Medicine (St Vincent's) - Theses

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    Mass spectrometry based examination of ADMA/DDAH modification in diabetes and cardiac disease
    Garlick, John ( 2016)
    Nitric Oxide (NO) is an important mediator in the prevention of monocyte recruitment and endothelial dysfunction, which drive the development of atherosclerosis. Asymmetric Dimethylarginine (ADMA) but not its isomer Symmetric Dimethylarignine (SDMA) has been identified as a circulating inhibitor of Nitric Oxide Synthase (NOS), with submiromolar increases of ADMA previously been demonstrated associated with an increase in cardiovascular disease risk. Currently ADMA has been measured with low reproducibility in most analytical techniques other than the gold standard HPLC-MS/MS. This work shows the separation that can be achieved with the emerging technique of Ion Mobility Spectrometry coupled with traditional mass spectrometry. Also using a more traditional HPLC based separation we were able to develop a mass spectrometry based measurement of metabolites ADMA, SDMA, Arginine, Ornithine and Citrulline critical in the NOS/ADMA pathway and the Urea cycle. This assay was used to measure changes across cardiac surgery within the circulation as well as generation from the myocardium. We demonstrated our technique had high reproducibility (>7% CV for ADMA) and showed that ADMA but not SDMA significantly decreases after cardiac surgery. This was the first such study to perform measurements of ADMA and SDMA alongside Urea cycle metabolites and with clinical data also collected will provide the foundation to further work. Our assay was also used in a cell culture model whereby DDAH the enzyme responsible for the majority (80%) of ADMA metabolism has previously been shown to have increased expression and activity with high glucose treatment. Our results demonstrate that ADMA is highly regulated compared to SDMA and the change with high glucose treatment does not correspond to a decrease in intracellular ADMA concentration or from the treatment media. Also tested was the efficacy of the proton pump inhibitor Omeprazole which has been recently demonstrated to inhibit DDAH in endothelial cells. This work demonstrated that Omeprazole does not inhibit DDAH within our HepG2 cell culture model in low or high glucose concentrations.
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    Contractile dysfunction of the heart in early diabetes
    Waddingham, Mark Thomas ( 2016)
    The prevalence of heart failure, especially heart failure with preserved ejection (HFpEF), is increasing annually in part due to an ageing population and the dramatically increased incidence of obesity, insulin resistance, prediabetes and diabetes worldwide. It is well established that patients with type-1 diabetes (T1DM) and type-2 diabetes (T2DM) are at a significantly increased risk of developing HFpEF. HFpEF is a progressive condition and its earliest manifestations are subtly impaired myocardial function that is termed diabetic cardiomyopathy (DCM). At present, there is only a limited understanding of the underlying pathophysiological mechanisms that drive the development of DCM and eventual HFpEF in early T1DM and T2DM. Therefore, the aim of this thesis was to further explore mechanisms that could drive the development of DCM and HFpEF using rat models of early T1DM and T2DM. Using synchrotron radiation as a source for small angle x-ray scattering (SAXS) in the in situ beating rat heart, we are able to measure actin-myosin cross-bridge (CB) dynamics in the entire cardiac cycle, in real time. In the first part of this thesis, we were able to demonstrate that chronic inhibition of the RhoA/Rho-kinase (ROCK) pathway with fasudil improved regionally impaired diastolic myosin head extension and depressed systolic mass transfer in the myocardium of rats with early T1DM. Further, we were also able to demonstrate that global left ventricular (LV) systolic performance was significantly improved in diabetic rats treated with fasudil. These results suggest that the activation of the ROCK pathway is involved in the development of early DCM in the context of T1DM. The Goto-Kakizaki (GK) rat is a non-obese model of spontaneous T2DM, which makes it a useful model to examine the effects of early T2DM on the myocardium without the added complication of obesity. Utilising the synchrotron radiation SAXS technique in the in situ beating heart preparation, we are able to demonstrate that young GK rats (10-12 weeks old) with early T2DM (prediabetes) exhibit impaired basal diastolic myosin head extension and reduced systolic myosin mass transfer in the deeper myocardial layer, the subendocardium. Interestingly, basal global cardiac function and β-adrenergic mediated positive inotropy was preserved in young GK rats. We speculate that a combination of cardiomyocyte hypertrophy and enhanced epicardial fibre function are the most likely mechanisms for the preserved global LV function in young GK rats. Diabetes is rarely seen in isolation in patients and commonly coexists with hypertension. The interaction of diabetes and hypertension is known to exacerbate myocardial dysfunction and accelerate the development of HFpEF, but the precise mechanisms remain elusive. The GK rat is a model of salt-sensitive hypertension induced by exposure to a high-salt diet (6% NaCl) for eight weeks. Thus, we examined if the interaction of prediabetes and salt-sensitive hypertension exacerbated myocardial dysfunction and accelerated the development of HFpEF in young GK rats. In GK rats exposed to a HS diet, we observed subtle declines in basal global diastolic and systolic LV function. Limited contractile reserve is a key feature of clinical HFpEF. Consistent with this, we were able to show that regional contractile reserve was limited at the fibre-level in the subepicardial and subendocardial fibre layers of the myocardium in GK rats maintained on a HS diet. These results suggest that limited contractile reserve at the fibre-level may be an early manifestation of HFpEF. In summary, this thesis has demonstrated that the ROCK pathway is involved in the evolution of DCM in T1DM, possibly by modulating actin-myosin interactions in the cardiac cycle. Our results also indicate that impaired CB dynamics is a feature of early T2DM DCM, although global cardiac function is preserved. Importantly, we have demonstrated that limited contractile reserve at the fibre-level may be an early manifestation of HFpEF in the presence of early T2DM and hypertension. Although further work is required to identify the specific molecular mechanisms that drive the impaired actin-myosin CB dynamics in early T1DM and T2DM, this thesis provides novel information of the pathophysiological features of contractile dysfunction of the heart in early diabetes.