TRPC1 and muscle stretch

This post is a follow up to a post to discussing 50 Hz PEMF activating sphingosine kinase 1. (Yang 2019) This post explores two things that PEMF and exercise have in common: the both activate TRPC1 Ca²⁺ channels and increase sphingosine-1-phosphate. This post also explores the interaction between TRPC1 and S1P for muscle health.

A companion YouTube video

Cordeiro 2019S1P review

The primary source of S1P is erythrocytes and platelets. S1P is important for skeletal muscle health.

One thing that needs to be made abundantly clear is that S1p is thought to act via many isoforms of Sphingosine-1-P heterotrimeric G protein coupled receptors. Many different isoforms and signaling pathways branching off

• S1P is involved in the control of Ca²⁺ release and resistance‐to‐fatigue, cell
• survivor, insulin sensitivity, and suppression of apoptosis. functions of S1P, such as the stimuli of growth ,
• suppression of apoptosis
• regulation of motility
• thermotolerance
• rearrangements in cytoskeletal structure
• metabolic functions in skeletal muscle contractile functions
• response to oxidative stress
• role in regulating cytochrome c oxidase activity and mitochondrial respiration.
• response to mitochondrial stress
• mitochondrial unfolded protein response
• S1P and attenuate fatigue development

The above is a collection of terms with references removed. It is highly recommended to follow up these phrases with a PubMed search. The caveat is always that there may be a therapeutic window of S1P.

Frustration: what about lipid allosteric effects?

So red blood cells, platelets, lipoprotein particles are the carriers of S1P? What is the partitioning when a RBC containing S1P floats by a membrane raft or an S1P receptor? Does S1P go to the membrane first? Are there binding sites on other proteins?

Baranowski 2015 exercise increases S1P

This post will not “deep dive” into this study. The S1P results point to an explanation as how reactive oxygen species may influence TRPC1 activity without reacting with any thiol group or such on TRPC1 itself.

• Some in the PEMF community claim PEMF is cellular exercise.
• 13 male athletes performed a 60-min exercise at 65 % of VO₂max
• Muscle biopsies and blood samples
• Exercise until exhaustion increased plasma S1P and sphinganine-1-phosphate (SA1P) concentration in the plasma (p < 0.05),
• Moderate-intensity exercise elevated only SA1P (p < 0.001).

Frustration: what about membrane biophysics?

Alonso and Goñi 2018 state that the trans double bond is needed to stabilized the membrane, as opposed to the cis configuration. Much is in the PubMed database on phytosphingosine.

Benavides Damm 2013 TRPC1 and Microgravity, opposite of stretch

Like secretion of adipokines from adipocytes, growth and differentiation of C2C12 myocytes into myotubules is known to be dependent on Ca²⁺ entry. Benaides Damm 2013 used a microgravity device to test the hypothesis that the stretch signal can come from gravity. This study related to muscle atrophy during the microgravity experience of space flight. TRPC1 conductance was shown to result in more TRPC1 expression in a “feed forward” mechanism. Insulin like growth factor (IGF1 transcripts were also increased. Portions of this figure were adapted from Yap 2019

parturition, lay example	TRPC1 Benavides Damm 2013
baby grows putting pressure on cervix	Mechanical stress opens TRPC1 cation channels
cervix sends message to brain	Ca2+ binds to calmodulin- and calcineurin-dependent phosphorylation events at several key junctures within the G1 and G2 phases
brain signals pituitary gland to release oxytocin into the blood.	myocyte enhancer binding factor 2 (MEF-2) regulated gene transcription.
Oxytocin causes uterus to contract pushing baby towards the cervix	Calcineurin increases regulator of mitochondria genesis PGC1α transcription
Cervix sends message to the brain	increase in transcripts for insulin like growth factor, mechanogrowth factor, and TRPC1 itself
More oxytocin released, baby is eventually pushed out of uterus	IGF-1 enhances transport of TRPC1 to cell membrane. Muscle continues to grow.

Two examples of feed forward mechanisms in biology

Frustration: too many stress responsive elements in biology

The above animation is overly simplistic. In addition to actin being connected to cortactin before it is connected to integrin in the rafts of the membrane rafts, integrin is also connected to the extracellular matrix. Is simply tugging on one of those elements sufficient to open a stress activated channel like TRPC1 or does a connection sometimes get broken entirely and do something different?

Lipid raft involvement, Kim 2010

Sarcolemma membrane rafts are isolated by virtue of their detergent insolubility that is based on the high cholesterol content.

Detergent-resistant lipid rafts were isolated from C2C12 myotubes and analyzed by capillary RPLC/MS/MS. Among the 327 proteins (or
protein groups) identified, 28% were categorized to the plasma membrane or raft proteins, 29% to mitochondria, 20% to microsomal proteins, 10% to other proteins, and 13% to unknown proteins. The localization of oxidative phosphorylation (OXPHOS) complexes in the sarcolemma lipid rafts was further confirmed from C2C12 myotubes by cellular fractionation, surface-biotin labeling, immunofluorescence, and lipid raft fractionation. After adding exogenous cytochrome c, the sarcolemma isolated from myotubes had an ability to consume oxygen in the presence of NADH or succinate. The generation of NADH-dependent extracellular superoxide was increased by inhibiting or downregulating OXPHOS I, III, and IV in myotubes, indicating that OXPHOS proteins are major sources for extracellular ROS in skeletal muscle. With all these data, we can conclude that OXPHOS proteins are associated with the sarcolemma lipid rafts during C2C12 myogenesis to generate extracellular ROS.

• Five days after C2C12 myoblast differentiation into myotubules, some electron transport chain complex proteins had increased 3-5x.
• These fractions could perform biochemistry of the mitochondria such as reduce cytochrome C and produce reactive oxygen species

complex, subunits ID’ed	protein subunit	coverage	sequenced peptides
I, ~30 ID’ed	ADH-ubiquinone oxidoreductase PDSW subunit NDUFB10	49%	8
II, just 1 reported	Succinate dehydrogenase (ubiquinone) flavoprotein subunit, HDHA	16%	4
III, 6 ID’ed	Ubiquinol-cytochrome c reductase complex core protein 2, UQCRC2	51%	26
IV, 9 ID’ed	Cytochrome c oxidase, subunit Va, Cox5a	47%	13
V, 11 ID’ed	ATP synthase b chain, ATP5b	62%	37

Note, the subunits in this table are the top scoring in terms of percent coverage of the entire subunit amino acid sequence. Kim 2010 notes prior detection of each of these electron transport chain subunits in other membrane rafts. The “take home” message is that the PEMF source of reactive oxygen species to gate TRPC1 in response to PEMF can come from a very close location.

Frustration: no other sarcolemma membrane raft proteome

Lee 2016 is a remarkable follow up Kim 2016 demonstrating the same occurrence in gastrocnemii from 8-week-old male mice. Generation of reactive oxygen species was not published in this study. Lee 2017, from the same laboratory, did demonstrate generation of reactive oxygen species by electron transport chain proteins in rafts from HepG2 cells and primary hepatocytes.

TRPC1 and S1P in C2C12 myoblasts

Formigli 2009. TRPC1 is expressed in C2C12 myoblasts and its silencing with siRNA also silences stretch activated Ca²⁺ entry. TRPC1 was also shown to localize to the lipid raft microdomains. Lipid raft domains (LRD) are essentially islands in the plasma membrane that are rich in cholesterol and sphingolipids. Sphingosine-1-phospate modulated TRPC1 activity as did the presence of the actin cytoskeleton. Actin disruption with cytochalasin decreased stretch activated channel activity.

Of note, the upregulation of TRPC1 and its recruitment into membrane microdomains by S1P represent novel mechanisms by which the bioactive lipid exerts its pro-myogenic action on these cells, linking SAC-opening to gene expression.

• Atopic force microscope gadgets were used to stretch the C2C12 myocytes while measuring the current through TRPC1 channels
• TRPC1 conductance normalized to membrane surface area
• Used silencing RNA to remove TRPC1
• Supplemented with 1 μM S-1-P,

treatment	(stretch-original)/original
scrambled RNA, positive control	1.9±0.6x, stretching boosts conductance, p<0.001
siTRPC1	0.1±0.01, P<0.001 compared to SCR control and no stretch. No TRPC1, stretch increases remaining conductance only a bit
scrambled RNA + S1P	2.9±0.5, p<0.01 compared to SCR and no stretch, add S1P and stretching really increases conductance.
siTRPC1+S1P	0.2±0.01, p<0.001 compared to SCR no stretch. Slightly bigger bump in conductance in absence of RPC1 but with S1P

• Cholesterol was depleted with methylcyclodextrin (MβCD, 2 mM), disrupting TRPC1 localization and store activated Ca²⁺ channel activity.
• TRPC1 co-immunoprecipitated with cortactin, an actin-binding protein expressed underneath the plasma membrane
• TRPC1 copurified with caveolin.
• Silencing TRPC1 decreased the sarcomeric actin isoform expression and S1P increased its expression.
• Over a three day period of myoblast differentiation, TRPC1 peaked 2.5x at 48 hours and decreased to only 1.5x at 72 hours.
• Differentiation medium with or without transforming growth factor TGFβ and S1P enhanced actin filaments and TRPC1 expression
• C2C12 cells were incubated 30 minutes with the intracellular Ca²⁺ stores inositol-3-phosphate receptor blocker, 2-aminoethoxydiphenil borate (2-APB). S1P still enhanced activated current and siTRPC1 still silenced it. The weak influence of 2-AP led the authors to include that store activated and stretch activated roles of TRPC1 might be occurring on a temporal basis given the role of S1P in both.

Frustration: direct, indirect, or combo effect of S1P?

This was a truly remarkable study. Was S1P acting as a S1P receptor agonist, a modulator of lipid raft biophysics, or a combination of both? This brings us to the previous report that 50 Hz 0.8mT PEMF activates sphingosine kinase 1.

Summary

From the first observation that 50 Hz, 0.8mT PEMF, additional observations were collected to support the hypothesis that TRPC1 modulation by PEMF is by way of increases i S1P

1. S1P is important in muscle health (Cordeiro 2019)
2. Like PEMF, exercise increases S1P (Baranowski 2015) The possibility is open that S1P may have biophysical effects (Alonso and Goñi 2018)
3. Microgravity suppresses stretch activated channel TRPC1 (Benavides Damm2013)
4. Mitochondrial electron transport chain proteins are found in C2C12 myoblasts that also contain sphingolipids. These rafts can be a source of reactive oxygen species. (Kim 2010, Lee 2016,2017)
5. TRPC1 is found in lipid rafts, is connected with actin, and is modulated by S1P

This PEMF / exercise skeletal muscle story would be so much nicer if we had a complete proteome of the sarcolemma membrane rafts demonstrating TRPC1 and other proteins that can generate reactive oxygen species in response to PEMF and stretch. The ROS need not modify TRPC1 directly but could also modify the lipids that gate it.

References

• Alonso A, Goñi FM. (2018)The Physical Properties of Ceramides in Membranes. Annu Rev Biophys. 2018 May 20;47:633-654. free paper
• Baranowski M, Błachnio-Zabielska AU, Charmas M, Helge JW, Dela F, Książek M, Długołęcka B, Klusiewicz A, Chabowski A, Górski J. (2015) Exercise increases sphingoid base-1-phosphate levels in human blood and skeletal muscle in a time- and intensity-dependent manner. Eur J Appl Physiol. 2015 May;115(5):993-1003 PMC free paper
• Benavides Damm T, Richard S, Tanner S, Wyss F, Egli M, Franco-Obregón A. (2013) Calcium-dependent deceleration of the cell cycle in muscle cells by simulated microgravity. FASEB J. 2013 May;27(5):2045-54… Sci-Hub free paper
• Cordeiro AV, Silva VRR, Pauli JR, da Silva ASR, Cintra DE, Moura LP, Ropelle ER. (2019) The role of sphingosine-1-phosphate in skeletal muscle: Physiology, mechanisms, and clinical perspectives. J Cell Physiol. 2019 Jul;234(7):10047-10059. Sci-Hub free paper
• Formigli L, Sassoli C, Squecco R, Bini F, Martinesi M, Chellini F, Luciani G, Sbrana F, Zecchi-Orlandini S, Francini F, Meacci E. (2009) Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation. J Cell Sci. 2009 May 1;122(Pt 9):1322-33. Sci-Hub free paper
• Kim BW, Lee JW, Choo HJ, Lee CS, Jung SY, Yi JS, Ham YM, Lee JH, Hong J, Kang MJ, Chi SG, Hyung SW, Lee SW, Kim HM, Cho BR, Min DS, Yoon G, Ko YG. (2010) Mitochondrial oxidative phosphorylation system is recruited to detergent-resistant lipid rafts during myogenesis. Proteomics. 2010 Jul;10(13):2498-515. doi: 10.1002/pmic.200900826. PMID: 20422640. Sci-Hub free paper
• Lee H, Kim SH, Lee JS, Yang YH, Nam JM, Kim BW, Ko YG. (2016) Mitochondrial oxidative phosphorylation complexes exist in the sarcolemma of skeletal muscle. BMB Rep. 2016 Feb;49(2):116-21. PMC free paper
• Lee H, Kim BW, Lee JW, Hong J, Lee JW, Kim HL, Lee JS, Ko YG. (2017) Extracellular reactive oxygen species are generated by a plasma membrane oxidative phosphorylation system. Free Radic Biol Med. 2017 Nov;112:504-514. PubMed
• Yang X, Ye A, Chen L, Xia Y, Jiang W, Sun W. (2019) Involvement of calcium in 50-Hz magnetic field-induced activation of sphingosine kinase 1 signaling pathway. Bioelectromagnetics. 2019 Apr;40(3):180-187. Sci-hub free paper