electrical stimulation and hind limb unloading

Many PEMF practitioners use PEMF when an animal, including humans, has an injured limb and loss of use of the associated skeletal muscle. Some may argue that PEMF resembles electrical stimulation via Faraday’s Law of Induction. PEMF seems to act on the ERK signaling cascade in cultured myoblasts. [1] Electrical muscle stimulation in a rat hind limb unloaded model worked via the JNK MAP kinase member rather than ERK and p38. ES also promoted the anabolic mTOR pathway. [2] A PEMF study with pre osteoblast fibroblasts looked at serum free conditions that would activate the ERK branch of the MAP kinase superfamily. A complicated PEMF waveform activated many of the same proteins as electrical stimulation. [3] We cannot say if PEMF is the same as EF or even if two different modalities of PEMF are the same.

100 Hz 1mT 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.

• Growth factor activated ERK, 30 minutes activates 3.5x control, more than 15 and 90-240 min
• Inflammatory cytokine activated p38 is not influenced that much by PEMF albeit small changes statistically significant.
• Stress activated JNK, changes are also small and maybe 10% from the appea4rance of things.

Fig. 2. MAPK/ERK pathway was activated by PEMF. Phosphorylation and total level of ERK, P38 and JNK in C2C12 myoblasts after different time exposure of PEMF. (B, Multiple relationship of activated ERK, P38 and JNK between PEMF groups and control groups. *p < 0.05, **p < 0.01, #p > 0.05 compared with the control group.

Summary of more figures

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]

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.

Hind limb unloading, 20 Hz electrostimulation [2]

DuPont and coworkers used 20 Hz on for 10 s and off for 20 s) was chosen to approximate soleus motor units during normal motor behavior. The stimulus had a burst duration of 0.2 ms and an intensity of 0.2–0.4 mA, producing visible repetitive plantar flexion movements.

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 electrostimulation. ,

Changes is muscle proteins Fig 1

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.

Fig 2

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 hind limb unloading. PEMF returned these glycolytic enzymes to control levels. TCA cycle enzyme malate dehydrogenase was increased by HU but brought down to the control level with ES. PEMF prevented the HU increase of adenylate kinase. :

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 Fig 4

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.

Differential activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway by HU and ES in the rat soleus muscle. A: representative Western blots showing the expression of whole protein level and phosphorylated (P) forms of

• AKT, PEMF didn’t do much to preventing the HU decrease in phosphorylation of this kinase
• glycogen synthase kinase-3β (GSK-3β), mammalian target of rapamycin (mTOR) phosphorylation decreases due to HU were not helped by ES
• 70-kDa ribosomal protein S6 kinase (p70S6K), 7-28 weeks HU reduced phosphorylation of this controlling enzyme by 90%. PEMF on top of 14 weeks HU increased phosphorylation to about 5x the control levels! p70S6K is a kinase downstream from mTOR. It helps regulate translation of messenger RNA into proteins.
• A similar trend, though not as great in magnitude, was seen for phosphorylation of 4E-binding protein 1 (4E-BP1), The absolute amounts of 4EBP1 was also decreased by HU but brought up by HU-14w and PEMF. 4EPB1 mediates messenger RNA translation into protein. It receives input from hormones, growth factors and other stimuli that signal through mitogen activated protein kinase and mTORC1.
• Transcription factor forkhead box O 1 (FOXO1) phosphorylation (activation) was decreased by HU but HU-14 + ES brought up to control levels. FOXO1 is a transcription factor that transcribes genes in response to insulin and oxidative stress. The implication is that something might have happened to the normal growth factor signaling that was compensated by small amounts of PEMF induced oxidative stress.
• The expression of total form of muscle-specific RING finger protein-1 (MuRF1) in soleus increased with the time of HU but brought down (HU 14 weeks) by ES. Under conditions of amino acid starvation Murf1 adds a ubiquitin tag that signals the cell to degrade that protein.

From Wikipedia author(s) on mTOR in muscle

Scroll down to the entry that discuses specifically protein synthesis in skeletal muscle.

“Overview of signaling and muscle proteins synthesis (MPS) responses induced by amino acids (AAs) and different contraction intensities. An increase in intracellular AAs leads to the activation of the mammalian target of rapamycin (mTORC1) and its associated downstream protein substrates: 4E‐binding protein 1 (4E‐BP1) and p70 ribosomal protein S6 kinase 1 (S6K1), promoting assembly of the pre‐initiation complex and mRNA translational efficiency. AA‐induced increases in MPS are transient and return back to baseline despite elevated AAs. Exercise prior to AA availability enhances protein synthetic responses which may persist for >24 h, resulting in greater net protein accretion. Resistance exercise (RE) favours stimulation of myofibrillar (myo) MPS through activation of the mTORC1 pathway, with repeated bouts leading to accumulation of contractile proteins and muscle hypertrophy. Endurance exercise (EE) favors stimulation of mitochondrial (Mito) protein synthesis through activation of 5′ AMP‐activated protein kinase (AMPK) and stimulation of proteins involved in mitochondrial biogenesis. Repeated performance of EE increases muscle mitochondrial content increasing oxidative capacity. Phospholipase D (PLD), phosphatidic acid (PA), adenosine monophosphate (AMP), adenosine triphosphate (ATP), peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α), eukaryotic translation initiation factor 4E (eIF4E), ribosomal protein S6 (RPS6), eukaryotic elongation factor 2 (eEF2). ↑ represents activation, and Τ represents inhibition. [134]”

134. Brook MS, Wilkinson DJ, Phillips BE, et al. (January 2016). “Skeletal muscle homeostasis and plasticity in youth and ageing: impact of nutrition and exercise”. Acta Physiologica. 216 (1): 15–41. PMC 4843955.

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 pulses of 14.9 Hz embedded with microbursts of 3,850 Hz, .

• MC3T3-E1 osteoblasts 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 decreasing 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 between 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 receptors 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