GelatinâPolyaniline Composite Nanofibers Enhanced Excitation

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Gelatin-Polyaniline Composite Nanofibers Enhanced ExcitationContraction Coupling System Maturation in Myotubes serge ostrovidov, Majid Ebrahimi, Hojae Bae, Hung Kim Nguyen, Sahar Salehi, Sang Bok Kim, Akichika Kumatani, Tomokazu Matsue, Xuetao Shi, Ken Nakajima, Shizu Hidema, Makoto Osanai, and Ali Khademhosseini ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b03979 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Gelatin-Polyaniline Composite Nanofibers Enhanced Excitation-Contraction Coupling System Maturation in Myotubes Serge Ostrovidov†,α,#, Majid Ebrahimi†,β,#, Hojae Bae§, #, Hung Kim Nguyen†, Sahar Salehi†,γ, Sang Bok Kim‡, Akichika Kumatani†,∆, Tomokazu Matsue†,∆, Xuetao Shiǁ, Ken Nakajima┴, Shizu Hidema○, Makoto Osanai╪,♪, Ali Khademhosseini†,§,α,¶,●,* †

WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan Department of Medicine, Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA β Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto ON M5S3G9, Canada § College of Animal Bioscience and Technology, Department of Bioindustrial Technologies, Konkuk University, Hwayang-dong, Kwangjin-gu, Seoul 143-701, Republic of Korea γ Lehrstuhl Biomaterialien, Fakultät für Ingenieurwissenschaften, Universität Bayreuth, Bayreuth 95440, Germany ‡ Department of Eco-Machinery system, Korea Institute of Machinery and Materials, Daejeon 305-343, Republic of Korea ∆ Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan ǁ National Engineering Research Centre for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006, PR China ┴ School of Materials and Chemical Technology, Tokyo Institute of Technology, Tokyo 152-8550, Japan ○ Graduate School of Agricultural Science, Department of Molecular and Cell Biology, Tohoku University, Sendai 981-8555, Japan ╪ Graduate School of Medicine, Department of Radiological Imaging and Informatics, Tohoku University, Sendai 980-8575, Japan ♪ Graduate School of Biomedical Engineering, Department of Intelligent Biomedical Systems Engineering, Tohoku University, Sendai 980-8575, Japan ¶ Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA ● Department of Physics, Faculty of Science, King Abdulaziz University, Jeddah 21569, Saudi Arabia1 α

*Corresponding author: Department of Medicine, Biomaterials Innovation Research Center, Division of Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Massachusetts 02139, USA Email address: [email protected] (Ali Khademhosseini) Phone: +1 617 388 9271; Fax: +1 617 768 8477 #

These authors contributed equally to this work.

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ABSTRACT In this study, composite gelatin-polyaniline (PANI) nanofibers doped with camphorsulfonic acid (CSA) was fabricated by electrospinning and used as substrates to culture C2C12 myoblast cells. We observed enhanced myotube formation on composite gelatin-PANI nanofibers compared to gelatin nanofibers, concomitantly with enhanced myotube maturation. Thus, in myotubes, intracellular organization, co-localization of the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), expression of genes correlated to the excitation-contraction (E-C) coupling apparatus, calcium transients, and myotube contractibility were increased. Such composite material scaffolds combining topographical and electrically conductive cues may be useful to direct skeletal muscle cell organization and to improve cellular maturation, functionality and tissue formation.

KEYWORDS Composite nanofibers, Polyaniline, Gelatin, Excitation-contraction coupling, Myotube maturation, Intracellular calcium, C2C12

1. INTRODUCTION Polymeric nanofibers can be fabricated by electrospinning and used as substrate for cell and tissue engineering.1 These nanofibers mimic components of the extra-cellular matrix (ECM) and when aligned induce cell alignment and allow the formation of anisotropic tissues, such as the skeletal muscle tissue.2 Numerous synthetic (e.g. Poly(ethylene glycol) (PEG), Poly(lactic acid)


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(PLA)) or natural (e.g. chitosan, silk, gelatin) polymers have been electrospun into nanofibrous scaffolds for tissue engineering applications.1 Among them gelatin has been often used for its cell supportive properties.3 However, gelatin has weak electrical conductivity, which is a limitation when using electroconductive cells such as skeletal muscle cells. One way to fabricate electroconductive nanofibers is by the integration of conductive nanoparticles, such as gold or carbon nanotubes, into the polymeric matrix.4 Another approach to fabricate electrically conductive nanofibrous scaffolds is to elctrospin a composite polymer made of a carrier polymer and an electrically conductive polymer (CP), such as

polypyrrole

(PPy),

polyaniline

(PANI),

polythiophene

(PT),

or

poly(3,4-

ethylenedioxythiophene) (PEDOT).5 These CPs have conjugated double bonds in their backbones, which allow the delocalization of the π electrons into a conduction band. The electrical conductivity of such polymers arises in the presence of a dopant, which can oxidize (ptype) or reduce (n-type) the polymer chains.6 The appearance of charges on the polymer chains induces instabilities, which are energetically compensated by local distortions in the polymer chain lattice, to surround and localize the charges.7 This type of charge localized by lattice distortion is called a polaron.8 When an electrical potential is applied to the CP, the dopant begins to move in the polymer, disrupting the stabilization process, and the polarons can move along the polymer chains, allowing electrical conduction. Among CPs, PANI is one of the most investigated. Depending on its oxidative state, PANI exist under three different insulating forms: The leucoemeraldine base (reduced form), the emeraldine base (half-oxidized form, PANI-EB), and the pernigraniline base (oxidized form).6 The emeraldine base is the most stable form at room temperature and is also the form with the highest electrical conductivity when doped into the conductive form of emeraldine salt (PANI-ES).6 It



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has been used in biological applications, such as actuators9, biosensors10, drug delivery11, and tissue engineering.12,13 Especially, some research groups have fabricated composite PANIpolymer nanofibers by electrospinning for tissue engineering applications. Thus, Chen et al. fabricated composite nanofibers of PANI-polycaprolactone (PCL) and used them as a scaffold for muscle tissue engineering.14 They observed enhanced myotube formation on PANI-PCL nanofibers compared to pure PCL nanofibers. Similarly, Jun et al. and Ku et al. observed enhanced myogenesis in myoblasts cultured on composite PCL-PANI nanofibers.15,16 Natural polymers, such as gelatin, have also been blended with PANI. Li et al. cultured H9c2 rat cardiac myoblasts on gelatin-PANI nanofibers and observed good cell attachment, proliferation and migration.17 Gelatin methacryloyl (GelMA)-PANI composite conductive hydrogels have also been fabricated, characterized, and used as scaffold with mesenchymal progenitor cells.18 In another study, Kim et al. fabricated a hybrid collagen film containing dispersed PANI nanofibers and showed that it can be used as a scaffold for culturing porcine skeletal muscle cells.19 Thus, numerous composite platforms have been fabricated with PANI and used as scaffolds for culturing different cell types, including skeletal muscle cells, and most of these studies have focused on scaffold characterization, biocompatibility, muscle cell proliferation and differentiation but not on the maturation and functional properties of the differentiated myotubes.12 Moreover, in studies using electrospun nanofibers, although such composite nanofibers are composed of at least three components (PANI, a polymer carrier, and a dopant), the effect of the dopant itself on cell culture is not usually reported.

In this study, we fabricated composite gelatin-PANI nanofibers by electrospinning and used them as substrates for skeletal muscle tissue engineering. After characterization of the


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nanofibers, we analyzed the effect of the composite polymer and of the dopant (CSA) on myogenesis; then, we focused on myotube maturation and functionality by studying the E-C coupling system.

2. EXPERIMENTAL SECTION 2.1. Materials Type A gelatin from porcine skin (300 bloom), dimethylformamide (DMF), camphorsulfonic acid

(CSA,

Aldrich

282146),

polyaniline

powder

(PANI,

Aldrich

328429),

and

penicillin/streptomycin (P/S) were purchased from Sigma-Aldrich (USA). Dulbecco’s modified Eagle’s medium (DMEM), Hanks’ balanced salt solution (HBSS), Dulbecco’s phosphatebuffered saline (DPBS), essential amino acid solution, non-essential amino acid solution (MEMNEAA), and horse serum (HS) were purchased from Gibco (USA). HEPES (4-(2-hydroxyethyl)1-piperazineethanesulfonic acid) was purchased from Dojindo (Japan), glutaraldehyde 25% aqueous solution (GTA) was purchased from Kanto-Chemical (Japan), and fetal bovine serum (FBS) was purchased from BioWest (USA). The 35-mm dishes were purchased from ThermoScientific (Denmark). Our electrospinning setup consisted of a DC high voltage power supply (Max Electronics, AMK-30 K06PBX, Japan), a syringe pump (World Precision Instruments, Aladdin syringe pump, USA), and two glass chemical coolers (on request).

2.2. Nanofibers fabrication by electrospinning Gelatin dissolves in water and is practically insoluble in most organic solvents, whereas PANI does not dissolve in water but in polar solvents, such as dimethyl sulfoxide (DMSO), DMF, and



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N-methyl-2-pyrrolidone (NMP), and strong protonic acids, such as sulfuric acid, methane sulfonic acid, and trifluoromethane sulfonic acid (Sigma-Aldrich PANI product information). In this study, gelatin 20% (w/v) and gelatin 20% (w/v) + CSA 5% (w/v) were dissolved in Milli-Q water at 50 °C on a hot plate for 15 min with stirring. Gelatin-CSA-PANI were dissolved in DMF 50%-Milli-Q water 50% to final concentrations of gelatin 20% + CSA 5% + PANI 5% or gelatin 20% + CSA 10% + PANI 10% at 50 °C on a hot plate for 15 min with stirring. The polymer mixture was sonicated (10 pulses) with a sonicator (QSonica, model Q55, USA) to reduce aggregates of PANI particles and to help the mixing. The blend polymer was then placed again on a hot plate at 50 °C with stirring for 10 min before loading in a 1 mL plastic syringe, which was placed in a glass chamber that was custom-designed to fit a 1 mL syringe (Terumo/SS-01T, Japan). This glass chamber was connected to a water bath via a perfusion circuit and was used as a warmer. As spinneret, we used a 20-gauge steel needle (Hoshiseido, Japan). To warm the needle, a glass chamber was secured on the needle using a piece of rubber and was connected to the water bath via a second perfusion circuit (Fig. 1A). The polymer solution was delivered at a flow rate of 10 µL/min by a syringe pump, and the nanofibers formed under a tension of 18 kV were harvested on an aluminum foil-based counter electrode placed 8 cm from the spinneret. To obtain aligned fibers, plastic bands (1 cm wide, 7 cm long) were placed on the aluminum foil and were bridged by the polymeric fibers attracted by the conductive aluminum parts (Fig. 1B). The nanofibers were then crosslinked overnight in GTA vapor and rinsed in Milli-Q water for 2 days at room temperature. Before their use in experiments, the nanofibers were sterilized under UV light on a clean bench for 10 min.


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Figure 1. Schematic of the electrospinning setup (A). Two glass chambers were used to warm the syringe and the needle, enabling electrospinning at a constant temperature (50 °C). On the aluminum foil used as a counter electrode, plastic film bands were used to induce fiber alignment and to harvest the fibers (B). FE-SEM images of the nanofibers electrospun obtained at 50 °C, and 18 kV (C)



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2.3. Characterization of the nanofibers by field emission-scanning electron microscopy (FE-SEM) The gelatin and gelatin composite nanofibers were examined by FE-SEM (JEM2100F, JEOL, Japan) after 30 s of platinum metallization in a sputter coater (JEOL, JFC1600, Japan) at 10 mA. The fiber diameters and alignment were measured from the FE-SEM images using the AxioVision Rel. 4.8 software package by analyzing 100 individual fibers.

2.4. Electrical conductivity measurements For each polymer solution, 900 µL of polymeric solution was added to the wells of a flat-bottom 24 well culture plate (Greiner bio-one, Germany) and left at room temperature (RT) overnight on a table for gelation. Then, the plate was left in a desiccator with GTA vapor for 2 days for crosslinking. Polymeric discs (1.5 cm diameter, 0.4 cm thickness) were harvested and rinsed in water for 1 day. Because the polymeric gels maintained their disc shape but swelled in water, the diameter and the thickness of the different discs were measured again, and these values were taken into account in the evaluation of the electrical conductivity. The electrical conductivity was measured by 4 probe measurement (Loresta GP MCP-T610, Mistubishi Chemical Analytic, Japan). All measurements were conducted under atmospheric conditions.

2.5. Fourier transform infrared (FTIR) spectroscopy FTIR spectra were recorded using an infrared spectrophotometer (FT-IR; JASCO, FT/IR-6600, Japan) with attenuated total reflection mode. The spectra were obtained with 32 scans per sample, ranging from 400 to 4000 cm-1.


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2.6. Atomic force microscopy (AFM) The mechanical properties of the different nanofiber types were assessed via nanomechanical mapping in Milli-Q water using a Bruker MultiMode™ atomic force microscopy (AFM, Japan) with a NanoScope V controller. The measurements were operated in force volume AFM mode, in which force-distance curves were obtained over randomly selected surface areas of 6 × 6 µm at a resolution of 64 × 64 pixels.2,20 AFM probes Cantilevers (OMCL-TR800PSA-1) were purchased from Olympus. The spring constant and curvature radius of these probes were calibrated to be ~0.5 N/m and ~25 nm, respectively. The Derjaguin, Muller, Toporov (DMT) model21, which takes into account a small adhesive interaction between the probe and nanofibers in water, was used to analyze the force-distance curves that yielded the mapping of the Young’s modulus on the surface of the nanofibers.2, 22

2.7. Cell cultures Murine C2C12 myoblast cells (before passages 5) from American Type Culture Collection (ATCC) were cultured in DMEM supplemented with 10% FBS, 1% P/S, and 20 mM HEPES in a humidified incubator at 37 ºC and 5% CO2. At ~70% confluency, the cells were harvested by trypsinization. A differentiation medium composed of DMEM supplemented with 2% HS, 1% P/S, and 20 mM HEPES was used to induce myotube formation. Cells were seeded on the fibers at 8 × 104 cells/cm2 and were left for 30 min in the incubator at 37 °C to attach. The fibers were then gently rinsed to remove non-adhered cells, and 2 mL of warm culture medium was added to each culture dish.



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2.8. Electrical pulses Plastic bands holding polymeric fibers were cut into 1 cm × 1 cm pieces and taped in 35-mm dishes. After 1 day of culture in growth medium and 6 days in differentiation medium, the samples were subjected to continuous square electrical pulses (6 V, 1 Hz, 1 ms duration) in stimulation medium (DMEM supplemented with 2% HS, 1% P/S, 20 mM HEPES, 1% MEMNEAA, and 2% MEM) for 1 day via an electronic stimulator (Ion Optix, C-pace EP, USA) and 6 pairs of carbon electrodes (Ion Optix, USA) with an electrode gap of 1.8 cm.

2.9. Immunostaining - Immunostaining of myosin heavy chain (MHC) in myotubes Myotubes were fixed in 4% (w/v) formaldehyde for 15 min, permeabilized with 0.3% (v/v) Triton X-100 for 5 min, and blocked with 5% bovine serum albumin (BSA) in DPBS for 20 min at 37 ºC. After removing the BSA solution, mouse monoclonal anti-fast skeletal myosin IgG antibody (abcam, ab-7784, 1:1000 dilution in DPBS with 0.1% BSA) was added, and the myotubes were incubated at 4 ºC overnight. The following day, the samples were stained with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen, A11001, 1:1000 dilution in DPBS with 0.1% BSA) and incubated at 37 ºC for 60 min. Myotubes having at least 3 cell nuclei were quantified on day 6 of culture in differentiation medium. The myotube length was determined using the AxioVision Rel. 4.8 software package (Zeiss, Germany). - Sequential immunostaining for DHPR and RyR visualization We followed the “double immunofluorescence-sequential protocol” from abcam company with slight modifications.23 Briefly, myotubes were fixed in 4% (w/v) formaldehyde for 15 min, permeabilized with 0.3% (v/v) Triton X-100 for 5 min, blocked with 5% BSA in DPBS for 30


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min at 37 ºC, and incubated at 4 ºC overnight with (the first primary antibody) mouse antiryanodin receptor (abcam, ab 2868, 1:500 dilution in DPBS with 0.1% BSA). The following day, the solution of the first antibody was removed and the samples were rinsed 3 times with 2 mL DPBS for 5 min for each wash. Then, the samples were stained with (the second antibody) Alexa Fluor 594 conjugated donkey anti-mouse IgG (Invitrogen, A21203, 1:1000 dilution in DPBS with 0.1% BSA) and left for 1 hour at RT in the dark. The samples were then rinsed 3 times with 2 mL DPBS for 5 min for each wash and blocked with 5% BSA in DPBS for 30 min at 37 ºC. The samples were then rinsed 2 times with 2 mL DPBS and incubated at 4 ºC overnight with (the second primary antibody) mouse anti-DHPR (CACNA1S; abcam, ab2862, 1:500 dilution in DPBS with 0.1% BSA). The following day, the solution was removed and the samples were rinsed 3 times with 2 mL DPBS for 5 min for each wash. Then, the samples were stained with Alexa Fluor 488 (green) conjugated goat anti-mouse IgG (Invitrogen, A11001, 1:1000 dilution in DPBS with 0.1% BSA) and left for 1 hour at RT in the dark. The samples were then rinsed 3 times with 2 mL DPBS. Then, 2 mL DPBS was added, and the samples were visualized under fluorescence microscopy (Zeiss, Germany).

2.10. Intracellular calcium measurement Intracellular [Ca2+] was evaluated using the acetoxymethyl (AM) ester of Fura-2 LR (Fura-2 Leakage Resistant, Calbiochem, USA) dissolved in dimethyl sulfoxide (DMSO, Dojindo, Japan) to obtain a 5 mM stock solution.24 200 µL HBSS based dye-loading solution containing 0.4 µL 10% Cremophor-EL and 0.8 µL 5 mM Fura-2 LR-AM were used for 35-mm diameter dish to stain the cells at day 6 of differentiation. The dish was incubated at RT in a small humidified chamber for 30 min, then the sample was rinsed 3 times with HBSS. The sample was then



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incubated for 15 min at 35 °C to allow the esterases in the cytosol to cleave the ester from Fura-2 LR and to free the carbonate groups. The sample was then transferred to the stage of an epifluorescent upright microscope (BX51WI, Olympus, Japan). Cells were placed under perfusion of HBSS at a constant temperature of 30 ± 1 °C and were electrically stimulated (square pluses, 6 V, duration 1 ms applied every 5 s, 10 s after start of recording) for the time of the video recording. Intracellular [Ca2+] changes were imaged with an X20, NA 1.00 water immersion objective (Olympus). Cells were alternatively excited at 340 and 380 nm using a filter changer (Lambda DG-4, Sutter Instruments, USA) with exposure times of 20 ms for each wavelength. The fluorescent signal with wavelength of 525 ± 23 nm was recorded (F340, F380) every 0.12 s for 2 min with a cooled EM CCD (DU 897 Ardor Technology, UK). All equipment was controlled by MetaFluor Software (Molecular Devices, USA). Image analysis was performed with MetaFluor Software and ImageJ. To obtain the transient rate quantification graph, we took the values of the fluorescence ratio (R=F340/F380) recorded every 0.12 s for 2 min from each imaged myotube and defined a baseline for each trace. To remove the noise we applied a threshold of + 0.006 to the baseline value and then counted the number of calcium transient peaks (fluorescence R > to baseline value + 0.006) recorded within the 2 min window.

2.11. Real-time quantitative reverse transcription-polymerase chain reaction (qRTPCR) qRT-PCR was performed on a Bio-Rad MYiQ2 two-color real-time PCR machine. Total RNA was extracted from C2C12 cells, at day 4 (for evaluation of genes correlated to cell attachment) and day 7 (for evaluation of genes correlated to DHPR and RyR) of differentiation, cultured on


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the different fiber types using a PureLink RNA Mini Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. Genomic DNA contamination was eliminated by a PureLink DNase Kit (Invitrogen, USA). Ten nanograms of purified RNA were used per PCR reaction. The one-step quantitative polymerase chain reaction (qPCR) was performed in 4 replicates using a SuperScript III Platinum SYBR green one-step qRT-PCR kit (Invitrogen, USA) and primers (listed in Table TS1 with PCR conditions). The relative gene expression was quantified by calculating the 2-∆CT values, where CT represents the cycle number at which an arbitrary threshold is reached and ∆CT = (CT targeted gene – CT GAPDH).

2.12. Statistical analysis The results are shown as the mean ± standard deviation. Student’s two-tailed unpaired t-test was used for comparative analysis. P < 0.05 was considered statistically significant. Each experiment was repeated three times.

3. RESULTS AND DISCUSSION 3.1. Fabrication and characterization of composite gelatin-PANI nanofibers Fig. 1C shows the FE-SEM images of the four types of aligned nanofibers obtained by electrospinning at 18 kV and 50 ºC. Fig. S1A shows the fiber diameter averages with 343, 315, 322, and 423 nm for gelatin 20%, gelatin 20% + CSA 5%, gelatin 20% + CSA 5% + PANI 5%, and gelatin 20% + CSA 10% + PANI 10% nanofibers, respectively. The morphology of the three first types of nanofibers appears similar. However, the morphology of gelatin 20% + CSA 10% + PANI 10% nanofibers was different, and the electrospun consisted of a mixture of nanofibers and nanoribbons with residual inserted PANI particles. When the electrostatic force overcomes



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the surface tension at the tip of the spinneret, a polymer jet is ejected from the Taylor cone towards the counter electrode. During its path, the solvent evaporates from the polymer jet, which elongates and decreases in diameter, resulting in a nanofiber with circular cross-section.25 However, depending on the polymer and the solvent used, a fiber skin forms first, then, if the solvent evaporates quickly, the fiber skin collapses, resulting in a fiber with a cross-section that shifts towards an oval and then flattens, resulting in the formation of nanoribbons with a flat central part lined by two polymeric tubes on their sides.26 To obtain a homogeneous solution of PANI, most studies mixed PANI with its solvent for several hours and sometimes filtered the final solution on Whatman paper to remove residual PANI particles.17,27 This process allows the production of homogeneous nanofibers with good shapes, but results in an unknown PANI concentration because residual PANI particles remain in the filter. In this study, we mixed the PANI + CSA solution for only 15 min and did not filter it before adding it to the gelatin carrier solution, but we sonicated the whole gelatin + CSA + PANI solution for 10 pulses to decrease aggregation of PANI particles. However, some PANI particle aggregates were still visibly inserted between the nanofibers of the gelatin 20% + CSA 5% + PANI 5% and gelatin 20% + CSA 10% + PANI 10% in the FE-SEM pictures. In a previous study, we developed an electrospinning setup to fabricate gelatin nanofibers at low and high temperatures using a glass chamber as a warmer to hold the syringe and a perfusion circuit.2 However, the needle was warmed by static warm water, which induced temperature fluctuations. In this study, we optimized our previous setup by using a second glass chamber as a warmer around the needle with its own perfusion circuit to maintain the polymer solution at a constant temperature (Fig. 1A). To obtain aligned nanofibers, we fabricated a counter electrode by covering an aluminum foil with a plastic film grid featuring alternating aluminum and plastic


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bands of 7 cm × 1 cm (Fig. 1B). Fig. S1B shows the percentages of aligned nanofibers within 10° angle with 64, 67, 69, and 54% for gelatin 20%, gelatin 20% + CSA 5%, gelatin 20% + CSA 5% + PANI 5%, and gelatin 20% + CSA 10% + PANI 10%, respectively. Excepted for gelatin 20% + CSA 10% + PANI 10% nanofibers which were a little different, the graphs (S1A, S1B) show that the percentages of alignment and the average diameters are in the same range for the three other types of nanofibers.

3.2. Electrical conductivity The electrical conductivity was evaluated by 4 probe measurement on gels with a disk shape crosslinked by GTA vapor for 2 days and rinsed in water for 1 day. The results show that the gels of gelatin 20% and gelatin 20% + CSA 5% were poorly conductive, with electrical conductivity of 5.1 × 10-7 S/cm and 9.1 × 10-7 S/cm, respectively. Alternatively, gels of gelatin 20% + CSA 5% + PANI 5% and gelatin 20% + CSA 10% + PANI 10% had substantial electrical conductivity of 4.2 × 10-3 S/cm and 2.2 × 10-3 S/cm, respectively. An additional factor in the polaronic structure of doped PANI, which affects its conductivity, is the conformation of the polymeric chains. PANI has a coil conformation before protonation, which is allowed by the nitrogen atoms separating each aromatic ring.28 However, after protonation with an acid, such as CSA, the PANI emeraldine base transforms into a polyconjugated polyradical cation salt stabilized by the dopant counteranions, and the repulsion between the positive charges created on each two nitrogen atoms induces a conformational change into an expanded coil structure that is extremely favorable to electron delocalization.6 This polymer conformation is strongly influenced by the dopant and also by the solvent. Xia et al. showed that when PANI + CSA is dissolved in N-methyl-2-pyrrolidone (NMP), chloroform, DMF, and benzyl alcohol, the polymer



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chains are in a coil conformation, whereas when it is dissolved in m-cresol, p-cresol, 2chlorophenol, 2-fluorophenol, and 3-ethylphenol, the polymer chains are in an expanded coil conformation.29 They reported that films of PANI + CSA have an electrical conductivity of approximately 0.1 S/cm when dissolved in chloroform (first type of solvent) and approximately 150 S/cm when dissolved in m-cresol (second type of solvent). Jeong et al. reported that electrospun (PANI + CSA)-poly(L-lactide-co-ε-caprolactone) (PLCL) fibers have an electrical conductivity ranging from 1.5 to 7.7 × 10-3 S/cm.30 Moreover, Li et al. fabricated gelatin-CSAPANI fibers in the solvent 1,1,1,3,3,3 hexafluoro-2 propanol (HFP) and reported an electrical conductivity of 1-2.1 × 10-2 S/cm.17 In one of the rare studies concerning the fabrication of pure PANI fibers by electrospinning, Cardenas et al. reported that the electrical conductivities of the fibers were on the order of 10-3 to 10-2 S/cm.31 Our results are in accordance with this range of values.

3.3. FTIR spectroscopy and mechanical characterization (Young’s modulus) The presence of PANI in the composite gelatin-CSA + PANI nanofibers can be detected visually because electrospun gelatin 20% was white whereas electrospun gelatin-CSA + PANI was greygreen, and the color darkened with increasing PANI concentration. We also evaluated the presence of PANI in the composite gelatin-PANI nanofiber using ATR-FTIR and present the part of the spectra from 400 cm-1 to 1900 cm-1. As shown in Fig. 2A the characterization method supports the presence of PANI in the nanofibers. Thus, peaks at 1115 cm-1, 1000 cm-1, 792 cm-1, and 669 cm-1 are clearly identified in the spectra of gelatin 20% + CSA 5% + PANI 5% and gelatin 20% + CSA 10% + PANI 10% nanofibers, whereas they are absent from the spectra of gelatin 20% and gelatin 20% + CSA 5% nanofibers. The peak at 1115 cm-1 is assigned to


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aromatic C−H in-plane bending in PANI32, the peak at 792 cm-1 is correlated to out-of-plane aromatic C−H bending vibration in PANI32, and the peak at 669 cm-1 is attributed to the out-ofplane C−H vibration mode in PANI33. At last, several peaks are common to all the spectra but their absorbances are different. Among them, the peak at 1635 cm-1 is assigned to C=C in the quinonoid rings of PANI14 and to the C=O bond in gelatin.6, 34

Because the stiffness of a material affects the cell behavior, we evaluated the microscopic surface properties of the different crosslinked nanofibers via AFM measurements in the presence of water and extracted the Young’s modulus of each fiber type from the recorded AFM nanomechanical maps.35,36 Fig. 2B shows a significant decrease in the Young’s modulus of the nanofibers when gelatin (1.05 MPa) was mixed with CSA 5%, CSA 5% + PANI 5%, or CSA 10% + PANI 10%, with Young’s modulus values of 0.50, 0.51, and 0.54 MPa, respectively. It has been reported that the addition of doped PANI to polymers usually impairs their mechanical properties.37,38 For instance, Jeong et al. observed a decrease in the Young’s modulus and tensile strength of (PANI + CSA)-PLCL blend nanofibers with increasing doped PANI concentration.30 However, some research groups have also reported an increase in stiffness (Young’s modulus, tensile modulus) with increasing PANI concentration in gelatin and gelatin-PCL blends.6,

17

However, in our experiment the change in the mechanical properties of the nanofibers due to the effect of CSA is observed, while the effects of PANI are not. Indeed, CSA hydrolyzed partially the gelatin carrier and affects more the bulk mechanical properties of the nanofibers, whereas the more localized effects of PANI at these concentrations are masked.



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Figure 2. Characterization and mechanical properties of the different nanofibers. FTIR analysis from 400 to 1900 cm-1 on dry samples (A) and Young’s modulus evaluation of gelatin 20%, gelatin 20% + CSA 5%, gelatin 20% + CSA 5% + PANI 5%, and gelatin 20% + CSA 10% + PANI 10% nanofibers in water by force volume AFM (B) (* p < 0.001 compared to other samples).


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3.4. Myotube formation After one day of culture in growth medium, we induced myoblast differentiation to myotubes by serum starvation with a low serum (2% HS) differentiation medium. At day 6 of differentiation, cells were fixed and stained with mouse monoclonal antibodies anti-fast skeletal myosin (MY32). As shown in Fig. 3A-D, the linear topography of the fibers and their alignment induced the alignment of the myoblasts by contact guidance and the formation of parallel myotubes. Myotubes formed on gelatin 20% + CSA 5%, gelatin 20% + CSA 5% + PANI 5%, and gelatin 20% + CSA 10% + PANI 10% nanofibers appear bigger and stronger than those formed on gelatin 20% nanofibers. Fig. 3E shows the myotube length quantification for the different culture conditions. Myotubes formed on gelatin 20% nanofibers have an average length of 308 µm. However, myotubes formed on gelatin 20% + CSA 5%, gelatin 20% + CSA 5% + PANI 5%, and gelatin 20% + CSA 10% + PANI 10% nanofibers were significantly longer, with average lengths of 511, 630, and 573 µm, respectively. In addition, we observed a synergy in myotube formation between the dopant (CSA) and PANI because myotubes formed on composite gelatin 20% + CSA 5% + PANI 5% and gelatin 20% + CSA 10% + PANI 10% nanofibers were significantly longer than those formed on gelatin 20% + CSA 5% nanofibers. However, myotubes formed on gelatin 20% + CSA 5% + PANI 5% and gelatin 20% + CSA 10% + PANI 10% nanofibers had similar trends (p = 0.06) in their lengths. We observed similar tendencies in the results of the myotube aspect ratio quantification (Fig. 3F). Thus, myotubes formed on gelatin 20% had an aspect ratio of 16 whereas those formed on gelatin 20% + CSA 5%, gelatin 20% + CSA 5% + PANI 5% and gelatin 20% + CSA 10% +



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PANI 10% nanofibers had significant higher aspect ratios of 20, 28, and 21, respectively (p < 0.001).

Figure 3. Myotube formation at day 6 of culture in differentiation medium. Fluorescence microscopy images of myotubes (stained with mouse anti-fast skeletal myosin antibody, revealed by Alexa Fluor 488 conjugated IgG secondary antibody) cultured on aligned electrospun gelatin 20% (A), gelatin 20% + CSA 5% (B), gelatin 20% + CSA 5% + PANI 5% (C), and gelatin 20%


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+ CSA 10% + PANI 10% (D) nanofibers. Myotube length quantification (E) and myotube aspect ratio quantification (F) (* p < 0.001).

Because CSA 5% had affected myotube formation by itself, we evaluated the effect of different concentrations of CSA on myotube formation by culturing myoblasts on gelatin 20% fibers + CSA 1.25, 2.5, and 5% in an addition set of experiments. We did not test gelatin 20% + CSA 10% because we were not able to produce nanofibers at this concentration under the same electrospinning conditions. The results (Fig. S2A) show that myotubes formed on gelatin 20% + CSA 1.25% and gelatin 20% + CSA 2.5% have average lengths similar to those formed on gelatin 20% (334, 329, and 353 µm, respectively); therefore, CSA 1.25% and CSA 2.5% have no effect on myotube formation. In contrast, myotubes formed on gelatin 20% + CSA 5% have an average length of 506 µm, and CSA 5% significantly enhanced the myotube formation. We also observed the synergy between CSA and PANI on myotube formation because myotubes formed on gelatin 20% + CSA 5% + PANI 5% were significantly longer (with an average length of 615 µm) than those formed on gelatin 20% + CSA 5%. Similarly, we observed (Fig. S2B) that myotubes formed on gelatin 20%, gelatin 20% + CSA 1.25%, and gelatin 20% + CSA 2.5% all have aspect ratios of 18. In contrast, myotubes formed on gelatin 20% + CSA 5% have a significantly higher aspect ratio of 21, which shows the positive effect of CSA on myotube formation. Again a marked effect of PANI on myotube formation was observed because myotubes formed on gelatin 20% + CSA 5% + PANI 5% have an aspect ratio of 27, which is significantly higher than the aspect ratio of myotubes formed on gelatin 20% + CSA 5%.



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To explain this increase in myotube formation on gelatin 20% + CSA 5% nanofibers compared to gelatin 20% nanofibers, we hypothesized that CSA partially hydrolyzed the gelatin, allowing the nanofibers to present more functional chemical groups to the cells, favoring better cell attachment. Thus, we analyzed, by qRT-PCR, the expression of genes correlated to cell attachment (focal adhesion kinase (FAK), integrin β1) and to myogenesis (myogenin) in C2C12 cultured for 1 day in growth medium and 4 days in differentiation medium on gelatin 20% nanofibers with different CSA concentrations (0, 1.25, 2.5, and 5% CSA) and gelatin 20% + CSA 5% + PANI 5% nanofibers (Fig. S2C). Taken together, the results showed similar trends in all three genes, with no effect on the gene expression of integrin β1, FAK, and myogenin when cells were cultured on gelatin 20% + CSA 1.25% and gelatin 20% + CSA 2.5%, a noticeable increase in gene expression when cells were cultured on gelatin 20% + CSA 5% nanofibers, and a significant increase in gene expression when cells were cultured on gelatin 20% + CSA 5% + PANI 5% compared to the gene expression in cells cultured on gelatin 20% nanofibers. This is in accordance with others studies which have also reported upregulation of myogenin, in addition to troponin T and MHC gene expression, induced by composite PLCL-PANI and PCL-PANI nanofibers.15,16

Importantly, several studies have also shown improved myotube formation when myoblasts (e.g. C2C12) were cultured on an electrically conductive scaffold without electrical stimulation.12, 39 Thus, Jun et al. cultured C2C12 cells on composite PLCL-PANI nanofibers and reported increased number, length, and coverage area of myotubes compared to cultures on PLCL nanofibers.16 In another study, Ku et al. cultured C2C12 cells on composite PCL-PANI nanofibers and found enhanced myotube length with increasing PANI concentration.15


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Furthermore, Yang et al. cultured C2C12 on nanopatterned poly urethane acrylate (PUA) substrate coated by gold and observed increased number of myotubes, length, width, and coverage area compared to cultures on bare PUA nanopatterned substrates.40 These different studies have established that it was not the mechanical properties of the substrates, but the electroconductivity of the substrates which was responsible for this increase in C2C12 myogenesis and myotube maturation in absence of external electrical pulse stimulations. Thus, Thrivikraman et al. cultured C2C12 without electrical pulse stimulations on hydroxyapatite (HA) substrates with increasing electroconductivity due to the addition of CaTiO3 and reported increase of myotube formation, length, diameter, as well as upregulation of myogenin gene expression with the increase in substrate conductivity.41 The reason why the myogenesis is increased on electroconductive substrates is not yet well understood. However, it has been proposed that a potential mechanism for the observed phenomenon may involve the calcium homeostasis with an increase in intracellular calcium level when myoblasts are cultured on electroconductive substrates.40,42,43 In line with this idea, we also observed higher intracellular calcium transients when myoblasts were cultured on composite gelatin-CSA + PANI nanofibers as exposed further.

3.5. Myotube maturation We next analyzed the A band formation in myotubes at day 4 of differentiation. Myotubes stained with mouse monoclonal antibodies anti-fast skeletal myosin (MY-32) showing a spatially segregated A band (dashed line under fluorescence microscopy) were counted positive, whereas myotubes without a mature A band (lines or dots under fluorescence microscopy) were counted negative. Fig. 4A-D showed improved A band formation when myotubes formed on gelatin 20%



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+ CSA 5% + PANI 5% and gelatin 20% + CSA 10% + PANI 10% fibers compared with myotubes formed on gelatin 20% + CSA 5% and gelatin 20% fibers. Thus, in the group of myotubes formed on gelatin 20% fibers (Fig. 4A), we observed few myotubes with a short portion of A bands formed, whereas most of the myotubes had stress fiber-like structures (lines). In the group of myotubes formed on gelatin 20% + CSA 5% fibers (Fig. 4B), we observed some myotubes with A bands. These A bands were mostly visible in the borders of the myotubes, whereas stress fiber-like structures were visible in the center of the myotubes. In contrast, in the group of myotubes formed on gelatin 20% + CSA 5% + PANI 5% (Fig. 4C) abundant myotubes had well-formed A bands, and these A bands were as long and visible in the borders as in the center of the myotubes. Similarly, in the group of myotubes formed on gelatin 20% + CSA 10% + PANI 10% fibers (Fig. 4D-F), we observed abundant myotubes with long A bands formed throughout the myotubes, and sometimes dashed patterns appeared in a more pronounced way, allowing them to be visible in phase-contrast microscopy (Fig. 4F is the phase-contrast image of Fig. 4D). We analyzed at least 100 myotubes per culture condition, and the quantification (Fig. 4E) showed a significant enhancement of A band formation in myotubes formed on gelatin 20% + CSA 5% + PANI 5% and gelatin 20% + CSA 10% + PANI 10% fibers with 34% and 37% of myotubes having A bands, respectively, compared with 16% and 11% of myotubes with A bands when the myotubes formed on gelatin 20% + CSA 5% and gelatin 20% fibers, respectively. The contractile activity depends on the precise spatial arrangement of myofibrils in myotubes. These myofibrils contain cytoplasmic proteins, such as myosin (thick filament) and actin (thin filament), arranged in repeated units, termed sarcomeres, which are delimited under microscope by two dark lines of dense proteins named Z lines.44 Between these Z lines are two light bands (I bands) of actin filaments separated by a dark band (A band) containing myosin filaments.


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A

Ge20%

Ge20% + CSA 5%

B

A bands

A bands C

Ge20% + CSA 5% + PANI 5%

A bands

D

Ge20% + CSA10% + PANI 10%

A bands

E

Ge20% + CSA 10% + PANI 10%

F A bands

Figure 4. “A bands” formation in myotubes at day 4 of culture in differentiation medium. Fluorescence microscopy images of myotubes (stained with mouse anti-fast skeletal myosin antibody, revealed by Alexa Fluor 488 conjugated IgG secondary antibody) cultured on aligned electrospun gelatin 20% (A), gelatin 20% + CSA 5% (B), gelatin 20% + CSA 5% + PANI 5% (C), and gelatin 20% + CSA 10% + PANI 10% (D) nanofibers. Few A bands were visible in the myotubes formed on gelatin and gelatin + CSA nanofibers, whereas abundant A bands were



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formed in myotubes cultured on composite gelatin-CSA-PANI nanofibers. Phase-contrast image showing the A bands (F) corresponding to the fluorescence image in (D). Quantification of A band formation in myotubes cultured on different nanofibers (E) (* p < 0.03).

Neighboring sarcomeres share a Z disk, which anchors the actin filaments and several other proteins. Under muscle contraction, the filaments of myosin slide along the actin filaments, shortening the sarcomeres. The myofibril assembly begins with the formation of a regular array of sarcomeres, which align in registry, giving the striated aspect of muscle myofibers. This progressive alignment of sarcomeres constitutes a maturation step with the formation of premyofibrils, nascent myofibrils and mature myofibrils, which has been described by Sanger in a premyofibril model of myofibrillogenesis.45 Therefore, myotube contractibility gradually increases in parallel with the intracellular organization of sarcomeres and myofibril maturation.46 Our results clearly showed that A band formation, and therefore the intracellular organization of myotubes, is faster when myotubes formed on composite gelatin-CSA + PANI fibers. This result is also confirmed by the fact that the A bands formed in myotubes cultured on gelatin 20% and gelatin 20% + CSA 5% were mainly at the borders of the myotubes, whereas they were found throughout the myotubes when the myotubes were cultured on composite gelatin-CSA + PANI fibers (5% and 10%). Indeed, it has been shown that sarcomere assembly always begins at the cell periphery; therefore, the first myofibrils (with formed A bands) are always observed close to the membrane on the borders of the myotubes.46 This is because in the lateral dimension, the sarcomeres nearest the sarcolemma interact at the edges of the Z lines with the integrins bounded to the ECM via protein complexes called costameres.47 This cell-material interaction is essential to maintaining sarcomere integrity and myofibrillogenesis by creating a certain and continuous


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tension across the myotubes. Considering the differences in the cells and culture conditions, our results of A band formation quantification are in the range of those presented by De Deyne.48

3.6. DHPR and RyR1 co-localization Because we observed myotube formation and intracellular organization enhancement when myoblasts were cultured on PANI nanofibers, we then focused on myotube functionality. We evaluated the maturation of myotubes formed on different nanofibers through the co-localization of the DHPR and RyR1 receptors. Fig. 5 shows representative merged fluorescence microscopy images of myotubes stained for DHPR (red) and RyR1 (green) when myotubes were formed on different fiber groups. We analyzed the co-localization of the two receptors in 20 myotubes formed on each fiber group using the Puncta Analyzer plug-in for ImageJ software. Puncta Analyzer was written by Bary Wark to analyze the puncta in the red and green channels separately and to produce a merged image with black dots corresponding to the co-localization of the two puncta (yellow in fluorescence) as the puncta number and other numerical parameters. We provide an example of the image analysis in Fig. S3 and refer to research on the use of this analysis method.49 Remarkably, we observed that the quantification of the DHPR and RyR1 receptor co-localization matches the myotube formation enhancement observed in Fig. 3. Thus, myotubes formed on gelatin 20% + CSA 5%, gelatin 20% + CSA 5% + PANI 5%, and gelatin 20% + CSA 10% + PANI 10% nanofibers have significantly more co-localized receptors at day 9 of culture in differentiation medium than those formed on gelatin 20% nanofibers, with 137, 207, 180 and 72 merged puncta detected/myotube, respectively. Furthermore, we observed a significantly higher co-localization of the two receptors in myotubes formed on gelatin 20% + CSA 5% + PANI 5% nanofibers compared to those formed on gelatin 20% + CSA 5%



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nanofibers. In muscle fibers, the conversion of an action potential into a mechanical force, inducing fiber contraction, is a process named E-C coupling. This E-C coupling is based on a biological structure named the triad, which is composed of sarcolemma invagination (T tubule) in close apposition with the membrane of two terminal cisternae containing calcium, which are an enlargement of the internal sarcoplasmic reticulum (SR).50 At the triadic junctions, clusters of four DHPRs form tetrads in the membrane of the T tubule, and each DHPR tetrad is positioned on a single RyR embedded in the membrane of the terminal cisternae.50 The DHPR serves as a voltage sensor because it contains segments of charged amino acids, which undergo conformational change under membrane depolarization.50 Because the DHPR is physically coupled to the RyR, this conformational change induces a conformational change in RyR, causing it to open, which allows the release of Ca2+ from the terminal cisternae into the cytosol.50 The Ca2+ ions bond to troponin C, inducing a conformation change of the troponin-tropomyosin complex, which uncovers the myosin binding site on the actin filaments, allowing myosin filaments to bond to actin filaments and then to slide on them.51 Interestingly, Das et al. showed that primary fetal rat myotubes developed several aspects of mature muscle, such as sarcomeric structure, E-C coupling apparatus, and MHC class switching, in a defined serum-free culture medium.52 In their culture system, the co-localization of DHPRs and RyRs in myotubes was observed after 30 days in culture and over a period of 60 more days in culture.


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Figure 5. Co-localization of the DHPR and RyR receptors at day 9 of culture in differentiation medium. Merged fluorescence microscopy images of the DHPRs stained in red and the RyRs stained in green in myotubes formed on aligned gelatin 20% (A), gelatin 20% + CSA 5% (B), gelatin 20% + CSA 5% + PANI 5% (C), and gelatin 20% + CSA 10% + PANI 10% (D)



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nanofibers. Quantification of the co-localization of the DHPR and RyR receptors (or puncta) in myotubes (E) (*, # p < 0.03).

Our results show that the myotube formation improvement on composite gelatin-CSA + PANI fibers is correlated with improved intracellular myotube organization, with more A band formation and more co-localization of the DHPR and RyR1 receptors, favoring the E-C coupling.

3.7. Gene expression correlated to DHPR and RyR We then analyzed, by qRT-PCR, the expression of genes correlated to DHPR and RyR in myotubes after 7 days in differentiation medium (Fig. 6). We observed an increase (nearly significant, p = 0.09 and 0.07) in the gene expression of RyR1 and RyR3 in myotubes formed on gelatin 20% + CSA 5% + PANI 5% nanofibers compared to myotubes formed on gelatin 20% nanofibers. This gene expression increase was significant when myotubes formed on gelatin 20% + CSA 5% + PANI 5% were compared to those formed on gelatin 20% + CSA 5% and gelatin 20% + CSA 10% + PANI 10% nanofibers. In mammals, RyR proteins are homotetramers with a molecular mass of approximately 2240 kDa, and three isoforms are produced by the genes RyR1, RyR2, and RyR3 and are expressed in skeletal muscle (RyR1 predominantly and RyR3) and in the heart (RyR2).53 We also observed a slight increase in gene expression of Cav1.1 in myotubes formed on gelatin 20% + CSA 5% + PANI 5% nanofibers compared to those formed on gelatin 20%. Cav1.1 gene expression was also significantly decreased in myotubes formed on gelatin 20% + CSA 5% and


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gelatin 20% + CSA 10% + PANI 10% compared to those formed on gelatin 20% + CSA 5% + PANI 5% nanofibers. Moreover, we observed a significant increase in the gene expression of β1.1 in myotubes formed on gelatin 20% + CSA 5% + PANI 5% nanofibers compared to myotubes formed on other fiber types. In skeletal muscle, the DHPR proteins are heteropentamer, with a transmembrane α1 subunit (α1S, 160 kDa), an extracellular α2 subunit (140 kDa), an intracellular β subunit (β1a, 54 kDa), and a membrane-associated γ (γ1, 30 kDa)

Figure 6. Relative expression of genes correlated to DHPR (Cav1.1, and β1.1) and RyR (RyR1, and RyR3) in myotubes at day 7 of differentiation, evaluated by qRT-PCR



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and δ (δ1, ~27 kDa) subunits.50 The α1S subunit serves as a modest L-type voltage-activated Ca2+ channel (Cav1.1) in T-tubule and, more importantly, is the voltage sensor of the E-C coupling in the triad.50 Thus, in skeletal muscle, E-C coupling depends on α1S, β1a and RyR1. Indeed, α1S has 4 membrane-inserted units, each having 6 transmembrane helices, and the fourth of which (segment S4) has charged amino acids, which constitute the voltage sensor, whereas the β1a subunit binds to loops I and II of α1S and RyR1, and the c-terminal of α1S binds to RyR1.50,54,55 Since we observed co-localization of the DHPR and RyR1 at day 9 of cell differentiation, we evaluated the activation and expression of genes correlated to DHPR and RyR earlier. Taken together, our results show a substantial expression of these genes at day 7 of cell differentiation, which fits and supports our previous results on the development and maturation of DHPR and RyR observed by immunostaining at day 9 of differentiation. Interestingly, a study by PietriRouxel et al. on mice linked a subpopulation of DHPR α1S subunits localized in the sarcolemma (at a different position than the T-tubules and triads) to the control of muscle mass and maintenance.56

3.8. Calcium measurements We next evaluated the functionality of the E-C coupling apparatus by monitoring intracellular calcium transients (Fig. 7A). Fig. 7B shows the Ca2+ oscillations under electrical stimulation in myotubes formed on the different nanofiber types after 6 days in differentiation medium. Remarkably, we observed in myotubes formed on gelatin 20% + CSA 5% + PANI 5% nanofibers very regular calcium transients with high and almost same amplitude, synchronously with the electrical pulse stimulation signal. In contrast, in myotubes formed on gelatin 20% we observed most of the time no calcium transient. However, for few myotubes we also observed


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spontaneous and anarchic calcium transients of small and irregular amplitudes often not synchronized with the electrical pulse signals. Similarly, myotubes formed on gelatin 20% + CSA 5% often showed no calcium transient, whereas few of them presented spontaneous calcium transients irregular in time and amplitude. At last, myotubes formed on gelatin 20% + CSA 10% + PANI 10% nanofibers showed some calcium transients with sharp peaks and high amplitude. The comparison of the calcium transient rate (Fig. 7C) between the myotubes formed on the different nanofibers showed a significant increase (p < 0.001) with 0.16 peak/s in myotubes formed on gelatin 20% + CSA 5% + PANI 5% nanofibers compared to all others groups. Myotubes formed on gelatin 20% and gelatin 20% + CSA 5% nanofibers had similar calcium transient rates of 0.006 and 0.005 peak/s, respectively. Furthermore, myotubes formed on gelatin 20% + CSA 10% + PANI 10% nanofibers showed a significant increase (P < 0.05) in calcium transient rate with 0.027 peak/s compared to the calcium transient rates of myotubes formed on gelatin 20% and gelatin 20% + CSA 5% nanofibers. In skeletal muscle cells, the cytosolic calcium concentration [Ca2+] is approximately 100 nM, whereas in the extracellular fluid, this concentration is 1 mM in terrestrial mammals and approximately 10 mM in seawater animals.57 This control over [Ca2+] is possible because cells remove calcium from the cytosol via calcium pumps, such as the plasma membrane ATPase, the sarcoplasmic/endoplasmic reticulum membrane ATPase (SERCA), the Na+/Ca2+ exchanger, and the mitochondria.58 Thus, during cell excitation, cytosolic [Ca2+] may increase up to 20 µM in less than 1 s; then, the SERCA pumps reuptake the calcium ions into the terminal cisternae and [Ca2+] returns to its basal level.54 Several types of Ca2+ signaling events exist (quark, spark, wave or calcium transient) in the cytosol and involve only one RyR, a group of RyRs, or several groups of RyRs, for different lengths of times and with different amplitudes.57 These calcium transients are important for



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skeletal muscle development because disrupting Ca2+ transients in Xenopus laevis embryos impaired myofibril organization and sarcomere assembly, whereas mutant mice with abolished RyR-mediated Ca2+ release have small myotubes, disrupted musculature, and die prenatally.59 It is also known that calcium influx is a prerequisite for membrane fusion of myoblasts during


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Figure 7. Fluorescence images of myotubes formed on the different nanofiber types and stained with Fura-2 LR-AM (A). Representative time course of calcium transients observed in myotubes formed on the different nanofiber types, when subjected to electrical stimulation (square pulses, 6V, 1 ms duration applied every 5 s, 10 s after start of recording) (B). Quantification of the calcium transient rate in myotubes formed on the different nanofiber types (C). (* p < 0.001 compared to all others samples, and # p < 0.05 compared to gelatin 20% and gelatin 20% + CSA 5% nanofibers).

differentiation, and that several proteins related to calcium are involved in myogenesis.60 Thus, the calcium-dependent phosphatase calcineurin plays an important regulatory role in early myogenesis by activating the myocyte-specific enhancer factor-2 (MEF2) and the myogenic regulatory factor MyoD.61 Calcineurin also promotes the formation of slow muscular fiber type by recruiting the nuclear factor of activated T cell (NFATc).62 A recent study by Nasipak et al. showed that the brahma related gene 1 (Brg1) chromatin remodeling enzyme is a calcineurin substrate, which integrates two antagonist calcium-dependent signaling pathways controlling myogenic differentiation.63 Other proteins involved in store-operated calcium entry (SOCE), such as the plasma membrane channel Orai 1, the stromal interaction molecule 1 (STIM1), which is the calcium sensor that gauges the level of calcium in the endoplasmic reticulum (ER) and in the SR, and the sarcolemmal transient receptor potential canonical type 1 (TRPC1) cationic channel, participate in the development of skeletal muscle cells.58 Indeed, the SR and the ER function as calcium stores in excitable and non-excitable cells, respectively. In skeletal muscle cells, Ca2+ release from the terminal cisternae located in the SR and entry into the cytosol is performed by the DHPR-RyR under voltage activation. In non-excitable cells, the release of Ca2+



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from the ER into the cytosol is under the activation of phospholipase C via plasma membrane receptor binding and its second messenger inositol 1,4,5-triphospate (IP3), which binds to the inositol triphosphate receptor (InsP3R) located in the ER and induces the release of calcium. This depletion in calcium induces STIM1 accumulation in ER-PM (plasma membrane) junctions, where it binds to Orai 1, activating the SOCE and the entry of Ca2+ from the extracellular medium via the SOC channel.58 Although in normal conditions, without depletion of calcium stores, the SOCE contribution to the increase in cytosolic [Ca2+] in skeletal muscle cells is less than 1%, it exists, and the proteins involved in SOCE are also present in myoblasts and myotubes.64 Stiber et al. observed that myotubes lacking STIM1 did not show SOCE and fatigued quickly. Moreover, mice lacking STIM1 have a myopathy and die prenatally.65 In another study, Louis et al. showed that myoblasts depleted in TRPC1 have reduced SOCE, significantly reduced Ca2+ transients, and a marked decrease in migration and fusion into myotubes.66 Similarly, inactivation of TRPC3 impaired myoblast fusion into myotubes.67 Furthermore, Toth et al. silenced the expression of SERCA1b in C2C12 using an ShRNA sequence and reported that the amount and the rate of Ca2+ released, as the reuptake of Ca2+, were significantly decreased, whereas the cells showed a significant decrease in proliferation and in the number of nuclei per myotube formed.68

Our results are in line with these studies because we observed enhanced myogenesis concomitantly with enhanced E-C coupling apparatus maturation. Thus, we showed that myotubes formed on composite gelatin 20% + CSA 5% + PANI 5% have high sarcomeric organization, enhanced length and myotube formation, high co-localization of DHPRs and RyRs, high gene expression of these two receptors, and strong and regular calcium transients under


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electrical stimulation compared to myotubes formed on gelatin 20% and gelatin 20% + CSA 5%. Furthermore, the fact that the calcium transient peaks were synchronized with the electrical stimulation showed that myotubes formed on composite gelatin 20% + CSA 5% + PANI 5% were mature. In contrast, the time courses of calcium transient for myotubes formed on gelatin 20% and gelatin 20% + CSA 5% were similar to a baseline and these myotubes were not mature. Although, myotubes formed on gelatin 20% + CSA 10% + PANI 10% also have high sarcomeric organization, enhanced length and myotube formation, and high co-localization of DHPRs and RyRs, they only showed some sharp calcium transients of high amplitude under electrical stimulation. We think that these myotubes needed to be trained under electrical stimulation to develop numerous and regular intracellular calcium transients synchronized with the electrical pulses. We believe that this slight disparity in myotube maturation compared to those formed on gelatin 20% + CSA 5% + PANI 5% nanofibers may be due to the topography of the gelatin 20% + CSA 10% + PANI 10% nanofibers, which is slightly different than that of gelatin 20% + CSA 5% + PANI 5% nanofibers, because the myotube formation and maturation results with gelatin 20% + CSA 10% + PANI 10% nanofibers usually showed similar trend (but not higher) to those obtained with gelatin 20% + CSA 5% + PANI 5% nanofibers.

3.9. Myotube contraction analysis We next analyzed the contraction of the myotubes under electrical stimulation (ES) in the different culture systems. After 6 days in differentiation medium, the different cell cultures were electrically stimulated with square pulses (6 V, 1 Hz, 1 ms duration) for 1 day; then, videos of myotube contraction (examples Movies S1) were recorded and analyzed. Fig. 8 shows the contractibility analysis of myotubes formed on gelatin 20%, gelatin 20% + CSA 5%, gelatin 20%



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+ CSA 5% + PANI 5%, and gelatin 20% + CSA 10% + PANI 10% nanofibers. We observed higher maturation and contractibility of myotubes formed on composite gelatin-CSA + PANI nanofibers compared to those formed on gelatin 20% or gelatin 20% + CSA 5% nanofibers. Thus, in the group of myotubes formed on gelatin 20% nanofibers, only a few myotubes were active under ES, and they had to be searched for using a microscope. The video analysis shows that the beating amplitude was low (approximately 6-8 arbitrary unit (A.U.)) and irregular (presence of small peaks). In the group of myotubes formed on gelatin 20% + CSA 5% nanofibers, several myotubes were beating under ES, and the video analysis also shows low and irregular amplitude contractions (approximately 6 A.U.) similarly to myotubes formed on gelatin 20% nanofibers. In the group of myotubes formed on gelatin 20% + CSA 5% + PANI 5% nanofibers, abundant myotubes were beating under ES, giving the feeling that the whole surface of the dish was beating under the microscope. The video analysis shows that the contraction amplitude was high (approximately 30 A.U.) and much more regular than in the previous groups.


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Figure 8. Myotube contraction analysis after 6 days in differentiation medium followed by 1 additional day with continuous electrical stimulation (square pulses, 6 V, 1 Hz, 1 ms duration).

Similarly, in the group of myotubes formed on gelatin 20% + CSA 10% + PANI 10% nanofibers, abundant myotubes were beating under ES, and the video analysis shows that the contraction amplitude was high (approximately 20-25 A.U.) and regular. It is well known that ES improves the speed and rate of myotube formation and also enhances myotube striation, maturation and contractibility.69 ES increased the speed of myotube maturation similarly in the different culture systems. Therefore, because at the beginning of ES, the myotubes on the different nanofiber types were in different states of maturation, after 1 day of ES, the same pattern was observed, with only a few mature and contractile myotubes in the gelatin 20% nanofiber cultures, more contractile myotubes in the gelatin 20% + CSA 5% nanofiber cultures and abundant contractile myotubes in the composite gelatin 20% + CSA + PANI (5% and 10%) cultures.

CONCLUSION In summary, we have shown improved myotube formation when myoblasts were differentiated on composite gelatin-CSA + PANI nanofibers compared to gelatin or gelatin + CSA nanofibers. In addition to this enhancement in myogenesis, we observed concomitant improvement in myotube maturation. Thus, in myotubes, the speed and the rate of A band formation were increased, showing better intracellular organization. Furthermore, the co-localization of the DHPR and RyR receptors in myotubes was improved, showing better myotube maturation and E-C coupling. Moreover, the gene expression of genes correlated to the E-C coupling was



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increased. In addition, the functionality of the myotubes was improved because we observed more calcium transients and contractions with higher amplitude and regularity when myotubes were electrically stimulated.

Supporting information: Nanofiber diameters and alignment; myotube length and aspect ratio for myotubes grew on gelatin 20% nanofibers with different concentrations of CSA (0, 1.25, 2.5, 5%); genes expression correlated to cell attachment and myogenesis in myotubes grew on gelatin 20% nanofibers with different concentrations of CSA (0, 1.25, 2.5, 5%); example of the co-localization of DHPRs and RyRs analysis using the Puncta Analyzer program; videos of contracting myotubes.

ACKNOWLEDGEMENTS This work was supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan. S. O., M. E., and H. B. designed the research, performed the experiments, analyzed the results, and wrote the manuscript. Akichika K. measured the electrical conductivity of the polymers under the supervision of T. M., and S. S. performed the FTIR spectra and corresponding analysis. S. B. K. conducted the analysis of myotube contraction. H. K. N. performed the AFM measurement under the supervision of K. N., and M. O. performed the intracellular calcium measurements and the corresponding results analysis. X. S. and S. H. helped to edit the manuscript. Ali K. supervised the whole project. All authors read the manuscript, commented on it and approved its content.


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