PEMF and complex II

Toda and coworkers 2022 investigated the effect of optimized extremely low frequency weak magnetic fields (Opti-ELF-WMF) on the mitochondria of mouse livers and in cultured hepatocytes. Their optimization protocol, aimed at reducing the mitochondrial mass, is the featured image at the top of the screen. They looked at protein and mRNA levels, enzyme activity, and mitophagy, the removal of defective mitochondria. Complex II seemed to contain the Opti-ELF-WMF antenna , perhaps in terms of Fe-S clusters in the SDHB subunit. The final panel looked at rotenone that acts on an Fe-S cluster of complex I. Exposed cells were more affected far more so than a complex II inhibitor that acts on the substrate binding site. Toda and coauthors proposed that ion cyclotron resonance and/or the radical pair mechanism.

Toda T, Ito M, Takeda JI, Masuda A, Mino H, Hattori N, Mohri K, Ohno K. Extremely low-frequency pulses of faint magnetic field induce mitophagy to rejuvenate mitochondria. Commun Biol. 2022 May 12;5(1):453. PMC free paper

Mitophagy, clearance of dying mitochondria

Mitochondria are usually “eaten” as soon as they stop working, most notably by not being able to maintain an outside positive membrane potential. Mitotracker Green is flow cytometry. Loss of mitochondrial mass in choosing the Opti-ELF-WMF conditions never meant a loss in average mitochondrial mass but the pulk total of all of them in each cell as they flowed by the detector.

This image is a hodge-podge of a variety of “Commons” images found on the Internet first appearing in another post about presenilin. This narrative starts in the upper left hand corner.

1. Loss of membrane is a sign mitochondria death. In comes Pink1 is a mitochondrial protein kinase that attaches phosphate groups to serine and threonines of PARK2 and ubiquitin.  PINK1 localize to the outer membrane of the mitochondria where it senses depolarization, i.e. a decrease in membrane potential.
2. PARK2 codes for Parkin, a ubiquitin ligase It is activated by Pink1 phosphorylation.
3. Ubiquitin is a protein of which polymers may be attached to larger proteins by ubiquitin ligases, these are essentially “eat me” tags.
4. Kazlauskaite et al. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem J. 2014 May 15;460(1):127-39. PMC free paper
5. The three sided box is the start of an autophagosome initiated by P62/ SQSTM1 is generally thought of as an adaptor protein, in the mitophagy process, that binds poly ubiquitination and light chain 3.
6. LC3, microtubule associated protein light chain 3 is associated with the fusion and expansion of the autophagosome around the dead mitochondria, upper right hand corner
7. LAMP2, lysosome associated membrane glycoprotein 2 is a protein on lysosome organelle membranes that mediates fusion with the autophagosome.
   1. Bottom panel. The dead mitochondria are digested. .

The abstract, Toda 2022

Now that we’ve covered mitophagy, this is a summary of what Toda 2022 did:

“Humans are frequently exposed to time-varying and static weak magnetic fields (WMF). However, the effects of faint magnetic fields, weaker than the geomagnetic field, have been scarcely reported. Here we show that extremely low-frequency (ELF)-WMF, comprised of serial pulses of 10 µT intensity at 1–8 Hz, which is three or more times weaker than the geomagnetic field, reduces mitochondrial mass to 70% and the mitochondrial electron transport chain (ETC) complex II activity to 88%. Chemical inhibition of electron flux through the mitochondrial ETC complex II nullifies the effect of ELF-WMF. Suppression of ETC complex II subsequently induces mitophagy by translocating parkin and PINK1 to the mitochondria and by recruiting LC3-II. Thereafter, mitophagy induces PGC-1α-mediated mitochondrial biogenesis to rejuvenate mitochondria. The lack of PINK1 negates the effect of ELF-WMF. Thus, ELF-WMF may be applicable for the treatment of human diseases that exhibit compromised mitochondrial homeostasis, such as Parkinson’s disease.“

Three or more times weaker than the geomagnetic field, eh? They used copper plates to shield the mice and cultured cells from ambient magnetic field sources. Most of their protocols seemed to be pretty standard and not worthy of comment in this post.

summary of figures

Fig. 1. Opti-ELF-WMF ⤊ liver electron transport chain (ETC) activities.

The time period was four weeks, the mitochondria were from mouse liver. 40% increases were seen in O₂ consumption and the mitochondrial membrane potential. Only complex IV activity increased significantly. Representative protein levels of complex I-V did not change. Long term, in whole mice, this Opti-ELF-WMF seemed to be pretty healthy. We are about to be told the rest of the story.

Fig. 2. Opti-ELF-WMF temporarily makes mitochondria sick

In cultured mouse AML12 hepatocytes that is! This was a 1-24 hour time course. Superoxide decreased a little in the 0-1 hr period. Mitochondrial mass, as measured by a fluorescence assay, decreased by about 40% in the 0-3 h period and slowly recovered. Mitochondrial membrane potential decreased by about 20% in the 0-6 h window and slowly recovered. Are these mitochondria even sick? Superoxide is a bad thing right? Less should be more in panel A yet cumulative mass and membrane potential are decreasing.

Fig 3. OptiELF-WMF 3 hr, mitochondria proteins take a hit, recover 12 hr

Mitochondria complexes may contain dozens of individual protein subunits that were resolved by gels and quantified by a technique called Western blotting. After 3hr representative proteins from each subunit had decreased by about 20-50%. By 12 hours protein levels were back to normal.

Fig 4 Opti-ELF-WMF induces mitophagy

We’re still looking at cultured cells and shorter time periods within the 3 h window. More Western blots quantify the relative protein amounts of Pink (the kinase), Parkin (the ubiquitin attachment), and LC3 (phagosome wrapper). Pink levels are the fist to climb in the first 90 minutes. LC3-II takes about 120 min to peak. Pink protein levels double while total Parkin levels stay about the same. The big change is most of the Parkin moves from the cytosol onto the mitochondria.

Fig. 5. Opti-ELF-WMF induced mitochondrial biogenesis.

Fig 3 data tell us that mitochondria proteins decrease and then come back. Fig 4 data suggest that the mitochondria proteins are disappearing in the mitophagy garbage disposal. Fig 5 looks at levels of transcription factors that are responsible for transcribing genes for new mitochondria proteins, hence the recovery in Fig 3. The most notable change was a more than doubling of protein levels of PPARα and a smaller change in PGC-1α. While reduced degradation is one explanation, the mostly likely explanation is that some unknown factor is binding to the PGC-1α promoter and increasing the transcription of the gene. Those mRNA get translated into protein. The big mystery is the unknown transcription factor for PPARα and PGC-1α. Yes, transcription factor gene transcription requires transcription factors!

The more important is their target genes revealed in Table 1. Gene Ontologies (GOs) suppressed by Opti-ELF-WMF in mouse hepatocyte-derived AML12 cells at 1 h by Gene Set Enrichment Analysis (GSEA). Wikipedia has a page on p-values vs q-values in analysis of gene expression. In general, the lower the number the higher the chance that the difference is real.

Table 1. Gene Ontologies (GOs) suppressed by Opti-ELF-WMF in mouse hepatocyte-derived AML12 cells at 1 h by Gene Set Enrichment Analysis (GSEA). I keep going back. Yes, I’m reading correctly. We see an in crease in protein amounts of transcription factors PPARα and PGC-1α at 12 h. A GO table at 12 hr would have been appropriate. The authors are attributing reduced protein on the reduced gene transcription and reduced protein level at 3 h, but not at 12 hr, in Fig3. Let us not forget the pretty much the same protein levels in Fig1d in whole mice after 4wk.

GO name	Size	p-value	q-value
GO_NADH_dehydrogenase activity	43	<1 × 10−6	0.0134
GO_respiratory chain activity	73	<1 × 10−6	0.0265
GO_respirasome	86	<1 × 10−6	0.0317
GO_ETC NADH to ubiquinone	52	<1 × 10−6	0.0387
GO_oxidoreducatase activity acting on quinone or similar compound as electron acceptor	50	<1 × 10−6	0.0465
GO_ATP synthesis coupled electron transport, aka Complex V, ATP synthase	90	<1 × 10−6	0.0492
GO_NADH dehydrogenase complex, aka complex I	47	<1 × 10−6	0.0713
GO_ETC, electron transport chain	106	<1 × 10−6	0.1468
GO_oxidative phosphorylation	132	<1 × 10−6	0.1661

It would be good to see these data at later time points. We don’t know if the initial protein losses are due to decreased transcription, mitophagy, or a combination of both.

Fig. 6. Opti-ELF-WMF interogating complexes I-IV

Mitochondrial electron transport chain complexes that is…

• Panel A tells us that only complex II activity was decreased, and just by about 5-10%.
• Panel B examined the four main components of complex II. Succinate:quinone reductase (SQR) was the only complex II enzyme activity to be significantly reduced in mouse liver homogenates exposed to Opti-ELF-WMF for 8 min. SQR is also known as SDHB, the Fe-S cluster center subunit of complex II.
• Panel C tells us that 3-nitropropionic acid, decreases cumulative mitochondria mass more in the treated mitochondria than in the controls. 3NP forms a covalent bond with the tricarboxylate binding site of SDHA preventing the conversion of succinate to fumarate. Huang 2006.
• Panel D Rotenone is a complex I inhibitor that inhibits the transfer of one of the Fe-S clusters to ubiquinone. The interesting thing is that the inhibition of complex II in panel A was significant but not that much in terms of absolute magnitude. At the higher doses of 3NP there was no difference between treated and control. However, at all doses of rotenone tested, there was a very large difference between treated and control. No inhibition of Complex I was seen.

Toda and coauthors speculated that the mechanism could be “radical pair” or ion cyclotron resonance. The former seems most attractive because the authors did not look at Ca²⁺ and the usual PEMF responsive ions that the right combination of frequency and field strength set resonating.

Whether FAD, Fe-S clusters, or something totally different, are the antenna of r Opti-ELF-WMF PEMF, this study suggests that the signal can be optimized for an antenna.