title: Adenovirus-mediated expression of myogenic differentiation factor 1 (MyoD) in equine and human dermal fibroblasts enables their conversion to caffeine-sensitive myotubes.
abstract: Several human and animal myopathies, such as malignant hyperthermia (MH), central core disease and equine recurrent exertional rhabdomyolysis (RER) are confirmed or thought to be associated with dysfunction of skeletal muscle calcium regulation. For some patients in whom the genetic cause is unknown, or when mutational analysis reveals genetic variants with unclear pathogenicity, defects are further studied through use of muscle histopathology and in vitro contraction tests, the latter in particular, when assessing responses to ryanodine receptor agonists, such as caffeine. However, since muscle biopsy is not always suitable, researchers have used cultured cells to model these diseases, by examining calcium regulation in myotubes derived from skin, following forced expression of muscle-specific transcription factors. Here we describe a novel adenoviral vector that we used to express equine MyoD in dermal fibroblasts. In permissive conditions, transduced equine and human fibroblasts differentiated into multinucleated myotubes. We demonstrate that these cells have a functional excitation-calcium release mechanism and, similarly to primary muscle-derived myotubes, respond in a dose-dependent manner to increasing concentrations of caffeine. MyoD-induced conversion of equine skin-derived fibroblasts offers an attractive method for evaluating calcium homeostasis defects in vitro without the need for invasive muscle biopsy.
authors.affiliation: Department of Clinical Sciences and Services, Royal Veterinary College, Royal College Street, London NW1 0TU, UK.
authors.forename: Marta
authors.initials: M
authors.lastname: Fernandez-Fuente
authors.affiliation: Universidad Francisco de Vitoria, Facultad de Ciencias Biosanitarias, 28223 Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Martin-Duque
authors.affiliation: Laboratoire TIRO, UMRE 4320, iBEB, DSV, Commissariat a' l'Energie Atomique, Nice, France.
authors.forename: Georges
authors.initials: G
authors.lastname: Vassaux
authors.affiliation: Department of Clinical Sciences and Services, Royal Veterinary College, Royal College Street, London NW1 0TU, UK.
authors.forename: Susan C
authors.initials: SC
authors.lastname: Brown
authors.affiliation: Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, Gower Street, London WC1E 6BT, UK.
authors.forename: Francesco
authors.initials: F
authors.lastname: Muntoni
authors.affiliation: Laboratory of Cell Electrophysiology, Heart Science Centre, Imperial College, Harefield Hospital, Hill End Road, Harefield, Middlesex UB9 6JH, UK.
authors.forename: Cesare M
authors.initials: CM
authors.lastname: Terracciano
authors.affiliation: Department of Clinical Sciences and Services, Royal Veterinary College, Royal College Street, London NW1 0TU, UK. Electronic address: rpiercy@rvc.ac.uk.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Piercy
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002478:Cells, Cultured; D020405:Dermis / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006736:Horses; D006801:Humans; D008297:Male; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D002110:Caffeine
keywords: Adenovirus; Calcium; Equine; Exertional rhabdomyolysis; Malignant hyperthermia; MyoD