title: Superoxide in skeletal muscle: sites that regulate intracellular changes during contractions and role in age related degeneration
Creators: name: Sakellariou, Yorgos
Publisher: name: University of Liverpool Repository
Year: 2014

description: Ageing is defined as an age-related increase in susceptibility to diseases and death and is a complex process that affects every major system at the molecular, cellular and organ level. Although the exact cause of ageing is unknown, there is significant evidence that oxidative stress plays a major role in the ageing process. Skeletal muscle produces oxidants from a variety of different sources with nitric oxide and superoxide being the primary radical species. Nitric oxide is regulated by the activity of nitric oxide synthases, however the sites that modulate changes in superoxide remain unclear. Skeletal muscle ageing is associated with a reduction in muscle mass and strength and leads to a significant vulnerability that opposes healthy ageing. Reports have indicated a positive correlation between tissue concentrations of oxidised macromolecules in skeletal muscle of old individuals, which implies the possible involvement of reactive species in the processes of skeletal muscle ageing. The role of oxidants in skeletal muscle ageing has also been extensively examined in different model organisms, which have undergone genetic manipulations and reports have shown that absence of Cu, Zn superoxide dismutase (SOD1) in homozygotic SOD1 knockout mice, induces an acceleration of skeletal muscle ageing phenotypic changes which further provides support for the implication of radical species in the processes of muscle ageing. The overall aim of the work carried out in this thesis was; i) to develop specific techniques to determine changes in superoxide within the cytosolic and mitochondrial compartment of skeletal muscle, ii) to identify the major sites for superoxide generation in skeletal muscle and iii) to identify the reactive species that are involved in the accelerated loss of muscle mass in the homozygotic SOD1 knockout mouse model and to characterize the changes in redox status and adaptive responses that occur in muscles from the SOD1 knockout mice. The results of carried out in this thesis indicated that the superoxide sensitive fluorescent probes dihydroethidium and MitoSOX Red were capable of selectively detecting changes in superoxide within the cytosolic and mitochondrial matrix of skeletal muscle, respectively. Specific pathway inhibitors and immunolocalisation techniques showed that the major sub-cellular sites contributing to cytosolic superoxide changes in skeletal muscle both at rest and during contractions were the NAD(P)H oxidases. Finally with the use of single isolated muscle fibres from the flexor digitorum brevis muscle, it was concluded that formation of peroxynitrite in muscle fibres was a major effect of lack of SOD1 in SOD1 null mice, which may contribute to fibre loss in this model. Techniques developed in this study to monitor real-time changes in superoxide in the mitochondrial and cytosolic compartment of muscle fibres provided a useful tool to i) examine the sub-cellular pathways that are involved in the regulation of superoxide at rest and during contractile activity in skeletal muscle and to ii) determine the role of superoxide in skeletal muscle degeneration observed in SOD1 knockout mice. These results may have widespread implications for the understanding of diverse scientific areas, including the responses of muscle to exercise training, age-related loss of muscle mass and function, as well as inflammatory or degenerative muscle diseases, such as the muscular dystrophies that are associated with increased levels of oxidative damage.