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The GIST

The Blog

 

(The GIST is about a third way down the page)

Exosome

An extracellular vesicle called an exosome bearing a heat shock protein. (Image by Guillaume-Pelletier, CC – Wikimedia Commons)

Extracellular vesicles (EVs) have been a relatively new introduction to the chronic fatigue syndrome (ME/CFS) field. They first popped up in 2018 and since then, at least 8 studies have surveyed them in ME/CFS. The Ludovic Giloteaux/Maureen Hanson NIH-funded group has led the way with 4 studies.

Ranging in size from 30-1,000 nanometers, extracellular vesicles are vanishingly small (a human hair is 80,000- 100,000 nanometers wide) packages that are regularly being emitted by our cells. They’re so tiny that it took the development of electron microscopes and ultracentrifuges to find them, but once they were found, they turned out to be everywhere and they pack a punch.

Our cells communicate with the rest of the body by emitting EVs filled with proteins, amino acids, lipids, DNA, and RNA. They can affect many processes in the body including immune and metabolic regulation. Because their composition reflects what’s happening in the moment, studies assess their protein (proteomics) content, gene expression (transcriptomics), etc., to get a snapshot of how the body is responding.

In “Dysregulation of extracellular vesicle protein cargo in female myalgic encephalomyelitis/chronic fatigue syndrome cases and sedentary controls in response to maximal exercise“, Giloteaux, in Maureen Hanson’s group, examined how exercise affects these strange communication devices in ME/CFS.

Their hypothesis was that studying EVs released during/after exercise would help inform what’s happens during exercise and what may be causing the post-exertional malaise people that ME/CFS experience.

Extracellular vesicle

How extracellular vesicles work. (Image by Yu_Jin_Lee_CC_4.0_Wikimedia_Commons)

The GIST

  • Our cells communicate with the rest of the body by emitting vanishingly small bags of proteins, amino acids, lipids, DNA, and RNA called extracellular vesicles (EVs). These EVs can affect many processes in the body including immune and metabolic regulation. Because their composition reflects what’s happening in the moment, studies assess their protein (proteomics) content, gene expression (transcriptomics), etc., to get a snapshot of how the body is responding.
  • It was no surprise then to see the Gilotreaux / Hanson team at Cornell use them to check out what happens when people with ME/CFS engage in a short bout of intense exercise.
  • They found that the EVs in the female ME/CFS patients were “highly disrupted” – and in a familiar way. Just as Hanson has shown has occurred with proteins, gene expression and metabolites, the EVs in the ME/CFS patients simply failed to respond. That is, far fewer EVs in the ME/CFS responded to the exercise than did the healthy controls, and when they responded, they often took longer to respond.
  • These findings fit a broad theme that, at the most basic of levels – the molecular level – ME/CFS patients’ bodies simply aren’t responding much to it. It’s as if they’re kind of ignoring that it’s happening at all. When they do respond, their response is also often off – suggesting that they’re responding in a deleterious way.
  • Pathway analyses indicated that the coagulation pathways were altered in the ME/CFS patients, which made sense given studies indicating the people with ME/CFS have 10x’s more microclots than normal.
  • Because muscles generate force by contracting, anything that disrupts the muscles’ ability to contract is going to impact one’s ability to exercise. Perhaps not surprisingly, a pathway analysis found that EVs involved in muscle contraction were in the top ten most altered pathways shortly after exercise in the people with ME/CFS.
  • While proteins involved in muscle contraction increased dramatically in the healthy controls after exercise, they were either reduced or did not increase in ME/CFS. Similarly, EVs associated with muscle-related tissue enrichment increased in the healthy controls but failed to do so in the people with ME/CFS.
  • Rob Wust’s recent long-COVID muscle biopsy exercise study appears to back up some of these findings. Wust found frequently damaged, and even dying, muscle tissue after exercise (in some patients), and an increased emphasis on the dirty and inefficient anaerobic energy production system.
  • A protein called clusterin (CLU) really stuck out for the researchers. It was the only protein that was both positively correlated with the degree of fatigue pre-exercise and with the amount of muscle and joint pain after exercise. It also decreased in abundance after exercise in the healthy controls but not in people with ME/CFS.
  • Clusterin  is involved in several processes that other studies suggest may be impaired in ME/CFS. It clears out cellular debris, helps remove old and damaged cells, and helps fold protein correctly. Clusterin levels were associated with increased muscle and joint pain and fatigue. The authors suggested that “this protein may have an important role in ME/CFS pathophysiology.”
  • Suggesting perhaps that a herpesvirus had reactivated, the researchers found evidence of upregulated immune pathways that respond to “antigen presentation”; i.e. the appearance of a pathogen or other substance that tweaks the immune system.
  • While nobody knows what is causing the strange lack of a molecular response to exercise, the fact that it’s been demonstrated to happen in some different compartments (gene expression, EVs, proteins, metabolites) in different studies suggests the group is on the right track.

The Study

The NIH-funded study involved 18 females with ME/CFS and 17 age and BMI-matched sedentary controls. Blood samples were collected immediately before an exercise test to exhaustion, and then 15 minutes and 24 hours later. We’ve seen exercise affect everything from gene expression, to proteins and lipids at the molecular level. Would that extend to the EVs?

A Failure to Respond

It did. Giloteaux et al. found the ME/CFS patients’ EVs were “highly disrupted” – and in a familiar way – compared to the sedentary but healthy controls (HC’s). The study found:

  1.  Fewer proteins in EVs 15 after exercise in patients compared to controls (Figure 4a).
  2.  Reduced protein expression in EVs after exercise in ME/CFS patients (63 increased EV proteins in ME/CFS vs 178 E in HCs).
  3.  Delayed response – a delayed increase in the abundance of several EV proteins after exercise.

The authors concluded that the ME/CFS patients exhibited a “failure to mount an adequate response to exercise at the molecular level.” The theme was a familiar one. In her exercise studies, Hanson’s group has found the same pattern with regard to proteins, metabolites, and gene expression in ME/CFS patients: for some reason, their systems are simply unable at the molecular level to respond to exercise.

Their systems may be responding in a way that’s injurious as well, but the broad theme is that, at the most basic of levels – the molecular level – ME/CFS patients’ bodies simply aren’t responding much to it. It’s as if they’re kind of ignoring that it’s happening at all. Given how stressful exercise is, that could explain a lot. One wonders how much problems with energy production at a systemic level could play a role in that.

Check out some reports from earlier blogs on Hanson’s team’s findings.

  • Gene expression – “As 102 genes in the HC” immune cells exploded into action, the genes in the immune cells of the ME/CFS patients lay low. They basically sat the exercise bout out – no significant changes in gene expression were found.”
  • Metabolites – “A small urine metabolomics study that found an explosion in altered metabolites (n=400) in healthy controls but no significant change in ME/CFS patients’ metabolites 24 hours after exercise later.”
  • Proteins – “Germain’s study found exercise triggered a much bigger change in the proteins found in the sedentary, but healthy, controls than in the ME/CFS patients. The healthy controls responded to the rigors of the second exercise test by scrambling their protein mix more. Lacking the same ability to do so, the ME/CFS patients did not.”

And now we can add EVs to the mix. Digging deeper into the study, the researchers found:

  • More EVs in ME/CFS – Despite the fact that the EVs in the ME/CFS patients didn’t respond as fully to exercise, apparently ME/CFS patients’ cells were shedding them more frequently as more EVs overall were found. The authors didn’t explain why this might be so but did note that higher concentrations of EVs have been found in other conditions, including Alzheimer’s and vascular conditions that affect the brain.

The Crazy Coagulation Cascade

blood clot.

Exercise triggers coagulation in everyone. The coagulation cascade triggered in ME/CFS was, however, quite different.

Looking at the pathways activated or not activated in the EVs, the researchers found that exercise triggered an increase in coagulation activity in both ME/CFS patients and the healthy controls but also that the coagulation pathways found were different in the people with ME/CFS.

Several coagulation cascade proteins (factors VIII and XIII AI, fibronectin) were significantly decreased 15 minutes later in ME/CFS. Likewise, several coagulation factors that have been associated with exercise in healthy humans (FN1, FGA, FGG or FGB) were not increased after exercise in the ME/CFS patients. In a disease characterized by increased levels of microclotting, the reduction in some parts of the coagulation cascade seemed surprising.

On the other hand, factors associated with fibrinogen (FGA, FGB and FGG), which forms the actual clots, were positively correlated with increased fatigue and PEM after exercise in ME/CFS. Likewise, proteins involved in platelets (platelet degranulation, platelet aggregation), as well as wound healing, were strongly correlated with the muscle pain in ME/CFS.

Similarly, increased levels of plasminogen – a new factor which raised a lot of interest in a recent ME/CFS study – were “strongly correlated” with the percentage of time spent reclining or lying down, which itself is, of course, associated with how severe ME/CFS is. The authors proposed that the increased levels of plasminogen reflected an increase in clot busing activity or fibrinolysis. The authors stated:

“The altered temporal profiles of clotting cascade factors in ME/CFS EVs post‐exercise reveals a disruption in the haemostatic balance between clot formation and fibrinolysis.”

The Plasmalogen Possibility for ME/CFS and Long COVID

That suggested, if I’m reading it right – that clot formation during exercise is being inhibited in ME/CFS (!) – just the opposite of what we might have expected. On the other hand it might reflect the attempts by the body to attack large numbers of, difficult-to-break-down clots. Whatever is happening, the study suggested, as others have, that blood clotting and coagulation are messed up in ME/CFS. Indeed, the authors wrote:

“Other recent reports corroborate the importance of coagulation processes in ME/CFS” – they noted that past studies have found a 10fold increase in microclots in ME/CFS.

Clotting, with all its factors, is a very complex process. Time will tell how EVs affect coagulation in ME/CFS, but this study suggests something has gone awry with that process in ME/CFS and EVs are playing a part in that.

The Muscles

muscles

Muscles generate force by contracting. The EVs suggested people with ME/CFS were having problems contracting their muscles properly.

Muscle contraction is kind of the cat’s meow when it comes to exercise. Because muscles generate force by contracting, anything that disrupts the muscles’ ability to contract is going to impact one’s ability to exercise. Perhaps not surprisingly, a pathway analysis found that EVs involved in muscle contraction were in the top ten most altered pathways shortly after exercise in the people with ME/CFS.

Myosin light chain factors help the muscles contract and help repair muscle damage after exercise. Of all the proteins found in the EVs of the healthy controls, they showed the largest increase after exercise. Myosin light chain factors (MYL9/MYL12A/MYL9/MYL16) in people with ME/CFS took a different tack however: they were either reduced or did not increase.

Similarly, while muscle-related tissue/protein enrichment was found after exercise in the healthy controls, no such enrichment was found in the people with ME/CFS. Increased levels of tropomyosin (TPM4), tropomodulin (TMOD3), and calmodulin (CALM2) from baseline to 24 h post‐exercise were strongly correlated with higher levels of muscle pain in ME/CFS.

In the end, the same pattern seen in EVs overall was found in the muscles – a failure to adequately respond to exercise. A decreased or delayed EV muscle response was associated with more symptoms in ME/CFS. While the authors didn’t say so, delayed muscle repair processes might result in more muscle damage/pain after exercise; i.e. PEM.

Note that Rob Wust, in his long COVID muscle biopsy study found that exercise produced similar findings including frequently damaged and even dying muscle tissue (in some patients), and an increased emphasis on the dirty and inefficient anaerobic energy production system. Wust is currently engaged in a similar study in ME/CFS.

Exercise Causes Muscle Damage and Energy Depletion in Long COVID

Trash Buildup? The Clusterin Factor

A protein called clusterin (CLU) really stuck out for the researchers. The only factor they devoted an entire section of the paper to, clusterin could play a central node in the fatigue/pain problems occurring in ME/CFS. It was the only protein that was both positively correlated with the degree of fatigue pre-exercise and with the amount of muscle and joint pain after exercise. It also decreased in abundance after exercise in the healthy controls but not in people with ME/CFS.

Clusterin is involved in a slew of cleanup processes that other studies suggest could be in play in ME/CFS. It clears out cellular debris, helps remove old and damaged cells, and helps fold protein correctly. In short, something like clusterin seems to make sense in a disease in which unusually shaped amyloid proteins (in the brain and clots) may be present, and problems with autophagy (mitochondrial cleanup) may be occurring. Since intense exercise always produces small injuries in the muscles, cleanup processes probably play an important role in the post-exercise period.

Plus, increased CLU levels have shown up in a variety of neurodegenerative and inflammatory diseases (inflammatory myopathy, rheumatoid arthritis) and have been linked with cognitive issues. It was no wonder that the authors reported:

“In our study, elevated CLU in EVs post‐exercise is associated with worse myalgia, arthralgia, and fatigue indicating that this protein may have an important role in ME/CFS pathophysiology.”

Immune Trouble

The stress of exercise could be reactivating herpesviruses or other pathogens which tend to reactivate during stress. To that end, the researchers found evidence of upregulated immune pathways that respond to “antigen presentation”; i.e. the appearance of a pathogen or other substance that tweaks the immune system.

Conclusion

Question Marks

ME/CFS patients fail to respond normally to exercise at the molecular level. The question is: why?

The Hanson group has done it again: for at least the fourth time, they’ve shown that, at the molecular level, people with ME/CFS are not only not responding normally to exercise but in key ways they simply aren’t responding at all. Whether one is measuring proteins, metabolites, gene expression or EVs, a normal response to exercise is, for some reason, not kicking in during exercise or afterwards.

While nobody knows what is causing that, the fact that it’s been demonstrated to happen in different compartments in different studies suggests the group is on the right track.

The EV study highlighted dysfunctions in some key factors at play in exercise including coagulation, muscle contraction, cleanup or trash removal, and immune issues.

It’s great to see the exertion problems in ME/CFS validated at a molecular level. The important next step would presumably involve trying to understand why, when the stress of exercise appears, ME/CFS systems are unable to respond normally.

The NIH’s Molecular Transducers of Physical Activity in Humans Program (MoTrPAC; Motor…Pac – get it? Kind of?) might provide some clues. The 2016 MoTrPac Initiative was designed to track “exercise’s impact on biological molecules” which, if I’m not mistaken, Hanson’s studies just did. Two the 19 original grants went to two Stanford researchers (Michael Snyder, Stephen Montgomery) to identify and characterize all the molecules that form during or after exercise, and Snyder has been involved in ME/CFS research.

Time will tell but Hanson’s group has got us off to a great start.

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