Background Mutations in the DJ-1 gene in dopaminergic neurones induce mitochondrial dysfunction and a genetic form of Parkinson’s disease (PD). Although DJ-1 is present in the heart its role there is currently unknown. We hypothesised that DJ-1 may be a novel target for cardioprotection. Methods and Results Overexpression of wild-type (WT) DJ-1 in the HL-1 cardiac cell line induced mitochondrial elongation, delayed the opening of the mitochondrial permeability transition pore (mPTP), and reduced cell death following simulated ischaemia-reperfusion injury (s I/R), effects which were absent in non-functional DJ-1 mutants. Adult murine hearts deficient in DJ-1 sustained larger myocardial infarcts following in vivo 45 minutes regional ischaemia and 24 hours reperfusion, and were partially resistant to ischaemic preconditioning (IPC), when compared to WT controls. DJ-1 deficient murine hearts displayed increased mitochondrial fragmentation, although there were no differences in mitochondrial function, myocardial ATP levels, or cardiac dimensions or function. Conclusion DJ-1 is a novel target for cardioprotection.

Factor XI (FXI) is a plasma glycoprotein that participates in the consolidation phase of blood coagulation and is important in the creation of a stable fibrin clot. Deficiency of FXI leads to an injury-related bleeding diathesis, which is notable for the variability in the bleeding tendency and the lack of a clear relationship between bleeding and FXI coagulant activity (FXI:C). A comprehensive understanding of the factors which influence the bleeding tendency in factor XI deficiency would enable a more structured evaluation of bleeding risk and would focus the treatment choices for individual patients. The purpose of this work is to comprehensively analyse physiological and genetic factors which modify the clinical phenotype of FXI deficiency. In this study, a comprehensive bleeding history was obtained from participating patients with known FXI deficiency (n=102) which was then independently scored. This clinical bleeding score was utilised to compare the following laboratory parameters between patients with and without a clinical bleeding tendency: aPTT, FXI:C, FXI:Ag, Blood group, von Willebrand factor levels, inherited thrombophilic traits, Thrombin generation (by subsampling and continuous methods) and TAFI levels (as evaluated by TAFI antigen and a clot lysis assay). The underlying genetic mutation causing the FXI deficiency was evaluated in all patients and where an existing Jewish mutation was not found, novel mutations were sought. Molecular modelling of mutations in the FXI gene was undertaken which required development of molecular models of the apple domains and serine protease domain of the FXI protein. In addition, genetic analysis of a polymorphism in a gene (ALG6) encoding a specific glucosyl transferase was undertaken due to the link between FXI deficiency and congenital disorders of glycosylation. Finally, the first clinical trial of the use of recombinant FVIIa in FXI deficiency was conducted as part of this study.

Myocardial revascularisation in the form of coronary artery bypass surgery (CABG) and percutaneous coronary interventions (PCI) are the two important treatment strategies in combating ischaemic heart disease. There have been tremendous improvements in many aspects of these revascularisation methods over the years. However both these forms of revascularisation can have a deleterious effect on the myocardium while its vascularity is re-established. This revascularisation induced myocardial injury is one of the prognostically important deleterious effects. Consequently, many therapeutic strategies are being studied which can cardioprotect and reduce peri-operative myocardial injury during CABG and peri-procedural myocardial injury during PCI. We evaluate Remote ischaemic preconditioning, a cardioprotective strategy, for its effect on myocardial revascularisation methods of PCI and CABG. In the PCI settings, the beneficial effect of remote ischaemic preconditioning in patients undergoing elective PCI has now been established. However its role in patients admitted with Non ST elevation acute coronary syndrome (NSEACS) and undergoing urgent PCI is still unknown. This group of patients particularly has increased peri-procedural myocardial injury due to their soft coronary plaque and thrombi. In the CABG settings, remote preconditioning has been shown reduce perioperative myocardial injury in non diabetic patients. There are no clinical studies looking at the beneficial effect of remote preconditioning in diabetic cohort of patients undergoing CABG. In animal models, diabetic myocardium is known to possess higher threshold for preconditioning, albeit still possible to precondition. As diabetic patients, are more vulnerable for myocardial injury during CABG and also tolerate the injury relatively poorly compared to their non diabetic counterparts, it is important to explore for cardioprotective strategies to be applied to this high risk diabetic population. The two clinical studies in this thesis evaluate the effect of remote ischemic preconditioning in a) reducing peri-procedural myocardial injury in patients with NSTEACS undergoing urgent percutaneous intervention and b) reducing peri-operative myocardial injury in type 2 diabetic patients undergoing coronary artery bypass surgery.

Coronary artery disease is by far the single biggest killer in the UK. Atherosclerosis is the underlying cause of coronary artery disease. Hypercholesterolemia is one of the major risk factors in the aetiopathogenesis of atherosclerosis. 3-Hydroxy Methyl Glutaryl CoA reductase (HMG CoA) inhibitors, known as statins, reduce cholesterol levels and offer mortality and morbidity benefit for those with coronary artery disease.The cardiovascular benefits from statins tend to be significant even in individuals with normal cholesterol levels which suggests that there are non-lipid lowering benefits of statins termed “pleiotropic” effects. Statins attenuate infarct size expansion due to lethal reperfusion injury either prior to ischaemia or at the onset of reperfusion by activating the pro-survival, Reperfusion Injury Salvage Kinase (RISK) pathway. As the duration of treatment acutely at the onset of reperfusion is too short to make any impact on cholesterol levels, it was hypothesized that the protection observed is due to a pleiotropic effect of the statins. The mechanism by which statins activate the RISK pathway is not entirely clear. Thus experiments with Langendorf perfused isolated rat heart preparation were used to obtain insights into potential mechanisms for statin’s pleiotropic effects in the setting of ischaemia-reperfusion by using Atorvastatin. Infarct size analysis and Western blot analysis were performed to measure cellular injury and protein activation respectively. Atorvastatin was shown to produce a reduction in infarct size when administered acutely during reperfusion at a dose of 50 micromol/l. Atorvatstatin has affinity to the glucocorticoid receptor at levels comparable to potent glucocorticoids. Glucocorticoids have also been shown to activate pro survival kinases. It was thus hypothesized that the acute cardiovascular effects of Atorvastatin may also be mediated by the glucocorticoid receptor, and would be blocked in the presence of the glucocorticoid receptor antagonist, RU486. However studies undertaken demonstarted inconclusive results as RU486 was itself shown to be protective and it would appear that it worked by an alternative mechanism. Since it is known that pre-ischaemic delivery of Atorvastatin also activates adenosine, an agent known to be cardioprotective. It was hypothesized that Atorvastatin administered during reperfusion may also protect the heart by an adenosine-dependent mechanism. However, despite the infarct sparing action of preischaemic administration of Atorvastatin being confirmed to be dependent on adenosine binding to its receptor, the protection of Atorvastatin administered during reperfusion was shown to be independent of adenosine. Finally, the acute effects of statins are believed to be due to their action on the HMG CoA reductase enzyme which results in a decrease in mevalonic acid and isoprenyl intermediates.The isoprenyl intermediates such as Rho A, are inhibitors of the various components of the RISK pathway. We were able to demonstrate for the first time that mevalonic acid abrogates the infarct size reduction of Atorvastatin administered during reperfusion. Western blot analysis did not reveal a significant reduction in active Rho A levels, however there was a trend towards a reduction and this reflected in the infarct size studies. Although not conclusive, these results would suggest that the acute cardiovascular effects of Atorvastatin, when given at reperfusion, were due to its pleiotropic effects on Rho A.

In order to explore the phenotypic heterogeneity of the myeloproliferative neoplasm essential thrombocythaemia (ET), the role of the JAK2 mutation V617F in the pathogenesis of the disease was investigated, in particular its relationship to myeloid clonality. The clinical, haematological and molecular characteristics of 133 ET patients were studied. JAK2 V617F was detected in 55 (41%) patients; a clonal X-chromosome inactivation pattern (XCIP) was found in 24 (39%) of the 62 evaluable female patients. There was no association between JAK2 mutational status and XCIP status or thrombotic risk, but higher JAK2 V617F mutant levels were noted in patients who had a thrombosis. A trend towards a higher thrombotic rate was observed in patients whose XCIP was clonal. In 10 untreated JAK2 V617F-positive ET patients, JAK2 WT thrombopoiesis was not suppressed despite the presence of a thrombocytosis, suggesting that the regulation of JAK2 WT thrombopoiesis was abnormal. Eleven patients were screened for the presence of more than one JAK2 V617F-positive population using an exonic SNP located near the mutation. In ten (91%) of these the mutation appeared to have been independently acquired on at least two occasions. Furthermore, XCIP analysis of JAK2 V617Fpositive erythroid colonies from six ET patients revealed that in one patient the V617F-positive populations were not derived from a single clonal population. An association between the reported JAK2 haplotype (known as ‘46/1’) and JAK2 V617F-positive ET patients was observed in the cohort studied. Methylation studies indicated that this haplotype introduced additional methylated sites near to the mutation locus, which may potentially affect conformation of the DNA and mutability of the JAK2 locus. Together, the studies reported in this thesis suggest that JAK2 V617F is not the initiating event at least in some cases of ET, and that its presence does not invariably indicate the presence of a monoclonal disorder.

Activation of the PI3K/AKT pathway protects the heart from ischaemia-reperfusion injury. Phosphatase and Tensin Homolog deleted on Chromosome10 (PTEN) is a negative regulator of this pathway. The hypothesis on which this thesis was based stated that inhibition of PTEN would confer protection against ischaemia-reperfusion injury. PTEN was reduced using: 1) a PTEN inhibitor, bpV(HOpic), 2) a mouse model of PTEN haploinsufficiency and 3) PTEN siRNA. The effects of PTEN reduction on ischaemia-reperfusion injury were investigated by using: 1) an isolated perfused heart model of ischaemia-reperfusion injury, 2) an isolated cardiomyocyte model of ROS induced mitochondria damage and 3) a cellular model of hypoxia-reoxygenation injury. No protection against ischaemia-reperfusion was observed in isolated perfused myocardium from C57BL/J6 mice, which were perfused with bpV(HOpic), or from PTEN+/-mice. Likewise, no protection against ROS induced mitochondrial damage was observed in isolated cardiomyocytes from the PTEN+/- mice. In these models an increase in AKT activity was recorded, however, this was not sufficient to confer cardioprotection. Similarly, H9c2 rat myoblast cells, silenced for PTEN expression using siRNA, were not protected against hypoxia-reoxygenation injury. Nevertheless, in isolated C57BL/J6 hearts perfused with bpV(HOpic) and in myocardium from PTEN+/- mice, when the PI3K/AKT pathway was stimulated by the cardioprotective intervention of ischaemic preconditioning a reduced threshold for protection was achieved. To conclude, the level of PTEN inhibition achieved in this study was not sufficient to bestow protection against simulated ischaemiareperfusion injury. However, it appears that reductions in PTEN can increase the sensitivity towards cardioprotection.

The background of the thesis is based on the conflicting results between bench and bedside regarding the susceptibility to myocardial infarction with old age and diabetes. In laboratories all over the world, strategies have been developed to protect the myocardium from this insult, including the use of ischaemic conditioning (short periods of ischaemia and reperfusion prior to or following lethal ischaemia) or the use of a variety of pharmacological agents. However, surprisingly, translating these effective cardioprotective treatments into the clinic has proved problematic. The main issue seems to be the fact that the experimental investigations have mainly used young, healthy animals while the human patients present often with a number of other risk factors, or comorbidities, such as type 2 diabetes and old age. Therefore the aim of this thesis was to investigate the susceptibility to ischaemia-reperfusion injury and the proficiency of cardioprotective strategies to protect the heart in the setting of ageing and type 2 diabetes. Utilizing a model of type 2 diabetes, the Goto-Kakizaki rat and its normoglycaemic control Wistar rat, within the range of 3 to 18 months of age, the Langendorff isolated heart model and in vivo coronary artery occlusion and reperfusion were employed to investigate the susceptibility to ischaemia-reperfusion injury. Mechanical or pharmacological cardioprotective strategies were also investigated in this setting and the mechanisms of the failed cardioprotection were examined further using in vitro techniques focusing on known pro survival signalling pathways within the myocardium. The ageing diabetic heart demonstrated an increased vulnerability to injury and was less amenable to protection by ischaemic conditioning. Pharmacological agents namely, metformin and sitagliptin appear to differentially protect the diabetic and non-diabetic heart, and this could be due to the underlying intracellular changes associated with ageing and diabetes.

Aim. To investigate the association between Gaucher disease (GD) and gammopathy. Introduction. Gaucher disease is a disorder characterised by deficiency in lysosomal glucocerebrosidase, foamy macrophages, cytopenias, bony lesions, organomegaly and a high incidence of monoclonal/polyclonal gammopathy. Abnormalities in the bone marrow microenvironment, including pseudo-Gaucher cells, have been reported in non-GD patients with myeloma. Methods. Gaucher gene analysis, co-culture, drug assays, western blotting, enzymatic assays, flow cytometry, immunofluorescence and cytotoxic assays. Results. GD patients had a high incidence of gammopathy (polyclonal/monoclonal). Serum biomarkers of macrophage burden were predictive of gammopathy and the Zimran severity score was higher in those with monoclonal than polyclonal gammopathy. Enzyme replacement therapy ameliorated polyclonal gammopathy and stabilised paraprotein levels. Gaucher mutations were not prevalent in Jewish patients with paraproteinaemia (8/77 patients). Non-GD patients with paraproteinaemia had normal serum chitotriosidase and monocyte glucocerebrosidase activity. GD monolayers (osteoclasts/macrophages) did not confer a proliferative or survival advantage on co-cultured myeloma cell lines. GD monolayers, compared to control cultures, reduced sensitivity to melphalan and this was contact dependent. Western blotting identified differences in the levels of Bim and Bcl-xL between myeloma cells harvested from control and GD monolayers. When cultured alone, GD monolayers generated more osteoclasts and this was enhanced by plasma cell coculture (contact independent). GD monolayers did not preferentially rescue myeloma cells pre-treated with doxorubicin from cell death. Patients with GD have decreased lymphocyte glucocerebrosidase activity, abnormal lipid trafficking and low peripheral NK (natural-killer), invariant NK and CD4+ve T-cell numbers. Invariant NK cells, GD derived, displayed impaired proliferation to α-galactosylceramide. Cytotoxicity assays, derived from the GD peripheral blood mononuclear cells, displayed inferior killing compared to control assays. NK killing assays were equivalent between controls and GD patients. Conclusion. This thesis presents novel data, which may confer an increased risk of gammopathy in GD.

Cardiac Resynchronisation Therapy (CRT) is an important adjunctive treatment for selected heart failure (HF) patients. With increasing numbers of devices being implanted, strategies to improve outcome in CRT patients are paramount. These were explored in three separate studies. Firstly, a retrospective cohort study of all CRT procedures (n=490) was undertaken to identify the incidence and type of early (≤ 90 days) and late complication (> 90 days) in different CRT populations. Overall, complication rates were low (early 9.4%; late 6.1%) and the LV lead remained stable over long-term follow up. Compared to patients with HF with ischaemic aetiology, patients with idiopathic dilated cardiomyopathy had an increased risk of complications (OR 2.76, 95%CI 1.04-7.31, p=0.04). Secondly, the use of simple infection control measures to reduce cardiac device infection (CDI) rates were explored. A retrospective audit (2004-2007, n=2779 procedures), introduction of an infection control protocol (November 2007) and a prospective audit (2007-2009 n=981 procedures) identified that CDI within 1-year of procedure reduced from 1.3% to 0.6% due to the new protocol, with an estimated cost saving of £132,190 per-year. Finally, a prospective study to explore the acute haemodynamic response (AHR) to CRT using single and multi-site pacing from within a single coronary sinus (CS) branch was performed (n=28). Major intra- and inter-individual variations in the 5 maximum rate of change in LV pressure (LV dP/dt max), depending on the LV pacing site(s), were noted. Selecting the best LV pacing configuration resulted in an increase in AHR of 9.9% compared to conventional biventricular pacing and an absolute increase of 36.3% from baseline. This study is the first to evaluate multi-site pacing from within a single CS tributary and supports an individually tailored approach to CRT. The findings from these studies inform potential strategies to improve patient outcomes in a growing CRT population.

Background: Coronary artery disease is the leading worldwide cause of death. Even despite restoration of blood flow following acute myocardial infarction, further myocardial damage is seen during the reperfusion phase. Pharmacological strategies to induce resistance to ischaemia reperfusion injury have been shown to share common pathways. The most important common signalling pathway involved is the Reperfusion Injury Salvage Kinase pathway, and in animal models this can be pharmacologically activated, resulting in inhibition of the opening of the mitochondrial permeability transition pore, with a resultant cardioprotective effect. Atorvastatin has been shown to act in this way in animal models of ischaemia-reperfusion injury, although this has not been demonstrated in humans. This thesis examines (a) the role of atorvastatin in acute protection from ischaemia-reperfusion injury in human myocardium, (b) the ability of high-dose atorvastatin to recapture cardioprotection in human myocardium in the setting of chronic statin therapy, and (c) the functionality of the mitochondrial permeability transition pore in human hypertrophic cardiomyopathy, and the role of atorvastatin in inhibiting pore opening following oxidative stress. Methods and Results: Using human models to simulate ischaemia-reperfusion injury we have demonstrated: (1) Human atrial myocardium can be protected from simulated ischaemia-reperfusion injury by treatment with atorvastatin at the time of reperfusion; (2) Cardioprotection against simulated ischaemia-reperfusion injury can be recaptured with high-dose atorvastatin; (3) The mitochondrial permeability transition pore is a critical determinant in cell death in hypertrophic cardiomyopathy, and its inhibition can be achieved using atorvastatin. Conclusion: The key aim of emergency reperfusion therapy in the setting of myocardial infarction is salvage of myocardium and preservation of cardiac function. These studies contribute to the field by exploring the cardioprotective effects of atorvastatin in human myocardium and delineating protective cascades involved. They offer a key translational step in our understanding of statin cardioprotection, and an insight into cardioprotection in hypertrophic cardiomyopathy.