Role of Factor Inhibiting HIF-1 alpha in cardiac oxygen sensing

Hypoxia-inducible factors (HIFs) are transcription factors present in all mammalian cells, and play a pivotal role in the molecular response to changes in oxygen availability. Regulation of HIF transcriptional activity is thought to be via 2 mechanisms, the best understood being constant HIF degradation. Upstream control is via prolyl hydroxylase enzymes, which are sensitive to changes in molecular oxygen concentration. The second, less well understood mechanism is via inhibition of activation of co-factors such as iron, via factor inhibiting HIF-1 (FIH1). Blocking of these co-factors potentially limit HIF-1 transcriptional activity, hence offering alternative regulation of HIF activity in response to low oxygen availability.

Little is known about how FIH1 functions in vivo, and the consequences for the intact organism. FIH1 may act in concert with prolyl hydroxylase enzymes to maintain HIF transcriptional activity throughout the physiological range of oxygen concentrations - FIH1 is known to remain active at very low oxygen concentrations, whereas the prolyl hydroxylase enzymes become inhibited.

This PhD is part of an established, well-funded research project which is investigating the role of FIH1 in cardiac function and metabolism - HIF1 controls transcription of a series of metabolic genes. Little is known about the role of FIH1 in the heart, and we have found that cardiac function is impaired with FIH ablation. FIH1-/- molecular physiology is consistent with a hypoxic phenotype, with abnormal calcium handling and upregulated carbohydrate metabolism. In light of this data, the PhD project will first investigate how FIH1 function controls metabolism, and how HIF stabilisation and transcription are affected by varying FIH function, by both manipulation in vivo and via siRNA cell culture techniques. The techniques described in the project rotation will be followed by more sophisticated measurements of cardiac function and metabolism within intact, beating heart. Of particular importance will be determining the partitioning of carbohydrate metabolism with FIH1 disruption, simultaneously measuring glycolytic flux and glucose oxidation, and central to how FIH1 potentially influences cardiac metabolism. This will be achieved using established 3H and 14C radiolabel techniques. The limited existing literature shows FIH1-/- mice are hypermetabolic. This root cause of this abnormality is potentially mitochondrial, although this has not been investigated. The mechanism by which the HIF system is activated is thought to be via signalling from mitochondria to a cytosolic intermediary and subsequent nuclear stabilisation of HIF. Therefore understanding the role mitochondria have within this signalling system is essential. The project will investigate the possibility that FIH1 ablation results in dissipation of the electrochemical gradient across the inner mitochondrial membrane, examining the role of mitochondrial uncoupling proteins. Mitochondrial morphology will be assessed using established funding for electron microscopy work. The metabolic and mitochondrial themes of the project will be developed concurrently with cell culture techniques that allow for FIH1 loss of function experiments with siRNA in beating CD-1 cardiomyocytes, to investigate the role of FIH1 in maintenance of HIF transcriptional activity at varying levels of hypoxia. Using this in vitro approach will allow fine control of hypoxia, to identify the relative contributions of prolyl hydroxylase enzymes and FIH1 to transcription in cardiac cells.