1 (β1) selective blocker metoprolol was used to investigate the role of the sympathetic nervous system (SNS) in transmitting the influence of DEP in the lung to the myocardium via β1 adrenoceptor activation. Administration of metoprolol (10 mg/kg, intraperotineal) at the time of DEP instillation into the lung was found to protect the heart from potentiation of ex vivo reperfusion injury. Metoprolol was also effective in reducing oxygen free radical generation from these hearts. The TRPV1 antagonist AMG 9810 was also used to study the role of TRPV1 receptors in mediating the priming influence of pulmonary DEP to the myocardium since activation of sensory receptors have been reported to modify sympathetic output via feedback to the central nervous system (Widdicombe et al., 2001). Coadministration of AMG 9810 (30 mg/kg) in vivo with DEP into the lung was found to prevent enhancement of ex vivo reperfusion injury associated with DEP instillation alone. Collectively these results have demonstrated that a single exposure of the lung to DEP leads to priming of the myocardium for I/R injury. Furthermore, this priming occurs via activation of a pulmonary sensory reflex that is likely to involve secondary activation of systemic β1 adrenoceptors.

Aims There is a growing body of evidence that some individuals are at risk of developing post-traumatic stress disorder (PTSD) after life-threatening cardiac events, such as myocardial infarction (MI) or cardiac arrest, which can result in distress, dysfunction and increased risk of mortality. In relation to this population, this thesis had two aims: to review the evidence regarding whether pain during MI predicts post-traumatic stress symptoms; and to explore the characteristics and impact of traumatic imagery experienced by individuals who develop symptoms of post-traumatic stress subsequent to MI or cardiac arrest. Methods A review of the evidence relating to pain as a potential risk factor for PTSD subsequent to MI is presented in the systematic review. The findings from a qualitative study investigating the characteristics of traumatic imagery and associated behaviours experienced by individuals who have symptoms of post-traumatic stress subsequent to MI or cardiac arrest, are presented in the journal article. Interpretative Phenomenological Analysis (IPA) was used to identify themes in the data. Results The systematic review indicated that there are mixed findings for pain as a risk factor for PTSD subsequent to MI. The limited number of studies in this area and significant methodological limitations within the existing evidence make it difficult to draw any firm conclusions with regard to the relationship between pain and PTSD post-MI. With regard to the qualitative study, the majority of imagery related to flashbacks of the event and were focused mainly on external experiences. Themes arising from the distressing flashback imagery included: loss of control; realisation of threat; negative impact on others; physical sensations; and actions of others. Imaginary elements and distortions were a feature of some traumatic imagery experienced, and non-flashback imagery connected with mortality was also experienced. Imagery was associated with avoidance behaviour and affected behaviour within relationships. Conclusions Findings from the systematic review indicate that further studies are warranted in this area to establish the link between pain and PTSD post-MI. These studies should seek to address methodological limitations of the current evidence by using a standardised pain measurement tool; adopting a prospective design; using a diagnostic tool to measure PTSD; ensuring PTSD is measured at least one month after the MI; assessing prior PTSD of non-cardiac origin; including a sufficient sample size and using an appropriate method of recruitment to improve generalisability. External experiences during a cardiac event are the main focus of traumatic visual imagery experienced by people with intrusive post-traumatic stress symptoms post cardiac event. Specific aspects of the cardiac event may be particularly distressing and these may be represented in post-traumatic visual imagery. Both gradual exposure and imagery rescripting techniques may be useful for reducing distress associated with the imagery, depending on the type of imagery experienced.

Background: Stem cells are a promising therapy for regeneration following myocardial infarction (MI). Another therapy currently under investigation for MI is glucagon-like peptide-1 (GLP-1), a natural incretin hormone that has cardio-protective properties, although a short half-life in vivo. GLP-1 CellBeads are a novel therapy, combining stem cells and GLP-1. Human mesenchymal stem cells (MSCs) were immortalised, engineered to secrete a fusion protein of GLP-1 and encapsulated in alginate. We have previously demonstrated that GLP-1 CellBeads significantly reduce infarct size and improve ejection fraction post-MI, but the underlying mechanisms are unclear. The therapy was assessed in an in vivo pig MI model and an in vitro cardiomyocyte ischaemia model. Methods: GLP-1 CellBeads were delivered to coronary artery branches in pigs, creating micro-infarcts, as determined by echocardiography. Cell-free beads (Beads) and CellBeads containing hMSCs without GLP-1 (Beads-MSC) were delivered as controls (n=3-5/group). Pigs were sacrificed one and four weeks post-MI. Tissue was analysed for: apoptosis, collagen, cardiomyocyte cross sectional area and myofibroblasts. The localised response around the beads was also measured using immunohistochemistry. Atomic force microscopy (AFM) was used to examine the ultra-structure of the collagen scar. The expression profiles of genes involved in collagen remodelling were measured using qRT-PCR. Viability of MSCs was measured using GFP-tagging and confirmed using qRT-PCR. To examine effects on apoptosis in vitro, human adult cardiomyocytes underwent ischaemia for 1 hour before incubation with: media conditioned with MSCs or MSC+GLP-1, GLP-1, Exendin-4 or media. Apoptosis and viability were measured at 24 and 48 hours respectively. Results: In the in vivo pig model, significant increases in apoptosis were observed in the infarct of all groups one week post-MI, with no differences between treatments. Despite decreased numbers of myofibroblasts, significantly more collagen was observed in MSC treated groups, with increased collagen fibril periodicity and a more organised collagen scar. The altered scar structure was reflected in differences in gene expression between groups, with an accelerated healing response in the MSC groups. However, significantly fewer myofibroblasts were observed in the MSC treated groups. Viability of MSCs was confirmed up to four weeks post-infusion, with GLP-1 secretion confirmed up to one week. In the in vitro ischaemia model, MSC+GLP-1 conditioned media significantly reduced cardiomyocyte apoptosis 24 hours post-ischaemia, compared to media alone. All agonists (GLP-1, MSC media and MSC+GLP-1 media) significantly improved viability compared to media alone 48 hours post-ischaemia. Conclusions GLP-1 CellBeads have a beneficial effect on healing following MI by significantly decreasing infarct size and improving ejection fraction post-MI. these benefits are associated with decreased cardiomyocyte apoptosis and altered collagen scar formation. The CellBeads act as local hubs for regeneration and are viable up to one month post-infusion. The effects observed are due to a combination of the GLP-1 and paracrine factors released from the hMSCs.

Ischaemic heart disease (IHD) is the leading cause of death worldwide. Currently, even optimal medical therapies do not attenuate deterioration of the left ventricular (LV) function completely. Stem cell therapies, and recently cardiac stem cell therapies, have emerged as potential novel treatments for IHD. However, clinical evidence from randomised controlled studies has shown mixed results. Thus understanding what patient-related factors may affect the therapeutic performance of the cells may help improving treatment outcomes. The studies described in this thesis aim to understand how cardiac progenitor cells (CPCs) can re-vascularise ischaemic myocardium and promote functional repair of the heart. Resident CPCs were isolated and expanded from the right atrial appendage of 68 patients following the ‘cardiosphere’ method (cardiosphere-derived cells or CDCs). They resemble mesenchymal progenitors as they lack the expression of endothelial and haematopoietic cell surface markers but express mesenchymal progenitor cell markers (e.g. CD105, CD90). Cell function was evaluated by support of angiogenesis, mesenchymal lineage differentiation potential in vitro, and improvement in heart function in vivo. Notably in vitro, CDC from different patients differed in their angiogenic supportive and differentiation potentials. In a rodent model of myocardial infarction (MI), transplantation of CDC reduced infarct size significantly (p<0.05). However, only those CDCs with a robust pro-angiogenic ability in vitro improved vessel density and heart systolic function (p<0.05) in vivo. A multiple regression model, which accounted for 51% of the variability observed, identified New York Heart Association (NYHA) class, smoking, hypertension, type of ischaemic disease and diseased vessel as independent predictors of angiogenesis. In addition, gene expression analyses revealed that differential gene expression of several extracellular matrix components (e.g. CUX1, COL1A2, BMP1 genes and microRNA-29b) could explain the differences observed in CDC’s vascular supportive function. In summary, this is the first description of variability in the pro-angiogenic and differentiation potential of CDCs and its correlation with their therapeutic potential. This study indicates that patient stratification may need to be included in the design of future trials to improve the efficacy of cell-based therapies.

Acute myocardial infarction (AMI) results in the activation of the innate immune system with monocytes playing critical roles in both the initial inflammation following myocardial ischaemia and subsequent recovery. Monocytes are a heterogeneous cell population and observations from experimental models demonstrate that immediately following myocardial injury, classical inflammatory monocytes, which are highly phagocytic, are recruited to ischaemic myocardium from the bone marrow and spleen and peak at 48 hours. This is followed by the recruitment of non-classical monocytes that are involved in repair and healing, peaking at day 5. The monocyte response in humans following AMI is currently poorly understood. Due to their central role in the pathogenesis of AMI, monocytes are attractive both as potential biomarkers to inform of extent of myocardial injury (and recovery) and also as therapeutic targets with the specific targeting of monocytes in experimental models resulting in reduced infarction size and improved LV remodelling. However, in spite of these promising results and our greater understanding of the pathogenesis of AMI, no immune-modulating therapeutic has been translated into routine clinical practice. We therefore hypothesized that characterisation of the monocyte response to AMI by flow cytometry and gene expression profiling in both experimental models and humans would give novel insights into underlying biological processes and function (both locally in the myocardium and systemically), identify novel therapeutic targets, enable their use as cellular biomarkers of disease, and test conservation between species validating the experimental model for future investigation. Classical inflammatory monocytes were found to significantly increase in the peripheral blood 48 hours following AMI in both mice and humans, with the magnitude of the monocyte response correlating with the extent of myocardial injury in both species. Gene expression profiling of peripheral circulating monocytes following AMI identified a number of candidate genes, biological pathways and upstream regulators that were conserved between species and that could represent novel therapeutic targets. Furthermore, in an experimental model of AMI, leukocytes appeared to have effects beyond the ischaemic myocardium, with leukocyte recruitment in remote myocardium and in kidneys associated with elevated inflammatory markers and endothelial activation.

Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs would be most beneficial if administered soon after infarction, thus the aim of this project was to optimize CSC culture conditions to enhance their therapeutic potential for myocardial infarction. CSCs were isolated and expanded in vitro via the formation of cardiospheres to give cardiosphere-derived cells (CDCs). Neonatal rat CDCs were found to be heterogenous, containing cells expressing the cardiac stem cell marker, c-Kit, pluripotent cell markers, Oct-4, Sox 2, Klf-4 and Nanog, and early cardiac specific differentiation markers, Nkx 2.5 and GATA 4. Administration of CDCs to the infarcted rat heart increased the cardiac ejection fraction by 9%, capillary density by 9% and reduced scar volume by 33%, compared to the non-treated group. The proliferation rates and the expression of c-Kit were significantly decreased in CSCs isolated from aged rats and after extended culture in vitro, so, CSC culture was optimized using hypoxic preconditioning. Under hypoxia, CDC proliferation rates were 1.7-fold greater, and larger cardiosphere clusters were formed. Hypoxic CDCs had an increased cardiac stem cell population, in that c-Kit was increased by 220% and CD90 and CD105 were decreased by 55% and 35%, respectively, compared to normoxic CDCs. Further, hypoxia induced the expression of CXCR-4 (~3.2-fold), EPO (~3.0-fold) and VEGF (~1.5-fold), indicating that hypoxic preconditioning may stimulate stem cell homing and neovascularization in the infarcted myocardium. Notably, hypoxic CDCs were able to switch to anaerobic glycolytic metabolism and had approximately 80% lower oxygen consumption, suggesting that they may be better adapted to survive within the hypoxic infarct scar, compared with normoxic CDCs. Culture of CDCs with hypoxia-mimicking prolyl-4-hydroxylase inhibitors (PHDIs) using DMOG, BIC and a novel compound, EDBA, induced similar effects to hypoxic culture by increasing c-Kit, EPO, VEGF, CXCR-4, decreasing CD90 and CD105 and increasing glycolytic metabolism. However, PHDI treatment for 24 hours did not alter CDC proliferation rates and cells died after 24 hours. In conclusion, CDCs are a potential cell source for therapy after myocardial infarction and their therapeutic potential can be enhanced using hypoxia or PHDI-preconditioning techniques.

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.

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.

Glucocorticoids can reduce infarct size when given immediately after myocardial infarction (MI) but are detrimental when administration is continued into the post-infarct healing phase. A number of experimental studies have shown that reduction of infarct expansion by enhancing blood supply to the infarct border reduces remodelling and improves heart function post-MI. Previous experiments from this laboratory have shown that mice unable to locally regenerate corticosterone due to deficiency in 11β-hydroxysteroid dehydrogenase type 1 (11HSD1) have an enhanced angiogenic response during myocardial infarct healing that is associated with improved cardiac function. We hypothesized that the enhanced angiogenic response in 11HSD1 knock out (-/-) mice would be preceded by augmented inflammation. Moreover this would be associated with improved cardiac function. This thesis aimed firstly to establish that murine cardiac phenotype was not influenced by 11HSD1 deficiency. 11HSD1-/- and C57Bl6 control mice had comparable cardiac structure and function. 11HSD1 expression was localised to fibroblasts and vascular smooth muscle cells in the myocardium. The second aim of this thesis was to characterise the healing response after MI in 11HSD1-/- mice compared to C57Bl6 mice. Neutrophil infiltration peaked 2 days after MI and was significantly enhanced in the 11HSD1-/- mice relative to C57Bl6 mice, despite comparable infarct size in both groups. This was followed by increased macrophage accumulation in the infarct border. Furthermore, in the 11HSD1-/- mice a greater proportion of macrophages were of the alternatively activated phenotype. Left ventricular expression of pro-angiogenic IL-8, but not VEGF, was increased. Cellular proliferation and vessel density at 7 days were greater in 11HSD1-/- compared to C57Bl6 hearts. This was associated with improved cardiac function 7 days post-MI. The third aim of this thesis was to determine whether the enhancement in vessel density and cardiac function was maintained beyond the initial wound healing phase. 11HSD1-/- mice retained the increased vessel density compared to C57Bl6 mice and these vessels were smooth muscle coated suggesting vessel maturation. This was associated with sustained improvement in cardiac function and modification of the scar characteristics. The final aim of this thesis was to establish whether the effect of the knock out could be recapitulated by administration of a small molecule inhibitor of 11HSD1 after MI. Oral administration of the 11HSD1 inhibitor had no effect on inflammation, angiogenesis and heart function as determined at 7 days post-MI relative to vehicle treated animals. In conclusion, the data confirm the enhancement in vessel density and cardiac function in 11HSD1-/- mice and demonstrate that this was preceded by enhanced inflammation. This was not due to an underlying cardiac phenotype or modification of the infarct size. Increased infiltration of alternatively activated macrophages may have been the source of pro-angiogenic factor, IL-8, which was also increased at the time of angiogenesis. Importantly the enhanced vessel density was retained 4 weeks after MI, these vessels were mature suggesting longevity and the improvement in cardiac function was retained. While pharmacological inhibition did not recapitulate the effect of the knock out this may have been due to route of administration. The data provides compelling evidence that further development and use of small molecule inhibitors of 11HSD1 may be of benefit post-MI.

Background: Diagnosis of acute coronary syndromes (ACS) in the Emergency Department (ED) is a topical and contentious issue. Current diagnostic techniques rely on hospital admission for troponin testing. Only a minority of those admitted prove to have ACS while unacceptable proportions of those discharged have unrecognised ACS. Aims: We aimed to evaluate the diagnostic and prognostic value of individual clinical findings and novel biomarkers in ED patients with suspected cardiac chest pain. We then aimed to derive a clinical decision rule (CDR) to potentially enable safe, immediate discharge of a proportion of patients from the ED while risk stratifying others to facilitate triage to an appropriate level of in-patient care. Methods: We recruited patients who presented to the ED with suspected cardiac chest pain. Variables that have previously been shown to predict diagnosis of acute myocardial infarction (AMI) or to predict outcome were prospectively recorded. Blood was drawn at presentation for levels of eight biomarkers. Patients underwent 12-hour troponin testing and were followed up for the composite primary outcome of AMI, death or urgent coronary revascularisation for six months. Variables that were univariate predictors (p<0.05) of outcome were entered into a multivariate analysis using recursive partitioning. Results: While many clinical findings and levels of all eight novel biomarkers were found to be significant predictors of outcome, none could be used individually to confirm or exclude ACS in the ED. We derived a nine-point CDR that combined clinical findings with biomarker levels to effectively stratify patients into four risk groups. 14.2% of patients were identified as being at ‘no risk’ and had a 0.0% outcome rate. The rule performed significantly better than two commonly used risk scores and may improve on triage decisions made in actual clinical practice. Conclusion: ACS remains a difficult diagnosis to confidently confirm or refute in the ED. Our CDR may help to avoid unnecessary hospital admissions while improving on triage decisions made for the remaining in-patients. Prospective validation of our findings is warranted.

Maternal mortality remains high in the US and pregnancy-associated myocardial infarction accounts for over 20% of maternal cardiac deaths. The relative risk of myocardial infarction during pregnancy is approximately 3 to 4-fold higher than the rates of age-matched non-pregnant individuals in the reproductive age group. During myocardial infarction, ischemia restricts blood flow to the myocardium, necessitating timely reperfusion therapy to restore blood flow. However, reperfusion therapy can exacerbate tissue damage. Our lab has demonstrated that cardiac vulnerability to ischemia reperfusion injury drastically increases in late pregnancy, and one bolus of intralipid at reperfusion reduces the infarct size in late pregnant rats. However, the mechanisms underlying the higher vulnerability of late pregnancy to ischemia reperfusion injury and the cardioprotective role of intralipid are unknown. In Chapter 2, we employ a rat model to investigate the reasons behind the heightened susceptibility of the heart in late pregnancy to ischemia-reperfusion injury. We identify microRNA-98-5p, whose expression increases during late pregnancy upon ischemia reperfusion injury, and promotes cardiomyocyte apoptosis, mitochondrial oxidative stress, and inflammation via its targets Stat3 and Pgc-1α. In an in vivo late pregnant ischemia-reperfusion injury rat model, we show the therapeutic potential of microRNA-98-5p inhibition at the onset of reperfusion. In late pregnant patients with acute myocardial infarction, plasma microRNA-98-5p was significantly higher than healthy late pregnancy and it was correlated with troponin levels. In Chapter 3, we study the therapeutic role of intralipid in attenuating ischemia-reperfusion injury in a rat model of late pregnancy. We demonstrate that the protective effects of intralipid are mediated by microRNA-122-5p. MicroRNA-122-5p exhibits its protective effects in late pregnancy by mitigating cardiomyocyte apoptosis and oxidative stress through its target Pkm2. In humans, plasma microRNA-122-5p levels were lower in healthy late pregnant compared to healthy non-pregnant individuals and even lower in late pregnant patients with acute MI and negatively correlated with troponin levels. In an in vivo late pregnant ischemia-reperfusion injury rat model, we show the therapeutic potential of microRNA-122-5p overexpression at the onset of reperfusion. Taken together, this dissertation offers insights into the heightened susceptibility of late pregnancy to ischemia-reperfusion injury and the cardioprotective role of intralipid.

Myocardial infarctions (heart attacks) are the most common cause of cardiac damage globally, leading to the significant health consequences and even death. Biomaterials, such as hydrogels, can serve as a better method to manage cardiac damage. However, an exploration of the current biomaterial and microneedle solutions shows a lack of directed drug delivery capabilities or conductivity. Given the density and conductivity of human cardiac tissue, these aspects are critical in improving the effectiveness of any biomaterial solution. Introduced in this thesis is the combination of both elements into a conductive hydrogel-based microneedle patch. We utilized a new, naturally derived biomaterial, tannic acid:PEDOT (poly(3,4-ethylenedioxythiophene), also known as TADOT. We then combined this with gelatin methacryoyl (GelMA) hydrogel to develop a biomaterial-based microneedle platform aimed to solve both issues. The formation of a tannic acid complex with PEDOT to form TADOT allows the hydrogel to be conductive, while the GelMA provided sufficient mechanical strength. With the anti-inflammatory characteristics of tannic acid, and the biocompatible nature of gelatin, the material is designed for better contribution to cardiac healing. We were able to engineer a microneedle patch based on TADOT-GelMA into microneedles which could penetrate porcine cardiac tissue, proving the potential for this biomaterial as a platform for cardiac repair applications. The conductive and mechanical characteristics of the TADOT-GelMA microneedles can allow it to serve as a delivery platform to be combined with other cardiac repair solutions.

Heart Failure, sometimes called Congestive Heart Failure, is a collection of pathological disruptions in which the heart is unable to pump blood properly. It affects over 6 million people in the United States, with the cost-per-patient at approximately $30,000 and the total cost estimated to be $160 billion by 2030. There is no single cause of heart failure, as it can manifest as a varying array of diseases, disorders, and syndromes. However, many of these underlying diseases result specifically from dysregulation of mechanically active cells called cardiac fibroblasts, which play a critical role in the remodeling of the extracellular matrix and provide a feedback mechanism to control force distributions within the myocardium via stiffening or softening the tissue. These forces, generated by cardiomyocytes, are then altered into one of many unique disease states. Cardiac fibrosis stimulates excessive collagen production, stiffening the ECM and resulting in Left Ventricle hypertrophy. Myocardial Infarction results in a localized region of dominant and activated fibroblasts, greatly upregulating ECM production to protect against loss of healthy tissue and disrupting electrical signaling propagation. The underlying mechanisms of fibroblast-mediated matrix remodeling and its effects on cardiomyocyte force generation are critical to our understanding of cardiovascular diseases. We have developed an in vitro 3D wound platform that can recreate biologically relevant mechanical conditions utilizing cardiac fibroblasts and cardiomyocytes. It does this by precisely balancing matrix stiffness properties, cell distributions, and electrical/mechanical stimulation. With the ability to create healthy left ventricular myocardial tissue, we also show the ability to produce diseased tissues that either mimic the mode of injury (MI) or upregulate fibroblast signaling pathways (cardiac fibrosis) to recreate an appropriate model of each pathology. This provides a unique platform that can create and characterize, with high precision, the biological and mechanical condition of diseased myocardial tissue for use in clinical or drug development applications.

Background: Myocardial infarction involves the three processes of atheroma development, plaque rupture and formation of a thrombus. Proliferation of smooth muscle cells and vasoconstriction are important aspects of atheroma formation and are influenced by the renin-angiotensin system. Angiotensin converting enzyme (ACE) pivotal to this system has recently been shown to be influenced by an insertion/deletion (I/D) polymorphism in the ACE gene. Patients homozygous for the D allele have the highest circulating levels. Inhibition of fibrinolysis through raised levels of plasminogen activator Inhibitor (PAI- 1) has also been shown to be under the control of a PAI-1 promoter single nucleotide insertion/deletion (4G/5G) polymorphism. Patients homozygous for the 4G allele have the highest levels of circulating PAI-1. Aims: The aim of this thesis, was to investigate the role of both polymorphisms in relation to atherothrombosis in subjects with coronary artery disease (CAD). Results: 609 Caucasian patients (420 males 189 females) admitted for angiography for known or suspected coronary artery disease were recruited from two centres. Patients were classified as having no significant coronary artery disease (20%), single (21%), double (21%) and triple vessel disease (38%) on the basis of 50% stenosis. Both the ACE genotype and the PAI-1 genotype were associated with their respective circulating levels (P= 0.0008) and (P = 0.0001) respectively. There was no relationship between the ACE genotype or levels with either the degree of coronary stenosis or a history of MI. In contrast, the 4G/4G genotype was significantly related to a history of myocardial infarction, an association which was stronger in the group with pre-existing significant atheroma. Conclusions: These data suggests that the ACE genotype-activity does not influence the atherothrombotic process, whereas the PAI-1 promoter polymorphism influences the development of myocardial infarction through its effects on thrombus formation in patients with pre-existing atheroma. Conclusions These data suggests that the ACE genotype-activity does not influence the atherothrombotic process, whereas the PAI-1 promoter polymorphism influences the development of myocardial infarction through its effects on thrombus formation in patients with pre-existing atheroma.

An accurate diagnosis of patients presenting with chest pain is required for appropriate patient management and to define prognosis. Current methods used to diagnose myocardial infarction were reviewed and an assessment of the biochemical markers currently available for identifying myocyte damage revealed a potential role for a more cardiac specific marker. Further review of current strategies to risk stratify patients with unstable angina identified a continuing problem between available investigations and predictive power for future cardiac events. An assay for the cardiac specific protein Troponin T (cTnT) has recently become available and its potential role in acute coronary syndromes was studied. The cardiac specificity of cTnT was compared with creatine kinase MB isoenzyme (CK- MB) concentration measurement for the differential diagnosis of elevated creatine kinase (CK) produced by arduous physical training in 219 Royal Marine Commandos with no evidence of cardiovascular disease. CK was elevated up to 22.6 times and CK-MB mass up to 6.6 times the upper reference limit. Only 2 individuals had detectable cTnT, neither exceeded 0.2 micrograms/litre. Cardiac Troponin T was considered to be superior and more cardiac specific than the other biochemical markers. The potential role of cTnT measurement in the differential diagnosis of suspected ischaemic myocardial damage was examined in 93 consecutive patients admitted to a Coronary Care Unit. The study showed that the cut off value should be 0.2 micrograms/litre, and that a single measurement at either 12-24 hours from admission or 12-48 hours from the onset of chest pain will confirm or exclude myocardial infarction. Cardiac Troponin T was also identified in a subset of patients with unstable angina and the prognostic potential and usefulness in risk stratification was examined. 183 patients admitted with unstable angina were followed for a median of 1032 days. The presence of cTnT in the serum when tested at 12-24 hours from admission in this syndrome identified a high risk subgroup that had a higher frequency of cardiac death. Its presence also identified a subgroup that had a higher frequency of coronary revascularisation, cardiac death or revascularisation and cardiac death or readmission with non fatal myocardial infarction as first events. Further studies were undertaken to try and identify a pathophysiological explanation for the adverse prognosis of this subgroup. The angiographic culprit lesion morphological findings were compared in 46 unstable angina patients during the repair phase of the syndrome. No major differences in the extent or severity of coronary artery disease, or the location or morphological findings of the culprit lesion between the cTnT positive and cTnT negative unstable angina patients were identified. Activation of coagulation on admission in unstable angina in relation to cTnT concentrations were studied in 96 patients by examining variations m prothrombin fragment 1+2 and fibrinogen concentrations. No significant differences were observed between the two unstable angina groups. Variations in Lipoprotein (a) concentrations in patients admitted with unstable angina in relation to cardiac Troponin T levels were examined. Lipoprotein (a) concentrations were significantly higher in the cTnT positive than in the cTnT negative unstable angina patients and these findings represent the first demonstration of a risk factor that may play a role in the pathogenesis of this acute coronary syndrome subgroup. The cardiac specificity and long diagnostic window of the biochemical marker cardiac Troponin T offers advantages over existing biochemical markers for the confirmation of myocardial infarction. The identification by its presence in the acute syndrome of unstable angina of a high risk subgroup justifies its routine measurement in clinical practice.

Early identification and treatment for the patient experiencing an ST elevation myocardial infarction is imperative. The American Heart Association and the American College of Cardiology have provided national guidelines to standardize the expectation of care for these patients. When organizations are not equipped to perform a percutaneous coronary intervention, the patient requires transfer. Delay in patient transfer leads to increased morbidity and mortality and poor patient outcomes. For this reason, it is essential that non-PCI capable hospitals have a standard protocol that is followed to expedite treatment and transfer of the patient to a higher level of care. This project evaluated the impact that instituting a STEMI protocol had on transfer times. A retrospective analysis of previous transfer times was utilized to compare if the institution of a protocol was effective at improving the transfer process.