Simulated microgravity attenuates myogenic differentiation via epigenetic regulations

Takuma Furukawa; Keiji Tanimoto; Takahiro Fukazawa; Takeshi Imura; Yumi Kawahara; Louis Yuge

doi:10.1038/s41526-018-0045-0

Simulated microgravity attenuates myogenic differentiation via epigenetic regulations

Furukawa, Takuma; Tanimoto, Keiji; Fukazawa, Takahiro; Imura, Takeshi; Kawahara, Yumi; Yuge, Louis 2018-05-23 00:00:00 www.nature.com/npjmgrav ARTICLE OPEN Simulated microgravity attenuates myogenic differentiation via epigenetic regulations 1 2 3 1 4 1,4 Takuma Furukawa , Keiji Tanimoto , Takahiro Fukazawa , Takeshi Imura , Yumi Kawahara and Louis Yuge The molecular mechanisms involved in myogenic differentiation are relatively well-known. Myogenic differentiation is regulated by the sequential activation of the basic helix-loop-helix myogenic regulatory transcription factors (MRFs), and biomechanical signals play an important role in the regulation of myogenesis. In this study, we sought to determine whether simulated microgravity culture using Gravite may affect myoblast differentiation and expression of MRF genes. Although rat myoblasts, L6 cells were differentiated to myotubes in an incubation period-dependent manner, myogenesis of L6 cells was signiﬁcantly attenuated under -3 simulated microgravity (10 G) conditions. Real-time Reverse transcription polymerase chain reaction (RT-PCR) showed that expressions of Myog, Myf6, Mef2c, Des, and Ckm under 1 G conditions increase in an incubation period-dependent manner, and that Myod1 expression was speciﬁcally observed to increase transiently in the early phase. However, expressions of Myod1 and Myog were signiﬁcantly inhibited under simulated microgravity conditions. To clarify the molecular mechanisms, L6 cells were treated −3 with 5-AzaC, and further incubated with differentiation medium under 1 G or 10 G conditions. The results showed differences in −3 expression levels of Myod1, Myog, and, as well as those of myotube thickness between 1 G and 10 G conditions, completely disappeared in this experimental condition. Modiﬁed HpaII tiny fragment enrichment by ligation-mediated PCR (HELP)-assay showed that kinetic changes of DNA methylation status were attenuated in simulated microgravity conditions. These results indicate that microgravity regulates myogenesis and Myod1 expression by controlling DNA methylation. npj Microgravity (2018) 4:11 ; doi:10.1038/s41526-018-0045-0 INTRODUCTION It is signiﬁcant that the muscle mass of astronauts after 2 weeks space ﬂight was diminished by up to 20%. Sarcopenia is deﬁned as an age-related loss of skeletal muscle DNA methylation is also known to be involved in regulation of mass and strength. Beginning with the 4th decade of life, and myogenic gene expression and myogenesis: The DNA- symptoms progress with age. If muscle mass accounts for up to demethylating agent 5-azacytidine induced myogenesis via 60% of body weight, pathological changes in skeletal muscle can 22–25 upregulated MyoD expression. Barrès et al. interestingly cause serious effects on older adults. Nevertheless, the therapeutic reported that acute exercise tends to reduce global methylation outcome of age-related skeletal muscle atrophy and weakness on the entire genome, resulting in activation of regulated genes in 2–4 remains unknown. The understanding of molecular mechan- 26 human skeletal muscle. Singh et al. demonstrated that simulated isms of myogenic differentiation process will result in better microgravity induced epigenetic changes of genome DNA in treatment outcomes, since impaired regulation of myogenic human lymphocytes through altered expression of DNMT1, differentiation is closely associated with age-related skeletal DNMT3a, and DNMT3b. However, the effects of space ﬂight or 5–8 muscle dysfunction. It is well known that myogenic differentia- simulated microgravity on epigenetic changes of MRF genes and tion is regulated by the sequential activation of the basic helix- resulting myogenesis remain unclear. Therefore, this study aims to loop-helix myogenic regulatory transcription factors (MRFs): MyoD, determine whether simulated microgravity affects myoblast 9–11 Myf5, myogenin, and MRF4 (Myf6). MyoD in particular, is differentiation, expression of MRF genes, and the status of DNA 12–14 involved in the commitment of cells to the myogenic lineage. methylation. It has been reported that exposure of myoblast or muscle satellite cells to mechanical uniaxial stretch and stretching by a magnetic ﬁeld, or electrical stimulation, upregulate MRFs expression, RESULTS 15–17 resulting in activation of myogenesis. Moreover, differentia- Morphological evaluation of myogenesis under microgravity tion of myoblasts was enhanced on highly aligned fullerene conditions whiskers scaffolds culture. In contrast, differentiation of myo- To evaluate myogenesis under simulated microgravity conditions, blast was suppressed in the microgravity culture for in vitro study L6 cells were cultured with differentiation medium under normal and spaceﬂight experiments, suggesting that biomechanical -3 1 G or microgravity (10 G) conditions for 1, 3, 5, or 7 days (Fig. 1a). 19,20 signals play an important role in the regulation of myogenesis. Cells were then observed by capturing images, and the maximum 1 2 Division of Bio-Environmental Adaptation Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan; Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan and Space Bio-Laboratories Co., Ltd, Hiroshima, Japan Correspondence: Keiji Tanimoto (ktanimo@hiroshima-u.ac.jp) or Louis Yuge (ryuge@hiroshima-u.ac.jp) Received: 27 September 2017 Revised: 18 April 2018 Accepted: 23 April 2018 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity attenuates myogenic T Furukawa et al. Fig. 1 Morphological evaluation of myogenesis under microgravity conditions. a Schematic representation of experimental time course is shown. L6 cells were cultured with growth medium for 2 days, and then replaced with differentiation medium. Cells were then cultured under -3 normal 1 G or 10 G conditions for 0, 12 h, 1, 3, 5, or 7 days, and observed by taking pictures, and the maximum transverse diameters of myotube cells (myotube thickness) were measured. b Representative microscopic images during myogenesis are shown. White arrow heads: myotube cell; white H-shaped bars: examples of the maximum transverse diameter of myotube cells; white scale bar: 300 μm. c The maximum transverse diameters of myotube cells were measured by image processing software ImageJ, and the mean value of 10 ﬁelds was calculated. Columns show the mean of three independent experiments; bars, SD. P values calculated with Student’s t test. (**P < 0.01) (n = 3) transverse diameters of myotube cells (myotube thickness) were Although transient increases of Myod1 expression under 1 G measured as indicated in Fig. 1b. As expected, L6 cells were conditions were observed from 12 h to Day1, those increases were differentiated to myotubes, resulting in an increase in the signiﬁcantly inhibited in microgravity conditions. Myog expres- thickness, under 1 G conditions in an incubation period- sions were also signiﬁcantly inhibited in microgravity conditions at dependent manner (Fig. 1c). Interestingly, myogenesis was the same points of time. Expression levels of Pax3, Pax7, and Myf5 signiﬁcantly attenuated under microgravity conditions on day 3, did not change under our study conditions. 5, and 7, although myotubes gradually increased (Fig. 1c). Effects of 5-Azacytidine treatment on myogenesis under Gene expression analyses of myogenesis-relating genes under microgravity conditions microgravity conditions Expression of Myod1 and Myog had previously been reported to 23,25,28 In order to clarify the molecular mechanisms of attenuation of be regulated by DNA methylation. In order to clarify the myogenesis under microgravity conditions, expression levels of molecular mechanisms of the decreased expression of Myod1 and myogenesis-related genes were evaluated using real-time RT-PCR Myog, and the signiﬁcance in myogenesis under microgravity (Fig. 2): Expression of Myog, Myf6, Mef2c, Des, and Ckm under 1 G conditions, L6 cells were then treated with DNA methylation conditions increased in an incubation period-dependent manner. inhibitor, 5-Azacytidine (5-AzaC), before incubation with npj Microgravity (2018) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; Simulated microgravity attenuates myogenic T Furukawa et al. Fig. 2 Gene expression analyses of myogenesis-related genes under microgravity conditions. For expression analysis, L6 cells were cultured -3 under normal 1 G or 10 G conditions for 0, 12 h, 1, 3, 5, or 7 days. Total RNA was extracted from cell pellets, and expression levels of myogenesis-relating genes were evaluated by real-time RT-PCR. Three independent measurements were averaged, and relative gene expression levels were calculated as a ratio against Actb expression for each experiment. Columns show the mean of three independent experiments; bars, SD. P values calculated with Student’s t test. (*P < 0.05, **P < 0.01) (n = 3) differentiation medium (Fig. 3a). To optimize concentration and HpaII sites (Fig. 4b). Since DNA methylation sensitive restriction treatment periods of 5-AzaC, L6 cells were ﬁrst treated with 1, 2, 5, enzyme HpaII cannot digest methylated DNA, but/although can or 10 μΜ of 5-AzaC for 4, 8, or 12 days. As a result, Myod1 digest unmethylated DNA, the primer set ampliﬁed methylated expression was observed to increase in a concentration- or DNA, but not unmethylated or digested DNA after incubation with treatment period-dependent manner (Fig. 3b). Of these condi- HpaII. Therefore, methylated DNA can be quantiﬁed by real-time tions, treatment with 5 μΜ of 5-AzaC for 12 days was conﬁrmed as PCR, resulting in evaluation of DNA methylation status. Genomic the effective condition for the following experiments. After DNAs were then isolated from L6 cell samples that had been treatment with 5 μΜ of 5-AzaC for 12 days, L6 cells were cultured as in Fig. 1, and incubated with HpaII for 12 h. Real-time incubated with growth medium for 2 days, and also with PCR demonstrated that relative amounts of PCR products differentiation medium for the indicated periods under 1 G or ampliﬁed from 1 G samples decreased gradually until Day3, and microgravity conditions. Real-time RT-PCR showed that the then increased until Day7 (Fig. 4c). It is striking that PCR products expression levels of Myod1 and Myog seemed to increase from microgravity samples also slightly decreased but to a smaller gradually in a treatment period-dependent manner, but the degree than those of 1 G, suggesting a retaining DNA methylation −3 difference between 1 G and 10 G conditions observed in Fig. 2, on Myod1 promoter (Fig. 4c). In order to clarify the underlying and disappeared in Fig. 3c. In this experimental condition, mechanisms, real-time RT-PCR analyses of methylation-related morphological evaluations were also performed as in Fig. 1. genes were performed using the same samples of cDNA as in Fig. Results showed that differences in myotube thickness between 2. Results showed that expressions of DNA methyltransferase, 1 G and microgravity conditions also completely disappeared, Dnmt1 and Dnmt3a, under 1 G conditions were decreased during although L6 cells were gradually differentiated to myotubes under the myogenic process (Fig. 4d), while Dnmt3b was not detected both conditions (Fig. 3d,e). (data not shown). Interestingly, expressions of Dnmt1 and Dnmt3a under microgravity conditions also decreased during the myo- genic process, but remained signiﬁcantly higher than those under DNA methylation in Myod1 gene under microgravity conditions 1 G conditions, suggesting they retained functions of methyl- To determine whether DNA methylation actually contributes to transferases (Fig. 4d). Expression levels of Tdg also decreased regulation of Myod1 expression, we use modiﬁed HELP-assay to during the myogenic process, but differences between 1 G and detect DNA methylation in the promoter region of Myod1 gene. microgravity conditions were smaller. Expression levels of Mbd2 UCSC Genome Browser (http://genome.ucsc.edu) showed that varied and there was no trend observed. MYOD1 gene virtually overlapped with CpG island, indicating very dense CpG sites, and the proximal promoter region contained Other regulation of Myod1 and Myog genes under microgravity actually methylated as shown in the results of reduced conditions representation bisulﬁte sequencing and methyl 450 K bead arrays (Fig. 4a). A primer set was designed for just upstream from the In order to clarify regulation mechanisms of expression of Myod1 transcription start site of Myod1, which contained 12 CpG or 3 and Myog genes during the myogenic process under 1 G and Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 11 Simulated microgravity attenuates myogenic T Furukawa et al. Fig. 3 Effects of 5-Azacytidine treatment on myogenesis under microgravity conditions. a Schematic representation of experimental time course is shown. L6 cells were cultured with 5 μM of 5-AzaC in growth medium for 12 days, with passage every 4 days. Cells were then seeded on culture ﬂasks and cultured in growth medium for 2 days. After medium replacement with differentiation medium, cells were cultured in −3 normal 1 G or 10 G conditions for 0, 12 h, 1, 3, 5, and 7 days. b Optimization of 5-azaC treatment was performed. L6 cells were treated with 1, 2, 5, or 10 μM of 5-AzaC for 4, 8, or 12 days, and expression levels of Myod1 gene were evaluated by real-time RT-PCR. Three independent measurements were averaged and relative gene expression levels were calculated as a ratio against Actb expression for each experiment. Columns show the mean of three independent experiments; bars, SD (n= 3). c Expression levels of Myod1 and Myog genes were evaluated by real-time RT-PCR. Three independent measurements were averaged and relative gene expression levels were calculated as a ratio against Actb expression for each experiment. Columns show the mean of three independent experiments; bars, SD (n = 3). d Representative microscopic images during myogenesis are also shown. White arrow heads: myotube cell; white H-shaped bars: examples of the maximum transverse diameter of myotube cells; white scale bar: 300 μm. e The maximum transverse diameters of myotube cells were measured using image processing software ImageJ, and the mean value of 10 ﬁelds was calculated. Columns show the mean of three independent experiments; bars, SD. (n = 3) microgravity conditions, further analyses of promoter activities were no differences in those levels under 1 G or microgravity and RNA stabilities were performed. First, the 1.47 kb DNA conditions (Supplementary Fig 2B). fragment of the Myod1 gene promoter region, or the 1.55 kb DNA fragment of the Myog gene promoter region, was ampliﬁed DISCUSSION and subcloned into the luciferase reporter plasmid pGL4.16 As a consequence of an aging population, the number of elderly (designated pGL4.16-Myod1 Pro1474 or pGL4.16-Myog Pro1546, who exhibit skeletal muscle atrophy and weakness are increasing, Supplementary Fig 1B). L6 cells were then transfected with these 30–32 becoming a concern of society. In order to achieve better promoter reporters and incubated for 1 day. Next, growth medium treatment outcomes, the understanding of the molecular was replaced with differentiation medium and incubated for one mechanisms of myogenesis is essential, since impaired myogen- more day under 1 G or microgravity conditions (Supplementary esis is closely associated with age-related skeletal muscle loss and Fig 1A). Dual-luciferase assays demonstrated that cloned regions 5–8 weakness. Previous reports have suggested that biomechanical had strong promoter activities compared to empty vector, but any signals play an important role in the regulation of myogenesis. differences in those activities under 1 G or microgravity conditions Speciﬁcally, exposure of myoblast and muscle satellite cells to were not observed (Supplementary Fig 1C). L6 cells were next mechanical stretching, electrical stimulation, or microgravity have cultured in growth medium for 2 days, and then cultured in been found to modify expression levels of MRFs, resulting in 15–17,19 differentiation medium for further a day. After adding 5 μg/ml of alteration of myogenesis. actinomycin D, cells were cultured under 1 G or microgravity In this study, we used our new generation 3D-clinostat ® -3 conditions (Supplementary Fig 2A). Real-time RT-PCR showed that “Gravite ” to obtain simulated microgravity conditions (10 G) for gene expression level of Myod1 had decreased gradually, while cell culture: We observed that myogenesis of rat myoblast cells (L6 those of Myog and Actb had not (Supplementary Fig 2B). There cells) cultured in Gravite were signiﬁcantly attenuated, indicating npj Microgravity (2018) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity attenuates myogenic T Furukawa et al. Fig. 4 DNA methylation in Myod1 gene under microgravity conditions. a Schematic structure of MYOD1 gene in UCSC Genome Browser showed that MYOD1 gene virtually overlapped with CpG island (upper rectangle drawn in red dotted line), indicating very dense CpG sites, and the proximal promoter region contained actually methylated sites (lower rectangle drawn in red dotted line). b The nucleic acid sequence of proximal promoter region in Myod1 gene is shown. Blue circle and arrow indicate transcription start site of Myod1 gene, red rectangles, CpG sites, blue rectangles, HpaII sites, and red arrows, primer sets for modiﬁed HELP-assay. c Methylated proximal promoter regions in Myod1 gene are quantiﬁed by real-time PCR. Three independent measurements were averaged and relative amounts of PCR products were calculated as the ratio against that of Set0 for each experiment. (n = 3). d Expression levels of methylation related genes were evaluated by real-time RT- PCR. Three independent measurements were averaged and relative gene expression levels were calculated as a ratio against Actb expression for each experiment. Columns show the mean of three independent experiments; bars, SD. P values calculated with Student’s t test. (*P < 0.05, **P < 0.01) (n = 3) successful creation of microgravity conditions (Fig. 1). L6 cells analysing biological and molecular mechanisms under micro- gravity conditions. cultured in differentiation medium under 1 G conditions normally To clarify the molecular mechanisms of decreased expression of differentiated to myotubes in an incubation period-dependent Myod1 and Myog, and their signiﬁcance in myogenesis under manner. However, myogenesis of L6 cells was signiﬁcantly microgravity conditions, L6 cells were ﬁrst treated with various attenuated under microgravity conditions from Day3 to Day7, doses of DNA methylation inhibitor (5-AzaC) for various incubation although myotubes gradually increased, indicating a retardation periods to optimize experimental conditions. As expected, Myod1 of myogenesis under microgravity conditions (Fig. 1). Since expression increased in a concentration- or a treatment period- myogenesis is regulated by the sequential activation of MRFs, dependent manner, although treatment with 10 μΜ of 5-AzaC expression levels of myogenesis-related genes were evaluated by showed slightly toxic effects. We thus decided to try treatment real-time RT-PCR. Results showed expressions of Myod1, Myog, with 5 μΜ of 5-AzaC for 12 days as an effective condition for the Myf6, Mef2c, Des, and Ckm under 1 G conditions were increased in following experiments. Real-time RT-PCR showed that expression an incubation period-dependent manner (Fig. 2). Among them, levels of Myod1 and Myog seemed to increase gradually in a Myod1 expression under 1 G conditions showed a unique transient treatment period-dependent manner, but the transient increase of increase in the early phase (from 12 h to Day1), suggesting Myod1 in the early phase, seen in Fig. 2, disappeared. And initiation of the myogenic process. And expressions of Myod1,as differences in expression levels of Myod1 and Myog between 1 G −3 well as Myog were strongly inhibited under microgravity condi- and 10 G conditions also disappeared. Furthermore, differences tions, suggesting that those inhibitions might have directly in myotube thickness between 1 G and microgravity conditions affected initiation of myogenesis as in Fig. 1. These signiﬁcant completely disappeared in this experimental condition, suggest- results suggest that our experimental model would be useful in ing that modifying DNA methylation status is essential to regulate Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 11 Simulated microgravity attenuates myogenic T Furukawa et al. suggesting the inhibitors’ potential as a molecular targeting therapy for muscle atrophy and weakness. Although this study mainly focused on biological viewpoints, further analyses includ- ing the inﬂuence of external physico-chemical parameters will probably provide new insight into the molecular mechanisms of myogenesis, and also the development of molecular targeting therapy for age-related skeletal muscle dysfunction in the clinical phase. MATERIALS AND METHODS Cell culture Rat myoblast cells, L6 cells, were obtained from Health Science Research Resources Bank (Osaka, Japan) and maintained with Dulbecco’s modiﬁed Eagle’s minimal essential medium-High glucose (DMEM-H) (NACALAI TESQUE, Inc., Kyoto, Japan) containing 10% fetal bovine serum (FBS; Fig. 5 Hypothetical model of regulation of myogenesis. Differentia- BioWhittaker, Verviers, Belgium), 100 U/ml penicillin, and 0.1 mg/ml tion signals regulate DNA methylation machinery, and especially, streptomycin (Sigma, St. Louis, MO, USA) as previously described. Cells the Myod1 gene in myoblast cells, resulting in sequential activation were cultured with growth medium for 2 days, and then replaced with of target genes and consecutive myogenic differentiation. Simu- differentiation medium containing DMEM-H, 2% FBS, 100 U/ml penicillin, lated microgravity attenuated this myogenesis by controlling DNA and 0.1 mg/ml streptomycin after reaching sub-conﬂuence. Cells were -3 ® methylation status of Myod1. This signal pathway was also then cultured under normal 1 G or 10 G conditions, using “Gravite ” controlled using DNA methylation inhibitor, 5-AzaC, treatment (Space Bio-Laboratories Co., Ltd., Hiroshima, Japan). For expression analysis, cells were cultured under normal 1 G (group 1 G) -3 −3 or 10 G conditions (group 10 G) for 0, 12 h, 1, 3, 5, or 7 days. Cells were myogenesis and expression of Myod1 and Myog under micro- then harvested and stored at −80˚C until use. L6 cells were also cultured with 5 μM of 5-Azacytidine (5-AzaC, Wako Pure Chemical Industries, Ltd., gravity conditions (Fig. 3d,e). These results suggest that DNA Osaka, Japan) in growth medium for 12 days, with passage every 4 days. methylation is a key factor in gravity-regulating myogenesis, and Cells were then seeded on 12.5 cm culture ﬂasks and cultured in growth possibly a molecular target for treatment of skeletal muscle medium for 2 days. After medium replacement with differentiation dysfunctions and Sarcopenia. −3 medium, cells were cultured in normal 1 G or 10 G conditions. UCSC Genome Browser indicated that MYOD1 gene virtually Morphological observations and harvesting were performed out at 0, overlapped with CpG island, indicating very dense CpG sites, and 12 h, 1, 3, 5, and 7 days, and stored at −80 °C until use. Methods were the proximal promoter region actually contained methylated sites performed in accordance with relevant regulations and guideline. (Fig. 4a). In fact, our modiﬁed HELP-assay showed that methylated Myod1 promoter had gradually decreased (PCR product Gravite decreased) until Day3, and then increased until Day7. These Microgravity conditions can be produced either by space ﬂight or by free kinetic changes in DNA methylation status apparently contributed fall; to simulate microgravity, we used a newly developed Gravite® (Space to increased expression of Myod1 in the initiation process of Bio-Laboratories Co., Ltd.), as previously patented (undifferentiated myogenesis. Furthermore, these kinetic changes in DNA methyla- pluripotent stem cell proliferation/differentiation regulation method and tion status were attenuated under microgravity conditions, system, Patent No. 8034616B2 (US), 2515552 (CA), 1577380 (EPC: GB, FR, indicating that microgravity regulated both myogenesis and DE, IT, SE), ZL02830112.9 (CN), and O731940 (KR), and GRAVITY Myod1 expression by controlling DNA methylation status. The CONTROLLER, Patent No. 623009 (JP), US9494949B2 (US) and granted in mRNA expressions of Dnmt1 and Dnmt3a gave further evidence EU). This device produces an environment similar to that of outer space −3 that expression levels in DNA methyltransferases might contribute (10 G) by rotating a sample around two axes, integrating the gravity vector with the temporal axis. This is accomplished by rotation of a to differences in DNA methylation status under microgravity chamber at the center of the device, resulting in uniform dispersion of the conditions. gravity vector within a spherical volume, with a constant angular velocity. Singh et al. reported that simulated microgravity increased −3 These speciﬁc conditions produced a simulated environment of 10 Gin expression levels of DNMT1, DNMT3a, and DNMT3b at 72 h, and 8 minutes actually measured by gravity acceleration sensor, and it was reduced them at 7 days in human T-lymphocyte cells. Also, the −3 33–35 deﬁned as simulated microgravity (10 G). methylation sensitive-random ampliﬁed polymorphic DNA (MS- RAPD) analysis revealed that simulated microgravity exposure Analysis of myogenesis resulted in DNA hypomethylation and mutational changes. In To evaluate myogenesis, cells were observed by taking pictures of 10 ﬁelds our study, with similar kinetics, increased levels of Dnmt1 and using inverted phase contrast microscope (Eclipse, Nikon, Japan) after Dnmt3a at 1-5 days and decreased levels at 7 days were observed, −3 incubation under 1 G or 10 G conditions for indicated periods. The but DNA methylation status should have opposite effects: maximum transverse diameters of myotube cells were measured by image Hypermethylation resulted in decreased expression of Myod1 processing software ImageJ, and the mean value of 10 ﬁelds was and retardation of myogenesis. Effects of Tdg kinetics were not calculated. consistent with the DNA methylation status. These differences might be due to differences in cell type, culture system, method of RNA preparation and real-time RT-PCR methylation detections, or target gene. Although our ﬁndings Total RNA was extracted from frozen cell pellets using the NucleoSpin seem consistent, further investigation will be necessary to clarify RNA II kit (MACHEREY-NAGEL, Düren, Germany) according to manufacturer the results. Other experiments to evaluate promoter activities and instructions. Two micrograms of total RNA extracted from each cell line mRNA stabilities have also supported the importance of epige- were reverse-transcribed using the High-Capacity cDNA Archive™ Kit netic regulation of Myod1 in myogenesis and MRF expression (Applied Biosystems, Foster City, CA, USA). A 1/200 dilution of the cDNA under altered gravity conditions. was subjected to real-time RT-PCR using primers (ﬁnal concentration of In conclusion, we demonstrated that simulated microgravity 200 nM each) and MGB probe (ﬁnal concentration of 100 nM, the Universal attenuated myogenesis by controlling DNA methylation status of Probe Library: UPL, Roche Diagnostics, Tokyo, Japan) (shown in S1 Table) Myod1 (Fig. 5). It is notable that a DNA methylation inhibitor sets with FastStart Universal Probe Master (ROX) (Roche Diagnostics) for attenuates the inhibitory effects of microgravity on myogenesis, quantitation of gene expressions with Actb as an internal housekeeping npj Microgravity (2018) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity attenuates myogenic T Furukawa et al. control. PCR reactions were carried out using 7500 Real-Time PCR System T.F., T.I., Y.K.; Project administration: K.T.; Resources: K.T., Y.K., L.Y.; Supervision: K.T. L.Y.; (Applied Biosystems) under the following standard conditions: Three Writing–original draft: T.F., K.T., T.F., T.I.; Writing–review and editing: K.T. and L.Y. independent measurements were averaged and relative gene expression levels were calculated as a ratio against Actb expression for each experiment. ADDITIONAL INFORMATION Supplementary information accompanies the paper on the npj Microgravity website (https://doi.org/10.1038/s41526-018-0045-0). DNA extraction and methylation analysis Genomic DNA was isolated from frozen cell pellets using the NucleoSpin Competing interests: L.Y. is a director of Space Bio-laboratories Co., Ltd. (SBL) and K. Tissue (MACHEREY-NAGEL) according to manufacturer instructions. Fifty ng Y. is a president of SBL. They share holding. The interest conﬂicts of this research have of genomic DNA in total 50 μl reaction mixture were digested with been approved by the Conﬂict of Interest Management Committee. By regularly methylation-sensitive HpaII at 37 °C for 12 h, and 2 μl of reaction mixture reporting of the research progress to the Conﬂicts of Interest Management were then subjected to real-time PCR using primer set (shown in S2 Table) Committee, we will maintain fairness regarding the interests of this research. The to amplify a fragment including CpG sites in Myod1 promoter region with remaining authors declare no competing interests. iTaq SYBR™ Green Supermix with ROX (BIO-RAD, Hercules, CA, USA). PCR reactions were carried out using 7500 Real-Time PCR System (Applied Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims Biosystems) under the following standard conditions: Three independent in published maps and institutional afﬁliations. measurements were averaged and relative gene expression levels were calculated as a ratio to sample DNA at Set0. REFERENCES Plasmid construction and luciferase reporter experiments 1. Metter, E. J., Conwit, R., Tobin, J. & Fozard, J. L. age-associated loss of power and strength in the upper extremities in women and men. J. Gerontol. Biol. Sci. 52, The 1.47 kb DNA fragment of the Myod1 gene (−1264 to +210 from the 267–276 (1997). transcriptional start site at +1) or the 1.55 kb DNA fragment of the Myog 2. Iolascon, G. et al. 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MyoD converts primary dermal ﬁbroblasts, chondroblasts, smooth Myod1, Myog, and Actb were analysed using real-time RT-PCR method as muscle, and retinal pigmented epithelial cells into striated mononucleated mentioned above. myoblasts and multinucleated myotubes (myogenesis/myoﬂbrils/desmin/master switch genes). Dev. Biol. 87, 7988–7992 (1990). 15. Chandran, R., Knobloch, T. J., Anghelina, M. & Agarwal, S. Biomechanical signals Statistical analysis upregulate myogenic gene induction in the presence or absence of inﬂamma- All statistical tests were performed using the StatView version 5.0 software tion. Am. J. Physiol. Cell Physiol. 293, 267–276 (2007). ® ® (SAS Institute Inc., NC, USA) and Microsoft Excel for Mac version 12.3.6. 16. Yuge, L. & Kataoka, K. Differentiation of myoblasts is accelerated in culture in a Student’s t-test was used to determine P-values (*P < 0.05, **P < 0.01). magnetic ﬁeld. Vitr. Cell. Dev. Biol. -Anim. 36, 383–386 (2000). 17. Kawahara, Y. et al. Novel electrical stimulation sets the cultured myoblast con- tractile function to ‘on’. Pathobiology 73, 288–294 (2006). Data availability 18. Minami, K. et al. Highly ordered 1D fullerene crystals for concurrent control of All relevant data are available from the corresponding author. macroscopic cellular orientation and differentiation toward large-scale tissue engineering. Adv. Mater. 27, 4020–4026 (2015). 19. Torgan, C. E., Burge, I. S. S., Collinsworth, I. A. M., Truskey, G. A. & Kraus, W. E. ACKNOWLEDGEMENTS Differentiation of mammalian skeletal muscle cells cultured on microcarrier We thank Prof. N. Hirohashi for his encouragement and Ms. C. Oda for her technical beads in a rotating cell culture system. Cell. Eng. 38, 583–590 (2000). support. A part of this work was carried out at the Analysis Center of Life Science at 20. Kulesh, D. A. et al. Space shuttle ﬂight (STS-45) of L8 myoblast cells results in the Hiroshima University. isolation of a nonfusing cell line variant. J. Cell Biochem 55, 530–544 (1994). 21. Clement, G. Musculo-skeletal system in space. Fundamentals of space medicine. (Kluwer Academic Publisher, Dordrecht, Netherlands, 2003). 22. Laker, R. C. & Ryall, J. G. DNA methylation in skeletal muscle stem cell speciﬁ- AUTHOR CONTRIBUTIONS cation, proliferation, and differentiation. Stem Cells Int. 2016, 5725927 (2016). Conceptualization: K.T., L.Y.; Data curation: T.F., K.T., T.F.; Formal analysis: T.F., K.T., T.F.; Funding acquisition: K.T., Y.K., L.Y.; Investigation: T.F., K.T., T.F.; Methodology: T.F., K.T., Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 11 Simulated microgravity attenuates myogenic T Furukawa et al. 23. Montesano, A., Luzi, L., Senesi, P. & Terruzzi, I. Modulation of Cell Cycle Pro- 33. Mitsuhara, T. et al. Simulated microgravity facilitates cell migration and neuro- gression by 5-Azacytidine Is Associated with Early Myogenesis Induction in protection after bone marrow stromal cell transplantation in spinal cord injury. Murine Myoblasts. Int J. Biol. Sci. 9, 391–402 (2013). Stem Cell Res. Ther. 4, 35 (2013). 24. Zhang, F., Pomerantz, J. H., Sen, G., Palermo, A. T. & Blau, H. M. Active tissue- 34. Kawahara, Y., Manabe, T., Matsumoto, M., Kajiume, T. & Yuge, L. LIF-free speciﬁc DNA demethylation conferred by somatic cell nuclei in stable hetero- embryonic stem cell culture in simulated microgravity. PLoS One 4, e6343 (2009). karyons. Proc. Natl Acad. Sci. 104, 4395–4400 (2007). 35. Yuge, L. et al. Simulated microgravity maintains the undifferentiated state and 25. Taylor, S. M. & Jones, P. A. Multiple new phenotypes induced cells treated with enhances the neural repair potential of bone marrow stromal cells. Stem Cells SAzacytidine in 10T 1/2 and 3T3 cells treated with 5-Azacytidine. Cell 17, 771–779 Dev. 20, 893–900 (2011). (1979). 26. Barrès, R. et al. Acute exercise remodels promoter methylation in human skeletal muscle. Cell Metab. 15, 405–411 (2012). Open Access This article is licensed under a Creative Commons 27. Singh, K. P., Kumari, R. & DuMond, J. W. Simulated microgravity-induced epige- Attribution 4.0 International License, which permits use, sharing, netic changes in human lymphocytes. J. Cell. Biochem. 111, 123–129 (2010). adaptation, distribution and reproduction in any medium or format, as long as you give 28. Zhong, X. et al. Ten-Eleven Translocation-2 (Tet2) is involved in myogenic dif- appropriate credit to the original author(s) and the source, provide a link to the Creative ferentiation of skeletal myoblast cells in vitro. Sci. Rep. 8, 43539 (2017). Commons license, and indicate if changes were made. The images or other third party 29. Khulan, B. et al. Comparative isoschizomer proﬁling of cytosine methylation: The material in this article are included in the article’s Creative Commons license, unless HELP assay. Genome Res. 16, 1046–1055 (2006). indicated otherwise in a credit line to the material. If material is not included in the 30. Greenlund, L. J. S. & Nair, K. S. Sarcopenia–consequences, mechanisms, and article’s Creative Commons license and your intended use is not permitted by statutory potential therapies. Mech. Ageing Dev. 124, 287–299 (2003). regulation or exceeds the permitted use, you will need to obtain permission directly 31. Cruz-Jentoft, A. J. et al. Sarcopenia: European consensus on deﬁnition and from the copyright holder. To view a copy of this license, visit http://creativecommons. diagnosis Report of the European Working Group on Sarcopenia in Older People. org/licenses/by/4.0/. Age Ageing 39, 412–423 (2010). 32. Cooper, C. et al. Frailty and sarcopenia: deﬁnitions and outcome parameters. © The Author(s) 2018 Osteoporos. Int. 23, 1839–1848 (2012). npj Microgravity (2018) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Microgravity Springer Journals http://www.deepdyve.com/lp/springer-journals/simulated-microgravity-attenuates-myogenic-differentiation-via-2YMewUgDyn

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Publisher: Springer Journals
Copyright: Copyright © 2018 by The Author(s)
Subject: Life Sciences; Life Sciences, general; Classical and Continuum Physics; Biotechnology; Immunology; Space Sciences (including Extraterrestrial Physics, Space Exploration and Astronautics) ; Applied Microbiology
eISSN: 2373-8065
DOI: 10.1038/s41526-018-0045-0
Publisher site: See Article on Publisher Site

Abstract

www.nature.com/npjmgrav ARTICLE OPEN Simulated microgravity attenuates myogenic differentiation via epigenetic regulations 1 2 3 1 4 1,4 Takuma Furukawa , Keiji Tanimoto , Takahiro Fukazawa , Takeshi Imura , Yumi Kawahara and Louis Yuge The molecular mechanisms involved in myogenic differentiation are relatively well-known. Myogenic differentiation is regulated by the sequential activation of the basic helix-loop-helix myogenic regulatory transcription factors (MRFs), and biomechanical signals play an important role in the regulation of myogenesis. In this study, we sought to determine whether simulated microgravity culture using Gravite may affect myoblast differentiation and expression of MRF genes. Although rat myoblasts, L6 cells were differentiated to myotubes in an incubation period-dependent manner, myogenesis of L6 cells was signiﬁcantly attenuated under -3 simulated microgravity (10 G) conditions. Real-time Reverse transcription polymerase chain reaction (RT-PCR) showed that expressions of Myog, Myf6, Mef2c, Des, and Ckm under 1 G conditions increase in an incubation period-dependent manner, and that Myod1 expression was speciﬁcally observed to increase transiently in the early phase. However, expressions of Myod1 and Myog were signiﬁcantly inhibited under simulated microgravity conditions. To clarify the molecular mechanisms, L6 cells were treated −3 with 5-AzaC, and further incubated with differentiation medium under 1 G or 10 G conditions. The results showed differences in −3 expression levels of Myod1, Myog, and, as well as those of myotube thickness between 1 G and 10 G conditions, completely disappeared in this experimental condition. Modiﬁed HpaII tiny fragment enrichment by ligation-mediated PCR (HELP)-assay showed that kinetic changes of DNA methylation status were attenuated in simulated microgravity conditions. These results indicate that microgravity regulates myogenesis and Myod1 expression by controlling DNA methylation. npj Microgravity (2018) 4:11 ; doi:10.1038/s41526-018-0045-0 INTRODUCTION It is signiﬁcant that the muscle mass of astronauts after 2 weeks space ﬂight was diminished by up to 20%. Sarcopenia is deﬁned as an age-related loss of skeletal muscle DNA methylation is also known to be involved in regulation of mass and strength. Beginning with the 4th decade of life, and myogenic gene expression and myogenesis: The DNA- symptoms progress with age. If muscle mass accounts for up to demethylating agent 5-azacytidine induced myogenesis via 60% of body weight, pathological changes in skeletal muscle can 22–25 upregulated MyoD expression. Barrès et al. interestingly cause serious effects on older adults. Nevertheless, the therapeutic reported that acute exercise tends to reduce global methylation outcome of age-related skeletal muscle atrophy and weakness on the entire genome, resulting in activation of regulated genes in 2–4 remains unknown. The understanding of molecular mechan- 26 human skeletal muscle. Singh et al. demonstrated that simulated isms of myogenic differentiation process will result in better microgravity induced epigenetic changes of genome DNA in treatment outcomes, since impaired regulation of myogenic human lymphocytes through altered expression of DNMT1, differentiation is closely associated with age-related skeletal DNMT3a, and DNMT3b. However, the effects of space ﬂight or 5–8 muscle dysfunction. It is well known that myogenic differentia- simulated microgravity on epigenetic changes of MRF genes and tion is regulated by the sequential activation of the basic helix- resulting myogenesis remain unclear. Therefore, this study aims to loop-helix myogenic regulatory transcription factors (MRFs): MyoD, determine whether simulated microgravity affects myoblast 9–11 Myf5, myogenin, and MRF4 (Myf6). MyoD in particular, is differentiation, expression of MRF genes, and the status of DNA 12–14 involved in the commitment of cells to the myogenic lineage. methylation. It has been reported that exposure of myoblast or muscle satellite cells to mechanical uniaxial stretch and stretching by a magnetic ﬁeld, or electrical stimulation, upregulate MRFs expression, RESULTS 15–17 resulting in activation of myogenesis. Moreover, differentia- Morphological evaluation of myogenesis under microgravity tion of myoblasts was enhanced on highly aligned fullerene conditions whiskers scaffolds culture. In contrast, differentiation of myo- To evaluate myogenesis under simulated microgravity conditions, blast was suppressed in the microgravity culture for in vitro study L6 cells were cultured with differentiation medium under normal and spaceﬂight experiments, suggesting that biomechanical -3 1 G or microgravity (10 G) conditions for 1, 3, 5, or 7 days (Fig. 1a). 19,20 signals play an important role in the regulation of myogenesis. Cells were then observed by capturing images, and the maximum 1 2 Division of Bio-Environmental Adaptation Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan; Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan and Space Bio-Laboratories Co., Ltd, Hiroshima, Japan Correspondence: Keiji Tanimoto (ktanimo@hiroshima-u.ac.jp) or Louis Yuge (ryuge@hiroshima-u.ac.jp) Received: 27 September 2017 Revised: 18 April 2018 Accepted: 23 April 2018 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity attenuates myogenic T Furukawa et al. Fig. 1 Morphological evaluation of myogenesis under microgravity conditions. a Schematic representation of experimental time course is shown. L6 cells were cultured with growth medium for 2 days, and then replaced with differentiation medium. Cells were then cultured under -3 normal 1 G or 10 G conditions for 0, 12 h, 1, 3, 5, or 7 days, and observed by taking pictures, and the maximum transverse diameters of myotube cells (myotube thickness) were measured. b Representative microscopic images during myogenesis are shown. White arrow heads: myotube cell; white H-shaped bars: examples of the maximum transverse diameter of myotube cells; white scale bar: 300 μm. c The maximum transverse diameters of myotube cells were measured by image processing software ImageJ, and the mean value of 10 ﬁelds was calculated. Columns show the mean of three independent experiments; bars, SD. P values calculated with Student’s t test. (**P < 0.01) (n = 3) transverse diameters of myotube cells (myotube thickness) were Although transient increases of Myod1 expression under 1 G measured as indicated in Fig. 1b. As expected, L6 cells were conditions were observed from 12 h to Day1, those increases were differentiated to myotubes, resulting in an increase in the signiﬁcantly inhibited in microgravity conditions. Myog expres- thickness, under 1 G conditions in an incubation period- sions were also signiﬁcantly inhibited in microgravity conditions at dependent manner (Fig. 1c). Interestingly, myogenesis was the same points of time. Expression levels of Pax3, Pax7, and Myf5 signiﬁcantly attenuated under microgravity conditions on day 3, did not change under our study conditions. 5, and 7, although myotubes gradually increased (Fig. 1c). Effects of 5-Azacytidine treatment on myogenesis under Gene expression analyses of myogenesis-relating genes under microgravity conditions microgravity conditions Expression of Myod1 and Myog had previously been reported to 23,25,28 In order to clarify the molecular mechanisms of attenuation of be regulated by DNA methylation. In order to clarify the myogenesis under microgravity conditions, expression levels of molecular mechanisms of the decreased expression of Myod1 and myogenesis-related genes were evaluated using real-time RT-PCR Myog, and the signiﬁcance in myogenesis under microgravity (Fig. 2): Expression of Myog, Myf6, Mef2c, Des, and Ckm under 1 G conditions, L6 cells were then treated with DNA methylation conditions increased in an incubation period-dependent manner. inhibitor, 5-Azacytidine (5-AzaC), before incubation with npj Microgravity (2018) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; Simulated microgravity attenuates myogenic T Furukawa et al. Fig. 2 Gene expression analyses of myogenesis-related genes under microgravity conditions. For expression analysis, L6 cells were cultured -3 under normal 1 G or 10 G conditions for 0, 12 h, 1, 3, 5, or 7 days. Total RNA was extracted from cell pellets, and expression levels of myogenesis-relating genes were evaluated by real-time RT-PCR. Three independent measurements were averaged, and relative gene expression levels were calculated as a ratio against Actb expression for each experiment. Columns show the mean of three independent experiments; bars, SD. P values calculated with Student’s t test. (*P < 0.05, **P < 0.01) (n = 3) differentiation medium (Fig. 3a). To optimize concentration and HpaII sites (Fig. 4b). Since DNA methylation sensitive restriction treatment periods of 5-AzaC, L6 cells were ﬁrst treated with 1, 2, 5, enzyme HpaII cannot digest methylated DNA, but/although can or 10 μΜ of 5-AzaC for 4, 8, or 12 days. As a result, Myod1 digest unmethylated DNA, the primer set ampliﬁed methylated expression was observed to increase in a concentration- or DNA, but not unmethylated or digested DNA after incubation with treatment period-dependent manner (Fig. 3b). Of these condi- HpaII. Therefore, methylated DNA can be quantiﬁed by real-time tions, treatment with 5 μΜ of 5-AzaC for 12 days was conﬁrmed as PCR, resulting in evaluation of DNA methylation status. Genomic the effective condition for the following experiments. After DNAs were then isolated from L6 cell samples that had been treatment with 5 μΜ of 5-AzaC for 12 days, L6 cells were cultured as in Fig. 1, and incubated with HpaII for 12 h. Real-time incubated with growth medium for 2 days, and also with PCR demonstrated that relative amounts of PCR products differentiation medium for the indicated periods under 1 G or ampliﬁed from 1 G samples decreased gradually until Day3, and microgravity conditions. Real-time RT-PCR showed that the then increased until Day7 (Fig. 4c). It is striking that PCR products expression levels of Myod1 and Myog seemed to increase from microgravity samples also slightly decreased but to a smaller gradually in a treatment period-dependent manner, but the degree than those of 1 G, suggesting a retaining DNA methylation −3 difference between 1 G and 10 G conditions observed in Fig. 2, on Myod1 promoter (Fig. 4c). In order to clarify the underlying and disappeared in Fig. 3c. In this experimental condition, mechanisms, real-time RT-PCR analyses of methylation-related morphological evaluations were also performed as in Fig. 1. genes were performed using the same samples of cDNA as in Fig. Results showed that differences in myotube thickness between 2. Results showed that expressions of DNA methyltransferase, 1 G and microgravity conditions also completely disappeared, Dnmt1 and Dnmt3a, under 1 G conditions were decreased during although L6 cells were gradually differentiated to myotubes under the myogenic process (Fig. 4d), while Dnmt3b was not detected both conditions (Fig. 3d,e). (data not shown). Interestingly, expressions of Dnmt1 and Dnmt3a under microgravity conditions also decreased during the myo- genic process, but remained signiﬁcantly higher than those under DNA methylation in Myod1 gene under microgravity conditions 1 G conditions, suggesting they retained functions of methyl- To determine whether DNA methylation actually contributes to transferases (Fig. 4d). Expression levels of Tdg also decreased regulation of Myod1 expression, we use modiﬁed HELP-assay to during the myogenic process, but differences between 1 G and detect DNA methylation in the promoter region of Myod1 gene. microgravity conditions were smaller. Expression levels of Mbd2 UCSC Genome Browser (http://genome.ucsc.edu) showed that varied and there was no trend observed. MYOD1 gene virtually overlapped with CpG island, indicating very dense CpG sites, and the proximal promoter region contained Other regulation of Myod1 and Myog genes under microgravity actually methylated as shown in the results of reduced conditions representation bisulﬁte sequencing and methyl 450 K bead arrays (Fig. 4a). A primer set was designed for just upstream from the In order to clarify regulation mechanisms of expression of Myod1 transcription start site of Myod1, which contained 12 CpG or 3 and Myog genes during the myogenic process under 1 G and Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 11 Simulated microgravity attenuates myogenic T Furukawa et al. Fig. 3 Effects of 5-Azacytidine treatment on myogenesis under microgravity conditions. a Schematic representation of experimental time course is shown. L6 cells were cultured with 5 μM of 5-AzaC in growth medium for 12 days, with passage every 4 days. Cells were then seeded on culture ﬂasks and cultured in growth medium for 2 days. After medium replacement with differentiation medium, cells were cultured in −3 normal 1 G or 10 G conditions for 0, 12 h, 1, 3, 5, and 7 days. b Optimization of 5-azaC treatment was performed. L6 cells were treated with 1, 2, 5, or 10 μM of 5-AzaC for 4, 8, or 12 days, and expression levels of Myod1 gene were evaluated by real-time RT-PCR. Three independent measurements were averaged and relative gene expression levels were calculated as a ratio against Actb expression for each experiment. Columns show the mean of three independent experiments; bars, SD (n= 3). c Expression levels of Myod1 and Myog genes were evaluated by real-time RT-PCR. Three independent measurements were averaged and relative gene expression levels were calculated as a ratio against Actb expression for each experiment. Columns show the mean of three independent experiments; bars, SD (n = 3). d Representative microscopic images during myogenesis are also shown. White arrow heads: myotube cell; white H-shaped bars: examples of the maximum transverse diameter of myotube cells; white scale bar: 300 μm. e The maximum transverse diameters of myotube cells were measured using image processing software ImageJ, and the mean value of 10 ﬁelds was calculated. Columns show the mean of three independent experiments; bars, SD. (n = 3) microgravity conditions, further analyses of promoter activities were no differences in those levels under 1 G or microgravity and RNA stabilities were performed. First, the 1.47 kb DNA conditions (Supplementary Fig 2B). fragment of the Myod1 gene promoter region, or the 1.55 kb DNA fragment of the Myog gene promoter region, was ampliﬁed DISCUSSION and subcloned into the luciferase reporter plasmid pGL4.16 As a consequence of an aging population, the number of elderly (designated pGL4.16-Myod1 Pro1474 or pGL4.16-Myog Pro1546, who exhibit skeletal muscle atrophy and weakness are increasing, Supplementary Fig 1B). L6 cells were then transfected with these 30–32 becoming a concern of society. In order to achieve better promoter reporters and incubated for 1 day. Next, growth medium treatment outcomes, the understanding of the molecular was replaced with differentiation medium and incubated for one mechanisms of myogenesis is essential, since impaired myogen- more day under 1 G or microgravity conditions (Supplementary esis is closely associated with age-related skeletal muscle loss and Fig 1A). Dual-luciferase assays demonstrated that cloned regions 5–8 weakness. Previous reports have suggested that biomechanical had strong promoter activities compared to empty vector, but any signals play an important role in the regulation of myogenesis. differences in those activities under 1 G or microgravity conditions Speciﬁcally, exposure of myoblast and muscle satellite cells to were not observed (Supplementary Fig 1C). L6 cells were next mechanical stretching, electrical stimulation, or microgravity have cultured in growth medium for 2 days, and then cultured in been found to modify expression levels of MRFs, resulting in 15–17,19 differentiation medium for further a day. After adding 5 μg/ml of alteration of myogenesis. actinomycin D, cells were cultured under 1 G or microgravity In this study, we used our new generation 3D-clinostat ® -3 conditions (Supplementary Fig 2A). Real-time RT-PCR showed that “Gravite ” to obtain simulated microgravity conditions (10 G) for gene expression level of Myod1 had decreased gradually, while cell culture: We observed that myogenesis of rat myoblast cells (L6 those of Myog and Actb had not (Supplementary Fig 2B). There cells) cultured in Gravite were signiﬁcantly attenuated, indicating npj Microgravity (2018) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity attenuates myogenic T Furukawa et al. Fig. 4 DNA methylation in Myod1 gene under microgravity conditions. a Schematic structure of MYOD1 gene in UCSC Genome Browser showed that MYOD1 gene virtually overlapped with CpG island (upper rectangle drawn in red dotted line), indicating very dense CpG sites, and the proximal promoter region contained actually methylated sites (lower rectangle drawn in red dotted line). b The nucleic acid sequence of proximal promoter region in Myod1 gene is shown. Blue circle and arrow indicate transcription start site of Myod1 gene, red rectangles, CpG sites, blue rectangles, HpaII sites, and red arrows, primer sets for modiﬁed HELP-assay. c Methylated proximal promoter regions in Myod1 gene are quantiﬁed by real-time PCR. Three independent measurements were averaged and relative amounts of PCR products were calculated as the ratio against that of Set0 for each experiment. (n = 3). d Expression levels of methylation related genes were evaluated by real-time RT- PCR. Three independent measurements were averaged and relative gene expression levels were calculated as a ratio against Actb expression for each experiment. Columns show the mean of three independent experiments; bars, SD. P values calculated with Student’s t test. (*P < 0.05, **P < 0.01) (n = 3) successful creation of microgravity conditions (Fig. 1). L6 cells analysing biological and molecular mechanisms under micro- gravity conditions. cultured in differentiation medium under 1 G conditions normally To clarify the molecular mechanisms of decreased expression of differentiated to myotubes in an incubation period-dependent Myod1 and Myog, and their signiﬁcance in myogenesis under manner. However, myogenesis of L6 cells was signiﬁcantly microgravity conditions, L6 cells were ﬁrst treated with various attenuated under microgravity conditions from Day3 to Day7, doses of DNA methylation inhibitor (5-AzaC) for various incubation although myotubes gradually increased, indicating a retardation periods to optimize experimental conditions. As expected, Myod1 of myogenesis under microgravity conditions (Fig. 1). Since expression increased in a concentration- or a treatment period- myogenesis is regulated by the sequential activation of MRFs, dependent manner, although treatment with 10 μΜ of 5-AzaC expression levels of myogenesis-related genes were evaluated by showed slightly toxic effects. We thus decided to try treatment real-time RT-PCR. Results showed expressions of Myod1, Myog, with 5 μΜ of 5-AzaC for 12 days as an effective condition for the Myf6, Mef2c, Des, and Ckm under 1 G conditions were increased in following experiments. Real-time RT-PCR showed that expression an incubation period-dependent manner (Fig. 2). Among them, levels of Myod1 and Myog seemed to increase gradually in a Myod1 expression under 1 G conditions showed a unique transient treatment period-dependent manner, but the transient increase of increase in the early phase (from 12 h to Day1), suggesting Myod1 in the early phase, seen in Fig. 2, disappeared. And initiation of the myogenic process. And expressions of Myod1,as differences in expression levels of Myod1 and Myog between 1 G −3 well as Myog were strongly inhibited under microgravity condi- and 10 G conditions also disappeared. Furthermore, differences tions, suggesting that those inhibitions might have directly in myotube thickness between 1 G and microgravity conditions affected initiation of myogenesis as in Fig. 1. These signiﬁcant completely disappeared in this experimental condition, suggest- results suggest that our experimental model would be useful in ing that modifying DNA methylation status is essential to regulate Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 11 Simulated microgravity attenuates myogenic T Furukawa et al. suggesting the inhibitors’ potential as a molecular targeting therapy for muscle atrophy and weakness. Although this study mainly focused on biological viewpoints, further analyses includ- ing the inﬂuence of external physico-chemical parameters will probably provide new insight into the molecular mechanisms of myogenesis, and also the development of molecular targeting therapy for age-related skeletal muscle dysfunction in the clinical phase. MATERIALS AND METHODS Cell culture Rat myoblast cells, L6 cells, were obtained from Health Science Research Resources Bank (Osaka, Japan) and maintained with Dulbecco’s modiﬁed Eagle’s minimal essential medium-High glucose (DMEM-H) (NACALAI TESQUE, Inc., Kyoto, Japan) containing 10% fetal bovine serum (FBS; Fig. 5 Hypothetical model of regulation of myogenesis. Differentia- BioWhittaker, Verviers, Belgium), 100 U/ml penicillin, and 0.1 mg/ml tion signals regulate DNA methylation machinery, and especially, streptomycin (Sigma, St. Louis, MO, USA) as previously described. Cells the Myod1 gene in myoblast cells, resulting in sequential activation were cultured with growth medium for 2 days, and then replaced with of target genes and consecutive myogenic differentiation. Simu- differentiation medium containing DMEM-H, 2% FBS, 100 U/ml penicillin, lated microgravity attenuated this myogenesis by controlling DNA and 0.1 mg/ml streptomycin after reaching sub-conﬂuence. Cells were -3 ® methylation status of Myod1. This signal pathway was also then cultured under normal 1 G or 10 G conditions, using “Gravite ” controlled using DNA methylation inhibitor, 5-AzaC, treatment (Space Bio-Laboratories Co., Ltd., Hiroshima, Japan). For expression analysis, cells were cultured under normal 1 G (group 1 G) -3 −3 or 10 G conditions (group 10 G) for 0, 12 h, 1, 3, 5, or 7 days. Cells were myogenesis and expression of Myod1 and Myog under micro- then harvested and stored at −80˚C until use. L6 cells were also cultured with 5 μM of 5-Azacytidine (5-AzaC, Wako Pure Chemical Industries, Ltd., gravity conditions (Fig. 3d,e). These results suggest that DNA Osaka, Japan) in growth medium for 12 days, with passage every 4 days. methylation is a key factor in gravity-regulating myogenesis, and Cells were then seeded on 12.5 cm culture ﬂasks and cultured in growth possibly a molecular target for treatment of skeletal muscle medium for 2 days. After medium replacement with differentiation dysfunctions and Sarcopenia. −3 medium, cells were cultured in normal 1 G or 10 G conditions. UCSC Genome Browser indicated that MYOD1 gene virtually Morphological observations and harvesting were performed out at 0, overlapped with CpG island, indicating very dense CpG sites, and 12 h, 1, 3, 5, and 7 days, and stored at −80 °C until use. Methods were the proximal promoter region actually contained methylated sites performed in accordance with relevant regulations and guideline. (Fig. 4a). In fact, our modiﬁed HELP-assay showed that methylated Myod1 promoter had gradually decreased (PCR product Gravite decreased) until Day3, and then increased until Day7. These Microgravity conditions can be produced either by space ﬂight or by free kinetic changes in DNA methylation status apparently contributed fall; to simulate microgravity, we used a newly developed Gravite® (Space to increased expression of Myod1 in the initiation process of Bio-Laboratories Co., Ltd.), as previously patented (undifferentiated myogenesis. Furthermore, these kinetic changes in DNA methyla- pluripotent stem cell proliferation/differentiation regulation method and tion status were attenuated under microgravity conditions, system, Patent No. 8034616B2 (US), 2515552 (CA), 1577380 (EPC: GB, FR, indicating that microgravity regulated both myogenesis and DE, IT, SE), ZL02830112.9 (CN), and O731940 (KR), and GRAVITY Myod1 expression by controlling DNA methylation status. The CONTROLLER, Patent No. 623009 (JP), US9494949B2 (US) and granted in mRNA expressions of Dnmt1 and Dnmt3a gave further evidence EU). This device produces an environment similar to that of outer space −3 that expression levels in DNA methyltransferases might contribute (10 G) by rotating a sample around two axes, integrating the gravity vector with the temporal axis. This is accomplished by rotation of a to differences in DNA methylation status under microgravity chamber at the center of the device, resulting in uniform dispersion of the conditions. gravity vector within a spherical volume, with a constant angular velocity. Singh et al. reported that simulated microgravity increased −3 These speciﬁc conditions produced a simulated environment of 10 Gin expression levels of DNMT1, DNMT3a, and DNMT3b at 72 h, and 8 minutes actually measured by gravity acceleration sensor, and it was reduced them at 7 days in human T-lymphocyte cells. Also, the −3 33–35 deﬁned as simulated microgravity (10 G). methylation sensitive-random ampliﬁed polymorphic DNA (MS- RAPD) analysis revealed that simulated microgravity exposure Analysis of myogenesis resulted in DNA hypomethylation and mutational changes. In To evaluate myogenesis, cells were observed by taking pictures of 10 ﬁelds our study, with similar kinetics, increased levels of Dnmt1 and using inverted phase contrast microscope (Eclipse, Nikon, Japan) after Dnmt3a at 1-5 days and decreased levels at 7 days were observed, −3 incubation under 1 G or 10 G conditions for indicated periods. The but DNA methylation status should have opposite effects: maximum transverse diameters of myotube cells were measured by image Hypermethylation resulted in decreased expression of Myod1 processing software ImageJ, and the mean value of 10 ﬁelds was and retardation of myogenesis. Effects of Tdg kinetics were not calculated. consistent with the DNA methylation status. These differences might be due to differences in cell type, culture system, method of RNA preparation and real-time RT-PCR methylation detections, or target gene. Although our ﬁndings Total RNA was extracted from frozen cell pellets using the NucleoSpin seem consistent, further investigation will be necessary to clarify RNA II kit (MACHEREY-NAGEL, Düren, Germany) according to manufacturer the results. Other experiments to evaluate promoter activities and instructions. Two micrograms of total RNA extracted from each cell line mRNA stabilities have also supported the importance of epige- were reverse-transcribed using the High-Capacity cDNA Archive™ Kit netic regulation of Myod1 in myogenesis and MRF expression (Applied Biosystems, Foster City, CA, USA). A 1/200 dilution of the cDNA under altered gravity conditions. was subjected to real-time RT-PCR using primers (ﬁnal concentration of In conclusion, we demonstrated that simulated microgravity 200 nM each) and MGB probe (ﬁnal concentration of 100 nM, the Universal attenuated myogenesis by controlling DNA methylation status of Probe Library: UPL, Roche Diagnostics, Tokyo, Japan) (shown in S1 Table) Myod1 (Fig. 5). It is notable that a DNA methylation inhibitor sets with FastStart Universal Probe Master (ROX) (Roche Diagnostics) for attenuates the inhibitory effects of microgravity on myogenesis, quantitation of gene expressions with Actb as an internal housekeeping npj Microgravity (2018) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity attenuates myogenic T Furukawa et al. control. PCR reactions were carried out using 7500 Real-Time PCR System T.F., T.I., Y.K.; Project administration: K.T.; Resources: K.T., Y.K., L.Y.; Supervision: K.T. L.Y.; (Applied Biosystems) under the following standard conditions: Three Writing–original draft: T.F., K.T., T.F., T.I.; Writing–review and editing: K.T. and L.Y. independent measurements were averaged and relative gene expression levels were calculated as a ratio against Actb expression for each experiment. ADDITIONAL INFORMATION Supplementary information accompanies the paper on the npj Microgravity website (https://doi.org/10.1038/s41526-018-0045-0). DNA extraction and methylation analysis Genomic DNA was isolated from frozen cell pellets using the NucleoSpin Competing interests: L.Y. is a director of Space Bio-laboratories Co., Ltd. (SBL) and K. Tissue (MACHEREY-NAGEL) according to manufacturer instructions. Fifty ng Y. is a president of SBL. They share holding. The interest conﬂicts of this research have of genomic DNA in total 50 μl reaction mixture were digested with been approved by the Conﬂict of Interest Management Committee. By regularly methylation-sensitive HpaII at 37 °C for 12 h, and 2 μl of reaction mixture reporting of the research progress to the Conﬂicts of Interest Management were then subjected to real-time PCR using primer set (shown in S2 Table) Committee, we will maintain fairness regarding the interests of this research. The to amplify a fragment including CpG sites in Myod1 promoter region with remaining authors declare no competing interests. iTaq SYBR™ Green Supermix with ROX (BIO-RAD, Hercules, CA, USA). 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Comparative isoschizomer proﬁling of cytosine methylation: The material in this article are included in the article’s Creative Commons license, unless HELP assay. Genome Res. 16, 1046–1055 (2006). indicated otherwise in a credit line to the material. If material is not included in the 30. Greenlund, L. J. S. & Nair, K. S. Sarcopenia–consequences, mechanisms, and article’s Creative Commons license and your intended use is not permitted by statutory potential therapies. Mech. Ageing Dev. 124, 287–299 (2003). regulation or exceeds the permitted use, you will need to obtain permission directly 31. Cruz-Jentoft, A. J. et al. Sarcopenia: European consensus on deﬁnition and from the copyright holder. To view a copy of this license, visit http://creativecommons. diagnosis Report of the European Working Group on Sarcopenia in Older People. org/licenses/by/4.0/. Age Ageing 39, 412–423 (2010). 32. Cooper, C. et al. Frailty and sarcopenia: deﬁnitions and outcome parameters. © The Author(s) 2018 Osteoporos. Int. 23, 1839–1848 (2012). npj Microgravity (2018) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

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