The ELF-WMF apparatus
The ELF-WMF device was manufactured by Mr. Kota Okada at the Technical Center of Nagoya University, Japan. The device had a round coil (1 cm height, 10 cm inner diameter, and 10.7 cm outer diameter, 50 turns of copper wire, and 0.29 mm diameter). It generated 1–16 ms pulsed magnetic fields of 0 to 300 µT at time-varying frequencies of 1–16 Hz every second. We used a 10 µT magnetic field of 4 m pulse width with increasing frequencies of 1, 2, 3, 4, 5, 6, 7, and 8 Hz every second (Opti-ELF-WMF), unless indicated otherwise. This condition maximizes the hysteresis of the electronic resistance of pure water 11, 12. Before and after each experiment, we confirmed the intensity of the magnetic flux using a pulse magnetic field meter (Aichi Micro Intelligent). To reduce the effects of an electromagnetic field generated by an incubator and the geomagnetic field, the ELF-WMF device and a culture dish were sandwiched by two 5-mm thick copper plates, and were placed in a humidified incubator with 5% CO2 at 37°C. The basal electromagnetic field was below 20 µT (200 mG) on the culture dish. Control samples were incubated in parallel under the same conditions in another incubator without ELF-WMF.
Exposure of wild-type mice to ELF-WMF
All the studies on mice were approved by the Animal Care and Use Committee of Nagoya University, and were conducted in accordance with the relevant guidelines. Seven-week-old C57BL6/N male mice were purchased from Japan SLC. Two ELF-WMF devices were placed in tandem beneath the mouse cage, and mice were housed in the cage with switch on (ELF-WMF group, n = 4) or off (control group, n = 4) for 4 weeks. The Opti-ELF-WMF condition stated above was applied to the ELF-WMF group.
Test for open-field locomotor activity in mice
Open-field locomotor activity was evaluated using a photometric actimeter (45 cm × 45 cm, IR Actimeter, Panlab). Fast and slow movements were monitored with a grid of infrared beams every 30 min for 24 h and were used as indices for locomotor activity. To examine the effects of ELF-WMF on the locomotor activity in mice, fast and slow movements were measured before (0 week) and after (4 weeks) exposure. All data were collected using the SEDACOM software (Panlab). Each mouse was tested individually and had no contact with other mice.
Isolation of mitochondria from the mouse liver
Mitochondria were isolated from the mouse liver, as described previously 13. Briefly, a piece of liver was rinsed, minced, and disrupted with a mitochondrial isolation buffer (70 mM sucrose [Wako], 210 mM mannitol [Sigma], 5 mM HEPES [Dojindo], 1 mM EGTA [Sigma], and 0.5% [w/v] fatty acid-free BSA [Sigma], pH7.2) using a homogenizer. The homogenate was centrifuged at 800 × g for 10 min at 4°C. The supernatant was then centrifuged at 8,000 × g for 10 min at 4℃. The pellet was suspended in the mitochondrial isolation buffer to obtain the mitochondrial fraction.
TMRM assay of the isolated mitochondria
The mitochondrial membrane potential of isolated mitochondria was analyzed using TMRM (T668, Thermo Scientific), following the procedure described previously 14. Briefly, the isolated mitochondria were incubated with 100 nM TMRM in the mitochondrial isolation buffer for 30 min at 37˚C in a humidified incubator. The signal intensities of TMRM were quantified using BD FACS Calibur (BD Biosciences).
Measurement of basal oxygen consumption rate of the isolated mitochondria
The basal oxygen consumption rate (OCR) of the isolated mitochondria (20 μg of mitochondrial proteins per well) isolated from the mouse liver was determined using the Seahorse XFp Extracellular Flux Analyzer (Agilent Technologies). The assay was conducted as described previously 15.
Cell culture
AML12 cells were purchased from ATCC and cultured in DMEM/F-12 medium (Gibco) with 10% fetal bovine serum (FBS, Thermo Scientific), dexamethasone (Wako), and insulin-transferrin-sodium selenite (Sigma). HeLa, HEK293, Neuro2a, and C2C12 cells were also purchased from ATCC, and were cultured in DMEM (Gibco) with 10% FBS. PINK1 KO HeLa cells were kindly provided by Dr. Richard J. Youle from the National Institute of Neurological Disorders and Stroke 16, and were cultured in DMEM (Gibco) with 10% FBS. Human iPS cells were purchased from Riken BRC and cultured in the StemFit medium (Ajinomoto).
MitoSOX, MitoTracker Green, and TMRM assays of cultured cells
AML12 cells exposed to ELF-WMF for the indicated time periods were washed with PBS. MitoSOX (M36008, Thermo Scientific) and MitoTracker Green (M7514, Thermo Scientific) were dissolved in Hank’s balanced salt solution (HBSS, Gibco) at 5 µM and 50 nM, respectively. TMRM was dissolved in the medium at 200 nM. Each dye was added to the cells and incubated for 30 min at 37˚C in a humidified incubator. The cells were then washed with PBS, trypsinized, resuspended in PBS, and harvested. Signal intensities of MitoSOX, MitoTracker Green, and TMRM were quantified using BD FACS Calibur.
Western blot analysis of cell lysates
Cells were lysed in PLC buffer containing 50 mM HEPES (pH 7.0), 150 mM NaCl, 10% glycerol, 1% TritonX-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 μg/μl aprotinin, 1 μg/μl leupeptin, 1 μg/μl pepstatin A, and 1 mM PMSF. The cell lysates were rotated at 4°C for 20 min and centrifuged at 17,900 × g at 4°C for 15 min. The supernatant was incubated at 37°C for 1 h to analyze the mitochondrial ETC complex proteins or at 95°C for 5 min to analyze other proteins in the sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue, and 2% 2-mercaptoethanol). For LC3-II, the lysates were separated by Tricine-SDS-PAGE on a 16% polyacrylamide gel 17. For the other proteins, the lysates were separated by Tris-SDS-PAGE on a 10%, 12%, or 14% SDS-polyacrylamide gel. The samples were then transferred to a polyvinylidene fluoride membrane (Immobilon-P, Millipore). Membranes were washed in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and blocked for 1 h at 24°C in TBS-T with 5% skimmed milk. The membranes were then incubated overnight at 4°C with specific antibodies, as indicated below. The membranes were washed with TBS-T and incubated with secondary anti-goat IgG (1:2000, sc-2094, Santa Cruz), anti-mouse IgG (1: 2000, LNA931V/AG, GE Healthcare), or anti-rabbit IgG (1: 2000, LNA934V/AE, GE Healthcare) antibody conjugated to horseradish peroxidase (HRP) for 1 h at 24°C. Immunoreactive signals were detected with the ECL western blotting detection reagents (GE Healthcare) and visualized using LAS 4000mini (GE Healthcare). Signal intensities were quantified using ImageQuant (GE Healthcare).
Antibodies for western blot analysis
The following specific antibodies were used for western blot analysis: anti-UQCRFS1 (1:1000 dilution, ab14746, Abcam), anti-NDUFS1 (1:1000, ab169540, Abcam), anti-VDAC1 (1:3000, ab14734, Abcam), OXPHOS cocktail (1:1000, ab110413, Abcam), anti-LC3 (1:1000, ab51520, Abcam), anti-PINK1 (1:500, ab23707, Abcam), anti-parkin (1:500, #4211, Cell Signaling Technology), anti-ubiquitin (1:1000, P4D1, BioLegend), anti-PGC-1α (1:1000, ab106814, Abcam), anti-PPARα (1:1000, GTX101098, GeneTex), anti-TFAM (1:1000, GTX103231, GeneTex), anti-ATP5A (1:1000, ab14748, Abcam), anti-SDHA (1:1000, GTX101689, GeneTex), anti-SDHB (1:2000, GTX104628, GeneTex), anti-SDHC (1:500, ab155999, Abcam), and anti-SDHD (1:500, ab189945, Abcam) antibodies.
Preparation of mitochondrial and cytosolic fractions of cultured cells
A mitochondria isolation kit (ab110170, Abcam) was used for the extraction of mitochondrial and cytosolic fractions according to the manufacturer’s protocols. After obtaining the mitochondrial fraction by centrifugation at 12,000 × g for 10 min at 4°C, the supernatant was used as the cytosolic fraction.
Detection of mitophagy in cultured cells
To detect mitophagy in AML12 cells, the Mitophagy Detection Kit (Dojindo Molecular Technologies) was used according to the manufacturer’s protocol. Briefly, Mtphagy Dye (Dojindo) dissolved in HBSS at 100 nM was added to the cells and incubated for 30 min at 37°C in a humidified incubator. After incorporation of Mtphagy Dye into AML12 cells, the cells were exposed to ELF-WMF for 120, 150, and 180 min, washed with PBS, trypsinized, resuspended in PBS, and harvested. Signal intensities of Mtphagy Dye were quantified using BD FACS Calibur (BD Biosciences). To visualize both mitophagy and lysosomes after exposure to ELF-WMF, Lyso Dye (Dojindo) dissolved in HBSS at 1 μM was also added to the cells and incubated for 30 min at 37°C in a humidified incubator. Images were obtained using a confocal microscope TiE-A1R (Nikon).
Enzyme assay for mitochondrial ETC complex (I, II, III, IV) activities of the mouse liver homogenates
Mitochondrial ETC complex activities were measured using homogenates of the liver excised from C57BL/6N mice. The ETC complex activity assay was performed as previously described 18. Briefly, the protein concentration of each sample was measured using a Pierce 660 nm protein assay reagent. The ETC complex activities of complexes I, II, III, and IV were estimated by determining the decrease in absorbance of NADH at 340 nm, the decrease in absorbance of 2, 6-dichlorophenolindophenol (DCPIP) at 600 nm, the increase in absorbance of reduced cytochrome c at 550 nm, and the decrease in absorbance of reduced cytochrome c at 550 nm, respectively, with NanoDrop ONEC (Thermo Scientific). The ETC complex activities were measured in the liver homogenate of mice that were exposed to either ELF-WMF or control in vivo. Similarly, the ETC complex activities were measured in the mouse liver homogenates before and after exposure to ELF-WMF for 10 min or less in vitro.
Enzyme assay for mitochondrial ETC complex II activity of the mitochondria isolated from the mouse liver
Mitochondria (10 μg protein) isolated from the liver of C57BL/6N mice were used to measure the mitochondrial ETC complex II activities (Fig. 6E). Assays for measuring the fractional and extended mitochondrial ETC complex II activities of SQR, SCR, and SDH were performed as previously described 18, 19. The mitochondrial ETC complex II activities were quantified before and after exposure to ELF-WMF for 8 min in vitro. The SQR, SCR, and SDH activities were measured by determining the decrease in absorbance of DCPIP at 600 nm, the increase in absorbance of reduced cytochrome c at 550 nm, and the decrease in absorbance of DCPIP at 600 nm, respectively, using NanoDrop ONEC (Thermo Scientific). The SDHA activity was quantified by modifying a method used to measure the SDH (SDHA and SDHB) activity. For measuring the SDHA activity, 10 μg of sonicated mitochondrial fraction was resuspended in 35 mM phosphate buffer (pH 7.3) supplemented with 0.3 mM KCN (Wako), 10 μg/ml antimycin A (Sigma), 4 mM succinate (Wako), 1.6 mM PMS (Sigma), and 40 μM DCPIP (Sigma). The SDHA activity was quantified before and after exposure to ELF-WMF for 8 min by determining the decrease in absorbance of DCPIP at 600 nm with NanoDrop ONEc (Thermo Scientific).
Measurement of mitochondrial mass in AML12 cells exposed to an inhibitor of mitochondrial ETC complex I or II
AML12 cells were cultured either with variable concentrations of rotenone (Tokyo Chemical Industry Co.), an inhibitor of mitochondrial ETC complex I, or 3-nitropropionic acid (Cayman Chemical), an inhibitor of mitochondrial ETC complex II, for 12 h. The cells were then exposed to Opti-ELF-WMF for 3 h. Mitochondrial mass was measured by MitoTracker Green, as described above.
RNA-sequencing and GSEA of AML12 cells
Total RNA was extracted from AML12 cells exposed to OPTI-ELF-WMF for 1 h using QuickGene-Mini80 (Kurabo) according to the manufacturer’s instructions. The extracted RNA was subjected to RNA-seq at Macrogen, Japan. Briefly, a sequencing library was prepared using the TruSeq Stranded mRNA kit (Illumina), and the library was read on an Illumina NovaSeq 6000 (150 bp paired-end reads). GSEA was conducted with the GSEA v4.1.0 software for Windows (https://www.gsea-msigdb.org/gsea/downloads.jsp) using the RNA-seq dataset. RNA-seq data were deposited in the gene expression omnibus (GEO) with an accession number GSE166811.
Statistical analysis
All values are presented as the mean ± SEM. For in cellulo studies, values were normalized to those of control cells, unless indicated otherwise. Statistical significance was estimated either by Student’s t-test, one-way ANOVA followed by Dunnett’s posthoc test, or false discovery rate of multiple Student’s t-tests. P-values less than 0.05 were considered statistically significant.