Cell culture and myogenic differentiation
Mouse skeletal myoblast C2C12 cells were purchased from the National Infrastructure of Cell Line Resource in China. Proliferating myoblasts were maintained in DMEM/high glucose medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) in a humidified CO2 incubator (5% CO2, 37 °C; HF90, Heal Force, Hongkong, China). For myogenic differentiation, myoblasts with 80%~90% confluence were induced by DMEM/high glucose medium supplemented with 2% horse serum (Hyclone, Logan, UT, USA).
Myogenic cells of pigs (n = 3) were isolated using preplate techniques from skeletal muscle according to our previous reports [28]. The cells were cultured in growth medium in a dish coated with collagen I (Sigma-Aldrich) at 37 °C and 5% CO2. The growth medium was composed of DMEM/F12 (Hyclone), 10% FBS (Gibco-BRL, Carlsbad, CA, USA), 2 mM glutamine (Gibco-BRL), and 5 ng/mL bFGF (Peptech, Burlington, MA, USA). As for myogenic induction, cells were cultured for 5 days in DMEM/F12 medium containing 2% horse serum (Hyclone).
Cellular Ca2+ concentration measurement
Ca2+ concentration in the cytoplasm or ER was measured using flow cytometry. Cells were collected, washed three times with PBS and HBS respectively, and then incubated in 5μg/mL Fluo-3 acetoxymethyl ester (Cayman, Ann Arbor, MI, USA) or Mag-fluo-AM (GENMED, Shanghai, China) for 30 min at 37 °C in dark. After three washes with PBS supplemented with 1% FBS, cells were resuspended in 200 μL PBS containing 1% FBS. Flow cytometry was carried out immediately using a FACS Calibur Cytometer and Image Cytometry software (BD, Franklin, NJ, USA). Calcium-bound Fluo-3 or Mag-fluo-AM has an emission maximum of 526 nm which was quantified by excitation with a 488-nm laser and signals were collected using a 530/30 nm band-pass filter. Each sample generated 20,000 live gated events. Debris, multicellularity, and dead cells were excluded by forward scatter (FSC) and side scatter. For detecting dynamic change of Ca2+ concentration of cells during myogenic differentiation, a blank control combined with a house-keeping control (the proliferative C2C12 cells) was used to correct the deviation caused by the loaded indicator amount and the voltage used in each measurement. Mean fluorescence intensity was determined from the entire cell population and then adjusted by relative cell size calculated according to FSC to represent Ca2+ concentration.
Cell viability and apoptosis assays
Cell vitality was detected using Cell Counting Kit-8 (CA1210, Solarbio, Beijing, China). According to the experimental protocol, cells were cultured in 96-well plates for 24 h, and then CCK-8 reagent was added at 100 uL per well. One hour later, the absorbance of culture medium was analyzed by microplate spectrophotometer. In addition, cell proliferation activity was also measured by Cell-Light™ EdU Apollo®488 Cell Tracking Kit (RIBOBIO, Guangzhou, China). After pre-cultured for 24 h in 96-well plates, cells were cultured continuously for another 2 h in new media supplemented with 50 µmol/L EdU reagent. Then cells were fixed by 4% paraformaldehyde, permeabilized by 0.2% Trutib X-100, and fluorescently-tagged with Hoechst3342 using nucleus staining methods. The newly proliferated cells were visualized by an Apollo reaction system. Cell proliferation rate was analyzed by ImageJ (v1.51h, National Institutes of Health, Bethesda, MD, USA).
Apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection Kit (Gene Protein Link, Beijing, China) according to the manufacturer's protocol. Briefly, cells were stained with a combination of Annexin V-FITC and propidium iodide in darkness for 15 min at room temperature, and then analyzed by the flow cytometry system.
RNA isolation and qRT-PCR
Cells used for total RNA extraction obtained from 3 separate experiments (different batches of cells and on different days). Total RNA was extracted from cells using HiPure Total RNA Mini Kit (Magen, Beijing, China), and then reverse-transcribed into cDNA using a PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, Osaka, Japan). Synthesized cDNA was used for RT-qPCR analysis by employing a quantitative real-time PCR kit (Takara, Osaka, Japan) with an AJ qTOWER 2.2 Real-Time PCR system (Analytik Jena AG, Jena, Germany) according to standard procedures. All samples were measured in triplicate. The primers used in the experiment were listed in Additional file 4: Table S1. For the comparison of RyR1 and RyR3 expression in cells, the amplification efficiency of their primers was used firstly to rectify the qRT-PCR cycle number. GAPDH was used as an internal control. Relative gene expression level was calculated by 2−ΔΔCt method.
Protein extraction and western blot analysis
The relative abundances of proteins related to ER stress, MAPK signaling pathway, and apoptosis were determined by Western Blot. Cell samples were collected and lysed in RIPA buffer (Huaxingbio, Beijing, China) composed of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, and 0.1% SDS, plus a Halt protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The homogenate was centrifuged at 14,000 × g for 15 min at 4°C and the supernatant was isolated for Western Blot analysis. Protein concentrations were determined using a BCA Protein Assay Kit (Huaxingbio, Beijing, China). Equal amounts of protein (30 μg), together with a pre-stained protein ladder (Thermo Fisher Scientific, Waltham, MA, USA), were electrophoresed on SDS polyacrylamide gel, electro- transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, OH, USA), and blocked for 1 h in 5% non-fat dry milk at room temperature in Tris-Buffered saline and Tween-20 (TBST; 20 mmol/L Tris-Cl, 150 mmol/L NaCl, 0.05 % Tween 20, pH 7.4). Samples were incubated with corresponding primary antibodies overnight at 4°C. After washing with TBST (pH 7.4), membranes were incubated with the secondary antibody (DyLight 800, Goat Anti-Rabbit IgG). Protein bands were detected with the Odyssey Clx kit (LI-COR, Lincoln, NE, USA) and quantified using an Alpha Imager 2200 (Alpha InnoTec, CA, USA). Relative protein expression was calculated by taking GAPDH as an internal standard. Information of antibodies used for Western Blot in this study was listed in Additional file 5: Table S2.
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde (PFA)/PBS for 30 min. After the neutralization of excess formyl group by 2 mg/mL glycine, cells were permeabilized by 0.2% Trutib X-100 in PBS for 10 min. After blocked with 3% BSA/PBS, cells were incubated with primary antibody (anti-RyR1, 1:300, MA3-925, Thermo Fisher Scientific, Waltham, MA, IL, USA; anti-myosin, 1:300, M4276, Sigma-Aldrich, Louis, MO, USA) overnight and then incubated with Fluorescein-Conjugated secondary antibody (ZF-0311, ZSGB-BIO, Beijing, China) at 1:100. Nuclei were stained with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). Finally, myotubes were visualized by an inverted fluorescence microscope.
Chemical blockers of Ca2+ channels
Two kinds of chemical blocker were used for the experimental treatments. DAN (Stock solution: 200 mM in DMSO, HY-12542A, MedChemExpress, South Brunswick, NJ, USA) was added as a final concentration of 10 μΜ in culture media. THA (ab120286), purchased from Abcam (Cambridge, UK), was used at a final concentration of 100 nM in culture media. Equal amounts of vehicle (DMSO) were used as the control. During the proliferating period, myoblasts were treated with chemical blocker for 48 h and then collected for further analysis. Upon myogenic induction, myoblasts were treated with chemical blocker for 5 days and then used for mRNA extraction and immunocytochemistry. Each treatment was conducted in three independently repeated experiments.
Small interfering RNA transfection
RNA interference of RyR1 (mouse, gene ID: 20190) was performed using a 21-base pair small interfering RNA (siRNA) duplex (designed and synthesized by IBSBIO, Shanghai, China). The sense strand nucleotide sequence for RyR1 siRNA was 5’-CCUGCUCUAUGAACUUCUAGC-3’ (sense strand) and 5’-UAGAAGUUCAUAGAGCAGGUU-3’ (anti-sense strand). A scrambled siRNA (siControl, sense strand: 5’-UUCUCCGAACGUGUCACGUTT-3’, anti-sense strand: 5’-ACGUGACACGUUCGGAGAATT-3’) with the same nucleotide composition as RyR1 siRNA but lacks significant sequence homology to the RyR1 was also designed as a negative control. Briefly, myoblasts were plated in a cell culture dish for 24 h, and then transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol with 100 nM siRNA. After transfection for 24 h, myogenic differentiation was induced in cells.
CRISPR/Cas9 gene-editing
Gene-edited myoblasts with RyR1-knockout were generated via the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system. The plasmid vectors expressing Cas9 protein and guide-RNA (gRNA) were designed and synthesized by Syngentech (Beijing, China). One gRNA among the three gRNA targeting RyR1was selected for using in further study according to their shearing efficiency. The sequences of gRNA-1, gRNA-2, and gRNA-3 are as follows: 5’-GGCGATGATCTCTATTCTTA-3’, 5’-TACAGCCCCTACCCCGGAGG-3’, and 5’-AGCTCAGGCCACCCACCTGA-3’, respectively. The transient transfection of CRISPR-Cas9 vectors was achieved by electroporation using Nucleofector Program B-032 (VCA-1003, Lonza, Basel, Switzerland). Infected myoblasts were selected by incubation with 200 μg/mL hygromycin for 1 week. The stable RyR1-knockout cell line was obtained through single cell clone techniques. Genotype identification and verification of putative off-target sites (Additional file 6: Table S3) were conducted via DNA-sequencing technology. The related primers were list in Additional file 7: Table S4.
Statistical analysis
Data were analyzed using t-test procedures of SAS software (Version 9.3, SAS Institute, Cary, NC, USA) and presented as mean ± S.E.M. The criterion for statistical significance was set at P < 0.05.