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2+ 2+ Background: Cytosolic Ca plays vital roles in myogenesis and muscle development. As a major Ca release channel of endoplasmic reticulum (ER), ryanodine receptor 1 (RyR1) key mutations are main causes of severe congenital myopathies. The role of RyR1 in myogenic differentiation has attracted intense research interest but remains unclear. Results: In the present study, both RyR1-knockdown myoblasts and CRISPR/Cas9-based RyR1-knockout myoblasts were employed to explore the role of RyR1 in myogenic differentiation, myotube formation as well as the potential mechanism of RyR1-related myopathies. We observed that RyR1 expression was dramatically increased during the 2+ late stage of myogenic differentiation, accompanied by significantly elevated cytoplasmic Ca concentration. Inhibition of RyR1 by siRNA-mediated knockdown or chemical inhibitor, dantrolene, significantly reduced cytosolic 2+ 2+ Ca and blocked multinucleated myotube formation. The elevation of cytoplasmic Ca concentration can effectively relieve myogenic differentiation stagnation by RyR1 inhibition, demonstrating that RyR1 modulates 2+ 2+ myogenic differentiation via regulation of Ca release channel. However, RyR1-knockout-induced Ca leakage led to the severe ER stress and excessive unfolded protein response, and drove myoblasts into apoptosis. 2+ Conclusions: Therefore, we concluded that Ca release mediated by dramatic increase in RyR1 expression is required for the late stage of myogenic differentiation and fusion. This study contributes to a novel understanding of the role of RyR1 in myogenic differentiation and related congenital myopathies, and provides a potential target for regulation of muscle characteristics and meat quality. 2+ Keywords: Apoptosis, Ca homeostasis, Endoplasmic reticulum stress, Myoblast fusion, Myogenic differentiation, RyR1 knockout * Correspondence: firstname.lastname@example.org Kai Qiu and Yubo Wang contributed equally to this work. State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 2 of 14 Introduction [24–26], and roles of ER stress and UPR pathways in Ryanodine receptor 1 (RyR1), located on the endoplas- skeletal muscle health and diseases receive increased re- mic/sarcoplasmic reticulum (ER/SR) membrane, is search attention . Therefore, we surmise that ER highly expressed in the skeletal muscle. It serves as a stress signaling is involved in RyR1-related muscle 2+ critical Ca channel in mediating intracellular flux of myopathies. 2+ Ca , triggering the contraction of the skeletal muscle. Myogenic differentiation and fusion play a pivotal role Various mutations or epigenetic changes in the RyR1 in the development and skeletal muscle maturation via gene have been demonstrated to associate with muscle forming mature multinuclear myofibers, impacting on myopathies including malignant hyperthermia and sev- meat quality in livestock . In this study, we employed eral congenital myopathies [1–4]. RyR1 crystal structure CRISPR/Cas9-based RyR1-KO myoblasts and siRNA- has been resolved using electron cryomicroscopy as a 6- mediated RyR1-knockdown myoblasts to explore the transmembrane ion channel with an EF-hand domain role of RyR1 in myogenesis and formation mechanism of 2+ for Ca -mediated allosteric gating and a huge cytoplas- RyR1-related myopathies. mic domain on top of each transmembrane domain [5– 7]. These studies contribute to the deep understanding Materials and methods of the structure, function and channel activity of RyR1. Cell culture and myogenic differentiation In particular, the impacts of two malignant Mouse skeletal myoblast C2C12 cells were purchased hyperthermia-associated mutations on the RyR1 3D from the National Infrastructure of Cell Line Resource structure were recently solved to gain insights into the in China. Proliferating myoblasts were maintained in pathogenesis [8, 9]. Diagnostic gene-sequencing has DMEM/high glucose medium (Hyclone, Logan, UT, been employed to avoid RyR1-related congenital myop- USA) supplemented with 10% FBS (Gibco, Carlsbad, athies, and several disease-modulating therapeutic strat- CA, USA) in a humidified CO incubator (5% CO , 2 2 egies and salvage therapies have been developed against 37 °C; HF90, Heal Force, Hongkong, China). For myo- RyR1-related myopathies [10–15]. genic differentiation, myoblasts with 80% ~ 90% conflu- 2+ It has been well known that cytoplasmic Ca mediates ence were induced by DMEM/high glucose medium myogenic differentiation and skeletal muscle develop- containing 2% horse serum (Hyclone, Logan, UT, USA). 2+ ment . As a major Ca release channel of endoplas- Myogenic cells of pigs (n = 3) were isolated using pre- 2+ mic reticulum (ER), RyRs-mediated Ca release plays a plate techniques from the skeletal muscle according to role in the morphogenesis of mammalian skeletal the protocol our lab established previously [29, 30]. The muscle, while most of RyR1 mutations present gain-of- cells were cultured in growth medium in a 100 mm dish function phenotype and result in leaking of the internal coated with collagen I (Sigma-Aldrich, Louis, MO, USA) 2+ Ca store . Notably, the proportion of pigs carrying at 37 °C and 5% CO . The growth medium was com- the single-base mutation of RyR1 unexpectedly increased posed of DMEM/F12 (Hyclone), 10% FBS (Gibco-BRL, during intensive breeding for lean meat in pigs, which Carlsbad, CA, USA), 2 mmol/L glutamine (Gibco-BRL), leads to the increased yield of abnormal meat character- and 5 ng/mL bFGF (Peptech, Burlington, MA, USA). As istics of pale, soft and exudative . However, the block for myogenic induction, cells were cultured for 5 d in of RyRs activity by ryanodine selectively retards fetal DMEM/F12 medium containing 2% horse serum myoblast differentiation, and lead to both physiological (Hyclone). and pathological consequences of muscle morphology 2+ and functions . It has been demonstrated that homo- Cellular Ca concentration measurement 2+ zygous RyR1-null mice died after birth and displayed Ca concentration in the cytoplasm or ER was mea- small limbs and abnormal skeletal muscle organization sured using flow cytometry. Briefly, cells were collected, [20, 21]. Therefore, we hypothesize that RyR1 is not only washed with PBS (phosphate buffered saline) and HBSS involved in causing congenital myopathies, but impli- (Hanks balanced salt solutions) subsequently, and then cated in myogenesis and subsequent muscle develop- incubated in 5 μg/mL Fluo-3 acetoxymethyl ester (Cay- ment. Therefore, the mechanisms of RyR1 action in man, Ann Arbor, MI, USA) or Mag-fluo-AM (GENM myogenesis need to be elucidated. ED, Shanghai, China) for 30 min at 37 °C in dark. After Endoplasmic/sarcoplasmic reticulum is responsible for three washes with PBS supplemented with 1% FBS, cells proper folding, processing, and trafficking of proteins were resuspended in 200 μL PBS containing 1% FBS. 2+ and plays an important role in cellular Ca homeostasis. Flow cytometry was carried out immediately using a 2+ Alterations in cellular Ca dynamics directly trigger ER FACS Calibur Cytometer and Image Cytometry software stress and activate the unfolded protein response (UPR) (BD, Franklin, NJ, USA). Calcium-bound Fluo-3 or Mag- [22, 23]. ER stress and resultant UPR modulation are im- fluo-AM has an emission maximum of 526 nm which plicated in various human diseases including sarcopenia was quantified by excitation with a 488-nm laser and Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 3 of 14 signals were collected using a 530/30 nm band-pass fil- were measured in triplicate. The primers used in the ex- ter. Each sample generated 20,000 live gated events. periment were listed in Table S1. To compare the Debris, multicellularity, and dead cells were excluded by mRNA expression of RyR1 and RyR3 in cells, the ampli- forward scatter (FSC) and side scatter. For detecting dy- fication efficiency of their primers was used to rectify 2+ namic change of Ca concentration of unfused cells the qRT-PCR cycle number. GAPDH (glyceraldehyde-3- during myogenic differentiation, a blank control com- phosphate dehydrogenase) was used as an internal con- bined with a house-keeping control (the proliferative trol. Relative gene expression level was calculated by −ΔΔCt C2C12 cells) was used to correct the deviation caused by 2 method . the loaded indicator amount and the voltage used in each measurement. Mean fluorescence intensity was de- Protein extraction and western blot analysis termined from the entire cell population and then ad- The relative abundances of proteins concerning ER justed by relative cell size calculated according to FSC to stress, MAPK signaling pathway, and apoptosis were de- 2+ represent Ca concentration. termined by Western Blot. Cell samples were collected and lysed in RIPA buffer (Huaxingbio, Beijing, China) Cell viability and apoptosis assays composed of 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L Cell viability was measured using Cell Counting Kit-8 NaCl, 1% NP-40, and 0.1% SDS, plus a Halt protease/ (CA1210, Solarbio, Beijing, China) according to the phosphatase inhibitor cocktail (Thermo Fisher Scientific, manufacturer’s guideline. Briefly, cells were cultured in Waltham, MA, USA). The homogenate was centrifuged 96-well plates for 24 h, and then CCK-8 reagent was at 14,000 × g for 15 min at 4 °C and the supernatant was added at 100 μL per well. One hour later, the absorbance isolated for Western Blot analysis. Protein concentra- of culture medium was analyzed by microplate spectro- tions were determined using a BCA Protein Assay Kit photometer. In addition, cell proliferation activity was (Huaxingbio, Beijing, China). Equal amounts of protein also measured by Cell-Light™ EdU Apollo®488 Cell (30 μg), together with a pre-stained protein ladder Tracking Kit (RIBOBIO, Guangzhou, China). After pre- (Thermo Fisher Scientific, Waltham, MA, USA), were cultured for 24 h in 96-well plates, cells were cultured electrophoresed on SDS polyacrylamide gel, electro- continuously for another 2 h in new media supple- transferred to a polyvinylidene difluoride membrane mented with 50 μmol/L EdU reagent. Then cells were (Millipore, Bedford, OH, USA), and blocked for 1 h in fixed by 4% paraformaldehyde, permeabilized by 0.2% 5% non-fat dry milk at room temperature in Tris- Trutib X-100, and fluorescently-tagged with Buffered saline and Tween-20 (TBST; 20 mmol/L Tris- Hoechst3342 using nucleus staining methods. The newly Cl, 150 mmol/L NaCl, 0.05% Tween 20, pH 7.4). Samples proliferated cells were visualized by an Apollo reaction were incubated with corresponding primary antibodies system. Cell proliferation rate was analyzed by ImageJ overnight at 4 °C. After washing with TBST (pH 7.4), (v1.51h, National Institutes of Health, Bethesda, MD, membranes were incubated with the secondary antibody USA). (DyLight 800, Goat Anti-Rabbit IgG). Protein bands Apoptosis was tested using an Annexin V-FITC/PI were detected with the Odyssey Clx kit (LI-COR, Lin- Apoptosis Detection Kit (Gene Protein Link, Beijing, coln, NE, USA) and quantified using an Alpha Imager China) according to the manufacturer’s protocol. Briefly, 2200 (Alpha InnoTec, CA, USA). GAPDH was taken as cells were stained with a combination of Annexin V- an internal standard to calculate relative protein expres- FITC and propidium iodide in darkness for 15 min at sion. Antibodies used for Western Blot in this study room temperature, and then analyzed by the flow cy- were listed in Table S2. tometry system. RNA isolation and qRT-PCR Immunocytochemistry Cells used for total RNA extraction obtained from 3 sep- Cells were fixed with 4% paraformaldehyde (PFA)/PBS arate experiments (different batches of cells and on dif- for 30 min. After the neutralization of excess formyl ferent days). Total RNA was extracted from cells using group by 2 mg/mL glycine, cells were permeabilized by HiPure Total RNA Mini Kit (Magen, Beijing, China), 0.2% Trutib X-100 in PBS for 10 min. After blocked with and then reverse-transcribed into cDNA using a Prime- 3% BSA/PBS, cells were incubated with primary anti- Script™ RT reagent Kit with gDNA Eraser (Takara, body (anti-RyR1, 1:300, MA3–925, Thermo Fisher Scien- Osaka, Japan). Synthesized cDNA was used for RT- tific, Waltham, MA, IL, USA; anti-myosin, 1:300, qPCR analysis by employing a quantitative real-time M4276, Sigma-Aldrich, Louis, MO, USA) overnight and PCR kit (Takara, Osaka, Japan) with an AJ qTOWER 2.2 then incubated with Fluorescein-Conjugated secondary Real-Time PCR system (Analytik Jena AG, Jena, antibody (ZF-0311, ZSGB-BIO, Beijing, China) at 1:100. Germany) according to standard procedures. All samples Nuclei were stained with DAPI (Thermo Fisher Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 4 of 14 Scientific, Waltham, MA, USA). Finally, myotubes were CAGGCCACCCACCTGA-3′, respectively. When the visualized by an inverted fluorescence microscope. myoblasts were grown to 80% ~ 90% confluency, 1 × 10 cells were collected and transfected with 2 μg CRISPR- 2+ Chemical blockers of Ca channels Cas9 vectors by electroporation using Nucleofector Pro- Two kinds of chemical blocker were employed in the ex- gram B-032 (VCA-1003, Lonza, Basel, Switzerland). In- perimental treatments. Dantrolene (DAN) (Stock solu- fected myoblasts were selected by incubation with tion: 200 mmol/L in DMSO, HY-12542A, 200 μg/mL hygromycin for 1 week. The stable RyR1- MedChemExpress, South Brunswick, NJ, USA), an in- knockout cell line was obtained through single cell clone hibitor of RyR1, was added as a final concentration of techniques. Genotype identification and verification of 10 μmol/L in culture media. Thapsigargin (THA, putative off-target sites (Table S3) were conducted via ab120286, Abcam, Cambridge, UK), an ER stress- DNA-sequencing technology. The related primers were inducing agent, was used at a final concentration of 100 list in Table S4. nmol/L in culture media. Equal amounts of vehicle (DMSO) were used as the control. During the proliferat- Statistical analysis ing period, myoblasts were treated with chemical Student’s t-test was used between two groups; one-way blockers, respectively, for 48 h and then collected for fur- or two-way ANOVA with Tukey’s test was used among ther analysis. Upon myogenic induction, myoblasts were multiple groups. Data are presented as mean ± SEM. treated with chemical blockers, respectively, for 5 d and The criterion for statistical significance was set at P < then used for mRNA extraction and immunocytochem- 0.05. istry. Each treatment was conducted in three independ- ently repeated experiments. Results 2+ Small interfering RNA transfection Cytoplasmic Ca dynamics and expression patterns of 2+ RNA interference of RyR1 (mouse, gene ID: 20190) ex- Ca channels during myogenic differentiation pression was performed using a 21-base pair small inter- Upon myogenic induction, myoblasts gradually fering RNA (siRNA) duplex (designed and synthesized expressed plenty of myosin from the d 0 to 6 (Fig. S1A). by IBSBIO, Shanghai, China). The sense strand nucleo- During the entire period (d 1–5) of myogenic differenti- 2+ tide sequence for RyR1 siRNA was 5′-CCUGCUCUAU ation, cytoplasmic Ca concentration (labeled by Fluo- GAACUUCUAGC-3′ (sense strand) and 5′-UAGAAG 3) of myoblasts was significantly increased (Fig. S1B and UUCAUAGAGCAGGUU-3′ (anti-sense strand). A C). Myf5 (myogenic factor 5) and MyoD1 (myogenic dif- scrambled siRNA (siControl, sense strand: 5′- ferentiation 1) expression were significantly increased on UUCUCCGAACGUGUCACGUTT-3′, anti-sense d 2 relative to d 0, then sharply decreased on d 6 and 8 strand: 5′-ACGUGACACGUUCGGAGAATT-3′) with and even lower than the initial level before myogenic in- the same nucleotide composition as RyR1 siRNA but duction (Fig. 1A and B). Meanwhile, MyoG (myogenin) lacks significant sequence homology to the RyR1 was expression showed continuous increase and reached a also designed as a negative control. Briefly, myoblasts plateau on d 4 (Fig. 1C). 2+ were plated in a 100 mm cell culture dish for 24 h, and As for Ca transporters, CAV1.1 (also known as CACN then transfected with 100 nmol/L siRNA using 16 μL Li- A1S, calcium voltage-gated channel subunit alpha1 S), pofectamine 3000 (Invitrogen, Carlsbad, CA, USA) in CRACR2B (calcium release activated channel regulator each dish. After transfection for 24 h, myogenic differen- 2B), ITPR1 (inositol 1,4,5-trisphosphate receptor type 1), tiation was induced in cells. and ORAI2 (ORAI calcium release-activated calcium modulator 2) whose expression patterns showed similar CRISPR/Cas9 gene-editing with Myf5 and MyoD1 (Fig. 1D-G). The expression pat- Gene-edited myoblasts with RyR1-knockout were gener- terns of RyR1 and STIM1 (stromal interaction molecule 1) ated via the Clustered Regularly Interspaced Short Palin- resembled MyoG well (Fig. 1H and I). Notably, RyR1 dromic Repeats (CRISPR)-CRISPR associated protein 9 mRNA expression increased more than 100-fold in myo- (Cas9) system. The plasmid vectors expressing Cas9 pro- blastic C2C12 cells upon myogenic induction. Consist- tein and guide-RNA (gRNA) were designed and synthe- ently, RyR1 protein expression was significantly increased sized by Syngentech (Beijing, China). One gRNA among upon myogenic induction (Fig. S1Dand E).In addition, 2+ the three gRNA targeting RyR1 was selected for using in the mRNA expression of ATP2A2 (ATPase ER/SR Ca further study according to their shearing efficiency. The transporting 2; Fig. 1J), ATP2B (ATPase plasma mem- 2+ sequences of gRNA-1, gRNA-2, and gRNA-3 are as fol- brane Ca transporting 1; Fig. 1K) and CRACR2A (cal- lows: 5′-GGCGATGATCTCTATTCTTA-3′,5′- cium release activated channel regulator 2A; Fig. 1L) were TACAGCCCCTACCCCGGAGG-3′, and 5′-AGCT significantly increased in myoblastic C2C12 cells on d 2 Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 5 of 14 Fig. 1 Relative mRNA expression of Myf5 (A), MyoD1 (B), MyoG (C), CAV1.1 (D), CRACR2B (E), ITPR1 (F), ORAI2 (G), RyR1 (H), STIM1 (I) ATP2A2 (J), ATP2B (K), and CRACR2A (L), on d 0, 2, 4, 6, and 8 during myogenic differentiation of C2C12 cells (n = 3). The data are presented as the mean ± SEM. *Represents significant difference with the value on d 0 (P < 0.05) during myogenic induction and maintained a plateau for Consistently, we also observed that RyR1 mRNA expression subsequent days. showed almost a 25-fold increase during myogenic differen- Since RyR3 mRNA expression level was less than 1/10 tiation, while the mRNA expression of RyR3 was not chan- that of RyR1 in C2C12 cells, the increase in RyR3 mRNA ex- ged in myogenic cells of pigs (Fig. S2C-E). pression level during myogenic induction was far below that 2+ of RyR1 (Fig. S2A and B). From this view, therefore, RyR1 RyR1-mediated elevation of cytoplasmic Ca rather than RyR3, another member of RYRs expressed in concentration is indispensable for myogenesis 2+ skeletal muscle, may exert a major role in mediating myo- As shown in Fig. 2A and B, cytoplasmic Ca concentra- genesis of myoblastic C2C12 cells can be attributed to. tion was significantly decreased upon treatment with Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 6 of 14 2+ Fig. 2 The effects of RyR1 inhibition on myogenic differentiation of C2C12 myoblasts. (A-B) The concentration of cytoplasmic Ca labeled by 2+ Fluo-3 treated with Dantrolene (DAN, 10 μmol/L, RyR1 inhibitor) for 48 h (n = 5). (C-D) The concentration of cytoplasmic Ca labeled by Fluo-3 in C2C12 cells transfected with siRyR1 for 72 h (n = 5). (E-F) The proteins expression of RyR1 in C2C12 cells after myogenic induction for 5 d with siRyR1 transfection (n = 3). (G-H) Immunostaining with myosin antibody of C2C12 cells after myogenic induction for 5 d with DAN treatment or siRyR1 transfection (n = 6). (I) Relative mRNA expression of related genes in C2C12 cells treated with DAN on d 2 (Myf5 and MyoD1) and 4 (MyoG and Mymk) of myogenic differentiation (n = 3). (J) Relative mRNA expression of RyR1, Myf5, MyoD1, MyoG, and Mymk in C2C12 cells transfected with siRyR1 for 72 h (n = 3). *Represents significant difference between the two groups (P < 0.05) dantrolene (DAN), an inhibitor of RyR1, which blocks induction, DAN effectively blocked MyoD1 expression 2+ the release of Ca from SR/ER . Moreover, RyR1 on d 2 and both MyoG and Mymk (myomaker, myoblast knockdown by siRNA significantly also decreased cyto- fusion factor) expression on d 4 (Fig. 2I). At the d 4 dur- 2+ plasmic Ca concentration of C2C12 cells (Fig. 2C and ing myogenic induction, siRyR1-knockdown significantly D). Consistently, the protein expression of RyR1 was ef- reduced RyR1, MyoG, and Mymk expression without im- fectively suppressed by siRNA interference (Fig. 2E and pact on Myf5 and MyoD1 expression (Fig. 2J). F). Functional constraints of RyR1 by either DAN or Thapsigargin (THA) treatment significantly increased 2+ siRyR1 dramatically inhibited the formation of multinu- cytoplasmic Ca concentration (Fig. 3Aand B), but did cleated myotubes (Fig. 2G and H). During myogenic not influence the mRNA expressions of myogenic-specific Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 7 of 14 2+ 2+ Fig. 3 THA-increased cytoplasmic Ca concentration was independent of RyR1. (A-B) The concentration of cytoplasmic Ca labeled by Fluo-3 in C2C12 cells treated with thapsigargin (THA) for 48 h (n = 3). (C) Relative mRNA expression of related genes in C2C12 cells treated with THA on d 2(Myf5 and MyoD1) and 4 (MyoG and Mymk) during myogenic differentiation (n = 3). (D-E) Immunostaining with RyR1 antibody of C2C12 cells after myogenic induction for 5 d treated with siRyR1 or THA (n = 5). The data are presented as the mean ± SEM. *Represents significant difference between the two groups (P < 0.05) −/− genes including Myf5, MyoD1, MyoG and Mymk (Fig. 3C). heterozygote of RyR1-knockout cells, named as RyR1 +/− RyR1-knockdown by siRNA interference was independent and RyR1 , were obtained by monoclonal cultivation of THA treatment (P <0.01 for siRNA treatment, Fig. 3D and identified via gene sequencing on the target site and and E). On d 4 during myogenic induction, siRyR1- putative off-target sites of gRNA-3. Relative to the wild −/− knockdown significantly increased Myf5 and MyoD1 type cells (WT), RyR1 showed the higher concentra- 2+ mRNA expression, while THA effectively eliminated alter- tion of Ca in the cytoplasm, but the lower level of 2+ −/− +/− ations in Myf5 and MyoD1 mRNA expressions induced by Ca in the ER (Fig. 5A-C). In RyR1 or RyR1 , the siRyR1-knockdown (P < 0.05 for siRNA treatment × THA mRNA expression of ATP2A2, ATP2B, CRACR2B, and treatment, Fig. 4A). Accordingly, myotube formation was ORAI1 was significantly increased, while the mRNA ex- significantly inhibited by siRyR1-knockdown, which was ef- pression of CAV1.1 and ORAI2 was decreased relative to fectively recovered by THA (P <0.01 for siRNA treatment WT (Fig. 5D). Cell proliferation (the proportion of EdU −/− +/− × THA treatment, Fig. 4B-D). cells) of RyR1 or RyR1 significantly declined rela- tive to WT (Fig. 5E and F), which was also demonstrated Effects of RyR1-knockout on cell proliferation and by the CCK-8 test (Fig. 5G). The mRNA expression of differentiation Myf5, MyoD1,and MyoG was significantly increased in −/− +/− RyR1 was successfully knockout by CRISPR/Cas-9 gene RyR1 or RyR1 relative to WT (Fig. 5H). editing system targeting Exon 18 of RyR1 via gRNA-3 which demonstrated the highest shearing efficiency RyR1-knockout triggered apoptosis among three designed gRNAs and resulted in the frame- On d 2 during myogenic induction, apoptosis instead of shift mutation of RyR1 (Fig. S3). Homozygote and myotube formation was significantly accelerated in both Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 8 of 14 2+ Fig. 4 The elevation of cytoplasmic Ca concentration is required for myoblast fusion into myotubes. (A) Relative mRNA expression of Myf5, MyoD1, MyoG, and Mymk after the treatments with siRyR1 or thapsigargin (THA) after myogenic induction for 4 d (n = 3). (B-C) Immunostaining with myosin antibody after myogenic induction for 5 d treated with siRyR1 or THA (n = 4). (D) The relative myotube area in B. The data are presented as the mean ± SEM. *Represents significant difference between the two groups (P < 0.05) −/− +/− RyR1 and RyR1 relative to WT (Fig. 6A and B). RyR1 protein expression pattern during myogenic The protein expression level of cyclin D1 and caspase-3 differentiation of myoblastic C2C12 cells provides a −/− +/− was significantly lowered in RyR1 or RyR1 , but the well-controlled model for investigations of RyR1 expression of cleaved caspase-3 was significantly in- function during myogenic differentiation . In the 2+ creased as compared with WT (Fig. 6C and D). current study, we observed that cytoplasmic Ca ER stress level of cells was evaluated, and both phos- concentration of C2C12 myoblasts was significantly phorylated and total protein expression of IRE1α and elevated during myogenic differentiation, which was −/− PERK were dramatically elevated in RyR1 and consistent with our previouslystudy in primarymyo- +/− 2+ RyR1 , while phosphorylation of EIF2α was signifi- genic cells of pigs . Cytoplasmic Ca dynamics cantly decreased (Fig. 6E and F). As to several apoptosis- is tightly regulated by various channels and trans- related proteins, the protein abundance of CHOP (also ports in cells . Relative to the extracellular −/− 2+ known as DDIT3), caspase-9, and caspase-12 in RyR1 matrix and ER, cytoplasmic Ca concentration is +/− or RyR1 was significantly increased relative to WT, maintained at very low levels (10–100 nmol/L) under 2+ while the protein expression of ERP44 and HSPA5 was resting conditions. Ca is released from the ER, the 2+ not influenced (Fig. 6G and H). main storage site of intracellular Ca ,through the transmembrane channels RyR1 and ITPR1 [39, 40]. 2+ Discussion Cytoplasmic Ca influx from extracellular matrix Skeletal muscle mass is maintained by myogenic differ- occurs through plasma membrane channels, such as entiation of myogenic progenitors and subsequent myo- CAV1, CAV2, and CAV3 . In maintaining the 2+ 2+ blast fusion , in which cytosolic Ca dynamics plays resting state, excessive amounts of cytoplasmic Ca 2+ a vital role . Particularly, myotube formation requires re-accumulates in the ER by SR/ER Ca -ATPase 2+ net Ca influx into myoblasts [34–36]. RyR1, serving as (SERCA, also called ATP2A) oris expulsedin 2+ 2+ a major Ca release channel of ER, has attracted the the external milieu by plasma membrane Ca - most intense research interests. However, the role of ATPase (PMCA, also called ATP2B) [43, 44]. Fur- RyR1 in myogenesis remains unclear. thermore, store-operated calcium entry (SOCE), Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 9 of 14 2+ 2+ Fig. 5 The effects of RyR1-knockout on cellular Ca dynamics and differentiation of C2C12 myoblasts. (A-C) The concentration of Ca in cytoplasm (labeled by Fluo-3, n = 3) and endoplasmic reticulum (labeled by Mag-fluo-AM, n = 5) of RyR1-KO cells. (D) The effect of RyR1-KO on 2+ the mRNA expression of Ca channels (n = 3). (E-G) Proliferation and viability of RyR1-KO cells measured by EdU staining or CCK-8 test (n = 10). (H) Relative mRNA expression of Myf5, MyoD1, and MyoG in RyR1-KO cells (n = 3). (I-J) The expression of proteins related to MAPK signaling in −/− +/− RyR1-KO cells (n = 3). RyR1 and RyR1 represent homozygote and heterozygote of RyR1-knockout respectively. The data are presented as the mean ± SEM. *Represents significant difference between the two groups (P < 0.05) 2+ 2+ mediated by STIM (ER Ca sensors), activates major Ca release channel of ER, the dramatic increase CRAC and ORAI channels located at plasma mem- of RyR1 expression should be responsible for the signifi- 2+ 2+ brane to maintain cellular Ca homeostasis [45, 46]. cant elevation of cytosolic Ca concentration. There- It has been well known that lineage commitment and fore, we deduced that dramatic increase of RyR1 differentiation of myoblasts are governed by the pro- expression is required for the myoblast fusion at later grammed expression and functional activation of myo- stage of myogenesis. In addition, CAV1.1 is a physio- genic regulatory transcription factors (MRFs)[47, 48]. In logical activator of RyR1 in the excitation–contraction this study, we discovered RyR1 mRNA expression was coupling of skeletal muscle . However, in this study, increased more than 100-fold in C2C12 cells and almost the expression of CAV1.1 was not significantly increased 25-fold in myogenic cells of pigs during myogenic differ- as RyR1 during myogenic differentiation, which indicated entiation along with the normal expression pattern of that RyR1 expression in myogenic cells is independent of 2+ Ca transporters as well as MRFs during myogenic dif- CAV1.1. ferentiation. In addition, the expression patterns of RyR1 Thapsigargin, an inhibitor of SERCA, which mediated 2+ showed similar with MyoG, which was increased in the the reuptake of cytoplasmic Ca into the sarcoplasmic 2+ late stage of myogenesis. It strongly indicates that RyR1 reticulum, was used to increase cytoplasmic Ca con- is not necessary for initial myogenic commitment, but centration. In the current study, the dose of THA was more for later stage of myogenic differentiation. far below that used in previous studies [50, 51] in order Notably, RyR1 restriction via either DAN treatment or to keep cell viability from the adverse effect of THA, and siRNA interference significantly decreased cytoplasmic guaranteed that THA did not affect the efficiency of 2+ Ca concentration, and then blocked formation of siRyR1-knockdown. As a result, THA treatment signifi- 2+ multi-nuclei myotubes and expression of MRFs.Asa cantly increased cytoplasmic Ca concentration but did Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 10 of 14 Fig. 6 Endoplasmic reticulum stress-associated apoptosis in C2C12 cells was induced by RyR1-knockout (n = 3). (A-B) Apoptosis detection of RyR1-null cells through flow cytometry. (C-D) The protein expression of Cyclin D1, caspase-3 (CASP3), and cleaved-CASP3 in RyR1-KO cells. (E-H) −/− +/− The expression of proteins related to ER stress or ER stress-induced apoptosis in RyR1-KO cells. RyR1 and RyR1 represent homozygote and heterozygote of RyR1-knockout, respectively. The data are presented as the mean ± SEM. *Represents significant difference between the two groups (P < 0.05) Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 11 of 14 not affect expression of myogenic specific genes. How- present study, the total and phosphorylated protein ex- ever, myotube formation was significantly inhibited by pressions of IRE1α and PERK were sharply increased in siRyR1-knockdown, but recovered by THA treatment, RyR1-KO cells, indicating that RyR1-knockout activated accompanied by the expression of MRFs. Therefore, serious ER stress. Furthermore, aggravated ER stress sig- 2+ RyR1-mediated elevation of cytoplasmic Ca concentra- nificantly increased expression of CHOP, caspase-9, and tion was indispensable for myotubes formation. caspase-12. Meanwhile, the positive effects of ERP44 and To further explore the roles of RyR1 in myogenic dif- HSPA5 against ER stress were not enhanced in RyR1- ferentiation and subsequent myoblast fusion, a model of KO myoblasts. In addition, phosphorylation levels of RyR1-KO myoblasts by CRISPR/Cas9 gene-editing was JNK and Erk1/2, whose activations are beneficial for the 2+ employed in this study. The RyR1 Ca release channel resistance to ER stress-induced apoptosis [59, 60], were is composed of macromolecular complexes consisting of also significantly reduced in RyR1-KO cells. Therefore, a homotetramer of 560-kDa RyR1 subunits that form we deduced that RyR1 deletion led to serious ER stress scaffolds for proteins that regulate channel function in- and excessive UPR than the tolerance thresholds of cells, cluding protein kinase A (PKA) and the phosphodiester- and accounted for the apoptosis of RyR1-KO myoblasts ase PDE4D3 (both of which are targeted to the channel upon myogenic induction. via the anchoring protein mAKAP), PP1 (targeted via In summary, dramatic increase in RyR1 expression is 2+ spinophilin), and calstabin1 (FKBP12) [52, 53]. In con- indispensable for myogenesis, and RyR1-mediated Ca trast to the result of siRNA interference, we observed release plays a critical role in myoblast fusion at the later 2+ 2+ higher concentration of Ca in the cytoplasm of RyR1- stage of myogenesis. RyR1-KO led to cytoplasmic Ca KO myoblasts, which suggested that considering the role elevation and enhanced myogenic differentiation poten- of RyR1 as a calcium channel, RyR1-KO could differ tial, while serious ER stress and excessive UPR over the 2+ from siRyR1 knockdown in terms of controlling Ca dy- tolerance thresholds of cells resulted by RyR1-KO trig- namics between ER and cytoplasm. The deletion of gered the process of apoptosis of myoblasts upon myo- RyR1 resulted in the dysfunction of the protein complex, genic induction. This study contributes to a novel 2+ losing the control of Ca flowing out of the ER and ac- understanding of the role of RyR1 in muscle develop- 2+ celerated Ca flooding into the cytoplasm from the ER, ment and related congenital myopathies, and provides a which also distorted the expression patterns of other potential target for regulation of muscle characteristics 2+ Ca transporters. This observation is consistent with and meat quality. previous studies that demonstrated both over-activation 2+ Abbreviations and mutations of RyR1 resulted in leakage of Ca from 2+ ATP2A2: ATPase ER/SR Ca transporting 2; ATP2B: ATPase plasma membrane 2+ the SR [13, 54]. Cytoplasmic Ca elevation directly acti- 2+ Ca transporting 1; Cas9: CRISPR associated protein 9; CASP3: Caspase-3; vated ER stress [22, 23], and has a strong influence on CAV1.1: Calcium voltage-gated channel subunit alpha1 S; CHOP: C/EBP homologous protein; CRACR2A: Calcium release activated channel regulator differentiation through oxidative signaling and G0/G1 2A; CRACR2B: Calcium release activated channel regulator 2B; cell cycle arrest . In the current study, expression of CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; Cyclin D1 was abolished and cell viability was decreased DAN: Dantrolene; DDIT3: DNA-damage inducible transcript 3; eIF2α: Eukaryotic translation initiation factor 2α; ER: Endoplasmic reticulum; in RyR1-KO cells, demonstrating that cell cycle and cell Erk1/2: Extracellular regulated protein kinases; ERP44: ER protein 44; viability were suppressed during the enhanced ER stress FKBP12: Calstabin1; FSC: Forward scatter; gRNA: Guide-RNA; GAPD 2+ caused by RyR1-KO mediated cytoplasmic Ca H: Glyceraldehyde-3-phosphate dehydrogenase; HSPA5: Heat shock protein 5; ITPR1: Inositol 1,4,5-trisphosphate receptor type 1; JNK: Stress-activated elevation. protein kinase/Jun-amino-terminal kinase; MRFs: Myogenic regulatory ER stress signaling gives rise to apoptosis . In the transcription factors; Myf5: Myogenic factor 5; Mymk: Myomaker; present study, myogenic differentiation potential of MyoD1: Myogenic differentiation 1; MyoG: Myogenin; ORAI2: ORAI calcium release-activated calcium modulator 2; OTS: Off-target sites; PKA: Protein RyR1-KO myoblasts reflected by MRFs expression was kinase A; RyR1: Ryanodine receptor 1; SOCE: Store-operated calcium entry; dramatically enhanced. However, the differentiation SR: Sarcoplasmic reticulum; STIM1: Stromal interaction molecule 1; TBST: Tris- process of RyR1-KO myoblasts was interrupted by apop- Buffered saline and Tween-20; THA: Thapsigargin; UPR: Unfolded protein response; WT: Wild type tosis, which indicated that RyR1 knockout make myo- blasts too fragile to undertake the stress of myogenic induction. UPR was a protective response for cells under Supplementary Information The online version contains supplementary material available at https://doi. stress, however, excessive or prolonged UPR can cause org/10.1186/s40104-021-00668-x. apoptosis . Caspase-3, belonging to a highly con- served family of cysteinyl aspartate-specific proteases, is 2+ Additional file 1: Fig. S1 Cytoplasmic Ca concentration of C2C12 an essential regulator of apoptosis. In the current study, cells during myogenic differentiation. (A) Immunostaining with myosin cleaved activation of caspase-3 was significantly stimu- antibody on d 0, 2, 4, and 6 during myogenic differentiation. (B) 2+ Cytoplasmic Ca signals of unfused C2C12 cells labeled by Fluo-3 on d lated in RyR1-KO cells, which was supported by a previ- 0–5 during myogenic differentiation (n = 3). (C) Quantitative results of ous study of RyRs-mediated ER stress . In the Qiu et al. Journal of Animal Science and Biotechnology (2022) 13:9 Page 12 of 14 2+ Research Center of Biological Feed, Institute of Feed Research, Chinese cytoplasmic Ca signals. (D-E) The proteins expression of RyR1 in C2C12 Academy of Agricultural Sciences, Beijing 100081, China. cells during myogenic differentiation (n = 3). GM: growth medium, repre- senting cells cultured in growth medium before myogenic induction; Received: 22 September 2021 Accepted: 9 December 2021 DM: differentiation medium, representing cells on d 4 during myogenic differentiation cultured in differentiation medium. The data are presented as the mean ± SEM. *Represents significant difference with the value on d 0(P < 0.05). References Additional file 2: Fig. S2 (A) The mRNA expression of RyR3 during 1. Maclennan DH, Duff C, Zorzato F, Fujii J, Phillips M, Korneluk RG, et al. 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Journal of Animal Science and Biotechnology – Springer Journals
Published: Feb 11, 2022
Keywords: Apoptosis; Ca2+ homeostasis; Endoplasmic reticulum stress; Myoblast fusion; Myogenic differentiation; RyR1 knockout
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