Primary Murine Myotubes as a Model for Investigating Muscular Dystrophy

Abstract

Muscular dystrophies caused by defects in various genes are often associated with impairment of calcium homeostasis. Studies of calcium currents are hampered because of the lack of a robust cellular model. Primary murine myotubes, formed upon satellite cell fusion, were examined for their utilization as a model of adult skeletal muscle. We enzymatically isolated satellite cells and induced them to differentiation to myotubes. Myotubes displayed morphological and physiological properties resembling adult muscle fibers. Desmin and myosin heavy chain immunoreactivity in the differentiated myotubes were similar to the mature muscle cross-striated pattern. The myotubes responded to electrical and chemical stimulations with sarcoplasmic reticulum calcium release. Presence of L-type calcium channels in the myotubes sarcolemma was confirmed via whole-cell patch-clamp technique. To assess the use of myotubes for studying functional mutation effects lentiviral transduction was applied. Satellite cells easily underwent transduction and were able to retain a positive expression of lentivirally encoded GFP up to and after the formation of myotubes, without changes in their physiological and morphological properties. Thus, we conclude that murine myotubes may serve as a fruitful cell model for investigating calcium homeostasis in muscular dystrophy and the effects of gene modifications can be assessed due to lentiviral transduction.