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

This research develops and tests a novel method to convert horse skin cells into muscle cells using a specific equine gene expression technique. The successful conversion provides an enhanced tool for studying horse muscle diseases.

Methodology

• The researchers began by identifying the gene sequence for equine myogenic differentiation 1 (eqMyoD), a gene involved in muscle cell differentiation. Total mRNA, a type of RNA that carries instructions from DNA for controlling the synthesis of proteins, was extracted from young horse skeletal muscle.
• This eqMyoD was then cloned into a plasmid vector containing an internal ribosomal entry site to guide protein synthesis, leading to the expression of either eqMyoD or a fluorescent protein (Enhanced Green Fluorescent Protein or EGFP).
• The team confirmed the transient expression of these molecules in horse fibroblasts (skin cells) and mouse-embryo-derived fibroblasts via immunocytochemical analysis and Western blotting.
• Following this, they created a lentiviral vector (a tool commonly used in molecular biology to deliver genetic material into cells) containing the same coding sequences.

Results

• The transformation of horse skin-derived cells into skeletal muscle cells (myotubes) was confirmed using various techniques, including immunohistochemical analysis, Western blotting, and periodic acid-Schiff staining (a staining method to detect polysaccharides, such as glycogen).
• They found that eqMyoD mRNA consisted of 960 base pairs and shared high homology (similarity in sequence) with the myogenic differentiation 1 gene in other mammals.
• The fluorescent EGFP confirmed the expression of a 53-kd (kilodalton, a unit of molecular weight) protein with mainly nuclear localization, thus demonstrating the protein expression within the cell nucleus.
• Lentiviral transduction (gene transfer) proved efficient, with about 80% of EGFP-positive cells transformed into multinucleated myotubes over 15 days. This was evident from the expression of muscle-specific proteins, including desmin, troponin-T, and sarcomeric myosin, and the storage of glycogen within the cell cytoplasm.

Conclusion

• The research successfully transformed primary equine fibroblasts into fusion-competent myoblasts (muscle cells) by lentiviral transduction of eqMyoD. This method presents a significant alternative to primary myoblast cultures for investigating cellular defects in horse muscle diseases, including recurrent exertional rhabdomyolysis and polysaccharide storage myopathy—two common muscle disorders affecting horses.