Dissociated Flexor Digitorum Brevis Myofiber Culture System-A More Mature Muscle Culture System

Gina Ravenscroft, Kristen Nowak, Connie Jackaman, S. Clement, M.A. Lyons, S. Gallagher, Tony Bakker, Nigel Laing

Research output: Contribution to journalArticlepeer-review

35 Citations (Scopus)

Abstract

Considerable knowledge regarding skeletal muscle physiology and disease has been gleaned from cultured myoblastic cell lines or isolated primary myoblasts. Such muscle cultures can be induced to differentiate into multinucleated myotubes that become striated. However they in general do not fully mature and therefore do not model mature muscle. Contrastingly, fresh and cultured dissociated adult mouse flexor digitorum brevis (FDB) myofibers have been studied for many years. We aimed to investigate the possibility of using the FDB myofiber culture system for drug screening and thus long-term cultures of enzymatically dissociated 171313 myofibers were established in 96-well plates. Ca2+ handling experiments were used to investigate the functional state of the myofibers. Imaging of intracellular Ca2+ during electric field stimulation revealed that calcium handling was maintained throughout the culture period of at least 8 days. Western blot and immunostaining analysis showed that the FDB cultures maintained expression of mature proteins throughout the culture period, including alpha-sarcoglycan, dystrophin, fast myosin heavy chain and skeletal muscle alpha-actin. The high levels of the fetal proteins cardiac alpha-actin and utrophin, seen in cultured C(2)C12 myotubes, were absent in the FDB cultures. The expression of developmentally mature proteins and the absence of fetal proteins, in addition to the maintenance of normal calcium handling, highlights the 171313 culture system as a more mature and perhaps more relevant culture system for the study of adult skeletal muscle function. Moreover, it may be a useful system for screening therapeutic agents for the treatment of skeletal muscle disorders.
Original languageEnglish
Pages (from-to)727-738
JournalCell Motility and the Cytoskeleton
Volume64
Issue number10
DOIs
Publication statusPublished - 2007

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