Delayed Dedifferentiation and Retention of Properties in Dissociated Adult Skeletal Muscle Fibers in Vitro

Abstract

Adult skeletal muscle fibers can be isolated and cultured but tend to dedifferentiate and sprout with time in culture. We examined isolated adult mouse flexor digitorum brevis muscle fibers under various culture conditions by monitoring maintenance of the same fibers at 2-d intervals using survival analysis. Fibers plated on laminin and cultured in serum-free media did not show sprouting and exhibited significantly (P < 0.0001) longer survival (median survival time, T(50) = 10.2 d) than fibers in serum-containing media (T(50) = 3.3 d). Cell proliferation was markedly suppressed in serum-free cultures. Multiple or delayed Ca(2+) transients in response to brief field stimulation were often observed in dedifferentiated fibers after several d in serum-containing media but were not observed in fibers in serum-free media. The addition of cytosine arabinoside to serum-containing cultures did not prolong fiber survival (P = 0.39) and did not eliminate sprouting but did greatly suppress proliferation of nonmuscle cells. Fibers cultured in agarose gel with serum exhibited small, bud-like extensions but no sprouts and did not survive as long (T(50) = 6.2 d) as fibers plated on laminin and cultured in serum-free media (T(50) = 10.2 d) did. These results demonstrate that both morphological and physiological properties of fibers become modified in serum-containing media but can be retained by culturing without serum.

Ca2+sparks and T tubule reorganization in dedifferentiating adult mouse skeletal muscle fibers

“…These changes, including loss of striations and the appearance of cytoplasmic sprouts coming out of the ends or sides of dedifferentiating fibers, can occur along the length of the fiber. The exact mechanisms underlying the observed dedifferentiation to a myotube-like structure are not known, but the morphological changes are more pronounced and enhanced by the presence of serum in the culture media (2). During this period of pronounced changes, at approximately five to seven days in serum-containing media, Ca 2ϩ spark activity increased 25-fold when compared with fibers only cultured for one day in serum-containing media.…”

“…Fibers cultured for up to 3 days in serumcontaining medium retain normal morphology and striated appearance. At 5-7 days in culture, many fibers begin to exhibit a change in the adult morphology, characterized by sprouting, fusion with mononuclear cells (presumably myoblasts), and loss of striations, generally beginning at their ends (2). Whether or not the morphological loss of striations and drastic changes in fiber shape observed in cultured fibers represent only fiber remodeling to the developmentally preceding terminal myotube stage, or whether this is true dedifferentiation to a precursor cell type (8,18), remains to be determined.…”

“…In addition to the pronounced morphological changes observed during fiber dedifferentiation in culture, functional changes were also evident. Multiple or delayed Ca 2ϩ transients in response to brief field stimulation were often observed in dedifferentiated fibers (2), indicating alterations in action potential initiation and possibly in excitation contraction (EC) coupling.…”

Measuring mitochondrial respiration in intact single muscle fibers

“…The use of this approach enables the investigator to make measurements with minimal disruption of the myofibers. Additionally, by using appropriate culture conditions, the intact single fibers can be maintained overnight in a standard CO 2 incubator for subsequent measures (4). In these experiments, intact adult muscle fibers were isolated and cultured overnight.…”

Structural and functional evaluation of branched myofibers lacking intermediate filaments

“…Myofibers were plated on extracellular matrix (ECM; Sigma E1270)-coated imaging dishes (P35G-1.0 -14-C, Matek) and incubated for 12-24 h at 37°C and 5% CO2 in DMEM supplemented with 0.2% BSA and 1 l/ml gentamycin before imaging or fixation with 4% paraformaldehyde or electron microscopy (EM) fixative, described below. FDB myofibers can be maintained in culture for over 10 days (5), but all of the myofibers in this study were imaged or fixed for immunolabeling within a 12-h period to avoid potential changes from prolonged incubation (27). Canato et al (6) suggest that FDB enzymatic dissociation may favor survival of a subset of the myofiber population, such that those fibers that are most fragile and/or necrotic do not survive dissociation and the first day in culture (6).…”