Ambient and supplemental magnetic fields promote myogenesis via a TRPC1-mitochondrial axis: evidence of a magnetic mitohormetic mechanism

Abstract

We show that both supplemental and ambient magnetic fields modulate myogenesis. A lone 10 min exposure of myoblasts to 1.5 mT amplitude supplemental pulsed magnetic fields (PEMFs) accentuated in vitro myogenesis by stimulating transient receptor potential (TRP)-C1-mediated calcium entry and downstream nuclear factor of activated T cells (NFAT)-transcriptional and P300/CBP-associated factor (PCAF)-epigenetic cascades, whereas depriving myoblasts of ambient magnetic fields slowed myogenesis, reduced TRPC1 expression, and silenced NFAT-transcriptional and PCAF-epigenetic cascades. The expression levels of peroxisome proliferator-activated receptor γ coactivator 1α, the master regulator of mitochondriogenesis, was also enhanced by brief PEMF exposure. Accordingly, mitochondriogenesis and respiratory capacity were both enhanced with PEMF exposure, paralleling TRPC1 expression and pharmacological sensitivity. Clustered regularly interspaced short palindromic repeats-Cas9 knockdown of TRPC1 precluded proliferative and mitochondrial responses to supplemental PEMFs, whereas small interfering RNA gene silencing of TRPM7 did not, coinciding with data that magnetoreception did not coincide with the expression or function of other TRP channels. The aminoglycoside antibiotics antagonized and down-regulated TRPC1 expression and, when applied concomitantly with PEMF exposure, attenuated PEMF-stimulated calcium entry, mitochondrial respiration, proliferation, differentiation, and epigenetic directive in myoblasts, elucidating why the developmental potential of magnetic fields may have previously escaped detection. Mitochondrial-based survival adaptations were also activated upon PEMF stimulation. Magnetism thus deploys an authentic myogenic directive that relies on an interplay between mitochondria and TRPC1 to reach fruition.-Yap, J. L. Y., Tai, Y. K., Fröhlich, J., Fong, C. H. H., Yin, J. N., Foo, Z. L., Ramanan, S., Beyer, C., Toh, S. J., Casarosa, M., Bharathy, N., Kala, M. P., Egli, M., Taneja, R., Lee, C. N., Franco-Obregón, A. Ambient and supplemental magnetic fields promote myogenesis via a TRPC1-mitochondrial axis: evidence of a magnetic mitohormetic mechanism.

Keywords: PGC-1α; calcineurin; mitochondriogenesis; pulsed electromagnetic fields; reactive oxygen species.

Conflict of interest statement

The authors acknowledge Dr. T. Benavides Damm [Swiss Space Center, Swiss Federal Institute of Technology (ETH Zurich)] for assisting with the simulated microgravity experiment shown in Fig. 10D, and Dr. Krzysztof Krawczyk (ETH Zurich) for assistance in conducting the fusion index histogram in Fig. 3H. The authors also acknowledge Zac Goh (iHealthtech, National University of Singapore) for the design of graphical abstract shown in Fig. 1. This study was financially supported by the European Space Agency Grant ESA-CORA-GBF (4000113883), the Fondation Suisse de Recherche sur les Maladies Musculaires, and the Lee Foundation, Singapore. J.F., C.B., C.N.L., and A.F.-O. are inventors on patent WO 2016/178631 A1, System and Method for Applying Pulsed Electromagnetic Fields, and J.F., C.N.L. and A.F.-O. are contributors to QuantumTx Pte. Ltd., which elaborates on the use of similar magnetic fields on human health (targeting muscle). The other authors declare no conflicts of interest.

Figures

Figure 1

Graphical abstract. Schematic representation of how magnetic fields activate a TRPC1-mitochondrial axis upstream of calcineurin/NFAT and PCAF genetic and epigenetic pathways, promoting myogenesis and mitochodriogenesis.

Figure 2

PEMF exposure stimulates in vitro myogenesis. A) [Ca2+]i following exposures to 0 mT (red), 1 mT (blue), or ionomycin [Iono; 1 μM; 30 (light green) and 300 min (green)] as indicated. B) Effect of 2-APB (10 μM) on [Ca2+]i in response to 0 mT (gray blue) or 1 mT (gray) PEMFs. Unstained cell distributions in black (A, B). C) Effect of PEMF exposure duration on proliferation. Data at each time point were normalized to their own respective control scenario (0 mT) that was treated identically to the experimental condition with the lone exception of no PEMF exposure. 2-APB (light blue) was applied during PEMF exposure. Inset: Effect of CsA (2 μM) on PEMF-induced proliferation; n = 3. D) PEMF efficacy window (Supplemental Fig. S3). E–G) Myotube formation following exposure to 0 (E), 1.5 (F), or 3 mT (G) PEMFs. Scale bar, 500 μm. H) Fusion frequency distribution for 0 (red), 1.5 (blue), and 3 mT (light blue) exposures; n = 3. I) CsA (2 μM) precluded myotube enhancement following 1.5 mT exposure; n = 10. All PEMF exposures were applied once <24 h postplating for 10 min, except for C as noted; 24 h post-PEMF exposure myoblasts were either counted (C, D) or allowed to differentiate (E–I) for 6 d. *P < 0.05, **P < 0.01 (with regard to 0 mT).

Figure 3

PEMF exposure enhances in vitro myogenesis when applied once before 24 h postplating. A) Resting [Ca2+]i at 24 or 48 h in culture; n = 4. B) PEMF-induced [Ca2+]i at 24 or 48 h; n = 4. Despite resting [Ca2+]i being depressed at 48 h (A), PEMFs remained capable of augmenting [Ca2+]i (B), indicating that TRPC1-mediated calcium entry was operational, albeit down-regulated at 48 h. C) Proliferative responses to 1 mT exposure at 24 or 48 h; n = 8 (inset: despite the absence of proliferative response at 48 h, Trpc1 transcript levels rose 1 h after exposure). D, E) Cyclin D1 (D) and MyoD (E) protein levels following PEMF exposure at 24 h on low-density (∼2500 cells/cm2) or high-density (∼8500 cells/cm2) cultures; n = 3 or 5, respectively. Protein was collected 24 h after PEMF exposure as indicated. Inset: anti-BrdU-FITC positive myoblasts 6 h after PEMF exposure. F, G) Effects of TTX (1 µM) on PEMF-mediated proliferation applied at 24 and 48 h, as indicated, in low-density (F) or high-density (G) myoblast cultures. H) Nuclei/myotube distribution for exposures to 1.5 mT at 24 (blue) or 48 h (black); 0 mT (red). *P < 0.05, **P < 0.01 (with regard to 0 mT, respectively) (A–G). I) Effect of repeated stimulation on differentiation on myoblast cultures exposed once (blue; 24 h) or consecutively for 4 d (hatched). Myoblasts cultures were exposed at the indicated times and then either analyzed for proliferation/calcium entry (A–G) or induced to differentiate for subsequent analysis (H–I). **P < 0.01, #P < 0.05 with regard to 0 or 1.5 mT, respectively (I). All PEMF exposures were 10 min.

Figure 4

Pharmacological evidence implicating TRPC1 in magnetoreception. A) Effect of 2-APB (10 μM; hatched) on PEMF-induced proliferation at 24 h (dark blue) and 48 h (black) after plating; n = 11 (24 h) and n = 7 (48 h). B) Effect of SKF-96365 (50 µM) on PEMF-induced proliferation; n = 5. C) Levels of [Ca2+]i following 10 min exposure to 0 (red) or 1 mT (blue) PEMFs in the absence (solid) or presence (hatched) of 10 mM MgCl2 (hexahydrate) added to the culture medium immediately before exposure; n = 3 ± sem). D) PEMF-modulated proliferation (Prolif; 1.5/0 mT) in response to no antibiotics (solid), penicillin/streptomycin (PS) (1%, hatched), streptomycin (Strepto; 100 mg/L), neomycin (Neo; 50 mg/L), or gentamycin (Genta; 50 mg/L) added to the culture medium at time of PEMF exposure; n = 4. Inset: effect of 10 min PS application and immediate removal 2 h before PEMF exposure; n = 3. E, F) PS (1%) (E) and Neo (50 mg/L) (F) prevented myotube formation following single PEMF exposure. G–L) Myotube formation without (G–I) and with (J–L) PS (1%) as indicated. Scale bar, 100 μm. M) PS and 2-APB attenuate 2-APB–sensitive PEMF-induced calcium entry; n = 2 × 8 wells. N, O) Myotube size distribution without (N) and with (O) CsA; correspondent to Fig. 2I. The red dashed lines indicate the previous 0 mT level. Unless otherwise stated, all data are generated from the means of independent experiments ± sd each pertaining to minimally the means of biological triplicates. All PEMF exposures were conducted for 10 min applied 24 h postplating, unless otherwise stated (48 h; A); 24 h post-PEMF exposure myoblasts were either counted (B–D) or allowed to differentiate (E, F) for 6 d. **P < 0.01 (with regard to 0 and 1.5 mT, black and red asterisks, respectively). All drugs were applied transiently to coincide with PEMF exposure and to avoid non-TRP channel–related tertiary effects. Specifically, 2-APB, SKF-96365, and antibiotics were added to culture medium before PEMF exposure and then replaced with age-matched control medium from sister cultures summarily thereafter, with the exception of D (inset), wherein PS (hatched) was added and removed before PEMF exposure.

Figure 5

Magnetic fields promote epigenetic MyoD. A, B) PEMF-modulated PCAF and MyoD protein levels at 24 (A) or 48 (B) h postplating. C) H3K9 histone acetylation levels after 1.5 mT PEMF exposure at 24 (top) or 48 (bottom) h postplating. D, E) PCAF protein and H3K9 acetylation levels after growth of myoblasts within a μ-metal box (D) or in the presence of PS (PS; 1%; for 72 h (E). F, G) Protein levels of TRPC1 and PCAF and proliferation after 72-h growth in a µ-metal box (F, µ+) or in the presence of PS (G, PS+) relative to controls (µ−, PS−); (n/condition). H) Effects of CsA (2 μM) over PEMF-modulated PCAF expression at 24 or 48 h. I) Nuclear and cytoplasmic distribution of NFATC1 and NFATC3 following 1.5 mT exposure (blue) at 25 h postplating (see also Supplemental Fig. S8); n = 6/condition. Data represent the means of n experiments ± sd), each derived from the means of triplicates. All PEMF exposures were 10 min. **P < 0.01 with regard to relative control.

Figure 6

PEMFs activate mitochondrial respiration upstream of myogenic enhancement. A) ROS (H2O2) production in response to PEMF exposure and normalized to that at 0 mT; n = 6. Inset: SKF-inhabitable ATP production 30 min following PEMF exposure (10 min at 1.5 mT); n = 3. B) ROS production in response to exposure to PEMFs or 400 µM tert-butyl hydroperoxide (tBHP; 1 h; green). C) Normalized proliferation (relative 0 mT) in response to PEMF exposure with and without N-acetylcysteine (NAC); n = 9. D) Percentage change in ROS production immediately after PEMF exposure with and without PS (PS; 1%) or SKF-96365 (SKF; 50 µM). Data shown indicate ROS quantification 6–8 min after reading commenced. E, F) Mitochondrial OCR in response to PEMF exposure without (E) and with (F) SKF-96365. G–J) OCR and ECAR in myoblasts plated at 20,000 (G and I, respectively) and 30,000 (H and J, respectively) per well. K, L) OCR (K) and ECAR (L) in myoblasts exposed to PEMFs in suspension and then plated at 40,000 myoblasts/well 5 h before measurement. The green- and gray-shaded arrows indicate points of addition of oligomycin (ATP synthase inhibitor; 1 µM) and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (mitochondrial proton ionophore; 1 µM), respectively. Unless otherwise stated, all PEMF exposures (blue) was applied for a duration of 10 min at an amplitude of 1.5 mT with (hatched) or without (solid) drugs as indicated; red represents correspondent control scenarios. **P < 0.01 with regard to correspondent 0 and 1.5 mT scenarios, respectively.

Figure 7

TRPC1 silencing precludes PEMF sensitivity. A) Western blots showing protein levels of TRPC1 and cyclin D1 following transfection with targeted (dsi1 and dsi2) and nontargeted Scr dsiRNAs. TRPC1 silencing decreased basal cyclin D1 levels by ∼10%, whereas PEMF exposure increased cyclin D1 levels by 15% in controls (Con, Scr), compared with negligible PEMF-induced changes in TRPC1-silenced myoblasts, dsi1 (3%) and dsi2 (−5%). B) Normalized protein abundance of TRPC1 relative to Con. C) Proliferative responses of TRPC1-silenced and Con myoblasts to PEMF exposure (blue, 1.5 mT) relative to unexposed (0 mT; red); n = 6. D) TRPM7 protein abundance following silencing using 1 dsiRNA (dsi1). The data (lane) from another ineffective dsiRNA was removed for clarity and is delineated by the black border. E) TRPM7-silenced (ds1) and nonsilenced (Scr) myoblasts exhibited normal PEMF-induced proliferative enhancements; n = 6. F) Cellular DNA content, reflecting cell number, following PEMF exposure in CRISPR/Cas9 TRPC1-silenced clones, c60 and c61, relative to wild-type clone, c20. G) CRISPR/Cas9 targeted deletion of TRPC1 exon 1 reduced TRPC1 expression by ∼40 and 60% in clones c60 and c61, respectively, relative to wild-type clone, c20. See Supplemental Fig. S9 for details of the CRISPR/Cas9 deletion of TRPC1 exon 1; n = 3. All PEMF exposures were for 10 min. *P < 0.05, **P < 0.01 (with regard to correspondent 0 mT or Con scenario).

Figure 8

Mitochondrial oxidative capacity parallels TRPC1 channel expression and magnetic sensitivity. A–C) Mitochondrial OCR in response to PEMF exposure in wild-type C2C12 myoblasts (A) and in CRISPR/Cas9 TRPC1-silenced clones c60 (B) and c61 (C) (also see Table 2). D–F) ECAR in wild-type C2C12 myoblasts (D) and in CRISPR/Cas9 TRPC1-silenced clones c60 (E) and c61 (F). G–I) Mitochondrial OCR from wild-type C2C12 myoblasts in response to 10 min exposures to 0 (G), 1.5 (H), or 3 mT (I) amplitude PEMFs in the absence (solid symbols) or presence (hatched symbols) of SKF-96365 (SKF; 50 µM) as indicated. J–L) ECAR in wild-type C2C12 myoblasts in response to 10 min exposures to 0 (J), 1.5 (K), or 3 mT (L) amplitude PEMFs in the absence (solid symbols) or presence (hatched symbols) of SKF (50 µM) as indicated.

Figure 9

PEMFs promote mitochondriogenesis and functional adaptations. A–D, F, G) Gene expression scatter plots at 24 and 48 h post-PEMF treatment for the indicated genes. E) Myogenin (MyoG) to MyoD ratio at 24 and 48 h following PEMF exposure. For gene expression at early time points, the highest value at 16 or 24 h was taken. Also see Table 3. H, I) Change in mitochondrial DNA relative to nuclear DNA 24 h post-PEMF for 16s rRNA/GAPDH (H) and cytochrome c oxidase subunit II (COXII)/GAPDH (I); n = 6 ± sd. J) Differential percentage of Trypan Blue positive (dead) cells after 24 and 48 h post-PEMF exposure; n = 7 ± sem). K, L) Proapoptotic (K) and antiapoptotic (L) protein expression at 24 and 48 h post-PEMF exposure as indicated; n = 12 ± sem. *P < 0.05, **P < 0.01 (with regard to correspondent 0 mT control scenario or as indicated by bar). All data represent the mean of n independent experiments each pertaining to the means of biological triplicates.

Figure 10

Evidence for magnetic specificity. A) Coincident µ-metal shielding or PS administered only during PEMFs exposure precluded proliferative response (Supplemental Fig. S1C); n = 6. B) Vibrating myoblasts at the same frequency and amplitude as they experience during PEMF exposure does not recapitulate the proliferative effects of PEMF exposure relative to nonvibrated, nonexposed myoblasts (red); n = 4. C) Exposing myoblasts to PEMFs while in suspension did not preclude a proliferative response; n = 4. D) PEMF exposure (1.5 mT; dark blue hatched) or 5 mM CaCl2 (green hatched) applied to C2C12 cultures for 10 min before placement into simulated microgravity (hatched) transiently rescued G2/M accumulation of myoblasts caused by gravitational mechanical unloading (µg; red hatched) (12), whereas 0.5 mT PEMFs (light blue hatched) did not; n = 3. All data are generated from the means of independent experiments ± sd each pertaining to minimally the means of triplicates. All PEMF exposures were for 10 min. **P < 0.01, *P < 0.05 [with regard to correspondent control scenarios (0 mT)].