Physiology - Theses
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ItemCharacterising cellular and molecular mechanisms of cardiac diastolic dysfunction( 2019)Background: Diastolic dysfunction is an important contributor to many cardiac pathologies including diabetic cardiomyopathy and heart failure with preserved ejection fraction. It is characterised by ventricular stiffness, inadequate filling of the ventricles and elevated ventricular pressure. In addition to extracellular influences, evidence suggests that a cardiomyocyte specific intrinsic stiffness may also be an important contributor to diastolic dysfunction, but the mechanisms are not well understood. This might be partly due to the lack of specific animal models available to study underlying mechanisms, in particular in HFpEF. The aim of this thesis was to evaluate cellular and molecular mechanisms of diastolic dysfunction in a model of type 1 diabetes and in a newly characterised model of HFpEF, the Hypertrophic Heart Rat (HHR). Research questions: Q1. Can measurement of in vitro intact cardiomyocyte stiffness be correlated with in vivo diastolic function to determine whether cellular stiffness contributes to cardiac diastolic dysfunction in pathological settings? (Chapter 2) Q2. What are the subcellular mechanisms that contribute to increased stiffness in a pathological model of diastolic dysfunction? (Chapter 3) Q3. Can the Hypertrophic Heart Rat be used as a novel rodent model of HFpEF and what is the underlying cardiomyocyte pathophysiology driving diastolic dysfunction in HFpEF? (Chapter 4) Methods: Type 1 diabetes was induced in Sprague Dawley rats using a single dosage of Streptozotocin. The Hypertrophic Heart Rat (HHR) was characterised and utilised as a model of HFpEF. Echocardiography was used to assess in vivo heart function in diabetic and HFpEF rats. Surface electrocardiogram recordings were performed to assess in vivo electrical activity in HFpEF rats. Cardiomyocytes were isolated by collagenase dissociation. Under loaded conditions glass fibers were attached (MyoTak) at the cell longitudinal surface, and paced cardiomyocytes (1Hz, 2.0mM Ca2+, 37°C) were serially stretched (011.2%, piezomotor). Force development and intracellular Ca2+ transients (Fura-2AM, 5µM) were simultaneously measured (Myostretcher, IonOptix). In the HHR, histological analysis was undertaken to evaluate collagen deposition. Intracellular Ca2+ and contractility was measured in single unloaded cardiomyocytes (4Hz, 2.0mM Ca2+, 37°C). Left ventricular tissue was homogenised and used for Western blot analysis of Ca2+ handling proteins. Results: A1. Validation of in vivo and in vitro methodologies for the measurement of cardiomyocyte and cardiac diastolic function along with confirmation that in vitro cardiomyocyte stiffness directly correlates to in vivo cardiac dysfunction. This verifies the contribution of cellular stiffness to cardiac diastolic dysfunction in the pathological setting. (Chapter 2) A2. Cardiomyocyte stiffness was shown to be an important contributor to diastolic dysfunction in the diabetic heart. The additive contribution of myofilament cooperativity reduction and slowed Ca2+ reuptake were found to be the subcellular mechanisms for the intracellular stiffness. (Chapter 3) A3. A new model of HFpEF was successfully characterised which closely mirrors clinical pathology without surgical or drug intervention. Animals display early mortality, with cardiac diastolic dysfunction, preserved ejection fraction and arrhythmias. The cardiomyocyte pathology was one of hypercontractility and Ca2+ overload, contrasting strongly with what has been reported in systolic failure leading to potential new therapeutic targets for HFpEF treatment. (Chapter 4) Conclusion: This thesis demonstrates that intact cardiomyocyte stiffness contributes directly to cardiac diastolic dysfunction, which was validated in two separate pathological models. Importantly, this is the first evidence that there is an increase in the slope of the end diastolic force length relation in intact diabetic cardiomyocytes indicating increased cellular stiffness. This was linked to changes in Ca2+ reuptake during relaxation and reduced myofilament cooperativity. In addition, a newly characterized model of HFpEF was described, along with cellular and molecular changes that are apparent in this model of diastolic dysfunction, providing new insight and potentially leading to new therapeutic targets to treat HFpEF. Taken together, this thesis advances the mechanistic understanding of the cellular and molecular mechanisms of diastolic dysfunction.
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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.
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ItemFructose and the heart: myocardial remodelling and functional responses( 2011)Context: Large population studies have demonstrated that high dietary sugar is associated with increased risk for type 2 diabetes and cardiovascular disease independent of body mass index. Specifically, fructose intake has been linked with the onset of insulin resistance and evidence is emerging that dietary fructose induces a specific cardiac pathology. Metabolism of fructose bypasses the phosphofructokinase regulatory step of glycolysis and high throughput may lead to distinct cellular disturbances. But whether fructose can have direct effects on cardiomyocytes is unknown. The heart may be especially vulnerable to the indirect (i.e. systemic insulin resistance) and direct (i.e. cardiomyocyte metabolic dysregulation via phosphofructokinase bypass) effects of fructose and requires investigation. Aims: This thesis aimed to investigate the cardiac-specific effects of high dietary fructose, specifically assessing whole heart morphology and signalling, and cardiomyocyte performance. Evaluation of cardiomyocyte capacity for fructose transport and utilisation was undertaken to assess the potential for high fructose intake to have direct effects on the heart. Specific questions of cardiac pathophysiological importance were addressed: 1. Do cardiomyocytes have the capacity to transport and utilise fructose? [Chp3]2. How does dietary fructose affect cardiac morphology and cell survival signalling? [Chp4]3. How does dietary fructose affect cardiomyocyte contractile function and Ca2+ handling? [Chp5]4. Does cardiac angiotensin II (AngII) upregulation interact with dietary fructose-induced cardiac signalling alterations? [Chp6] Methods: Detailed in vitro studies manipulating glucose/fructose substrate were used to functionally demonstrate that rodent cardiomyocytes have the capacity to utilise fructose. Dietary fructose-induced cardiac pathology was evaluated in mice with histological, biochemical, molecular, and cellular techniques. Assessment of dietary fructose cardiac effects in the presence of an underlying predisposition for renin-angiotensin system upregulation utilised the cardiac-specific angiotensinogen overexpressing mouse model. Results: The major overall findings of this thesis are: 1. The fructose-specific transporter, GLUT5, is expressed in adult rat ventricular cardiomyocytes, and functional cardiomyocyte fructose utilisation is evident. Fructose reversed the 2-deoxyglucose(2DG)-induced slowing of the Ca2+ transient time to peak: 2DG: 29.0±2.1ms vs. glucose: 23.6±1.1ms vs. fructose + 2DG: 23.7±1.0ms; p<0.05). 2. A high fructose diet induces a specific cardiac pathology associated with systemic insulin resistance. This cardiac pathology is characterised by: • myocardial autophagy activation (46% increase in LC3BII:I ratio; 47% increase in p62) but no evidence of apoptosis induction • cardiac remodelling (28% increased collagen deposition with no change in heart size)
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ItemExercise and GLUT4 expression in type 2 diabetes( 2010)Peripheral insulin resistance is characterised by reduced insulin-stimulated glucose uptake in skeletal muscle and adipose tissue, and the condition represents one of the earliest hallmarks in the development of type 2 diabetes (T2D). In patients with T2D, protein expression of the insulin-stimulated glucose transporter, GLUT4, is reduced in adipose tissue, but preserved in skeletal muscle. Transgenic studies in rodents provide evidence that overexpression of GLUT4 selectively in either skeletal muscle or adipose tissue enhances whole-body insulin action. Since skeletal muscle accounts for the majority of insulin-stimulated glucose disposal, the effect of adipose tissue GLUT4 on insulin sensitivity is thought to be secondary to an altered secretion of adipokines which affect insulin action in muscle, in the context of a ‘metabolic crosstalk’ between insulin sensitive tissues. Increasing GLUT4 expression in skeletal muscle and adipose tissue could be an effective therapy in the treatment of insulin resistance and T2D. Exercise training increases GLUT4 protein expression in skeletal muscle of patients with T2D. This adaptation occurs in the face of enhanced insulin sensitivity, and results from the cumulative and transient increase in GLUT4 mRNA following each acute exercise bout. Less is known regarding the regulation of skeletal muscle GLUT4 expression by a single bout of exercise in patients with T2D, or the effect of exercise training on GLUT4 expression in adipose tissue. The primary aim of the studies undertaken for this thesis was to enhance understanding of exercise-mediated GLUT4 expression in skeletal muscle and adipose tissue of patients with T2D. The first investigation determined the effect of a single bout of exercise on skeletal muscle GLUT4 mRNA, and the signalling pathways which regulate GLUT4 expression, in patients with T2D and healthy control volunteers, matched for age and BMI. Increased (p<0.05) expression of GLUT4 and PGC-1α mRNA, together with increased (p<0.05) phosphorylation of AMPK and p38 MAPK was observed following exercise in patients with T2D, to a similar extent as in age- and BMI-matched control subjects. These findings lead to the conclusion that exercise-mediated regulation of GLUT4 expression is normal in patients with T2D. The second investigation of this thesis sought to identify the effect of a 4 week exercise training program on skeletal muscle and adipose tissue GLUT4 expression in patients with T2D. It was found that exercise training increased (p<0.05) GLUT4 protein expression by ~36% and ~20% in adipose tissue and skeletal muscle, respectively. These adaptations occurred in the absence of changes in insulin sensitivity or plasma levels of adipokines, adiponectin and resistin. Accordingly, the third study of this thesis sought to identify novel adipokines that regulate peripheral glucose metabolism in an adipocyte model of GLUT4 overexpression. Amyloid precursor protein (APP) was reduced (p<0.05) in culture media of GLUT4 overexpressing adipocytes, and the APP cleavage product, amyloid-beta (Aβ), reduced (p<0.05) insulin-stimulated Akt phosphorylation in L6 myocytes in vitro. These observations lead to the conclusion that increased adipose tissue GLUT4 expression may influence whole body glucose metabolism through reduced levels of Aβ. The primary aim of the final study undertaken was to identify novel changes in the abundance of proteins in skeletal muscle following exercise training in patients with T2D, including proteins of glucose metabolism, which may regulate of GLUT4 expression. Exercise training altered the abundance of several proteins involved in energy metabolism, as well as some novel proteins which may play a role in cytoskeleton interactions with mitochondria. In summary, this thesis demonstrated that skeletal muscle from patients with T2D responds normally to an acute exercise bout in terms of increased GLUT4 mRNA expression. In addition, it was shown that exercise training increased GLUT4 protein expression, not only in skeletal muscle, but also in adipose tissue of patients with T2D. This is significant because adipose tissue GLUT4 overexpression enhances insulin sensitivity. Data from this thesis suggest that improvements in insulin sensitivity may be secondary to altered secretion of Aβ from adipose tissue. Collectively, the findings provide a number of therapeutic targets for the treatment of insulin resistance and T2D.