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Title: The role of the mitochondrial permeability transition pore in cardiac myocyte cell death
Author: Benton, Eleanor
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2004
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Ischaemia-reperfusion injury, sustained when the blood and oxygen supply is removed and then reinstated, is a common cause of cardiac tissue damage and therefore a popular area of scientific research. It is commonly thought that the mitochondria play an important role in mediating ischaemia-reperfusion injury. Particular attention has been focused on the mitochondrial permeability transition pore. This has many times been shown to open only during the reperfusion phase disrupting the mitochondrial transmembrane potential and therefore the synthesis of ATP. This study endeavours to examine the activity of the pore in rat neonatal primary cardiomyocytes during cell death. Models of apoptotic and necrotic cell death are established, as both have been implicated in ischaemia-reperfusion injury. An in vitro model of ischaemia- reperfusion injury is set up and an in vivo model of ischaemia-reperfusion injury is also examined. Many studies have shown that inhibition of cyclophilin D can successfully block the opening of the mitochondrial pore. In this study, the cyclophilin inhibitor cyclosporin A is applied to determine the importance of cyclophilin D as an attenuator of ischaemia-reperfusion injury. The resulting effect on cell viability is analysed by various assays. Changes in the activity of the mitochondrial permeability transition pore are also monitored using a fluorescent mitochondrial transmembrane potential-sensitive dye. A kinetic assay is established to measure the peptidyl prolyl cis-trans-isomerase activity of cyclophilin D, which is believed to have a role in regulation of the pore. Additionally ATP synthesis is measured by a luciferase-based assay and free radical production is monitored using a radical-sensitive dye during cell death. The effect of cyclosporin A on all measured parameters is examined. The results presented in this study show that the importance of the mitochondrial permeability transition pore can vary widely according to the prevailing conditions. However, the data obtained in both isolated cardiomyocytes and cardiac tissue broadly uphold the concept of the major impact of mitochondrial permeability transition pore induction during ischaemia-reperfusion injury and strongly support the hypothesis of a significant role for cyclophilin D in the molecular mechanism of the pore.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available