Metabolic adaptation and fragility in healthy 3-D in vitro skeletal muscle tissues exposed to [CFS] and Long COVID-19 sera, 2025, Mughal+

[Hutan]

Tissue Weakness and Fragility increases with exposure along with Mitochondrial Fragmentation

For this experiment, we used tissues obtained from the same batch of encapsulation to avoid variability in handling and exposed them to LC-19 and control sera for 48, 96 and 144 hours.

So, to test the tissue strength, they used some of the engineered muscle tissue and applied LC and control sera for the three different lengths of time. Note, no ME/CFS sera in this experiment.

The diseased tissues were weaker as evidenced by a lower T50% compared to the controls (Figure 6a)

I think this is interesting. They weren't testing tear strength of the tissue here, they were testing the ability of the muscle to contract (as they did before).

Figures 6A-C

A) Relative absolute force at 50 Hz for Control. The dotted line indicates the time taken for the force to drop to 50% of its peak value under sustained tetanic stimulation of 50 Hz (T50%)
B) Relative absolute force at 50 Hz for LC-19 tissues over time.
C) Brightfield images of progressive muscle exposure to Control and LC sera


In A, the blue lines are the control at the different serum exposure times. B, with the orange lines, is the same for the LC serum. The lines are the force exerted by the tissue when stimulated. Note that the charts are of relative force, for that tissue and that exposure. So, all the experiments achieve 100% of the force at the peak, just because that is how the chart is set up. But, tissue with longer exposure to the LC serum cannot sustain the force as long.

Figure 6c shows examples of the tissue, with the LC tissue looking less robust over time.

Tissue survival decreased sharply with time for both diseased and control groups, but the decline was two-fold higher for the former than the latter.

If you look at Figure 6f, I'm not sure that there are really differences in mitochondrial branching between groups. The differences between groups don't seem to be significant. The very small samples sizes don't help.

We then analyzed mitochondrial morphology and quantified mitochondrial networks for each time point. Our data indicated a decline in mitochondrial branching and mean branch length. The mitochondria not only assumed the familiar globular geometry observed during fragmentation but also toroidal conformations indicating changes in mitochondrial membrane potential at 144 hours of patient serum exposure (Figure 6F, G).

The toroidal conformations are typically observed with FCCP administration at high dosages due to depolarization of the mitochondrial membrane, resulting in a drop in mitochondrial membrane potential and uncoupling of mitochondrial oxidative phosphorylation and ATP synthesis (47). These findings confirmed mitochondrial stress induced by systemic stress factors coupled with myotube atrophy at longer exposures. Our results of prolonged exposure to patient sera further warrant investigation due to small sample size but signpost at progressive deterioration of muscle structure, function and mitochondrial energy production, mimicking the conditions observed in patients.

There are some photos of the cells with the mitochondria looking more odd in the LC and ME/CFS samples. This is a bit shaky, and the authors seem to acknowledge that, noting the very small sample sizes. It does look though, that the muscle tissue is less able to handle the stress of being incubated with LC or ME/CFS serum rather than healthy serum.

That's the last of the results.

 

[wigglethemouse]

Thanks for the breakdown of the results in the paper. For something this detailed in a pilot study I think it's hard to have many samples.

In the recent Stanford Vishnu Shankar paper they were able to add more samples between preprint and publication. It sure would be nice given the promising findings if funding could be found to add more samples & testing for some of the more promising results.

EDIT: Oops. This does not seem to be a preprint.

 

[jnmaciuch]

Your explanation helps me understand what I should be looking for, but I'm still not really seeing it.

Yeah it’s the weakest finding in the paper by far. I had a classmate with more IHC experience try to point out mitochondrial branching to me once and still couldn’t fully see the differences they were talking about. I sort of see it in Fig 5, there’s more “Y” shapes throughout. Though the CFS image is way sparser overall, seems like a poor representative image to pick.

I’m always a little skeptical of things that require such a trained eye to pick out, but if the phenomenon really does exist and you just need to know what you’re looking for, then you need to have all the images fully blinded and randomized, ideally with multiple people annotating every image or at least the same person doing everything.

The methods don’t mention anything like that—I’m hoping it’s because it’s so obvious that they didn’t bother to mention it. Though I’ve been disappointed in the past. It’s a minor finding that can’t be interpreted easily anyways, so I think it’s probably not worth the attention.

 

[Hutan]

Yes, I don't want to end my posts on this paper negatively on that mitochondrial branching. All up, I like the idea of growing muscle tissue and exposing it to ME/CFS and LC serum. I'm glad this technology has been brought to the ME/CFS puzzle.

I think the findings about muscle strength and the ability to sustain contractions are really interesting and I'd like to see that work replicated quickly.

I'm not so sure about the Seahorse analysis or the mitochondrial morphology findings.

Hopefully the team will get funding to build on this work.

 

[ryanc97]

Again, refer to the Daratumumab thread. I suspect it could be AABs that are doing the damage. And where do the AABs come from? The LLPCs, which come from B-cells that differentiate.

 

[Grigor]

I think the findings about muscle strength and the ability to sustain contractions are really interesting and I'd like to see that work replicated quickly.

Both the lead as well as the senior author responded to my post on LinkedIn and so did Rob Wüst. If I understand correctly Rob Wüst and Jeroen den Dunnen are trying to (partly) replicate the findings.

Rob: Thank you for finding this one, Anil! It's published in a very exotic/unknown magazine which concerns me a bit. We can now verify the results

Me: Rob Wüst That's for sure. Perhaps verifying will become something like actual replicating? Although of course you are already short of hands and probably short of subsidies.

Rob: Anil van der Zee Yes, we're already doing this. Together with Jeroen den Dunnen and Amelie Bos, PhD, as part of PCNN | Post-COVID Network Netherlands WP6

"We developed 3D in vitro skeletal muscle tissues to map muscle adaptations to patient sera over time. Short exposures (48 hours) to patient sera led to a significant reduction in muscle contractile… | Anil van der Zee | 17 comments

"We developed 3D in vitro skeletal muscle tissues to map muscle adaptations to patient sera over time. Short exposures (48 hours) to patient sera led to a significant reduction in muscle contractile strength. Transcriptomic analysis revealed the upregulation of glycolytic enzymes, disturbances...

www.linkedin.com

I remember Jeroen den Dunnen speaking about culturing muscles cells and creating "PEM in a dish" in Berlin. Since he is slowly also focusing on muscle cells, this is where they'll try to replicate these findings as well? I should ask.

At 11:28

 

[DMissa]

Yeah it’s the weakest finding in the paper by far. I had a classmate with more IHC experience try to point out mitochondrial branching to me once and still couldn’t fully see the differences they were talking about. I sort of see it in Fig 5, there’s more “Y” shapes throughout. Though the CFS image is way sparser overall, seems like a poor representative image to pick.

I’m always a little skeptical of things that require such a trained eye to pick out, but if the phenomenon really does exist and you just need to know what you’re looking for, then you need to have all the images fully blinded and randomized, ideally with multiple people annotating every image or at least the same person doing everything.

The methods don’t mention anything like that—I’m hoping it’s because it’s so obvious that they didn’t bother to mention it. Though I’ve been disappointed in the past. It’s a minor finding that can’t be interpreted easily anyways, so I think it’s probably not worth the attention.

I'm not sure how it was done in the paper but you can quantify these things objectively with imaging software now

 

[Sean]

I remember Jeroen den Dunnen speaking about culturing muscles cells and creating "PEM in a dish" in Berlin.

There are few things I want more in this world than a robust in vitro model for PEM. That really would be a game changer for research and for us.

 

[jnmaciuch]

I'm not sure how it was done in the paper but you can quantify these things objectively with imaging software now

Yeah that's what I was trying to look for in the methods--it says that the mitochondrial morphology macro for Fiji was used. It gives the impression of being "semi-automated" but can't say for sure since I haven't used it.

 

[hotblack]

I also wonder how this relates to the study by @chillier which found no difference in oxygen consumption rate when ME/CFS serum was added to myoblasts. Do these studies contradict each other?

This was where my thoughts went immediately. If we assume both studies are sound I wondered if rather than contradicting each other could the two studies work together to help narrow down what the factor could be?

 

[V.R.T.]

I remember Jeroen den Dunnen speaking about culturing muscles cells and creating "PEM in a dish" in Berlin.

The question is have they created PEM in a dish or found the factor that causes ME/CFS exercise intolerance and muscle weakness/fatiguability? As opposed to delayed PEM.

 

[DMissa]

Yeah that's what I was trying to look for in the methods--it says that the mitochondrial morphology macro for Fiji was used. It gives the impression of being "semi-automated" but can't say for sure since I haven't used it.

We have a student in the lab doing this, I sat down with her and went through it a few days ago, actually. It was pretty objective and automated. That mito morphology package was part of the workflow, i just dont remember the full suite she was using. So it's possible this work here is also objective and automated

 

[DMissa]

There is a lot of talk in this thread about muscle culture and seahorse etc, I haven't gotten to read the paper yet but useful things to look into would be a) whether activity matched controls b) passaging/handling of the cultures (primary muscle cell phenotypes change with doublings - is that what has been used? havent looked in detail at what was cultured) c) absolute basal oxygen consumption rate values not being negative or close to 0 - if reported as normalised to cell number or stained DNA fluorescence or total protein this might be harder to glean d) not a huge amount of variability after ionophore injection (if used, ia assume so) this can cause cells to swell up and detach which confounds measurements, many aspects of the protocol have to be optimised to avoid it such as instrument mixing protocol, choice or presence of adherence matrix, incubation times, etc

sorry to offload homework, under the pump

my interactions with the lead author leave me confident that pitfalls would have been avoided but yes those are the first things that spring to mind when it comes to evaluating this kind of work. there are others but too technical to explain briefly

 

[DMissa]

I also wonder how this relates to the study by @chillier which found no difference in oxygen consumption rate when ME/CFS serum was added to myoblasts. Do these studies contradict each other?

is it the same same cell type and what are the differences in how they were handled would be my questions (have absolutely no time to look)

 

[jnmaciuch]

We have a student in the lab doing this, I sat down with her and went through it a few days ago, actually. It was pretty objective and automated. That mito morphology package was part of the workflow, i just dont remember the full suite she was using. So it's possible this work here is also objective and automated

Good to know!

 

[hotblack]

I’m only just digging into this in more detail and others have I think raised most of the questions that popped into my head already. But a couple of other thoughts

Could the difference be that this is investigating a muscle tissue preparation (with a longer setup time) which could then include secreted extracellular matrix components, vs multiple but independent and disorganised muscle cells in a well? Abnormal signalling and pathological cell effects might require ECM components.

Interesting. I wondered what differences there could be between the myoblast culture and this sort of grown muscle model, I know nothing about this area but it would be interesting to understand more and of the differences could tell us something interesting about the mechanism.

What I can’t figure out is what would make this study resemble the Fluge et al. results when the Ryback study tried to mimic the Fluge study’s protocol as much as possible. The best idea I have so far is that this study and the Fluge study included participants in active PEM, but no way to really assess that.

Would this effect or serum factor only being present in active PEM tell us anything? I also wonder how this fits into the narrative in this paper of the whole process being modelled (they seem to imply they are triggering PEM by putting strain on the muscle).

Sample size has been mentioned, presumably the small sample sizes in both Fluge and this paper mean they may have picked up something which is only present in a subset of ME/CFS patients but not all?

I don’t think we have any attempts to correlate with symptoms severity or other patient characteristics here, probably impossible with the small sample sizes but could be interesting and is something Ryback et al were able to do.

What would the differences in concentration mean and why so different between the papers? Is there a ‘standard’ concentration used for these sort of experiments or one which most closely matches real world conditions?

As with a number of these papers I come back to the idea that it may have found something but it’s not clear what that is. The narrative weaved distracts and my lack of knowledge of the area doesn’t help glean what the data is telling us. Seems interesting though!

 

[wigglethemouse]

The Seahorse testing only used a small number of patients. Talking to researchers who've measured PBMC's, and the Rybeck myoblast paper showed, individual results are either high, low, or average. If you use a low number of patients the mean could be anywhere.

So I don't think we should read too much into that part of the paper right now.

 

[wigglethemouse]

I remember Jeroen den Dunnen speaking about culturing muscles cells and creating "PEM in a dish" in Berlin. Since he is slowly also focusing on muscle cells, this is where they'll try to replicate these findings as well? I should ask.

Thanks for posting the video again. This was really good. That team is creative, smart, adaptable, and able to get things done. I liked how he had complete answers to questions, none of this "I'll need to get back to you". And he collaborates and recognises others work. I really enjoyed the talk.

 

[Dude]

Cort’s take on the study:

"Something in the Blood" Again! ME/CFS and Long COVID Serum Locks the Muscles Down - Health Rising

Geoff’s Narration The GIST Muscles on a Chip Whoa, another muscle study. We recently reported on a proteomic study suggesting that muscle repair problems contribute to post-exertional malaise in ME/CFS. Now, we have a direct muscle test – and even better – we get to see whether something in the […]

www.healthrising.org

 

[V.R.T.]

Cort’s take on the study:

"Something in the Blood" Again! ME/CFS and Long COVID Serum Locks the Muscles Down - Health Rising

Geoff’s Narration The GIST Muscles on a Chip Whoa, another muscle study. We recently reported on a proteomic study suggesting that muscle repair problems contribute to post-exertional malaise in ME/CFS. Now, we have a direct muscle test – and even better – we get to see whether something in the […]

www.healthrising.org

I'm not a huge fan of Cort's 'everything is true even conflicting things' approach to MECFS science but the below quote sounds quite a good idea to me:

What we need is a serious effort to break the logjam: a multi-center, blinded, consortium-based serum/plasma “challenge” study that handles the samples in the same way, uses standardized assays, determines if a dose-response pattern is present, identifies a specific component that’s causing trouble, and then removes it to see if the same response occurs. Surely we have enough evidence to support a major study like that.

This study would test a variety of tissues (platelets, endothelial NO, BBB endothelial activation, cardiomyocytes, 3D muscle) against a range of agents (IgG, EVs/exosomes, complement, etc.). Once it was clear which component affected which tissue, researchers would attempt to identify the active agent; i.e., which antibodies, EVs, proteins, or small molecules were causing the problem. Then the suspect factor would be introduced and removed multiple times to assess the robustness of the finding.

If we did that, we would know if something in the blood is contributing to ME/CFS or long COVID, and if it was, we would have a treatment target, and the opportunity to directly affect patients.