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Assessment of the transformation of equine skin–derived fibroblasts to multinucleated skeletal myotubes following lentiviral-induced expression of equine myogenic differentiation 1

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  • 1 Comparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Hawkshead Ln, Hertfordshire AL9 7TA, England
  • | 2 Dubowitz Neuromuscular Centre, Hammersmith Hospital, Imperial College London, DuCane Rd, London W12 0NN, England
  • | 3 Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108
  • | 4 Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108
  • | 5 Dubowitz Neuromuscular Centre, Hammersmith Hospital, Imperial College London, DuCane Rd, London W12 0NN, England
  • | 6 Division of Molecular Neuroendocrinology, National Institute for Medical Research, Mill Hill, London NW7 1AA, England
  • | 7 Randall Division of Cell and Molecular Biophysics, King's College London, Strand, London WC2 2LS, England
  • | 8 Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108
  • | 9 Dubowitz Neuromuscular Centre, Hammersmith Hospital, Imperial College London, DuCane Rd, London W12 0NN, England
  • | 10 Dubowitz Neuromuscular Centre, Hammersmith Hospital, Imperial College London, DuCane Rd, London W12 0NN, England
  • | 11 Comparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Hawkshead Ln, Hertfordshire AL9 7TA, England
  • | 12 Dubowitz Neuromuscular Centre, Hammersmith Hospital, Imperial College London, DuCane Rd, London W12 0NN, England

Abstract

Objective—To develop a reliable method for converting cultured equine skin–derived fibroblasts into muscle cells.

Sample Population—Equine skin–derived fibroblasts.

Procedures—The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin–derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid–Schiff staining.

Results—eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T, and sarcomeric myosin and by cytoplasmic storage of glycogen.

Conclusions and Clinical Relevance—Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy.

Abstract

Objective—To develop a reliable method for converting cultured equine skin–derived fibroblasts into muscle cells.

Sample Population—Equine skin–derived fibroblasts.

Procedures—The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin–derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid–Schiff staining.

Results—eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T, and sarcomeric myosin and by cytoplasmic storage of glycogen.

Conclusions and Clinical Relevance—Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy.

Contributor Notes

Supported by the Horserace Betting Levy Board.

Address correspondence to Dr. Piercy.