40] Use of myoblast cultures to study mitochondrial myopathies

Abstract

This chapter describes the procedures used for (1) direct cloning of primary human muscle satellite cells, (2) immunofluorescent staining of myoblasts for fluorescence-activated cell sorting (FACS), and (3) analysis of mitochondrial translation products in myoblasts and myotubes. Mutations in mitochondrial DNA (mtDNA) that affect mitochondrial protein translation (large-scale deletions, tRNA point mutations) are associated by many clinical entities classified as mitochondrial encephalomyopathies. Skeletal muscle fibers in patients with these diseases contain heteroplasmic mixtures of wild-type and mutant mtDNAs, inhomogeneously distributed along their length. Skeletal muscle fibers are postmitotic, multinuclear syncytia that cannot be grown in tissue culture. They are surrounded by mononuclear, undifferentiated myoblasts called “satellite cells” that may proliferate in culture. Although a number of different cell types are often available to study the expression of pathogenic mtDNA mutations, two important advantages are gained by using primary myoblast cultures. First, myoblasts can be induced to fuse in culture to form multinucleate myotubes that terminally differentiate and contract, thus, making it possible to study the effects of growth versus terminal differentiation on the expression of mtDNA mutations. Second, because satellite cells are dormant most of the time, the turnover of mitochondria and mtDNA is extremely slow as compared to that in differentiated muscle fibers. Thus, the relative proportions of mutant and wild-type mtDNAs in the satellite cell population reflects the degree of heteroplasmy that existed in the myoblast precursor population during embryological development.