MyoD−/− Satellite Cells in Single-Fiber Culture Are Differentiation Defective and MRF4 Deficient

Abstract

MyoD-deficient mice are without obvious deleterious muscle phenotype during embryogenesis and fetal development, and adults in the laboratory have grossly normal skeletal muscle and life span. However, a previous study showed that in the context of muscle degeneration on a mdx (dystrophin null) genetic background, animals lacking MyoD have a greatly intensified disease phenotype leading to lethality not otherwise seen in mdx mice. Here we have examined MyoD−/− adult muscle fibers and their associated satellite cells in single myofiber cultures and describe major phenotypic differences found at the tissue, cellular, and molecular levels. The steady-state number of satellite cells on freshly isolated MyoD−/− fibers was elevated and abnormal branched fiber morphologies were observed, the latter suggesting chronic muscle regeneration in vivo. Single-cell RNA coexpression analyses were performed for c-met, m-cadherin, and the four myogenic regulatory factors (MRFs.) Most mutant satellite cells entered the cell cycle and upregulated expression of myf5, both characteristic early steps in satellite cell maturation. However, they later failed to normally upregulate MRF4, displayed a major deficit in m-cadherin expression, and showed a significant diminution in myogenin-positive status compared with wildtype. MyoD−/− satellite cells formed unusual aggregate structures, failed to fuse efficiently, and showed greater than 90% reduction in differentiation efficiency relative to wildtype. A further survey of RNAs encoding regulators of growth and differentiation, cell cycle progression, and cell signaling revealed similar or identical expression profiles for most genes as well as several noteworthy differences. Among these, GDF8 and Msx1 were identified as potentially important regulators of the quiescent state whose expression profile differs between mutant and wildtype. Considered together, these data suggest that activated MyoD−/− satellite cells assume a phenotype that resembles in some ways a developmentally "stalled" cell compared to wildtype. However, the MyoD−/− cells are not merely developmentally immature, as they also display novel molecular and cellular characteristics that differ from any observed in wild-type muscle precursor counterparts of any stage.

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