This post is an ad for a Moodle.com free course I’d like to create explaining this PEMF publication. I’ve taken a few online courses and learned so much from the discussion than from the actual course material. This post previews what I know about the methods used to generate the hypothesis that PEMF ameliorates the atherosclerosis process via inhibition of the TRPV4 Ca2+ channel. Is this true? Let’s explore the hypothesis together!
Cheng H, Zhang Q, Zhong W, Li H, Wang L, Wang S, He C, Fu C, Wei Q. Pulsed electromagnetic fields inhibit atherosclerosis by regulating pyroptosis through membrane tension-mediated mechanosensitive channels. Signal Transduct Target Ther. 2025 Nov 28;10(1):388. PMC free paper
This publication has some good points and some problems. My goal is to guide the reader through the paper as they may not be familiar with all of the methodology. The biggest problems is that Cheng 2025 exposed whole mice and isolated cultured endothelial cells to PEMF. We biohackers are not going to perform whole human/companion animal PEMF exposure. We are not isolated cells but collections of interacting different cell types. Perhaps all academic endeavors are a mix of strengths and weaknesses. We acknowledge both and build upon them. Contact me if you’d like to participate on a discussion of the entire publication in a course like format. We will all be students of each other.
Atherosclerosis background
Say Cheng 2025 has some application to commercially available PEMF devices, this is an example of a large vessel that is prone to peripheral artery disease atherosclerosis. Wikipedia’s piece on thefemoral artery:
“The common femoral artery (CFA) is located between the inferior margin of the inguinal ligament, and the branching point of the deep femoral artery. Its first three or four centimetres are enclosed, with the femoral vein, in the femoral sheath.” The CFA basically lives in the upper thigh. The CFA is also prone to calcification that cannot easily be resolved with a stent. An extensive review of the literature was conducted by vascular surgeons from NYU Langone Health Long Island and Stonybrook who concluded that blasting plaques with sound waves, lithotripsy, is a way around stents that cannot be placed in the common femoral artery (Ye 2025). Ye and the other vascular surgeons claim that the gold standard is endarterectomy, cutting the plaque out. Wikipedia authors do a good job of explaining the basics of the surgery and the problems elaborated by Ye 2025. Shockwavemedical.com is one of the few sources of the actual soundwave frequencies I could find. Their waveform is about 14 microseconds in length, or about 71 kHz ultrasound.
Ye IB, Giannopoulos S, Kokkosis AA, Jasinski PT, Labropoulos N. Challenging the No-Stent Zone: Intravascular Lithotripsy for Common Femoral Artery Disease. J Clin Med. 2025 Sep 15;14(18):6492. PMC free paper
Cheng 2025 Materials and Methods, sample “lesson”
Cheng 2025 is about the intersection of inflammasomes (no adenosine), mitochondria (no complex IV), and TRPV4 Ca2+ channels that are distinct from what PEMF hacks think of voltage gated Ca2+channels. I’m taking screen captures of the techniques and give some background of what we are looking at. The strength of the conclusions of a study rest on the reliability of the methods.
Lipid Deposits
Oil Red O simply stains lipids red. H&E staining detects a variety of different cellular structures. Hematoxylin stains calcium deposits red while eosin cytoplasm and the lipid parts of plaques pink. Cheng 2025 is the first PEMF paper to my knowledge demonstrate prevention of lipid build up anywhere. It is noted that they did not look for their oxidized low density lipoprotein binding to their cell culture model.
Membrane tension and the cytoskeleton
Flipper basically detects changes in membrane fluidity. Think fats in the frig versus fats at room temperature. Cheng 2025 did a bang up job characterizing lipids in the atherosclerotic plaques yet did not characterize the cell membrane lipid content that can affect membrane tension/fluidity. This course does mention the effect of PEMF on ceramide from acid sphingomyelinase.
Wang Y, Li X, Sun L, Feng B, Sun W. Acid sphingomyelinase mediates 50-Hz magnetic field-induced EGF receptor clustering on lipid raft. J Recept Signal Transduct Res. 2016 Dec;36(6):593-600. Pub Med Sci-Hub free
Filamentous actin is a protein polymer that looks and acts like cables maintaining the structure of the cell, often called the cytoskeleton. Ca2+regulates many aspects of the cytoskeleton. Cheng 2025 produced some amazing PEMF changes in F-actin in these atherosclerosis models.
Atomic Force Microscopy
The many applications of atomic force microscopy are described in great detail by Wikipedia authors. Nothing in this page suggests that trawling the surface of the cell activates stretch activated ion channels.
Stewart AP, Smith GD, Sandford RN, Edwardson JM. Atomic force microscopy reveals the alternating subunit arrangement of the TRPP2-TRPV4 heterotetramer. Biophys J. 2010 Aug 4;99(3):790-7. PMC free paper
This group was using AFM to map the subunit composition of these heterotetramers. No mention was made if probing these channels resulted in gating.
These things said, what does the surface of the aorta look like around the lipid deposits stained with Oil Red? What happens when a scientist trawls through a fat deposit?
Electrophysiology
The interesting thing is that the authors are measuring all of the currents in these whole cells in physiological inside and outside solutions. Thing get crazy because intracellular Ca2+ can influence the gating of other ion channels including TRP family members via Ca2+-calmodulin. A Ca2+ binding protein. The types of patch clamping link. Whole cell patch clamping is the easiest even if it gets all ion channels in the cell. I’ve never patch clamped any cell with any technique. I’ve been in the room on the outside of a Faraday cage to shield the system from EMFs. Prior exposure of the cells to PEMF would have been nice. Patch clamping during PEMF exposure could probably have been impossible.
These probes that become fluorescent when they bind Ca2+ have been around for a long time. The are made cell membrane permeable by attaching a hydrophobic group connected by an ester bond. Esterases inside the cell remove the group trapping the fluorescent probe inside the sell. This is why Cheng 2025 were washing three times with PBS, to remove extra unincorporated dye.
The Mitochondria
The Seahorse is a “respirometer” that measures oxygen consumption at complex IV where O2 is reduced to H20, and “non-mitochondrial” O2 consumption. Many biohackers are familiar with the “lore” that PEMF boosts the flux of electrons through complex IV and promotes the notion that superoxide generation from active mitochondria is part of normal business and that PEMF might promote ROS generation.
JC-1 is fluorescent compound used to detect losses in the mitochondrial membrane potential that target the mitochondria to be “eaten” by lysosomes. The eating of dysfunctional mitochondria, mitophagy, is considered to be a healthy process as the dysfunctional mitochondria generate reactive oxygen species. Wikipedia’s page on mitophagy is pretty good if you scroll down to the part on Pink1 and Parkin, yes named after Parkinson’s disease. Pink1 accumulates in the mitochondria that have lost their membrane potential, i.e. have become dysfuncional. Parkin comes along and attaches “eat me” ubiquitin tags that target mitocondria to the lysosomes, cellular stomachs. Mitophagy is important in energy demanding tissues like the brain. The Wikipedia have a few things to say about mitophagy and inflammasomes. Mitochondrial DNA can activate inflammasomes.
Inflammasomes and Western Blotting
Wikipedia has a good piece on Western blotting. Basically one homogenizes cells or tissue in a detergent called SDS that you might also be in your shampoo. Proteins in mixtures are separated by size in porous gel slabs by applying a voltage gradient. The SDS gives the proteins a negative, anionic, charge. Anions go to the anode. The smaller the protein, the faster it moves through the gel. The proteins in the gel are transferred to a sicky membrane. Bare spots on the sticky membranes are blocked with dissolved powdered milk. Antibody combinations are used to detect the inflammasome proteins.
Cytokines, components of the inflammasomes, and ion channels were measured based on the binding of antibodies raised in the first animal, like a rabbit, injected with the protein, and then an antibody raised in a second animal, like a goat, against immunoglobins from a rabbit. The anti-rabbit goat antibodies are tagged with a probe for detection. Many times scientists take the easy route and measure the messenger RNA, mRNA, transcripts and assume they get translated into proteins equally. These protein based methods don’t tell us if more protein is the result of more gene transcription and translation, or less degradation. Genomics and proteomics approach in combination are generally required to get the full picture.
Human clinical data
Plasma is blood minus the cells. Cheng 2025 digested the whole proteins with the pancreatic enzyme trypsin. The peptides were separated by a combination of size and charge. The Orbitrap Astral was used to measure the size of the peptides and presumably the size of the fragments when the peptides were collided and allowed to decay into pieces. Proteins can be identified quickly based on the size of the fragments. Databases can be searched to correlate the proteins with a particular function. It almost as if this analysis was part of another study and Cheng 2025 just threw it in to bulk up their study. What really irritates me from a PEMF biohacker perspective is that they could have done the same sort of plasma proteomic analysis to ask if there were any differences in proteins in the ND, HFD, and HFD+PEMF mice. They were exposing the whole mice to PEMF, not just the aorta. Maybe something in the circulation far removed from the aorta was being released by PEMF and then influencing fat deposits in the aorta.
NLRP3 knock down
Short hairpin RNA, shRNA, is a means of silencing target genes, in this case the NLRP3 subunit of inflammasomes. AAV, adeno-associated viruses are used for gene therapy. The mCherry fluorescent probe that goes along with the shRNA and informs the scientists that the transfection was successful. We still have the same problem that streptomycin is a TRP channel inhibitor. I like that knocking down is done in cultured cells. I don’t much like the endothelial cell specific knock down in whole mice.
Experimental cell culture groups
Most of these compounds are agonists or antagonist of one type or another used in experiments. Penicillin-streptomycin is a common antibiotic combination used in cell culture experiments. This is very problematic!
Franco-Obregón A, Tai YK. Are Aminoglycoside Antibiotics TRPing Your Metabolic Switches? Cells. 2024 Jul 29;13(15):1273. PMC free paper
“Critically, aminoglycoside antibiotics (AGAs) have been shown to block the capacity of TRP channels to conduct calcium entry into the cell in response to a wide range of developmental stimuli of a biophysical nature, including mechanical, electromagnetic, thermal, and chemical.” And, as near as we care tell, these endothelial cells were not subjected to any kind of mechanical stress.
Experimental mouse groups
The Cherry-NC is a fluorescent protein marker to tell the group that the transfection was in the targeted cells was successful. This seems to be more precise than the double knockout that can affect multiple organ systems. I’m going to assume that the “no drug” control mice got injected with saline such that the stress of handling could be controlled for. Are Nigericin, HC, HY, and GSK safe and non-toxic at these doses? I don’t honestly know. We have to trust the reviewers to know these things. I had to do a key word search to figure out that all the mice were males. It would be interesting to know the lipid composition of this high fat diet as Cheng 2025 performed “lipidomics on the atherosclerotic plaques of these animals. Are the plaques what was in the diet in the first place?
Here is an image generated from one of my favorite resources: Proteinatlas.org.
The mice used in the Cheng 2025 PEMF study, ApoE-/-, were genetically engineered to not process fats properly. The -/- means that they lack both maternal and paternal copies of the APOE gene that codes for apolipoprotein E, a protein involved in transport of fats and cholesterol in the brain. Many of you are aware of the APOE4 variant being associated with Alzheimer’s Disease in this summary ApoE cartoon from protein Atlas.org. Imperfect or not, this APOE-/- mouseis used a lot in published scientific literature.
Raber J, Akana SF, Bhatnagar S, Dallman MF, Wong D, Mucke L. Hypothalamic-pituitary-adrenal dysfunction in Apoe(-/-) mice: possible role in behavioral and metabolic alterations. J Neurosci. 2000 Mar 1;20(5):2064-71. PMC free paper
This is more a adrenal cortex cortisol type thing.
DoctorLib.org has a good piece the adrenal glands. The frustrating thing is that the adrenal cortex and medulla, producer of adrenaline and noradrenaline, are of different embryonic origin.
Blue ML, Williams DL, Zucker S, Khan SA, Blum CB. Apolipoprotein E synthesis in human kidney, adrenal gland, and liver. Proc Natl Acad Sci U S A. 1983 Jan;80(1):283-7. PMC free paper It looks like APOE is mostly an adrenal cortex thing and maybe not a medulla → adrenaline thing. I don’t know at this point.
The PEMF device
44. Huang, L. et al. Enhanced effect of combining bone marrow mesenchymal stem cells (BMMSCs) and pulsed electromagnetic fields (PEMF) to promote recovery after spinal cord injury in mice. MedComm 3, e160 (2022). PMC free paper 50 Hz, 1 mT, 1 h/day.
45. Peng, L. et al. Pulsed electromagnetic fields increase angiogenesis and improve cardiac function after myocardial ischemia in mice. Circ. J. 84, 186–193 (2020). PMC free paper 30 Hz 3 mT 45 min a day in a pen pigs were allowed to move around in.
46. Ross, C. L., Ang, D. C. & Almeida-Porada, G. Targeting Mesenchymal Stromal Cells/Pericytes (MSCs) With Pulsed Electromagnetic Field (PEMF) Has the Potential to Treat Rheumatoid Arthritis. Front. Immunol. 10, 266 (2019). PMC free paper Many different field and frequency combinations were covered in Table 1 of this review.
- normal chow diet
- high fat diet for 15 weeks
- (PEMF I group) 1.5 Hz/0.5 mT/60 min/3 weeks
- (PEMF II group) 2.15 Hz/1.5 mT/60 min/3 weeks
- (PEMF III group) 3.30 Hz/3.0 mT/60 min/3 weeks
Supplementary Figure 10. PEMFs generator
a. The PEMFs generator for in vivo experiments consists of a power supply, a pulse signal generator, a pulse signal amplifier, and two circular Helmholtz coils. These two circular Helmholtz coils exhibit identical thermal effects, enabling them to generate and provide an electromagnetic field with uniform distribution inside. Mouse cages were placed within the coils, with five mice housed in each cage. The generated hertz (frequency) and gauss (magnetic field intensity) levels were measured around the circumference of the cages.
b. For the PEMFs generator used in in vitro experiments, the Helmholtz coils were installed inside a cell incubator. Thermal effects were excluded by monitoring the incubator temperature. Cell culture dishes or well plates were placed inside the coils, and the generated hertz and gauss levels were assessed around the perimeter of the culture vessels.
Discussion question:
I’m searching for ways to make a reductionist cell culture approach and a whole animal exposure relevant to PEMF biohacker that only exposed an affected part of the whole animal or person. As a biohacker, what are your thoughts? Do you have experience in these techniques and the hardware? What are you thoughts that you’d like to share with fellow learners?
If you’d like to go though Cheng 2025 results as a group of fellow learners, contact me.
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