Pharmacological manipulation of hypoxic signalling as a therapeutic target to improve metabolism and function in the type 2 diabetic heart

The work presented in this thesis investigates the metabolic and functional benefits of metabolic modulatory treatment strategies in a rodent model of type 2 diabetes. The first strategy, using a fatty acid translocase (FAT/CD36) inhibitor was investigated, in an attempt to reduce fatty acid oxidation and force glucose uptake and glycolysis. A second strategy, using prolyl hydroxylase domain (PHD) inhibitors, was investigated, to activate hypoxia inducible factor 1 alpha (HIF-1–) to stimulate glycolysis and inhibit fatty acid oxidation. Type 2 diabetes was induced using high fat feeding and low dose streptozotocin in male Wistar rats. Hearts were excised for Langendorff perfusion, to assess substrate metabolism and cardiac function in the intact contracting organ. The use of ex vivo treatment with fatty acid uptake inhibitor sulfo-N-succinimidyl oleate (SSO) was investigated using a protocol of acute hypoxia and reoxygenation. The use of in vivo treatment with the prolyl hydroxylase domain inhibitors, dimethyloxalylglycine (DMOG) and Molidustat, (BAY85-3934) was investigated using a protocol of acute low-flow ischaemia and reperfusion. Diabetic hearts showed a significant impairment in functional recovery following both hypoxia-reoxygenation and ischaemia-reperfusion, and this was associated with lower glycolytic rates, and higher fatty acid oxidation rates, compared with controls. Metabolomics analysis revealed non-uniform changes to the Krebs cycle intermediates in diabetes, with a depletion of intermediates in the first span of the cycle, and accumulation of intermediates within the second span. When perfused hearts were given SSO just four minutes prior to the onset of hypoxia, a significant improvement in functional recovery was seen in diabetic hearts, which displayed similar recovery levels to control hearts. This improvement was associated with decreased fatty acid oxidation rates, increased glycolytic rates, and decreased triacylglyceride accumulation following hypoxia. Diabetic hearts from rats given three doses of DMOG (40 mg/kg) in vivo showed significantly improved functional recovery following ischaemia-reperfusion. However, this was not accompanied by the predicted improvement in susbtrate metabolism. No significant changes were seen in glycolytic rate, instead fatty acid oxidation was found to be significantly increased in DMOG-treated diabetics. Changes in several metabolites found through metabolomics suggested the metabolic effect seen with DMOG treatment was likely due to off-target effects on other –-ketoglutaratedependent enzymes. No evidence was found for significant upregulation of HIF-1 signalling with DMOG treatment, other than increased VEGFA mRNA. Additionally, hearts from control rats that received DMOG recovered to a much lesser extent than untreated control hearts following ischaemia-reperfusion. Diabetic hearts from rats treated with five daily oral doses of Molidustat (5 mg/kg) in vivo also showed significantly improved functional recovery following ischaemia-reperfusion. This was paired with increased baseline glycolytic rates, and decreased fatty acid oxidation rates. Molidustat treatment resulted in significantly increased blood haemoglobin levels in both control and diabetic rats, suggesting stimulation of HIF-1 signalling, although no increases in HIF-1– protein or downstream target glucose transporter 1 (GLUT1, SLC2A1) were found in cardiac tissue from treated rats. This work has validated the potential of metabolic modulation as a therapeutic avenue for the treatment of the diabetic heart, harnessing substrate metabolism as a driving force for functional improvement, and to improve function under stress conditions. We have shown that metabolic modulation improves the diabetic heart’s ability to recover in acute hypoxia, and that hypoxic signalling upregulation improves recovery in ischaemia. This provides a new opportunity for the investigation and development of drugs which can selectively target metabolism in the heart to relieve the long-term maladaptation caused by diabetes, allowing it to regain its inherent metabolic flexibility, which is critical for survival under stress.