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Title: Investigating the biochemical basis of muscle cell dysfunction in chronic fatigue syndrome
Author: Rutherford, Gina
ISNI:       0000 0004 6352 5541
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2016
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Chronic fatigue syndrome/ Myalgic Encephalomyelitis (CFS/ME) is a debilitating disorder of unknown aetiology and is characterised by severe disabling fatigue in the absence of an alternative diagnosis. Historically, there has been a tendency to draw psychological explanations for the origin of fatigue. However, this model is at odds with patient descriptions of their fatigue, with many citing difficulty in maintaining muscle activity due to perceived lack of energy and discomfort. In vivo studies have revealed profound and sustained intracellular acidosis following a standardised exercise protocol, suggestive of underlying bio-energetic abnormality and pointing towards an over-utilisation of the lactate dehydrogenase pathway. Similarly, a recent in vitro pilot investigation reported aberrantly low intracellular pH in CFS/ME patient myoblast samples when compared to healthy controls. Remarkably, intracellular pH in CFS/ME myoblasts was normalised to control level following treatment with pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) , suggesting bio-energetic dysfunction in CFS/ME may be modifiable and therefore treatable. In this thesis, in vitro approaches were used to investigate possible mechanisms leading to muscle dysfunction and the fatigue phenotype exhibited in CFS/ME. Validation work was performed to assess the capacity of a novel pH responsive nanosensor system to measure intracellular pH in CFS/ME patient myoblast cells. The work was unable to reliably detect any acidosis in CFS/ME cells, or any difference between CFS/ME and control cells. In addition, DCA did not modify intracellular pH in either CFS/ME or control cells. The fluorescent pH responsive dye 2’7’-bis (2-carboxyethyl)-5 (6) carboxyfluorescein (BCECF) was used to measure intracellular pH at rest, following electrical pulse stimulation (EPS) and after treatment with DCA in myoblast and differentiated myotube cells. Intracellular pH did not differ between CFS/ME patient and control cells at rest or post-EPS. In addition, treatment with DCA did not modify pH in either CFS/ME patient or control cells. Glycolytic function was assessed via a combination of extracellular flux analysis (XF) and through the measurement of cellular L-lactate concentration. XF analysis revealed extracellular acidification rate (ECAR) measurements for all glycolytic ii parameters to be comparable in CFS/ME patient muscle samples when compared to controls. Additionally, DCA did not alter ECAR in either group. L-lactate concentration was elevated at rest of post-EPS in CFS/ME cells compared to controls. DCA did not modify L-lactate concentration in either sample group. Mitochondrial function was assessed via extracellular flux analysis. Bio-energetic function was investigated by manipulating glucose substrate availability in the assay medium. Basal oxygen consumption rate (OCR) was reduced in CFS/ME myoblasts under hypoglycaemic conditions compared to control cells, however this was not observed in CFS/ME myotubes. ATP-linked OCR was reduced in CFS/ME myoblasts under hyperglycaemic conditions compared to control cells but was not observed in CFS/ME myotube cells. There was no difference between CFS/ME and control cells for any of the other mitochondrial parameters tested. A direct real-time electrochemical approach was used to monitor superoxide (O2.- ) generation in CFS/ME cells following ethanol stimulation and lactic acidification of the assay medium. O2.- generation was not elevated in CFS/ME cells compared to controls following ethanol stimulation or lactic acidification. The in vitro muscle culture approaches reported in this thesis have enabled the investigation of the biochemical basis of muscle cell dysfunction in patients with CFS/ME. It is possible to conclude there to be no evidence of impaired muscle function in CFS/ME patients. Additionally, there was no impairment found in PDK enzyme function. Therefore, it can be determined that bioenergetic function is normal in CFS/ME patients and cannot be attributed to the excessive peripheral muscle fatigue phenotype frequently exhibited.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available