Understanding the genesis of revertant muscle fibers using a novel muscle dystrophic reporter mouse (DmdEGFP-mdx)
Final Report Abstract
Duchenne muscular dystrophy (DMD), a fatal hereditary neuromuscular disorder, is caused by mutations that disrupt the open reading frame of the DMD gene and prevent the expression of the dystrophin protein. Nevertheless, some rare dystrophin-positive fibers, termed “revertant fibers” (RFs), can be found alone or in clusters in an otherwise dystrophin-negative muscle background. This phenomenon is seen in both patients and the mdx mouse model of the disease. RFs express an internally deleted, truncated form of dystrophin protein, which is presumed to be protective for muscle cell function. However, the revertant event is far too rare to be of clinical relevance. Researchers have postulated that secondary events (e.g. somatic mutations) must have occurred that would result in the re-establishment of the reading frame. Aim of our study was understand the molecular and cellular origin and development of spontaneously occurring revertant fibers, in which the expression of dystrophin is restored despite the presence of a Dmd germline mutation (stop-mutation). We addressed this question by generating a transgenic dystrophin reporter mouse model for the disease: the DmdEGFP-mdx mouse. This model provided us for the first time with the technical capability to directly visualize and detect RFs in vivo without staining. Additionally, we were able to isolate intact RFs together with their attached satellite cells by identifying the fibers that expressed the EGFP-dystrophin fusion protein. We were able to demonstrate the expression of revertant dystrophin in skeletal and cardiac muscle without antibody staining at the correct subcellular position. Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibers. We isolated single revertant fibers with their attached satellite cells and cultured them. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant dystrophin in isolated fibers ex vivo. We successfully extracted mRNA from individual single revertant fibers and performed single fiber RNA sequencing. For the first time we were able to show that each RF expressed a distinct shorter in-frame RNA transcript and thus showed a specific exon skipping pattern, which differed between different non-neighboring RFs. We were able to culture muscle stem cells from revertant fibers, however we failed to establish clonal cultures and therefore we could not provide evidence of whether the revertant event originated in muscle stem cells. Hence, we were not able to elucidate the exact molecular mechanism behind RFs. Nevertheless, we established the first suitable model to study revertant fibers in vivo and ex vivo, and we provide the foundation and the animal model for further experiments aimed to elucidate the RF phenomenon. Understanding the RF biology will lead to a better understanding of the molecular pathogenesis of DMD and could contribute to the development of novel therapies exploiting the patient cells’ own mechanism to restore dystrophin expression.
Publications
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Live‐imaging of revertant and therapeutically restored dystrophin in the DmdEGFP‐mdx mouse model for Duchenne muscular dystrophy. Neuropathology and Applied Neurobiology, 46(6), 602-614.
Petkova, M. V.; Stantzou, A.; Morin, A.; Petrova, O.; Morales‐Gonzalez, S.; Seifert, F.; Bellec‐Dyevre, J.; Manoliu, T.; Goyenvalle, A.; Garcia, L.; Richard, I.; Laplace‐Builhé, C.; Schuelke, M. & Amthor, H.
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Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle. Proceedings of the National Academy of Sciences, 120(2).
Morin, Adrien; Stantzou, Amalia; Petrova, Olga N.; Hildyard, John; Tensorer, Thomas; Matouk, Meriem; Petkova, Mina V.; Richard, Isabelle; Manoliu, Tudor; Goyenvalle, Aurélie; Falcone, Sestina; Schuelke, Markus; Laplace-Builhé, Corinne; Piercy, Richard J.; Garcia, Luis & Amthor, Helge
