Cell cycle behavior and MyoD expression in response to T3 differ in normal and mdx dystrophic primary muscle cell...

Since mdx limb muscle regeneration in vivo is accompanied by rapid myoblast proliferation and differentiation compared to normal, we tested the possibility that proliferation and differentiation were differentially regulated in normal and mdx dystrophic muscle cells. Cell cycle behavior, MyoD expression, and the effects of thyroid hormone (T3) treatment were examined in primary cultures. Using a 4-hour pulse time for bromodeoxyuridine (BrdU) incorporation during S-phase, the phases of the cell cycle (early S, late S, G(2)/M, and G(0)/G(1)) were separated by 2-colour fluorescence (BrdU/PI) analysis using flow cytometry. The G(0)/G(1)-early S and the late S-G(2)/M transitions were examined under the influence of T3 in cycling normal and mdx muscle cell cultures over a 20-hour time period. Myogenesis and differentiation were assessed morphologically and by immunostaining for MyoD protein. Mdx cultures had fewer cells in G(0)/G(1) at 20 hours and more cells in early and late S-phase compared to normal cultures. T3 significantly increased the proportion of normal cells in early S-phase by 20 hours, and reduced the proportions in G(2)/M phase. Over the same time interval in parallel cultures, the proportion of MyoD+ normal cells decreased significantly. In the absence of T3, mdx cell cultures showed greater proportions of cells in S-phase than normal cultures, and similar increases in S-phase and loss of MyoD expression over time. However, mdx cultures had no change in the proportion that were MyoD+ during T3 treatment. The results confirm that T3 in primary cultures increased proliferation and prevented the de-differentiation of mdx cells to a greater degree than was typical of normal cells. The different susceptibilities to T3-related shifts between proliferation and differentiation observed in vitro support the idea that committed mdx myoblasts may be more activated and proliferative than normal myoblasts during regeneration in vivo.