PEMF eNOS PI3K

This post looks at Li 2015, a delightfully simple publication that explores the use of a very specific type of PEMF to counter the tissue damage that proceeds femoral artery excision in a mouse model of peripheral artery disease. While this is not the exact same as a branch of the femoral artery being occluded by atherosclerotic plaques in humans, it is a very nice study for a short post to focus on some key enzymes that could be linked to an elusive “PEMF antenna.”

Li RL, Huang JJ, Shi YQ, Hu A, Lu ZY, Weng L, Wang SQ, Han YP, Zhang L, Hao CN, Duan JL. Pulsed electromagnetic field improves postnatal neovascularization in response to hindlimb ischemia. Am J Transl Res. 2015 Mar 15;7(3):430-44. PMC free paper

The featured image is meant to allow the reader to follow along the summary of the figures in this publication.

Basic protocol

• Mice were anesthetized before a longitudinal incision was cut in the left hindlimb. The femoral artery was completely excised from its proximal origin to the point where it bifurcated into the popliteal and saphenous arteries including all branches of the femoral artery.
• PEMF was generated by a Chinese device: asymmetric fields consisted of 4.5 ms pulses at 30 ± 3 Hz, with a magnetic flux density increasing from 0 to 5 mT in 400 μs.
• Human umbilical vein endothelial cells (HUVEC cells were used for cell migration, wound healing, apoptosis, and tube forming assays.

Summary of figures

Figure 1 These mice have hind limb skin lesions just like human patients. PEMF greatly decreased ischemia in both controls and the ischemia treatment.

Figure 2 showed some intra red temperature analysis that suggested that the affected areas might be colder than normal tissue. The authors did not give any equations on how they derived their ratio that ranged from 0.25 to 1.0 on the day of the surgery. PEMF had little influence at this time but significantly improved the temperature by day 14.

Figure 3 presented data from tissue sections of muscle stained with a marker for capillaries. PEMF did nothing to increase vascularization of sham operated muscle. Ischemia increased the capillary density with or without PEMF. PEMF in the ischemic model increased capillary density almost twice that of ischemia alone.

Figure 4 presented the expression of vascular endothelial growth factor (VEGF) PEMF had no influence on protein levels of this factor in sham operated animals but greatly enhanced the VEGF in the ischemic model. The authors also examined the phosphorylation activation of Akt1 and endothelial nitric oxide synthase (eNOS) PEMF greatly increased the phosphorylation of these enzymes only in the ischemic rats.

figure	sham	sham + PEMF	ischemia	ischemia + PEMF	comment
Fig 1 Ishemia score	1.1	0.2	2.8	0.6	isch PEMF same as sham PEMF
Fig 2 Isch/norm temp ratio	0.9	0.95	0.5	0.75	14 days post surgery, no diff 6 d
Fig 3 capillary density CD31+	20%	20%	30%	50%	hypoxia + PEMF angiogenesis?
Fig 4 VEGF, p-Akt and p-eNOS	1	1	3	6	looked at protein compare control

The first four figures suggested that PEMF was augmenting some sort of neovascularization. The authors went to a cell culture system and determined if one or more 8 min cycles of 30 ± 3 Hz, 5 mT performed best. 

Figure 5 examined the affect of the number of PEMF cycles on HUVEC toxicity and proliferation. No evidence of toxicity was observed however there seemed to be an increase in proliferation with increased PEMF cycles.

Figure 6 In going from 1-4 cycyles there was a progressive decrease in the number of cells under going apoptosis (programmed cell death). Cyle 4 improvements were reversed by eNOS inhibitor L-NAME and PI3-K inhibitor LY294002.

Figure 7 documented data from the cell culture model of wound healing. The healing increased as a function of the number of PEMF cycles but was inhibited with L-NAME and LY294002. Four cycles was better than 1-3 cycles and no treatment.

Figure 8 looked at the length of blood vessel like tubes that these HUVEC cells form. The tube length was increased as a function of the number of PEMF cycles. eNOS and PI3-K inhibitors prevented the PEMF effect.

Figure 9 Increasing cycles of PEMF promoted VEGF released from HUVECs. At the same time, the same set of experiments, the phosphorylation of Akt and eNOS also significantly increased.

Figure 10, The four cycles of PEMF effects on VEGF, p-Akt, and p-eNOS were neutralized by eNOS and PI3 kinase inhibitors L-NAME and LY294002 .

Ca²⁺ calmodulin: eNOS, PI3K, and Akt, a PEMF antenna?

Even though Li 2015 did not look at Ca²⁺, it is still a very nice paper. Binding to Ca²⁺-calmodulin is one thing that these three enzymes have in common. For some users of PEMF, voltage gated Ca²⁺ channels are the antenna of PEMF. This site has favored reactive oxygen species from the FAD-cryptochrome radical pair mechanism that results in the gating of TRPC1 Ca²⁺ channels. I found an X-ray crystal structure of an eNOS peptide bound to Ca²⁺-calmodulin.

eNOS

A quote associated with the crystal structure 1NIW

“The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible.”

PI3Kα

Zhang M, Li Z, Wang G, Jang H, Sacks DB, Zhang J, Gaponenko V, Nussinov R. Calmodulin (CaM) Activates PI3Kα by Targeting the “Soft” CaM-Binding Motifs in Both the nSH2 and cSH2 Domains of p85α. J Phys Chem B. 2018 Dec 13;122(49):11137-11146. PMC free paper

“PI3Kα is a key lipid kinase in the PI3K/Akt pathway. Its frequent oncogenic mutations make it a primary drug target. Calmodulin (CaM) activates PI3Kα independently of extracellular signals, indicating a significant role in oncogenic PI3Kα activation. Here, we reveal the atomic-scale structures of CaM in complexes with the nSH2 and cSH2 domains of the regulatory p85α subunit of PI3Kα, and illustrate how CaM activates PI3Kα by targeting the “soft 1–5–10” CaM-binding motifs in both nSH2 and cSH2 domains. Experiment observed CaM binding cSH2 first, followed by nSH2 binding hours later. CaM typically prefers binding helical peptides. Here we observe that, unlike in cSH2, the CaM-binding motif in nSH2 populates a mixed β-sheet/α-helix/random coil structure. The population shift from a β-sheet toward CaM’s favored α-helical conformation explains why the nSH2 domain needs a longer time for CaM binding in the experiments. The “soft” CaM-binding motifs in both nSH2 and cSH2 domains establish strong CaM–PI3Kα interactions, collectively facilitating PI3Kα activation. This work uncovers the structural basis for CaM-driven PI3Kα activation.”

Akt

Weako J, Jang H, Keskin O, Nussinov R, Gursoy A. The structural basis of Akt PH domain interaction with calmodulin. Biophys J. 2021 May 18;120(10):1994-2008. doi: 10.1016/j.bpj.2021.03.018. Epub 2021 Mar 26. PMC free paper.

“Akt plays a key role in the Ras/PI3K/Akt/mTOR signaling pathway. In breast cancer, Akt translocation to the plasma membrane is enabled by the interaction of its pleckstrin homology domain (PHD) with calmodulin (CaM). At the membrane, the conformational change promoted by PIP3 releases CaM and facilitates Thr308 and Ser473 phosphorylation and activation. Here, using modeling and molecular dynamics simulations, we aim to figure out how CaM interacts with Akt’s PHD at the atomic level. Our simulations show that CaM-PHD interaction is thermodynamically stable and involves a β-strand rather than an α-helix, in agreement with NMR data, and that electrostatic and hydrophobic interactions are critical. The PHD interacts with CaM lobes; however, multiple modes are possible. IP4, the polar head of PIP3, weakens the CaM-PHD interaction, implicating the release mechanism at the plasma membrane. Recently, we unraveled the mechanism of PI3Kα activation at the atomistic level and the structural basis for Ras role in the activation. Here, our atomistic structural data clarify the mechanism of how CaM interacts, delivers, and releases Akt—the next node in the Ras/PI3K pathway—at the plasma membrane.”

Li 2015 was a very nice and simple study that made a good case for the use of PEMF to encourage neovascularization when blood flow is cut off to the hind limb of a mouse model. They did not come close to establishing an “antenna” for 4.5 ms pulses at 30 ± 3 Hz, with a magnetic flux density increasing from 0 to 5 mT in 400 μs. Human peripheral artery disease is bit more complicated than just a reduction of blood flow to the affected limb.