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Rutherford Ph.D. Thesis: Investigating the biochemical basis of muscle cell dysfunction in CFS

Discussion in 'Latest ME/CFS Research' started by Dolphin, Aug 8, 2017.

  1. Dolphin

    Dolphin Senior Member

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    Source: Newcastle University
    Date: April 2016
    URL: https://theses.ncl.ac.uk/dspace/handle/10443/3497
    https://theses.ncl.ac.uk/dspace/bitstream/10443/3497/1/Rutherford, G. 2016.pdf


    Investigating the biochemical basis of muscle cell dysfunction
    in chronic fatigue syndrome
    --------------------------------------------------------------
    Gina Rutherford
    - Institute of Cellular Medicine, Newcastle University


    Abstract

    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 (O_2-) generation in CFS/ME cells following ethanol stimulation and lactic acidification of the assay medium. O_2- 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.

    --------
    (c) 2016 Newcastle University
     
  2. Adamm

    Adamm

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    Hi dolphin could you help me on what this is trying to prove/disaprove?
     
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  3. Orla

    Orla Senior Member

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    <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.>

    I haven't read this properly yet, but just a few things that stood out. I don't like the use of the expression perceived lack of energy, as it might be implying that we have a perception problem only (this is how this expression is often used in relation to ME/CFS. I know she might also mean that the problem is in the brain and not in the muscle) and also "discomfort" is massively understating PEM.

    It sounds like the study didn't find any significant abnormalities, but this is overstating it I think

    <It is possible to conclude there to be no evidence of impaired muscle function in CFS/ME patients.>

    Surely it is only safe to conclude that from her studies that the specific things she tested for are not abnormal in the specific group she tested, and so therefore (at most) these things might not be of significance in terms of aetiology in ME/CFS?
     
  4. Valentijn

    Valentijn Senior Member

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    The author discusses PEM and different criteria, then chooses a criteria which doesn't require PEM:
    Apparently she didn't get the memo about PACE:
    We can probably thank Action for ME for a lot of the mess:
    There's a lot of data in there. It looks like 1-3 patients were used where listed, but there's other experiments where they aren't listed. A lot of the differences are pretty big as seen on the graphs, but non-significant due to lack of power.
     
  5. lansbergen

    lansbergen Senior Member

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    What medium did they use to culture the cells?
     
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  6. Valentijn

    Valentijn Senior Member

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    There's multiple processes used for various tests. For glycotic function and lactate they say:
    This is a good indication of "non-significant" findings that have a drastic difference between patients and controls, as the controls stay in the normal range (under 2.2) and the patients exceed it:
    lactate.jpg

    There's also a lot of odd graph scaling. Eg, showing numbers scrunched together on a large pH scale, even though results will only ever be on a very small portion of it, and small differences are very significant in determining serious pH problems.
     
  7. Simon

    Simon

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    I've got quite a lot of sympathy for Gina Rutherford here: no one devotes long years to a PhD in the hope of a null result. This is Julia Newton's group, of course, and I've seen past presentations by Gina reporting promising early results from her work that were hailed by many, including me (AfME YouTube page has them, I think). Kudos to her for pursuing these and checking if the findings held up or not. The work on myotubules, growing muscle fibres in the lab and electrically-stimulating them, is innovative and potentially a good model (as well as technically very difficult).

    That said, the claim that there is no muscle dysfunction is overstated - I wonder if this was a big claim to justify all that work and a PhD? All that can be said is the techniques found nothing, and that's assuming the methodology was sound (I've no idea, and certainly don't have the energy to look at it).

    That's a big concern: if the study failed because it was too small that needs to be stated (or better, redone with a big enough sample, and PEM-confirmed patients).

    added: Just as big a concern is that you need 20-30 subjects to have a chance of a representative sample; with so few subjects there's a fair chance the patients are very unrepresentative and so don't reveal so much. There again, it must be hard to do things like growing muscles in the lab at any scale.

    It's all a bit frustrating.
     
    Last edited: Aug 11, 2017
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  8. trishrhymes

    trishrhymes Senior Member

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    I agree with Simon that a lot of good work was done by this student, and she clearly learned a lot of techniques that could be applied to a larger study if there were funding. It really demonstrates the importance of using much larger samples. With such tiny samples of patients, and with the possibility that the definition used means some (or all) may not have PEM, this seems a wasted opportunity.

    Several of the graphs, like the one @Valentijn has posted, seem at a glance to show quite big differences between ME patients and controls, however, with such tiny samples, you need enormous differences to achieve statistical significance. What a pity if this apparent lack of significance means there may not be further funding to try this with a bigger and better defined sample.
     
  9. lansbergen

    lansbergen Senior Member

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    What would healthy fetal bovine serum do to diseased me cells?
     
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  10. MeSci

    MeSci ME/CFS since 1995; activity level 6?

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    :rolleyes:

    and chick embryo extract. ???
     
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  11. Esther12

    Esther12 Senior Member

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    I agree.

    While there are so few solid leads, and likely a number of different subgroups to be disentagled, I wonder if it's a mistake for PhD students to be focussing on CFS?

    The uncritical citing of PACE is shitty though.
     
  12. JaimeS

    JaimeS Senior Member

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    We now have multiple researchers telling us the patient's serum/plasma matters. Putting them in some other medium is significant.
     
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  13. lansbergen

    lansbergen Senior Member

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    Thought so. Thanks
     
  14. Snowdrop

    Snowdrop Rebel without a biscuit

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    But of course that relevant research was not done in the UK (to my knowledge) so therefore rendered irrelevant as a result?
     
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  15. daisybell

    daisybell Senior Member

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    I think this paragraph should read...it is possible to conclude there to be no evidence of impaired muscle function in muscle extracted from patients with CFS/ME when using in vitro muscle culture approaches. However, it cannot be determined that bio energetic function is normal in patients.
     
  16. alicec

    alicec Senior Member

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    Foetal bovine serum and things like chick embryo extract are standard ingredients for tissue culture of many cell types. The cells simply won't survive without them.

    The effect of patient serum is a separate question. If this were to be investigated then it would be an addition to the culture. The fetal bovine serum etc would still be necessary.
     
  17. lansbergen

    lansbergen Senior Member

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    Why not testing the fresh cells and later the cultured cells?
     
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  18. alicec

    alicec Senior Member

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    Isolating the pure myoblast cells from a tissue sample is complex and involves culturing the cells for several days under conditions which favour the myoblasts and not contaminating cells such as fibroblasts.
     
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  19. sdmcvicar

    sdmcvicar

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    Likely lack of an established lab protocol or workflow for taking patient samples, processing and then immediately testing them. It's a weakness in practically all biomedical research, because attempting to test samples immediately requires greater resources (transport, experiment prepped and ready to go), introduces additional variables (when did you get the sample? How much transport time between there and the lab? Temperature and sterility controls?), and generally requires collaboration of clinical and research personnel. There's also the issue of expansion (culturing the cells to allow them to multiply), which is usually done so that you can run multiple different tests, or multiple replicates for a single test, since your sampling may not give you a sufficient number of cells to give you a measurable response in biochemical tests.
     
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  20. lansbergen

    lansbergen Senior Member

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    Thank you
     
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