Muscle injury was induced by a single intramuscular (IM) injection of 20 μl of the myotoxic agent notexin (12.5 μg/ml, Latoxan, Valence, France), diluted in PBS, (or with 20 μl PBS as a control) into the gastrocnemius muscle of experimental mice. Briefly, WT and Nogo-KO mice (3 mice per group) were anesthetized after which both hind limbs were shaved. Notexin was injected into the right leg muscle, while the muscle of the left leg served as a control and was injected with PBS. Three days after notexin injection, the mice were euthanized and the gastrocnemius muscle was surgically isolated, as previously described [12, 67]. The gastrocnemius muscle was cut in half as a cross-section, fixed in 4% paraformaldehyde (PFA) overnight, and subsequently transferred to 30% sucrose in PBS for 24 hours. Using optimum cutting temperature (OCT) medium, the samples were embedded in a cryo block for histological analysis. The remaining half of the muscle sample was immediately frozen in liquid nitrogen for molecular analysis. The sample was subsequently stored at -80 °C until further analysis. All animal studies were approved by the Institutional Animal Use and Care Committee of Kyungpook National University, Daegu, Republic of Korea (KNU 2017-0023 and 2018-0074).
Mouse model of Duchenne muscular dystrophy
Mice were housed under the appropriate conditions with 12-hour light cycles and were supplied with water and food in accordance with the regulations of the Kyungpook National University animal facility. For the DMD animal model, male WT mice (C57BL/6J, 12 weeks of age, n = 10) and male mdx mice (C57BL/10ScSn-Dmdmdx/J, 12 weeks of age, n = 4) were used. Mdx mice were a gift from Jacques Tremblay (CHUQ Research Center, Quebec City, Canada), and C57BL/6J mice were purchased from Japan SLC, Incorporated. (Hamamatsu, Japan), as previously described .
Induction of endoplasmic reticulum (ER) stress using tunicamycin
Healthy ten-week-old mice were housed under the appropriate conditions and were divided into the following 4 groups: WT mice without tunicamycin treatment (n = 3), WT mice treated with tunicamycin (n = 4), Nogo-KO mice without tunicamycin treatment (n = 4), and Nogo-KO mice treated with tunicamycin (n = 5). Tunicamycin was administered at a single dose of (1 µg/kg) via intraperitoneal (IP) injection. Muscles were harvested 24 hours after injection. Tunicamycin was prepared in DMSO and diluted in PBS to reduce the toxicity of DMSO in mice.
Muscle biopsies were obtained from patients at the Pusan National University Yangsan Hospital (Yangsan, Republic of Korea, Institutional review board No. 05-2018-045) and Yonsei University College of Medicine (Seoul, Republic of Korea, Institutional review board No. 3-2018-0060) after approval from the Medical School Ethical Committee. Muscle samples were harvested after diagnosis, and informed consent was obtained from all patients for the scientific use of their muscle biopsy specimens. The samples from patients with myopathy and those with DMD were collected according to the patient’s age (22 months, 2 years, 5 years, 5 years 1 month, 5 years 7 months, 15 years, 20 years, 46 years, 57 years, and 81 years). Four muscle biopsies were obtained for each group from age-matched healthy control patients (18 years, 26 years, 41 years, and 42 years). qPCR and immunoblot analyses were performed on all muscle biopsy samples.
C2C12 cell culture
The murine myoblast cell line (C2C12) was cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (P/S). Cells were cultured in a humidified incubator containing 5% CO2 at 37 °C. C2C12 cells were grown until they were 60%–70% confluent. Cells were then sub-cultured and grown for another 48 hours. Finally, the cells were differentiated in 2% horse serum for 3 days, as previously described [69, 70].
Recombinant adenovirus and si-RNA transfection of C2C12 cells
Ad-Nogo-A and Ad-CHOP were purchased from Vector Biolabs (Malvern, PA, USA). The small interfering (si) RNAs against Nogo-A and CHOP (si-cram, si-Nogo-A, and si-CHOP) were purchased from Bioneer Research (Seoul, Republic of Korea) and were transfected into cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For transfections, the cells were plated in 60-mm dishes at a density of 1 x 105 cells in DMEM without antibiotics and allowed to grow for 24 hours. When the cells became 40%–50% confluent, the cells were transfected according to the manufacturer’s instructions.
Isolation, culture, and activation of bone marrow-derived macrophages
Macrophages were obtained from bone marrow with several modifications, as previously described [7, 71]. Briefly, bone marrow cells were obtained by flushing the femur and tibia of 8-week-old C57BL/6 (WT) and Nogo-knockout (KO) mice (n = 6). The femur and tibia were washed with 70% ethanol and then with PBS. Sterile scissors were used to cut both the knee and hip joints. The ends of the femur and tibia bones were also cut to obtain macrophages from the bone marrow. The bone marrow was flushed out in a 50-ml Falcon tube using a 26 gauge syringe and sterile PBS. The sample was then centrifuged at 3,000 g for 5 minutes at 4 °C after which the cells were suspended in RPMI 1640 medium containing 15% conditioned medium from the L929 cell line as a source of macrophage colony stimulating factor (M-CSF). Cells were incubated for seven days and treated with lipopolysaccharide (LPS), an M1 inducer (100 ng/ml), or IL-4, an M2 inducer (20 ng/ml) for 24 hours.
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
Total RNA was extracted from the gastrocnemius muscles of mice and from BMDM using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Gene expression was measured by quantitative real-time polymerase chain reaction (qPCR) using SYBR Green with low ROX (Enzynomics, catalog no. RT500S) [72, 73]. Relative quantification of the target gene was determined by normalizing expression to that of the housekeeping gene GAPDH, which served as a control. The primer sequences used in this study are listed in Table S1. qPCR data were analyzed using a CFX Connect Real-Time System (Bio-Rad).
Western blot analysis
Proteins were isolated from BMDM and analyzed by immunoblotting, as previously described . Briefly, the protein concentration in the samples was measured, samples were prepared in SDS and sample loading buffer, and heated for 10 minutes at 95 °C. Proteins were separated using 10% SDS-PAGE and immunoblotted onto membranes. The membranes were blocked with 1% bovine serum albumin (BSA) for 1 hour and incubated with primary antibodies, including those against Nogo-A (Abcam, catalog no. ab62024), CHOP (Santa Cruz, catalog no. sc-71136), β-actin (Cell Signaling Technology, catalog no. 8457s), and GAPDH (Cell Signaling Technology, catalog no. 2118), overnight at 4 °C. After a 1-hour incubation with HRP-labeled secondary antibodies (Anti-rabbit-HRP, Cell Signaling Technology, catalog no. 7074s and Anti-mouse-HRP, Cell signaling Technology, catalog no. 7076s), the proteins were detected using enhanced chemiluminescence (ECL, SuperSignal West Dura Extended Duration Substrate, catalog no. 34076) in an Amersham Imager 680 (GE Healthcare, Life Sciences). Blots were quantified using ImageJ software.
Immunofluorescence (IF) assay
The immunofluorescence assay was performed with modifications, as previously described . Briefly, cryosections and BMDM were washed with tris-buffered saline (TBS) and fixed in 4% paraformaldehyde (PFA) for 10 minutes. After washing, the samples were permeabilized with TBST (0.2% Triton X-100 in TBS) for 10 minutes and washed three times with TBS for 5 minutes. Samples were blocked using 2% BSA after which the cells were incubated with primary antibodies, including rabbit anti-Nogo-A (Abcam, catalog no. ab62024), mouse anti-CD68 (Santa Cruz Biotechnology, catalog no. ab955), mouse anti-iNOS (Santa Cruz Biotechnology, ab49999), mouse anti-CD206 (Santa Cruz Biotechnology, catalog no. sc-58986), mouse anti-CHOP (Santa Cruz Biotechnology, sc-71136), and mouse anti-calnexin (Novus Biologicals, catalog no. NB300518), at 4 °C overnight. The cells were then washed 3 times with TBS for 5 minutes each wash. Samples were incubated with secondary antibodies (donkey anti-mouse immunoglobulins (Alexa Fluor 488, Abcam, catalog no. ab150105) and donkey anti-rabbit immunoglobulins (Alexa Fluor 555, Abcam, catalog no. ab150066) for 1 hour in the dark. The samples were mounted using ProLong™ Gold Antifade reagent containing DAPI to visualize the nuclei (Cell Signaling Technology, catalog no. 8961s) and were analyzed by confocal microscopy (ZEISS).
Gastrocnemius muscles from mice samples were rapidly fixed with 5% sucrose in 4% paraformaldehyde (PFA) for 24 hours and subsequently transferred into 30% sucrose in PBS for 24 hours. Samples were embedded in OCT compound for cryopreservation. Cryosections of 5 µm thick tissues were cut for histological analysis. Sections of muscle were stained with hematoxylin and eosin (H&E). Stained tissue sections were visualized using a light microscope.
Flow cytometry analysis
BMDM were incubated for seven days and stimulated with lipopolysaccharide (LPS) (100 ng/ml) or IL-4 (20 ng/ml) for 24 hours. BMDM were collected and washed twice in PBS and centrifuged at 1500 g for 3 minutes. Cells were incubated at 37 °C with primary antibodies against iNOS (Abcam, catalog no. ab49999) and CD206 (santa cruz, catalog no. sc-58986) for 1 hour and were then washed with PBS. Finally, the cells were incubated with fluorochrome-labeled secondary antibodies in PBS for 30 minutes. After three washes in PBS, the cells were analyzed by flow cytometry.
A migration assay was performed with modifications, as previously described . Transwell chambers (6.5 mm diameter and 8 μm pore size) were obtained from Corning (catalog no. 3422). BMDM were harvested and suspended in RPMI supplemented with 10% FBS at a concentration of 2 × 104 cells/well. Cells were seeded in serum-free medium into the upper chamber of a 24-well plate. The lower chambers were filled with RPMI medium containing 10% FBS. Cells were incubated overnight. Cells that had migrated to the reverse side of the Transwell membrane were fixed in 4% PFA and permeabilized with absolute methanol. Cells were stained with H&E, and non-migrated cells were removed using cotton swabs at which point the cells that had migrated were counted using a light microscope.
BMDM were stimulated with Alexa Fluor 488-labeled zymosan fluorescent bioparticles (catalog no. z-23373). For flow cytometry, the BMDM were washed twice in PBS. Adherent cells were detached as a result of incubation with trypsin-EDTA for 5 minutes in the incubator and were subsequently centrifuged at 1500 g for 3 minutes. Cells were placed in the incubator and given 30 minutes to internalize the zymosan particles. Noninternalized particles were removed by three washes in cold PBS. The harvested cells were then washed and fixed in 4% paraformaldehyde. Cells were washed twice with PBS, placed in a FACS tube, and were immediately examined by flow cytometry.
Statistical analysis was performed using GraphPad Prism 6.01 (GraphPad Software) program. Statistical significance was determined using Student’s t-test. Data are expressed as means and standard error of the mean (SEM). The statistical significance of data is denoted on the graphs by asterisks (*), with P values of *P < 0.05, **P < 0.01, and ***P < 0.001.