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Nanopattern surface improves cultured human myotube maturation

Nanopattern surface improves cultured human myotube maturation Background: In vitro maturation of human primary myoblasts using 2D culture remains a challenging process and leads to immature fibers with poor internal organization and function. This would however represent a valuable system to study muscle physiology or pathophysiology from patient myoblasts, at a single-cell level. Methods: Human primary myoblasts were cultured on 800-nm wide striated surface between two layers of Matrigel, 2+ and in a media supplemented with an inhibitor of TGFβ receptor. Gene expression, immunofluorescence, and Ca measurements upon electrical stimulations were performed at various time points during maturation to assess the organization and function of the myotubes. Results: We show that after 10 days in culture, myotubes display numerous functional acetylcholine receptor clusters and express the adult isoforms of myosin heavy chain and dihydropyridine receptor. In addition, the myotubes are internally well organized with striations of α-actinin and STIM1, and occasionally ryanodine receptor 1. We also 2+ demonstrate that the myotubes present robust Ca responses to repetitive electrical stimulations. Conclusion: The present method describes a fast and efficient system to obtain well matured and functional 2+ myotubes in 2D culture allowing thorough analysis of single-cell Ca signals. 2+ Keywords: Human primary myoblasts, Cell alignment, Myotube maturation, Acetylcholine receptor clusters, Ca signals Background organization of muscle fibers, and even if important pro- Skeletal myofibers are large multinucleated cells with an ex- gresses were accomplished over the years, it remains challen- ceptional level of internal organization dedicated to produce ging to obtain well differentiated and matured myofibers strength upon nerve activation. Acetylcholine, released from in vitro. Several models of myogenesis were designed to gen- nerve terminals, induced membrane depolarization (action erate muscle tissue that could be eventually engrafted in 2+ potential) that is sensed by voltage-gated Ca channels, damaged/diseased muscles [1], to provide suitable models to Cav1.1, also called dihydropyridine receptor (DHPR). The study pathophysiological mechanisms of the disease [2, 3], or conformational change of DHPR is transmitted to the ryano- to understand the various steps of muscle formation [4, 5]. 2+ dine receptor 1 (RyR1), a Ca channel localized at the ter- The sources of cells used to perform in vitro culture are di- minal cisternae of sarcoplasmic reticulum (SR). The opening verse, such as C2C12 mouse cell line or primary myoblasts 2+ of RyR1 leads to a strong release of Ca from the SR and of murine or human origin. As well, cultures derived from eventually muscle contraction, an overall process called exci- human iPSC are used with promising outcomes [6, 7]. In the tation–contraction (EC) coupling. Because of thesizeand recent years, several studies have highlighted the benefits of establishing 3D compared with 2D culture. Indeed, 3D ap- proaches promote the level of maturation and contractility of * Correspondence: maud.frieden@unige.ch myotubes/myofibers and allow to keep cells longer in cul- Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland ture. The improved internal architecture, the presence of © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Brunetti et al. Skeletal Muscle (2021) 11:12 Page 2 of 14 acetylcholine receptor (AChR) clusters, and the higher con- Table 1 Cell culture media tractility are among the main advantages of 3D versus 2D Concentration Reference culture [8]. However, 2D cultures are likely more appropriate Growth medium (GM) for single-cell level analysis, but myotubes differentiated on F-10 (Ham’s) ThermoFisher 31550-023 2D frequently do notmatureproperlywithoften alack of BSA 0.5 mg/ml Sigma 05482 striations and the appearance of small/broken cells [6][8]. Insulin 0.04 mg/ml I-9278 One cue demonstrated to promote a higher level of myotube Creatine powder 1 mM Fluka 27900 maturation is the cell alignment. Indeed, skeletal muscles are composed of very long and aligned fibers, an organization Dexamethasone 0.39 μg/ml Sigma D1756 that could be recapitulated, at least partially, in culture. To Gentamycin 5 μg/ml Sigma G1272 promote such alignment, self-fabricated substrates are usu- EGF 10 ng/ml Corning 354001 ally created, which are nonetheless complicated to establish FBS 15% Lot 4205662K and require special lab equipment [9–11]. Fetuin 0.5 mg/ml Sigma F-3385 In the present study, we describe an in vitro matur- Sodium pyruvate 100 μg/ml Sigma P4562 ation system that uses striated surface to culture human primary myoblasts. We report evidence of clear benefi- Uridine 50 μg/ml Sigma U3003 cial effects of growing and differentiating muscle cells on Differentiation medium (DM) a striated surface, both at structural and functional DMEM Thermo Fisher 41965-039 levels. We show that on this surface, myocytes display BSA 0.5 mg/ml Sigma 05482 rapidly a high level of internal organization and numer- Insulin 0.01 mg/ml Sigma I5500 ous AChR clustering. Finally, this culture model allows Creatine powder 1 mM Fluka 27900 thorough analysis of single-cell robust and repetitive 2+ Ca transients elicited by electrical field stimulations. Gentamycin 10 μg/ml Thermo Fisher 15710-049 Sodium pyruvate 100 μg/ml Sigma P4562 Methods Uridine 50 μg/ml Sigma U3003 Cell culture Horse serum 1% Amimed 2-05F26-I Human primary myoblasts were isolated from semiten- dinous muscle samples obtained after orthopedic surgery (surgical waste) on patients without known muscular differentiation, the TGFβ inhibitor was removed, and a diseases. All samples were collected anonymously after thick layer of Matrigel diluted at 1:3 in DM was added obtaining a written consent and approval by the Univer- to limit cell detachment [13], and then half of the sity of Geneva (protocol CCER no. PB_2016-01793 (12- medium was changed every 2 days. 259) accepted by the Swiss Regulatory Health Author- ities and approved by the “Commission Cantonale Immunofluorescence d'Ethique de la Recherche” from the Geneva Cantonal Human myotubes were fixed in PBS with 4% parafor- Authorities, Switzerland). The purification of myoblasts maldehyde, permeabilized and blocked in PBS contain- was performed as previously described [12]. ing 0.3% of Triton X-100 and 5% goat serum. Proteins of Myoblasts were seeded either on a conventional dish interest were revealed by incubation of specific primary TM with a flat surface (FluoroDish , Cat n°FD34-100; antibodies overnight at 4 °C, followed by incubation of WPI), or on a nanopattern surface (Anisotropic Nano- fluorophore-conjugated secondary antibodies for 75 min Fabricated Surface, ANFS, Nanosurface Biomedical), that at room temperature. Primary and secondary antibodies has ridges and grooves of 800-nm and 600-nm deep. and their dilution are listed in Table 2. Nuclei were Both surfaces were coated with Matrigel (Corning Matri- stained using ProLong® Gold Antifade Reagent with gel Basement Membrane Matrix Growth Factor Re- DAPI (ref. P36931, Life Technologies). To detect acetyl- duced, Phenol Red Free, #356321) diluted at 1:100 in choline receptors, α-bungarotoxin (α-BTX, B13422, Invi- Ham's F-10 (ThermoFisher) as previously described [13]. trogen) was added together with the secondary Cells were expanded in a growth medium (GM; see antibodies. Images were acquired either with a widefield Table 1), and when they reached around 90% of con- AxioImager M2 microscope (widefield microscopy, fluency, the differentiation was triggered by replacing Zeiss, Germany) through a 20× objective (EC Plan- GM with a differentiation medium (DM; see Table 1). Apochromat 20×/ 0.8), or with a confocal Nikon A1r At the time, cells were placed in DM, and during the spectral microscope (Nikon; Japan) through a 60× ob- first 3–4 days in differentiation, the medium was supple- jective (1.4 CFI Plan Apo Lambda). Three random fields mented with 10 μM of TGFβ receptor I inhibitor were acquired for each dish, with a minimum of two (SB431542, #S4317, Sigma). After 4 days in dishes per experimental condition. Clusters of AChR Brunetti et al. Skeletal Muscle (2021) 11:12 Page 3 of 14 Table 2 List of primary and secondary antibodies Dilution Reference Primary antibodies Mouse anti-(sarcomeric) α-actinin 1:500 Sigma A7811 Mouse anti-Pax7 1:100 DSHB C Mouse anti-RyR1 1:500 DSHB 34C Rabbit anti-MEF2C 1:500 Cell Signaling 5030S Rabbit anti-STIM1 (C-terminal) 1:500 Sigma S6197 Rabbit anti-MyoD1 1:200 Cell Signaling D8G3 Secondary antibodies: Alexa Fluor® 488-conjugated goat anti-mouse IgG (H + L) 1:1000 Life Technologies A11029 Alexa Fluor® 546-conjugated goat anti-mouse IgG (H + L) 1:1000 Life Technologies A11030 Alexa Fluor® 546-conjugated goat anti-rabbit IgG (H + L) 1:1000 Life Technologies A11030 bigger than 5 μm were considered for analysis. Analysis added at the excitation side to limit phototoxicity (694/SP of the images were performed using ImageJ software. BrightLine HC shortpass filter, AHF analysentechnik AG, For display purposes, some pictures were rotated and Germany). Acetylcholine (10 μM, A2661, Sigma-Aldrich) rescaled to have the same resolution. or electrical stimulations (see below) were applied to elicit 2+ Ca responses. Image acquisition was performed at 3.3 Nuclei segmentation analysis Hz with the VisiWiew software, version 4.4.0.11 (Visitron Nuclei were detected based on their DAPI fluorescent Systems, Puchheim, Germany). The fluorescence intensity channel using Cellpose 0.1.0.1 pretrained model [14], was expressed as F/Fo to normalize the data. with automated detection of the diameter. Then the seg- mentation masks and the acquired images were proc- Electric field stimulations essed with Matlab 2020b. Briefly, myocyte enhancer A chamber containing platinum electrodes (RC-37FS, factor 2C (MEF2C) or Myogenic differentiation 1 Cat.64-0366, Warner Instruments) was inserted into the (MyoD)-positive nuclei were analyzed if their individual culture dish. For the experiments performed with cells area was between 40 and 200 μm . Nuclei were consid- grown on the nanopattern surface, the electrodes were ered as part of a cluster if their nearest neighbor dis- oriented in a transversal direction compared with the tance was below 3 μm border-to-border. For each long axis of the cells. The chamber was connected to an isolated nucleus (i.e., not part of a cluster), orientation in electric field stimulator (A310 Accupulser, World Preci- the field of view and eccentricity (that informed about sion Instruments), where the parameters of the stimula- the shape of the nuclei) were computed. Nuclei detec- tion were set up, and to an electric field isolator (A385 tion and segmentation was performed by Nicolas Liau- Stimulus isolator, World Precision Instruments) to sup- det from the Bioimaging core facility of the University of ply a constant current. Geneva (https://www.unige.ch/medecine/bioimaging). Myotubes were stimulated with repetitive bursts of 1 s at 10 Hz every 10 s (each stimulation lasts 2 ms), at 70 Calcium measurements V for 3 min. Three to four rounds of stimulations were Myotubes were loaded with Cal520-AM (5 μM, AAT Bio- performed on each cell culture. Stimulations were per- quest®) and 0.1% Pluronic F-127 (Invitrogen, cat. No formed on a medium containing (mM) 135 NaCl, 5 KCl, 2+ P3000MP), in the dark at 37 °C in Ca -containing solu- 1 MgCl , 10 Hepes, 10 glucose, 0.050 EGTA, and 2 2+ tion. Following an incubation of 90 min, cells were washed CaCl , pH 7.4 (NaOH). The analysis of Ca transient and kept for 30 min to allow de-esterification of the dye. parameters was performed with MatLab (R2020b). Fluorescence was recorded using a Zeiss Axio Observer A1 microscope equipped with a Lambda XL illumination Quantitative and conventional real-time quantitative system (Sutter Instrument, Novato, CA, USA). The excita- polymerase chain reaction (RT-PCR) tion wavelength was 480 nm (ET480/20×; Chroma), and RNA was isolated using TRI Reagent Solution (AM9738, emission was collected through a T505lpxr dichroic mir- Invitrogen™) from myoblasts, myotubes differentiated for 4 ror (Chroma) and a 510WB40 filter (Omega Optical) by a and 10 days, and from adult muscles, according to the manu- cooled 16-bit CMOS camera (pco.Edge sCMOS, Visitron facturer’s instructions. Quantification of the samples, quality Systems, Puchheim, Germany). An attenuation filter was control, reverse transcription, and real-time quantitative Brunetti et al. Skeletal Muscle (2021) 11:12 Page 4 of 14 polymerase chain reaction (RT-qPCR) were all performed at used. DNA fragments of Cav1.1 splice variant, Cav1.1a theiGE3Genomic Platform oftheUniversityofGeneva (adult; 350 bp) and Cav1.1e (embryonic; 250 bp), were (https://ige3.genomics.unige.ch/). Following the measure- separated in 2.2% agarose gel electrophoresis. Each DNA ment of sample concentration, quality of the RNA was deter- fragment was further sequenced by Fasteris DNA Se- mined using the Agilent RNA 6000 Nano Kit and analyzed quencing Service (Fasteris SA, Switzerland). with the Agilent 2100 Bioanalyzer instrument (Agilent Tech- nologies, Germany). From the total RNA, 0.5 μgwas reverse Statistics transcribed with the PrimeScript™ RT reagent kit (TaKaRa, For the analysis of nuclei clusters and shape, statistical ana- Bio Company, Japan) according to the manufacturer’sin- lysis based on Wilcoxon rank sum tests were conducted on struction. The expression of genes listed in Table 3 was eval- the median number of nuclei in clusters and on the median uated during the differentiation and maturation of the eccentricity of each acquisition. The null hypothesis between myotubes and compared with the expression in the adult tis- nuclei cultured on nanopattern and flat surfaces was consid- sue. RT-qPCR was performed on 7900HT instrument (Ap- ered as rejected if the p values were below the significant plied Biosystems™). Raw threshold-cycle (Ct) values obtained level of 0.05. For all other experiments, data are mean ± with SDS 2.2 (Applied Biosystems™)wereimportedinto SEM, and the statistically significant differences were deter- * ** *** Excel. Normalization factor and fold changes were calculated mined using a Student t test, where p <0.05, p <0.01, **** using the GeNorm method [15]. The level of gene expression p < 0.001, and p < 0.00001. was normalized for two housekeeping genes (B2M coding for β2 microglobulin and EEF1A1). Fold changes obtained Results for each condition were normalized to the adult tissues. Myotube differentiation on flat versus striated surfaces For conventional PCR, cDNA from myotubes differen- We first evaluated the benefit of growing and differenti- tiated for 4 and 10 days and from adult muscle were ating human primary myoblasts on a nanopattern Table 3 List of primers Proteins Names Sequences Genes CACNA1S Cav1.1α1s 26-35F CAC CTC CTC CTA CTT TGA ATA 26-35R AGA ACT TCC CAA AGC CCA GA E27F GCT CAT GGC CTT CAA GG ex31R TGA CGA TGA GCA GAG CC RYR1 RyR1 ex-102-103-27F TGG CCA TCA TCC AGG GTC T ex-102-103-77R GGT CTC GGA GCT CAC CAA AAG ATP2A1 SERCA1 ex-15-16-29F CAG TGG CTG GCT CTT CTT CC ex-15-16-79R GCA CCC ACA TAG CCC CC ATP2A2 SERCA2 ex-3V-871F CCT TGA GGA CTC TGC CAA CTT T ex-3V-921R ACG AAG GTC AGA TTG GTC TCA TATT MYH1 MyHC-2X (fast) ex-39-40-48F CAA GCT GAA GAA GCG GAG GA ex-39-40-98R GCG GAA TTT GGA GAG GTT GAC MYH2 MyHC-2A (fast) ex-37-38-64F AAA CTG GAG GCC AGG GTA CG ex-37-38-114R TTG CTC ACT CTC AAC CTC TCC TT MYH3 MyHC-3 7F TCA GAA GCC GAT TCT ACA TGG AC (embryonic) 57R ACA ACT TAG CGG CAC TTG GG MYH7 MyHC-7 (slow) ex-37-38-66F AGG AGC TCA CCT ACC AGA CGG ex-37-38-116R GCA GCC GCA GCA GGT TT Housekeeping genes β2-microglobulin TGC TCG CGC TAC TCT CTC TTT TCT GCT GGA TGA CGT GAG TAA AC EEF1A1 AGC AAA AAT GAC CCA CCA ATG GGC CTG GAT GGT TCA GGT A Brunetti et al. Skeletal Muscle (2021) 11:12 Page 5 of 14 surface (ANSF, see Methods section) compared with percentage of MEF2C positive nuclei were similar be- those on a flat surface. We analyzed the level of myotube tween the two surfaces (Fig. 1b, c). However, differentiation after 4 days in DM, by immunolabeling organization of myotubes was notably different. On the the cells with α-actinin and the transcription factor flat surface, myotubes were mainly randomly oriented, MEF2C (Fig. 1a). The fusion index (number of nuclei while on the nanopattern surface, they were tightly within myotubes/total number of nuclei) and the aligned parallel to the striations (Fig. 1a). The position A MEF2C/ -actinin/DAPI Flat ANFS B C 60 60 0 0 Flat ANFS Flat ANFS Median number of nuclei D Flat ANFS Flat (n=39) ANFS (n=35) Isolated nuclei eccentricity Flat (n=39) ANFS (n=35) Orientation (°) Orientation (°) Fig. 1 Nanopattern surface promotes myotube and nuclei alignment. a Immunofluorescence images of 4-day-old myotubes on flat (left panel) or nanopatterned (ANFS, right panel) surface. Myotubes were stained using antibodies against α-actinin (green) and MEF2C (red) together with a DAPI staining (blue). Scale bar: 50 μm. b,c Quantification of fusion index (b) and MEF2C-positive nuclei (c). Data were obtained from four independent experiments, and each dot is the mean of 3 fields/dish. Error bars are Mean ± SEM. d Nuclei detection based on a pretrained model on flat (left panel) and on nanopattern (right panel) surface. Each cluster of nuclei is depicted with the same color code. e Corresponding distribution of the nuclei orientation on flat (left panel) and on nanopattern surface (right). f–g Median number of nuclei in cluster (f) and median eccentricity of the isolated nuclei (g). Perfect round nuclei would have a value of 0, and elongated nuclei have an eccentricity value closer to 1. Each dot is the median value of a **** -5 **** field, from four to five independent experiments. Statistical analysis is based on Wilcoxon rank sum test: p =5.76 × 10 < α =0.05, z = 4.023; p = -13 1.54 × 10 < α =0.05, z = − 7.384 Fusion Index (%) Probability MEF2C Nuclei (%) Probability Eccentricity Nuclei median number Brunetti et al. Skeletal Muscle (2021) 11:12 Page 6 of 14 and the shape of the nuclei were also different (Fig. 1d, myotubes on the flat surface grow in all directions, e): nuclei were frequently found as aggregates of up to they are less sensitive to an optimal orientation, and 20 nuclei on flat surface (median value between 2 and this could not explain the low percentage of respond- 7), while the aggregates contained less nuclei (up to 8) ing cells that we observed on the flat surface. Hence, on the nanopattern surface (median from 2 to 4; Fig. 1f), this increased response to electrical field stimulations and the nuclei displayed predominantly a linear arrange- strongly suggests that myotubes grown on a striated ment along the axis of the cells (Fig. 1e). As well, nuclei pattern are not only better organized but are also of cells grown on the nanopattern were more elongated functionally more mature. (eccentricity close to 1), as compared with nuclei of cells grown on a flat surface (Fig. 1g). As dispersion and Gene expression during maturation on striated surface alignment of myonuclei are reminiscent of the sequence The poor myotube organization and their progressive of events taking place during myotube maturation [16], detachment on a flat substrate prevent keeping our cul- these results suggested that the maturation of myotubes tures for more than 4 to 5 days. On the contrary, myo- is more advanced when grown on a striated surface than tubes adhere better when cultured on nanopattern, and on a flat surface. after addition of a second thick layer of Matrigel, we To further characterize the differentiation process on could keep them for up to 10–12 days in culture and both surfaces, we used immunostaining against the tran- study accurately their maturation. Under these condi- scription factors paired box 7 (Pax7) and MyoD to evalu- tions, the width of the myotubes remained stable over ate the proportion of reserve cells in our two cultures time (19.7 ± 1.1 μm to 19.8 ± 0.7 μm at 4 and 10 days, (Supplemental Fig. 1A). During the early step of differenti- respectively; data not shown). ation in vitro, reserve cells escape the terminal differenti- To obtain a broad overview of the myotube maturation ation and commit to a quiescent state [17]. These cells + - achieved on nanopattern surface, we analyzed several param- express Pax7, but not MyoD (Pax7 /MyoD ), while myo- 2+ eters, the first one being the gene expression of Ca -hand- tubes express MyoD and downregulate Pax7 (Pax7 / 2+ ling proteins (Cav1.1α1s, RyR1, and sarco-endoplasmic Ca MyoD ). We did not observe a difference between sur- ATPase (SERCA)1/2) and myosin heavy-chain isoforms, by faces in the percentage of Pax7-positive cells (Supplemen- RT-qPCR in cultured myotubes (at 4 and 10 days) and, for tal Fig. 1B), indicating that the striated surface did not comparison, in adult muscle sample. As expected, the ex- interfere with the establishment of reserve cells. MyoD- pression of all these genes increased between myoblasts and positive nuclei were, however, slightly more frequent on differentiated myotubes, while their expression remained the striated surface (Supplemental Fig. 1C). Hence, the stable between 4 and 10 days in culture (Fig. 3a). Transcript nanopatterned striated surface seems to favor specifically levels of CACNA1S, RYR1, MYH7 (encoding the isoform myotube maturation but not the entire process of expressed in slow fibers), and ATP2A2 (encoding SERCA2) myogenesis. were less than 10-fold lower in myotubes compared with 2+ those in adult fibers. On the contrary, genes encoding pro- Ca response to electrical field stimulation on flat versus teins abundant in fast fibers (MYH1, MYH2, and ATP2A1) striated surfaces were between 10- and more than 100-fold less expressed in We then investigated whether aligned myotubes ob- myotubes than in adult tissue. Transcript levels of MYH3 tained from the striated surface were functionally (coding the embryonic myosin heavy chain) slightly de- more advanced compared with those grown on a flat 2+ creased between 4- and 10-day old myotubes, and they were surface. To this end, we recorded Ca responses after higher in myotubes than in the adult tissue (Fig. 3a). In paral- electrical field stimulation to assess the level of EC lel, we assessed the expression of the embryonic and adult coupling. Four-day-old myotubes were electrically isoforms of the Cav1.1α1s subunit, namely the Cav1.1e and stimulated by repetitive bursts (1 s at 10 Hz, every 10 the Cav1.1a, respectively. Immature myotubes express pre- s) for 3 min. Myotubes grown on either surface 2+ dominantly the Cav1.1e isoform (that lacks exon 29), while it responded by transient cytosolic Ca elevations after represents less than 10% of the transcripts in differentiated stimulation. To note, while less than 45% myotubes 2+ muscle [18]. Using conventional PCR analysis, we detected cultured on a flat substrate displayed Ca elevation two bands corresponding to the embryonic and adult iso- upon stimulation, this percentage increased to more forms of Cav1.1 both at 4 and 10 days, while on the adult tis- than 95% when the myotubes were grown on nano- sue, only the Cav1.1a was expressed (Fig. 3b). Sequencing pattern (Fig. 2a, b). We cannot exclude that a small the PCR product confirmed that the lower band found in percentage of myotubes grown on a flat surface did 2+ myotubes lacks exon 29 (Fig. 3c). Furthermore, the ratio of not display a Ca response to electrical field stimula- Cav1.1e and Cav1.1a expression decreased between 4- and tion because of a suboptimal orientation of the cell 10-day old myotubes (Fig. 3d), highlighting a shift towards compared with the electrodes. However, as the Brunetti et al. Skeletal Muscle (2021) 11:12 Page 7 of 14 2+ Fig. 2 The nanopattern surface improves Ca responses to electric field stimulation. a Four-day-old myotubes were loaded with 5 μM Cal520- 2+ AM to detect changes in cytosolic Ca concentration upon 1 min of electric field stimulation (bursts of 1 s at 10 Hz, every 10 s). Representative recordings of myotube responses on flat and striated surfaces (ANFS). The fluorescence intensity was expressed as F/F . b Cell responses to stimulations were classified based on the amplitude of the first peak: non-responders, amplitude < 0.5; responders, amplitude > 0.5. Bar charts represent the percentage of each category of responses on the two surfaces, flat and nanopattern (ANFS). Data were obtained from four independent experiments the expression of the adult Cav1.1 channel with time in Supplemental figure 2). Over time, the typical perpen- culture. dicular alignment of α-actinin appeared first (at around 4 days) followed by STIM1 (at 8–10 days) and eventually 2+ Organization of muscle-specific proteins and Ca RyR1 (Fig. 4c–e). The striations of α-actinin and STIM1 responses were observed in a consistent way, while the RyR1 dou- Skeletal muscles have a highly organized internal archi- blets were seldom obtained (in about 20% of the cul- tecture with striated pattern of most of the muscle- tures) after 10 days of myotube maturation. We further specific proteins. To evaluate how mature are myotubes noticed that on myotubes at that stage, the nuclei were grown on the nanopattern surface, we performed immu- mostly localized at the cell periphery (Fig. 4f). Import- 2+ nostaining of α-actinin, RyR1, and the SR Ca sensor antly, already after 6 days in DM, the myotubes dis- stromal interaction molecule 1 (STIM1) after 10–12 played frequently spontaneous contractions, in around days of differentiation. As shown on Fig. 4a, the staining 30–40% of the cultures (Movie 1). of all these proteins appeared striated, and the distance Next, we analyzed the presence of AChR clusters at between two z lines was of around 2.7 μm, close to what different times of maturation. Both number and size of is found in the adult human muscle (2.5 μm; Fig. 4b and clusters significantly increased within 2, 4, and 10 days Brunetti et al. Skeletal Muscle (2021) 11:12 Page 8 of 14 CACNA1S RYR1 ATP2A1 ATP2A2 10 10 ns 10 10 ns ns 1 1 1 0.1 0.1 0.1 0.01 0.01 0.01 0.1 0.001 0.001 0.001 0.0001 0.0001 0.0001 0.01 MYH1 MYH2 MYH3 MYH7 (fast) (fast) (embryonic) (slow) 10 10 100 ns ns ns ns 1 1 0.1 0.1 0.1 10 0.01 0.01 0.01 0.001 0.001 0.001 1 0.0001 0.0001 0.0001 0.00001 0.00001 0.00001 0.1 B C 400 bp Cav 1.1a Cav 1.1e 200 bp Cav1.1a Cav1.1e Adult Muscle D4 D10 Fig. 3 (See legend on next page.) D0 D4 D10 Adult Muscle D0 D4 D10 Adult Muscle D0 D4 D10 Adult Muscle D0 D4 D10 Adult Muscle D0 D4 D10 Adult Muscle D0 D4 D10 AdultMuscle D0 D4 D10 Adult Muscle D0 D4 D10 Adult Muscle Relativeexpression (AU fold) Relativeexpression (AU fold) Ratio(Cav1.1e/Cav1.1a) Relative expression (AU fold) Relative expression (AU fold) Relative expression(AU fold) Relative expression (AU fold) Relative expression (AU fold) Relativeexpression (AU fold) Brunetti et al. Skeletal Muscle (2021) 11:12 Page 9 of 14 (See figure on previous page.) Fig. 3 Expression of EC coupling-associated proteins and myosin heavy chain during myotube maturation. a Transcript levels of genes encoding for proteins involved in muscle contraction and EC coupling were quantified by RT-qPCR in adult muscle (three donors) and in myoblasts (D0), 4-day-old (D4), and 10-day-old (D10) myotubes (cultures from five donors). CACNA1S (Cav1.1), RYR1 (RyR1), ATP2A1 (SERCA1), ATP2A2 (SERCA2), MYH1 (MyHC-2X), MYH2 (MyHC-2A), MYH3 (MyHC-3 embryonic) and MYH7 (MyHC-7 slow). Transcript levels are relative to adult muscle transcript content. Results are mean ± SEM. A.U., arbitrary units. Each dot represents one independent experiment. b PCR products of the two variants of Cav1.1, embryonic (Cav1.1e) and adult (Cav1.1a) detected in 4- and 10-day-old myotubes and in human adult muscle. M, marker of molecular weights. c PCR product sequencing of CACNA1S exon 29, specific of the adult isoform of Cav1.1 (Cav1.1a) but absent in the embryonic isoform (Cav1.1e). d Quantification of the ratio between Cav1.1e and Cav1.1a expressions in 4- and 10-day-old myotubes. Error bars represent mean ± SEM of culture (Fig. 5a–d). The shape of the clusters is [21]. In addition, we added during the initial 4 days of known to change during maturation, with the emergence differentiation an inhibitor of TGFβ receptor, a proced- of branched structures with time [19]. We detected only ure that was shown to improve cell fusion [22], MYH ex- very few clusters with such rearrangement of the AChR pression, and sarcomere organization [23, 24]. after 10 days of maturation, most of the clusters being As expected, the myotubes grown on a striated surface oval-shaped. However, AChR were functional, as shown were very well aligned, with nuclei being also more elon- 2+ by the rapid cytosolic Ca elevation elicited by applica- gated, initially aligned in the middle of the myotubes tion of 10 μM acetylcholine (Fig. 5e–g). By comparison, while moving at the cell periphery with time in culture, on the flat surface at day 4 of differentiation, the AChR hence recapitulating the progression of nuclei movement were more dispersed on the myotubes and did not form occurring in vivo [16]. Yet, we did not notice differences, proper clusters (Supplemental Figure 3). In addition, neither in fusion index nor in percentage of MEF2C- 2+ only 64% of the myotubes displayed a Ca response positive nuclei, between flat and grooved surfaces after 4 when stimulated with 10 μM ACh, compare with the days of differentiation. This is somehow in contradiction 97% of responding myotubes grown on the nanopattern with what was previously reported, where differentiation surface (Supplemental Figure 3). performed on striated surfaces enhanced the fusion 2+ Lastly, we assessed Ca responses to electrical stimu- index [20, 21]. It should be pointed out, however, that lations. Four- and 10-day-old myotubes were stimulated human primary cells cultured in our laboratory has a for 3 min with repetitive bursts every 10 s (Fig. 6a). The high fusion index (around 60%), higher than what was 2+ amplitude and the decay time of the first cytosolic Ca reported in the abovementioned studies. In line with the elevation were very similar at early and later stages of fusion index, the percentage of Pax7-positive cells was maturation (Fig. 6b, c). As well, the amplitude of the last also similar between the two surfaces. Hence, our cul- transient compared with the first one (percentage of ture system did not favor the initial steps of differenti- 2+ remaining Ca ), that depicts the sustained nature of the ation but presents the advantage to promote cell response was also not significantly different between the alignment and to maintain cells for longer time in cul- two time points (Fig. 6d). Hence, myotubes already dis- ture allowing their maturation. played robust EC coupling after 4 days when cultured We analyzed then several parameters during 10–12 days on nanopattern striated surface. in culture: the expression level of muscle-specific genes, 2+ the internal organization of the myotubes, and their Ca Discussion responses to ACh and electrical stimulations, in order to We reported here a simple in vitro culture model of hu- provide a broad overview of the processes taking place man primary myoblasts that presents greater level of over time, both morphologically and functionally. myotube maturation compared with commonly used 2D Although the myotube diameter remained stable dur- cultures. ing time in culture, the number and size of AChR clus- Our culture model combined various procedures that ters increased between 2 and 10 days of maturation. At foster myotube maturation. First, cells were grown be- 10 days of differentiation, almost all myotubes displayed tween two layers of Matrigel that provided an extracellu- clusters of AChR, which reflects a greater maturation of lar matrix limiting cell detachment, as previously myotubes compared with conventional 2D cultures reported [13]. Second, cells were grown on a striated where only a minority of myotubes have AChR clusters pattern surface that favors alignment and maturation of after 2 weeks in culture [8]. The clusters remained how- myotubes [11, 20, 21]. This surface is a fabricated pat- ever essentially homogenous, without a sign of lacunarity tern with grooves and ridges of 800-nm width and 600- or pretzel structures, like it was reported in 3D culture nm depth, dimensions that were previously described to [8] or on surfaces coated with various laminin [25], mor- favor myotube maturation [20], even if larger striations phological changes that reflect the maturation of AChR (up to 1600 nm) also led to an enhanced maturation clusters [19, 26]. AChRs were however functional as Brunetti et al. Skeletal Muscle (2021) 11:12 Page 10 of 14 2+ shown by the Ca elevations elicited by ACh stimula- tion. The amplitude of the response was akin to the one elicited by electrical stimulation, in line with what was reported on human 3D myobundles [27]. On the con- trary, 4-day-old myotubes grown on a flat surface have a diffuse pattern of AChR and not all myotubes responded 2+ to ACh stimulation by a Ca elevation. Interestingly, the genes expressed predominantly in slow fibers (MYH7 and ATP2A2) were expressed in 10- day-old myotubes at levels closer to adult tissue, than the ones expressed in fast fibers (MYH1&2 and ATP2A1). Hence, even if there was a mixed expression of genes from slow and fast fibers, like it is frequently observed in cultured myotubes [28, 29][7], myotubes appear to mature preferentially towards a slow pheno- type. This slow phenotype resembles the one observed during the establishment of a human muscle cell line HMCL-7304 [30], with a higher abundance of slow MYH. This slow phenotype is however different to what is observed in vivo when regeneration happens in the ab- sence of innervation, where fibers follow what is called the “default pathway” toward a fast phenotype [31]. The reasons of this maturation towards a slow phenotype are unclear, but this may, at least partially, explain the kinet- 2+ ics of the Ca responses triggered by ACh or electrical stimulation that we measured. Indeed, even if myotubes 2+ in culture are known to have slower Ca recovery phase than adult fibers [32], we were anticipating a faster decay time associated with enhanced maturation, which was 2+ not the case. Certainly, the slow Ca kinetics of the re- sponse also reflects the fact that the system did not suffi- 2+ ciently mature to have an obvious impact on the Ca repumping efficiency. During muscle development, a splice variant of Cav1.1 lacking exon 29, namely Cav1.1Δ29 or Cav1.1e is expressed, characterized by dis- tinct biophysical properties compared with the adult 2+ Cav1.1 isoform: a higher Ca current amplitude to- gether with a 30-mV left shift of the activation potential [18, 33]. The expression of Cav1.1e was also reported in human myotubes in culture, leading us to evaluate its expression. Our results show that the embryonic Cav1.1e is expressed both at 4 and 10 days, and interestingly, the Fig. 4 Organization of muscle-specific proteins and of nuclei. ratio between the two, embryonic and adult isoforms, Representative images of 10-day-old myotubes cultured on nanopattern decreased with time in culture, confirming an enhanced surface (a) and adult human muscle tissue (b) immunolabeled for α-actinin fiber maturation at 10 days. (green) and STIM1 (red) (upper lines) and for RyR1 (green) and STIM1 (red) (lower lines). Scale bar: 5 μm. c–e Organization of α-actinin, STIM1, and We also assessed the organization level of several pro- RyR1 during maturation. Representative images of 4-, 10-, and 12-day-old teins over time in culture. As already reported [34], the myotubes immunolabeled for α-actinin (c) STIM1 (d), and RyR1 (e). At 4 transverse alignment of α-actinin occurred rapidly after days, α-actinin is already perpendicularly oriented, followed at 10 days by the induction of differentiation. Indeed, after 4 days in STIM1, and in some cells by RyR1 at 12 days. Scale bar: 5 μm. f culture, a substantial number of cells displayed this typ- Representative image of 12-day-old myotubes immunolabeled with STIM1 together with DAPI. White arrows point to peripheral nuclei. Scale bar: ical arrangement, and the striations get sharper with lon- 20 μm ger time in culture. The distance between two Z lines in 10-day-old myotubes was around 2.7 μm, close to the size of adult human sarcomeres (2.5 μm). Furthermore, Brunetti et al. Skeletal Muscle (2021) 11:12 Page 11 of 14 -actinin/ -BTX/DAPI Day 2 Day 4 Day 10 Day 4 Day 10 Day 2 BC D 0 0 D2 D4 D10 D2 D4 D10 D2 D4 D10 E F G Amplitude 10 µM ACh 10 µM ACh 6 6 4 4 3 2 2 0 0 0 102030405060 0 102030405060 0 D4 D10 Time (s) Time (s) 2+ Fig. 5 Organization of AChR clusters during maturation and Ca response to acetylcholine. a Representative images of 2-, 4-, and 10-day-old myotubes cultured on nanopattern surface and immunolabeled for α-actinin (red) and α-bungarotoxin (α-BTX, green) together with a DAPI staining (blue). Lower panels are enlargement of the white rectangles. Scale bar: 20 μm. b, c, d Quantification of the average number of acetylcholine receptors (AChR) clusters per field (b), AChR cluster size (c), and of the average numbers of AChR clusters per myotube (d). Data were obtained from three independent experiments. Error bars are mean ± SEM. e−f Myotubes were loaded with 5 μM Cal520-AM to measure 2+ 2+ changes in cytosolic Ca , and stimulated with 10 μM of acetylcholine (ACh). Traces represent the mean ± SEM of Ca responses of myotubes differentiated for 4 (e) and 10 days (e). g Statistical evaluation of the amplitude of the peak following ACh stimulation. Error bars are mean ± SEM 2+ after 7–8 days in culture, the SR resident Ca sensor human cells cultured in 2D. Advanced maturation were STIM1 also appeared striated, with an important level of obtained with human iPSC but after a significantly lon- colocalization on the Z lines. This localization of STIM1 ger time (50 days to obtain titin striations [6], or after 2 is consistent with findings obtained in muscle adult tis- weeks to obtain α-actinin striations of myotubes derived sue, where STIM1 was reported to be present both at a from induced myogenic progenitor cells [7]. Hence, the region corresponding to the I band (around the Z lines), advantage of growing the myotubes on a striated surface, as well as on the longitudinal SR [35][36, 37]. A proper beside the improvement of cell adhesion, is that myo- RyR1 localization in doublet was however infrequently tubes remain thin and elongated which likely promotes obtained, even after 12 days of maturation. On the few their internal organization and thus maturation. cases where such matured arrangement of RyR1 was ob- Finally, we assessed the ability of myofibers to elicit cyto- 2+ served, it always took place after STIM1 was perpen- solic Ca transients upon electrical field stimulation. Already dicularly organized. Even if RyR1 doublets were seldom at 4 days of culture, electrical stimulations resulted in large 2+ observed, the high level of internal organization achieved Ca transients of almost all myotubes, highlighting a func- within 10 days was to our knowledge not obtained with tional EC coupling. This time frame is rapid if we compared F/F #of AChR clusters per field AChR cluster size (um ) F/F #ofAChR clusters per myotube F/F 0 Brunetti et al. Skeletal Muscle (2021) 11:12 Page 12 of 14 2+ 2+ Fig. 6 Ca transients upon electric field stimulation. a Representative traces of cytosolic Ca transients generated by repeated burst of 1 s at 10 Hz, every 10 s for 3 min in 4-day (left panel) and 10-day (right panel)-old myotubes, differentiated on nanopattern surface. Statistical evaluation of 2+ the amplitude (b) and the decay time (c) of the first Ca transient, and the percentage of remaining calcium at the end of 3-min stimulation (d). Data were obtained from four independent experiments, and each dot is a mean of 4−6 myotubes on a field. Error bars are mean ± SEM it to the 8 days needed to obtain 50% of myotubes this rapid and easy-to-perform 2D culture, which does responding to electrical stimulation with classical 2D not require sophisticated technology, presents certainly culture of human cells [32]. We analyzed at 4 and 10 great advantages for further single-cell studies designed 2+ days of culture three parameters out of these Ca re- to investigate the processes of skeletal muscle cordings, the amplitude of the first transient, the kin- maturation. 2+ etic of the Ca recovery phase, and the sustained Abbreviations nature of the signals during the 3-min stimulation. GM: Growth medium; DM: Differentiation medium; SR: Sarcoplasmic None of these parameters significantly changed be- reticulum; EC: Excitation–contraction; MEF2C: Myocyte enhancer factor 2C; MyoD: Myogenic differentiation 1; Pax7: Paired box 7; RyR1: Ryanodine tween 4 and 10 days, showing that even if the cells receptor 1; DHPR: Dihydropyridine receptor; STIM1: Stromal interaction displayed a better internal organization at 10 days, molecule 1; ACh: Acetylcholine; AChR: Acetylcholine receptor; α-BTX: α- 2+ 2+ this did not correlate with an improved Ca signals. Bungarotoxin; SERCA: Sarco-endoplasmic Ca ATPase This is however in agreement with the expression levels of RYR1, CACNA1S, and ATP2A1/2 that remain Supplementary Information The online version contains supplementary material available at https://doi. stable during the maturation period we analyzed. org/10.1186/s13395-021-00268-3. Conclusions Additional file 1: Movie 1. Spontaneous myotube contraction on 6- Overall, we proposed a 2D culture system of human day old culture on nanopattern surface. myotubes that reach after 10 days a high level of matur- Additional file 2: Supplemental Figure 1. The reserve cell population ation, with the presence of numerous functional AChR is not affected by the surface pattern. (A) Immunofluorescence of nuclei of 4-day old myotubes on Flat (upper panel) or nanopattern surface clusters, the expression of adult MYH, increased adult (ANFS, lower panel). Cell nuclei were stained using an antibody against CACNA1S isoform expression, and a high level of in- Pax7 (in green), MyoD (in red) and all nuclei were counterstained with ternal fiber organization. Further improvements are DAPI (in blue). Scale bar: 50 μm. Images shown are representative of six independent experiments. (B, C) Percentage of Pax7 positive nuclei (B) however required to obtain myofibers that resemble and MyoD positive nuclei (C). Error bars are Mean ± SEM. more the adult organization and function. Nevertheless, Brunetti et al. Skeletal Muscle (2021) 11:12 Page 13 of 14 embryonic stem cell-derived skeletal myocytes in vitro. Proc Natl Acad Sci. Supplementary Figure 2. Length of the sarcomere on culture myo- 2014;111(22):8275–80. https://doi.org/10.1073/pnas.1322258111. tubes and on adult tissue. (A-B) Representative images of 10-day old 5. Roman W, Martins JP, Carvalho FA, Voituriez R, Abella JVG, Santos NC, et al. myotubes cultured on nanopattern surface (A) and adult human muscle Myofibril contraction and crosslinking drive nuclear movement to the tissue (B) Scale bar: 10 μm. (C-D) Corresponding line profiles (white dot- periphery of skeletal muscle. Nat Cell Biol. 2017;19(10):1189–201. https://doi. ted line) of α-actinin normalized fluorescence intensity. Supplemental 2+ org/10.1038/ncb3605. Figure 3. Organization of AChR clusters and Ca response to acetylcho- 6. 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Nanopattern surface improves cultured human myotube maturation

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Abstract

Background: In vitro maturation of human primary myoblasts using 2D culture remains a challenging process and leads to immature fibers with poor internal organization and function. This would however represent a valuable system to study muscle physiology or pathophysiology from patient myoblasts, at a single-cell level. Methods: Human primary myoblasts were cultured on 800-nm wide striated surface between two layers of Matrigel, 2+ and in a media supplemented with an inhibitor of TGFβ receptor. Gene expression, immunofluorescence, and Ca measurements upon electrical stimulations were performed at various time points during maturation to assess the organization and function of the myotubes. Results: We show that after 10 days in culture, myotubes display numerous functional acetylcholine receptor clusters and express the adult isoforms of myosin heavy chain and dihydropyridine receptor. In addition, the myotubes are internally well organized with striations of α-actinin and STIM1, and occasionally ryanodine receptor 1. We also 2+ demonstrate that the myotubes present robust Ca responses to repetitive electrical stimulations. Conclusion: The present method describes a fast and efficient system to obtain well matured and functional 2+ myotubes in 2D culture allowing thorough analysis of single-cell Ca signals. 2+ Keywords: Human primary myoblasts, Cell alignment, Myotube maturation, Acetylcholine receptor clusters, Ca signals Background organization of muscle fibers, and even if important pro- Skeletal myofibers are large multinucleated cells with an ex- gresses were accomplished over the years, it remains challen- ceptional level of internal organization dedicated to produce ging to obtain well differentiated and matured myofibers strength upon nerve activation. Acetylcholine, released from in vitro. Several models of myogenesis were designed to gen- nerve terminals, induced membrane depolarization (action erate muscle tissue that could be eventually engrafted in 2+ potential) that is sensed by voltage-gated Ca channels, damaged/diseased muscles [1], to provide suitable models to Cav1.1, also called dihydropyridine receptor (DHPR). The study pathophysiological mechanisms of the disease [2, 3], or conformational change of DHPR is transmitted to the ryano- to understand the various steps of muscle formation [4, 5]. 2+ dine receptor 1 (RyR1), a Ca channel localized at the ter- The sources of cells used to perform in vitro culture are di- minal cisternae of sarcoplasmic reticulum (SR). The opening verse, such as C2C12 mouse cell line or primary myoblasts 2+ of RyR1 leads to a strong release of Ca from the SR and of murine or human origin. As well, cultures derived from eventually muscle contraction, an overall process called exci- human iPSC are used with promising outcomes [6, 7]. In the tation–contraction (EC) coupling. Because of thesizeand recent years, several studies have highlighted the benefits of establishing 3D compared with 2D culture. Indeed, 3D ap- proaches promote the level of maturation and contractility of * Correspondence: maud.frieden@unige.ch myotubes/myofibers and allow to keep cells longer in cul- Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland ture. The improved internal architecture, the presence of © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Brunetti et al. Skeletal Muscle (2021) 11:12 Page 2 of 14 acetylcholine receptor (AChR) clusters, and the higher con- Table 1 Cell culture media tractility are among the main advantages of 3D versus 2D Concentration Reference culture [8]. However, 2D cultures are likely more appropriate Growth medium (GM) for single-cell level analysis, but myotubes differentiated on F-10 (Ham’s) ThermoFisher 31550-023 2D frequently do notmatureproperlywithoften alack of BSA 0.5 mg/ml Sigma 05482 striations and the appearance of small/broken cells [6][8]. Insulin 0.04 mg/ml I-9278 One cue demonstrated to promote a higher level of myotube Creatine powder 1 mM Fluka 27900 maturation is the cell alignment. Indeed, skeletal muscles are composed of very long and aligned fibers, an organization Dexamethasone 0.39 μg/ml Sigma D1756 that could be recapitulated, at least partially, in culture. To Gentamycin 5 μg/ml Sigma G1272 promote such alignment, self-fabricated substrates are usu- EGF 10 ng/ml Corning 354001 ally created, which are nonetheless complicated to establish FBS 15% Lot 4205662K and require special lab equipment [9–11]. Fetuin 0.5 mg/ml Sigma F-3385 In the present study, we describe an in vitro matur- Sodium pyruvate 100 μg/ml Sigma P4562 ation system that uses striated surface to culture human primary myoblasts. We report evidence of clear benefi- Uridine 50 μg/ml Sigma U3003 cial effects of growing and differentiating muscle cells on Differentiation medium (DM) a striated surface, both at structural and functional DMEM Thermo Fisher 41965-039 levels. We show that on this surface, myocytes display BSA 0.5 mg/ml Sigma 05482 rapidly a high level of internal organization and numer- Insulin 0.01 mg/ml Sigma I5500 ous AChR clustering. Finally, this culture model allows Creatine powder 1 mM Fluka 27900 thorough analysis of single-cell robust and repetitive 2+ Ca transients elicited by electrical field stimulations. Gentamycin 10 μg/ml Thermo Fisher 15710-049 Sodium pyruvate 100 μg/ml Sigma P4562 Methods Uridine 50 μg/ml Sigma U3003 Cell culture Horse serum 1% Amimed 2-05F26-I Human primary myoblasts were isolated from semiten- dinous muscle samples obtained after orthopedic surgery (surgical waste) on patients without known muscular differentiation, the TGFβ inhibitor was removed, and a diseases. All samples were collected anonymously after thick layer of Matrigel diluted at 1:3 in DM was added obtaining a written consent and approval by the Univer- to limit cell detachment [13], and then half of the sity of Geneva (protocol CCER no. PB_2016-01793 (12- medium was changed every 2 days. 259) accepted by the Swiss Regulatory Health Author- ities and approved by the “Commission Cantonale Immunofluorescence d'Ethique de la Recherche” from the Geneva Cantonal Human myotubes were fixed in PBS with 4% parafor- Authorities, Switzerland). The purification of myoblasts maldehyde, permeabilized and blocked in PBS contain- was performed as previously described [12]. ing 0.3% of Triton X-100 and 5% goat serum. Proteins of Myoblasts were seeded either on a conventional dish interest were revealed by incubation of specific primary TM with a flat surface (FluoroDish , Cat n°FD34-100; antibodies overnight at 4 °C, followed by incubation of WPI), or on a nanopattern surface (Anisotropic Nano- fluorophore-conjugated secondary antibodies for 75 min Fabricated Surface, ANFS, Nanosurface Biomedical), that at room temperature. Primary and secondary antibodies has ridges and grooves of 800-nm and 600-nm deep. and their dilution are listed in Table 2. Nuclei were Both surfaces were coated with Matrigel (Corning Matri- stained using ProLong® Gold Antifade Reagent with gel Basement Membrane Matrix Growth Factor Re- DAPI (ref. P36931, Life Technologies). To detect acetyl- duced, Phenol Red Free, #356321) diluted at 1:100 in choline receptors, α-bungarotoxin (α-BTX, B13422, Invi- Ham's F-10 (ThermoFisher) as previously described [13]. trogen) was added together with the secondary Cells were expanded in a growth medium (GM; see antibodies. Images were acquired either with a widefield Table 1), and when they reached around 90% of con- AxioImager M2 microscope (widefield microscopy, fluency, the differentiation was triggered by replacing Zeiss, Germany) through a 20× objective (EC Plan- GM with a differentiation medium (DM; see Table 1). Apochromat 20×/ 0.8), or with a confocal Nikon A1r At the time, cells were placed in DM, and during the spectral microscope (Nikon; Japan) through a 60× ob- first 3–4 days in differentiation, the medium was supple- jective (1.4 CFI Plan Apo Lambda). Three random fields mented with 10 μM of TGFβ receptor I inhibitor were acquired for each dish, with a minimum of two (SB431542, #S4317, Sigma). After 4 days in dishes per experimental condition. Clusters of AChR Brunetti et al. Skeletal Muscle (2021) 11:12 Page 3 of 14 Table 2 List of primary and secondary antibodies Dilution Reference Primary antibodies Mouse anti-(sarcomeric) α-actinin 1:500 Sigma A7811 Mouse anti-Pax7 1:100 DSHB C Mouse anti-RyR1 1:500 DSHB 34C Rabbit anti-MEF2C 1:500 Cell Signaling 5030S Rabbit anti-STIM1 (C-terminal) 1:500 Sigma S6197 Rabbit anti-MyoD1 1:200 Cell Signaling D8G3 Secondary antibodies: Alexa Fluor® 488-conjugated goat anti-mouse IgG (H + L) 1:1000 Life Technologies A11029 Alexa Fluor® 546-conjugated goat anti-mouse IgG (H + L) 1:1000 Life Technologies A11030 Alexa Fluor® 546-conjugated goat anti-rabbit IgG (H + L) 1:1000 Life Technologies A11030 bigger than 5 μm were considered for analysis. Analysis added at the excitation side to limit phototoxicity (694/SP of the images were performed using ImageJ software. BrightLine HC shortpass filter, AHF analysentechnik AG, For display purposes, some pictures were rotated and Germany). Acetylcholine (10 μM, A2661, Sigma-Aldrich) rescaled to have the same resolution. or electrical stimulations (see below) were applied to elicit 2+ Ca responses. Image acquisition was performed at 3.3 Nuclei segmentation analysis Hz with the VisiWiew software, version 4.4.0.11 (Visitron Nuclei were detected based on their DAPI fluorescent Systems, Puchheim, Germany). The fluorescence intensity channel using Cellpose 0.1.0.1 pretrained model [14], was expressed as F/Fo to normalize the data. with automated detection of the diameter. Then the seg- mentation masks and the acquired images were proc- Electric field stimulations essed with Matlab 2020b. Briefly, myocyte enhancer A chamber containing platinum electrodes (RC-37FS, factor 2C (MEF2C) or Myogenic differentiation 1 Cat.64-0366, Warner Instruments) was inserted into the (MyoD)-positive nuclei were analyzed if their individual culture dish. For the experiments performed with cells area was between 40 and 200 μm . Nuclei were consid- grown on the nanopattern surface, the electrodes were ered as part of a cluster if their nearest neighbor dis- oriented in a transversal direction compared with the tance was below 3 μm border-to-border. For each long axis of the cells. The chamber was connected to an isolated nucleus (i.e., not part of a cluster), orientation in electric field stimulator (A310 Accupulser, World Preci- the field of view and eccentricity (that informed about sion Instruments), where the parameters of the stimula- the shape of the nuclei) were computed. Nuclei detec- tion were set up, and to an electric field isolator (A385 tion and segmentation was performed by Nicolas Liau- Stimulus isolator, World Precision Instruments) to sup- det from the Bioimaging core facility of the University of ply a constant current. Geneva (https://www.unige.ch/medecine/bioimaging). Myotubes were stimulated with repetitive bursts of 1 s at 10 Hz every 10 s (each stimulation lasts 2 ms), at 70 Calcium measurements V for 3 min. Three to four rounds of stimulations were Myotubes were loaded with Cal520-AM (5 μM, AAT Bio- performed on each cell culture. Stimulations were per- quest®) and 0.1% Pluronic F-127 (Invitrogen, cat. No formed on a medium containing (mM) 135 NaCl, 5 KCl, 2+ P3000MP), in the dark at 37 °C in Ca -containing solu- 1 MgCl , 10 Hepes, 10 glucose, 0.050 EGTA, and 2 2+ tion. Following an incubation of 90 min, cells were washed CaCl , pH 7.4 (NaOH). The analysis of Ca transient and kept for 30 min to allow de-esterification of the dye. parameters was performed with MatLab (R2020b). Fluorescence was recorded using a Zeiss Axio Observer A1 microscope equipped with a Lambda XL illumination Quantitative and conventional real-time quantitative system (Sutter Instrument, Novato, CA, USA). The excita- polymerase chain reaction (RT-PCR) tion wavelength was 480 nm (ET480/20×; Chroma), and RNA was isolated using TRI Reagent Solution (AM9738, emission was collected through a T505lpxr dichroic mir- Invitrogen™) from myoblasts, myotubes differentiated for 4 ror (Chroma) and a 510WB40 filter (Omega Optical) by a and 10 days, and from adult muscles, according to the manu- cooled 16-bit CMOS camera (pco.Edge sCMOS, Visitron facturer’s instructions. Quantification of the samples, quality Systems, Puchheim, Germany). An attenuation filter was control, reverse transcription, and real-time quantitative Brunetti et al. Skeletal Muscle (2021) 11:12 Page 4 of 14 polymerase chain reaction (RT-qPCR) were all performed at used. DNA fragments of Cav1.1 splice variant, Cav1.1a theiGE3Genomic Platform oftheUniversityofGeneva (adult; 350 bp) and Cav1.1e (embryonic; 250 bp), were (https://ige3.genomics.unige.ch/). Following the measure- separated in 2.2% agarose gel electrophoresis. Each DNA ment of sample concentration, quality of the RNA was deter- fragment was further sequenced by Fasteris DNA Se- mined using the Agilent RNA 6000 Nano Kit and analyzed quencing Service (Fasteris SA, Switzerland). with the Agilent 2100 Bioanalyzer instrument (Agilent Tech- nologies, Germany). From the total RNA, 0.5 μgwas reverse Statistics transcribed with the PrimeScript™ RT reagent kit (TaKaRa, For the analysis of nuclei clusters and shape, statistical ana- Bio Company, Japan) according to the manufacturer’sin- lysis based on Wilcoxon rank sum tests were conducted on struction. The expression of genes listed in Table 3 was eval- the median number of nuclei in clusters and on the median uated during the differentiation and maturation of the eccentricity of each acquisition. The null hypothesis between myotubes and compared with the expression in the adult tis- nuclei cultured on nanopattern and flat surfaces was consid- sue. RT-qPCR was performed on 7900HT instrument (Ap- ered as rejected if the p values were below the significant plied Biosystems™). Raw threshold-cycle (Ct) values obtained level of 0.05. For all other experiments, data are mean ± with SDS 2.2 (Applied Biosystems™)wereimportedinto SEM, and the statistically significant differences were deter- * ** *** Excel. Normalization factor and fold changes were calculated mined using a Student t test, where p <0.05, p <0.01, **** using the GeNorm method [15]. The level of gene expression p < 0.001, and p < 0.00001. was normalized for two housekeeping genes (B2M coding for β2 microglobulin and EEF1A1). Fold changes obtained Results for each condition were normalized to the adult tissues. Myotube differentiation on flat versus striated surfaces For conventional PCR, cDNA from myotubes differen- We first evaluated the benefit of growing and differenti- tiated for 4 and 10 days and from adult muscle were ating human primary myoblasts on a nanopattern Table 3 List of primers Proteins Names Sequences Genes CACNA1S Cav1.1α1s 26-35F CAC CTC CTC CTA CTT TGA ATA 26-35R AGA ACT TCC CAA AGC CCA GA E27F GCT CAT GGC CTT CAA GG ex31R TGA CGA TGA GCA GAG CC RYR1 RyR1 ex-102-103-27F TGG CCA TCA TCC AGG GTC T ex-102-103-77R GGT CTC GGA GCT CAC CAA AAG ATP2A1 SERCA1 ex-15-16-29F CAG TGG CTG GCT CTT CTT CC ex-15-16-79R GCA CCC ACA TAG CCC CC ATP2A2 SERCA2 ex-3V-871F CCT TGA GGA CTC TGC CAA CTT T ex-3V-921R ACG AAG GTC AGA TTG GTC TCA TATT MYH1 MyHC-2X (fast) ex-39-40-48F CAA GCT GAA GAA GCG GAG GA ex-39-40-98R GCG GAA TTT GGA GAG GTT GAC MYH2 MyHC-2A (fast) ex-37-38-64F AAA CTG GAG GCC AGG GTA CG ex-37-38-114R TTG CTC ACT CTC AAC CTC TCC TT MYH3 MyHC-3 7F TCA GAA GCC GAT TCT ACA TGG AC (embryonic) 57R ACA ACT TAG CGG CAC TTG GG MYH7 MyHC-7 (slow) ex-37-38-66F AGG AGC TCA CCT ACC AGA CGG ex-37-38-116R GCA GCC GCA GCA GGT TT Housekeeping genes β2-microglobulin TGC TCG CGC TAC TCT CTC TTT TCT GCT GGA TGA CGT GAG TAA AC EEF1A1 AGC AAA AAT GAC CCA CCA ATG GGC CTG GAT GGT TCA GGT A Brunetti et al. Skeletal Muscle (2021) 11:12 Page 5 of 14 surface (ANSF, see Methods section) compared with percentage of MEF2C positive nuclei were similar be- those on a flat surface. We analyzed the level of myotube tween the two surfaces (Fig. 1b, c). However, differentiation after 4 days in DM, by immunolabeling organization of myotubes was notably different. On the the cells with α-actinin and the transcription factor flat surface, myotubes were mainly randomly oriented, MEF2C (Fig. 1a). The fusion index (number of nuclei while on the nanopattern surface, they were tightly within myotubes/total number of nuclei) and the aligned parallel to the striations (Fig. 1a). The position A MEF2C/ -actinin/DAPI Flat ANFS B C 60 60 0 0 Flat ANFS Flat ANFS Median number of nuclei D Flat ANFS Flat (n=39) ANFS (n=35) Isolated nuclei eccentricity Flat (n=39) ANFS (n=35) Orientation (°) Orientation (°) Fig. 1 Nanopattern surface promotes myotube and nuclei alignment. a Immunofluorescence images of 4-day-old myotubes on flat (left panel) or nanopatterned (ANFS, right panel) surface. Myotubes were stained using antibodies against α-actinin (green) and MEF2C (red) together with a DAPI staining (blue). Scale bar: 50 μm. b,c Quantification of fusion index (b) and MEF2C-positive nuclei (c). Data were obtained from four independent experiments, and each dot is the mean of 3 fields/dish. Error bars are Mean ± SEM. d Nuclei detection based on a pretrained model on flat (left panel) and on nanopattern (right panel) surface. Each cluster of nuclei is depicted with the same color code. e Corresponding distribution of the nuclei orientation on flat (left panel) and on nanopattern surface (right). f–g Median number of nuclei in cluster (f) and median eccentricity of the isolated nuclei (g). Perfect round nuclei would have a value of 0, and elongated nuclei have an eccentricity value closer to 1. Each dot is the median value of a **** -5 **** field, from four to five independent experiments. Statistical analysis is based on Wilcoxon rank sum test: p =5.76 × 10 < α =0.05, z = 4.023; p = -13 1.54 × 10 < α =0.05, z = − 7.384 Fusion Index (%) Probability MEF2C Nuclei (%) Probability Eccentricity Nuclei median number Brunetti et al. Skeletal Muscle (2021) 11:12 Page 6 of 14 and the shape of the nuclei were also different (Fig. 1d, myotubes on the flat surface grow in all directions, e): nuclei were frequently found as aggregates of up to they are less sensitive to an optimal orientation, and 20 nuclei on flat surface (median value between 2 and this could not explain the low percentage of respond- 7), while the aggregates contained less nuclei (up to 8) ing cells that we observed on the flat surface. Hence, on the nanopattern surface (median from 2 to 4; Fig. 1f), this increased response to electrical field stimulations and the nuclei displayed predominantly a linear arrange- strongly suggests that myotubes grown on a striated ment along the axis of the cells (Fig. 1e). As well, nuclei pattern are not only better organized but are also of cells grown on the nanopattern were more elongated functionally more mature. (eccentricity close to 1), as compared with nuclei of cells grown on a flat surface (Fig. 1g). As dispersion and Gene expression during maturation on striated surface alignment of myonuclei are reminiscent of the sequence The poor myotube organization and their progressive of events taking place during myotube maturation [16], detachment on a flat substrate prevent keeping our cul- these results suggested that the maturation of myotubes tures for more than 4 to 5 days. On the contrary, myo- is more advanced when grown on a striated surface than tubes adhere better when cultured on nanopattern, and on a flat surface. after addition of a second thick layer of Matrigel, we To further characterize the differentiation process on could keep them for up to 10–12 days in culture and both surfaces, we used immunostaining against the tran- study accurately their maturation. Under these condi- scription factors paired box 7 (Pax7) and MyoD to evalu- tions, the width of the myotubes remained stable over ate the proportion of reserve cells in our two cultures time (19.7 ± 1.1 μm to 19.8 ± 0.7 μm at 4 and 10 days, (Supplemental Fig. 1A). During the early step of differenti- respectively; data not shown). ation in vitro, reserve cells escape the terminal differenti- To obtain a broad overview of the myotube maturation ation and commit to a quiescent state [17]. These cells + - achieved on nanopattern surface, we analyzed several param- express Pax7, but not MyoD (Pax7 /MyoD ), while myo- 2+ eters, the first one being the gene expression of Ca -hand- tubes express MyoD and downregulate Pax7 (Pax7 / 2+ ling proteins (Cav1.1α1s, RyR1, and sarco-endoplasmic Ca MyoD ). We did not observe a difference between sur- ATPase (SERCA)1/2) and myosin heavy-chain isoforms, by faces in the percentage of Pax7-positive cells (Supplemen- RT-qPCR in cultured myotubes (at 4 and 10 days) and, for tal Fig. 1B), indicating that the striated surface did not comparison, in adult muscle sample. As expected, the ex- interfere with the establishment of reserve cells. MyoD- pression of all these genes increased between myoblasts and positive nuclei were, however, slightly more frequent on differentiated myotubes, while their expression remained the striated surface (Supplemental Fig. 1C). Hence, the stable between 4 and 10 days in culture (Fig. 3a). Transcript nanopatterned striated surface seems to favor specifically levels of CACNA1S, RYR1, MYH7 (encoding the isoform myotube maturation but not the entire process of expressed in slow fibers), and ATP2A2 (encoding SERCA2) myogenesis. were less than 10-fold lower in myotubes compared with 2+ those in adult fibers. On the contrary, genes encoding pro- Ca response to electrical field stimulation on flat versus teins abundant in fast fibers (MYH1, MYH2, and ATP2A1) striated surfaces were between 10- and more than 100-fold less expressed in We then investigated whether aligned myotubes ob- myotubes than in adult tissue. Transcript levels of MYH3 tained from the striated surface were functionally (coding the embryonic myosin heavy chain) slightly de- more advanced compared with those grown on a flat 2+ creased between 4- and 10-day old myotubes, and they were surface. To this end, we recorded Ca responses after higher in myotubes than in the adult tissue (Fig. 3a). In paral- electrical field stimulation to assess the level of EC lel, we assessed the expression of the embryonic and adult coupling. Four-day-old myotubes were electrically isoforms of the Cav1.1α1s subunit, namely the Cav1.1e and stimulated by repetitive bursts (1 s at 10 Hz, every 10 the Cav1.1a, respectively. Immature myotubes express pre- s) for 3 min. Myotubes grown on either surface 2+ dominantly the Cav1.1e isoform (that lacks exon 29), while it responded by transient cytosolic Ca elevations after represents less than 10% of the transcripts in differentiated stimulation. To note, while less than 45% myotubes 2+ muscle [18]. Using conventional PCR analysis, we detected cultured on a flat substrate displayed Ca elevation two bands corresponding to the embryonic and adult iso- upon stimulation, this percentage increased to more forms of Cav1.1 both at 4 and 10 days, while on the adult tis- than 95% when the myotubes were grown on nano- sue, only the Cav1.1a was expressed (Fig. 3b). Sequencing pattern (Fig. 2a, b). We cannot exclude that a small the PCR product confirmed that the lower band found in percentage of myotubes grown on a flat surface did 2+ myotubes lacks exon 29 (Fig. 3c). Furthermore, the ratio of not display a Ca response to electrical field stimula- Cav1.1e and Cav1.1a expression decreased between 4- and tion because of a suboptimal orientation of the cell 10-day old myotubes (Fig. 3d), highlighting a shift towards compared with the electrodes. However, as the Brunetti et al. Skeletal Muscle (2021) 11:12 Page 7 of 14 2+ Fig. 2 The nanopattern surface improves Ca responses to electric field stimulation. a Four-day-old myotubes were loaded with 5 μM Cal520- 2+ AM to detect changes in cytosolic Ca concentration upon 1 min of electric field stimulation (bursts of 1 s at 10 Hz, every 10 s). Representative recordings of myotube responses on flat and striated surfaces (ANFS). The fluorescence intensity was expressed as F/F . b Cell responses to stimulations were classified based on the amplitude of the first peak: non-responders, amplitude < 0.5; responders, amplitude > 0.5. Bar charts represent the percentage of each category of responses on the two surfaces, flat and nanopattern (ANFS). Data were obtained from four independent experiments the expression of the adult Cav1.1 channel with time in Supplemental figure 2). Over time, the typical perpen- culture. dicular alignment of α-actinin appeared first (at around 4 days) followed by STIM1 (at 8–10 days) and eventually 2+ Organization of muscle-specific proteins and Ca RyR1 (Fig. 4c–e). The striations of α-actinin and STIM1 responses were observed in a consistent way, while the RyR1 dou- Skeletal muscles have a highly organized internal archi- blets were seldom obtained (in about 20% of the cul- tecture with striated pattern of most of the muscle- tures) after 10 days of myotube maturation. We further specific proteins. To evaluate how mature are myotubes noticed that on myotubes at that stage, the nuclei were grown on the nanopattern surface, we performed immu- mostly localized at the cell periphery (Fig. 4f). Import- 2+ nostaining of α-actinin, RyR1, and the SR Ca sensor antly, already after 6 days in DM, the myotubes dis- stromal interaction molecule 1 (STIM1) after 10–12 played frequently spontaneous contractions, in around days of differentiation. As shown on Fig. 4a, the staining 30–40% of the cultures (Movie 1). of all these proteins appeared striated, and the distance Next, we analyzed the presence of AChR clusters at between two z lines was of around 2.7 μm, close to what different times of maturation. Both number and size of is found in the adult human muscle (2.5 μm; Fig. 4b and clusters significantly increased within 2, 4, and 10 days Brunetti et al. Skeletal Muscle (2021) 11:12 Page 8 of 14 CACNA1S RYR1 ATP2A1 ATP2A2 10 10 ns 10 10 ns ns 1 1 1 0.1 0.1 0.1 0.01 0.01 0.01 0.1 0.001 0.001 0.001 0.0001 0.0001 0.0001 0.01 MYH1 MYH2 MYH3 MYH7 (fast) (fast) (embryonic) (slow) 10 10 100 ns ns ns ns 1 1 0.1 0.1 0.1 10 0.01 0.01 0.01 0.001 0.001 0.001 1 0.0001 0.0001 0.0001 0.00001 0.00001 0.00001 0.1 B C 400 bp Cav 1.1a Cav 1.1e 200 bp Cav1.1a Cav1.1e Adult Muscle D4 D10 Fig. 3 (See legend on next page.) D0 D4 D10 Adult Muscle D0 D4 D10 Adult Muscle D0 D4 D10 Adult Muscle D0 D4 D10 Adult Muscle D0 D4 D10 Adult Muscle D0 D4 D10 AdultMuscle D0 D4 D10 Adult Muscle D0 D4 D10 Adult Muscle Relativeexpression (AU fold) Relativeexpression (AU fold) Ratio(Cav1.1e/Cav1.1a) Relative expression (AU fold) Relative expression (AU fold) Relative expression(AU fold) Relative expression (AU fold) Relative expression (AU fold) Relativeexpression (AU fold) Brunetti et al. Skeletal Muscle (2021) 11:12 Page 9 of 14 (See figure on previous page.) Fig. 3 Expression of EC coupling-associated proteins and myosin heavy chain during myotube maturation. a Transcript levels of genes encoding for proteins involved in muscle contraction and EC coupling were quantified by RT-qPCR in adult muscle (three donors) and in myoblasts (D0), 4-day-old (D4), and 10-day-old (D10) myotubes (cultures from five donors). CACNA1S (Cav1.1), RYR1 (RyR1), ATP2A1 (SERCA1), ATP2A2 (SERCA2), MYH1 (MyHC-2X), MYH2 (MyHC-2A), MYH3 (MyHC-3 embryonic) and MYH7 (MyHC-7 slow). Transcript levels are relative to adult muscle transcript content. Results are mean ± SEM. A.U., arbitrary units. Each dot represents one independent experiment. b PCR products of the two variants of Cav1.1, embryonic (Cav1.1e) and adult (Cav1.1a) detected in 4- and 10-day-old myotubes and in human adult muscle. M, marker of molecular weights. c PCR product sequencing of CACNA1S exon 29, specific of the adult isoform of Cav1.1 (Cav1.1a) but absent in the embryonic isoform (Cav1.1e). d Quantification of the ratio between Cav1.1e and Cav1.1a expressions in 4- and 10-day-old myotubes. Error bars represent mean ± SEM of culture (Fig. 5a–d). The shape of the clusters is [21]. In addition, we added during the initial 4 days of known to change during maturation, with the emergence differentiation an inhibitor of TGFβ receptor, a proced- of branched structures with time [19]. We detected only ure that was shown to improve cell fusion [22], MYH ex- very few clusters with such rearrangement of the AChR pression, and sarcomere organization [23, 24]. after 10 days of maturation, most of the clusters being As expected, the myotubes grown on a striated surface oval-shaped. However, AChR were functional, as shown were very well aligned, with nuclei being also more elon- 2+ by the rapid cytosolic Ca elevation elicited by applica- gated, initially aligned in the middle of the myotubes tion of 10 μM acetylcholine (Fig. 5e–g). By comparison, while moving at the cell periphery with time in culture, on the flat surface at day 4 of differentiation, the AChR hence recapitulating the progression of nuclei movement were more dispersed on the myotubes and did not form occurring in vivo [16]. Yet, we did not notice differences, proper clusters (Supplemental Figure 3). In addition, neither in fusion index nor in percentage of MEF2C- 2+ only 64% of the myotubes displayed a Ca response positive nuclei, between flat and grooved surfaces after 4 when stimulated with 10 μM ACh, compare with the days of differentiation. This is somehow in contradiction 97% of responding myotubes grown on the nanopattern with what was previously reported, where differentiation surface (Supplemental Figure 3). performed on striated surfaces enhanced the fusion 2+ Lastly, we assessed Ca responses to electrical stimu- index [20, 21]. It should be pointed out, however, that lations. Four- and 10-day-old myotubes were stimulated human primary cells cultured in our laboratory has a for 3 min with repetitive bursts every 10 s (Fig. 6a). The high fusion index (around 60%), higher than what was 2+ amplitude and the decay time of the first cytosolic Ca reported in the abovementioned studies. In line with the elevation were very similar at early and later stages of fusion index, the percentage of Pax7-positive cells was maturation (Fig. 6b, c). As well, the amplitude of the last also similar between the two surfaces. Hence, our cul- transient compared with the first one (percentage of ture system did not favor the initial steps of differenti- 2+ remaining Ca ), that depicts the sustained nature of the ation but presents the advantage to promote cell response was also not significantly different between the alignment and to maintain cells for longer time in cul- two time points (Fig. 6d). Hence, myotubes already dis- ture allowing their maturation. played robust EC coupling after 4 days when cultured We analyzed then several parameters during 10–12 days on nanopattern striated surface. in culture: the expression level of muscle-specific genes, 2+ the internal organization of the myotubes, and their Ca Discussion responses to ACh and electrical stimulations, in order to We reported here a simple in vitro culture model of hu- provide a broad overview of the processes taking place man primary myoblasts that presents greater level of over time, both morphologically and functionally. myotube maturation compared with commonly used 2D Although the myotube diameter remained stable dur- cultures. ing time in culture, the number and size of AChR clus- Our culture model combined various procedures that ters increased between 2 and 10 days of maturation. At foster myotube maturation. First, cells were grown be- 10 days of differentiation, almost all myotubes displayed tween two layers of Matrigel that provided an extracellu- clusters of AChR, which reflects a greater maturation of lar matrix limiting cell detachment, as previously myotubes compared with conventional 2D cultures reported [13]. Second, cells were grown on a striated where only a minority of myotubes have AChR clusters pattern surface that favors alignment and maturation of after 2 weeks in culture [8]. The clusters remained how- myotubes [11, 20, 21]. This surface is a fabricated pat- ever essentially homogenous, without a sign of lacunarity tern with grooves and ridges of 800-nm width and 600- or pretzel structures, like it was reported in 3D culture nm depth, dimensions that were previously described to [8] or on surfaces coated with various laminin [25], mor- favor myotube maturation [20], even if larger striations phological changes that reflect the maturation of AChR (up to 1600 nm) also led to an enhanced maturation clusters [19, 26]. AChRs were however functional as Brunetti et al. Skeletal Muscle (2021) 11:12 Page 10 of 14 2+ shown by the Ca elevations elicited by ACh stimula- tion. The amplitude of the response was akin to the one elicited by electrical stimulation, in line with what was reported on human 3D myobundles [27]. On the con- trary, 4-day-old myotubes grown on a flat surface have a diffuse pattern of AChR and not all myotubes responded 2+ to ACh stimulation by a Ca elevation. Interestingly, the genes expressed predominantly in slow fibers (MYH7 and ATP2A2) were expressed in 10- day-old myotubes at levels closer to adult tissue, than the ones expressed in fast fibers (MYH1&2 and ATP2A1). Hence, even if there was a mixed expression of genes from slow and fast fibers, like it is frequently observed in cultured myotubes [28, 29][7], myotubes appear to mature preferentially towards a slow pheno- type. This slow phenotype resembles the one observed during the establishment of a human muscle cell line HMCL-7304 [30], with a higher abundance of slow MYH. This slow phenotype is however different to what is observed in vivo when regeneration happens in the ab- sence of innervation, where fibers follow what is called the “default pathway” toward a fast phenotype [31]. The reasons of this maturation towards a slow phenotype are unclear, but this may, at least partially, explain the kinet- 2+ ics of the Ca responses triggered by ACh or electrical stimulation that we measured. Indeed, even if myotubes 2+ in culture are known to have slower Ca recovery phase than adult fibers [32], we were anticipating a faster decay time associated with enhanced maturation, which was 2+ not the case. Certainly, the slow Ca kinetics of the re- sponse also reflects the fact that the system did not suffi- 2+ ciently mature to have an obvious impact on the Ca repumping efficiency. During muscle development, a splice variant of Cav1.1 lacking exon 29, namely Cav1.1Δ29 or Cav1.1e is expressed, characterized by dis- tinct biophysical properties compared with the adult 2+ Cav1.1 isoform: a higher Ca current amplitude to- gether with a 30-mV left shift of the activation potential [18, 33]. The expression of Cav1.1e was also reported in human myotubes in culture, leading us to evaluate its expression. Our results show that the embryonic Cav1.1e is expressed both at 4 and 10 days, and interestingly, the Fig. 4 Organization of muscle-specific proteins and of nuclei. ratio between the two, embryonic and adult isoforms, Representative images of 10-day-old myotubes cultured on nanopattern decreased with time in culture, confirming an enhanced surface (a) and adult human muscle tissue (b) immunolabeled for α-actinin fiber maturation at 10 days. (green) and STIM1 (red) (upper lines) and for RyR1 (green) and STIM1 (red) (lower lines). Scale bar: 5 μm. c–e Organization of α-actinin, STIM1, and We also assessed the organization level of several pro- RyR1 during maturation. Representative images of 4-, 10-, and 12-day-old teins over time in culture. As already reported [34], the myotubes immunolabeled for α-actinin (c) STIM1 (d), and RyR1 (e). At 4 transverse alignment of α-actinin occurred rapidly after days, α-actinin is already perpendicularly oriented, followed at 10 days by the induction of differentiation. Indeed, after 4 days in STIM1, and in some cells by RyR1 at 12 days. Scale bar: 5 μm. f culture, a substantial number of cells displayed this typ- Representative image of 12-day-old myotubes immunolabeled with STIM1 together with DAPI. White arrows point to peripheral nuclei. Scale bar: ical arrangement, and the striations get sharper with lon- 20 μm ger time in culture. The distance between two Z lines in 10-day-old myotubes was around 2.7 μm, close to the size of adult human sarcomeres (2.5 μm). Furthermore, Brunetti et al. Skeletal Muscle (2021) 11:12 Page 11 of 14 -actinin/ -BTX/DAPI Day 2 Day 4 Day 10 Day 4 Day 10 Day 2 BC D 0 0 D2 D4 D10 D2 D4 D10 D2 D4 D10 E F G Amplitude 10 µM ACh 10 µM ACh 6 6 4 4 3 2 2 0 0 0 102030405060 0 102030405060 0 D4 D10 Time (s) Time (s) 2+ Fig. 5 Organization of AChR clusters during maturation and Ca response to acetylcholine. a Representative images of 2-, 4-, and 10-day-old myotubes cultured on nanopattern surface and immunolabeled for α-actinin (red) and α-bungarotoxin (α-BTX, green) together with a DAPI staining (blue). Lower panels are enlargement of the white rectangles. Scale bar: 20 μm. b, c, d Quantification of the average number of acetylcholine receptors (AChR) clusters per field (b), AChR cluster size (c), and of the average numbers of AChR clusters per myotube (d). Data were obtained from three independent experiments. Error bars are mean ± SEM. e−f Myotubes were loaded with 5 μM Cal520-AM to measure 2+ 2+ changes in cytosolic Ca , and stimulated with 10 μM of acetylcholine (ACh). Traces represent the mean ± SEM of Ca responses of myotubes differentiated for 4 (e) and 10 days (e). g Statistical evaluation of the amplitude of the peak following ACh stimulation. Error bars are mean ± SEM 2+ after 7–8 days in culture, the SR resident Ca sensor human cells cultured in 2D. Advanced maturation were STIM1 also appeared striated, with an important level of obtained with human iPSC but after a significantly lon- colocalization on the Z lines. This localization of STIM1 ger time (50 days to obtain titin striations [6], or after 2 is consistent with findings obtained in muscle adult tis- weeks to obtain α-actinin striations of myotubes derived sue, where STIM1 was reported to be present both at a from induced myogenic progenitor cells [7]. Hence, the region corresponding to the I band (around the Z lines), advantage of growing the myotubes on a striated surface, as well as on the longitudinal SR [35][36, 37]. A proper beside the improvement of cell adhesion, is that myo- RyR1 localization in doublet was however infrequently tubes remain thin and elongated which likely promotes obtained, even after 12 days of maturation. On the few their internal organization and thus maturation. cases where such matured arrangement of RyR1 was ob- Finally, we assessed the ability of myofibers to elicit cyto- 2+ served, it always took place after STIM1 was perpen- solic Ca transients upon electrical field stimulation. Already dicularly organized. Even if RyR1 doublets were seldom at 4 days of culture, electrical stimulations resulted in large 2+ observed, the high level of internal organization achieved Ca transients of almost all myotubes, highlighting a func- within 10 days was to our knowledge not obtained with tional EC coupling. This time frame is rapid if we compared F/F #of AChR clusters per field AChR cluster size (um ) F/F #ofAChR clusters per myotube F/F 0 Brunetti et al. Skeletal Muscle (2021) 11:12 Page 12 of 14 2+ 2+ Fig. 6 Ca transients upon electric field stimulation. a Representative traces of cytosolic Ca transients generated by repeated burst of 1 s at 10 Hz, every 10 s for 3 min in 4-day (left panel) and 10-day (right panel)-old myotubes, differentiated on nanopattern surface. Statistical evaluation of 2+ the amplitude (b) and the decay time (c) of the first Ca transient, and the percentage of remaining calcium at the end of 3-min stimulation (d). Data were obtained from four independent experiments, and each dot is a mean of 4−6 myotubes on a field. Error bars are mean ± SEM it to the 8 days needed to obtain 50% of myotubes this rapid and easy-to-perform 2D culture, which does responding to electrical stimulation with classical 2D not require sophisticated technology, presents certainly culture of human cells [32]. We analyzed at 4 and 10 great advantages for further single-cell studies designed 2+ days of culture three parameters out of these Ca re- to investigate the processes of skeletal muscle cordings, the amplitude of the first transient, the kin- maturation. 2+ etic of the Ca recovery phase, and the sustained Abbreviations nature of the signals during the 3-min stimulation. GM: Growth medium; DM: Differentiation medium; SR: Sarcoplasmic None of these parameters significantly changed be- reticulum; EC: Excitation–contraction; MEF2C: Myocyte enhancer factor 2C; MyoD: Myogenic differentiation 1; Pax7: Paired box 7; RyR1: Ryanodine tween 4 and 10 days, showing that even if the cells receptor 1; DHPR: Dihydropyridine receptor; STIM1: Stromal interaction displayed a better internal organization at 10 days, molecule 1; ACh: Acetylcholine; AChR: Acetylcholine receptor; α-BTX: α- 2+ 2+ this did not correlate with an improved Ca signals. Bungarotoxin; SERCA: Sarco-endoplasmic Ca ATPase This is however in agreement with the expression levels of RYR1, CACNA1S, and ATP2A1/2 that remain Supplementary Information The online version contains supplementary material available at https://doi. stable during the maturation period we analyzed. org/10.1186/s13395-021-00268-3. Conclusions Additional file 1: Movie 1. Spontaneous myotube contraction on 6- Overall, we proposed a 2D culture system of human day old culture on nanopattern surface. myotubes that reach after 10 days a high level of matur- Additional file 2: Supplemental Figure 1. The reserve cell population ation, with the presence of numerous functional AChR is not affected by the surface pattern. (A) Immunofluorescence of nuclei of 4-day old myotubes on Flat (upper panel) or nanopattern surface clusters, the expression of adult MYH, increased adult (ANFS, lower panel). Cell nuclei were stained using an antibody against CACNA1S isoform expression, and a high level of in- Pax7 (in green), MyoD (in red) and all nuclei were counterstained with ternal fiber organization. Further improvements are DAPI (in blue). Scale bar: 50 μm. Images shown are representative of six independent experiments. (B, C) Percentage of Pax7 positive nuclei (B) however required to obtain myofibers that resemble and MyoD positive nuclei (C). 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