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Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells
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Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells Vaikundamoorthy Ramalingam, Inho Hwang* Department of Animal Science, Chonbuk National University, Jeonju, 561-756, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 October 2019 Received in revised form 21 November 2019 Accepted 4 December 2019 Available online xxx
In the present study, we investigated the effect of monodispersed zinc oxide (ZnO) nanoparticles on the proliferation, myogenic differentiation and regulation of associated myogenic marker genes. The asprepared ZnO nanoparticles had the hexagonal wurtzite structure with maximum absorption at 355 nm and the band gap energy was found to be 3.21 eV. The electron microscopy analysis showed that the ZnO nanoparticles are spherical in shape with an average size range between 10 to 15 nm. The XRD analysis confirmed the hexagonal structure of ZnO nanoparticles and the Raman spectroscopic analysis showed the vibrations of the zinc lattice and oxygen vibration of ZnO nanoparticles. The effect of ZnO nanoparticles on myogenic differentiation was analyzed using C2C12 cells and the results showed the nanoparticles supported the proliferation with negotiable cytotoxic activity. Moreover, the ZnO nanoparticles were significantly enhanced the myoblasts into myotube formation through upregulating the myogenic markers such as myosin heavy chain, MyoD, MyoG genes. The further analysis demonstrated that the ZnO nanoparticles regulates the non-apoptotic effect of caspases and calpain family proteins in respond to the enhancement of myogenic differentiation. Together, the ZnO nanoparticles provide an additional evidence for the role of nanomaterials in skeletal muscle repair and tissue regeneration engineering. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: ZnOnanoparticles Characterization Myoblasts Myogenic differentiation Molecular mechanism
Introduction In adult human body, about 40%–45% of mass is skeletal muscles which play an important role in generating voluntary movement. The genesis of skeletal muscle during development and postnatal life is a pattern to control and regulate the stem cell and progenitor cell mechanism, heredity specification and terminal differentiation [1]. The development of skeletal muscles is a complicated process that encompasses the growth of myoblasts and the differentiation of myofibers. Myogenesis is a
Abbreviations: ZnO, Zinc oxide; HR-TEM, High resolution – transmission electron microscope; SEM, Scanning electron microscope; EDX, Energy-dispersive X-ray spectroscopy; SAED, Selected area electron diffraction; JCPDS, Joint committee on powder diffraction standards; XRD, X-ray powder diffraction; qRT-PCR, Quantitative real time – polymerase chain reaction; RIPA, Radioimmunoprecipitation; SDS-PAGE, sodium dodecyl sulfate - polyacrylamide gel electrophoresis; PVDF, Polyvinylidene fluoride; CAPN, Calpain; MyHC, Myosin heavy chain; CASP, Caspase; MyoD, myoblast determination protein 1; MyoG, Myogenin; TNF-α, tumor necrosis factor α. * Corresponding author. E-mail address: [email protected] (I. Hwang).
quite coordinated process that comprises the regulation of myogenic genes, arrest of the cell cycle and terminal fusion of mononucleated myotubes into multinucleated myotubes [2]. The myogenesis is accompanied by the transcriptional upregulation of myogenic differentiation and other myogenic differentiation marker genes [3]. The process of myogenesis is regulated by various hormones, cell cycle regulators, inhibitors of myoblast differentiation, myotube formation and myogenic markers (MyoD, Myf-5, MRF-4 and myogenin) are called as myogenic regulatory factors [4,5]. It was well documented in the C2C12 myoblasts cells that the exogenous tumor necrosis factor α (TNF-α) prevents the formation of myotubes by down regulating the myogenic markers gens such as MyoD, myogenin (MyoG), myosin heavy chain (MyHC) via NF-kB activation [6]. Moreover, the programmed cell death is also playing critical role in the differentiation, in which the caspases (3/9) and ER stress are required for the activation of differentiation, consequently, to form myotubes [7]. Depends on the duration and strong enzymatic activity, the caspase enzymes decide the cells either undergoes differentiation or death [8]. The C2C12 cells isolated from murine skeletal muscle cells is often used as model to investigate the muscle regeneration and differentiation. In the present study, the C2C12
https://doi.org/10.1016/j.jiec.2019.12.004 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004
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cells was used to study the response of skeletal muscle to zinc oxide (ZnO) nanoparticles for myogenic differentiation into myotubes. In the modern world, the growth of nanotechnology is phenomenal, and it has an extensive report on application in medical and industrial field. Especially, the impact of metallic nanoparticles on the proliferation and differentiation of normal, cancer, and stem cells is well-studied. Among various metal nanoparticles, the ZnO has various benefits specially in the human system, in which it maintains the cytosolic zinc concentration and controls the signaling pathways involved in normal cell development, differentiation and death [9]. In other hand, the zinc deficiency is serious problem in worldwide and accounting ~2 billion people are at risk by showing various symptoms including but not limited to diarrhea, immune system damage, neurological deficits and reduced reproductive development [10–12]. Moreover, zinc is essential for cellular homeostasis and also serves as a signaling molecule that regulates the number of cellular signal transduction processes. Hence, the present study aims to prepare zinc oxide nanoparticles and analyzing the effect of ZnO nanoparticles in regulating the myogenic differentiation factors to induce the myotube formation using gene and protein expression analysis.
of ZnO nanoparticles was investigated using scanning electron microscope (SIGMA model, CASL ZEISS, German) operated with EDX mapping. The Raman spectroscopic analysis (Renishaw, UK) was performed with laser power set to ~50 mW to avoid thermal damage to the particles. Cell culture and maintenance The mouse myoblast C2C12 cells were procured from American Type Culture Collection (ATCC-CRL: 1772). The cells were incubated in a humidified incubator with 5% CO2 at 37 C. The complete growth medium (GM) for C2C12 myoblast was Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Biological Industries) and 1% Penicillin-Streptomycin-Glutamine (100X, Gibco). The differentiation medium (DM) for C2C12 cells was DMEM containing 2% horse serum (HS, Gibco) and 1% Penicillin-Streptomycin-Glutamine. In vitro cytotoxic activity of ZnO nanoparticles
The precursor ferric chloride hexahydrate (FeCl3.6H2O), sodium hydroxide, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, DMSO (cell culture grade), MTT (dimethyl thiazolyl tetrazolium bromide), 1x phosphate buffer saline (PBS), glucose and glutamine were purchased from HiMedia Laboratories, Seoul, Republic of Korea. The primary and secondary antibodies used in the present study was purchased from Abcam technologies, USA.
The in vitro cytotoxic activity of ZnO nanoparticles against murine myoblast C2C12 cells was investigated using MTT assay [14]. Briefly, the C2C12 myoblast cells at a density of 15,000 cells/ well were seeded into a 96-well plate with complete growth medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine (100X, Gibco). After 12 h of incubation, the cells were treated with different concentration of ZnO nanoparticles (01000 mg/ml) and again incubated for 7 days. The culture medium was replaced by fresh complete growth medium for every 24 h and the viability of the cells were measured at predetermined intervals (2, 5 and 7 days). The cytotoxic activity of ZnO nanoparticles were measured after washing the cells with 1X PBS and 10 ml of MTT solution was added to each well. After incubation for 4 h in the dark, the absorbance was measured at 490/630 nm and IC50 was calculated.
Preparation of ZnO nanoparticles
Induction of myogenesis
The ZnO nanoparticles was synthesized using wet chemical method as per the method described previously [13]. Initially, 1.98 g of zinc acetate dihydrate was added to 40 ml of methanol and the solution was kept for reflux to dissolve completely. The solution (30 ml) was mixed with 0.72 g of sodium hydroxide by dropwise and refluxed for 48 h. The resulting solution was centrifuged at 10,000 rpm for 30 min to obtain a white precipitate and the precipitate washed with water for several times to improve the particle homogeneity. Further, the resulted precipitate was again washed with 1:1 ethanol/acetone solution and dried under vacuum for overnight. The dried white powdered ZnO nanoparticles was used for further characterization and biological applications.
The mouse myoblast C2C12 cells were obtained from ATCC and the cells were cultured in complete DMEM medium supplemented with 20% fetal bovine serum (FBS) (growth medium, GM) and 100 mg/ml penicillin/streptomycin used as control. The myoblast
Materials and methods Chemicals and reagents
Characterization of ZnO nanoparticles There are numerous methods and techniques were employed to characterize the ZnO nanoparticles such as crystalline phase, compositional analysis, particle size measurements, particle morphology and surface composition of ZnO nanoparticles. The crystalline structure of ZnO nanoparticles was measured using powder X-ray diffraction (XRD) measurements (Philips X’Pert Pro MPD, UK) with Kα radiation in the 2u range from 10 to 80 . For size and morphology, a transmission electron microscope (TEM) was used (Leica EM UC 6 Ultramicrotome & Nikon Trinocular Microscope Model E200, Leica Camera AG – Germany) to obtain an average particle size by measuring the size of 50–100 particles. The surface morphology and composition associated on the surface
Fig. 1. UV-spectroscopic and DRS-UV spectroscopic (inset) analysis of ZnO nanoparticles prepared using wet-chemical approach.
Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004
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Fig. 2. Scanning electron microscopic analysis showed the aggregation of ZnO nanoparticles (a & b), the elemental composition was analyzed using EDS spectrum (c) and the SAM-nanoprobe analysis used for Edax mapping of Zn (e) and O (f) arrangement on the surface of ZnO nanoparticles (g).
cells at a density of 50,000 cells/well were grown in differentiation medium containing glutamine-free DMEM with high glucose (25 mM), 2% horse serum, 1% of penicillin-streptomycin, 10 mg/ml insulin (Sigma–Aldrich Co. LLC., St-Louis, MO, USA), 10 mg/ml doxycycline (Sigma–Aldrich Co. LLC.), and 0, 1, 2, 4 mM l-glutamine (Thermo Fisher Scientific Inc.) respectively for 7 days, to induce myogenesis. The medium was replaced every 2 days. In mean time, the ZnO nanoparticles was used to treat the C2C12 cells cultured in the complete growth medium to study the effect on the myogenic differentiation and the morphological differences was observed at predetermined times under phase contrast microscope [15]. Quantitative real time PCR analysis qRT-PCR was performed to investigate the myogenic gene expression levels upon treatment with ZnO nanoparticles in C2C12 cells. Initially, the total RNA was isolated from 5 days treated cells using TRIzol Reagent (Life technologies, USA). The cDNA was synthesized using First Strand cDNA synthesis kit (Thermo
Scientific, USA) and qPCR was performed with a 20 mL reaction system comprising 10 mL of SYBR Green Realtime PCR Master Mix, 0.8 mL of each of the forward and reverse primers (200 mM), 2 mL of cDNA and 6.4 mL of distilled water. All the reactions were run in iCycler iQ Real-time PCR (Bio-Rad laboratories, USA) using the protocol: 95 C for 60 s to denature; 40 cycles of 95 C for 15 s to anneal, 60 C for 15 s to elongate, and 72 C for 45 s to extension. The 2DDCt method was used to determine the relative mRNA abundance in the control and treated C2C12 cells as described earlier [16]. The following primers were used: CAPN1-F (5'-CCCTCAATGACACCCTCC-3'), CAPN1-R (5'-TCCACCCACTCACCAAACT-3'), CAPN2-F (5'-GAGGACATGCACACCATTGGCTTCG-3'), CAPN2-R (5'-TCCTCGCTGATGTCAATCTGGTCAATGTTG-3'), CASP3F (5'-ACTGGAAAGCCGAAACTC-3') CASP3-R (5'-GCAAGCCATCTCCTCATC-3'), CASP7-F (5'-TGAGGAGGACCACAGCAA-3'), CASP7-R (5'-GGGTGTCACGCCATCTTT-3'), MyHC-F (5'-GTCCAAGTTCCGCAAGGT-3') and MyHC-R (5'-CCACCTAAAGGGCTGTTG-3'). Expression amounts was normalized to β-actin and represented as fold change.
Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004
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Fig. 3. The size and shape of the ZnO nanoparticles was calculated using transmission electron microscope (a & c), the particle size distribution was measured using gaussian fit (b & d) and the SAED pattern of ZnO nanoparticles (e).
Western blotting After the myoblast cells were cultured in high glucose medium and treated with ZnO nanoparticles for 5 days, the cells were harvested and the whole cell lysates were prepared with RIPA buffer supplemented with EDTA-free protease inhibitor cocktail and phosphatase inhibitors. The cell lysates were incubated on ice for 30 min and sonicated for every 10 min to release the protein from chromatin. The cell lysates were centrifuged at 12,000 rpm for 5 min to obtain the protein. Equal volume of protein was separated by SDS-PAGE, proteins were transferred to PVDF membrane and immunoblotted as defined previously [17]. The enhanced ECL solution was used to obtain the signals of the
interested protein using Molecular Imager Gel Doc system (Bio-Rad, Hercules, CA) with quantity one software. Caspases-3/7 assay The effect of ZnO nanoparticles in the effect of caspases was quantified using a enzymatic activity assay according to the instructions of the manufacturer (Abcam, Germany). After treatment of C2C12 cells with ZnO nanoparticles for 24 h, the cells were washed with 1X PBS and centrifuged. The cell pellets were collected, lysed (cold lysis buffer) and quantified for total protein by Bradford assay. The samples containing 200 mg of total protein were used for caspase 3/7 enzymatic activity by adding 2x
Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004
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reaction buffer. Further, 50 ml of caspase substrate was added and incubated at room temperature for 4 h in dark. Finally, the absorbance of the sample was read at 405 nm in a microplate reader [18]. Statistical analysis All the data were expressed as mean standard deviation of triplicates. Statistical analysis was made using GraphPad Prism5 software using one-way analysis of variance (ANOVA) when only one variable was considered and the statistical significance is indicated as follow: *p < 0.05, **p < 0.01. Results and discussion Preparation of ZnO nanoparticles Initially, the synthesis of ZnO nanoparticles was confirmed by the formation of white precipitate and further it was proved using UV spectroscopic analysis. The results showed the strong spectrum at 355 nm (Fig. 1) due to the surface plasmon resonance of ZnO nanoparticles and it attributed to the electron fluctuation and conversion from the valence band to transmission band upon communication with the light of specific wavelength. Previously, it was reported that the strong absorption peak at 355 nm indicates the monodispersed nature of ZnO nanoparticles [19] and the surface plasmon resonance occurs between 320 to 370 nm which is a characteristic feature of ZnO nanoparticles [20]. Further, the energy gap (bandgap) was determined by DRS-UV analysis and the results showed that the optical energy bandgap of ZnO nanoparticles was found to be 3.21 eV (Fig. 1, inset) through αhv = A(hv—Eg)n, where a is the absorption coefficient, hy represents the energy of the photon, A is the proportionality constant and varies depends on the material, and n represents the index. The 3d orbitals of the Zn and the 2p orbitals of the O atoms has large discrepancy at the bandgap energy 12 eV and 6.5 eV respectively [21]. Hence, the predicted and theoretically proved bandgap for ZnO nanoparticles was 3.22 eV [22] which is good agreement with the bandgap energy of ZnO nanoparticles obtained in the present study.
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Fig. 3. Most of the ZnO nanoparticles were spherical in shape with size range between 10 to 15 nm and the well distribution of nanoparticles were also observed. The wet chemical mediated preparation of ZnO nanoparticles showed roughly spherical in shape and uniform size range between 7 to 10 nm and addition of water during synthesis is control the size of ZnO nanoparticles [13]. The average size of the ZnO particles was found to be 13 nm which measured from over 50 nanoparticles have been fitted with Gaussian to obtain the mean diameter. Also, the particles were tended to low degree of agglomeration which could be corresponded to the high surface area of ZnO nanoparticles [24]. The hexagonal nature of ZnO nanoparticles was confirmed with SAED analysis and the results showed the lattices at (101) and (110) corresponding with JCPDS file no. 36-1451. The crystalline structure and purity of as-prepared ZnO nanoparticles was further investigated using XRD measurements and the results showed in Fig. 4a. There are seven strong intensity peaks were observed at 2u of 31.9, 34.5, 36.4, 47.6, 56.7, 62.9 and 68.04 which indexed to the (100), (002), (001), (101), (102), (110), (103) and (112) crystal planes of ZnO nanoparticles, respectively (JCPDS No.: 36-1451). There are no additional intense peaks associated to any other impurities were observed in the sample which confirms the purity of ZnO nanoparticles. The d-spacing
Characterization of ZnO nanoparticles The corresponding SEM images of ZnO nanoparticles synthesized from Zn(OAc)2 are shown in Fig. 2a & b which showed the particles are spherical morphology with monodispersed in nature and well distributed due to the larger surface energy. Further the elemental composition analysis showed that the ZnO nanoparticles composed of 75% of Zn and 25% of O atoms (Fig. 2c) confirming that the prepared ZnO nanoparticles was pure. The scanning augur microscopy (SAM) nanoprobe analysis used to document the elemental mapping of ZnO nanoparticles and the results showed that the surface of ZnO nanoparticles covered with Zn and O atoms (Fig. 2d-2 g) which significantly associated with Edx spectrum analysis. The Edx mapping also confirms the uniform distribution of Zn and O on the surface of ZnO nanoparticles and the Zn background in the Edx signal is due to the residual Zn on the surface and also the significant O atom signal suggests the prepared nanoparticles is a consequence of ZnO formation. Previously it has been reported that the high peak intensity of Zn and O atoms was clearly observed which revealing the purity phase of ZnO and the absence of other elements affirms the synthesized ZnO nanoparticles are possess good stoichiometry in the agreement with the chemical composition of respective materials [23]. The morphology and size of the ZnO nanoparticles was observed using TEM analysis and the results were shown in
Fig. 4. The XRD spectrum (a) and the Raman spectroscopic (b) analysis of ZnO nanoparticles.
Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004
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Fig. 5. The FTIR spectrum analysis of ZnO nanoparticles.
values for the (100), (002) and (101) planes was found to be 0.28, 0.26 and 0.24 nm, respectively and these results confirmed that the prepared ZnO nanoparticles are hexagonal structure. The crystallites size of as synthesized ZnO particles was estimated by the intense of the peak observed in the XRD pattern using the Scherrer formula (D = Kl / β Cos u) [25] and the size was found to be 13 nm which correlated with size measured using TEM analysis. Further, the ZnO nanoparticles were analyzed for Raman spectroscopic analysis to confirm the crystallinity structural characterization. The Raman spectrum of ZnO nanoparticles showed two key characteristic optical bands for ZnO located at ~99 and~480 cm1 are attributed to the E2 (low) and E2 (high) modes respectively (Fig. 4b). The peak at~99 cm1 is ascribed to vibrations of the zinc lattice and ~480 cm1 is dominantly assigned to the oxygen vibration of ZnO nanoparticles [26]. The vibrations of zinc and oxygen is corresponded to the distinct asymmetry lattice disorder and to the phonon-phonon interactions [27]. Moreover, the FTIR analysis of ZnO nanoparticles showed the vibrational peak at 437 cm1 corresponding to ZnO nanoparticles which shows the stretching vibration of Zn O and the peaks at 1604 and 3512 cm1 are assigned to symmetric C¼O stretching and vibrations of hydroxyl groups from precursor and water molecules (Fig. 5). Therefore, based on the above experimental results, it is reasonable to believe that the ZnO nanoparticles can be successfully prepared by wet chemical approach. ZnO nanoparticles in myogenic differentiation
Fig. 6. Cytotoxic activity of ZnO nanoparticles was investigated against mouse myoblast C2C12 cells using in vitro MTT assay. The results were taken in triplicates and expressed as mean standard deviation (*p < 0.05, **p < 0.01).
Zn is an essential trace metal and nutrient is required for normal physiological functions in all forms of life, hence, the cellular Zn homeostasis is tightly controlled [28]. In the present study, the wet chemical mediated prepared ZnO nanoparticles was used to induce the myogenic differentiation in mouse myoblast C2C12 cells. Initially, the in vitro cytotoxic activity of ZnO nanoparticles was assessed using MTT assay and the results showed that the ZnO nanoparticles cause the cell growth in concentration dependent manner at 2, 5 and 7 days. At higher concentration (1000 mg/ml), the ZnO nanoparticles inhibits the 40% of cell death and there is not much inhibition was observed up to 400 mg/ml concentration of ZnO nanoparticles after 7 days of treatment (Fig. 6). However, the slight cytotoxic activity was observed at 2 and 5 days at higher
Fig. 7. The effect of ZnO nanoparticles on the morphological changes from myoblast to myotube formation was observed using phase contrast microscopic analysis. The results were compared with the C2C12 cells cultured in differentiation medium containing high concentration of glucose.
Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004
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Fig. 8. (a) The expression of myogenic marker proteins MyoD, MyoG, Casp3 in ZnO nanoparticles treated C2C12 cells for 2 days and 5 days and the relative expression was analysed using Image J software (https://imagej.nih.gov/ij/download.html).
concentration (1000 mg/ml) of ZnO nanoparticles against C2C12 cells. Previously, it has been reported that the high concentration of ZnO nanoparticles (5 mg/ml) induce cytotoxic activity by altering the ROS level in the mitochondria and consequent effect on the enzymatic activity in C2C12 cells [29]. The myogenesis covers a number of physiological and molecular changes that are connected with cellular energy, muscle growth and movement, in which the minimum level of Zn is essential [30]. Hence, the effect of ZnO in myogenic differentiation of C2C12 cells was studied and the variation in the cellular morphology was observed predetermined time intervals. After culture the C2C12 cells for overnight, the cells were exposed to ZnO nanoparticles and high glucose medium (positive control) for 7 days. The myogenic differentiation was analyzed by observing the cellular morphology at 0 days, 2 days, 5 days and 7 days period of interval (Fig. 7). The significant difference in the cellular morphology was detected between the cells cultured in the normal medium and ZnO nanoparticles treatment. A difference in myotube formation was observe from 5 days between control and ZnO nanoparticles treated and high glucose medium cultured C2C12 cells, while the length of the myotubes were increased at 7 days treatment compared with control C2C12 cells. Previously, about 60% of total systemic Zn is utilized and used in skeletal muscle and myoblast differentiation [28], however, the Zn deficient medium inhibits the myogenic differentiation in mouse C2C12 cells and chicken embryonic cells [31]. The regulation of myogenic differentiation is largely controlled by the myogenic basic helix-loop-helix- family of transcriptional factors which controls the expression of various myogenic marker genes [32]. So, in the present study the genes regulated the myogenic differentiation upon the treatment with ZnO nanoparticles at different time intervals was quantified using qRT-PCR analysis. The results showed that the 2 days treatment with ZnO nanoparticles showed moderate expression of myogenic markers such as MyoD and MyoG and the expression was increased with increase of treatment period from 2 days to 5 days (Fig. 8). Previously, the significant increase in the expression of MyoG mRNA during cultured with differentiating medium and reached highest level in middle differentiation stage, but the later stages showed relatively decreased expression of MyoG and MyHC proteins [33,34]. The activation of myogenic regulatory factors (MRFs) including MyoD and MyoG also controls the expression of various muscle specific genes including myosin heavy chain (MyHC) for maturation of muscle fibers [35]. The MyHC gene expression was upregulated after 5 days treatment with ZnO nanoparticles and minimal expression of MyHC was observed at 2 days treatment while the expression of MyHC was higher in the
C2C12 cells cultured in high glucose medium compared with ZnO nanoparticles (Fig. 8). The elevated expression of MyHC and its isoforms were observed during different stages of cellular differentiation and fusion of myoblasts into multinucleated myotubes [36]. The MRFs are not only controls the myogenic differentiation, also the caspases are play a crucial role in the myogenesis and organogenesis during development [37,38]. The activation of caspase 3 signaling not only leads to apoptosis of the cell but similarly activates the non-apoptotic process of differentiation which is still unclear. In the present study, the treatment of ZnO nanoparticles attenuates the expression of caspase 3 and caspase 7 mRNA during myogenic differentiation, compared with control C2C12 cells (Fig. 9). The caspase3/7 enzymatic activity also provide supporting evidence for upregulation of caspases during myogenic differentiation and also the C2C12 cells cultured in high glucose medium showed high level expression of caspases than the control cells (Fig. 10). Earlier, there are several protein kinases which are target of caspases also required for myogenesis, for example, the caspase targeted ERK5 regulates the development of cell fusion in skeletal muscle differentiation [39,40].
Fig. 9. The effect of ZnO nanoparticles and high glucose containing differentiation medium on expression of myogenic markers mRNA expression upon treatment for 5 days. The results were taken in triplicates and expressed as mean standard deviation (*p < 0.05, **p < 0.01).
Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004
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Acknowledgement Authors acknowledges the Rural Development Administration, Republic of Korea, for the financial support under the research program of next generation Biogreen 21 (PJ013169) and the Korea MAFRA and IPET for the financial support through Hanwoo Export Program (PN. 618002-5). References
Fig. 10. The effect of ZnO nanoparticles on the enzymatic activity of Caspase 3/7 in C2C12 cells.
In other hand, the calpain (CAPN) family proteins, a Ca2+ regulated cysteine proteases play crucial role in cellular proliferation and myoblast fusion by the inadequate proteolytic damage of various substrates [41]. In the present study, the myoblast C2C12 cells treated with ZnO nanoparticles for 5 days showed higher expression of CAPN1 and CAPN2 mRNA gene expression during myogenic differentiation compared with 2 days treated and control C2C12 cells (Fig. 9). The expression of CAPN1 or m-calpain in skeletal muscle is linked with Ca2+ signaling including myogenesis and cell differentiation through controlling and preventing the degradation of proteins associated with sarcolemma and playing as protective role in skeletal muscle [42,43]. However, the CAPN2 is encompassed in the regulation of the MyoD mRNA by inducing its destabilization and leading to myoblast to quiescence in the later stages of myogenic differentiation [44]. Conclusion The present work demonstrated that the ZnO nanoparticles is play a crucial role in mouse skeletal muscle myoblast cell differentiation through regulating key marker genes. Initially, the ZnO nanoparticles were synthesized using wet chemical approach and confirmed with various spectroscopic and microscopic techniques as the synthesized ZnO particles are hexagonal structure with size range between 10 to 15 nm. The ZnO nanoparticles significantly enhanced the myogenic differentiation of C2C12 myoblasts to myotube formation through promoting the MyHC gene expression and regulates the key myogenic marker genes such as MyoD and MyoG. Also, the ZnO nanoparticles significantly enhanced the non-apoptotic effect of CASP3, CASP7 and calpain family proteins during myogenic differentiation. Together, the present study demonstrates a key role of ZnO nanoparticles in differentiation of myoblast cells into myotube formation and affords the basis of future research connecting with muscle cell proliferation, differentiation and Zn homeostasis. Conflicts of interest
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Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004
Journal of Industrial and Engineering Chemistry xxx (2019) xxx–xxx
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Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells Vaikundamoorthy Ramalingam, Inho Hwang* Department of Animal Science, Chonbuk National University, Jeonju, 561-756, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 October 2019 Received in revised form 21 November 2019 Accepted 4 December 2019 Available online xxx
In the present study, we investigated the effect of monodispersed zinc oxide (ZnO) nanoparticles on the proliferation, myogenic differentiation and regulation of associated myogenic marker genes. The asprepared ZnO nanoparticles had the hexagonal wurtzite structure with maximum absorption at 355 nm and the band gap energy was found to be 3.21 eV. The electron microscopy analysis showed that the ZnO nanoparticles are spherical in shape with an average size range between 10 to 15 nm. The XRD analysis confirmed the hexagonal structure of ZnO nanoparticles and the Raman spectroscopic analysis showed the vibrations of the zinc lattice and oxygen vibration of ZnO nanoparticles. The effect of ZnO nanoparticles on myogenic differentiation was analyzed using C2C12 cells and the results showed the nanoparticles supported the proliferation with negotiable cytotoxic activity. Moreover, the ZnO nanoparticles were significantly enhanced the myoblasts into myotube formation through upregulating the myogenic markers such as myosin heavy chain, MyoD, MyoG genes. The further analysis demonstrated that the ZnO nanoparticles regulates the non-apoptotic effect of caspases and calpain family proteins in respond to the enhancement of myogenic differentiation. Together, the ZnO nanoparticles provide an additional evidence for the role of nanomaterials in skeletal muscle repair and tissue regeneration engineering. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: ZnOnanoparticles Characterization Myoblasts Myogenic differentiation Molecular mechanism
Introduction In adult human body, about 40%–45% of mass is skeletal muscles which play an important role in generating voluntary movement. The genesis of skeletal muscle during development and postnatal life is a pattern to control and regulate the stem cell and progenitor cell mechanism, heredity specification and terminal differentiation [1]. The development of skeletal muscles is a complicated process that encompasses the growth of myoblasts and the differentiation of myofibers. Myogenesis is a
Abbreviations: ZnO, Zinc oxide; HR-TEM, High resolution – transmission electron microscope; SEM, Scanning electron microscope; EDX, Energy-dispersive X-ray spectroscopy; SAED, Selected area electron diffraction; JCPDS, Joint committee on powder diffraction standards; XRD, X-ray powder diffraction; qRT-PCR, Quantitative real time – polymerase chain reaction; RIPA, Radioimmunoprecipitation; SDS-PAGE, sodium dodecyl sulfate - polyacrylamide gel electrophoresis; PVDF, Polyvinylidene fluoride; CAPN, Calpain; MyHC, Myosin heavy chain; CASP, Caspase; MyoD, myoblast determination protein 1; MyoG, Myogenin; TNF-α, tumor necrosis factor α. * Corresponding author. E-mail address: [email protected] (I. Hwang).
quite coordinated process that comprises the regulation of myogenic genes, arrest of the cell cycle and terminal fusion of mononucleated myotubes into multinucleated myotubes [2]. The myogenesis is accompanied by the transcriptional upregulation of myogenic differentiation and other myogenic differentiation marker genes [3]. The process of myogenesis is regulated by various hormones, cell cycle regulators, inhibitors of myoblast differentiation, myotube formation and myogenic markers (MyoD, Myf-5, MRF-4 and myogenin) are called as myogenic regulatory factors [4,5]. It was well documented in the C2C12 myoblasts cells that the exogenous tumor necrosis factor α (TNF-α) prevents the formation of myotubes by down regulating the myogenic markers gens such as MyoD, myogenin (MyoG), myosin heavy chain (MyHC) via NF-kB activation [6]. Moreover, the programmed cell death is also playing critical role in the differentiation, in which the caspases (3/9) and ER stress are required for the activation of differentiation, consequently, to form myotubes [7]. Depends on the duration and strong enzymatic activity, the caspase enzymes decide the cells either undergoes differentiation or death [8]. The C2C12 cells isolated from murine skeletal muscle cells is often used as model to investigate the muscle regeneration and differentiation. In the present study, the C2C12
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cells was used to study the response of skeletal muscle to zinc oxide (ZnO) nanoparticles for myogenic differentiation into myotubes. In the modern world, the growth of nanotechnology is phenomenal, and it has an extensive report on application in medical and industrial field. Especially, the impact of metallic nanoparticles on the proliferation and differentiation of normal, cancer, and stem cells is well-studied. Among various metal nanoparticles, the ZnO has various benefits specially in the human system, in which it maintains the cytosolic zinc concentration and controls the signaling pathways involved in normal cell development, differentiation and death [9]. In other hand, the zinc deficiency is serious problem in worldwide and accounting ~2 billion people are at risk by showing various symptoms including but not limited to diarrhea, immune system damage, neurological deficits and reduced reproductive development [10–12]. Moreover, zinc is essential for cellular homeostasis and also serves as a signaling molecule that regulates the number of cellular signal transduction processes. Hence, the present study aims to prepare zinc oxide nanoparticles and analyzing the effect of ZnO nanoparticles in regulating the myogenic differentiation factors to induce the myotube formation using gene and protein expression analysis.
of ZnO nanoparticles was investigated using scanning electron microscope (SIGMA model, CASL ZEISS, German) operated with EDX mapping. The Raman spectroscopic analysis (Renishaw, UK) was performed with laser power set to ~50 mW to avoid thermal damage to the particles. Cell culture and maintenance The mouse myoblast C2C12 cells were procured from American Type Culture Collection (ATCC-CRL: 1772). The cells were incubated in a humidified incubator with 5% CO2 at 37 C. The complete growth medium (GM) for C2C12 myoblast was Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Biological Industries) and 1% Penicillin-Streptomycin-Glutamine (100X, Gibco). The differentiation medium (DM) for C2C12 cells was DMEM containing 2% horse serum (HS, Gibco) and 1% Penicillin-Streptomycin-Glutamine. In vitro cytotoxic activity of ZnO nanoparticles
The precursor ferric chloride hexahydrate (FeCl3.6H2O), sodium hydroxide, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, DMSO (cell culture grade), MTT (dimethyl thiazolyl tetrazolium bromide), 1x phosphate buffer saline (PBS), glucose and glutamine were purchased from HiMedia Laboratories, Seoul, Republic of Korea. The primary and secondary antibodies used in the present study was purchased from Abcam technologies, USA.
The in vitro cytotoxic activity of ZnO nanoparticles against murine myoblast C2C12 cells was investigated using MTT assay [14]. Briefly, the C2C12 myoblast cells at a density of 15,000 cells/ well were seeded into a 96-well plate with complete growth medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine (100X, Gibco). After 12 h of incubation, the cells were treated with different concentration of ZnO nanoparticles (01000 mg/ml) and again incubated for 7 days. The culture medium was replaced by fresh complete growth medium for every 24 h and the viability of the cells were measured at predetermined intervals (2, 5 and 7 days). The cytotoxic activity of ZnO nanoparticles were measured after washing the cells with 1X PBS and 10 ml of MTT solution was added to each well. After incubation for 4 h in the dark, the absorbance was measured at 490/630 nm and IC50 was calculated.
Preparation of ZnO nanoparticles
Induction of myogenesis
The ZnO nanoparticles was synthesized using wet chemical method as per the method described previously [13]. Initially, 1.98 g of zinc acetate dihydrate was added to 40 ml of methanol and the solution was kept for reflux to dissolve completely. The solution (30 ml) was mixed with 0.72 g of sodium hydroxide by dropwise and refluxed for 48 h. The resulting solution was centrifuged at 10,000 rpm for 30 min to obtain a white precipitate and the precipitate washed with water for several times to improve the particle homogeneity. Further, the resulted precipitate was again washed with 1:1 ethanol/acetone solution and dried under vacuum for overnight. The dried white powdered ZnO nanoparticles was used for further characterization and biological applications.
The mouse myoblast C2C12 cells were obtained from ATCC and the cells were cultured in complete DMEM medium supplemented with 20% fetal bovine serum (FBS) (growth medium, GM) and 100 mg/ml penicillin/streptomycin used as control. The myoblast
Materials and methods Chemicals and reagents
Characterization of ZnO nanoparticles There are numerous methods and techniques were employed to characterize the ZnO nanoparticles such as crystalline phase, compositional analysis, particle size measurements, particle morphology and surface composition of ZnO nanoparticles. The crystalline structure of ZnO nanoparticles was measured using powder X-ray diffraction (XRD) measurements (Philips X’Pert Pro MPD, UK) with Kα radiation in the 2u range from 10 to 80 . For size and morphology, a transmission electron microscope (TEM) was used (Leica EM UC 6 Ultramicrotome & Nikon Trinocular Microscope Model E200, Leica Camera AG – Germany) to obtain an average particle size by measuring the size of 50–100 particles. The surface morphology and composition associated on the surface
Fig. 1. UV-spectroscopic and DRS-UV spectroscopic (inset) analysis of ZnO nanoparticles prepared using wet-chemical approach.
Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004
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Fig. 2. Scanning electron microscopic analysis showed the aggregation of ZnO nanoparticles (a & b), the elemental composition was analyzed using EDS spectrum (c) and the SAM-nanoprobe analysis used for Edax mapping of Zn (e) and O (f) arrangement on the surface of ZnO nanoparticles (g).
cells at a density of 50,000 cells/well were grown in differentiation medium containing glutamine-free DMEM with high glucose (25 mM), 2% horse serum, 1% of penicillin-streptomycin, 10 mg/ml insulin (Sigma–Aldrich Co. LLC., St-Louis, MO, USA), 10 mg/ml doxycycline (Sigma–Aldrich Co. LLC.), and 0, 1, 2, 4 mM l-glutamine (Thermo Fisher Scientific Inc.) respectively for 7 days, to induce myogenesis. The medium was replaced every 2 days. In mean time, the ZnO nanoparticles was used to treat the C2C12 cells cultured in the complete growth medium to study the effect on the myogenic differentiation and the morphological differences was observed at predetermined times under phase contrast microscope [15]. Quantitative real time PCR analysis qRT-PCR was performed to investigate the myogenic gene expression levels upon treatment with ZnO nanoparticles in C2C12 cells. Initially, the total RNA was isolated from 5 days treated cells using TRIzol Reagent (Life technologies, USA). The cDNA was synthesized using First Strand cDNA synthesis kit (Thermo
Scientific, USA) and qPCR was performed with a 20 mL reaction system comprising 10 mL of SYBR Green Realtime PCR Master Mix, 0.8 mL of each of the forward and reverse primers (200 mM), 2 mL of cDNA and 6.4 mL of distilled water. All the reactions were run in iCycler iQ Real-time PCR (Bio-Rad laboratories, USA) using the protocol: 95 C for 60 s to denature; 40 cycles of 95 C for 15 s to anneal, 60 C for 15 s to elongate, and 72 C for 45 s to extension. The 2DDCt method was used to determine the relative mRNA abundance in the control and treated C2C12 cells as described earlier [16]. The following primers were used: CAPN1-F (5'-CCCTCAATGACACCCTCC-3'), CAPN1-R (5'-TCCACCCACTCACCAAACT-3'), CAPN2-F (5'-GAGGACATGCACACCATTGGCTTCG-3'), CAPN2-R (5'-TCCTCGCTGATGTCAATCTGGTCAATGTTG-3'), CASP3F (5'-ACTGGAAAGCCGAAACTC-3') CASP3-R (5'-GCAAGCCATCTCCTCATC-3'), CASP7-F (5'-TGAGGAGGACCACAGCAA-3'), CASP7-R (5'-GGGTGTCACGCCATCTTT-3'), MyHC-F (5'-GTCCAAGTTCCGCAAGGT-3') and MyHC-R (5'-CCACCTAAAGGGCTGTTG-3'). Expression amounts was normalized to β-actin and represented as fold change.
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Fig. 3. The size and shape of the ZnO nanoparticles was calculated using transmission electron microscope (a & c), the particle size distribution was measured using gaussian fit (b & d) and the SAED pattern of ZnO nanoparticles (e).
Western blotting After the myoblast cells were cultured in high glucose medium and treated with ZnO nanoparticles for 5 days, the cells were harvested and the whole cell lysates were prepared with RIPA buffer supplemented with EDTA-free protease inhibitor cocktail and phosphatase inhibitors. The cell lysates were incubated on ice for 30 min and sonicated for every 10 min to release the protein from chromatin. The cell lysates were centrifuged at 12,000 rpm for 5 min to obtain the protein. Equal volume of protein was separated by SDS-PAGE, proteins were transferred to PVDF membrane and immunoblotted as defined previously [17]. The enhanced ECL solution was used to obtain the signals of the
interested protein using Molecular Imager Gel Doc system (Bio-Rad, Hercules, CA) with quantity one software. Caspases-3/7 assay The effect of ZnO nanoparticles in the effect of caspases was quantified using a enzymatic activity assay according to the instructions of the manufacturer (Abcam, Germany). After treatment of C2C12 cells with ZnO nanoparticles for 24 h, the cells were washed with 1X PBS and centrifuged. The cell pellets were collected, lysed (cold lysis buffer) and quantified for total protein by Bradford assay. The samples containing 200 mg of total protein were used for caspase 3/7 enzymatic activity by adding 2x
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reaction buffer. Further, 50 ml of caspase substrate was added and incubated at room temperature for 4 h in dark. Finally, the absorbance of the sample was read at 405 nm in a microplate reader [18]. Statistical analysis All the data were expressed as mean standard deviation of triplicates. Statistical analysis was made using GraphPad Prism5 software using one-way analysis of variance (ANOVA) when only one variable was considered and the statistical significance is indicated as follow: *p < 0.05, **p < 0.01. Results and discussion Preparation of ZnO nanoparticles Initially, the synthesis of ZnO nanoparticles was confirmed by the formation of white precipitate and further it was proved using UV spectroscopic analysis. The results showed the strong spectrum at 355 nm (Fig. 1) due to the surface plasmon resonance of ZnO nanoparticles and it attributed to the electron fluctuation and conversion from the valence band to transmission band upon communication with the light of specific wavelength. Previously, it was reported that the strong absorption peak at 355 nm indicates the monodispersed nature of ZnO nanoparticles [19] and the surface plasmon resonance occurs between 320 to 370 nm which is a characteristic feature of ZnO nanoparticles [20]. Further, the energy gap (bandgap) was determined by DRS-UV analysis and the results showed that the optical energy bandgap of ZnO nanoparticles was found to be 3.21 eV (Fig. 1, inset) through αhv = A(hv—Eg)n, where a is the absorption coefficient, hy represents the energy of the photon, A is the proportionality constant and varies depends on the material, and n represents the index. The 3d orbitals of the Zn and the 2p orbitals of the O atoms has large discrepancy at the bandgap energy 12 eV and 6.5 eV respectively [21]. Hence, the predicted and theoretically proved bandgap for ZnO nanoparticles was 3.22 eV [22] which is good agreement with the bandgap energy of ZnO nanoparticles obtained in the present study.
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Fig. 3. Most of the ZnO nanoparticles were spherical in shape with size range between 10 to 15 nm and the well distribution of nanoparticles were also observed. The wet chemical mediated preparation of ZnO nanoparticles showed roughly spherical in shape and uniform size range between 7 to 10 nm and addition of water during synthesis is control the size of ZnO nanoparticles [13]. The average size of the ZnO particles was found to be 13 nm which measured from over 50 nanoparticles have been fitted with Gaussian to obtain the mean diameter. Also, the particles were tended to low degree of agglomeration which could be corresponded to the high surface area of ZnO nanoparticles [24]. The hexagonal nature of ZnO nanoparticles was confirmed with SAED analysis and the results showed the lattices at (101) and (110) corresponding with JCPDS file no. 36-1451. The crystalline structure and purity of as-prepared ZnO nanoparticles was further investigated using XRD measurements and the results showed in Fig. 4a. There are seven strong intensity peaks were observed at 2u of 31.9, 34.5, 36.4, 47.6, 56.7, 62.9 and 68.04 which indexed to the (100), (002), (001), (101), (102), (110), (103) and (112) crystal planes of ZnO nanoparticles, respectively (JCPDS No.: 36-1451). There are no additional intense peaks associated to any other impurities were observed in the sample which confirms the purity of ZnO nanoparticles. The d-spacing
Characterization of ZnO nanoparticles The corresponding SEM images of ZnO nanoparticles synthesized from Zn(OAc)2 are shown in Fig. 2a & b which showed the particles are spherical morphology with monodispersed in nature and well distributed due to the larger surface energy. Further the elemental composition analysis showed that the ZnO nanoparticles composed of 75% of Zn and 25% of O atoms (Fig. 2c) confirming that the prepared ZnO nanoparticles was pure. The scanning augur microscopy (SAM) nanoprobe analysis used to document the elemental mapping of ZnO nanoparticles and the results showed that the surface of ZnO nanoparticles covered with Zn and O atoms (Fig. 2d-2 g) which significantly associated with Edx spectrum analysis. The Edx mapping also confirms the uniform distribution of Zn and O on the surface of ZnO nanoparticles and the Zn background in the Edx signal is due to the residual Zn on the surface and also the significant O atom signal suggests the prepared nanoparticles is a consequence of ZnO formation. Previously it has been reported that the high peak intensity of Zn and O atoms was clearly observed which revealing the purity phase of ZnO and the absence of other elements affirms the synthesized ZnO nanoparticles are possess good stoichiometry in the agreement with the chemical composition of respective materials [23]. The morphology and size of the ZnO nanoparticles was observed using TEM analysis and the results were shown in
Fig. 4. The XRD spectrum (a) and the Raman spectroscopic (b) analysis of ZnO nanoparticles.
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Fig. 5. The FTIR spectrum analysis of ZnO nanoparticles.
values for the (100), (002) and (101) planes was found to be 0.28, 0.26 and 0.24 nm, respectively and these results confirmed that the prepared ZnO nanoparticles are hexagonal structure. The crystallites size of as synthesized ZnO particles was estimated by the intense of the peak observed in the XRD pattern using the Scherrer formula (D = Kl / β Cos u) [25] and the size was found to be 13 nm which correlated with size measured using TEM analysis. Further, the ZnO nanoparticles were analyzed for Raman spectroscopic analysis to confirm the crystallinity structural characterization. The Raman spectrum of ZnO nanoparticles showed two key characteristic optical bands for ZnO located at ~99 and~480 cm1 are attributed to the E2 (low) and E2 (high) modes respectively (Fig. 4b). The peak at~99 cm1 is ascribed to vibrations of the zinc lattice and ~480 cm1 is dominantly assigned to the oxygen vibration of ZnO nanoparticles [26]. The vibrations of zinc and oxygen is corresponded to the distinct asymmetry lattice disorder and to the phonon-phonon interactions [27]. Moreover, the FTIR analysis of ZnO nanoparticles showed the vibrational peak at 437 cm1 corresponding to ZnO nanoparticles which shows the stretching vibration of Zn O and the peaks at 1604 and 3512 cm1 are assigned to symmetric C¼O stretching and vibrations of hydroxyl groups from precursor and water molecules (Fig. 5). Therefore, based on the above experimental results, it is reasonable to believe that the ZnO nanoparticles can be successfully prepared by wet chemical approach. ZnO nanoparticles in myogenic differentiation
Fig. 6. Cytotoxic activity of ZnO nanoparticles was investigated against mouse myoblast C2C12 cells using in vitro MTT assay. The results were taken in triplicates and expressed as mean standard deviation (*p < 0.05, **p < 0.01).
Zn is an essential trace metal and nutrient is required for normal physiological functions in all forms of life, hence, the cellular Zn homeostasis is tightly controlled [28]. In the present study, the wet chemical mediated prepared ZnO nanoparticles was used to induce the myogenic differentiation in mouse myoblast C2C12 cells. Initially, the in vitro cytotoxic activity of ZnO nanoparticles was assessed using MTT assay and the results showed that the ZnO nanoparticles cause the cell growth in concentration dependent manner at 2, 5 and 7 days. At higher concentration (1000 mg/ml), the ZnO nanoparticles inhibits the 40% of cell death and there is not much inhibition was observed up to 400 mg/ml concentration of ZnO nanoparticles after 7 days of treatment (Fig. 6). However, the slight cytotoxic activity was observed at 2 and 5 days at higher
Fig. 7. The effect of ZnO nanoparticles on the morphological changes from myoblast to myotube formation was observed using phase contrast microscopic analysis. The results were compared with the C2C12 cells cultured in differentiation medium containing high concentration of glucose.
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Fig. 8. (a) The expression of myogenic marker proteins MyoD, MyoG, Casp3 in ZnO nanoparticles treated C2C12 cells for 2 days and 5 days and the relative expression was analysed using Image J software (https://imagej.nih.gov/ij/download.html).
concentration (1000 mg/ml) of ZnO nanoparticles against C2C12 cells. Previously, it has been reported that the high concentration of ZnO nanoparticles (5 mg/ml) induce cytotoxic activity by altering the ROS level in the mitochondria and consequent effect on the enzymatic activity in C2C12 cells [29]. The myogenesis covers a number of physiological and molecular changes that are connected with cellular energy, muscle growth and movement, in which the minimum level of Zn is essential [30]. Hence, the effect of ZnO in myogenic differentiation of C2C12 cells was studied and the variation in the cellular morphology was observed predetermined time intervals. After culture the C2C12 cells for overnight, the cells were exposed to ZnO nanoparticles and high glucose medium (positive control) for 7 days. The myogenic differentiation was analyzed by observing the cellular morphology at 0 days, 2 days, 5 days and 7 days period of interval (Fig. 7). The significant difference in the cellular morphology was detected between the cells cultured in the normal medium and ZnO nanoparticles treatment. A difference in myotube formation was observe from 5 days between control and ZnO nanoparticles treated and high glucose medium cultured C2C12 cells, while the length of the myotubes were increased at 7 days treatment compared with control C2C12 cells. Previously, about 60% of total systemic Zn is utilized and used in skeletal muscle and myoblast differentiation [28], however, the Zn deficient medium inhibits the myogenic differentiation in mouse C2C12 cells and chicken embryonic cells [31]. The regulation of myogenic differentiation is largely controlled by the myogenic basic helix-loop-helix- family of transcriptional factors which controls the expression of various myogenic marker genes [32]. So, in the present study the genes regulated the myogenic differentiation upon the treatment with ZnO nanoparticles at different time intervals was quantified using qRT-PCR analysis. The results showed that the 2 days treatment with ZnO nanoparticles showed moderate expression of myogenic markers such as MyoD and MyoG and the expression was increased with increase of treatment period from 2 days to 5 days (Fig. 8). Previously, the significant increase in the expression of MyoG mRNA during cultured with differentiating medium and reached highest level in middle differentiation stage, but the later stages showed relatively decreased expression of MyoG and MyHC proteins [33,34]. The activation of myogenic regulatory factors (MRFs) including MyoD and MyoG also controls the expression of various muscle specific genes including myosin heavy chain (MyHC) for maturation of muscle fibers [35]. The MyHC gene expression was upregulated after 5 days treatment with ZnO nanoparticles and minimal expression of MyHC was observed at 2 days treatment while the expression of MyHC was higher in the
C2C12 cells cultured in high glucose medium compared with ZnO nanoparticles (Fig. 8). The elevated expression of MyHC and its isoforms were observed during different stages of cellular differentiation and fusion of myoblasts into multinucleated myotubes [36]. The MRFs are not only controls the myogenic differentiation, also the caspases are play a crucial role in the myogenesis and organogenesis during development [37,38]. The activation of caspase 3 signaling not only leads to apoptosis of the cell but similarly activates the non-apoptotic process of differentiation which is still unclear. In the present study, the treatment of ZnO nanoparticles attenuates the expression of caspase 3 and caspase 7 mRNA during myogenic differentiation, compared with control C2C12 cells (Fig. 9). The caspase3/7 enzymatic activity also provide supporting evidence for upregulation of caspases during myogenic differentiation and also the C2C12 cells cultured in high glucose medium showed high level expression of caspases than the control cells (Fig. 10). Earlier, there are several protein kinases which are target of caspases also required for myogenesis, for example, the caspase targeted ERK5 regulates the development of cell fusion in skeletal muscle differentiation [39,40].
Fig. 9. The effect of ZnO nanoparticles and high glucose containing differentiation medium on expression of myogenic markers mRNA expression upon treatment for 5 days. The results were taken in triplicates and expressed as mean standard deviation (*p < 0.05, **p < 0.01).
Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004
G Model JIEC 4888 No. of Pages 8
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V. Ramalingam, I. Hwang / Journal of Industrial and Engineering Chemistry xxx (2019) xxx–xxx
Acknowledgement Authors acknowledges the Rural Development Administration, Republic of Korea, for the financial support under the research program of next generation Biogreen 21 (PJ013169) and the Korea MAFRA and IPET for the financial support through Hanwoo Export Program (PN. 618002-5). References
Fig. 10. The effect of ZnO nanoparticles on the enzymatic activity of Caspase 3/7 in C2C12 cells.
In other hand, the calpain (CAPN) family proteins, a Ca2+ regulated cysteine proteases play crucial role in cellular proliferation and myoblast fusion by the inadequate proteolytic damage of various substrates [41]. In the present study, the myoblast C2C12 cells treated with ZnO nanoparticles for 5 days showed higher expression of CAPN1 and CAPN2 mRNA gene expression during myogenic differentiation compared with 2 days treated and control C2C12 cells (Fig. 9). The expression of CAPN1 or m-calpain in skeletal muscle is linked with Ca2+ signaling including myogenesis and cell differentiation through controlling and preventing the degradation of proteins associated with sarcolemma and playing as protective role in skeletal muscle [42,43]. However, the CAPN2 is encompassed in the regulation of the MyoD mRNA by inducing its destabilization and leading to myoblast to quiescence in the later stages of myogenic differentiation [44]. Conclusion The present work demonstrated that the ZnO nanoparticles is play a crucial role in mouse skeletal muscle myoblast cell differentiation through regulating key marker genes. Initially, the ZnO nanoparticles were synthesized using wet chemical approach and confirmed with various spectroscopic and microscopic techniques as the synthesized ZnO particles are hexagonal structure with size range between 10 to 15 nm. The ZnO nanoparticles significantly enhanced the myogenic differentiation of C2C12 myoblasts to myotube formation through promoting the MyHC gene expression and regulates the key myogenic marker genes such as MyoD and MyoG. Also, the ZnO nanoparticles significantly enhanced the non-apoptotic effect of CASP3, CASP7 and calpain family proteins during myogenic differentiation. Together, the present study demonstrates a key role of ZnO nanoparticles in differentiation of myoblast cells into myotube formation and affords the basis of future research connecting with muscle cell proliferation, differentiation and Zn homeostasis. Conflicts of interest
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Please cite this article in press as: V. Ramalingam, I. Hwang, Zinc oxide nanoparticles promoting the formation of myogenic differentiation into myotubes in mouse myoblast C2C12 cells, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.12.004