Distinct Roles of Nfatc1 and Nfatc4 in Human Primary Myoblast

RESEARCH ARTICLE Distinct roles of NFATc1 and NFATc4 in human primary myoblast differentiation and in the maintenance of reserve cells Julie Perroud1, Laurent Bernheim1, Maud Frieden2 and Stephane Koenig1,*

ABSTRACT (MRF) family that, together with the myocyte enhancer factor 2 – Ca2+ signaling plays a key role during human myoblast differentiation. (MEF2; which has several different isoforms, MEF2A MEF2D) Among Ca2+-sensitive pathways, calcineurin is essential for myoblast transcription factor, regulate the expression of the late markers differentiation and muscle regeneration. Nuclear factor of activated of differentiation like embryonic myosin heavy chain (MYH3, T-cell (NFAT) transcription factors are the major calcineurin targets. hereafter denoted MyHC), which is usually expressed after the We investigated the expression and the role of each NFAT gene fusion of myoblasts into myotubes. We previously described the long during human primary myoblast differentiation. We found that isoform of stromal interaction molecule 1 (STIM1L) as a main actor of 2+ three NFAT isoforms are present, NFATc1, NFATc3 and NFATc4. the process called store-operated Ca entry (SOCE) in muscle. Its Importantly, while their mRNA expression increases during expression is also induced only during human myoblast differentiation differentiation, NFATc1 is more highly expressed in myotubes, (Darbellay et al., 2011), and it is thus used as a late marker of ∼ whilst NFATc4 is specifically maintained in reserve cells. NFATc3 is differentiation. Terminal fusion is achieved after 2 to 3 days. At this ∼ present in both cell types, although no specific role during myoblast time, 80% of myoblasts have fused into multinucleated myotubes differentiation was observed. Knockdown of either NFATc1 or whereas 20% of cells remain mononucleated. These non-fusing cells NFATc4 affects the differentiation process similarly, by decreasing are called reserve cells (Yoshida et al., 1998). Reserve cells, as with the expression of late differentiation markers, but impairs myotube muscle stem cells, are quiescent and show the characteristic expression formation differently. Whereas NFATc1 knockdown strongly reduced of Pax7 (Pawlikowski et al., 2009). 2+ the number and the surface area of myotubes, NFATc4 knockdown Previous studies have all demonstrated that Ca modulations increased the surface area of myotubes and reduced the pool of play a key role during the onset of human myoblast differentiation 2+ reserve cells. We conclude that NFAT genes have specific roles in (Tu et al., 2016). Although the exact roles of endogenous Ca 2+ myotube formation and in the maintenance of the reserve cell pool signals are still not understood, different well-known Ca signal- during human postnatal myogenesis. generating proteins were shown to be required during myoblast differentiation. Concerning human muscle differentiation, we KEY WORDS: NFATc1, NFATc4, Human, Myoblast, Differentiation, could show that STIM1, STIM2 and Orai1 proteins are strongly Reserve cells involved, and that the very first event described so far during the differentiation process is an influx of Ca2+ caused by the INTRODUCTION activation of a SOCE (Darbellay et al., 2010, 2009). This Ca2+ Adult human skeletal muscle is able to regenerate. This remarkable influx, reinforced by myoblast hyperpolarization, activates the capacity is due to resident muscle stem cells, called satellite cells, Ca2+-sensitive Ser/Thr phosphatase calcineurin, which in turn which are found on muscle fibers between the basal lamina and participates in the induction of the differentiation program through the sarcolemma. Satellite cells are activated after muscle lesion: the expression of myogenic transcription factors (Konig et al., they proliferate as myoblasts, differentiate and then fuse into 2006). Others have also described the importance of calcineurin multinuclear cells called myotubes. Myotubes can then give rise to activation for the initiation of differentiation (Abbott et al., 1998; new myofibers or are able to regenerate damaged ones (Morgan and Friday et al., 2000; Nasipak et al., 2015) and muscle regeneration Partridge, 2003). This differentiation process can be mimicked ex vivo (Sakuma et al., 2003) in murine myoblasts. Calcineurin signaling by using a well-established in vitro model based on clonal culture of was first described in T lymphocytes as a regulator of the primary human myoblasts (Baroffio et al., 1993). Human primary transcription factors of the nuclear factor of activated T-cell myoblast differentiation can be separated into three sequential steps: (NFAT) family (Rao et al., 1997). Upon dephosphorylation by initiation, genotypic differentiation (expression of muscle-specific calcineurin, NFAT proteins can translocate to the nucleus where genes) and phenotypic differentiation (fusion). Genotypic they activate target genes. The NFAT family possess five different differentiation is characterized by the induction of early genes among which four are regulated by Ca2+/calcineurin signaling differentiation markers, like myogenin (Wright et al., 1989). The (NFATc1, NFATc2, NFATc3 and NFATc4), while NFAT5 is myogenin transcription factor belongs to the muscle regulatory factor regulated by osmotic stress. NFAT proteins are not necessarily expressed all together nor in the same cell type amongst many cell 1Department of Basic Neurosciences, University Medical Center, Rue Michel types, and they play different roles in cell proliferation, Servet 1, 1211 Geneva 4, Switzerland. 2Department of Cell Physiology and differentiation and survival (Crabtree and Olson, 2002; Hogan Metabolism, University Medical Center, Rue Michel Servet 1, 1211 Geneva 4, Switzerland. et al., 2003; Horsley and Pavlath, 2002). By using the luciferase reporter plasmid 9NFAT-Luc, we have already shown that *Author for correspondence ([email protected]) calcineurin activity increases during human primary myoblast S.K., 0000-0002-0801-279X differentiation (Konig et al., 2006), although the precise role of each NFAT protein remains unclear. In addition, an NFAT-independent

Received 26 October 2016; Accepted 25 July 2017 role for calcineurin has also been proposed (Friday and Pavlath, Journal of Cell Science

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2001). The purpose of the present work was to clarify the role with the expression of specific muscle markers i.e. myogenin, of NFAT isoforms in human primary myoblast differentiation. MEF2C, MyHC and STIM1L. Fig. 2 shows, as expected, that the We found that three of the NFAT genes are expressed in human induction of early markers (myogenin and MEF2C) is detected at primary myoblasts: NFATc1, NFATc3 and NFATc4. Their mRNA the onset of differentiation, while expression of late markers (MyHC expression increased during differentiation, with NFATc1 and STIM1L) is only observed in fused myotubes. Consistent with expression being stronger in myotubes and NFATc4 being more this, these late markers are almost absent in reserve cells. These highly expressed in reserve cells. Silencing of both NFATc1 and patterns of expression were also studied at the mRNA level by using NFATc4 through siRNA downregulated the expression of late the NanoString Technologies’ nCounter analysis system, which markers of differentiation, MyHC and STIM1L. Furthermore, allowed us to measure the expression of several genes at the same although NFATc1 knockdown inhibited myotube formation, time (Fig. S1A,B). This technology, although less sensitive, NFATc4 knockdown induced the development of abnormally confirmed roughly the pattern of expression of NFAT genes as large myotubes. Conversely, downregulation of NFATc3 did not well as of the various differentiation markers. Furthermore, Pax7, a affect the differentiation process. Taken together, these results marker of satellite cells (Jacquemin et al., 2007; Pawlikowski et al., demonstrate that both NFATc1 and NFATc4 are essential for the 2009), is detected in reserve cells but not in myotubes (Fig. S1C). correct differentiation of human primary myoblasts. Interestingly, EGFR, whose expression is strongly downregulated in myotubes (Leroy et al., 2013), also appeared to be a marker of RESULTS reserve cells, a result that is in accordance with EGFR expression in Expression of NFAT genes during human primary myoblast quiescent satellite cells (Charville et al., 2015). differentiation Taken together, these results show that NFATc1, NFATc3 and We first investigated the expression of the different NFAT genes NFATc4 are expressed during myoblast differentiation. NFATc1, during the differentiation of human primary myoblasts by while already expressed during proliferation, has a pattern of performing quantitative PCR (qPCR). We focused on the expression close to an early marker of differentiation (e.g. MEF2C). expression of NFATc1, NFATc2, NFATc3 and NFATc4 mRNAs, NFATc3 is expressed all along the proliferation process, with a as they are regulated by Ca2+/calcineurin signaling. We gathered sustained expression in both myotubes and reserve cells. Finally, several different RNA samples: in proliferating conditions (growth NFATc4 is expressed in proliferating myoblasts with a profile of medium, GM), during the indicated time course within the first 24 h expression close to that of EGFR. Its expression is maintained in of differentiation (differentiation medium, DM), and once fusion the early stages of differentiation as well as in the reserve cell was achieved, the latter on both separated myotubes and reserve population. cells (DM48 h and 72 h). Fig. 1 shows that NFATc1, NFATc3 and NFATc4 mRNAs are present in human myogenic cells, and that Knockdown of NFATc1 and NFATc4 inhibits the expression their expression profiles are markedly different during the of late myogenic markers chronology of differentiation. NFATc1 slowly increases its As NFAT genes are differently expressed in human myogenic cells, expression during differentiation although, surprisingly, after 2 we hypothesized that they could play distinct roles during human days of differentiation, NFATc1 is preferentially expressed in myoblast differentiation. To evaluate the role of each NFAT gene in myotubes rather than in reserve cells. By contrast, NFATc4 human myoblast differentiation, we tested different siRNAs (small expression increases during the first 6 h of differentiation and interference RNAs) against NFATc1, NFATc3 or NFATc4 mRNAs remains mostly present only in reserve cells. The expression of in order to identify, for each NFAT gene, an efficient siRNA with NFATc3 is stable between myoblasts, myotubes and reserve cells. minimal cross-reaction with the other NFAT mRNAs. Myoblasts Finally, we could not detect NFATc2 in any condition, although its were transfected with siRNAs and differentiation was initiated at expression has been described in human adult muscle and is confluency. Total RNA was extracted after 2 days of differentiation proposed to have a role in murine myoblast myogenesis (Hoey et al., and analyzed by qPCR to determine the mRNA expression of NFAT 1995; Horsley et al., 2003). genes. Results obtained with these siRNAs are presented in Fig. 3. The complex expression profile observed for NFATc1, NFATc3 By this approach, we could evaluate the impact of NFATc1, and NFATc4 mRNAs prompted us to compare NFAT expression NFATc3 and NFATc4 silencing on the genotypic differentiation of

Fig. 1. NFATc1, NFATc3 and NFATc4 expression during human primary myoblast differentiation. RNA samples were analyzed for NFATc1, NFATc2, NFATc3 and NFATc4 mRNAs. RNA was extracted from proliferating myoblasts (GM), from myoblasts at the indicated times during differentiation (DM), from myotubes (MT) that formed after 48 h and 72 h in DM, and from reserve cells (RC) formed after 48 h and 72 h in DM. Results of qRT-PCR are expressed as fold change normalized to housekeeping genes followed by normalization to the GM condition. Results are expressed as means±s.e.m. (n=4). A.U., arbitrary units. Journal of Cell Science

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Fig. 2. Precise chronology of protein expression of four differentiation markers during human primary myoblast differentiation. Proteins were extracted in different conditions: during proliferation (GM), at the indicated times during differentiation (DM), and in isolated myotubes (MT) and reserve cells (RC) after 48 h and 72 h in DM. Protein expression of the differentiation markers myogenin, MEF2C, MyHC and STIM1L were analyzed by western blotting. (A) Quantification of western blot experiments, normalized to α-tubulin expression. Results are expressed as means±s.e.m. (n=4). (B) Representative blots of the four proteins together with α-tubulin expression as a loading control. Membranes were cut horizontally to allow several antibody detections. Images were cropped to mask lanes of non-studied samples. A.U., arbitrary units. human myoblasts. We observed that, while downregulation of silencing reduces MyHC and STIM1L expression by 40% and NFATc1 and NFATc4 has a clear impact on myoblast differentiation 83%, respectively, compared to the 14% and 43% decrease after (Fig. 4A,C), NFATc3 silencing did not show any significant NFATc4 silencing. Furthermore, it is important to note that the change in the expression of the tested differentiation markers effect of NFATc1 silencing on MyHC expression can be rescued by (Fig. 4B). Unexpectedly, although calcineurin inhibition almost overexpressing NFATc1. Indeed, the expression of a mutated abolished the expression of both early and late differentiation EGFP–NFATc1 construct that is able to resist siNFATc1 silencing markers (Fig. 4D), the decrease of NFATc1 or NFATc4 expression without modification of the protein sequence, increased the did not affect the expression of early markers. Indeed, NFATc1 and expression of MyHC to normal levels (Fig. S2A). In line with NFATc4 knockdown decreased the expression of late markers of these observations, larger myotubes were obtained after EGFP– differentiation, MyHC and STIM1L, without significantly affecting NFATc1 overexpression, reinforcing the role of NFATc1 in the early markers myogenin and MEF2C (Fig. 4A,C). It is, however, myoblast fusion (Fig. S2B). worth noting that NFATc1 silencing more efficiently reduces the These results suggest that NFATc1 and NFATc4 are both late differentiation process than NFATc4 silencing. NFATc1 implicated during the late steps of myoblast differentiation (24 h Journal of Cell Science

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Fig. 3. Specific knockdown of NFATc1, NFATc3 and NFATc4 with siRNAs. Myoblasts were transfected in growth medium with either siControl, siNFATc1 no. 5, siNFATc3 no. 3 or siNFATc4 no. 4. After 48 h of differentiation, NFATc1, NFATc3 and NFATc4 mRNA expression was quantified by qRT-PCR. The fold change is normalized to housekeeping genes followed by normalization to the siControl condition (gray line on graphs). Results are expressed as means±s.e.m. (n=4–7). **P≤0.01; ****P≤0.0001; ns, not significant (P>0.05). A.U., arbitrary units. after the onset of differentiation), while the early steps are also change after NFATc1 knockdown, while a significant increase is controlled by calcineurin but probably via NFAT-independent observed after NFATc4 silencing (Fig. 6B). This increase was mechanisms. expected and reflects an increased fusion. On the other hand, as shown in Fig. 6B, NFATc1 silencing leads to an increase in the NFATc3 knockdown does not impede primary human percentage of mononucleated MEF2-positive cells (the proportion myoblast differentiation of MEF2-positive nuclei outside myotubes increased from Fig. 4B illustrates that genotypic differentiation was not impacted by 22% for siControl to 48% for siNFATc1). Thus, myoblasts still NFATc3 knockdown. This result was unexpected as others have differentiate but do not fuse. These results show that the fusion described a role for NFATc3 in mouse myogenesis and mouse process rather than the process of differentiation is affected by muscle differentiation (Delling et al., 2000; Kegley et al., 2001). NFATc1 silencing. To strengthen our results, we first confirmed the efficiency of Together, these experiments suggest that even if both siNFATc1 siNFATc3, at the protein level by western blotting. Fig. 5A clearly and siNFATc4 similarly downregulated STIM1L and MyHC shows that after 2 days of siNFATc3 transfection in proliferation expression, NFATc1 and NFATc4 silencing differentially affects conditions (GM), NFATc3 protein expression was strongly reduced. the process of myotube formation, and especially myotube fusion. Moreover, this inhibition was conserved after 2 additional days in differentiation conditions (DM). Since NFATc3 did not affect Knockdown of NFATc4 decreases the pool of reserve cells genotypic differentiation, we then evaluated a putative role of without affecting the proportion of Pax7-expressing cells NFATc3 on the phenotypic differentiation (number, size or Reserve cells are quiescent non-fusing cells that do not express morphology) of the formed myotubes. To this end, we transfected muscle differentiation markers but share some markers and myoblasts with siNFATc3, and after 2 days of differentiation, cells properties of satellite cells. Their stemness is marked by the were immunostained for MEF2 proteins and MyHC. Fig. 5B expression of the transcription factor Pax7, which is also expressed illustrates that, after NFATc3 knockdown, myotubes form as in by satellite cells. Reserve cells, as can satellite cells, are able to give control conditions. These observations confirm that NFATc3 is rise to a de novo myoblast population (Baroffio et al., 1996; Yoshida neither implicated in the genotypic differentiation nor in the et al., 1998). We already demonstrated a stronger NFATc4 phenotypic differentiation of human myoblasts. expression in reserve cells as compared to myotubes (Fig. 1), and a decrease of the reserve pool by twofold after NFATc4 knockdown NFATc1 and NFATc4 knockdown differentially affects the (Fig. 6B). Considering these results, we wondered whether NFATc4 phenotypic differentiation of human primary myoblasts could have a role in the maintenance of Pax7 expression in reserve We next investigated the effect of NFATc1 and NFATc4 silencing cells. Pax7 expression was assessed in reserve cells after 2 days of on the phenotypic differentiation of human myoblasts. MEF2 differentiation and, as illustrated in Fig. 7A,B, Pax7-positive cells and troponin T immunostainings at 2 days after the onset of are decreased by twofold after NFATc4 knockdown. Keeping in differentiation were performed following either NFATc1 or mind that NFATc4 knockdown also decreases by twofold the NFATc4 silencing (troponin T immunostaining was used for reserve cell pool (Fig. 6), we can deduce that the proportion of these experiments as NFATc1 and NFATc4 knockdown reduces reserve cells expressing Pax7 is not modified after NFATc4 MyHC expression). Fig. 6 shows that both NFATc1 and NFATc4 knockdown. knockdown strongly impacted phenotypic differentiation, although It has been shown that NFATc4 is required for neuronal survival by remarkably the two knockdowns have opposite effects. Indeed, after controlling the expression of pro-survival genes (Benedito et al., NFATc1 knockdown, we observe a robust inhibition of myotube 2005), and Fig. 1 shows that NFATc4 is upregulated in the reserve formation characterized by a strong reduction in their surface area, cell population. The reduction of the reserve cell pool observed after 13,000 µm2 instead of 63,000 µm2, and the incorporation of half the NFATc4 knockdown could thus be due either to an augmentation of number of nuclei into myotubes. On the other hand, NFATc4 myoblast fusion or to a loss of reserve cells (or both). However, as knockdown increases strongly the size of myotubes leading to a NFATc4 knockdown does not significantly decrease the total mean cell surface area of 90,000 µm2, associated with a significant (myotubes and reserve cells) number of nuclei (Fig. 7C), we increase in the number of nuclei incorporated into myotubes. The conclude that the decrease of reserve cells after NFATc4 knockdown percentage of nuclei expressing the early marker MEF2 did not is only due to an increase of myoblast fusion into myotubes. Journal of Cell Science

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Fig. 4. Knockdown of NFATc1 and NFATc4 decreases expression of late differentiation markers. (A–C) Myoblasts were transfected in growth medium with either siControl (siCtl), siNFATc1 no. 5, siNFATc3 no. 3 or siNFATc4 no. 4 (denoted siNc1, 3, 4). At confluency, myoblasts were induced to differentiate. (D) Myoblasts transfected with siControl were passaged in differentiation medium supplemented with the calcineurin inhibitors (Cn Inhib.) FK506 and Cyclosporin A (both at 8 μM). (A–D) After 48 h of differentiation, myogenin, MEF2C, MyHC and STIM1L expressions were analyzed by western blotting. Left panels: quantifications are expressed as the ratio to α-tubulin expression normalized to the siControl condition, which was set to 100. Results are expressed as means±s.e.m. (n=3–8). Arrows represent the appearance of differentiation marker expression during the course of differentiation. Right panels, representative blots of the four proteins with α-tubulin expression as a loading control. Membranes were cut horizontally to allow several antibody detections. Images were cropped to mask lanes of non-studied samples. NFATc1 knockdown and calcineurin inhibition conditions share the same control bands, as they were blotted on the same membrane. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001. A.U., arbitrary units.

DISCUSSION qRT-PCR, we observed that three out of the four Ca2+-sensitive NFAT expression NFAT isoforms are expressed in proliferating myoblasts: NFATc1, In the present study, we assessed the role of each NFAT family NFATc3 and NFATc4. We also observed that, for each of them, member during human primary myoblast differentiation. By using mRNA expression increased during the differentiation process. Journal of Cell Science

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Fig. 5. Knockdown of NFATc3 does not impede human primary myoblast phenotypic differentiation. Myoblasts were transfected in growth medium with siControl (siCtl) or siNFATc3 no. 3 (siNc3). At confluency, myoblasts were induced to differentiate. (A) NFATc3 expression was analyzed by western blotting in proliferation medium at 2 days post transfection, and after culture in DM for 48 h. Left, quantification of experiments normalized to α-tubulin expression, followed by normalization to siControl at 48 h DM (set to 100). Results are expressed as means± s.e.m. (n=3). Right, representative blot of NFATc3 expression with α-tubulin expression as a loading control. Membranes were cut horizontally to allow several antibody detections. Images were cropped to mask lanes of non-studied samples. (B) Cells were fixed after 48 h of differentiation and immunostained for MEF2 proteins (red) and MyHC (green). Nuclei were stained with DAPI (blue). Scale bar: 50 μm. A.U., arbitrary units.

Conversely, NFATc2 was not detected in human primary with CsA impedes myoblast differentiation and fusion (Abbott myoblasts, neither during proliferation nor during differentiation. et al., 1998; Friday et al., 2000; Konig et al., 2006; Sakuma et al., NFATc1 was more highly expressed in myotubes while NFATc4 2003). The inhibition of myoblast differentiation obtained with CsA was more highly expressed in reserve cells. NFATc3 showed a is associated with a strong reduction of the expression of the early strong expression in both cell types. Hoey et al. (1995) were the first marker myogenin, which suggests that calcineurin is important to to describe NFAT expression in adult human skeletal muscle. As initiate the differentiation process (Friday et al., 2000). Accordingly, determined by northern blotting, they showed all four NFAT the present work shows that calcineurin inhibition almost entirely isoforms were present, with a higher expression of NFATc1 and abolishes myogenin and MEF2 expression and, therefore, myoblast NFATc3, and a weaker expression of NFATc2 and NFATc4. Our differentiation. The very strong impact of calcineurin inhibition observation that NFATc1 and NFATc3 are strongly expressed in on myoblast differentiation could be explained by the presence myotubes correlates well with the strong expression in mature adult of additional downstream targets other than NFAT. It has been skeletal muscle. Likewise, the weak NFATc4 expression in shown that calcineurin inhibition blocks chromatin remodeling at myotubes is in line with the low expression in adult skeletal the onset of myoblast differentiation and therefore reduces the muscle. Concerning NFATc2, its absence in human cultured expression of myogenic genes (Nasipak et al., 2015). In addition, myotubes could imply a role later during fiber maturation or in that study showed that initiation of myoblast differentiation requires mature muscle homeostasis. dephosphorylation by calcineurin of the Brg1 ATPase (also known as SMARCA4) of the SWI/SNF chromatin-remodeling enzyme Calcineurin-dependent differentiation family. All these results are in agreement with our previous work The Ca2+-sensitive pathway calcineurin plays an important role in showing that the induction of differentiation depends on Ca2+ muscle regeneration. In vivo studies demonstrated that inhibition of signaling (Darbellay et al., 2009). Interestingly, Friday et al. (2000) calcineurin strongly impaired muscle repair. Indeed, mice or rats proposed that NFAT activity is not required for the initiation of treated with the calcineurin inhibitor Cyclosporine A (CsA) exhibit differentiation of the L6 muscle cell line, as they observed that impaired muscle regeneration associated with a decrease in muscle inhibition of NFAT by GFP–VIVIT did not inhibit myogenin and weight and cross-sectional area (Abbott et al., 1998; Sakuma et al., MyHC expression. VIVIT is an inhibitory peptide preventing

2003). Consistently, several in vitro studies showed that treatment calcineurin–NFAT interaction without interfering with calcineurin Journal of Cell Science

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Fig. 6. Knockdown of NFATc1 and NFATc4 differentially affect the phenotypic differentiation of human primary myoblasts. (A) Myoblasts were transfected in growth medium with either siControl, siNFATc1 no. 5 or siNFATc4 no. 4. At confluency, myoblasts were induced to differentiate. Immunostaining was performed after 48 h of differentiation against MEF2 proteins (red) and troponin T (green). Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (B) Quantification of immunofluorescence experiments. The surface area of myotubes (µm2) was analyzed with Metamorph software. The proportion of MEF2- positive nuclei, nuclei outside myotubes and total nuclei were manually calculated with an ImageJ tool. Results are expressed as means±s.e.m. (n=3–45). *P≤0.05. phosphatase activity (Yu et al., 2007). However, different studies in will lead to Brg1 activation and to chromatin remodeling, allowing different species, including the present study, propose that each myogenin expression; and then at a second later time point that will NFAT protein could be specifically and differentially active during trigger NFAT activation. In this case, this later NFAT activation step the various stages of myoblast differentiation (Abbott et al., 1998; could require stronger calcineurin activation. This hypothesis is Delling et al., 2000). Thus, myoblast differentiation could require supported by our previous work showing that NFAT activity 2+ successive calcineurin activations: once at an earlier time point, that depends on a Kir2.1-induced hyperpolarization that increases Ca Journal of Cell Science

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Fig. 7. Knockdown of NFATc4 affects neither the proportion of Pax7-positive cells in reserve cells nor reserve cell survival. (A) Myoblasts were transfected in growth medium with either siControl or siNFATc4 no. 4. At confluency, myoblasts were induced to differentiate. Immunostaining was performed after 48 h of differentiation against Pax7 (green). Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (B,C) Quantification of immunofluorescence experiments. Pax7-positive nuclei (B) and total nuclei (C) were manually counted with an ImageJ tool. Results are expressed as means±s.e.m. (n=3–6). *P≤0.05; ns, not significant (P>0.05).

entry and that takes place 6 h after induction of differentiation expected for NFATc2. However, our results are strengthened by (Konig et al., 2006, 2004). another study in a murine model suggesting that NFATc1 is implicated in muscle regeneration as a downstream effector of A specific role for each NFAT isoform during myogenesis nuclear receptor interaction protein (NRIP) (Chen et al., 2015). In The roles of different NFAT isoforms have been studied by the use this work, it is proposed that NRIP, as an activator of both the of NFAT-knockout mice models. NFATc1−/− is lethal, although calcineurin–NFATc1 and CaMKII pathways, plays a role in conditional knockout in skeletal muscle causes a defect in fiber myotube formation. We recently published that STIM1L typing but no reduction in muscle mass, suggesting a role for downregulation induces a decrease in the number of nuclei per NFATc1 in late fiber maturation (Ehlers et al., 2014). NFATc2−/− myotube (Antigny et al., 2017). Hence, it is unlikely that inhibition mice exhibit a skeletal muscle growth impairment, with defects in of myoblast fusion after NFATc1 downregulation would be the recruitment and/or fusion of myogenic cells with nascent consequence of a lower STIM1L expression, as downregulation of multinucleated muscle cells (Horsley et al., 2001). NFATc3−/− either NFATc1 or NFATc4 leads to a similar reduction of late gene mice show defects in primary fiber formation during embryogenesis expression, although each is associated with specific and different (Kegley et al., 2001). Finally, to our knowledge, NFATc4−/− has phenotypes. It is more likely that these phenotypic alterations are the never been shown to be associated with an abnormal skeletal muscle direct consequence of NFAT downregulation. Nevertheless, our phenotype (Graef et al., 2001). In our model, NFATc3 knockdown work strengthens previous results showing that at least one isoform has no effect on postnatal human myoblast differentiation, an of NFAT, NFATc1, is implicated in myoblast fusion and myotube observation that could be explained by a role of NFATc3 restricted growth. to embryonic myogenesis. We also observed a strong effect of both NFATc1 and NFATc4 knockdown on expression of late Reserve cells differentiation markers and on myotube formation. However, more We describe for the first time the endogenous expression of a importantly, we showed that NFATc1 and NFATc4 have a specific specific NFAT gene in reserve cells during differentiation. We could effect that can be associated with the specific pattern of NFATc1 show that NFATc4 is expressed in proliferating myoblasts, and that (myotube) and NFATc4 (reserve cell) expression. We found that its later expression is maintained only in reserve cells. The NFATc1 knockdown decreased myoblast fusion, inducing a downregulation of NFATc4 during the differentiation process reduction of myotube size whereas NFATc1 overexpression has induces a decrease in the number of reserve cells associated with the opposite effect, an increase of myotube size. Judging from the an increase in myotube size. Based on these results, we propose a results obtained with transgenic mice, this role would have been role for NFATc4 in the maintenance of the reserve cell population. Journal of Cell Science

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Fig. 8. Schematic representation of the NFATc1 and NFATc4 roles during human primary myoblast differentiation. NFATc1 and NFATc4 are both required for normal expression of the late differentiation markers MyHC and STIM1L. Absence of NFATc1 impairs myotube formation, whereas absence of NFATc4 increases myotube size by decreasing the reserve cell pool.

A specific calcineurin–NFAT pathway has been suggested to exist were repeated at least three times to ensure the reproducibility of our data, in murine reserve cells by Friday and Pavlath (2001). Indeed, they and n numbers correspond to the number of different clonal populations observed that the overexpression of NFATc1 increased Myf5 used. A total of 19 different clonal populations were used. For calcineurin inhibition, the drugs FK506 and Cyclosporine A (Abcam Biochemicals) expression, a protein expressed in reserve cells but not in myotubes. μ Although little is known concerning the quiescent state of satellite were both diluted in DM at 8 M. cells or reserve cells and their activation, the roles of Notch, Pax7 and Myotube and reserve cell isolation MyoD have emerged recently as being essential. Indeed, the After 2 days of differentiation, myotubes can be separated from reserve constitutive activation of Notch promotes self-renewal of satellite cells. The medium is first removed and cells are rinsed with PBS. Cells are cells by increasing Pax7 expression (Bröhl et al., 2012; Wen et al., then incubated for 3 min with Trypsin-EDTA. Cells are observed under a 2012). Conversely, inhibition of Notch signaling induces direct light microscope to ensure that during this time only myotubes detach. fusion of the reserve cells with myotubes leading to muscle Myotubes are quickly and gently harvested in a tube containing GM. They hypertrophy (Kitzmann et al., 2006). Prox1, a transcription factor are allowed to sediment during 5 min and then as much of the supernatant as expressed in satellite cells, is implicated in muscle differentiation possible is removed. At the same time, a second incubation with trypsin- through its interaction with the Notch and NFAT pathways (Kivelä EDTA is performed on the remaining cells during 3 to 5 min. The reaction is et al., 2016). Prox1 expression is induced during differentiation and stopped with GM and cells are filtered on a 20 µm cell-strainer. This step activates NFATc1-3 signaling while Prox1 silencing triggers Notch1 allows reserve cells to be isolated and removes any remaining myotubes. Myotubes and reserve cells are then centrifuged, rinsed twice in PBS and activation. Taken together, these results strengthen the notion that the treated for protein or RNA extraction. Notch and the NFATc1–NFATc3 pathways are mutually exclusive during muscle regeneration. Our results propose specific roles for siRNA-mediated knockdown NFATc1 and NFATc4 during muscle regeneration, and thereby open Transfections were realized by incubating myoblasts in suspension with a new and unexpected perspectives concerning NFATc4 implication in solution containing a ratio of 1 ml Opti-MEM® (Gibco), 6 µl Lipofectamine® the self-renewal of quiescent muscle cells. RNAiMax (Invitrogen) and 40 pmol siRNA (Qiagen). The number of cells per volume of transfection solution was similar in all transfection assays. Confluence Conclusion was reached between 2 and 4 days in GM. Downregulation by siRNAs was still The purpose of our study was to better understand the role of the optimal after 4–6 days of transfection. For all experiments described in this study, calcineurin–NFAT pathway during human myoblast differentiation. the siNFATc1 no. 5 (target sequence 5′-CACCATGTCTGGGAGATGGAA-3′, Qiagen) was used for NFATc1 knockdown, the siNFATc3 no. 3 (target sequence Calcineurin is described to have a broad action that is essential for ′ ′ the initiation and the maintenance of myoblast differentiation. We 5 -CCGGGAGACTTCAATAGATGA-3 , Qiagen) was used for NFATc3 knockdown, the siNFATc4 no. 4 from Qiagen (target sequence 5′-CACCGTAG- showed, in addition, that expression of the late differentiation GTACTGAGTACAA-3′, Qiagen) was used for NFATc4 knockdown, and the markers (MyHC and STIM1L) are NFAT dependent, and that negative control siRNA (target sequence 5′-AATTCTCCGAACGTGTCAC- NFATc1 and NFATc4 have specific roles during some crucial steps GT-3′, Qiagen) was used as a negative control. of human muscle regeneration (Fig. 8). NFATc1 is important for the growth of myotubes while NFATc4 is essential for the maintenance Plasmid transfection of the pool of quiescent reserve cells. Myoblasts were transfected by electroporation using the Amaxa Nucleofector II device. Briefly, two million myoblasts were pelleted, MATERIALS AND METHODS suspended in 100 μl of the provided buffer (NHDF Nucleofector Solution, Cell culture VPD-1001, Lonza) supplemented with 2 μg of plasmid and electroporated Clonal cultures of human primary myoblasts were expanded from single using the program P-022. EGFPC1-huNFATc1EE-WT was Addgene satellite cells as previously described (Leroy et al., 2013). Human muscle plasmid #24219 (deposited by from Jerry Crabtree) (Beals et al., 1997). samples were obtained from orthopedic surgery on patients without known An siRNA-insensitive form of this construct was obtained by point mutation muscular diseases. All samples were collected anonymously, with written using the primers: 5′-CGGTCAGTTTTCGCCTCCATTTCCCATACGT- consent, as described in our ethics consent approved by the University GATGGCCATCTGGGGCTTTCTC-3′ and 5′-GAGAAAGCCCCAGAT- Hospital of Geneva Research Committee on the use of humans as GGCCATCACGTATGGGAAATGGAGGCGAAAACTGACCG-3′. experimental subjects (Protocol 05-078). Cells were expanded in a serum- rich medium called growth medium (GM), and differentiation is initiated at NanoString Technologies’ nCounter system confluency in a serum-deprived differentiation medium (DM), media RNA extraction was performed with TRIzol reagent (Life Technologies) composition is as in Leroy et al., 2013. All human myoblasts used in the according to the manufacturer’s protocol (phenol–chloroform RNA study had achieved between 20 and 30 divisions before experiments. Assays isolation). Then, experiments were all performed at the iGE3 Genomics Journal of Cell Science

3091 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3083-3093 doi:10.1242/jcs.198978

Platform of the University of Geneva (http://www.ige3.unige.ch/genomics- 1:10,000) antibodies. Secondary antibodies used were goat anti-mouse-IgG platform.php). 1 mg of RNA per sample was hybridized with multiplexed (H+L) HRP conjugate (#1721011, Bio-Rad, 1:6000) and goat anti-rabbit-IgG NanoString probes and samples were processed according to the published (H+L) HRP conjugate (#7074, Cell Signaling, 1:6000). procedure (Geiss et al., 2008). Five housekeeping genes were provided by NanoString Technologies. Barcodes were counted for 1150 fields of view Immunofluorescence per sample. For each sample, background correction was applied by Cells were fixed in PBS 4% paraformaldehyde, permeabilized in PBS 0.25% subtracting the value of the mean plus two standard deviations of the Triton X-100, and then blocked in PBS with 2% goat serum and 2% Tween negative controls. Values obtained that were less than 1 were set to 20. Proteins of interested were detected by incubation of specific primary 1. Positive controls were used as quality assessment: the ratio between the antibodies overnight at 4°C, followed by incubation of fluorophore- highest and the lowest positive control average among samples was conjugated secondary antibodies together with DAPI (0.3 µM; confirmed to be below 3. Then, using the geNorm algorithm Sigma-Aldrich) for 1 h at room temperature. Primary antibodies used were: (Vandesompele et al., 2002), counts for target genes were normalized anti-MEF2A-C-D (rabbit Ab, clone C-21, Santa Cruz Biotechnology, 1:200), with the geometric mean of reference genes (β2-microglobulin, HPRT1, anti-MyHC (mouse mAb, MF20, Developmental Studies Hybridoma Bank, TBP and EEF1A1 human genes were selected as the most stable). 1:200), anti-troponin T (mouse mAb, Clone JLT-12, Sigma-Aldrich, 1:200), and anti-Pax7 (mouse mAb, Developmental Studies Hybridoma Bank, 1:250; qRT-PCR deposited to the DSHB by A. Kawakami). Secondary antibodies used were The same RNA samples used for the nCounter system were used for Alexa Fluor® 488-conjugated goat anti-mouse IgG (H+L) (#A11034, Life qRT-PCR experiments. Reverse transcription and qPCR experiments were Technologies, 1:1000) and Alexa Fluor® 546-conjugated goat anti-rabbit IgG all performed at the iGE3 Genomics Platform of the University of (H+L) (#A11035, Life Technologies, 1:1000). Images were acquired using a Geneva (http://www.ige3.unige.ch/genomics-platform.php). After measuring Zeiss Axioskop 2 microscope (multi-channel fluorescence imaging in wide sample concentrations, 0.5 µg of total RNA was reverse-transcribed with the field microscopy) for siNFATc3, and using a Leica SP5 microscope PrimeScript™ RT reagent Kit (TaKaRa) according to the manufacturer’s (Confocal Laser Scanning Microscopy) for siNFATc1 and siNFATc4. Six instructions. The four NFAT genes (NFATc1– NFATc4) and two human random fields were acquired with a 40× objective in each condition. Analysis housekeeping genes (β2-microglobulin and human TBP) were assessed. of myotube surface area was carried out using Metamorph 6.3r7 software PCR was performed at the Platform on an SDS 7900 HT instrument (Molecular Devices Corp., Visitron Systems GmbH, Puchheim, Germany). (Applied Biosystems). Raw threshold-cycle (Ct) values obtained with SDS Analysis of Pax7- or MEF2-positive cells and the count of total nuclei were 2.2 (Applied Biosystems) were imported into Excel. Normalization factor assessed manually using the Cell Counter plugin of ImageJ Software. and fold changes were calculated using the GeNorm method (Vandesompele et al., 2002). Fold changes obtained for each clone and Statistical analysis each condition were then normalized to the siControl or GM conditions. GraphPad Prism Software was used for all the statistical analyses. Results Primers used for RT-PCR were: Hs β2-microglobulin forward, 5′-TGCT- are expressed as the mean±s.e.m. of n observations. An ANOVA or t-test CGCGCTACTCTCTCTTT-3′, reverse, 5′-TCTGCTGGATGACGTGAG- was used as comparison tests. Experiments were considered parametric TAAAC-3′; Hs TBP forward, 5′-GCCCGAAACGCCGAATATA-3′, and paired. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001; ns, not reverse, 5′-CGTGGCTCTCTTATCCTCATGA-3′; Hs NFATc1 forward, significant (P>0.05). 5′-CCCAGATGGCCACCATGT-3′, reverse, 5′-AGGTCCCGGTCAGTT- TTCG-3′; Hs NFATc2 forward, 5′-GGAGGCCACCCTGTGGTT-3′, Acknowledgements reverse, 5′-CAGAGGCTTGTTTTCCATGTAGC-3′; Hs NFATc3 forward, We thank the iGE3 Genomics Platform of the University of Geneva (http://www.ige3. 5′-TCGCGGCCTGCAGATC-3′, reverse, 5′-AAAGATGTAAATGGATG- unige.ch/genomics-platform.php) for their collaboration in the qRT-PCR and CACAATCAT-3′;HsNFATc4forward,5′-CATCCTACAGACCGGGCCT- nCOunter experiments, and the Developmental Studies Hybridoma Bank (University 3′; reverse, 5′-CCTGTGGTACCCCTAGTCTCAGG-3′. of Iowa) for providing antibodies against Pax7 and MyHC. We thank P. Brawand for technical assistance, Dr Thomas Laumonier for helpful discussions, and Dr Paula Nunes-Hasler for her language editing services and helpful discussion. Western blotting Protein extraction was performed using a CHAPS/NP40 cell lysis buffer Competing interests (0.50% CHAPS, 0.50% Nonidet NP-40, 50 mM Tris-HCl pH7.5, 150 mM The authors declare no competing or financial interests. NaCl, 5 mM EDTA, 10% glycerol) supplemented with phosphatase and protease inhibitor cocktails (PhosSTOP and cOmplete Mini from Roche). Author contributions Protein concentrations were obtained with a Bradford-based method Conceptualization: J.P., L.B., M.F., S.K.K.; Methodology: J.P., M.F., S.K.K.; (Protein Assay Dye Reagent from Bio-Rad). Total proteins were prepared Validation: S.K.K.; Formal analysis: J.P., S.K.K.; Investigation: J.P.; Writing - original in a 5% β-mercaptoethanol Laemmli buffer. The same quantities of proteins draft: J.P., L.B., M.F., S.K.K.; Writing - review & editing: L.B., M.F., S.K.K.; (10 µg to 20 µg depending on experiments) were separated on 8% SDS- Supervision: L.B., M.F., S.K.K.; Project administration: S.K.K.; Funding acquisition: PAGE gels and then transferred onto nitrocellulose membranes. Membranes L.B., M.F. were blocked with a PVA solution (Polyvinyl Alcohol 146–186k MW, 0.01%), and then sequentially blotted with specific primary antibodies and Funding ̈ secondary horseradish peroxidase (HRP)-conjugated antibodies, diluted in This work was supported by the Schweizerischer Nationalfonds zur Forderung der Wissenschaftlichen Forschung (Swiss National Science Foundation, grant number 5% bovine serum albumin (BSA) in TBST (0.1% Tween 20, 20 mM Tris- 310030-166313); Stiftung für die Erforschung der Muskelkrankheiten and the HCl pH 7.5, and 137 mM NaCl) and 5% non-fat milk in TBST respectively. Fondation Marcel Levaillant. Western blotting was revealed by chemiluminescence (western-lightning plus ECL, Perkin Elmer; Hyperfilm MP, Amersham Biosciences). Band Data availability © intensities were analyzed with the gel analysis plugin of ImageJ Software. The datasets supporting the conclusions of this article are all included within the α-Tubulin was used as a loading control for a first normalization, and then article and within its additional figures. each condition was normalized to the siControl or GM conditions. Primary antibodies used were: anti-myogenin (Mouse mAb, clone F5D, BD Supplementary information Pharmingen, 1:1500), anti-MEF2A (rabbit Ab, #9736 Cell Signaling, Supplementary information available online at 1:750), anti-MEF2C (rabbit mAb XP®, #D80C1, Cell Signaling, 1:1500), http://jcs.biologists.org/lookup/doi/10.1242/jcs.198978.supplemental anti-MyHC (mouse mAb, MF20, Developmental Studies Hybridoma Bank, References 1:2000; deposited to the DSHB by D.A. Fischman), anti-STIM1 (rabbit Ab, Abbott, K. L., Friday, B. B., Thaloor, D., Murphy, T. J. and Pavlath, G. K. (1998). AB9870, Millipore, 1:2000), anti-NFATc3 (rabbit Ab, #4998, Cell Signaling, Activation and cellular localization of the cyclosporine A-sensitive transcription

1:1000) and anti-αTubulin (mouse mAb, clone DM1A, Sigma-Aldrich, factor NF-AT in skeletal muscle cells. Mol. Biol. Cell 9, 2905-2916. Journal of Cell Science

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