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An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS patients 9/2019

Pyrrhus

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Would elevated proton leak explain burning muscles?

YES.

The leaking H+ ions (AKA protons) would trigger the Acid-sensing Ion Channels (ASICs) on the nearby nociceptors, causing the sensation of muscle soreness.

Many people incorrectly assume that it is lactic acid that triggers the ASICs to cause the sensation of muscle soreness. But lactic acid is quickly removed from the muscle tissue and transferred to the liver.
 
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Pyrrhus

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This is the final version of the paper published by MDPI/International Journal of Molecular Sciences on 6 Feb 2020
https://www.mdpi.com/1422-0067/21/3/1074

Note that there is a SIGNIFICANT change in the final version compared to the preliminary version posted at the beginning of this thread:

Paul Fisher 2020 said:
Received: 26 January 2020

Commenter: Paul Fisher

Comment:
This 1st version of this manuscript contains an error which we would like to alert readers to: During revision of the paper, we discovered an error in the spreadsheet formula calculating the mitochondrial membrane potential. This is calculated by dividing one quantity by another. However the original formular had been entered into the spreadsheet the wrong way round so that instead of "a" being divided by "b", "b" was divided by "a". The result was that instead of the mitochondrial membrane potential being higher in ME/CFS lymphoblasts it was actually lower. The error has been corrected in a revised version of the article which has been submitted to Preprints. The authors would like to apologize for this embarrassing calculation error. Best regards to all readers Paul Fisher on behalf of all authors.
 
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Pyrrhus

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The paper suggests three hypotheses to explain why the Complex V isn't working:

What might cause such a mitochondrial Complex V inefficiency? Three possibilities are a mutation affecting one of the Complex V subunits or assembly proteins, a dysregulation of Complex V, or an elevation of the relative use of the proton motive force for other purposes (“proton leak”) making less available for ATP synthesis.

It is important to note that the authors did NOT say that there was anything fundamentally wrong with Complex V, more commonly known as "ATP Synthase".

It merely said that ATP Synthase (Complex V) was operating inefficiently, most likely due to a leaky mitochondrial membrane. (proton leak)

A leaky mitochondrial membrane is clearly the most logical explanation for the inefficiency of ATP Synthase (Complex V) given the author's January 2020 disclosure that the mitochondrial membrane potential was actually LOWER in ME/cfs compared to controls, which would directly impair the efficiency of ATP Synthase (Complex V).
 

gbells

Improved ME from 2 to 6
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It merely said that ATP Synthase (Complex V) was operating inefficiently, most likely due to a leaky mitochondrial membrane. (proton leak)

This sounds like blocked apoptosis. The mitochondria want to self destruct their membranes but are being inhibited so the cell ends up immortalized. Some cell apoptosis triggers are firing to trigger it but blocked checkpoints are aborting it mid stream. The energy demand ramps up the energy pathways.

The release of proteins from the intermembrane space of mitochondria is one of the pivotal events in the apoptotic process, which can lead to the activation of caspases and the ultimate demise of the cell.

Henry-Mowatt, J., Dive, C., Martinou, JC. et al. Role of mitochondrial membrane permeabilization in apoptosis and cancer. Oncogene 23, 2850–2860 (2004). https://doi.org/10.1038/sj.onc.1207534
 

bread.

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This sounds like blocked apoptosis. The mitochondria want to self destruct their membranes but are being inhibited so the cell ends up immortalized. Some cell apoptosis triggers are firing to trigger it but blocked checkpoints are aborting it mid stream. The energy demand ramps up the energy pathways.



Henry-Mowatt, J., Dive, C., Martinou, JC. et al. Role of mitochondrial membrane permeabilization in apoptosis and cancer. Oncogene 23, 2850–2860 (2004). https://doi.org/10.1038/sj.onc.1207534

do keep us alive or the virus that infects the cell alive?
 

Pyrrhus

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This sounds like blocked apoptosis.

Perhaps blocked apoptosis or maybe just poor mitochondrial health due to some cellular stress that probably increases the intracellular Ca2+ concentration.

Note that the author's 2021 follow-up paper noted:
Missailidis et al 2021 said:
The activity of the major cellular energy stress sensor, AMPK, was elevated but the increase did not reach statistical significance.
 
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Inara

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YES.

The leaking H+ ions (AKA protons) would trigger the Acid-sensing Ion Channels (ASICs) on the nearby nociceptors, causing the sensation of muscle soreness.

Many people incorrectly assume that it is lactic acid that triggers the ASICs to cause the sensation of muscle soreness. But lactic acid is quickly removed from the muscle tissue and transferred to the liver.
Yes. I read a paper about that. Was quite a revelation to me. (I had to laugh then: Everybody in the fitness world talks about lactic acid, and I imagined trying to explain how it really is.)

Thanks for your reply, @Pyrrhus!
 

Inara

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A leaky mitochondrial membrane is clearly the most logical explanation for the inefficiency of ATP Synthase
This is about a H+ leak through the inner mitochondrial membrane? I think that implicates a leak via the outer membrane, too? Would the mitochondria associated membrane play a role in any way, here, too? Like, will the proton leak affect the MAM (and so other structures)? Or can some form of dysregulation (which one?) via the MAM lead to a proton leak?
 

Inara

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or maybe just poor mitochondrial health due to some cellular stress that probably increases the intracellular Ca2+ concentration.
But the authors found no difference in ROS between normal and ME cells? A disrupted calcium homeostasis would lead to increased ROS.
 

Pyrrhus

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This is about a H+ leak through the inner mitochondrial membrane? I think that implicates a leak via the outer membrane, too?


Excellent question. Yes, the authors are referring to a H+ leak in the inner mitochondrial membrane, not the outer mitochondrial membrane. They hypothesize that this leak is due to H+ ions "sneaking through" protein/solute transporters in the inner mitochondrial membrane:
For example, the levels of key proteins in the mitochondrial protein import complexes were elevated in the ME/CFS proteomes, as were the levels of multiple SLC25 transporters in the inner mitochondrial membrane (Figure S8). Such supporting transport pathways contribute to the so-called “proton leak”, which refers to depletion of the PMF by mitochondrial transport processes other than ATP synthesis by Complex V.


But these protein/solute transporters must exist in the outer mitochondrial membrane as well, if the cargo is to be transported from the cytoplasm across both mitochondrial membranes, into the mitochondrial matrix.

Of course, there may be quite another explanation for why there are "leaky" mitochondrial membranes...

Would the mitochondria associated membrane play a role in any way, here, too?

Interesting question. If you're talking about the Endoplasmic Reticulum-Mitochondria associated membrane, I don't know how that might contribute to any H+ leak.

But the authors found no difference in ROS between normal and ME cells? A disrupted calcium homeostasis would lead to increased ROS.

First, for the benefit of others, here is what the authors say about Reactive Oxygen Species (ROS):
Missailidis et al said:
Another indicator of abnormalities in electron transport is the level of reactive oxygen species (ROS). ROS are produced by electron “leakage” to molecular oxygen at the point where electrons are normally passed to Complex III from either Complex I or II. ROS production can be increased either by an increased flux of electrons through the electron transport chain or by a “downstream” blockage that diverts the electron flow. We therefore measured the levels of ROS in patient and control lymphoblasts and found no change in intracellular ROS levels in the ME/CFS cells when compared with controls (Figure 3C, Figure S5 Panel 2C). This is consistent with the insignificant changes in basal respiration rate and also suggests that the electron transport chain (ETC) is functionally normally

The answer to your question would probably depend upon the details of the calcium disruption. Oxidative stress due to Reactive Oxygen Species (ROS) can certainly lead to an increased Ca2+ concentration, but there may be other causes. If anyone is interested in the interactions between mitochondria and Ca2+ concentrations, this review may be interesting:

Mitochondria and calcium: from cell signalling to cell death (Duchen, 2000)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2270168/

Hope this helps.
 
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WantedAlive

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This sounds like blocked apoptosis.
Perhaps blocked apoptosis or maybe just poor mitochondrial health due to some cellular stress that probably increases the intracellular Ca2+ concentration.

Rather than blocked apoptosis, it could instead be insufficient efferocytosis: the failure to clear apoptotic cells by phagocytic cells. High cell turnover is indicated in ME/CFS which might suggest increased apoptosis. Excessive apoptosis, insufficient or defective efferocytosis occurs in many chronic inflammatory and autoimmune diseases (COPD, cystic fibrosis, RA, SLE, etc). The release of undegraded apoptotic bodies into circulation is highly inflammatory.

Interestingly, the phagocyte engulfment capacity is enhanced by lower mitochondrial membrane potential, and mitochondrial uncoupling protein UCP2 is upregulated to achieve this [1]. Additionally, multiple uptake of apoptotic cells by macrophages triggers Drp1 induced mitochondrial fission [2], any impairment here is associated with excessive mitochondrial calcium sequestration.

In @HTester thread on thermogenesis, I’d suggested UCP1 might cause the H+ proton lean in Fisher’s study, this futile cycle involved in thermogenesis (related to why PwME run cold). Robert Phair told me he’d asked Fisher this in 2019 - evidently UCP1 wasn’t implicated. However, I failed to ask him about UCP2. Or perhaps it’s some calcium cycling mechanism.

Other mechanisms of this proton leak are of course possible. An excessive rise in mitochondrial calcium, a rise in mitochondrial pH, maybe the H+/Ca+ exchanger is implicated…who knows.
 

gbells

Improved ME from 2 to 6
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Perhaps blocked apoptosis or maybe just poor mitochondrial health due to some cellular stress that probably increases the intracellular Ca2+ concentration.
Rather than blocked apoptosis, it could instead be insufficient efferocytosis: the failure to clear apoptotic cells by phagocytic cells. High cell turnover is indicated in ME/CFS which might suggest increased apoptosis. Excessive apoptosis, insufficient or defective efferocytosis occurs in many chronic inflammatory and autoimmune diseases (COPD, cystic fibrosis, RA, SLE, etc). The release of undegraded apoptotic bodies into circulation is highly inflammatory.

I think it's blocked apoptosis rather than phagocytosis but feel free to prove me wrong and cure millions of ME patients.

I did an extensive history and know the pathogenesis of my own illness. It started with EBV mononucleosis, followed by HHV-6 and coxsackievirus 6B and possibly a bacterial infection from a flea bite from a feral cat. All of these things block apoptosis and I'm improving using immunotherapy so it supports the hypothesis. At 12 years ME with an activity scale of 6 I'm doing ok when I started at a 2 with light and sound sensitivity and mental issues (anxiety, depression).
 
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sb4

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In @HTester thread on thermogenesis, I’d suggested UCP1 might cause the H+ proton lean in Fisher’s study, this futile cycle involved in thermogenesis (related to why PwME run cold).
Perhaps I am misunderstanding. Shouldnt proton leak and UCP1 result in high temperature not low?
 

Inara

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If you're talking about the Endoplasmic Reticulum-Mitochondria associated membrane
I think my thoughts were this: Might there be a signaling dysfunction from the ER via the MAM to the mitochondrion - if the ER "leaks" too much calcium into the mitochondrion e.g., this could increase H+ proton leak? Or does a proton leak in any way "attack" the MAM, amongst others, so that this leads to increased oxidative stress, e.g. via ER stress (and/or a disturbed autophagy)?

Or is it possible that the leaked H+ do not cross the outer mitochondrial membrane? Would this lead to apoptosis? And if so, wouldn't the number of mitochondria be reduced, or wouldn't ATP be decreased?

But - if calcium signaling plays a role here, could this explain the viral onset that is observed in many pwME? Especially herpesviruses (and some bacteria I guess) "address" the ER calcium signaling pathways to get their DNA into the cell. But how, then, does this lead to an on-going disrupted calcium signaling which *might* be behind the H+ leak? Ok, if calcium channels are dysfunctional (e.g. due to a mutation; but I doubt this is the case in ME). Could these viruses cause a long-time calcium leak from the ER? What else could?

Or could a dysfunction in voltage-gated calcium channels be behind a proton leak? What would be the mechanism then?

There will be several possibilities why a proton leak from the mitochondrion could occurr.
 
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WantedAlive

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Perhaps I am misunderstanding. Shouldnt proton leak and UCP1 result in high temperature not low?

Good thinking @sb4 Firstly, thermogenesis occurs in adipose tissue, so there may be no connection at all with Fishers’ study of lymphocytes. It’s just speculation. To answer your question though, in trauma, disease or infection, a thermogenic response is common, but the response can be hyperthermic or hypothermic.

PwME seem to have a lower set point for shivering thermogenesis, evidence of activated adipose non-shivering thermogenesis, and run slightly hypothermic. A regulated decrease of body temperature is known as anapyrexia. Hypoxia can induce anapyrexia thought mediated on the central nervous system by lactate, adenosine and nitric oxide among others. Anapyrexia reduces oxygen consumption, increases the affinity of haemoglobin for oxygen, and conserves the energy cost of hypoxia. A lower temperature can affect pH also, but I’m not sure if half a degree would have much effect. A lower temperature can also reduce inflammation. So overall, a thermoregulation change is a disease tolerance strategy, whether high or low.

I can only speculate therefore, that the non-shivering thermogenesis is for temperature maintenance in balance with other mediators, whether hypothermic or hyperthermic. How it might link with Fisher’s study and mitochondrial proton leak, if it even does, is still very unclear.
 

Inara

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Or is it possible that the leaked H+ do not cross the outer mitochondrial membrane?
To answer this question in part:

The flow of electrons along the electron transport chain results in the translocation of H+ ions across the inner mitochondrial membrane (IMM) from the matrix to the intermembrane space (IMS). This process contributes to create a large mitochondrial membrane potential (ΔΨmt) that, besides its key role in ATP synthesis, produces a large driving force for Ca2+ uptake into the mitochondrial matrix...
https://www.frontiersin.org/articles/10.3389/fphys.2018.00791/full

This doesn't mean the extra H+ do not cross the mitochondrial outer membrane, it means H+ stimulates Ca2+ uptake (amongst others, from the ER).
 

Inara

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if the ER "leaks" too much calcium into the mitochondrion e.g., this could increase H+ proton leak?
It seems it does. But it would increase ROS, too, and the authors found ME lymphoblasts don't produce more ROS compared to normal ones.
See e.g. https://www.frontiersin.org/articles/10.3389/fphys.2019.01142/full
The paper is about a RYR1 that leaks calcium; they found a higher Ca2+ uptake into mitochondria and higher ROS levels. Ok, one has to stress that the specific RYR1 channel in the paper leaks a lot more calcium than the normal channel, to my knowledge.

Does this exclude a calcium signaling issue behind the H+ leak?

How is it possible to have more H+ without more ROS? Or maybe is the experiment in Fisher et al's paper not "fine" enough?
 

WantedAlive

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See e.g. https://www.frontiersin.org/articles/10.3389/fphys.2019.01142/full
The paper is about a RYR1 that leaks calcium; they found a higher Ca2+ uptake into mitochondria and higher ROS levels. Ok, one has to stress that the specific RYR1 channel in the paper leaks a lot more calcium than the normal channel, to my knowledge.

@Inara You feature an interesting paper here, on 'malignant hyperthermia'. I mentioned malignant hyperthermia in context of 'UCP1 independent thermogenesis' in the thermoregulation thread with Robert Phair: Thermoregulation and body temperature | Page 2 | Phoenix Rising ME/CFS Forums, upon learning UCP1 wasn't the cause of the proton leak.
So UCP1 independent thermogenesis leaves creatine-cycling or more likely calcium-cycling generated thermogenesis. That's moving toward 'malignant hyperthermia' phenotype, which is interesting because exercise can trigger malignant hyperthermia in susceptible individuals, and a mild MH might resemble something similar seen in ME/CFS exercise studies.

I've got a boat load of papers to read since then to learn about this, so it's too early for me to make any helpful contribution but I just wanted to say there might be something worth exploring here - some sort of infection-induced calcium cycling malignancy in susceptible individuals could be implicated in this mitochondrial impairment.

Thanks for posting that paper!
 

Pyrrhus

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How is it possible to have more H+ without more ROS? Or maybe is the experiment in Fisher et al's paper not "fine" enough?

It sounds like your question is "Is it possible that Fisher et al failed to detect an increase in Reactive Oxygen Species (ROS), a finding which might help explain the H+ leak?"

First, note that the author's 2021 follow-up paper noted:
Missailidis et al 2021 said:
The activity of the major cellular energy stress sensor, AMPK, was elevated but the increase did not reach statistical significance.

So there may be some minor oxidative stress from ROS. But there may also be another type of cellular stress that leads to a leaky mitochondrial membrane...