Myoblasts ERK PEMF

The goal of this post was to come to some sort of understanding as to who, why, and if we even care if pulsed electromagnetic frequency (PEMF) therapy increases phosphorylation (activation of extracellular signal regulated kinases (ERK). Kinases are enzymes that attach phosphate groups to the -OH groups of serine, threonine, and tyrosine amino acid side chains. This sequential tagging is called signal transduction. This matter is being investigated because if a dog is being treated with PEMF to improve bone and muscle health of an injured limb, what happens if the dog has cardiac hypertrophy? Sure, preventing muscle atrophy would be good, but we don’t want the dog’s heart increasing in mass more. In this investigation, it was included that ERK phosphorylation is increased in PEMF of cultured myoblasts [1] and electrical muscle stimulation in a rat hindlimb unloaded model. [2] On the other hand, PEMF fires through PI3 kinase in serum deprived osteoblasts.

PEMF and cultured myoblasts [1]

For PEMF exposure, C2C12 cells were exposed to a sinusoidal 100 Hz PEMF with the density of 1 mT in a cell incubator. The magnetic field was generated by a pair of 60-turn Helmholtz coils. The coils were 300 mm in diameter, 122.5 mm apart. Culture dishes or plates were placed in the center of the coils. Cells of control groups were cultured in another incubator under the same conditions, without PEMF exposure.

1. Sight improvements in cell viability

The authors used antibodies against cell surface proteins indicating viability and programmed cell death to assess the health of the cultured cells.

2. Kinase pathways involved

The kinase pathways added to Xu Figure 2 came from this immunology review. All of the Mitogen Activated Protein Kinase (MAPK) family members are activated by phosphorylation on tyrosine residues (Y) by other upstream kinases. Kinases attach phosphates (green blobs). The cells were cultured with fetal bovine serum that should contain growth factors.

Summary of figures not shown

Figure 3 of the Xu publication added a MEK inhibitor and demonstrated a lack of phosphorylation response to 100 Hz PEMF in the myoblasts. Figure 4 demonstrated that ERK phosphorylation is required for PEMF induced myoblast proliferation. [1]

One of the frustrating aspects of PEMF research is lack of mechanistic definition. Xu and coauthors cited a publication by Li and others on primary cultured rat dorsal root ganglion cells. Here PEMF exposure (50 Hz, 1 mT) was shown to activate Ca²⁺ influx through L type voltage gated calcium channels in such a way as to activate the phosphorylation of ERK and increased transcription of brain derived neurotropic factor (BDNF). Is there any evidence that ERK is activated by any sort of muscle stimulation? This leads us to a 2011 publication examining the signaling pathways in response to electrical muscle stimulation used to prevent muscle atrophy in humans.

ERK, p38, JNK, hind limb unloading, electrostimulation [2]

Then, the overall aim of Dupont and coworkers was to define activation patterns of signaling targets of both PI3K-AKT and MAPK pathways during disuse muscle plasticity. Th is study was divided into two phase:

1. They analyzed in slow soleus and fast EDL muscles the time course of muscle atrophy (muscle mass), contractile phenotype as measured by myosin heavy chain type, and metabolic profile over the course of four weeks. Signal transduction kinases were AKT, GSK-3β, mTOR, p70S6K, and MAPK members p38, JNK, and ERK. Transcription factors included 4E-BP1,FOXO1 and MuRF1 .
2. The second objective was to counteract one of these modifications during hind limb unloading (HU) through chronic low frequency electostimulation. ,

Changes is muscle proteins

This post is going to summarize the Western blot analyses of phosphorylation (activation) status of protein kinases. Hindlimb unloading increased the myosin heavy chain IIb and IIc at 7, 14, and 28 weeks. [2] ES plus HU at 14 weeks restored the relative proportions of IIa and I back to levels of the control at 14 weeks.

The authors separated proteins by size via what are called SDS PAGE gels. Protein bands that were increased were excised and identified via a technique called mass spectrometry. A few protein band stood out as increasing due to HU:

These results were confirmed with protein specific antibodies using a technique called Western blotting. ES prevented these changes at 14 weeks. Cytosolic malate is sort of the odd one out because malate dehydrogenase is traditionally inside the mitochondria and part of the Krebs/ TCA cycle that generates NADH that feeds into the electron transport chain. The enzyme to watch is adenylate kinase that takes two ADP to make an AMP and ATP. ATP is the “energy currency” of the cell. Cells “spend” ATP to perform energy requiring biological functions.

Signal transduction protein kinases

An image of nutrient sensing was from Cell Metabolism. AMPK is activated when the ratio of AMP to ATP increases as may occur during exercise or decreased ATP production. Somewhere, entwined in this mystery, is how a kinase like ERK that is usually activated by hormonal growth factors may be activated by PEMF and electrostimulation.

Bypassing receptor tyrosine kinases, serum free [3]

One of the question marks in both these studies, cell culture, and in vivo, is the role of growth factors in ERK signaling. several years prior Patterson and coauthors had serum starved pre-osteoblasts to separate the influence of PEMF from growth factor signaling. [3]

Note the use of 14.9 Hz and 3,850 Hz, on either side of the 100 Hz in the cultured myoblast study. [1]

• MC3T3-E1 oseoblasts were serum-starved for 16 h prior to one hour PEMF exposure. for1h.The amount of p-p70 S6 kinase in 25 mg of cell culture pellet was significantly greater than non exposed cells (p=0.014).
• PEMF exposure for 10,30, and 60 min resulted in significantly increased
  mTOR phosphorylation compared to control samples (P=0.043, 0.013, and 0.043, respectively). By 60 min the p-mTOR was about 2.5x the control. Each exposure consisted of three replicates.
• PEMF exposure increased S6 phosphorylation by about 32x in the 30 to 140 minute time window with levels decreaseing by 180 min.
• Pretreatment of MC3T3-E1 cells for 30 min with 20 mM LY294002, a
  selective PI-3 kinase inhibitor, reduced the basal and PEMF-dependent phosphorylation of ribosomal protein S6 by 85%
• Similar phenomenon concerning the link beween PEMF and mTOR and S6 phosphorylation were observed in fibroblasts and a more differentiated osteoblast line.
• ERK phosphorylation was not found to be responsive to PEMF in by these authors.
  
  

Does PEMF and/or electrical stimulation of rat hind limb also increase the flux through the RAF/MAPK/ERK arm of the receptor tyrosine kinase growth factor signaling? We just do not know at this point. This post still does not answer the original question of whether PEMF might be safe for treating broken bones or injured muscle of dogs with cardiac hypertrophy. We are not going to deprive our dogs’ serum of growth factors. We can hypothesize that PEMF makes growth factor recptors more available for both branches of the pathway.

References

1. Xu H, Zhang J, Lei Y, Han Z, Rong D, Yu Q, Zhao M, Tian J. Low frequency pulsed electromagnetic field promotes C2C12 myoblasts proliferation via activation of MAPK/ERK pathway. Biochem Biophys Res Commun. 2016 Oct 7;479(1):97-102.  Sci-HUb free paper
2. Dupont E, Cieniewski-Bernard C, Bastide B, Stevens L. Electrostimulation during hindlimb unloading modulates PI3K-AKT downstream targets without preventing soleus atrophy and restores slow phenotype through ERK. Am J Physiol Regul Integr Comp Physiol. 2011 Feb;300(2):R408-17. free paper
3. Patterson TE, Sakai Y, Grabiner MD, Ibiwoye M, Midura RJ, Zborowski M, Wolfman A. Exposure of murine cells to pulsed electromagnetic fields rapidly activates the mTOR signaling pathway. Bioelectromagnetics. 2006 Oct;27(7):535-44. Sci-Hub free paper