Comparison of Gene Expression Patterns Associated with Myostatin Using Transcriptomic Data of the Myogenic C2C12 Cell Line and Skeletal Muscle Tissue

Document Type : Research Articles

Authors

Mitra Riasi

Ali Javadmanesh

Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

https://doi.org/10.22067/jcmr.2025.90066.1101

Abstract

TGF-β group member myostatin (MSTN) inhibits the growth and differentiation of skeletal muscle. It is unknown how total inhibition of MSTN restricts the hyperproliferation of muscle cells, though. This study aimed to determine the differentially expressed genes and biological pathways associated with myostatin in mouse C2C12 myogenic cells and skeletal muscle tissue, utilizing high-throughput mRNA expression data. In both tissue and cell line, two experimental groups were compared, including wild-type vs. MSTN-Knockout. Transcriptome data were extracted from GEO and analyzed using the LIMMA package in the R environment and GEO2R. Significantly differentially expressed genes were considered as adjusted p-value < 0.05 with log fold change > |0.5|. Lastly, the possible biological pathways were examined with the KEGG database, and the protein-protein interaction (PPI) network was created using Cytoscape software. To find key genes in the PPI network, a topological analysis was conducted using the Network Analyzer tool in Cytoscape software. The total number of differentially expressed genes was 14549 for the C2C12 cell line and 45267 for mouse skeletal muscle tissue; among them, 235 and 1425 transcripts were significant with an adjusted p-value <0.05. The comparison between these two DEG lists showed that 49 genes were common between the myogenic C2C12 cell and skeletal muscle tissue. Additionally, three biological pathways between the C2C12 cell line and the skeletal muscle tissue were in common, and they were associated with the decrease of MSTN gene expression, including: hypertrophic cardiomyopathy, Axon guidance, and dilated cardiomyopathy. These pathways were all related to muscle tissue. Despite the large number of DEGs, only 49 were in common in tissue and cell line; this could indicate that comparing a tissue with its relevant cell line at the transcriptome level might not be precise enough to draw a solid conclusion. This is due to the nature of cell heterogeneity of most tissues, including skeletal muscle.

Keywords

Myostatin

mouse

Gene network

C2C12 cell line

tissue muscle

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References

Ahmad F., Seidman J. and Seidman C. E. (2005) The genetic basis for cardiac remodeling. Annual review of genomics and human genetics 6: 185-216.

Birnbaum K. D. (2018) Power in numbers: single-cell RNA-seq strategies to dissect complex tissues. Annual review of genetics 52, 203-221.

Bogdanovich S., Krag T. O., Barton E. R., Morris L. D., Whittemore L.-A., Ahima R. S., et al. (2002) Functional improvement of dystrophic muscle by myostatin blockade. Journal of Nature 420(6914): 418-421.

Bowen T. S., Schuler G. and Adams V. (2015) Skeletal muscle wasting in cachexia and sarcopenia: molecular pathophysiology and impact of exercise training. Journal of cachexia, sarcopenia and muscle 6(3): 197-207.

Cassar-Malek I., Passelaigue F., Bernard C., Léger J. and Hocquette J.-F. (2007) Target genes of myostatin loss-of-function in muscles of late bovine fetuses. Journal of BMC Genomics 8(1): 1-11.

Ekblom R. and Galindo J. (2011) Applications of next generation sequencing in molecular ecology of non-model organisms. Heredity 107(1): 1-15.

Freire P. P., Fernandez G. J., Cury S. S., De Moraes D., Oliveira J. S., De Oliveira G., et al. (2019) The pathway to cancer cachexia: microRNA-regulated networks in muscle wasting based on integrative meta-analysis. International Journal of Molecular Sciences 20(8):1962.

Gorji A. E., Roudbari Z., Sadeghi B., Javadmanesh A. and Sadkowski T. (2019) Transcriptomic analysis on the promoter regions discover gene networks involving mastitis in cattle.  Microbial Pathogenesis 137: 103801.

Güller I. and Russell A. P. (2010) MicroRNAs in skeletal muscle: their role and regulation in development, disease and function. The Journal of Physiology 588(21): 4075-4087.

Hentzen E. R., Lahey M., Peters D., Mathew L., Barash I. A., Fridén J., et al. (2006) Stress‐dependent and‐independent expression of the myogenic regulatory factors and the MARP genes after eccentric contractions in rats. The Journal of Physiology 570(1): 157-167.

Huang P., Pang D., Wang K., Xu A., Yao C., Li M., et al. (2019) The possible role of complete loss of myostatin in limiting excessive proliferation of muscle cells (C2C12) via activation of microRNAs.  International Journal of Molecular Sciences 20(3):643.

Javadmanesh A. and Riasi M. (2023) Analysis of genes related to myostatin and muscle growth using mRNA expression data of C2C12 muscle cell line. In The 10th National and 2nd International Congress on animal science congress of Iran.

Kambadur R., Sharma M., SmithT. P. and Bass J. J. (1997) Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Journal of Genome Research 7(9): 910-915.

Kemp T., Sadusky T., Saltisi F., Carey N., Moss J., Yang S., et al. (2000) Identification of Ankrd2, a novel skeletal muscle gene coding for a stretch-responsive ankyrin-repeat protein. Genomics 66(3): 229-241.

Kojic S., Medeot E., Guccione E., Krmac H., Zara I., Martinelli V., et al. (2004) The Ankrd2 protein, a link between the sarcomere and the nucleus in skeletal muscle. Journal of Molecular Biology 339(2):313-325.

Lafzi A., Moutinho C., Picelli S. and Heyn H. (2018) Tutorial: guidelines for the experimental design of single-cell RNA sequencing studies. Nature Protocols 13(12): 2742-2757.

Maeda Y. (2019) P. 135Chemokine CXCL12 as an intrinsic modulator of skeletal muscle mass in healthy humans. Neuromuscular Disorders 29: S87.

Martinelli G., Olivari D., Re Cecconi A., Talamini L., Ottoboni L., Lecker S., et al. (2016) Activation of the SDF1/CXCR4 pathway retards muscle atrophy during cancer cachexia. Oncogene 35(48): 6212-6222.

McKoy G., Hou Y., Yang S. Y., Vega Avelaira D., Degens H., Goldspink G. and Coulton, G. R. (2005) Expression of Ankrd2 in fast and slow muscles and its response to stretch are consistent with a role in slow muscle function.  Journal of Applied Physiology 98(6): 2337-2343.

Miao Y., Yang J., Xu Z., Jing L., Zhao S. and Li X. (2015) RNA sequencing identifies upregulated kyphoscoliosis peptidase and phosphatidic acid signaling pathways in muscle hypertrophy generated by transgenic expression of myostatin propeptide. International Journal of Molecular Sciences 16(4): 7976-7994.

Miryala S. K., Anbarasu A. and Ramaiah S. (2018) Discerning molecular interactions: a comprehensive review on biomolecular interaction databases and network analysis tools. Gene 642: 84-94.

Mutryn M. F., Brannick E. M., Fu W., Lee W. R. and Abasht B. (2015) Characterization of a novel chicken muscle disorder through differential gene expression and pathway analysis using RNA-sequencing. BMC Genomics 16(1): 1-19.

Riasi M., Mozaffari Jovin S. and Javadmanesh A. (2024) The effect of modification of DNA interference on myostatin gene expression in mice. Journal of Genetics 103: 02.

Riasi M., Mozaffari Jovin S. and Javadmanesh A. (2022) Effect of Intramuscular and Intraperitoneal Injections of conjugated MSTN-siRNA-cholesterol on Inhibition of Myostatin Gene expression. J Journal of Cell Molecular Research 14(1): 20-27.

Saedi N., Aminafshar M., Chamani M., Honarvar M. and Javadmanesh A. (2022) Expression pattern and network visualization of genes involved in milk persistency in bovine mammary tissue.  Agriculture Natural Resources 56(1): 23–34.

Saliani M., Jalal R. and Javadmanesh, A. (2022) Differential expression analysis of genes and long non-coding RNAs associated with KRAS mutation in colorectal cancer cells. Scientific reports 12(1), 7965.

Sicinski P., Geng Y., Ryder-Cook A. S., Barnard E. A., Darlison M. G. and Barnard P. J. (1989) The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 244(4912): 1578-1580.

Soleimani S., Sekhavati M. H. and Javadmanesh A. (2019) Sequencing and Bioinformatic Investigation of Introducing a Repressive Micro-RNA Target Sites in the 3'UTR of Myostatin Gene in some Indigenous Sheep Breeds of Iran. J Iranian Journal of Animal Science Research 11(1): 111-119.

Tsukamoto Y., Senda T., Nakano T., Nakada C., Hida T., Ishiguro N., et al. (2002) Arpp, a new homolog of carp, is preferentially expressed in type 1 skeletal muscle fibers and is markedly induced by denervation. Laboratory Investigation 82(5): 645-655.

Wagner K. R., McPherron A. C., Winik N. and Lee S. J. (2002) Loss of myostatin attenuates severity of muscular dystrophy in mdx mice. J Annals of Neurology: Official Journal of the American Neurological Association the Child Neurology Society 52(6): 832-836.

Welle S., Cardillo A., Zanche M. and Tawil R. (2009) Skeletal muscle gene expression after myostatin knockout in mature mice. Physiological Genomics 38(3): 342-350.

Whittemore L.-A., Song K., Li X., Aghajanian J., Davies M., Girgenrath S., et al. (2003) Inhibition of myostatin in adult mice increases skeletal muscle mass and strength. Biochemical Biophysical Research Communications 300(4): 965-971.

Zhao B., Li E. J., Wall R. J. and Yang J. (2009) Coordinated patterns of gene expressions for adult muscle build-up in transgenic mice expressing myostatin propeptide. BMC Genomics 10(1): 1-16.