Volume: 3, 2024
3rd International PhD Student’s Conference at the University of Life Sciences in Lublin, Poland:
ENVIRONMENT – PLANT – ANIMAL – PRODUCT
Abstract number: A032
DOI: https://doi.org/10.24326/ICDSUPL3.A032
Published online: 24 April 2024
ICDSUPL, 3, A032 (2024)
Paracrine effect of fibroblasts on the course of myogenesis of bovine muscle satellite cells in vitro
Karolina Zygmunt1*, Katarzyna Piórkowska1, Wojciech Witarski1
1 Department of Animal Molecular Biology, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland
* Corresponding author: karolina.zygmunt@iz.edu.pl
Abstract
Fibroblasts, connective tissue cells rich in a large content of extracellular matrix containing proteins such as collagen or fibronectin, stimulate the myogenesis process through cell-cell interactions. However, few studies have focused on understanding their paracrine effects on muscle satellite cells (MuSC) myogenesis. The study aims to examine changes in the expression of muscle cell markers – Pax7 and genes from the MRF (Myogenic Regulator Factors) family at the molecular level and myosin at the protein level in coculture of bovine MuSC and fibroblasts through paracrine interactions. MuSC culture was carried out on 6-well coculture plates with 0.4 μm inserts on which fibroblasts were seeded. The proliferation stage was carried out for five days in DMEM medium supplemented with 20% Fetal Bovine Serum. Then, the differentiation stage was initiated by changing medium to DMEM supplemented with 10% Horse Serum, and the culture was maintained for 72 hours. After each experimental stage, RNA for RT-qPCR analysis and protein for ELISA analysis were isolated. The obtained results indicate a significantly higher expression of Myf5 and MyoD genes in the group maintained in coculture at the proliferation stage (p=0.017 and p=0.03, respectively), which are involved in the early stage of myogenic differentiation (mainly at the proliferation stage), giving the ability to further differentiation of MuSC. Moreover, higher expression of Myf6 and MyoG genes – involved in the differentiation – was observed in the group cocultured with fibroblasts. At the differentiation stage, significantly lower levels of Pax7 and Myf5 gene expression were observed in the group maintained in coculture (p = 0.028 and p = 0.003, respectively). MuSCs expressing Pax7 gene are mainly involved in maintaining a pool of undifferentiated satellite cells, with frequent co-expression of Myf5 gene. ELISA analysis indicated a significantly higher level of myosin protein expression at the proliferation stage in the group maintained in coculture (p=0.01). In summary, the paracrine effect of fibroblasts increases MuSC differentiation through higher expression of genes involved in muscle cell differentiation at the proliferation stage and lower expression of genes involved in the early stage of myogenesis at the differentiation stage. However, transcriptome analysis to examine other MuSC markers’ expression seems appropriate to confirm the obtained results.
Keywords: muscle satellite cells, fibroblasts, coculture, genes expression, myogenesis
How to cite
K. Zygmunt, K. Piórkowska, W. Witarski, 2024. Paracrine effect of fibroblasts on the course of myogenesis of bovine muscle satellite cells in vitro. In: 3rd International PhD Student’s Conference at the University of Life Sciences in Lublin, Poland: Environment – Plant – Animal – Product. https://doi.org/10.24326/ICDSUPL3.A032