Physiology - 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.