Brown AE, Jones DJ, Walker M, Newton JL
Institute of Cellular Medicine, William Leech Building, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom
This research was supported by ME Research UK and by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre based at Newcastle upon Tyne Hospitals NHS Foundation Trust and Newcastle University.
Post exertional muscle fatigue is a key feature in Chronic Fatigue Syndrome (CFS). Abnormalities of skeletal muscle function have been identified in some but not all patients with CFS. To try to limit potential confounders that might contribute to this clinical heterogeneity, we developed a novel in vitro system that allows comparison of AMP kinase (AMPK) activation and metabolic responses to exercise in cultured skeletal muscle cells from CFS patients and control subjects.
Skeletal muscle cell cultures were established from 10 subjects with CFS and 7 age-matched controls, subjected to electrical pulse stimulation (EPS) for up to 24h and examined for changes associated with exercise.
In the basal state, CFS cultures showed increased myogenin expression but decreased IL6 secretion during differentiation compared with control cultures. Control cultures subjected to 16h EPS showed a significant increase in both AMPK phosphorylation and glucose uptake compared with unstimulated cells. In contrast, CFS cultures showed no increase in AMPK phosphorylation or glucose uptake after 16h EPS. However, glucose uptake remained responsive to insulin in the CFS cells pointing to an exercise-related defect. IL6 secretion in response to EPS was significantly reduced in CFS compared with control cultures at all time points measured.
EPS is an effective model for eliciting muscle contraction and the metabolic changes associated with exercise in cultured skeletal muscle cells. We found four main differences in cultured skeletal muscle cells from subjects with CFS; increased myogenin expression in the basal state, impaired activation of AMPK, impaired stimulation of glucose uptake and diminished release of IL6. The retention of these differences in cultured muscle cells from CFS subjects points to a genetic/epigenetic mechanism, and provides a system to identify novel therapeutic targets.
Comment by ME Research UK
In the historical literature, the hallmark of myalgic encephalo-myelitis (ME) was marked muscle fatigability often in response to minor degrees of exercise. Muscle cramps, fasciculations (twitching) and extreme muscle tenderness were also common findings. As Dr Melvin Ramsay said in the Postgraduate Medical Journal in 1978, “This was sometimes obvious as the patients winced even on light palpitation of the affected muscle; but much more frequently it took the form of minute foci of muscle tenderness which had to be carefully sought and for no ostensible reason were generally found in the trapezii and gastrocnemii.” Today, patients diagnosed with ME/CFS frequently highlight the importance of peripheral fatigue – such as impairment of muscle power – in their experience of illness.
Research in other diseases has highlighted important biological mechanisms that appear to underlie muscle fatigue, and since 2006 ME Research UK has provided the pilot funding for many distinct projects at the University of Newcastle to explore the role of these mechanisms in ME/CFS (see programmes of research). In one of these studies, magnetic resonance scanning of peripheral muscle (a scanning technique which looks at the way in which muscle is working) revealed significant abnormalities in the handling of acid within muscle – suggesting that acid build-up during exercise in ME/CFS patients may be due to an impairment of muscle cells and their function. To explore these and other interesting leads, ME Research UK awarding further funding in 2009 to Prof David Jones and Prof Julia Newton to undertake in vitro studies based on primary assay and culture of muscle cells (myocytes) derived from ME/CFS patients and healthy controls following establishment of the techniques using existing myocyte cell lines. The first scientific paper from this series of investigations has just been published in the journal PLoS ONE (download the full paper), and it certainly makes fascinating, if complicated, reading.
For these experiments, the authors examined cultures of isolated skeletal muscle cells (obtained by needle biopsy of the vastus lateralis muscle) from 10 people with ME/CFS and 7 age-matched controls. Electrical pulse stimulation (EPS) was applied for up to 24h to simulate an ‘exercise challenge’ by inducing contraction in the cultured myotubes, so that the effect of ‘exercise’ directly on the cells themselves could be observed. As the researchers point out, the attraction of using the muscle cell cultures is that “they are subject to the same standardised conditions, so that any differences that emerge between the patients and control cultures will reflect changes… in the cultured cells”, rather than, say, the many intra-personal or intra-group differences which can complicate clinical studies.
The main findings were that, compared with unstimulated cells, cultures from the healthy group had significant increased levels of AMP-activated protein kinase (AMPK) phosphorylation and glucose uptake after a full 16 hours of ‘exercise’ simulated by EPS, while cultures from ME/CFS patients showed no such increases. In addition, the secretion of interleukin 6 (which is involved in inflammation) in response to EPS was significantly reduced in ME/CFS compared with the control cell cultures. Finally, even without ‘exercise’, the expression of myogenin (which co-ordinates skeletal muscle development and repair) was higher in muscle cell cultures from ME/CFS patients than in the control cultures.
The two defects found in the cultured skeletal muscle cells of ME/CFS patients – impaired activation of AMPK and impaired stimulation of glucose uptake – are particularly intriguing. The fact that the ME/CFS cultures were unable to increase the rate of glucose uptake in response to ‘exercise’ most probably reflects the impaired activation of AMPK, a complex enzyme. All living cells must maintain a high ratio of ATP to ADP if they are to survive. In animal cells ADP and phosphate are converted to ATP (equivalent to charging a battery), while cellular processes obtain their energy by converting ATP to ADP and phosphate (discharging the battery). The rates of these energy-requiring processes in the cell are balanced almost perfectly, and this balance is achieved by sophisticated regulatory systems in cells. The AMPK system plays a key role in these as a sensor of cellular energy status. As the authors point out, the lack of activation of AMPK during ‘exercise’ in muscle cells from ME/CFS points to a muscle abnormality at the level of AMPK (which is normally activated during muscle contraction) or in other regulatory enzymes further up the biochemical pathway, and they plan to investigate these as well using the experimental in vitro muscle system they have already developed.
Overall, the evidence from this important study points to an exercise-related, primary abnormality in the muscle of ME/CFS patients which, because it was observed in cultured isolated muscle cells, may well have a genetic or epigenetic basis. Exciting results without a doubt.
And there is an interesting coda to this story. In 2003, ME Research UK hosted a Workshop at the University of Dundee in which one of the speakers was Professor Grahame Hardie of the Wellcome Trust Biocentre. His talk was on “Management of cellular energy by AMPK” (download the report), and he ended with the words, “Studies on muscle, including work with McArdle’s disease patients who have a typical history of exercise intolerance and myoglobinuria, have shown that dysregulation of the pathways in which AMPK is involved may be a contributing factor in muscle fatigue. While it is too early to postulate a direct role for AMPK dysregulation in the pathogenesis of ME/CFS, researchers into this illness should be encouraged to consider this possibility.” These results from the team at the University of Newcastle suggest that his comments all these years ago were prescient indeed.