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

[SNT Gatchaman]

Metabolic adaptation and fragility in healthy 3-D in vitro skeletal muscle tissues exposed to Chronic Fatigue Syndrome and Long COVID-19 sera.

Spoiler: Authors

Sheeza Mughal; Félix Andújar-Sánchez; Maria Sabater-Arcis; Glória Garrabou; Joaquim Fernández-Solà; Jose Alegre-Martin; Ramon Sanmartin Sentañes; Jesus Castro-Marrero; Anna Esteve-Codina; Eloi Casals; Juan M Fernández-Costa; Javier Ramón-Azcón

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Long COVID-19 (LC-19) are complex conditions with no diagnostic markers or consensus on disease progression. Despite extensive research, no in vitro model exists to study skeletal muscle wasting, peripheral fatigue, or potential therapies.

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 in calcium homeostasis, hypertrophy, and mitochondrial hyperfusion. Structural analyses confirmed myotube hypertrophy and elevated mitochondrial oxygen consumption in ME/CFS. While muscles initially adapted by increasing glycolysis, prolonged exposure (96–144 hours) caused muscle fragility and fatigue, with mitochondria fragmenting into a toroidal conformation.

We propose that skeletal muscle tissue in ME/CFS and Long COVID-19 progresses through a hypermetabolic state, leading to severe muscular and mitochondrial deterioration. This is the first study to suggest such transient metabolic adaptation.

Web | PDF | Biofabrication | Open Access

 

[Hoopoe]

Figure 1 shows some abnormalities with a very good separation of controls and patients. This could be promising.

Structural analyses confirmed myotube hypertrophy and elevated mitochondrial oxygen consumption in ME/CFS. While muscles initially adapted by increasing glycolysis, prolonged exposure (96–144 hours) caused muscle fragility and fatigue, with mitochondria fragmenting into a toroidal conformation.

The idea that upon exposure to patient serum, cells can mount an initially successful compensation in response to some signal, which is later followed by dysfunction might explain some of the contradictory findings in ME/CFS research. It's good to see more research looking at things over time.

I'm curious if WASF3 would show up in some form. It's not mentioned in the text.

 

[DMissa]

There is a lot in here that will take me a very long time to parse in detail, but I met Sheeza last year in Oxford and I was impressed with her technical understanding. Good to see the work accepted

 

[SNT Gatchaman]

Recommended reading for all able. Prior to DecodeME this could be the paper the year. I’ve read it through once and it seems to hit ”a lot of notes.”

 

[Hutan]

Peripheral fatigue, a decline in muscle’s ability to contract and generate force due to internal causative factors and independent of changes in the brain or spinal cord, is one of the many hallmarks of ME/CFS and LC-19. Patients report high fatiguability which is the tendency of their muscles to tire and lose strength quickly, exercise intolerance and an overall reduced exercise capacity confirmed by clinical studies (3, 4).

Recent advances in 3D bioengineered in vitro skeletal micro-physiological systems replicate patient-specific physiological responses and enable the evaluation of pathophysiological responses to circulating systemic factors, such as autoantibodies, circulating cytokines and metabolic or redox toxins present in patient sera. These systems offer a non-invasive testing platform to accurately model conditions without bringing any discomfort to the patient.

Our work aims to understand the pathomechanism of two idiopathic conditions: ME/CFS and LC-19 by using 3D skeletal muscle tissues developed from immortalized human muscle progenitor cells. These mature and well-differentiated tissues were then exposed to sera from ME/CFS, Long COVID-19 and healthy donors for short and long exposures. Exposing tissues to patient sera allowed us to deliver actual systemic insults to muscles in a controlled environment without confounding factors such as physical deconditioning.

There are quite a few questionable statements about peripheral things and hypotheses in the Introduction. But, I'm keen to get on to the good stuff. Great to hear that they had healthy controls, as well as ME/CFS and Long Covid cohorts.

 

[Hutan]

@Snow Leopard

2.1. Tissues exhibit contractile weakness at short exposure to patient sera

Sorry about the watermark. Note that cohort size is small - 4 controls, 4 CFS, 5 Long Covid.
These experiments were after the in vitro muscle tissue was exposed to serum for 48 hours.
the differences do look impressive.

Consequently, at a higher frequency of 50 Hz, these tissues demonstrated significantly compromised force of contraction as indicated by a much shorter time to drop to 50 % relaxation (Figure 1L). A functional muscle is capable to sustain an increase in its force of contraction proportionally with the stimulus until a certain limit is reached, beyond which depletion of the energy reserves or changes in structural integrity induce impairment (24).

For LC-19 diseased tissues, this limit was reached at a lower stimulating frequency compared to the control and CFS samples. At 25 Hz, a majority of LC-19 tissues appeared to show peak performance following which they could no longer maintain their maximum force against the stimulus (Figure 1K).

 

[Sean]

Interesting. Hope they can replicate it in a much larger sample.

 

[V.R.T.]

What was the sample size here, out of curiosity?

 

[Hutan]

I'm just jumping ahead to Methods to check that the cohorts are not wildly different on confounders.

Age, sex (all female) and BMI matched.
ME/CFS - ICC, specialist confirmed diagnosis
LC - confirmed Covid-19 infection, also ICC with PEM, at least 3 months after the acute infection
Sedentary donors

That's all sounding fine.

For construct treatment experiments, serum biosamples were randomly assigned to the control or treatment groups. Investigators performing data analysis were blinded to the study hypotheses.

I think that means that the various serum samples were allocated randomly to the fabricated muscle. They don't say that the investigators were blinded to which cohort a sample came from, so it doesn't sound as though the assessment was really blinded. They didn't know which cohort was meant to perform worse, but I don't think it would have been hard to make a guess about that.

 

[Hutan]

What was the sample size here, out of curiosity?

Note that cohort size is small - 4 controls, 4 CFS, 5 Long Covid.

My impression so far is that there wasn't replication of the samples, so just one fabricated muscle with one serum sample from participant A; one fabricated muscle with one serum sample from participant B.
Edit - as per jnmaciuch's comment, the legend of Figure 1 tells us that there were 3 to 7 replicates of each serum sample. The figures have white dots presumably showing the replicates, and so the coloured dot must be the mean result for each participant donor.

Of course it would have been nicer with more muscle tissue replicates for each participant, and complete blinding of the samples. But, I've no doubt that this was quite a bit of work as it was. Just a bit more blinding would have been great, e.g. to really ensure that the best looking muscle tissue wasn't assigned to the healthy controls.

 

[V.R.T.]

What are the implications if this study replicates? If the factor in the serum causing these responses can be identified and removed, will cells and muscles return to normal? The paper calls these effects transient, so one would hope so...

And would this finding gel or clash with the Edwards/Cliff/Cambridge hypothesis @Jonathan Edwards?

 

[jnmaciuch]

Very excited to see this out. Will have to spend time combing through it.

 

[Hoopoe]

How difficult would it be to get an estimate of the size of the serum factors responsible for these effects?

How would a researcher try to find out what in the serum is causing the effects?

Is it possible that the effects are due to something important missing, rather than something harmful being present?

If we had an idea of the type of serum factor(s), would that help significantly in figuring out which cells might be making these serum factor(s)?

 

[ME/CFS Science Blog]

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?

 

[jnmaciuch]

My impression so far is that there wasn't replication of the samples, so just one fabricated muscle with one serum sample from participant A; one fabricated muscle with one serum sample from participant B.

Of course it would have been nicer with more muscle tissue replicates for each participant, and complete blinding of the samples. But, I've no doubt that this was quite a bit of work as it was. Just a bit more blinding would have been great, e.g. to really ensure that the best looking muscle tissue wasn't assigned to the healthy controls.

The specific figure legends list multiple serum replicates per sample--and many of the figures show (hard to see) white dots which I'm assuming corresponds to the individual replicate values. So I'm guessing that the colored dots are meant to be the mean for replicates from the same sample

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?

Perhaps. Like others have already noted, it's a very small sample size, so I'm going to take everything with a grain of salt compared to the Ryback study. The protocols also have differences between this study, the Ryback study, and the original Fluge study--different serum concentrations, incubation conditions, length of time spent exposed to serum, etc. I can't speak to exactly how much any of those factors could be expected to alter the results but they should be kept in mind.

 

[Hutan]

The specific figure legends list multiple serum replicates per sample--and many of the figures show (hard to see) white dots which I'm assuming corresponds to the individual replicate values. So I'm guessing that the colored dots are meant to be the mean for replicates from the same sample

Thanks jnmaciuch. I've amended my post above. 3 to 7 replicates for each participant's serum, and with the individual results of each replicate shown on the charts still showing good clustering - that makes the findings quite a lot more robust.

 

[Hutan]

2.2. Transcriptomics reveal Metabolic Plasticity, drop in Mitochondrial Fission and altered calcium homeostasis at short exposure.

To investigate the underlying mechanistic changes in diseased tissues that caused contractile impairment, transcriptomic analyses of the short exposure tissue samples was performed using total RNA Sequencing. Interestingly, the Multidimensional Scaling (MDS) analyses indicated that both ME/CFS and LC-19 treatments clustered together with no significant Differentially Expressed Genes (DEGs) (Figure 2A). There were, however, several DEGs when ME/CFS and LC-19 tissues were compared to Controls (Figure 2B, C). We further performed Gene Ontology (GO) Enrichment Analyses between both patient groups and controls, to identify dysregulated pathways and Gene Set Enrichment Anlaysis (GSEA) between ME/CFS and LC-19 to identify any underlying differential biological tendencies

They say 'short exposure', but these are the samples exposed to teh serum for 48 hours I think, the ones that showed the decrease in the muscle tissue's ability to contract. They didn't find differentially expressed genes when comparing the ME/CFS and LC serum treated samples. They did find some differences between the ME/CFS and LC samples compared to the controls.

It looks as though they compared the ME/CFS samples against the controls, and the LC samples against the controls, and got a similar amount of difference. Again, the charts look convincing.

There's basically no difference between the ME/CFS and LC samples - the difference between that comparison and the comparisons between each of the disease samples and the controls is astonishing. ('Astonishing' usually makes me suspicious, especially in small cohorts. It would be so good if this study actually holds up.)

 

[Hutan]

Figure 3 gives the GO enrichment analyses - so the pathways that the differentially expressed genes relate to.

ME/CFS versus controls

According to this GO profile, in ME/CFS skeletal muscle samples there is an upregulation of genes involved in protein translation, extracellular matrix, and developmental processes, while genes involved in basic metabolic, transcriptional, and organelle functions are generally downregulated. This could indicate an environment characterized by chronic stress, impaired metabolic activity, and ongoing or maladaptive tissue remodeling.

LC versus controls

Similar to ME/CFS vs Control comparison, a broad downregulation was observed for genes associated with cellular and nuclear functions

These findings indicate a pronounced shift in LC-19 tissue toward enhanced mitochondrial fatty acid metabolism, electron transport, and protein synthesis processes

Mitochondrial complexes and dynamics

Gene expression data from RNA Sequencing indicated that transcript levels corresponding to Mitochondrial Respiratory Chain complexes were dysregulated in diseased groups, highlighting a compensatory response (Figure 4I).

The upregulation was comparatively more for the LC-19 samples compared to ME/CFS. The same trend was observed for mitochondria encoded genes and those encoded in the nuclear genome. Mitochondrial Dynamin Related Protein 1 (DNM1L) transcript levels were downregulated indicating a drop in mitochondrial fission (Figure 3I). This observation in conjunction with the upregulation of Mitofusin- 2 (MFN2) and SMYD1 signposts towards adaptive changes in mitochondrial performance in favor of mitochondrial network fusion. Furthermore, upregulation in TCA cycle and Glycolytic gene expression for ME/CFS and LC-19 compared to controls indicates cellular adaptation against increased energy demands.
]
In the RNA Sequencing analysis, we also observed indication of mitochondrial apoptosis through the upregulated expression of AIFM1 (Apoptosis-Inducing Factor, Mitochondria Associated 1) and ENDOG (Endonuclease G). AIFM1 is involved in caspase-independent apoptosis and translocated from the mitochondria to the nucleus upon apoptotic stimulation. It has also been implicated in maintaining mitochondrial OXPHOS (25). Similar to AIFM1, ENDOG is also a pro-apoptotic mitochondrial protein. We also observed a downregulation in HTRA2 (High Temperature Requirement A2) which suggested compromised mitochondrial protein quality control and accumulation of damaged or misfolded proteins (26).

Validation of differentially expressed genes
It's worth noting that there looks to be only three participants per cohort. It's possible that some results for other participants were left out. But even so, there are some clear differences.


SMYD1

A lysine methyl transferase specific for striated muscles, SET and MYND Domain Containing 1 (SMYD1) plays an important role in muscle differentiation and mitochondrial bioenergetics including stabilization of respiratory complexes and cristae formation (27, 28). It was found to be upregulated in ME/CFS and LC-19 tissues compared to the controls (Figure 4A,I). SMYD1 gain-of-function has been previously associated with upregulation of mitochondrial respiration as a protective mechanism against injury (27).

ATP2A1

Furthermore, the levels of ATP2A1 which codes for SERCA1 Ca2+-ATPase were also upregulated (Figure 4B, I). Predominantly present in type II fast twitch muscle fibers, this pump is a key regulator of relaxation dynamics of a striated muscle. Found in the sarcoplasmic reticulum (SR) of muscle cells it pumps the calcium ions from the cytoplasm into the SR, allowing muscle relaxation post contraction and restocking ions for the next contraction (29).

Downregulation in ATP2B4 (calcium ATPase isoform 4) observed in RNA-Seq, (responsible for removing intracellular calcium ions against the large gradients), indicates high calcium sequestering (Figure 10I). Coupled with an upregulation in ATP2A1 levels, this indicates a disturbance in calcium homeostasis. Increase of calcium sequestering by ATP2A1 in the sarcoplasmic reticulum has been related to a disturbance in mitochondrial function and an inducer of fatigue. Increased mitochondrial calcium can trigger the production of ROS through the Electron Transport Chain, particularly the levels of superoxide radicals (30, 31).

Figure 4l


There's a lot here.

SOD2

Downregulation of SOD2 (superoxide dismutase 2) was observed in diseased tissues, confirming the previously reported data (8). Coupled with an increase in calcium sequestering, a decrease in SOD2 levels could signal oxidative stress (Figure 4I).

FHL1

Four and-a-half LIM protein 1 (FHL1) has been implicated in inducing myotube hypertrophy (32). It was found to be overexpressed in the diseased tissues (Figure 4C, I).

 

[jnmaciuch]

Thanks for your detailed analysis as always @Hutan. I agree the ME/CFS vs. C-19 volcano plot in Fig 2 is striking compared to the others. My instinct tells me that you should be seeing more low p-value genes than that just by chance—though I can’t say for sure without running a simulation on their data. The other two plots in that figure looks like what I would expect from a small cohort in terms of distribution and the amount of genes that pass the p-value and logFC cutoffs.

The RT-PCR validation for gene expression is a strong point of the paper, and necessary in my opinion for this small of a sample size.

I’d be really interested to get access to the whole data set if that’s going to be released with publication—I’m sure there were some other potentially interesting top hits that weren’t explored because they were outside the scope of the paper.

 

[SNT Gatchaman]

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?

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.

Briefly, for encapsulation, the human muscle precursor cells were trypsinized and resuspended in skeletal muscle growth medium. The cells were encapsulated at a density of 2.5×107 cells mL −1 in 30% v/v Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning), 2 U mL −1 thrombin from human plasma (Sigma-Aldrich) and 4 mg mL−1 fibrinogen from human plasma (Sigma-Aldrich). During hydrogel casting, care was taken to avoid bubbles and cold plasticware was used to prevent polymerization of Matrigel. The mixture was spread as homogenously as possible between the pillars without grazing the surface. After hydrogel introduction all tissues were incubated at 37°C for 30 min to allow for matrix compaction before adding skeletal muscle growth medium supplemented with 1 mg mL −1 of 6-amino-caproic acid (ACA, SigmaAldrich). The hence formed tissues were allowed to grow for 2 days after which differentiation was initiated for another 6 days by replacing growth medium with differentiation medium (DM) supplemented with 1 mg mL −1 ACA (DM/ACA). Subsequently, half of the DM/ACA was replenished every 2 days to maintain tissue survival.

Indistinguishable mitochondrial phenotypes after exposure of healthy myoblasts to myalgic encephalomyelitis or control serum (2025) —

Human skeletal muscle myoblasts were obtained from Lonza (#CC-2580, lot number 21TL138913). Cell culture was commenced and maintained according to the manufacturer’s protocols using SkGM-2 Medium (CC-3244) (bioscience.lonza.com/lonza_bs/GB/en/download/product/asset/29428). Myoblasts were kept below passage number 10 for all experiments, as reported in Fluge et al (2016).

HSMM were seeded at 8,000 cells per well and kept in a 37°C incubator with 5% CO2 overnight. The following day, cells were washed once with PBS and media changed to serum-free HSMM media supplemented with 20% serum from either a pwME or a healthy control, with media and serum refreshed on day 3. Detailed protocols can be found in the pre-registration (https://osf.io/qwp4v, 02/08/2024). On day 6, myoblasts underwent a mitochondrial stress test, performed as per the manufacturer’s protocols.