Ezrin regulated myoblast differentiation/fusion and muscle ber specialization through PKA-NFAT-MyoD/MEF2csignalling pathway

Backgorund:Neuromuscular diseases are a kind of nervous system diseases that have a high disability rate.Ezrin’ role in skeletal muscle has not been identied. This study aims to conrm the effect and mechanism of Ezrin on myoblast differentiation and fusion, myotube size, and myober type. Method:By using immunoassaying and western blot analyses, Ezrin, MyHC,MEF2c, MyoG, PKAα/β/γ, PKA reg Iα, PKA reg IIβand NFATc1-c4 were detected in myoblast cells treated with Ad-Ezrin or Ad-shEzrin. Real-time PCR were used to evaluate MyoD, Myf5, MyHC-I , MyHC-IIa/b and MyHC-IIx in myoblast cells. PKA inhibitor H-89 or PKAreg I activator N 6 -Bz-cAMP were added into medium to conrm their relationship between Ezrin and PKA during myoblast differentiation/fusion. In vitro, Ad-NFATc1/c2 or Ad-shNFATc3/c4 were respectively transfected into C2C12 cells, myoblast differentiation/fusion, myotube size and myober type were assessed by using immunostaining of MyHC, MEF2c and MyoG. In vivo, transfection of Ad-Ezrin into gastrocnemius and soleus muscles for 7 days, the numbers of MyHC-1 postivemyobers were analyzed after immunostaining of MyHC-1. Results: Ezrin expression were time-dependently increased during myoblast differentiation/fusion. Knockdown of Ezrin by shRNA delayed myoblast differentiation and fusion in a time dose-dependent pattern, as shown by immunostaining of MyHC. Conversely, over-expression of Ezrin by adenovirus time-and dosage-dependently promoted myoblastdifferentiation/fusion, and muscle ber specialization characterized by increased MyHC I and MyHCIIa/b. Forced expression of Ezrin did not alter PKA, and PKAreg II α levels, but altered the levels of PKAreg I α/β, Myf5 and MyoD, and leading to the accumulation of MyoG+/MEF2c+ nuclei. By contrast, Ezrin knockdown signicantly decreased the PKA reg I/II ratio and MyoG+/MEF2c+ nuclei. The PKA inhibitor H-89 remarkably abolished the benecial effect of overexpressingEzrin on the numbers of MyHC+ myotubes and MyoG+/MEF2c nuclei. These opposite changes mediated by knocking down Ezrin were almost eliminated by PKAreg I activator N 6 -Bz-cAMP. Furthermore, over-expression of NFATc2 or knockdown of NFATc4reversed the inhibitory effect of Ezrin knockdown on myoblast differentiation/fusion, resulting in the recovery of the numbers ofMyoG+/MEF2c+ nucleiin3-nuclei + myotubes. Meanwhile, overexpression of Ezrin specically induced type I muscle ber specialization, which was associated with increased levels of NFATc1/c2. Furthermore, in vivo transfection ofAd-Ezrin into gastrocnemius and soleus muscles increased the numbers of MyHC-1 postivemyobers. By contrast, knockdown of NFATc4resulted in the recovery to normal levels of MyHC-2b in Ezrin-knockdown myoblast cells, attributingtoregainingMyoDand MEF2c expression. Conclusions: Ezrin trigger myoblast differentiation and fusion, myotube size, and alters muscle ber specialization through PKA-NFAT-MyoD/MEF2C signalling pathway.


Introduction
Neuromuscular diseases are a kind of nervous system diseases that have a high disability rate, causing long-term mental and economic burden to the society, family, and the individual 1 . Muscle biopsy pathology is the most effective for the diagnosis of myopathy. Studies found that the pathological basis of muscular atrophy and muscle weakness is the degeneration and necrosis of muscle bers. Skeletal muscles have the ability to regenerate to prevent the loss of muscle mass and maintain normal shape and function 2 . It is, therefore, crucial to study the characteristics of muscle ber regeneration and their associatedfactors for the prevention and cure of neuromuscular diseases 3 .
Muscle satellite cells conduce to the need for physiological self-renewal and the repair of pathological injury 4 . In ammatory reactions are one of the pathologies that result in myopathy,includingpolymyositis and dermatomyositis 5 . A recent study has shown that a more robust in ammatory area within the skeletal muscle demonstrated features of high Ezrin expression, a member of the Ezrin/radixin/moesin (ERM) proteins family. Published data supplied evidence that Ezrin could play a crucial role in transferring extracellular signal molecules into the skeletal proteins, activating the corresponding signal pathways, regulating the cell morphology, adhesion, phagocytosis, movement, and angiogenesis [6][7][8][9][10] . However, the role and mechanism of Ezrin in muscle satellite cells are still unclear.
Traditionally, the activated protein kinase A(PKA) has been linked to the unique phenomenon of myoblast differentiation/fusion and myotube formation, ascribing to the alteration in PKA regulatory subunit I (PKA RI) under normal differentiation condition 11 . Our previous study has shown that it abated the ratio of PKA RI/RII in myoblast cells, resulting in the postponement of myoblast differentiation and fusion 12 . Further evidence shows that ERM proteins act as PKA-anchoring proteins and sequester PKA close to its target proteins for their effective phosphorylation and functional regulation 13 . NFATs (Nuclear factor of activated T cells) activation mediated by PKA plays crucial role in myoblast differentiation and fusion, myotube size, and altered muscle ber specialization 4,13 . In this study, knockdown of Ezrin by shRNA reduced the numbers of MyoG/MEF2C-positive cells and myotube number/size while decreasing the ratio of PKA RI/RII, causing the increased expression of NFATc2/c3/c4, suggesting that Ezrin triggered myoblast differentiation and fusion, myotube size, and altered muscle ber specialization through PKA-NFAT-MEF2C signalling pathway. Method C2C12 myoblast culture and differentiation induction C2C12 myoblast cells were inoculated in 75-cm 2 culture dishesand cultured with proliferation medium (PM) containing high glucose DMEM (HG-DMEM) supplemented with 10% FBS at 37℃and 5% CO 2 .
When the con uence of the cells reached 75%, the PM was replaced with differentiation medium (MD) containing HG-DMEM supplemented with 2% horse serum (HS) to induce C2C12 myoblast cell differentiation. Traits of myotube formation from myoblast differentiation were observed daily under a microscope 14 .
Overexpression or Knockdown of Ezrin in vitro Construction of Ezrin overexpression and short hairpin RNA (shRNA) adenoviral vector were prepared as previously described 15 . To con rm the role of Ezrin on myoblast cells,Ad-Null, Ad-shEzrin, or Ad-Ezrin (1 × 10 9 pfu) were added into the corresponding culture dishesa day before adding the differentiation medium. These cells were then replaced with the differentiation medium for further observation.

Overexpression or Knockdown of NFATs in vitro
Construction of NFATc1/c2 overexpression adenoviral vector were prepared as previously described 15 . The gene accession number of overexpressing-NFATc1/c2 is NM_172390 and NM_173091, respectively.
Construction of NFATc3/c4 short hairpin RNA (shRNA) adenoviral vector were prepared as previously described 15 . These overexpression adenoviral vectors containing Ad-NFATc1, Ad-NFATc2, Ad-shNFATc3 and Ad-shNFATc4were obtained from Vigenebio.To con rm the role of NFATc3 or NFAtc4 on myoblast cells, the addition of Ad-shCtrl, Ad-shNFATc3, or Ad-shNFATc4 (1 × 10 9 pfu) into correspondingculture dish one day before the ISO was performed. And then these cells were replaced with differentiation medium for further observation.
Immuno uorescence Staining C2C12 myoblast differentiotion was determined by immuno uorescence staining. Primary monoclonal and polyclonal antibodies against MEF2C (#5030s, 1:200, CST), MyHC (sc-20641, 1:150, Santa Cruze) were added into each well in every group and incubated for 12 h at 4 °C. The cells were washed with PBS 3 times for 15 min and incubated with appropriative uorescent dye-labeled secondary antibodies (Jackson Lab, 1:500, USA) at 25℃ for 2 h. The nuclei were stained with DAPI (Molecular Probes). The images for each group were photographed under Nikon 80i uorescence microscope 16 .

Myoblast Differentiation
After myoblast cells were treated under DM for the indicated time, the differentiated myoblast cells were stained for MyoG or MEF2C using the primary polyclonal antibody MyoG (sc-12732, 1:150, Santa Cruze) or MEF2C (5030S, 1:400, CST) and appropriative TRITC-labeled secondary antibody (Jackson Lab, 1:500, USA). The nuclei were stained with DAPI. C2C12 myoblast cells with only 1-2 nucleuseswithin a cellular structure were evaluated withMyoG or MEF2C staining.TheMyoG + or MEF2C + cells were de ned as the differentiated cells that did not fuse to form myotubes. Myoblast cells with 3 or morenucleusesin the structure of a cell were de ned as myotubes. The number of double-positive nuclei under high power eld (HPF, 50 µm) were analyzed afterdouble staining of MyoG/DAPI or MEF2C/DAPI. Two individuals who did not know the results evaluated the images using Image J (Java) software (National Institutes of Health, USA).

Myoblast Fusion And Myotube Morphology
The differentiated myoblast cells were stained for MyHC with the primary polyclonal antibody MyHC (rabbit anti-mice antibody, sc-20641, 1:150, Santa Cruze) and appropriative TRITC or FITC-labeled secondary antibody (Jackson Lab, 1:500, USA). C2C12 myoblast cells with only 1-2 nucleuses within a cellular structure were evaluated by MyHC staining, indicating that the MyHC + cells were de ned as the differentiated cells without mutually fusing to myotube. Myoblast cells with 3 or more nucleuses in the structure of a cell were de ned as myotube. The nuclei were stained with DAPI.
To analyze myotube size, we divided the cells into 2 groups, including short myotube with 3 ~ 5 myoblast fusion and long myotube with more than 5 myoblast fusions. Morphology was assessed by myotube length, area (grouped less than 200 µm and more than 200 µm), and the number of myotubes (grouped 3 5 nuclei or more than 5 nuclei myoblast fusion) under high-power magni cation 15,17 . Two individuals who did not know the results evaluated the images using Image J (Java) software (National Institutes of Health, USA).

Quantitative RT-PCR
Total RNA from C2C12 myoblast cells was obtained using TRIzol (Invitrogen, Life Technologies) and transcribed into cDNA using the SuperScript II cDNA kit (Invitrogen, Life Technologies). Quantitative PCR was carried out using SYBR green PCR master mix (Thermo Fisher Scienti c, Applied Biosystems, CN) in Real-Time PCR System (RotorGene 6000, Qiagen, Germany). The transcript levels of the gene of interest in each group were normalized to GAPDH levels 18 . The primers used are listed in Table 1. Table 1 The sequences of primers of qPCR.

Gene
Forward Reverse qPCRs were performed to identi ed satellite cell differentiation and muscle bers traits by using the speci c primers of satellite cell differentiation markers including MyoD and MyoG, type I muscle ber makers like MyHC1, and type II muscle ber makers such as MyHC2a, MyHC2b, and MyHC2X.

Statistical analysis
Data of quantitative and semi-quantitative analysis presented are mean ± SD.Paired or unpaired Student's t-test determined statistical signi cance between the two groups.One-way ANOVA was used to compare the results for more than two experimental groups to specify the differences between groups.P < 0.05 is considered meaningful.

Results
Ezrin is expressed in C2C12 cells during myoblast differention and fusion To con rm the role of Ezrinin C2C12 myoblast cells, we determined the changes ofEzrin expression during myoblast differentiation and fusion by western blot. We found that the expression ofEzrin gradually increased, reaching its peak on day 4 of myoblast differentiation (Fig. 1).This expression is accompanied byC2C12 myoblast cell alterations, that time-dependently differentiated into mature muscle cells,formingmyotubes characterized by MyHC positive staining under differentiation medium containing 2% HS-DMEM. These results indicated that Ezrin could play an essential role in myoblast differentiation and fusion.

MYOG/MEF2C Involved Ezrin-mediated Myoblast Differentiation And Fusion
Since MyoG and MEF2C play a crucial role in the initiation and later of myoblast cells differentiation 20 , we used MyoG and MEF2C staining to con rm the relationship between Ezrin and myoblast differentiation. Our results showed that following treatment of Ad-Ezrin,the number and percentage of MyoG + nuclei were higher than that of MEF2C + nuclei within the myoblast during C2C12 myoblast differentiation (Fig. 3A-2G). Knockdown of Ezrin by shRNA markedly reduced the percentage of MyoG + and MEF2C + nuclei cells in C2C12 myoblast cells (Fig. 3A-2G).Moreover, the percentage of MEF2C + nuclei was lower than that of MyoG + nuclei in Ezrin-knockdownC2C12 myoblast cells (Fig. 3C-2G).These results indicated thattheknockdown of Ezrincouldinhibit the initiation and late phase of myoblast differentiation through MyoG and MEF2C, especially MyoG.
Ezrin regulated myoblast differentiation and fusion through PKA signalling pathway Previous results have shown that PKA and PKAreg I/II ratio play crucial roles in controlling myoblast differentiation and fusion 11,12 . To con rm if Ezrin's role in myoblast differentiation and fusion was involved in PKA signalling pathway, we used western blot to detect PKA signaling (Fig. 4A-4G). Our results revealed that, the overexpression of Ezrin did not alter PKAreg II levels, but it signi cantly increased the levels of PKAα,PKAreg Iα,and PKAreg Iβ, resulting in an increased PKAreg I/II ratio. By contrast, knockdown of Ezrin by shRNA signi cantly reduced PKAreg Iαand PKAreg Iβlevels, but it did not alterPKAreg II levels, resulting in a decreased PKAreg I/II ratio (Fig. 4A-4G).

PKA Involved Ezrin-mediated Myoblast Differentiation And Fusion
PKA activity is found to have effects on myoblast differentiation and fusion 11,12 . Combining with the above results that the overexpression or knockdown of Ezrinaffected myoblast differentiation and fusion by altering PKA activity, we treated C2C12 cells with PKA inhibitor (H-89). We found that PKA inhibitor (H-89)abolished the bene cial role of Ezrin-mediated C2C12 myoblast differentiation and fusion. By contrast, both PKA activator and cAMP analogues reversed the inhibitory effects of Ezrin knockdown onC2C12 myoblast differentiation and fusion (Fig. 5A-5D). These results indicated that the effect of Ezrinduring myoblast differentiation and fusion could be associated with PKA signalling.
Ezrin regulated myoblast differentiation and fusion through PKA-MyoG/MEF2C signalling pathway Indeed, myotube is formed by the fusion of differentiated myoblasts, which is characterized by three (3 + ) or morenuclei in the structure of a cell 15,17 . As shown in Fig. 6A-6J, we found that knockdown of Ezrin by shRNA markedly decreased the percentage of MyoG + and MEF2C + nuclei in less than 3 cells and 3 + myotubes, and these effects could be abolished by PKA activator. By contrast, overexpression of Ezrin substantially increased the number of MyoG + or MEF2C + nucleus in less than 3 and 3 + myotubes. However, the speci c changes were almost entirely cancelled by the PKA inhibitor (Fig. 6C-6J). These results indicated that Ezrin participated in C2C12 myoblast differentiation and fusion with coordination of MyoG and MEF2C.
Ezrin regulated myoblast differentiation and fusion through NFATc2/c4-MyoG/MEF2C signaling pathway Since MyoG and MEF2Cinvolved in the ontrol of the initiation and later stage of myoblast differentiation, respectively 26 . As shown in Fig. 8A and sFigure3A-3F, we found that knockdown of Ezrin by shRNA markedly decreased the numbers and percentage of MyoG + and MEF2C + nuclei in less than 3-nuclei cells and 3-nuclei + myotubes, and these effects could be abolished by Ad-NFATc2 or Ad-shNFATc4. In addition, Ad-NFATc1 or Ad-shNFATc3 reversed the numbers of MEF2C + nuclei in 3-nuclei + myotubes. These results indicated that Ezrin participated in C2C12 myoblast differentiation and fusion with coordination of MyoG and MEF2C, which were associated with NFATc2/c4, at least in part.

Discussion
In this study, we made three novel observations. Firstly,we found that theEzrin expression has a timedynamic characteristic during myoblast differentiation and fusion.Secondly, Ezrin signi cantly controlled myoblast differentiation and fusion. And lastly, Ezrin regulated C2C12 myoblast differentiation, fusion, and myotube type formationviaPKA RI-NFAT-MyoD/MEF2C signalling pathway.
Ezrin belongs to the ERM family of proteins that play structural and regulatory roles in the assembly and stabilization of plasma membrane interactions through their ability to interact with transmembrane proteins and the cytoskeleton 13 . As one of ERM proteins, Ezrinactivated the intracellular signal pathways through transferring extracellular signal molecules into actin cytoskeleton, affecting several key cellular processes, including membrane dynamics, cell adhesion, cell survival, motility, and determination of cell shape [6][7][8][9][10] . Indeed, these cellular processes were associated with myoblast differentiation and fusion 21 . Forthe rst time, we found that Ezringradually increased during myoblast differentiation/fusion, and rapidly decreased when formed myotubes approached and/or reached maturity. Furthermore, overexpression of Ezrin signi cantly accelerated the process of myoblast differentiation and myotube formation. Conversely, this process was delayed by the knockdown ofEzrin. These results suggest that Ezrin could act as a crucial factor during skeletal muscle regeneration and restoration in physiological and pathological conditions.
Previous studies have shown that the overexpression of PKA inhibited myogenic differentiation, contributing to HDAC4 phosphorylation and the transcriptional repression of muscle-speci c genes by the myogenic regulators Myf-5 and MyoD 22,23,24,25 . Furthermore, ERM proteins,includingEzrin, are reported toact as PKA-anchoring proteins and sequester PKA close to its target proteins for their effective phosphorylation and functional regulation 13 . However, our results did not show anyapparentchanges in expressions of Myf-5 and MyoD in overexpressingEzrin in myoblast cells, but showed increased numbers of MyoGandMEF2C-positive myoblast cells and myotubes. Actually, MyoG and MEF2C play an essential role in the initiation and later stage of myoblast differentiation, respectively 26 .Recent reports have also associated PKA RI and RII to myoblast differentiation and myotube formation 11,12 . Of interest, we found that overexpressingEzrin markedly increased PKA RI levels, leading to an increased PKA RI/RII ratio, accompanied by an acceleration of myoblast differentiation and fusion. Furthermore, the knockdown of Ezrin, resulting in a lower PKA RI/RII ratio, inhibitory effects observed during myoblast differentiation/fusion could be reversed by the PKA RI activator. More importantly, PKA RI activator almost completely recovered the numbers of MyoG or MEF2C positive myotubes. Therefore, Ezrin could promotemyoblast differentiation/fusionviaPKA RI/RII-MyoG/MEF2C signalling pathway.
Existing data have shown that NFATs act as a crucial player in myoblast differentiation and fusion, especially myo ber speci cation [26][27][28][29][30][31][32] . Moreover, NFATs activities are frequently regulated by the PKAcalcineurinsignalling pathway during cell differentiation 33,34,35,36 . Indeed, NFATc2 primarily controlled myoblast recruitment and myoblast fusion 37,38,39,40 . Similarly, we found that the inhibitory effect of myoblast fusion by knockdown of Ezrin could be restored by the overexpression of NFATc2, but not NFATc1. In line with stimulatory effect of constitutively active NFATc1(caNFATc1) on MyHC-1 expresison 41 , the increased NFATc1 levels by overexpression of Ezrin induced MyHC-1 expression. However, enhanced NFATc1 levels by Ad-Ezrin did not decrease MyHC-2b as caNFATc1 did, but keeping its normal levels. Meanwhile, Ad-Ezrin reduced NFATc3/c4 levels in myoblast cells, but maintaining MyHC-2a expression, which was different from the inhibitory role of knockdown for NFATc3 and NFATc4 in the expression of MyHC-2a 30 . These different changes could be related with the increased levels of NFATc2 by Ad-Ezrin, because NFATc2 acted as important role in MyHC-2a and MyHC-2X expressions 37,40,41 . In a word, altered NFATs signaling by Ezrinaffectedmyoblast differentiation/fusion, especiallytype-1 and type-2amuscle ber specialization.
In relation to muscle ber type specialization, slow muscle specialization and fast to slow myo ber-type switch need the coordination of MEF2C and MyoD 39,40,41,42,43 respectively. In this study, the overexpression ofEzrinincreased the MEF2C levels within the nucleus while decreasingthelevels of MyoD in myoblast cells, resulting in the increased MyHC-1 expressions, accompanied by normal levels of MyHC-2a, MyHC-2b and MyHC-2X. The knockdown of Ezrinreduced MEF2C + myoblast cells numbers while increasing MyoDexpressions, leading to special changes that the levels MyHC-2a and MyHC-2b were obviously increased. Previous reports have shown that NFATc2 could perform speci c role in MyHC-2a and MyHC-2X expression with synergistic effect of MyoD while NFATc1 promoted MyHC-1 expression through inhibiting MyoD signaling 37,40 . More, NFATc4 has shown the inhibitory role in myoblast fusion 38 , and appears to mostly contribute to fast muscle ber formation, especially MyHC-2b 30 . The overexpression of NFATc4 affects the differentiation process by decreasing the expression of late differentiation markers, including MEF2c, and impairs myotube formation 30,38 . We found that these similar effects mediated by the knockdown of Ezrin could be reversed by knockdown of NFATc4. Thus, Ezrincould regulate myoblast fusion and type IIb muscle ber specialization through NFATc4-MEF2C signalling pathway.

Declarations
Ethics approval and consent to participate Not applicable.

Competing interests
The authors declare that they have no competing interests.

Availability of Data and material
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