School of Biomedical Sciences - Theses
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ItemPathology of glycogen excess in diabetic cardiomyopathy( 2017)Background: Diabetic cardiomyopathy is a distinct cardiac pathology and the underlying mechanisms are unknown. Elevated glycogen content has been observed in the diabetic human myocardium, first recorded 80 years ago, suggesting that despite impaired glucose uptake cardiomyocytes accumulate glycogen. Anecdotal evidence of glycogen accumulation in the diabetic myocardium has since been recorded in the literature but a systematic investigation of this paradoxical phenomenon has not been conducted. Glycogen storage diseases demonstrate that increased cardiac glycogen is associated with severe functional deficits, and therefore the observed glycogen ‘excess’ in diabetic hearts may be an important and novel agent of pathology in diabetic cardiomyopathy. Aim: This body of work aimed to systematically investigate the role myocardial glycogen accumulation in diabetic cardiomyopathy, with a focus on glycophagy, a glycogen-specific autophagy process. Key metabolic signaling pathways (insulin, AMPK, β-adrenergic) were interrogated to investigate their therapeutic potential. The four experimental questions addressed in this thesis are: 1. Does myocardial glycogen accumulation contribute to functional deficits in the diabetic heart? (Chapter 2) 2. What glycogen processing mechanisms are disrupted and may be associated with glycogen accumulation in the diabetic myocardium? (Chapter 2) 3. Do simulated hyperglycemic and hyperinsulinemic conditions mediate cardiomyocyte glycogen accumulation? (Chapter 3) 4. Can key metabolic signaling pathways (AMPK, β-adrenergic signaling) be exploited to degrade excess cardiomyocyte glycogen? (Chapter 4) Methods: Type 1 diabetes (T1D) was induced in male Sprague-Dawley rats using Streptozotocin. C57Bl/6 mice were fed a high fat diet to induce obesity and insulin resistance – a state of early type 2 diabetes (T2D). Human atrial tissue from diabetic patients were examined for glycogen content. Echocardiography was conducted to assess functional outcomes in diabetic animals. Neonatal rat ventricular cardiomyocytes were cultured in extracellular high glucose (30mM) and insulin (1nM) and/or had suppressed GABARAPL1 expression (siRNA, siGABARAPL1). Influence of β-adrenergic or AMPK activation was assessed using isoproterenol (100µM, 1 hour) or AICAR (30µM, 30 minutes), respectively. Glycogen content in cardiac tissue homogenates and cell lysates was measured via enzymatic assay. Molecular markers of key signaling pathways were investigated using immunoblotting, immunohistochemistry and qPCR. Results: Some of the overall findings of this investigation are that: 1. Myocardial glycogen accumulation in in vivo models of insulin resistance and progressed T1D is associated with diastolic and systolic dysfunction. 2. Myocardial glycogen accumulation is associated with decreased GABARAPL1 lipidation, suggesting a disruption in glycophagosome scaffold processing in the insulin resistant mouse and diabetic human myocardium. This finding was also established in vitro where a suppression of GABARAPL1 mRNA induced cardiomyocyte glycogen excess. 3. High extracellular glucose (simulated hyperglycemia) only increases cardiomyocyte glycogen content in the presence of insulin and is associated with increased expression levels of the glycophagy adapter protein STBD1 in vitro. 4. In vitro activation of β-adrenergic signaling mediates a reduction in cardiomyocyte glycogen via activation of glycogen phosphorylase when glycophagy is disrupted (siGABARAPL1). In vitro activation of AMPK signaling decreases cardiomyocyte glycogen induced by disrupted glycophagy (siGABARAPL1), but is not effective in modulating glycogen loading induced by high extracellular glucose (simulated hyperglycemia). This study identifies glycogen accumulation as a novel agent of pathology in the development of diabetic cardiomyopathy, associated with a disruption in glycophagy. It is the first to show that cardiac dysfunction is linked with myocardial glycogen accumulation. In a glycophagy compromised setting, AMPK and β-adrenergic signaling may provide potential therapeutic targets to rescue cardiac glycogen excess. An increased understanding of the complex signaling pathways mediating glycogen synthesis and storage in early diabetes may provide a platform for the development of cardiac specific, targeted therapeutic interventions in diabetes.
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ItemDiabetic cardiomyopathy: sex-specific aspects of functional, structural and molecular remodelling( 2017)Background: Clinical studies have revealed increased cardiovascular risk in diabetic patients, which is substantially elevated in women. Perplexingly, while there has been extensive experimental effort in characterising cardiac dysfunction in the progression of diabetic cardiomyopathy, studies investigating sex differences are limited. A mechanistic understanding of sexual dimorphism in diabetic cardiomyopathy remains to be achieved. Aim: The aim of this Thesis was to examine female susceptibility to cardiac pathology in type 1 diabetes (T1D). In particular, this thesis focussed on examining cardiac responses to diabetes (functional and molecular) under basal conditions, during ischemia and with increased cardiac renin angiotensin system (RAS) signalling. The four experimental questions addressed in this thesis are: 1. Are systemic and cardiac T1D phenotypes different between males and females? [Chapter 3] 2. Is there an accentuated female vulnerability to ischemia reperfusion injury in T1D? [Chapter 4] 3. Are there sex-specific changes in cell death, autophagy and metabolism associated with diabetes? [Chapter 5] 4. Does cardiac RAS upregulation interact with sex-specific cardiac responses in T1D? [Chapter 6] Methods: A wide range of in vivo, ex vivo and molecular strategies were employed to characterise the role of sex differences in a streptozotocin (STZ)-induced T1D mouse model. Echocardiographic assessment was performed to examine T1D-induced functional and structural deficits in vivo. Ex vivo isolated heart perfusion analysis was used to characterise the role of sex differences in ischemia-reperfusion injury and recovery in T1D. The mechanistic basis of T1D-induced cardiac pathology was evaluated with various histological, biochemical and molecular techniques. Molecular findings from T1D models were also compared against changes from type 2 diabetic (T2D) mouse models (lean and obese). Finally, the role of RAS in exacerbating the T1D phenotype was assessed using a cardiac-specific angiotensinogen overexpressing mouse model. Results: The overall findings of this thesis are: 1. Although the extent of hyperglycaemia and increase in glycated haemoglobin (HbA1c) was less marked in female T1D in comparison to male T1D, diastolic dysfunction was evident in female T1D, but not in male T1D mice. 2. In males, diabetic hearts showed greater reperfusion recovery associated with reduced cardiac glycogen levels post-ischemia, suggesting better glycogen utilisation during ischemia, compared to male controls. In contrast, despite an earlier onset of ischemic contracture, the reperfusion recovery and glycogen levels were unchanged in female T1D hearts, compared to female control hearts. 3. GABARAPL1, a gene responsible for lysosomal breakdown of glycogen, was upregulated in T1D male hearts, whereas genes from conventional glycogen breakdown pathways (glycogen phosphorylase and glycogen debranching enzyme) were increased in female T1D. In addition, a pronounced increase in expression of genes from macro-autophagy pathway (protein bulk degradation) and apoptotic cell death pathway genes were observed in female T1D but not male T1D hearts. Interestingly, in lean and obese T2D mice, contrasting cardiac gene expression responses were observed in glycogen metabolic and macro-autophagy pathways. 4. With elevated cardiac AngII, T1D-induced cardiac functional and structural changes were exacerbated in males, but these changes were not apparent in females. Conclusion: Collectively, the novel findings in this thesis have contributed new knowledge to the literature on sex-specific attributes of diabetic cardiomyopathy. This study is the first demonstration that a less pronounced hyperglycaemic response in T1D female mice is associated with more marked functional cardiac pathology. This female vulnerability may be partially attributed to a preferential slower/inefficient processing of glycogen and heightened cell death pathology, evidenced from pronounced autophagic drive in female T1D mice. A sex-specific role for cardiac RAS in exacerbating the T1D phenotype has also been identified. The findings in this thesis support a case for sex-specific progression of diabetic cardiac pathology.