Implementation of a system evaluating the contractile force of electrically stimulated myotubes from wrinkles formed on an elastic substrate

Animals

Adult C57BL/6N mice (8-12 weeks old, male) were housed in a cage in a temperature-controlled room at 24°C with a 12 h light-dark cycle (4:00 a.m. to 4:00 p.m. ). All animal experiments were approved by the Tokyo Metropolitan University Animal Care Committee (approval number A28-7, A29-14, A30-8, and A31-20) and performed in accordance with relevant guidelines and regulations . This study was conducted in accordance with ARRIVE guidelines.

Cell culture

Extensor digitorum longus (EDL) muscles were isolated from C57BL/6 N mice. Satellite cells were prepared as previously described.41 with some modifications. Briefly, EDL was digested with 0.2% collagenase type I (Worthington, Lakewood, NJ, USA) in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at 37°C. Isolated myofibers were collected and digested with Accutase (Innovative Cell Technologies, San Diego, CA, USA) for 10 min at 37°C. The digested myofibers were then inoculated onto 150 mm culture dishes coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) in a growth medium composed of DMEM, without glucose (Thermo Fisher Scientific) supplemented with 30% of FBS (511–98.175, FUJIFILM Wako Pure Corporation Chemical, Osaka, Japan), 1% GlutaMAX (Thermo Fisher Scientific), 1% Chicken Embryo Extract (US Biological, Marblehead, MA, USA), 10 ng/ml bFGF and 1% antibiotic (Thermo Fisher Scientific) at 37˚C with 5% CO2 to form myoblasts on culture dishes42. Myoblasts were reseeded at a density of 3.0 × 104 cells per well on Matrigel-coated silicone substrates fixed to the bottom of 2-well chambers (Thermo Fisher Scientific) (see “contractile force assay” section). 1 day after seeding, the medium was replaced with a differentiation medium composed of GlutaMAX high glucose DMEM supplemented with 5% horse serum (16,050,130, Thermo Fisher Scientific) and 1% antibiotic . Half the amount of medium was replaced daily. Myotubes were used for contractile force testing 5 or 7 days after differentiation.

LLC cells (kind of a gift from Dr. Hata, Y. Tokyo Medical and Dental University) were cultured in a growth medium consisting of glucose-rich DMEM supplemented with 10% horse serum (16,050,130, Thermo Fisher Scientific) and 1% antibiotics. The medium was changed every 2 days to maintain the cells.

Atrophic and hypertrophic myotubes

To produce the dexamethasone-induced atrophic myotubes, the myotubes were treated with 100 mM dexamethasone (Sigma Aldrich, MO, USA) in the differentiation medium on the 5th day of differentiation and cultured for 48 h. Control cells were treated with 0.1% (v/v) dimethylsulfoxide (DMSO; Sigma Aldrich) in the differentiation medium. To produce cancer cachexia-induced atrophic myotubes, CLLs and myoblasts were cultured separately in the differentiation medium for 48 h at 2.5 × 106 100mm cells/plate; the conditioned medium was then collected. CLL conditioned medium was mixed with fresh 50% (v/v) differentiation medium and used as cachexia medium. Myotube conditioned medium was mixed with fresh 50% (v/v) differentiation medium and used as control medium43. Myotubes were treated with cancer cachexia medium on day 3 of differentiation and cultured for 4 days. Control cells were treated with control medium. To produce the hypertrophic myotubes induced by IGF-1, the myotubes were treated with 100 ng/mL of IGF-1 (Sigma Aldrich) in the differentiation medium from the start of differentiation for 7 days. Control cells were treated with 0.1% (v/v) 10 mM Tris-HCl in differentiation medium.

contractile force test

Contractile force of myotubes was assessed using a deformable silicone substrate technique19.20 with some modifications. Parts A and B of CY 52–276 (Dow Corning TORAY, Tokyo, Japan) were mixed at a weight ratio of 1.4:1 to form a silicone gel. The gel was coated on an 18 mm × 18 mm cover glass (Matsunami Glass, Osaka, Japan) using a spin coater (K-359S1; Kyowariken, Tokyo, Japan) at 500 rpm for 10 s and 1500 rpm for 30 s and baked at 60˚C for 20 h in an oven to harden the gel (Tokyo Garasu Kikai, Tokyo, Japan). The gel surface was then treated with an oxygen plasma (2 mA, 100 V, 20 Pa, 1 min) (SEDE-GE; Meiwafosis, Tokyo, Japan). The substrate was fixed to the bottom of 2-well chambers and covered with Matrigel for 30 minutes before cell seeding. Myoblasts were seeded on the substrate and allowed to differentiate for 5 or 7 days. The medium was replaced with differentiation medium supplemented with 10 mM HEPES (Nacalai tesque, Kyoto, Japan) immediately before myotube contraction. The 2-well chamber was connected to a carbon electrode (Uchida Denshi, Tokyo, Japan) and an electrical pulse generator (Uchida Denshi). The myotubes were stimulated with electrical pulses with a current of 20 mA at 1 Hz, each cycle consisting of a duration of 20 ms followed by an interval of 980 ms. Wrinkle formation was observed under a phase-contrast microscope (ECLIPSE Ti; Nikon, Tokyo, Japan) at 200× magnification and recorded as a movie in 12 areas on the same substrate, acquired at 16 frames per second.

Evaluation of the relationship between strength and wrinkles

A certain magnitude of mechanical force was applied to the fixed myotubes using a flexible glass needle as previously described.20. The needle was fabricated from a glass rod using a glass electrode puller (P-1000; Sutter Instrument, CA, USA). The needle tip was designed to have a diameter of approximately 5 µm. The bending stiffness of the needle was determined at 465 nN/µm; this was measured from the displacement of the needle tip when a constant force was applied to the needle using an atomic force microscopy cantilever with precisely calibrated stiffness ( OMCL-TR400PB-1; Olympus, Tokyo, Japan). Myotubes on day 5 of differentiation were fixed with 1% glutaraldehyde in PBS for 10 min at room temperature and washed with 30 mmol/L glycine in PBS. The needle was pricked into a fixed myotube using a micromanipulator (MWO-3; Narishige, Tokyo, Japan) to apply mechanical force, and observed under a phase-contrast microscope. Needle deflection was measured as the distance between needle tips during and after force application. The force applied to the myotubes was calculated as the product of the bending stiffness (nN/µm) and the deflection (µm) of the needle. All data were subjected to outlier tests and data excluding outliers were plotted graphically.

Extraction of wrinkles generated by the contraction of myotubes

The wrinkles generated by the contractile force of the myotubes were automatically extracted from the phase-contrast film using the envelopment algorithm in ImageJ-Fiji. Movies of myotube contraction were converted to image sequences. In order to identify the clear regions corresponding to the cellular contours (therefore not to the wrinkles), a morphological operation in gray levels and binarization was carried out on a frame showing relaxed myotubes. The identified area has been zeroed in the images. This process is also necessary to exclude some wrinkles formed in non-cellular areas on the substrate due to attachment of non-cellular materials. Wrinkles generated exclusively by myotube contraction were then extracted by subtracting the relaxed image from the images where the myotube was contracted, leaving a signal only where there were wrinkles. Finally, the extracted wrinkles were skeletonized into line segments; wrinkles consisting of less than 20 pixels have been removed as noise. The total length of wrinkles in the image in pixels was plotted over time.

Myotube diameter measurement

Images of myotubes were acquired at 100× magnification prior to contractile force testing. Myotube diameters were measured in all cells from 5 random fields of view per substrate using NIS-Elements (Nikon).

Western blot

Myotubes were washed with PBS and harvested with 200 µL of lysis buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM tetrabasic sodium pyrophosphate, 1 mM ethylenediaminetetraacetic acid (pH 8.0 ), 1 mM sodium orthovanadate, 1% Nonidet P-40 , 10 mM sodium fluoride, 150 mM sodium chloride, 10 mg/L leupeptin, 1 mM phenylmethylsulfonyl fluoride, 5 mg/mL aprotinin, benzamidine 3 mM and 10 mM beta glycerophosphate. Harvested cells were sonicated and centrifuged at 13,000 xg for 15 min at 4°C, and the supernatant was used for immunoblotting. The protein concentration of the supernatant was determined by the Bradford protein assay. Cell lysates were separated by 8–10% polyacrylamide-sodium dodecyl sulfate gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were cut according to molecular weight and blocked with Tris Buffered Saline containing 0.1% Tween 20 and 5% skimmed milk powder. Membranes after blocking were incubated with the primary antibodies Myosin heavy chain I (MHC I; 1:1000, Sigma), Myosin heavy chain II (MHC II; 1:1000, Sigma) or glyceraldehyde -3-phosphate dehydrogenase (GAPDH; 1:3000, Cell Signaling, MA, USA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Buckinghamshire, UK Uni) for 1 h at room temperature. Blots were used for detection with enhanced chemiluminescence (PerkinElmer, MA, USA), analyzed with ImageQuant LAS 4000 mini (GE Healthcare), and quantified using ImageQuant TL (GE Healthcare).

Statistics

Data are presented as mean ± SEM. An unpaired Student’s t test was performed to assess the statistical differences between the two groups and the values ​​of PP

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