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 (ϳ80%), myogenin mRNA leveled down to ϳ70%. MyoD and desmin mRNA levels in controls and dysferlin-deficient cultures were similar. The reporter luciferase assay demonstrated a low myogenin activity in dysferlindeficient 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.

FHL1 mutations that cause clinically distinct human myopathies form protein aggregates and impair myoblast differentiation

“…Myoblast fusion is an essential step in muscle growth during development and for the regeneration of mature muscle (Pavlath and Horsley, 2003). Impaired myoblast fusion is observed in several myopathies (de Luna et al, 2006;Merrick et al, 2009;Vesa et al, 2009). Relative to wild-type HA-FHL1, which enhanced myoblast differentiation, the expression of RBM, SPM or XMPMA mutant HA-FHL1 did not promote increased myoblast fusion and myotube size in vitro.…”

Genetic ablation of complement C3 attenuates muscle pathology in dysferlin-deficient mice

“…Dysferlin has been shown to play roles in various cellular activities of muscle including membrane repair (5,(11)(12)(13)(14)(15), muscle differentiation (46), muscle regeneration, and cytokine release (18), all of which may rely on the function of dysferlin in promoting membrane fusion. It is still not clear why dysferlinopathy shows such a broad clinical spectrum and such extensive muscle inflammation.…”

Full‐length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells

“…Dysferlin is a 230-kDa protein that is abundantly expressed in skeletal and cardiac muscles and plays a central role in sarcolemmal repair [6]. Based on protein sequence analysis, it was classified as a member of the 'ferlins' family, sharing a common multiple-motif C2 domain and one transmembrane domain in the C-terminal part of the protein [7][8][9]. To date, the reported DYSF gene mutations include point mutations, small deletions and insertions, distributed throughout the entire coding sequence.…”