The role of oxysterols in cardiac dysfunction in diabetes

BACKGROUND. There is currently a global epidemic of diabetes which is responsible for disability and early death. For example, in the UK around 4 million people (1 in 16) have type 2 diabetes. Most people with diabetes will die from cardiovascular disease which affects both the coronary circulation and the heart itself. We are interested in the role that oxysterols play in diabetic cardiac dysfunction.

Oxysterols are oxidation products of cholesterol that have been shown to be involved in the pathogenesis of many diseases including Alzheimer’s disease, non-alcoholic fatty liver disease and cancer. Recently, tissue levels of various oxysterols have been shown to be dramatically altered in diabetes [1]. This fits with other data showing increased expression of the cytochrome P 450 family of enzymes (responsible for enzymatic oxysterol synthesis) and enhanced ROS production (responsible for direct chemical oxidation of cholesterol) in the diabetic state. Oxysterols are not just by-products of cholesterol metabolism. Cholesterol is a major component of cell membranes and even at low concentrations, oxysterols can act as biologically active substances that regulate processes including inflammation and programmed cell death [1].

In the cell membrane, cholesterol is not uniformly distributed, but is concentrated in microdomains known as lipid rafts. Caveolae are a specialised form of invaginated lipid raft found in high density in cells of the cardiovascular system, including the cardiac muscle cell (myocyte), see our recent review [2]. Caveolae act as meeting places for components of signal transduction cascades; they assemble and co-ordinate the activity of many receptors (including the β-adrenoceptors, insulin receptors), G proteins and their effectors (enzymes, ion channels, exchangers). Many important cellular events are initiated in caveolae, and these structures influence key processes in the cardiac myocyte ranging from contractile function to cell survival [2].

The lipid environment of membrane proteins can critically affect their function. Our pilot data shows that oxidation of membrane cholesterol and direct application of oxysterols modifies nitric oxide production and the contractile response to stimulation of β1 and β2 adrenoceptors [3]. These are all caveolae-dependent processes.

HYPOTHESIS AND AIMS: Our hypothesis is that oxysterols mediate aspects of contractile dysfunction in diabetes through dysregulation of caveolar control. However, we do not yet know how the repertoire of oxysterols changes in the membranes of cardiac myocytes in diabetes or the detail of how oxysterols affect the structure and function of caveolae. This is the aim of this project. We will measure changes in oxysterols and mediators of oxysterol production (CYP450 and ROS), describe the impact of diabetes on the structure and protein organisation of caveolae, and assess the impact on myocyte signalling pathways and contractile function. We can test for a causative role of particular oxysterol species by exposing healthy cells to oxysterols to mimic changes seen in diabetes.

APPROACH: The supervisory team has expertise in caveolar and lipid control of cardiac signalling (Calaghan), diabetes (Filippi), cardiac function and ROS (Steele) and oxysterol regulation of the cardiac cell (from our international collaborator in Moldova, Dr Roman Ursan).

We will use experimental models of diabetes in rodents (high fat feeding, db/db mice) and human cardiac tissue from patients with diabetes. Training will be given in a broad range of experimental techniques including HPLC-mass spectrometry, membrane fractionation, protein chemistry, confocal microscopy, electron microscopy and measurement of myocyte contractility and intracellular calcium.

EXPECTED VALUE OF RESULTS: Together these data will unravel a novel mechanism by which diabetes contributes to cardiovascular morbidity and mortality. Having shown how changes in oxysterols in diabetes affect key signalling pathways in the cardiac cell, we can develop ways of ameliorating oxysterol damage. Of note, the work will have general relevance to dysfunction of other tissues in diabetes and to other diseases where oxysterol levels are altered.