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

[Inara]

Hey, thanks @WantedAlive. I don't know anything about UCP1, so I should take a look at it. Like in your case, there are several papers waiting...

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?"

Well, actually no, I didn't mean that. After reading your post (again very helpful!) I realized I made a mistake: I didn't pay enough attention to the fact that I am mostly reading biochemistry papers - whereas in medical papers you look at differences between a patient and normal cohort, so you need to apply statistics. In biochemistry papers (please note I'm not a biochemist, so this may be trivial), my impression is it plays a central role which agents are used; and, obviously, this improved over the years so that now they can see and measure things which they couldn't before. (Also, I made the experience that knowledge about what these agents exactly do is fundamental. I met med people doing biochemistry stuff, and when they encountered something different to what they already knew they didn't realize it was a different situation, applied their method and made their - false - conclusion. But afterwards they realized their error and corrected their statement. Uhm...not saying this is what Fisher et al did!) So it was rather a question about: Could they use other agents and measurement methods to refine their findings? Or is what they used already "state of the art"? And would it be beneficial, now that they know there are differences between normal and patient, to research patient cells in more detail, without statistics? Or is that stupid?

So you're absolutely right:

The activity of the major cellular energy stress sensor, AMPK, was elevated but the increase did not reach statistical significance.

which might mean

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...

 

[sometexan84]

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.

and @Pyrrhus

Here is some info on how persistent Enterovirus potentially causes disruption of calcium signaling and regulation...

• 2B protein from Coxsackie disrupts calcium homeostasis. It modulates the calcium regulation in infected cells. This allows for control of apoptosis and virus release (viral replication), as well as the downregulation of host antiviral immune response.
  • Enterovirus controls apoptosis through the Ca2+ regulation, where low Ca2+ levels provide conditions for viral replication, and high Ca2+ levels lead to viral release.
  • Intracellular calcium participates in cell signaling, mitochondrial function, and cell death
  • Modulation of RYR1 (ryanodine receptor) via 2B protein is hypothesized.

 

[Pyrrhus]

and @Pyrrhus

Here is some info on how persistent Enterovirus potentially causes disruption of calcium signaling and regulation...

• 2B protein from Coxsackie disrupts calcium homeostasis. It modulates the calcium regulation in infected cells. This allows for control of apoptosis and virus release (viral replication), as well as the downregulation of host antiviral immune response.
  • Enterovirus controls apoptosis through the Ca2+ regulation, where low Ca2+ levels provide conditions for viral replication, and high Ca2+ levels lead to viral release.
  • Intracellular calcium participates in cell signaling, mitochondrial function, and cell death
  • Modulation of RYR1 (ryanodine receptor) via 2B protein is hypothesized.

Thanks @sometexan84 for bringing up the extremely important enteroviral viroporin 2B and its multiple effects on the cell via intracellular calcium! Good stuff!

Here's some more information on the enteroviral 2B protein, along with a great diagram showing how the 2B viroporin raises the intracellular Ca2+ level:
https://forums.phoenixrising.me/thr...n-shut-off-by-enterovirus.81997/#post-2307893

(You'll note in the diagram that the enteroviral 2B protein directly pokes holes in the mitochondria!)

 

[WantedAlive]

This Complex V inefficiency was also observed in New Zealander Warren Tate's research group: [A.M. Helliwell et al.] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641803/

In this study I observe they indicate upregulated UCP2 (EDIT: incorrect see below suggests down-regulated), an uncoupling protein which I referred to earlier in this thread being a possibility. I'm excited to see this confirmed. Fisher had previously ruled out UCP1 as the cause of complex V impairment but UCP2's involvement was unknown until this study. UCP2 is widely expressed in adipose tissue, muscle, heart, lung, spleen, thymus, immune cells, and vascular cells. Upregulated UCP2 could very likely be the cause of the mitochondrial proton leak causing Complex V inefficiency, if it is the question is why? One possibility discussed earlier in this thread involved phagocytosis supporting the high cell turnover findings in ME/CFS...

Interestingly, the phagocyte engulfment capacity is enhanced by lower mitochondrial membrane potential, and mitochondrial uncoupling protein UCP2 is upregulated to achieve this [1].

This study describes UCP2, it appears to have protective mechanisms:
[Giorgia Pierelli et al.] Uncoupling Protein 2: A Key Player and a Potential Therapeutic Target in Vascular Diseases: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661070/

UCP2, unlike UCP1, appears to contribute mainly to the regulation of ATP [16], mitochondrial membrane potential (Δψ) [17], cellular calcium homeostasis [17], cell survival [18], lipid metabolism [19], and the generation of ROS [19, 20].

Overview of UCP2 localization and function. UCP2 is located within the inner mitochondrial membrane where it uncouples proton flux with the consequent regulation of ATP synthesis. ROS activates UCP2. The latter, through a negative feedback mechanism, decreases ROS and protects vascular cells from the oxidative stress-dependent damage. See text for details. OMM: outer mitochondrial membrane; IMS: intermembrane space; IMM: inner mitochondrial membrane.

 

[Pyrrhus]

In this study I observe they indicate upregulated UCP2, an uncoupling protein which I referred to earlier in this thread being a possibility. I'm excited to see this confirmed. Fisher had previously ruled out UCP1 as the cause of complex V impairment but UCP2's involvement was unknown until this study. UCP2 is widely expressed in adipose tissue, muscle, heart, lung, spleen, thymus, immune cells, and vascular cells. Upregulated UCP2 could very likely be the cause of the mitochondrial proton leak causing Complex V inefficiency, if it is the question is why? One possibility discussed earlier in this thread involved phagocytosis supporting the high cell turnover findings in ME/CFS...

Thanks for bringing up the uncoupling proteins UCP1 and UCP2!
Investigating their function would certainly be relevant to the mitochondrial membrane leak found in this study.

You reference this study by Warren Tate's group:
https://forums.phoenixrising.me/thr...emic-dysfunctions-helliwell-et-al-2020.81953/

This is what they have to say about UCP2:

Clusters of differential methylation in the regulatory regions of genes UCP2, LONP1 and NDUFA11 related to mitochondrial function identified in this study suggest impaired functioning. UCP2 is an uncoupling protein capable of dissipating the proton gradient generated by NADH-powered pumping of protons into the inter-membrane space with associated links to energy metabolism efficiency [32]. It has been positively associated with reactive oxygen species (ROS) and reduces their generation making it important for cellular protection. As the enhancer for this gene was hyper-methylated, it indicates ME/CFS patients may exist in a state of impaired energy metabolism in addition to being more vulnerable to ROS.

(emphasis added)

So, the results from Warren Tate's group suggest that UCP2 may possibly be down-regulated, since its enhancer appeared to be hyper-methylated. This down-regulation might tend to increase the efficiency of ATP Synthase (Complex V), not decrease it.

EDIT: Clarified conclusion

 

[WantedAlive]

So, the results from Warren Tate's group suggest that UCP2 may possibly be down-regulated, since its enhancer appeared to be hyper-methylated. This down-regulation would tend to minimize a mitochondrial membrane leak, not exacerbate it.

Thanks @Pyrrhus I must confess my ignorance on methylation status with expression! I've clearly jumped to conclusions from this comment: "UCP2 is an uncoupling protein capable of dissipating the proton gradient generated by NADH-powered pumping of protons into the inter-membrane space with associated links to energy metabolism efficiency. It has been positively associated with reactive oxygen species (ROS) and reduces their generation making it important for cellular protection. As the enhancer for this gene was hyper-methylated, it indicates ME/CFS patients may exist in a state of impaired energy metabolism in addition to being more vulnerable to ROS."

I can see how this comment might be interpreted two ways with regards to ROS, but the "impaired energy metabolism" seemed to be imply UCP2. But if UCP2 is down regulated, what is affecting Complex V...still a damn mystery? I was hoping for some clarity!!!

 

[WantedAlive]

Do we know...are Angiotensin 2 (AngII) levels high in ME/CFS?
A 2012 study found high levels AngII in 'some' POTS patients, and a 1992 study found 80% of ME/CFS patients had high ACE(2?) levels [Me-Pedia]. We know the RAAS system is broken in ME/CFS with low renin and ALD, all this is well explained in HR's recent blog on it in context with Covid. The blog quotes "The high Ang II levels could be contributing to/causing the increased sympathetic nervous system (fight/flight) activity, narrowed blood vessels, the low blood volume, and inflammation".

However, nowhere official can I find if AngII levels are high in ME/CFS. If it is, could it be linked to this Complex V inefficiency?
Angiotensin 2 reduces Complex V (ATP Synthase)
AngII reduced ATP-synthase (complex V) expression by 68% in this study. Although they say it had no effect on other complexes, looking at the chart AngII appeared to upregulate Complex I and III compared to control. It involves mitochondrial angiotensin receptors.

 

[Consul]

https://www.healthrising.org/blog/2022/03/27/novel-approach-mitochondria-chronic-fatigue-syndrome/