Temporal expression of regulatory and structural muscle proteins during myogenesis of satellite cells on isolated adult rat 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 analyzed myogenesis of satellite cells on intact fibers isolated from adult rat muscle. In this culture model, satellite cells are maintained in their in situ position underneath the fiber basement membrane. In the present study patterns of satellite cell proliferation, expression of myogenic regulatory factor proteins, and expression of differentiation-specific, cytoskeletal proteins were determined, via immunohistochemistry of cultured fibers. The temporal appearance and the numbers of cells positive for proliferating cell nuclear antigen (PCNA) or for MyoD were similar, suggesting that MyoD is present in detectable amounts in proliferating but not quiescent satellite cells. Satellite cells positive for myogenin, alpha-smooth muscle actin (alpha SMactin), or developmental sarcomeric myosin (DEVmyosin) appeared following the decline in PCNA and MyoD expression. However, expression of myogenin and alpha SMactin was transient, while DEV-myosin expression was continuously maintained. Moreover, the number of DEVmyosin + cells was only half of the number of myogenin + or alpha SMactin + cells--indicating, perhaps, that only 50% of the satellite cell descendants entered the phase of terminal differentiation. We further determined that the number of proliferating satellite cells can be modulated by basic FGF but the overall schedule of cell cycle entry, proliferation, differentiation, and temporal expression of regulatory and structural proteins was unaffected. We thus conclude that satellite cells conform to a highly coordinated program when undergoing myogenesis at their native position along the muscle fiber.

Fig. 1

Micrographs of fiber cultures maintained in basal medium and reacted via indirect immunofluorescence with an antibody against PCNA (a′–c′), an antibody against MyoD (d′), and antibody against myogenin (e, f). (a–d) Fluorescent micrographs of the same fibers in a′–d′, respectively, stained with DAPI to highlight all nuclei in the micrographic field. (e and f) Three parallel micrographs demonstrating a phase micrograph of an individual fibers (top), a micrograph of the fiber nuclei stained with DAPI (middle), and a micrograph of the fiber reacted with an antibody against myogenin (bottom). Fibers reacted with anti-PCNA or anti-MyoD were maintained in culture for 2 days; fibers reacted with anti-myogenin were maintained in culture for 3 days. Bar, 68 μm for a, a′, b, b′, and e; 43 μm for c, c′, d, d′, and f. Please note that not all positive nuclei or cells on the fibers are in the same focal plane; therefore, not all positive nuclei or cells are in focus in the different micrographs.

Fig. 2

Micrographs of fiber cultures maintained in basal medium and reacted via indirect immunofluorescence with an antibody against DEVmyosin (a′–c′, d) and an antibody against αSMactin (e). (a–c) Fluorescent micrographs of the same fibers in a′–c′, respectively, stained with DAPI to highlight all nuclei in the micrographic field; arrows in c and c′ indicate two fused myosin-positive cells. (d) Parallel phase and fluorescent (anti-myosin stain) micrographs; arrows in d point out the position of the two myosin-positive cells. Fibers reacted with anti-myosin or anti-actin antibodies were maintained in culture for 5 or 3 days, respectively. Bar, 68 μm for a, a′, and e; 43 μm for panels b, b′, c, c′, and d. Please note that not all positive nuclei or cells on the fibers are in the same focal plane; therefore, not all positive nuclei or cells are in focus in the different micrographs.

Fig. 3

Micrographs of fiber cultures isolated from adult rat and maintained in medium containing 10% horse serum and 5% chicken embryo extract for 8 days. (a and a′) Phase and fluorescent micrographs of cultures reacted with the antibody against myogenin via indirect immunofluorescence. (b and b′) Phase and fluorescent micrographs of cultures reacted with the antibody against αSMactin via indirect immunofluorescence. In both cases cultures resemble myogenic mass cultures with mononucleated cells and myotubes; remnants of the original fibers are present as well. Bar, 43 μm.

Fig. 4

Temporal appearance of cells or nuclei positive for PCNA, MyoD, myogenin, αSMactin, and DEVmyosin on cultured rat fibers. Left two panels describe results with fibers maintained in basal medium only. Right two panels describe results with fibers maintained in basal medium to which bFGF was added at 2 ng/ml. Medium (±bFGF) was changed daily. Cells or nuclei positive for the different proteins were revealed with appropriate antibodies employing indirect immunofluorescence. Fiber cultures were routinely monitored using a 25X objective. Numbers reflect averages for 10 fibers, but routinely for each time point, within an individual experiment, a minimum of 30 fibers were analyzed in two or three culture plates. Results are based on several experiments and standard deviation is based on the different experiments. Standard deviation within parallel plates of the same experiments was no more than 5%. See Fig. 5 for the total number of fibers analyzed to construct the time points in the various panels of Fig. 4.

Fig. 5

Analysis of the frequency of fibers containing a specific number of PCNA+ nuclei when fibers are cultured in basal medium in the absence or presence of 2 ng/ml bFGF. Left and right columns describe results in the absence or presence of bFGF, respectively. Total number of fibers for each analysis is indicated in the figures. Data in this figure are the same ones contributing to Fig. 4.

Fig. 6

Analysis of the number of satellite cells on isolated rat fibers following the addition of an antibody that can block the activity of bFGF. Antibody and control IgG were added at 100 μg/ml and bFGF was added at 2 ng/ml. Fiber cultures were exposed to additives continuously (with a change of basal medium ± additives at 24 hr) and fixed 48 hr following initial culturing. Results are based on two independent experiments and each treatment within each experiment was performed on two or three parallel culture plates. Standard deviation between experiments is not shown but was less than 5–10%.