Proliferative Dynamics and the Role of FGF2 During Myogenesis of Rat Satellite Cells on Isolated Fibers

Satellite cells are situated between the basement membrane and the plasma membrane of the myofiber and are thought to be the primary source of myogenic cells in postnatal and adult skeletal muscle [8, 36; reviewed in 14, 27, 52]. Individual myofibers become encased by a basement membrane during late embryogenesis and it is at that stage that the distinction of satellite cells by their morphology and location is first possible [reviewed in 27, 63]. In vivo studies analyzing the incorporation of 3H-thymidine into satellite cell nuclei and, subsequently, into fiber nuclei, indicated that at least some of the satellite cells are proliferative in young animals, contributing additional nuclei to the fibers [40]. In the adult, satellite cells are mitotically quiescent but can reinitiate proliferative activity in response to injury, and their progeny eventually fuse into preexisting or new myofibers [15, 33, 50, 51, 54]. Muscle trauma involving fiber death and wound-related processes is not the only condition that leads to satellite cell proliferation; activation of these precursors also occurs in response to stresses such as stretch and compensatory hypertrophy, exercise, and denervation [4, 19, 55, 56, 61; summary in 3, 10, 52]. Myogenic cultures of cells isolated from normal juvenile or adult postnatal muscle are commonly presumed to be cultures of satellite cells [1, 7, 67]. In such in vitro models the myogenic precursors become mitotically active upon culturing and their progeny eventually differentiate and fuse into multinucleated fibers [23, 30, 64, 66]. A number of growth factor families including fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF) have been shown to be involved in the proliferation and differentiation of myogenic cells in such cultures of tissue dissociated satellite cells [1, 2, 17, 18, 22, 28, 31, 60, 70; reviewed in 27, 63]. These cell culture studies contribute immensely to our current understanding of the regulation of satellite cells. However, the association between the satellite cells and their neighboring fibers is not maintained in these cultures. Such interactions between the satellite cells and the myofibers could be important for maintaining the satellite cell in a quiescent or proliferative state [11, 12]. Gaining further understanding regarding the regulation of satellite cell proliferation in vivo can provide further insight for improving the transfer of donor myoblasts into skeletal muscle for cell-mediated gene therapy. We have adopted the rat single fiber system pioneered by Bekoff and Betz [6] and further developed by Bischoff [9, 10] in order to more critically evaluate myogenesis of adult satellite cells in their native position without the complexity of the intact tissue. In this model the fibers are isolated intact, retaining their few satellite cells in the original site underneath the basement membrane. These fiber-associated satellite cells can be induced to proliferate on the isolated fibers as was initially demonstrated by incorporation of 3H-thymidine and autoradiography [9, 10, 12]. More recently, we reported on the utilization of indirect immunofluorescence to trace myogenesis of satellite cells in the isolated fiber model [68]. We demonstrated that myogenesis of satellite cells in their native position follows a highly coordinated, multistep program of regulatory and structural protein expression. As the satellite cells enter the cell cycle their nuclei become positive for proliferating cell nuclear antigen (PCNA) [68]. PCNA is an auxiliary protein to DNA polymerase δ whose levels correlate with DNA synthesis during the cell cycle, becoming maximal during the S phase [5, 13]. We also analyzed the expression of the myogenic regulatory factor proteins MyoD and myogenin by the fiber associated satellite cells. MyoD and myogenin are members of the basic-helix-loop-helix family of myogenic transcription factors [20, 24, 62]. The four known members of this family are thought to be important in myogenic determination and in the progression from proliferation to differentiation during myogenesis [reviewed in 37, 41, 42, 59, 71]. We showed that nuclei of quiescent satellite cells and of the myofiber itself do not express detectable levels of MyoD or myogenin proteins but that activated satellite cells transit through a MyoD-myogenin expression program. In brief, our study demonstrated that the PCNA+ satellite cells coexpress the myogenic regulatory factor protein MyoD and that this dual expression is transient - following about 24 hours in the PCNA+/MyoD+ state the cells become negative for both PCNA and MyoD but are found positive for myogenin. The myogenin+ state is also transient, diminishing after 24 hours. Upon transiting into the myogenin+ state the satellite cells additionally become positive for smooth muscle α-actin and about 50% of these cells are detectable with an antibody against a developmental form of sarcomeric myosin. The expression of smooth muscle α-actin is transient and follows the same kinetics of myogenin expression but the expression of sarcomeric myosin is stably maintained [68]. We additionally demonstrated that FGF2 (basic FGF) can enhance the number of proliferating satellite cells (PCNA+/MyoD+ cells) without suppressing the subsequent transition of the cells into the differentiating, myo-genin+ state [68]. Collectively, our study suggested that FGF2 may be involved in cell cycle entry and/or proliferation of satellite cells but that regardless of the presence of the mitogen, the cells rapidly withdraw from the cell cycle and differentiate into the myogenin+ phenotype. The present study was conducted to analyze in finer details the influence of FGF2 on proliferation of adult rat satellite cells in the isolated fiber model. The study was especially designed to analyze whether the response of satellite cells to FGF2 is limited to specific time in culture. We concluded that maximal response to FGF (as determined by the number of PCNA+ cells) can be reached if FGF2 is added no later than 15-18 hours following culture establishment. Also, a subpopulation of satellite cells remains (or just becomes positive for) PCNA+ at 72 hours in culture only if the cultures are exposed constantly (i.c., up to 72 hours) to FGF2. Taken together, the study support the proposal that FGF enhances the number of satellite cells capable of undergoing proliferation. However, a prolonged maintenance of the proliferative state may require the addition (or removal) of yet unknown agents.