Medicine (St Vincent's) - Theses
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ItemThe role of the coronary vasculature and myocardium in the pathogenesis of diabetic cardiomyopathy( 2012)The prevalence of diabetes is increasing worldwide. This poses a significant threat to human health, as diabetes is associated with an increased risk of mortality due to cardiovascular disease. In particular, diabetic patients develop diabetic cardiomyopathy (DCM), characterised by impaired cardiac muscle contraction and relaxation, leading to left ventricle (LV) muscle stiffness and congestive heart failure. Previous studies suggest that changes in the coronary vasculature and cardiac subcellular function may account for the progression to DCM, however as yet this has not been assessed in vivo. Synchrotron radiation (SR) now makes possible novel imaging and diffraction techniques, to investigate the role these mechanisms play in the early development of DCM, where clinical intervention is most efficacious. To assess coronary function in vivo we validated the use of SR imaging to detect and quantify regional differences in resistance microvessel calibre. In type 1 diabetic rodents we found that although endothelium-dependent and –independent vasodilatory responses in individual coronary vessels are preserved, following inhibition of NO and PGI2 production, there is evidence of localised focal and segmental constrictions. This demonstrates, for the first time, localised coronary microvascular endothelial dysfunction in early-stage type 1 diabets (T1D). Contributing to this diabetic coronary impairment is the RhoA/Rho-kinase (ROCK) pathway, which had previously been shown to play a role in endothelial dysfunction and coronary vasospasm. Our data further support a role for ROCK in early diabetic coronary dysfunction, as following nitric oxide synthase/cyclooxygenase blockade, ROCK inhibition greatly reduced regional segmental constrictions and completely alleviated persistent focal stenoses in diabetic animals. Together, these findings provide strong evidence that early vascular dysfunction may contribute to the development of DCM. In addition, although characterised by global cardiac impairment, the role subcellular changes in the sarcomere play in DCM progression is not known. SR, as a source for small-angle X-ray diffraction, allows the assessment of cardiomyocyte cross-bridge dynamics (CB) and myosin interfilament lattice spacing in situ and in real time. Using SR, our data shows that in early T1D, CB dynamics are abnormal in the beating hearts and this is directly related to impaired LV function. The change in CB dynamics is caused by myosin head displacement from actin filaments, but notably is not related to estimated sarcomere length or myofilament order. SR X-ray diffraction thus provides a robust method to assess cardiac CB dynamics in situ and for the first time we provide evidence that impairment in the regulation of myosin head extension in T1D hearts contributes to DCM. Currently 85-90% of diabetics have T2D and it is therefore critical that these coronary microvascular and cardiac subcellular impairments in T1D are explored in T2D. As such, rodent models which account for the environmental factors important in the human development of DCM are required. We conducted a comprehensive characterisation of cardiac function and structure in diet-induced rodent models of obesity, insulin resistance and T2D, and uncovered mild systolic dysfunction in fructose fed and mild diastolic dysfunction in high fat fed rodents. Furthermore, we demonstrated mild contractile dysfunction in high fat fed low dose streptozotocin rodents. The characterisation of only mild cardiac dysfunction, in spite of the lengthy time course used, suggests further refinement is required to achieve more robust DCM models. In summary, through the validation of novel SR imaging and diffraction techniques our data has confirmed a role for coronary microvascular dysfunction, via the ROCK pathway and cardiac subcellular impairment, via reduced myosin head extension, in the development of DCM. In addition, further studies investigating rodent models of T2D and DCM are required. These findings provide a strong basis for the future development of novel therapies aimed at preventing and/or reversing the decline in cardiac function associated with diabetes.
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ItemFibrosis in experimental diabetic nephropathy and cardiomyopathy: effects of FT011, a novel anti-fibrotic intervention( 2011)It is estimated that by 2025, 366 million people worldwide will be diagnosed with diabetes mellitus (DM); this brings with it the potential for an increase in the prevalence of diabetic nephropathy (DN) and cardiomyopathy (DCM). Despite current management for diabetes, DN is the leading cause of end stage of kidney disease (ESKD) for renal replacement therapy, and DCM is associated with a higher incidence of chronic heart failure (CHF). Pathological fibrosis is a hallmark of progressive renal and cardiac disease leading to ESKD and CHF. Growth factors such as transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) have been consistently implicated in the fibrogenesis in DN and DCM. Therefore, strategies inhibiting the bioactivities of these cytokines are becoming valuable anti-fibrotic therapeutic targets. Tranilast is one of series compounds that have been shown to inhibit actions of TGF-β and PDGF. To optimise the anti-fibrotic effects of tranilast, FT011 (Fibrotech Therapeutics, Pty. Ltd, Melbourne, Australia) is a newly synthesized compound based on cinnamoyl core structure of tranilast. The hypothesis of the thesis is that targeted treatment for fibrosis with FT011 will attenuate functional and structural manifestations of injury in experimental DN and DCM. The aims of this thesis were to firstly evaluate the inhibitory effects of FT011 on collagen synthesis and cell proliferation in vitro and in vivo models, and then to explore therapeutic effects of FT011 in experimental DN and DCM. In cultured rat mesangial cells, FT011 inhibited TGF-β1 and PDGFBB induced collagen production and cell proliferation in a dose dependent manner with no evidence of cell toxicity. Consistent with these actions, treatment of anti-Thy1 nephritis with FT011 attenuated matrix accumulation, mesangial phenotypic changes, mesangial cell proliferation and glomerular macrophage infiltration. These findings suggest a promising profile of FT011 for its potential use as an anti-inflammatory and anti–fibrotic drug. Progressive DN is characterised by glomerulosclerosis and tubulointerstitial fibrosis leading to capillary rarefaction with consequent loss of renal parenchyma and function. Both early and late intervention with FT011 in diabetic Ren-2 rats, a clinically predictive experimental model of DN, prevented development of albuminuria, tubulointerstitial fibrosis, and glomerulosclerosis. In addition, treatment of diabetic Ren-2 rats with FT011 was associated with a reduction in loss of glomerular capillary endothelial cells, interstitial macrophage accumulation and tubular cell apoptosis. These findings prove the hypothesis that targeted treatment for fibrosis with FT011 would attenuate renal functional and structural injury in experimental DN. Pathologically, DCM is associated with microvascular disease and characterised by myocyte hypertrophy, apoptosis and accumulation of interstitial matrix. These structural changes ultimately lead to heart dysfunction. Treatment of diabetic Ren-2 rats with FT011 attenuated systolic and diastolic dysfunction associated with reduction in interstitial fibrosis and myocyte hypertrophy. These findings prove the hypothesis that anti-fibrotic and anti-hypertrophic therapy with FT011 would attenuate heart dysfunction in DCM. The anti-fibrotic effects of FT011 observed in diabetic kidney and heart are independent of changes in high blood pressure and glucose, and the anti-fibrotic therapeutic efficacy of FT011 is greater than previous studies with tranilast. In addition, treatment with FT011 reduced ERK1/2 MAP kinase phosphorylation in both the diabetic kidney and heart. These findings suggest that anti-fibrotic mechanism actions of FT011 are different from blood pressure and glucose lowering agents and may be attributable to events that are more down stream signalling pathways of TGF-β, although the precise mode of action for FT011 was not determined in the present thesis. In conclusion, the findings of the present thesis advocate the use of strategies that target treatment for fibrosis by inhibiting bioactivities of pro-fibrotic cytokines delays the progression of DN and DCM. These findings yield important preclinical information in terms of the potential utility of FT011 as a novel anti-fibrotic therapy in the treatment of DN and DCM.