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Journal Club: WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome

What’s The Deal With ME/CFS?

For decades, a mystery syndrome has struck down up to 130 million people worldwide. Crushing fatigue, difficulty in thinking clearly, muscle aches, exhaustion after exercise that takes forever to go away, difficulty sleeping and more. This constellation of symptoms is referred to as Myalgic Encephalomyelitis (ME) or Chronic Fatigue Syndrome (CFS).

Generally believed to be related to post-viral syndrome, people who develop this illness find themselves trapped in a cycle of fatigue and exhaustion that prevents them from going about their daily business.

Why are ME/CFS patients so tired all the time? What is making their muscles ache? Numerous research projects have examined muscles of ME/CFS sufferers, including taking biopsies and blood tests, but mostly results have been inconclusive or negative.

A common misunderstanding, is that when researchers do a clinical test and they get no answer, it means there’s nothing wrong. While sometimes this might be the case, if you don’t know what to test for, you will not find it. In reality, sometimes researchers solve a mystery by happy accident. This is one such serendipitous event.

While investigating Li-Fraumeni syndrome—a rare type of genetic cancer, Dr. Paul Hwang and his team noticed a difference between the mitochondrial proteins in a CFS/ME patient and those of her dad and brother. This caught their attention because we don’t know much about ME/CFS and its underlying biology.

This post will take you through the published results, explaining what the researchers found and what the evidence for their conclusions was.

If you have questions or thoughts, let use know in the comments below!

What are the researchers claiming?

  1. In the ME/CFS patient they examined, muscles didn’t make energy well and took a long time to refill their fuel supplies.
  2. Some ME/CFS patients had more WASF3 proteins than they should in their mitochondria.
  3. WASF3 caused mitochondria to malfunction by interfering with the conversion of glucose and oxygen into energy.
  4. Stressing the protein making compartment of cells made them produce more WASF3.
  5. Taking away WASF3 helped the mitochondria to make energy.

What’s the evidence for these claims?


Claim 1: In the ME patient they examined, her muscles didn’t make energy well and took a long time to refill their fuel supplies.

During a cancer research project, a Li-Fraumeni Syndrome patient was experiencing uncomfortable cramping, tired muscles. Strangely, her brother and father, who had the same genetic mutation, had no muscle complaints. After they ruled out cancer-related causes of the symptoms, the research team wondered if it might be linked to her ME/CFS diagnosis.

Muscle Testing

They started off looking at what was going on in her muscles, versus those of her dad and brother. When we use our muscles, the fuel that the cells use to help them contract and relax contains phosphorus so we can tell whether a cell is making energy correctly by how much phosphorous it uses.

The researchers dosed the family and some non-CFS/ME having volunteers with a tiny amount of radio-labelled phosphocreatine and set them off to exercise. They then used a specialized MRI machine to measure how much of the radio-labelled phosphorous was used to refill the muscles’ energy stores.

The woman used up far less phosphorous than the volunteers and her family members—this indicated that it was taking her muscle cells a lot longer to refuel after using up their existing energy stores.

The scientists also noticed that her blood lactate was high even at rest—could this be a sign that her mitochondria were having a hard time making enough energy?

How do we power our muscles?
Our muscles work by individual muscle cells contracting and expanding. Just like any movement, we need to supply energy to change from one state to another. Normally we use our mitochondria to make ATP.

ATP is the energy molecule that powers all our cellular processes. But when we exercise our muscles quickly run out of oxygen and glucose, we still need to make ATP.

Luckily for us, we keep stores of phosphocreatine and glycogen (modified glucose) in our muscles for just this scenario. When we need to keep using our muscles, a series of chemical reactions convert phosphocreatine and glycogen into ATP and lactate.

When we finish exercising our muscle cells have to replenish their stock of phosphocreatine ready for the next time, so we use creatine and phosphorous to rebuild our supply. Yep, that’s the same creatine you put in a shake before you work out, the idea is that it will help your muscles recover faster.

Lactate is a waste product that we can use as a marker for how much fuel our muscles consumed. Lactate is sent out of the cells and carried to the liver for processing. If you have a lot of lactate in your blood, it suggests that your muscles have had to dig into their emergency energy stores. If you didn’t do any exercise, it can indicate that your mitochondria are having trouble processing oxygen and glucose into energy.

Claim 2: Some ME/CFS patients have more WASF3 proteins than they should in their mitochondria.

The researchers took cell samples from the woman with Li-Fraumeni Syndrome and the volunteers. They made a list of proteins that we know are involved in mitochondrial function. Then, they did tests to see whether there was a difference between the energy making proteins in her cells, and the cells of people without ME/CFS. The woman’s test results showed that a protein called WASF3 (that genetic screens had previously implied might be involved in ME) was unusually high in her samples, compared to those of the others.

What’s WASF3?

Wiskott-AldrichSyndrome Protein Family Member 3 (WASF3) is a protein that helps to make changes in a cell’s shape. It helps to join other proteins together and often shows up in metastasizing cancer cells because it helps the cells to rearrange their protein skeleton and crawl around. Researchers have taken an interest in it because targeting it might slow or prevent the spread of cancer cells. The woman had 40% more WASF3 in her cells than the control group, and importantly, she had 34% less of the protein MTCO1- a protein used by mitochondria to make energy.

Once they had identified WASF3 as an interesting contender for the cause of the problem, the team did a deep dive into what it does. They started off by testing what effect WASF3 has on mitochondrial processes.

What happens inside mitochondria?

Mitochondria convert oxygen and glucose into energy through a series of chemical reactions that create the fuel molecule ATP. This is called “respiration.”

We use ATP to power the enzymes that keep us alive and functioning. For example, when your muscles do work, the molecular machines that make individual muscle cells contract need ATP to work.


If you can’t make ATP in large quantities and fast, you don’t have enough fuel to operate your muscles, so you feel weak or tired.

A fascinating element to ATP production is that it’s basically taking electrons off metabolites of glucose, passing them through a cascade of respiratory enzymes, ending up with giving them to oxygen and turning it into water. You could think of it as similar to an electric current—electrons moving from one place to another.

Breaking and making the chemical bonds releases energy, we store that energy in ATP. Some respiratory enzymes even have propellers for generating ATP!

Claim 3: WASF3 causes mitochondria to malfunction by interfering with the conversion of glucose and oxygen into energy.

Cells In a Dish

The scientists took a cell sample from the woman and grew it in a dish. They then used a targeted gene expression blocker to reduce the amount of WASF3 protein in the cell. This dramatically improved the cells’ ability to respire. They measured how much oxygen the cells were using before targeting WASF3, and how much they were using after. When they reduced WASF3, the cells were far better at consuming oxygen—a sign that they were happily making energy. The same thing happened when they blocked WASF3 in muscle cells.

When they turned the tables and powered up WASF3 production in muscle cells, they saw that the cells were no longer able to use up oxygen efficiently, and they were making fewer important mitochondrial proteins. This means that WASF3 interferes with the process of making energy from oxygen and glucose. The same effect happened in other cells too, so this energy-sapping process might not be exclusive to muscles.

We all know that sometimes results you get in a test tube don’t translate to the real world, so the next step was to see what WASF3 does in a whole body.

Mouse Model

The researchers genetically modified a family of mice to have more copies of the WASF3 gene. This increases the amount of WASF3 protein but only in places and at times when WASF3 would normally turn up.

The mice had the same experience as the original Li-Fraumeni Syndrome patient. They had more WASF3 and less MTCO1 in their muscles, they used a lot less oxygen, a lot less glucose and they were very, very tired after even a little exercise. When the team examined the mice’s muscles, they discovered that just like human ME/CFS patients, under the microscope they looked completely normal. Staining the muscle cell architecture to see if the mice had abnormal muscle fibres showed no difference, and all the usual important proteins for making the muscle contract were present and accounted for.

Now that they knew that WASF3 was interfering with a cell’s ability to turn glucose and oxygen into energy, it was time for the researchers to probe what WASF3 was actually doing.

How Did WASF3 Interfere with Respiration?

They started off by looking for WASF3 protein in the mitochondria of the WASF3 over producing muscles cells. They did this by popping the cells to let all their contents out into a tube, and separating the mitochondria out into another tube.

The team then used gel electrophoresis (kind of chromatography that uses an electric charge) to separate out all the proteins in the mitochondria, and all the proteins in the rest of the cell. The proteins were transferred them onto a membrane, and probed with labelled antibodies to see what was there -a western blot. Western blots give us a way to visualize and identify what proteins are in a mixture and how big they are. They found that the mitochondria had a high concentration of WASF3 compared to other parts of the cell. In cells making normal amounts of WASF3, the WASF3 protein was not found in such high amounts in the mitochondria.

Interestingly, they noticed that where WASF3 levels were high, the ratios of proteins involved in transferring electrons to oxygen were wrong. Somehow, WASF3 was interfering an important part of the mitochondrial machinery.


The researchers then looked to see what proteins WASF3 was attracted to by adding modifications to WASF3 that could put a mark on proteins it came close to. It turns out that WASF3 was interacting with the mitochondrial protein complex that helps electrons flow to oxygen molecules.

WASF3 was making it more difficult for a crucial part of the electron transfer apparatus to form.

Claim 4: Stressing the protein making compartment of the cells makes them produce more WASF3.

Finally, the scientists tested what triggers a muscle cell to make more WASF3 than they should. Other researchers had found that people with ME/CFS and rheumatic disorders often show signs of endoplasmic reticulum (ER) stress. With this in mind, the team made an educated guess that cells might make extra WASF3 when the ER is stressed.

The scientists acquired muscle biopsy samples from seven more ME/CFS patients and tested them to see which proteins their mitochondria contained. Using Western blots, they found that where WASF3 showed up so did ER stress markers. They discovered that the ME/CFS patients had higher levels of WASF3, lower levels of MTCO1 (a sign that oxygen wasn’t being metabolised well) and higher levels of the ER stress protein, PERK.

What’s more, they found that when they treated cells with poison that stressed the ER, WASF3 production shot up. When they took away the stress and babied the cells, WASF3 production dropped.

Claim 5: Taking away WASF3 helped the mitochondria to make energy.

Next the researchers added anti-ER stress drugs to cells taken from the original ME/CFS patient. They observed that proteins that show up when the ER was under stress were made in smaller amounts and they found less WASF3. The mitochondria in these cells started working better, using more oxygen, when the WASF3 and ER stress went away.

What Does All This Mean?

While the researchers only looked at a few patients, these results are pretty encouraging. If WASF3 over production is a common feature in patients who suffer from post-exertional malaise (a symptom that shows up in Long Covid/PASC patients too) it would solve a long standing mystery.


The question that has puzzlers doctors for so long has been “your muscles look completely normal! There’s nothing wrong with them, so why do you keep saying they are weak?”

Simply put, they were looking for answers in the wrong place. It wasn’t the muscle parts of the cells that weren’t working, it was the energy source.

Now that we have a possible cause of PEM, researchers should be able to start looking at ways to lower WASF3 or to stop it from entering the mitochondria. One approach would be to start looking at ER stress in ME/CFS patients.

This research has a long road to travel before it makes it into the clinic, but it’s a great start. Fingers crossed this is the first in a new generation of PEM studies!

References

  1. Wang PY, Ma J, Kim YC, et al. WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome. Proc Natl Acad Sci U S A. 2023;120(34):e2302738120. doi:10.1073/pnas.2302738120
Joanna Mulvaney PhD
Joanna Mulvaney PhD
Joanna Mulvaney worked as a bench researcher for much of her career before transitioning to science communication. She completed a PhD in developmental biology focusing on cell signaling in cardiogenesis at the University of East Anglia, Norwich, UK, before moving on to study axial skeleton development and skeletal myogenesis at King’s College London and regeneration of auditory cells in the ear at University of California San Diego Medical School, USA and Sunnybrook Research Institute, Toronto, Canada. When it comes to scientific information, her philosophy is: make it simple, make it clear, make it useful.
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2 COMMENTS
  1. What does fuel supply/energy stores mean?
    Several times you reference this but what is the actual molecule(s)

    • Hi Barclay, the energy molecule is ATP, made by the mitochondria from glucose metabolites and Oxygen. Remember the formula Glucose + Oxygen —> ENERGY+ water + carbon dioxide?
      In this scenario glucose donates its electrons to oxygen and in the process of breaking bonds energy is produced. That energy is stored in ATP a molecule that cells use to operate their proteins. So for regular ATP production the fuel is glucose metabolites.

      When you run out of oxygen and/or glucose to power the ATP making process, your muscles use a different form of fuel to make ATP: phosphocreatine and glycogen.

      These molecules are long term emergency stores that take a while to be processed and transported around the body. If your muscles find it difficult to use Oxygen to make muscles they are constantly tapping into their glycogen and phoshocreatine reserves. This makes it a much slower prorcess to refill the stores sitting in their cells for emergency.

      It’s like if you start using a savings account to pay your bills- it takes a lot longer to replace those savings if you have to keep dipping in cos there’s no money in your current account.

      Did this this help? Happy to answer any questions I can!

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