Expression of MyoD and m-Cadherin in C2C12 cells: Differentiation induced by cell-to-cell contact vs reduction in growth factors Bridgitte Garcia, Carita Chan, Emily Yap, Jessy Celine Santos, Muhammad Mohsin, Naomi Oviahon, Laura Atkinson Mount Royal University - Faculty of Science and Technology Research Day 2022 INTRODUCTION METHODS ● C2C12 cells derived from murine muscle tissue is commonly used to study myogenesis. ● Cell differentiation is part of myogenesis and can be induced through cell-to-cell contact or reduction of growth factors (GF) Project Rationale: There is wide variation in cell culture protocols across the literature which differ greatly from American Type Culture Collection (ATCC) recommendations, despite the specific effects behind each culture condition not being clearly defined. Research Question: How will differentiation be induced by cell-to-cell contact vs reduction of GF effect C2C12 cells? Biomarkers of myogenesis: ● MyoD is translocated to the nucleus to act as a master regulator and transcription factor of muscle-specific genes MB 80% confluency Control 10% FBS Day 0 100% confluency Figure 1. Process of myogenesis as illustrated by Zammit et al. (2006). Day 4 Day 7 10% HS at 100% confl. 2% HS at 80% confl. 2% HS at 100% confl. Fetal Bovine Serum (FBS) Horse Serum (HS) ● M-Cadherin is localized to the plasma membrane to mediate cell fusion prior to myotube formation Cell culture Sampling Samples were fixed at each sampling day of differentiation (MB, D0, D4 and D7) and permeabilized RESULTS RNA was isolated from cell pellets and assessed for quality and quantity Sample Preparation Fig 1. Phase contrast images of control myoblasts and experimental day 7 myotubes. The top panel is a myoblast plate grown in 10% FBS. The 3 bottom panels show day 7 myotubes grown in 2% HS at 80% confluency (largest myotubes, contraction observed), 2% HS at 100% confluency, and 10% HS at 100% confluency. All images were obtained using a mobile device at 100x magnification. The purpose of these images was to visualize the effects of cell-to-cell contact and reduction of growth factors on myotube formation. High Capacity cDNA RT kit Immunostaining Reverse Transcription (RT) to synthesize cDNA Visualization / Quantification Fluorescence Microscopy MyoD was observed and is a master transcriptional regulator of muscle-specific genes. M-Cadherin was observed and mediates cell fusion, allowing myotubes to form. qPCR and Pfaffl Primer Efficiencies m-Cadherin MyoD primer efficiency is 163% (E= 1.636) M-cadherin primer efficiency is 124% (E=1.24) Melt peak Fig 2. MyoD expression relative to GAPDH expression in control and experimental groups at myoblast, days 0, 4, and 7. Gene expression was measured using quantitative PCR and the Pfaffl method. The expression of MyoD was normalized to the expression of MyoD in MB. Control group had a n = 9, while all experimental groups had a n = 3, except for 2% HS at 80% confluency group had n=2. Mean +/- SEM: control: 0.944; 4.585 10%HS,100%con.: 0.787; 1.941 2% HS, 80% con.:0.826; 2.154 2% HS, 100% con.: 0.759; 2.040 Image from: https://www.thermofisher.com/ca/en/hom e/brands/thermo-scientific/molecular-biolo gy/molecular-biology-learning-center/molec ular-biology-resource-library/spotlight-articl es/basic-principles-rt-qpcr.html Average Cq over log dilution factor MyoD SYBR Green Supermix Both MyoD and m-Cadherin only had one melt peak, indicating that only one product was amplified. DISCUSSION & CONCLUSIONS Morphology Fig 3. M-Cadherin expression in relation to GAPDH expression in control and experimental groups at myoblast, days 0, 4, and 7. Gene expression was measured using quantitative PCR and the Pfaffl method. The expression of m-Cadherin was normalized to the expression of m-Cadherin in MB. Control group had n = 9 and all experimental groups had n = 3, except for 2% HS at 80% confluency group had n=2. Mean +/- SEM: control: 0.937; 4.212 10%HS,100%con.: 0.663; 1.742 2% HS, 80% con.: 0.780; 1.912 2% HS, 100% con.: 0.663; 1.742 ● Earlier induction (i.e. 80% confluency) enhanced myotube formation ● Higher serum concentrations (i.e. 10% HS) promotes myotube formation but in less coordinated pattern Relative Gene Expression ● MyoD: unexpected to peak on D4 ○ Expression should stay stable throughout myogenesis ○ More information is needed from localization assessment ● m-Cadh: consistent expression trend with literature (i.e. peak at D4) ○ Promoted levels of cell fusion on D4 were observed ○ m-Cadh mediates cell interactions prior to fusion ● 2% HS at 80% confluency: the better culture condition out of all three ○ produced thicker myotubes and gene expression data coincides with observed morphology ○ Earlier induction (i.e. 80% confluency) using adult serum at higher concentrations (i.e. 10% HS) may enhance myotube formation Conclusion This study shows that varying culture conditions at the onset of differentiation will have a significant effect on both the morphology and gene expression of C2C12 cells. Many myogenesis studies utilized culture conditions that deviated from what ATCC had recommended, which proves that data comparability across the literature involving C2C12 cell myogenesis to be a challenge. ACKNOWLEDGEMENTS REFERENCES We thank Lindsay Leahul and Dr. Adrienne Benediktsson for their help and support throughout this project. Zammit, P. S., Partridge, T. A., & Yablonka-Reuveni, Z. (2006). The skeletal muscle satellite cell: the stem cell that came in from the cold. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 54(11), 1177–1191. https://doi.org/10.1369/jhc.6R6995.2006 All Rights Reserved by ThermoFisher for visual aid for RT-cDNA and qPCR [RT-cDNA]. (n.d.). ThermoFisher. Retrieved on Mar 12, 2022, from https://www.thermofisher.com/ca/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/basic-principles-rt-qpcr.ht ml [qPCR]. (n.d.). ThermoFisher. Retrieved on Mar 12, 2022, from https://www.thermofisher.com/ca/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/basic-principles-rt-qpcr.ht ml Bibliography Burattini, S., Ferri, P., Battistelli, M., Curci, R., Luchetti, F., & Falcieri, E. (2009). C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. European Journal of Histochemistry, 48(3), 223–234. https://doi.org/10.4081/891 Blais, A., Tsikitis, M., Acosta-Alvear, D., Sharan, R., Kluger, Y., & Dynlacht, B. D. 2005. An initial blueprint for myogenic differentiation. Genes & Development, 19(5), 553-569.http://doi.org/10.1101/gad.1281105 Charrasse, S., Comunale, F., Grumbach, Y., Poulat, F., Blangy, A., & Gauthier-Rouvière, C. (2006). RhoA GTPase regulates m-cadherin activity and myoblast fusion. Molecular Biology of the Cell, 17(2), 749-759. http://dx.doi.org/10.1091/mbc.E05-04-0284 Chuang, H. N., Hsiao, K. M., Chang, H. Y., Wu, C. C., & Pan, H. (2014). The homeobox transcription factor Irxl1 negatively regulates MyoD expression and myoblast differentiation. The FEBS Journal, 281(13), 2990–3003. https://doi.org/10.1111/febs.12837 Choi, Song, Y. J., & Lee, H. (2015). The histone demethylase KDM4B interacts with MyoD to regulate myogenic differentiation in C2C12 myoblast cells. Biochemical and Biophysical Research Communications, 456(4), 872–878. https://doi.org/10.1016/j.bbrc.2014.12.061 Cifuentes-Diaz, C., Nicolet, M., Alameddine, H., Goudou, D., Dehaupas, M., Rieger, F., & Mège, R. (1995). M-cadherin localization in developing adult and regenerating mouse skeletal muscle: possible involvement in secondary myogenesis. Mechanisms of Development, 50(1), 85–97. https://doi.org/10.1016/0925-4773(94)00327-J Dedieu, S., Mazeres, G., Cottin, P., & Brustis, J.-J. (2002). . International Journal of Developmental Biology. Retrieved April 8, 2022, from http://www.ijdb.ehu.es/web/paper/11934152 Delgado, I., Huang, X., Jones, S., Zhang, L., Hatcher, R., Gao, B., & Zhang, P. (2003). Dynamic gene expression during the onset of myoblast differentiation in vitro. Genomics, 82(2), 109–121. https://doi.org/10.1016/s0888-7543(03)00104-6 Ferri, P., Barbieri, E., Burattini, S., Guescini, M., D'Emilio, A., Biagiotti, L., ... & Falcieri, E. (2009). Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. Journal of cellular biochemistry, 108(6), 1302-1317. https://doi-org.libproxy.mtroyal.ca/10.1002/jcb.22360 Florini, J. R., Ewton, D. Z., & Coolican, S. A. (1996). Growth hormone and the insulin-like growth factor system in myogenesis. Endocrine Reviews, 17(5), 481–517. https://doi.org/10.1210/edrv-17-5-481 Grabowska, I., Szeliga, A., Moraczewski, J., Czaplicka, I., & Brzóska, E. (2011). Comparison of satellite cell-derived myoblasts and C2C12 differentiation in twoand three-dimensional cultures: Changes in adhesion protein expression. Cell Biology International, 35(2), 125-133. https://doi.org/10.1042/cbi20090335 Hayashi, & Yonekura, S. (2019). Thermal stimulation at 39°C facilitates the fusion and elongation of C2C12 myoblasts. Animal Science Journal, 90(8), 1008–1017. https://doi.org/10.1111/asj.13227 Hollnagel, A., Grund, C., Franke, W.W., & Arnold, H.H. (2002). The cell adhesion molecule m-cadherin is not essential for muscle development and regeneration. Molecular and Cellular Biology, 22(13), 4760-4770. https://doi.org/10.1128/mcb.22.13.4760-4770.2002 Hsiao, S. P., & Chen, S. L. (2010). Myogenic regulatory factors regulate M-cadherin expression by targeting its proximal promoter elements. The Biochemical journal, 428(2), 223–233. https://doi.org/10.1042/BJ20100250 Kramerova, I., Kudryashova, E., Wu, B., & Spencer, M.J. (2006). Regulation of the m-cadherin-B-catenin complex by calpain 3 during terminal stages of myogenic differentiation. Molecular and Cellular Biology, 26(22), 8437-8447. https://dx.doi.org/10.1128%2FMCB.01296-06 Moran, J. L., Li, Y., Hill, A. A., Mounts, W. M., & Miller, C. P. (2002). Gene expression changes during mouse skeletal myoblast differentiation revealed by transcriptional profiling. Physiological Genomics, 2002(10), 103–111. https://doi.org/10.1152/PHYSIOLGENOMICS.00011.2002/SUPPL_FILE/DESCRIPTIONS.DOCX Ríos, R., Carneiro, I., Arce, V. M., & Devesa, J. (2002, May 25). Myostatin regulates cell survival during C2C12 myogenesis. Biochemical and Biophysical Research Communications. Retrieved April 9, 2022, from https://www.sciencedirect.com/science/article/pii/S0006291X00941597 [RT-cDNA and qPCR]. (n.d.). ThermoFisher. Retrieved on Mar 12, 2022, from https://www.thermofisher.com/ca/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/sp otlight-articles/basic-principles-rt-qpcr.html Sun, L., Trausch-Azar, J. S., Ciechanover, A., & Schwartz, A. L. (2005). Ubiquitin-Proteasome-mediated Degradation, Intracellular Localization, and Protein Synthesis of MyoD and Id1 during Muscle Differentiation. The Journal of Biological Chemistry, 280(28), 26448-26456. http://doi.org/0.1074/jbc.M500373200 Tanaka, Sato, K., Yoshida, T., Fukuda, T., Hanamura, K., Kojima, N., Shirao, T., Yanagawa, T., & Watanabe, H. (2011). Evidence for cell density affecting C2C12 myogenesis: possible regulation of myogenesis by cell-cell communication. Muscle & Nerve, 44(6), 968–977. https://doi.org/10.1002/mus.22224 Tannu, N. S., Rao, V. K., Chaudhary, R. M., Giorgianni, F., Saeed, A. E., Gao, Y., & Raghow, R. (2004). Comparative Proteomes of the Proliferating C 2 C 12 Myoblasts and Fully Differentiated Myotubes Reveal the Complexity of the Skeletal Muscle Differentiation Program* □ S. Molecular and Cellular Proteomics, 3, 1065–1082. https://doi.org/10.1074/mcp.M400020-MCP200 Wang, Hao, Y., & Alway, S. E. (2011). Suppression of GSK-3β activation by M-cadherin protects myoblasts against mitochondria-associated apoptosis during myogenic differentiation. Journal of Cell Science, 124(Pt 22), 3835–3847. https://doi.org/10.1242/jcs.086686 Wu, W., Ren, Z., Chen, C. et al. Subcellular localization of different regions of porcine Six1 gene and its expression analysis in C2C12 myoblasts. Mol Biol Rep 39, 9995–10002 (2012). https://doi-org.libproxy.mtroyal.ca/10.1007/s11033-012-1868-5 Zammit, P. S., Partridge, T. A., & Yablonka-Reuveni, Z. (2006). The skeletal muscle satellite cell: the stem cell that came in from the cold. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 54(11), 1177–1191. https://doi.org/10.1369/jhc.6R6995.2006