RyR1-mediated moderate endoplasmic reticulum stress is required for myogenic differentiation of myoblasts

Background Cytosolic Ca 2+ plays vital roles in myogenesis and muscle development. Key mutations of ryanodine receptor 1 (RyR1), a major Ca 2+ release channel of endoplasmic reticulum (ER), are main causes of severe congenital myopathies. The role of RyR1 in myogenic differentiation has attracted intense research interest, however, it remains unclear. Methods This study employed RyR1-knockdown myoblasts and CRISPR/Cas9-based RyR1-knockout myoblasts cells to explore the role of RyR1 in myogenic differentiation, myotube formation as well as the potential mechanism of RyR1-related myopathies. and related congenital potential target related tissue

RyR1 attracts intense interest in the eld of medicine because it is a Ca 2+ channel of great clinical signi cance. The crystal structure of RyR1 has been determined by electron cryomicroscopy to be a 6transmembrane ion channel with an EF-hand domain for Ca 2+ -mediated allosteric gating and a huge cytoplasmic domain on top of each transmembrane domain [10][11][12]. Ca 2+ signaling is important for myogenic gene expression and skeletal muscle differentiation [13]. RyRs-mediated Ca 2+ release plays a role in the histogenesis of mammalian skeletal muscle, and the block of RyRs selectively inhibits fetal myoblast differentiation [14]. Mutations in RyR1 that results in leaking of the internal Ca 2+ store can have both physiological and pathological consequences [15].
A naturally occurring single-base mutation of RyR1 closely was associated with malignant hyperthermia of pigs but has been particularly enriched in genetic selections for muscle growth rate and lean body mass [16]. Homozygous RyR1-null mice died after birth and displayed small limbs and abnormal skeletal muscle organization [17,18]. Presumably, RyR1 is not only associated with myopathies, but is implicated in myogenesis and subsequent muscle development. However, the mechanisms of RyR1 action in myogenesis have not been elucidated.
Alterations in cellular Ca 2+ dynamics directly trigger ER stress and activate the unfolded protein response (UPR) [19,20]. ER/SR is a membrane-bound organelle in mammalian cells that is responsible for proper folding, processing, and tra cking of proteins and also plays an important role in cellular Ca 2+ homeostasis. ER stress and UPR modulation are implicated in various human diseases including sarcopenia [21][22][23]. In recent decades, roles of ER stress and UPR pathways in skeletal muscle health and disease have received increased research attention [24]. In addition to induction of apoptosis, ER stress positively in uences myogenic differentiation and myo ber formation [25,26]. Therefore, we surmise that ER stress signaling is involved in RyR1-mediated muscle development.
Satellite cells and myoblasts play a pivotal role in the repair and maintenance of skeletal muscle by differentiating into mature myocytes [27]. In this study, we hypothesized that RyR1-mediated Ca 2+ dynamics delicately balance ER stress-induced apoptosis and myoblast differentiation. To test this theory, we employed RyR1-KO myoblasts established by CRISPR/Cas9 gene-editing and RyR1-weaken cells to explore the role of RyR1 in myogenic differentiation and formation mechanism of RyR1-related myopathies.

Cellular Ca 2+ concentration measurement
Ca 2+ concentration in the cytoplasm or ER was measured using ow 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-uo-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-uo-AM has an emission maximum of 526 nm which was quanti ed by excitation with a 488-nm laser and signals were collected using a 530/30 nm band-pass lter. 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 Ca 2+ 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 uorescence intensity was determined from the entire cell population and then adjusted by relative cell size calculated according to FSC to represent Ca 2+ 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 xed by 4% paraformaldehyde, permeabilized by 0.2% Trutib X-100, and uorescentlytagged 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). 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 signi cant sequence homology to the RyR1 was also designed as a negative control. Brie y, 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.

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 signi cance was set at P < 0.05.

Results
Cytoplasmic Ca 2+ dynamics and expression patterns of Ca 2+ channels during myogenic differentiation Upon myogenic induction, myoblasts gradually expressed plenty of myosin from the day 0 to 6 (Additional le 1: Figure S1A) and protein expression of RyR1 was signi cantly increased at day 6 (Additional le 1: Figure S1B, C). During the entire period (day 1-5) of myogenic differentiation, cytoplasmic Ca 2+ concentration (labeled by Fluo-3) of myoblasts was signi cantly increased (Additional le 1: Figure S1D, E).
Myf5 (myogenic factor 5) and MyoD1 (myogenic differentiation 1) expression were signi cantly increased on day 2 relative to day 0, then sharply decreased on day 6 and 8 and even lower than the initial level during myogenic induction ( Figure 1A, B). MyoG (myogenin) expression showed continuous increase and reached a plateau on day 4 ( Figure 1C).
As for Ca 2+ transporters, CAV1.1 (also known as CACNA1S, calcium voltage-gated channel subunit alpha1 S), CRACR2B (calcium release activated channel regulator 2B), ITPR1 (inositol 1,4,5-trisphosphate receptor type 1), and ORAI2 (ORAI calcium release-activated calcium modulator 2) showed similar expression pattern with Myf5 and MyoD1 ( Figure 1D-G). The expression patterns of RyR1 and STIM1 (stromal interaction molecule 1) matched MyoG expression pattern well. Notably, RyR1 mRNA expression showed greater 100-fold increase ( Figure 1H, I), while RyR3 mRNA expression was less than 1/10 of RyR1 in C2C12 cells, and the increase in RyR3 mRNA expression was far below that of RyR1 during myogenic differentiation (Additional le 2: Figure S2A, B), therefore, the major role of RyRs involved in myogenesis of C2C12 cells can be attributed to RyR1. Consistently, RyR1 mRNA expression showed almost a 25-fold increase during myogenic differentiation, while the mRNA expression of RyR3 was not changed in myogenic cells derived from pigs (Additional le 2: Figure S2C-E). In addition, the mRNA expression of ATP2A2 (ATPase ER/SR Ca 2+ transporting 2; Figure 1J), ATP2B (ATPase plasma membrane Ca 2+ transporting 1; Figure 1K), and CRACR2A (calcium release activated channel regulator 2A; Figure 1L) was signi cantly increased on day 2 and maintained a plateau on day 2-8 during myogenic induction of C2C12 cells.
As shown in Figure 2A, B, cytoplasmic Ca 2+ concentration was signi cantly decreased by treatment with dantrolene (DAN), an inhibitor of RyR1, which blocks the release of Ca 2+ from SR/ER [29]. RyR1 knockdown by siRNA signi cantly decreased cytoplasmic Ca 2+ concentration of C2C12 cells ( Figure 2C, D). The protein expression of RyR1 was effectively suppressed by siRNA interference (Figure 2E, F). Functional constraints of RyR1 realized by either DAN or siRyR1 dramatically inhibited the formation of multinucleated myotubes of myoblasts ( Figure 2G, H). Upon myogenic induction, DAN effectively blocked MyoD1 expression on day 2 and both MyoG and Mymk (myomaker, myoblast fusion factor) expression on day 4 ( Figure 2I). At the day 4 during myogenic induction, siRyR1-knockdown signi cantly reduced RyR1, MyoG, and Mymk expression without effects on Myf5 and MyoD1 expression ( Figure 2J).

RyR1-induced ER stress is indispensable for myogenic differentiation
During myogenic differentiation of C2C12 myoblasts, the mRNA expression of protein disul de isomerases including ERP44 (ER protein 44), PDIA3 (protein disul de isomerase associated 3), and PDIA4 (protein disul de isomerase associated 4) which are present in ER, was signi cantly increased relative to those before differentiation ( Figure 3A). Endoplasmic membrane proteins ATF6 (activating transcription factor 6) and EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), considered as typical ER stress markers, also showed signi cantly higher mRNA expression ( Figure 3B). Furthermore, the mRNA expression of apoptosis-and heat stress-related proteins including BAX (BCL2-associated X protein), caspase-12, DDIT3 (DNA-damage inducible transcript 3), HSP90B1 (heat shock protein 90, beta, member 1), and HSPA5 (heat shock protein 5) was also signi cantly stimulated during myogenic differentiation ( Figure 3C). Similarly, signi cantly increased mRNA expression of protein disul de isomerases, ERP44 and PDIA3, were also observed in primary myogenic cells derived from skeletal muscle of pigs during myogenic differentiation relative to those before differentiation ( Figure 3D). Simultaneously, mRNA expression of ER stress markers, ATF6 and EIF2AK3, increased signi cantly ( Figure 3E). The mRNA expression of apoptosis-and heat stress-related proteins including caspase-9, DDIT3, and HSPA5 was also signi cantly stimulated ( Figure 3F). The phosphorylation of IRE1α and PERK was signi cantly reduced by DAN treatment compared with the control, while EIF2α was not altered ( Figure 3G, H). For MAPK signaling ( Figure 3I, J), phosphorylation level of Erk1/2 (extracellular regulated protein kinases) was signi cantly increased by DAN treatment, while JNK (stress-activated protein kinase/Jun-aminoterminal kinase) was decreased.
THA treatment signi cantly increased cytoplasmic Ca 2+ concentration ( Figure 4A, B), but did not in uence the mRNA expressions of myogenic-speci c genes including Myf5, MyoD1, MyoG and Mymk ( Figure 4C). RyR1-knockdown realized by siRNA interference was independent on THA treatment ( Figure  4D, E). As shown in Figure 3F, G, on day 4 during myogenic induction, RyR1-silencing signi cantly decreased phosphorylation of IRE1α, PERK, and EIF2α. Meanwhile, THA did not in uence IRE1α and EIF2α phosphorylation, but tended to increase PERK phosphorylation in myoblasts. Obviously, THA recovered the decreased phosphorylation level of IRE1α, PERK, and EIF2α caused by siRyR1-knockdown.
On day 4 during myogenic induction, siRyR1-knockdown signi cantly increased Myf5 and MyoD1 mRNA expression but decreased MyoG and Mymk expression. Alterations in Myf5, MyoD1, MyoG and Mymk mRNA expressions caused by siRyR1-knockdown were effectively eliminated by THA treatment. In addition, mRNA expression of Myf5, MyoD1, MyoG and Mymk was not in uenced by TAH in myoblasts with absence of siRyR1-knockdown ( Figure 5A). Accordingly, THA treatment did not affect myogenic differentiation of myoblasts with absence of siRyR1-knockdown. In particular, myotube formation was signi cantly inhibited by siRyR1-knockdown, which was effectively recovered by THA ( Figure 5B, C).

Effects of RyR1-knockout on cell proliferation and differentiation
As shown in Additional le 3: Figure S3, the frame-shift mutation of RyR1 was realized by the CRISPR/Cas-9 gene editing system targeting Exon 18 of RyR1 via gRNA-3, which showed the highest shearing e ciency among three designed gRNA. Homozygote and heterozygote of RyR1-knockout cells, named as RyR1 -/and RyR1 +/-, were obtained by monoclonal cultivation and identi ed via gene sequencing on the target site and putative off-target sites of gRNA-3. Relative to the wild type cells (WT), RyR1 -/showed higher Ca 2+ concentration in cytoplasm, but lower Ca 2+ level in the ER ( Figure 6A-C). The mRNA expression of ATP2A2, ATP2B, CRACR2B, and ORAI1 was signi cantly increased, while CAV1.1 and ORAI2 expression were decreased in RyR1 -/or RyR1 +/relative to WT ( Figure 6D).
Cell proliferation viability (the proportion of EdU + cells) of RyR1 -/or RyR1 +/signi cantly declined relative to WT ( Figure 6E, F), which was also demonstrated by the CCK-8 test ( Figure 6G). As shown in Figure 6H, the mRNA expression of Myf5, MyoD1, and MyoG was signi cantly increased in RyR1 -/or RyR1 +/relative to WT. RyR1-knockout signi cantly reduced phosphorylation of Erk1/2 and JNK, as well as the total protein of JNK ( Figure 6I, J).

Apoptosis triggered by RyR1-knockout
On day 2 during myogenic induction, apoptosis instead of myotube formation was accelerated in both RyR1 -/and RyR1 +/-. The apoptosis rate of RyR1 -/and RyR1 +/was signi cantly increased relative to WT ( Figure 7A, B). The protein level of cyclin D1 and caspase-3 was signi cantly lower in RyR1 -/or RyR1 +/-, but the expression of cleaved caspase-3 was signi cantly increased as compared with WT ( Figure 7C, D).
ER stress level in RyR1 -/and RyR1 +/was evaluated by Western Blot ( Figure 7E). Both phosphorylated and total protein expression of IRE1α and PERK were dramatically elevated by RyR1-knockout, while phosphorylation of EIF2α was signi cantly decreased ( Figure 7F). As to several apoptosis-related proteins, the protein abundance of CHOP (also known as DDIT3), caspase-9, and caspase-12 in RyR1 -/or RyR1 +/was signi cantly increased relative to WT, while the expression of ERP44 and HSPA5 was not in uenced ( Figure 7G, H).

Discussion
Skeletal muscle mass is maintained by myogenic differentiation of myogenic progenitors [30], in which cytosolic Ca 2+ regulation plays a vital role [28]. Myotube formation requires net Ca 2+ in ux into myoblasts [31][32][33]. RyR1, serving as a major Ca 2+ release channel of ER, has attracted the most intense research interest among intracellular Ca 2+ channels. However, the role of RyR1 in myogenic differentiation remains unclear.
RyR1 protein expresses during differentiation in C2C12 cells which provides an appropriate model for investigations of RyR1 function during myogenic differentiation [34]. In the current study, we observed that cytoplasmic Ca 2+ concentration of C2C12 myoblasts was signi cantly elevated during myogenic differentiation. K Qiu, D Xu, L Wang, X Zhang, N Jiao, L Gong, et al.
[28] also observed increased Ca 2+ concentration in primary myogenic cells of pigs. Cellular Ca 2+ is tightly regulated by channels and transports [35]. Relative to the extracellular matrix and ER, cytoplasmic Ca 2+ concentration is maintained at very low levels (10-100 nM) under resting conditions. Ca 2+ is released from the ER, the main storage site of intracellular Ca 2+ , through the transmembrane channels RyR1 and ITPR1 [36,37]. Cytoplasmic Ca 2+ in ux from extracellular matrix occurs through plasma membrane channels, CAV1, CAV2, and CAV3 [38]. To maintain the resting state, excessive amounts of cytoplasmic Ca 2+ re-accumulates in the ER by SR/ER Ca 2+ -ATPase (SERCA, also called ATP2A) [39] or is expulsed in the external milieu by plasma membrane Ca 2+ -ATPase (PMCA, also called ATP2B) [40,41]. Furthermore, store-operated calcium entry (SOCE), mediated by STIM (ER Ca 2+ sensors), activates CRAC and ORAI channels located at plasma membrane to maintain cellular Ca 2+ homeostasis [42,43].
Lineage commitment and differentiation of myoblasts are governed by the programmed expression and functional activation of myogenic regulatory transcription factors (MRFs) [44,45]. In this study, we clearly demonstrated the expression pattern of Ca 2+ transporters as well as MRFs during myogenic differentiation and especially discovered RyR1 expression was increased more than 100-fold in C2C12 cells and almost 25-fold in myogenic cells of pigs during myogenic differentiation. CAV1.1 is a physiological activator of RyR1 in the excitation-contraction coupling of skeletal muscle [46]. In this study, the expression of CAV1.1 was not signi cantly increased as RyR1 during myogenic differentiation, which indicated that RyR1 expression in myogenic cells is not activated by the expression of CAV1.1 but probably induced by the surroundings, such as myogenic medium and cellular density. Notably, RyR1 restriction via either DAN treatment or siRNA interference signi cantly decreased cytoplasmic Ca 2+ concentration, and then blocked formation of multi-nuclei myotubes and expression of MRFs. As a major Ca 2+ release channel of ER, the dramatic increase of RyR1 expression should be responsible for the signi cant elevation of cytosolic Ca 2+ concentration. Therefore, we deduced that dramatic increase of RyR1 expression is required for myogenic differentiation.
Alterations in Ca 2+ dynamics can trigger ER stress and activation of the UPR [19,20]. In the current study, expressions of protein disul de isomerases in the ER including ERP44, PDIA3, and PDIA4, directly re ecting the oxidation state of ER [47], and were signi cantly increased during myogenic differentiation.
Moreover, we observed that ER transmembrane proteins including protein kinase RNA-like ER kinase (PERK/EIF2AK3) and activating transcription factor 6 (ATF6), involved in the UPR process as sensors [48], were elevated upon myogenic induction. Therefore, ER stress signaling was activated by the dramatic increase of RyR1 expression in myoblasts during myotube formation.
IRE1α, PERK and ATF6 function as UPR stress sensors. UPR transmits information about the proteinfolding status in the ER lumen to the nucleus and cytosol, and buffers uctuations in unfolded protein load via UPR stress sensors. Upon activation of ER stress, PERK phosphorylates initiation factor eukaryotic translation initiator factor 2α (eIF2α) and attenuates general protein synthesis [48]. ATF6transmitted ER stress signaling results in apoptosis during muscle development [26]. ER stress is always accompanied by activation of a series of stress-induced protein phosphorylation involved in the MAPK signaling pathways. For example, JNK protein kinases can be activated by the coupling of ER stress and transmembrane protein kinase IRE1 [49]. In addition, the activation of the Erk1/2 pathway is important for cells to avoid apoptosis caused by physical stress [50]. In our study, inhibition of RyR1 function by DAN treatment signi cantly reduced phosphorylation levels of IRE1α, PERK, and JNK proteins, but obviously increased Erk1/2 phosphorylation, demonstrating that DAN-induced RyR1 function restriction effectively and speci cally alleviated ER stress.
Thapsigargin (THA), a well-characterized ER stress-inducing agent, was used to trigger ER stress through inhibiting microsomal Ca 2+ -ATPase, disrupting cellular Ca 2+ homeostasis, and accumulating unfolded proteins in the ER lumen [51]. In the current study, the dose of THA was far below that used in previous studies [52,53] to gain a moderate ER stress status, and guaranteed that THA did not in uence the e ciency of siRyR1-knockdown. As a result, THA treatment did not trigger severe ER stress compared with the control. Furthermore, THA treatment signi cantly increased cytoplasmic Ca 2+ concentration but did not affect expression of myogenic speci c genes. We observed that ER stress alleviated by RyR1 suppression was dramatically intensi ed by THA addition. At the same time, myotube formation inhibited by siRyR1-knockdown was also recovered by THA treatment, accompanied by the expression of MRFs. Therefore, RyR1-mediated ER stress was indispensable for myogenic differentiation.
C/EBP homologous protein (CHOP) encoded by DDIT3 gene is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factor, whose expression is stimulated during ER stress [54]. Caspase-12 is speci cally activated during ER stress-mediated apoptosis especially caused by the disruption of ER Ca 2+ dynamic [55]. Caspase-9 activates apoptotic signals when mitochondria are damaged [56]. The localization of BAX from cytoplasm to mitochondria is a necessary process of apoptosis [57]. The HSPA5 gene encodes the binding immunoglobulin protein (BiP), a member of the heat shock protein 70 (HSP70) family localized in the ER lumen. Once ER stress overwhelms, BiP can initiate the UPR and decrease unfolded/misfolded protein load of the ER to assist cell survival by avoiding the activation of apoptosis machinery [58,59]. HSP90B1 (also called GRP94 or gp96), located in the ER, plays important roles in stabilizing and folding other proteins [60]. ERP44, a ER folding assistant of the thioredoxin family, is induced during ER stress for protein quality control [61]. In our study, myogenic induction not only increased expression of proteins related to ER stress-induced apoptosis, including DDIT3, caspase-12, and BAX, but also enhanced expression of proteins that mitigate ER stress, such as ERP44, HSP90B1, and HSPA5. Under the pressure of ER stress-induced apoptosis, cells express high levels of heat stress proteins (HSPA12A, HSP90B1, HSPA4, HSPA5 and HSPA6) to increase their adaptability [62]. Therefore, we deduced that a certain degree of ER stress-induced apoptosis was triggered during myogenic differentiation but balanced by the enhanced protection mechanism against ER stress per se.
To further explore the roles of RyR1 in myogenic differentiation and muscle development, a model of ER stress signaling can give rise to myogenic differentiation and apoptosis [25]. In the present study, differentiation potential of RyR1-KO myoblasts re ected by MRFs expression was dramatically enhanced. However, the differentiation process of RyR1-KO myoblasts was interrupted by programmed cell death through apoptosis during myogenic induction, which indicated that RyR1 knockout reduced the capacity of cells to adapt to the surroundings, such as the differentiation medium containing 2% horse serum. UPR was a protective response for cells under stress, while excessive or prolonged UPR can cause apoptosis [67]. Caspase-3, belonging to a highly conserved family of cysteinyl aspartate-speci c proteases, is an essential regulator of apoptosis. In the current study, cleaved activation of caspase-3 in RyR1-KO cells was signi cantly stimulated, which is supported by a previous study of RyRs-mediated ER stress [68]. The total and phosphorylated protein expressions of IRE1α and PERK were sharply increased in RyR1-KO cells, indicating that RyR1 deletion activated serious ER stress through ER Ca 2+ leakage. Furthermore, aggravated ER stress signi cantly increased expression of CHOP, caspase-9, and caspase-12.
Nonetheless, the positive effects of ERP44 and HSPA5 against ER stress were not enhanced in RyR1-KO myoblasts. In addition, phosphorylation levels of JNK and Erk1/2, whose activations are bene cial for the resistance to ER stress-induced apoptosis [49,50], were also signi cantly reduced in RyR1-KO cells.
Therefore, we deduced that RyR1 deletion led to more serious ER stress and excessive UPR than the tolerance thresholds of cells, and accounted for the apoptosis of RyR1-KO myoblasts upon myogenic induction.

Conclusions
Page 13 /29 In summary, dramatically increased RyR1 expression which activated ER stress signaling through increased cytoplasmic Ca 2+ was absolutely indispensable for myogenic differentiation. We discovered a novel role of RyR1 acting as a double-edged sword in myogenic differentiation by swaying the fate of myoblasts between differentiation and apoptosis. Our study contributes to the knowledge of the role of RyR1 in myogenic differentiation and related congenital myopathies, and provides a potential target to regulate muscle regeneration and related tissue engineering.