Dissecting the Sex-Specific Relationship Between Muscle Damage and Regenerative Myofiber Characteristics in Cardiotoxin-Induced Mouse Models
DOI:
https://doi.org/10.58445/rars.4165Keywords:
Muscle Regeneration, Cardiotoxin, Biological Sex, Muscle Damage, Histological AnalysisAbstract
Skeletal muscle regeneration following acute injury relies on highly coordinated interactions between inflammatory signaling and muscle tissue responses. Following myofiber necrosis, immune cells, including regulatory T cells, infiltrate the damaged ECM and secrete growth factors like Amphiregulin (Areg), which can promote satellite cell (SC) proliferation and muscle repair. However, how biological sex and Areg genotype are associated to influence these regenerative processes remains poorly defined. We examined whether the relationship between tissue damage and histological measures of regeneration differed according to sex and Areg genotype. It was hypothesized that in a mouse model of CTX‑induced inflammatory skeletal muscle injury, female and male mice will differ in the relationship between damaged area and regenerative myofiber characteristics (size and central nuclei), reflecting sex‑specific inflammatory and reparative responses. Twenty-seven mice were divided across four experimental cohorts: wild-type (WT) males, WT females, Areg knockout (KO) males, and KO females. Following cardiotoxin (CTX)-induced injury, Tibialis Anterior sections were harvested at day 5, stained with Wheat Germ Agglutinin (WGA) and DAPI, imaged, and analyzed using FIJI to quantify morphometric parameters. Pearson correlation analyses were performed separately for each group. A strong, statistically significant inverse correlation between percent damaged area and median CSA was observed only in WT males (r= –0.89, p = 0.019). Conversely, the percent damaged area positively correlated with the percent central nuclei in WT females (r = 0.87, p = 0.011) and KO females (r = 0.83, p = 0.041). The remaining correlations showed directional trends but did not reach statistical significance (p > 0.05). These findings suggest that the relationships among tissue damage, myofiber CSA, and central nucleation do not vary uniformly across biological sex and Areg genotypes. Consequently, relying on a single histological marker is insufficient, highlighting the need for multivariate assessment criteria when evaluating muscle regeneration.
References
Bahri, O. A., Naldaiz-Gastesi, N., Kennedy, D. C., Wheatley, A. M., Izeta, A., & McCullagh, K. J. A. (2019). The panniculus carnosus muscle: A novel model of striated muscle regeneration that exhibits sex differences in the mdx mouse. Scientific Reports, 9(1), 15964.
Burzyn, D., Kuswanto, W., Kolodin, D., Shadrach, J. L., Cerletti, M., Jang, Y., Sefik, E., Tan, T. G., Wagers, A. J., Benoist, C., & Mathis, D. (2013). A special population of regulatory T cells potentiates muscle repair. Cell, 155(6), 1282–1295.
Choi, W., Lee, J., Lee, J., Lee, S. H., & Kim, S. (2019). Hepatocyte Growth Factor Regulates Macrophage Transition to the M2 Phenotype and Promotes Murine Skeletal Muscle Regeneration. Frontiers in Physiology, 10, 914.
Deasy, B. M., Lu, A., Tebbets, J. C., Feduska, J. M., Schugar, R. C., Pollett, J. B., Sun, B., Urish, K. L., Gharaibeh, B. M., Cao, B., Rubin, R. T., & Huard, J. (2007). A role for cell sex in stem cell-mediated skeletal muscle regeneration: female cells have higher muscle regeneration efficiency. The Journal of Cell Biology, 177(1), 73–86.
Deprez, A., Orfi, Z., Rieger, L., & Dumont, N. A. (2023). Impaired muscle stem cell function and abnormal myogenesis in acquired myopathies. Bioscience Reports, 43(1). https://doi.org/10.1042/BSR20220284
Feno, S., Munari, F., Gherardi, G., Reane, D. V., D’Angelo, D., Viola, A., Rizzuto, R., & Raffaello, A. (2023). Myonecrosis Induction by Intramuscular Injection of CTX. Bio-Protocol, 13(1), e4587.
Fu, X., Li, S., Jia, M., Yang, W., & Hu, P. (2023). Induction of Skeletal Muscle Injury by Intramuscular Injection of Cardiotoxin in Mouse. Bio-Protocol, 13(9), e4668.
Gama, J. F. G., Romualdo, R. D., de Assis, M. L., de Oliveira, L. M., Quírico-Santos, T., Alves, L. A., & Lagrota-Candido, J. (2022). Role of Regulatory T Cells in Skeletal Muscle Regeneration: A Systematic Review. Biomolecules, 12(6). https://doi.org/10.3390/biom12060817
Guardiola, O., Andolfi, G., Tirone, M., Iavarone, F., Brunelli, S., & Minchiotti, G. (2017). Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection. Journal of Visualized Experiments : JoVE, 119. https://doi.org/10.3791/54515
Hardy, D., Besnard, A., Latil, M., Jouvion, G., Briand, D., Thépenier, C., Pascal, Q., Guguin, A., Gayraud-Morel, B., Cavaillon, J.-M., Tajbakhsh, S., Rocheteau, P., & Chrétien, F. (2016). Comparative Study of Injury Models for Studying Muscle Regeneration in Mice. PloS One, 11(1), e0147198.
Kapuscinski, J. (1995). DAPI: a DNA-specific fluorescent probe. Biotechnic & Histochemistry : Official Publication of the Biological Stain Commission, 70(5), 220–233.
Kostrominova, T. Y. (2011). Application of WGA lectin staining for visualization of the connective tissue in skeletal muscle, bone, and ligament/tendon studies. Microscopy Research and Technique, 74(1), 18–22.
Pizza, F. X., & Buckley, K. H. (2023). Regenerating Myofibers after an Acute Muscle Injury: What Do We Really Know about Them? International Journal of Molecular Sciences, 24(16). https://doi.org/10.3390/ijms241612545
Rigamonti, E., Zordan, P., Sciorati, C., Rovere-Querini, P., & Brunelli, S. (2014). Macrophage plasticity in skeletal muscle repair. BioMed Research International, 2014, 560629.
Wang, Y., Lu, J., & Liu, Y. (2022). Skeletal Muscle Regeneration in Cardiotoxin-Induced Muscle Injury Models. International Journal of Molecular Sciences, 23(21). https://doi.org/10.3390/ijms232113380
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