| We previously identified Xin, a novel actin-binding protein, to be upregulated during the early phases of skeletal muscle regeneration. Here I demonstrate the presence of Xin protein in primary satellite cells and study the effects of reducing endogenous Xin expression on the functional parameters of satellite cell progeny (myoblasts) and on the expression of various proteins within these cells. To localize Xin in the satellite cell, single muscle fibers were harvested from C57BL/6 mice and immunohistochemically double-stained for Xin and the satellite cell marker Syndecan-4. To determine the function of Xin within myoblasts, C2C12 myoblasts were infected with adenoviral Xin shRNA and proliferation, migration and differentiation were assessed. Inhibition of endogenous Xin expression resulted in significant increases in cell proliferation 2 and 3 days post-infection (16 +/- 2.8% and 26 +/- 2.8% increases for days 2 and 3, respectively, P < 0.05 for both days), significant increases in migratory capacity as measured by two different assays (20 +/- 1.2% and 27 +/- 16.4% increases for scratch and chamber migration assays, respectively, P < 0.05 for both assays), and no observable differences in post-differentiation myotube morphology versus control cells. Western analysis of markers of differentiation in Xin-deficient myoblasts showed a significant increase in myosin heavy chain expression after 4 days of differentiation and no change in myoglobin expression either 2d or 4d after the change to differentiation media versus control cells. Additional Western analysis of cytoskeletal proteins in the absence of Xin indicated a significant increase in filamin C expression with no change in either desmin or Homer expression versus control cells. These findings are the first to demonstrate the presence of Xin in the satellite cell and that alterations in Xin expression modify satellite cell function and protein expression. |
