Muscle regeneration after high-dose radiation exposure: therapeutic potential of Hedgehog pathway modulation?

Abstract

Purpose: Intentional or accidental exposure of relatively large as well as localized areas of the skin to ionizing radiation can lead to severe damage of many of its cellular components and cutaneous radiation syndrome. Patients can be treated with an invasive surgical procedure coupled with autologous cell therapy. However, this approach remains perfectible, especially for muscle repair. Indeed, a severe underlying muscle defect persists, in particular because of the damage to the satellite cells which ensure muscle regeneration. To overcome these shortcomings, a solution could be to develop new therapeutic strategies based on pharmacological treatments to improve post-irradiation muscle regeneration. In this study, we focus on the Hedgehog signaling pathway as a target, due to its involvement in myogenesis.

Materials and methods: To evaluate the benefit of the pro-myogenic Hedgehog signaling pathway modulation, recombinant Sonic Hedgehog (rShh; agonist) or Cyclopamine (antagonist) were used in a stable cell line of mouse C2C12 myoblasts exposed to radiation (X-rays; 5 Gy). Our in vitro studies were carried out under either proliferation or differentiation conditions. Proliferation, migration, survival (apoptosis) and expression of myogenic genes/proteins were evaluated.

Results: A high dose of radiation was shown to exert a serious negative impact in our in vitro model of mouse muscle progenitors after irradiation in proliferation or differentiation conditions. Interestingly, Hh pathway stimulation by rShh promotes the proliferation of myoblasts and their survival while its blockade by Cyclopamine significantly increases cell differentiation toward mature myotubes.

Conclusion: These data suggest that, after irradiation, the sequence of activation and inhibition of the Hh pathway could allow rescue and proliferation of satellite cells, followed by their differentiation to regenerate new fibers. On the basis of these encouraging in vitro results, the second phase of our study will involve the in vivo validation of this treatment in a new murine model of ultra-localized muscle irradiation.

Keywords:

• Cutaneous radiation syndrome
• muscle regeneration
• satellite cells
• modulation of Hedgehog pathway

Acknowledgments

We wish to express our deep thanks to the French Direction Générale de l’Armement (Paris, France) for granting our work, to IRBA Molecular Biology Unit headed by Dr Laure BARBIER for helping us in the development of RT-qPCR experiments, to IRBA Imaging Unit headed by Dr Anne-Laure FAVIER for making the cell imaging platform available, to Mrs Véronique CHASTRES for her precious advice in statistics and to Mr Daniel HENKEL for his excellent and very quick work in correcting the translation of the manuscript.

Disclosure statement

The authors report no conflict of interest. They are entirely responsible for the content of the paper.

Additional information

Funding

The author(s) stated that they received funding from the French Direction Générale de l'Armement (Paris, France) for the work reported in this article.

Notes on contributors

E. Rota Graziosi

Emmanuelle Rota Graziosi is a French engineer specialized in chemistry, biology and health. She has also obtained a double master’s degree in Medical and Translational Chemistry, University of Montpellier, France. For one year now, she has been completing a PhD in the Radiobiology Unit of the French Armed Forces Biomedical Research Institute (IRBA), Brétigny-sur-Orge, France.

S. François

Sabine François, PhD, is a specialist of cell therapy optimization for regenerative medicine, especially in cases of lesions induced by ionizing radiation. She is a project leader and develop new therapeutic approaches to treat acute radiation syndrome in the Radiobiology Unit of IRBA, Brétigny-sur-Orge, France. She is also part of the Joint Research Unit UMR1296 of French National Institute for Health and Medical Research (INSERM, Lyon, France) and French Armed Forces Biomedical Research Institute (IRBA, Brétigny-sur-Orge, France).

J. Pateux

Jérôme Pateux is a paramedical technician in IRBA, Brétigny-sur-Orge, France. He works both in the Radiobiology Unit where he is a specialist in the analysis of localized high-dose radiation effects and in the Radiation Biological Dosimetry Laboratory where he is in charge of the identification of new biomarkers for post-irradiation biological dosimetry.

M. Gauthier

Michel Gauthier is a technician specialized in gene identification and therapy in genetic diseases. He has also worked to develop therapeutic strategies for the treatment of chronic inflammatory bowel diseases. From 2020, he is a paramedical technician in the Radiobiology Unit of IRBA, Brétigny-sur-Orge, France.

X. Butigieg

Xavier Butigieg is a paramedical technician in the Imaging Unit of IRBA, Brétigny-sur-Orge, France. He is a specialist in various specific techniques like histology, bright field and fluorescence microscopy, scanning and transmission electron microscopy on the state-of-art microscopy platform of the Institute.

M. Oger

Myriam Oger, PhD, is a biomedical researcher specialized in Sciences and Technology of Information in IRBA, Brétigny-sur-Orge, France. She is a specialist in image acquisition, processing and analysis. Her fields of expertise are in microscopical and 3D microtomography images, using FIJI software and Python libraries.

M. Drouet

Michel Drouet, MD, PhD, is currently Head of Radiation Biological Effects Department and Head of Chemical, Biological, Radiobiological and Nuclear Risks Division in IRBA, Brétigny-sur-Orge, France. He is also part of the Joint Research Unit UMR1296 of French National Institute for Health and Medical Research (INSERM, Lyon, France) and French Armed Forces Biomedical Research Institute (IRBA, Brétigny-sur-Orge, France).

D. Riccobono

Diane Riccobono, MD, PhD, worked as a medical officer for two years. She performed a PhD specialized in radiobiology, in the development of advanced therapy medicinal products based on mesenchymal stromal cells and in inflammation pathways analysis after irradiation. She is also specialized in radioprotection and CBRN threat. Since 2018, Dr Diane RICCOBONO is in charge of the Radiobiology unit of IRBA Brétigny-sur-Orge, France, and is part of the Joint Research Unit UMR1296 of French National Institute for Health and Medical Research (INSERM, Lyon, France) and French Armed Forces Biomedical Research Institute (IRBA, Brétigny-sur-Orge, France).

N. Jullien

Nicolas Jullien, PhD, is a specialist in pharmacology, radiobiology and in the development of advanced therapy medicinal products based on the use of mesenchymal stromal cells obtained from bone marrow or adipose tissue. He is a biomedical researcher in the Radiobiology unit of IRBA Brétigny-sur-Orge, France.