title: Absence of dysferlin alters myogenin expression and delays human muscle differentiation "in vitro".
abstract: Mutations in dysferlin cause a type of muscular dystrophy known as dysferlinopathy. Dysferlin may be involved in muscle repair and differentiation. We compared normal human skeletal muscle cultures expressing dysferlin with muscle cultures from dysferlinopathy patients. We quantified the fusion index of myoblasts as a measure of muscle development and conducted optic and electronic microscopy, immunofluorescence, Western blot, flow cytometry, and real-time PCR at different developmental stages. Short interference RNA was used to corroborate the results obtained in dysferlin-deficient cultures. A luciferase reporter assay was performed to study myogenin activity in dysferlin-deficient cultures. Myoblasts fusion was consistently delayed as compared with controls whereas the proliferation rate did not change. Electron microscopy showed that control cultured cells at 10 days were fusiform, whereas dysferlin-deficient cells were star-shaped and large. After 15 days the normal multinucleated appearance and structured myofibrils were not present in dysferlin-deficient cells. Strikingly, myogenin was not detected in myotubes from dysferlin-deficient cultures using Western blot, and mRNA analysis showed low levels (p < 0.05) compared with controls. Flow cytometry and immunofluorescence also showed reduced levels of myogenin in dysferlin-deficient cultures. When the dysferlin gene was knocked down ( approximately 80%), myogenin mRNA leveled down to approximately 70%. MyoD and desmin mRNA levels in controls and dysferlin-deficient cultures were similar. The reporter luciferase assay demonstrated a low myogenin activity in dysferlin-deficient cultures. These results point to a functional link between dysferlin and myogenin, and both proteins may share a new signaling pathway involved in differentiation of skeletal muscle in vitro.
authors.affiliation: Servei de Neurologia i Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, 08025 Barcelona, Spain.
authors.forename: Noemí
authors.initials: N
authors.lastname: de Luna
authors.affiliation: Servei de Neurologia i Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, 08025 Barcelona, Spain.
authors.forename: Eduard
authors.initials: E
authors.lastname: Gallardo
authors.affiliation: Centro de Investigaciones Príncipe Felipe and Instituto Cavanilles, Universidad de Valencia, 46980 Valencia, Spain.
authors.forename: Mario
authors.initials: M
authors.lastname: Soriano
authors.affiliation: Servei de Neurologia i Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, 08025 Barcelona, Spain.
authors.forename: Raúl
authors.initials: R
authors.lastname: Dominguez-Perles
authors.affiliation: Servei de Neurologia i Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, 08025 Barcelona, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: de la Torre
authors.affiliation: Servei de Neurologia i Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, 08025 Barcelona, Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Rojas-García
authors.affiliation: Centro de Investigaciones Príncipe Felipe and Instituto Cavanilles, Universidad de Valencia, 46980 Valencia, Spain.
authors.forename: Jose M
authors.initials: JM
authors.lastname: García-Verdugo
authors.affiliation: Servei de Neurologia i Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, 08025 Barcelona, Spain. Electronic address: iilla@santpau.es.
authors.forename: Isabel
authors.initials: I
authors.lastname: Illa
mesh_terms: D019002:CD56 Antigen / Q000096:biosynthesis; D016022:Case-Control Studies; D002454:Cell Differentiation; D002478:Cells, Cultured; D000073939:Dysferlin; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology*; D009124:Muscle Proteins / Q000235:genetics* / Q000502:physiology*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D009132:Muscles / Q000378:metabolism; D009136:Muscular Dystrophies / Q000235:genetics*; D018008:Myogenin / Q000096:biosynthesis*; D015398:Signal Transduction
chemical_list: D019002:CD56 Antigen; C114839:DYSF protein, human; D000073939:Dysferlin; D008565:Membrane Proteins; D009124:Muscle Proteins; D018008:Myogenin