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

Abstract

Myogenic precursors in adult skeletal muscle (satellite cells) are mitotically quiescent but can proliferate in response to a variety of stresses including muscle injury. To gain further understanding of adult myoblasts, we are analyzing myogenesis of satellite cells on fibers isolated from adult rat muscle. In this culture model, satellite cells are maintained in their in situ position underneath the fiber basement membrane. Employing two different approaches to monitor proliferation of satellite cells on isolated fibers (autoradiography following ³H-thymidine incorporation and immunofluorescence of cells positive for proliferating cell nuclear antigen (PCNA)), we show in the present study that satellite cells initiate cell proliferation at 12 to 24 hours following fiber culture establishment and that cell proliferation is reduced to minimal levels by 60 to 72 hours in culture. Maximal number of proliferating cells is seen at 36 to 48 hours in culture. These PCNA+ satellite cells transit into the differentiated, myogenin+ state following about 24 hours in the proliferative state. Continuous exposure of the fiber culture to FGF2 (basic FGF; added at the time of culture establishment) leads to a 2 fold increase in the number of PCNA+ cells by 48 hours in culture but the overall schedule of proliferation and transition into the myogenin+ state is not affected. Delaying the addition of FGF2 until 15 to 18 hours following the initiation of the fiber culture does not reduce its effect. However, the addition of FGF2 at 24 hours or later results in a progressive reduction in the number of proliferating satellite cells. Exposure of fiber cultures to transforming growth factor β (TGFβ1) leads to a reduction in the number of proliferating cells in both the absence or presence of FGF2. We propose that FGF2 enhances the number of proliferating cells by facilitating the recruitment of additional satellite cells from the quiescent state. However, satellite cells on isolated fibers conform to a highly coordinated program and rapidly transit from proliferation to differentiation regardless of the presence of FGF. The identification of agents that can prolong the proliferative state of satellite cells when the cells undergo myogenesis in their native position by the intact myofiber might be useful in improving myoblast transplantation into skeletal muscle for cell-mediated gene therapy.