title: Regulation of glucose transport in cardiomyocytes
Creators: name: Bowman, Peter Ronald Thomas
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: Metabolism, diabetic cardiomyopathy, stem cell derived cardiomyocytes, GLUT4, glucose uptake, insulin resistance.

description: Major common complications of diabetes such as myocardial infarction arise from the onset of vascular disease. However, there is also evidence of a direct impairment of cardiac contractile function in diabetic individuals in the absence of atherosclerosis and hypertension, termed diabetic cardiomyopathy (DCM). This is characterised by early diastolic dysfunction that progresses to systolic dysfunction and heart failure through a pathological remodelling process. The earliest identified mechanism underlying this disease is the onset of metabolic perturbations such as cardiac insulin resistance. However, currently there are no specific treatments available, partly due to the lack of an appropriate experimental model with which translational research could be performed. iPSC-CM are a recently developed technology, whereby human dermal fibroblasts can be reliably harvested, dedifferentiated into a pluripotent form, and then differentiated into cardiomyocytes. These cells have an established intracellular calcium handling system and contractile capacity, however are generally considered to be at a foetal stage of development. A key aim of this project was to characterise the metabolic phenotype of these cells, in order to assess their potential suitability as the basis of a novel cellular model of DCM. Specifically, it was investigated if these cells exhibited robust insulin stimulated glucose uptake through insulin sensitive intracellular trafficking of the glucose transporter GLUT4, as impairment of this response is a central feature of any diabetic model. After adaptation of a [3H]-2-deoxyglucose uptake assay to a 96-well plate format, and optimisation of experimental factors, it was determined that iPSC-CM could not display robust insulin (or IGF-1) stimulated glucose uptake. Inhibition of the spontaneous contractile capacity of these cells did not induce a response upon subsequent insulin stimulation. iPSC-CM were found to express and activate central insulin signalling molecules such as Akt and Erk1/2, and also possess elements of the GLUT4 trafficking machinery such as the SNARE proteins Syntaxin 4 and SNAP23. However, the critically limiting factor identified was an approximate 10-fold lower expression of GLUT4 in iPSC-CM compared to primary adult cardiomyocytes, accompanied by strong expression of GLUT1. This data was supported by the finding that inhibition of GLUT4 had no impact on glucose uptake in iPSC-CM, whereas inhibition of GLUT1 significantly reduced uptake by ~50%. This phenotype suggests that iPSC-CM are also at a foetal-like stage of development with regards to their metabolic capacity, and are currently not suitable for modelling DCM. Subsequently, initial interventions based upon the literature were implemented in order to try and increase iPSC-CM GLUT4 content. However, neither increasing metabolic reliance upon fatty acid (rather than glucose) nor exposure to triiodothyronine were successful. In contrast, Lipofectamine 2000 mediated transfection of a customised GLUT4 plasmid facilitated a reliable 3-5 fold increase in iPSC-CM GLUT4 content. This increased basal glucose uptake, however did not induce an insulin response. It was concluded that a further increase in expression levels may be required. Finally, it was demonstrated that iPSC-CM are highly amenable to lentiviral mediated infection, and initial steps were taken towards the generation of a virus targeting the overexpression of GLUT4. Additionally, SNARE proteins are essential in facilitating insulin stimulated GLUT4 expression at the plasma membrane. Therefore they represent a possible mechanism by which cardiac insulin resistance could occur in disease states such as DCM. On account of this, the expression of a wide range of SNARE protein isoforms was assessed in cardiac lysates generated from 2 diabetic mouse models (db/db and high fat diet induced). The expression of SNAP29 and VAMP5 were found to differ in lysates from the high fat diet model, although the role of these proteins in GLUT4 trafficking is unclear. In contrast, in the more severe diabetic db/db model GLUT4 protein content was found to be significantly reduced, but SNARE protein content was unaffected. Finally, there is also an established link between glycemic control and both the risk of developing and subsequent prognosis for myocardial infarction (MI). There is a line of evidence suggesting that cardiac insulin sensitivity may also be highly relevant in this disease context. Accordingly, it was demonstrated that cardiomyocytes isolated from a clinically relevant 8-12 weeks post-MI rabbit model exhibited impaired insulin stimulated glucose uptake. This strengthens the association between MI and cardiac metabolic parameters. However, insulin stimulated phosphorylation of Akt, GLUT4 levels, and SNARE protein expression were unaffected post-MI. Therefore future work must identify both the underlying mechanism and clinical relevance of this finding.