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

Since mdx limb muscle regeneration in vivo is accompanied by rapid myoblast proliferation and differentiation compared to normal, we tested the possib ility that proliferation and differentiation were differentially regulated in normal and mdx dystrophic muscle cells. Cell cycle behavior, MyoD expres sion, and the effects of thyroid hormone (T3) treatment were examined in pr imary cultures. Using a 4-hour pulse time for bromodeoxyuridine (BrdU) inco rporation 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) anal ysis using flow cytometry. The G(0)/G(1)-early S and the late S-G(2)/M tran sitions were examined under the influence of T3 in cycling normal and mdx m uscle cell cultures over a 20-hour time period. Myogenesis and differentiat ion were assessed morphologically and by immunostaining for MyoD protein. M dx cultures had fewer cells in G(0)/G(1) at 20 hours and more cells in earl y 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 th e proportions in G(2)/M phase. Over the same time interval in parallel cult ures, 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 My oD expression over time. However, mdx cultures had no change in the proport ion that were MyoD+ during T3 treatment. The results confirm that T3 in pri mary cultures increased proliferation and prevented the de-differentiation of mdx cells to a greater degree than was typical of normal cells. The diff erent susceptibilities to T3-related shifts between proliferation and diffe rentiation observed in vitro support the idea that committed mdx myoblasts may be more activated and proliferative than normal myoblasts during regene ration in vivo. (C) 2000 Wiley-Liss, Inc.