The role of myonuclei in muscle regeneration: An in vitro study

It is well established that during muscle regeneration, the satellite cells which are in a state of mitotic arrest, can initiate cell division to produce myoblasts which subsequently fuse to form myotubes. However, whether myonuclei, contained within damaged myotubes, or “freed” as a result of the trauma, play any role in muscle regeneration remains unresolved. In myogenic cultures, it is possible to obtain renewed myogenesis when initial cultures are sub-cultured. The aim of this study, was to obtain evidence of the participation by myonuclei of primary cultures in myogenesis which occurs subsequently in secondary cultures. In culture, myonuclei can be labelled with H³-thymidine and their ultimate fate, either as “free” myonuclei or myonuclei associated with disrupted myotubes can be followed unequivocally. Three types of experiments are performed: (i) Primary myogenic cultures containing only myotubes are subcultured. (ii) Primary myogenic cultures containing myotubes with labelled myonuclei are disrupted and subcultured. (iii) Primary myogenic cultures containing myotubes with unlabelled myonuclei are mixed with labelled mononucleated myogenic cells and sub-cultured. In all instances no evidence of myogenesis from myonuclei is obtained. It is concluded that myonuclei, which were rendered postmitotic during myogenesis, remain so when muscle is disrupted and cannot re-enter the mitotic cycle.     

Biosynthesis of laminin and fibronectin by rat satellite cells during myogenesis in vitro

The biosynthesis of fibronectin and laminin was studied in satellite cells cultured from adult rat muscles before (day 4) and after fusion and formation of myotubes (day 14) using (35S) methionine as a tracer. The kinetics of incorporation into immunoprecipitable fibronectin and laminin were recorded at intervals from 1.5 to 24 hours of incubation with the tracer from the cells, the pericellular matrix and the culture medium. The rate of synthesis of fibronectin and laminin expressed as dpm/microgram DNA were constant from the mononucleated cell to the myotube state. Both glycoproteins were detected in the cells and in the pericellular matrix. When the results were expressed as the percentage of incorporation into total protein, major changes could be observed in the early phase of the kinetic studies in the cells and the pericellular matrix. Both showed an increase from the mononucleated myoblast to myotube, suggesting that an increasing fraction of total protein biosynthesis is directed towards these two extracellular matrix glycoproteins. At the same time, there was a decrease in the secretion into the medium of freshly synthesized radiolabeled fibronectin and laminin. Our results confirm the synthesis of varying ratios of both extracellular matrix macromolecules by undifferentiated mononucleated myogenic cells as well as myotubes.     

Proliferating satellite cells express acidic fibroblast growth factor during in vitro myogenesis.

Recent in vitro studies have indicated that the proliferation of satellite cells, which are involved in muscular regeneration in vivo, is stimulated by exogenous addition of fibroblast growth factor (FGF). We present evidence that satellite cell cultures produce acidic, but not basic FGF. Acidic or basic FGF content was measured by enzyme immunoassay on cellular extracts after partial purification by heparin-Sepharose chromatography. During maximal cell proliferation, the level of acidic fibroblast growth factor (aFGF) was increased over fivefold from the values obtained before plating. aFGF content drastically dropped at the postmitotic stage to almost the threshold of detection, and remained weak as differentiation was completed. The immunolocalization of aFGF using highly purified anti-aFGF antibodies confirmed these results and indicated that aFGF was cytoplasma- or membrane-associated. Our work suggests that an endogenous production of aFGF by satellite cells may trigger cell proliferation by an intra- or autocrine mechanism, and therefore play an important role in muscular regeneration.     

Temporal appearance of satellite cells during myogenesis.

In this study, differences between fetal and adult myoblasts in clonal and high density culture have been used to determine when adult myoblasts can first be detected during avian development. The results indicate that avian adult myoblasts are apparent as a distinct population of myoblasts during the midfetal stage of development. Three different criteria were used to differentiate fetal and adult myoblasts and demonstrate when adult myoblasts become a major proportion of the myoblast population: (1) differences in slow myosin heavy chain 1 (MHC1) isoform expression, (2) initiation of DNA synthetic activity, and (3) average myoblast length. Fetal chicken (ED10-12) pectoralis muscle (PM) myoblasts form myotubes that express slow MHC1 after prolonged culture, while adult chicken PM myoblasts do not. Fetal avian myoblasts were active in DNA synthesis and large when first isolated, reaching peak rates of synthesis by 24 hr in culture, while adult myoblasts were inactive in DNA synthesis and small when first isolated, only reaching peak rates of DNA synthesis and size at 3 days of incubation. A dramatic decrease in the percentage of muscle colonies with fibers that expressed slow MHC1 was observed between the midfetal stage and hatching in the chicken, along with a corresponding decrease in myoblast DNA synthetic activity and average length during this same period in both the chicken and the quail. Myoblast activity and average length increased again 3-4 days posthatch and a small transient increase in the number of slow MHC1-expressing clones was also associated with the massive growth of muscle that occurs in the neonatal bird. We conclude that adult myoblasts are present as a distinct population of myoblasts at least as early as the midfetal stages of avian development.     

Characterization of myogenesis from adult satellite cells cultured in vitro

We describe several characteristics of in vitro myogenesis from adult skeletal muscle satellite cells from the rat and several amphibian species. The timing of cell proliferation and fusion into myotubes was determined, and in urodeles, myogenesis from satellite cells was clearly demonstrated for the first time. Growth factors are known to stimulate satellite cell proliferation. Acidic FGF mRNA was present in rat satellite cells during proliferation but it was not detected in myotubes. Fibronectin was synthesized in satellite cells during proliferation and expelled into the extracellular medium when the myotubes differentiated. We suggest that fibronectin plays a part in the formation of myotubes, as this process was inhibited by anti-fibronectin IgG. Adult satellite cells might differ from fetal myoblasts since they were observed to exhibit the opposite response to a phorbol ester (TPA) to that of the myoblasts. We therefore examined the possibility that the different levels of protein kinase C activity and different phorbol ester binding characteristics in the two cell types account for these opposite responses. Our results suggest that the difference is not connected with the phorbol ester receptor but might be caused by events subsequent to protein kinase C activation. Localized extracellular proteolytic activity might have a role in cell mobilization and/or fusion when satellite cells are activated. We showed that the content of plasminogen activators, chiefly urokinase, was larger in tissues from slow twitch muscles which regenerate more rapidly than fast muscles. The urokinase level rose sharply in cultures when cells fused into myotubes, and was twice as high in slow muscle cells as in fast ones. We also found that, in vitro, slow muscle satellite cells displayed greater myogenicity, but that phorbol ester inhibited their mitosis and myogenicity. We conclude that satellite cells acquire characteristics which differentiate them from myoblasts and correspond to the fast and slow muscles from which they originate.     

Nerve activity-independent regulation of skeletal muscle atrophy: role of MyoD and myogenin in satellite cells and myonuclei

Electrical activity is thought to be the primary neural stimulus regulating muscle mass, expression of myogenic regulatory factor genes, and cellular activity within skeletal muscle. However, the relative contribution of neural influences that are activity-dependent and -independent in modulating these characteristics is unclear. Comparisons of denervation (no neural influence) and spinal cord isolation (SI, neural influence with minimal activity) after 3, 14, and 28 days of treatment were used to demonstrate whether there are neural influences on muscle that are activity independent. Furthermore, the effects of these manipulations were compared for a fast ankle extensor (medial gastrocnemius) and a fast ankle flexor (tibialis anterior). The mass of both muscles plateaued at approximately 60% of control 2 wk after SI, whereas both muscles progressively atrophied to <25% of initial mass at this same time point after denervation. A rapid increase in myogenin and, to a lesser extent, MyoD mRNAs and proteins was observed in denervated and SI muscles: at the later time points, these myogenic regulatory factors remained elevated in denervated, but not in SI, muscles. This widespread neural activity-independent influence on MyoD and myogenin expression was observed in myonuclei and satellite cells and was not specific for fast or slow fiber phenotypes. Mitotic activity of satellite and connective tissue cells also was consistently lower in SI than in denervated muscles. These results demonstrate a neural effect independent of electrical activity that 1) helps preserve muscle mass, 2) regulates muscle-specific genes, and 3) potentially spares the satellite cell pool in inactive muscles.     

Glycosphingolipid biosynthesis during myogenesis of rat L6 cells in vitro

Summary Glycosphingolipid biosynthesis was examined using [³H]-galactose as a precursor as rat L6 myoblasts fused to form multinucleated myotubes. Incorporation of label into neutral glycolipids decreased steadily as the population of myotubes increased, so that final biosynthesis was one-half that observed with myoblasts (p < 0.02). Conversely, ganglioside biosynthesis doubled during myoblast confluency (p < 0.02) and then decreased as myotubes formed. Qualitatively, L6 cells synthesized large amounts of ganglioside GM3 during all myogenic phases. The major neutral glycosphingolipid products were lactosylceramide and paragloboside (nLcOse₄Cer). Few changes in TLC autoradiographic patterns were noted during differentiation, with the exception of a slight decrease in ganglioside GM1. The results indicate that the biosynthesis of glycosphingolipids is tightly regulated during myogenesis in vitro and suggest a role for membrane gangliosides in muscle cell differentiation.Abbreviations GM1 II³NeuAc-GgOse₄Cer - GM3 II³NeuAc-GgOse²Cer - MG4 IV³NeuAc-nLcOse₄Cer - MG6 VI³NeuAc V⁴Gal-IV³GlcNAc-nLcOse⁴Cer - TLC Thin-Layer Chromatography - DMEM Dulbecco's Modified Eagles' Medium     

Growth and cell kinetics of human hair papilla cells in vitro. An autoradiographic and flow cytometric study

Hair papilla, a distinct specialized dermal compartment, plays a fundamental role in the biology of hair growth. Recently some attention has been focused on hair papilla cells (HPC) as possible targets for drugs influencing the hair growth. Isolation and cultivation of the HPC facilitates screening for such drugs. In the present work, growth and cell kinetics of human occipital scalp follicle HPC have been studied in vitro. HPC grow according to a Gompertz function, i.e. with considerable growth delay long before becoming confluent cultures, due probably to elongation of the potential doubling time (Tpot) and to a parallel increasing cell loss rate. The [3H]dT labelling index of the HPC strongly depends on the age of the subculture; the cycle time being about 4 days. A potential doubling time of about 93 h, indicative of growth fraction (GF) = 1, and a duration of S phase and G2 + M phase of about 8 h each were found by the combined application of continuous labelling with [3H]dT and DNA flow cytometry.     

Fate of microtubule-organizing centers during myogenesis in vitro

Microtubule organization and nucleation were studied during in vitro human myogenesis by immunocytology that used monoclonal and polyclonal antitubulin antibodies and a rabbit nonimmune serum that reacts with human centrosomes. In myoblasts, we observed a classical microtubule network centered on juxtanuclear centrosomes. Myotubes possessed numerous microtubules organized in parallel without any apparent nucleation centers. Centrosomes in these cells were not associated one to each nucleus but were often clustered in the vicinity of nuclei groups. They were significantly smaller than those of the mononucleated cells. The periphery of each nucleus in myotubes was labeled with the serum that labels centrosomes suggesting a profound reorganization of microtubule-nucleating material. Regrowth experiments after Nocodazole treatment established that microtubules were growing from the periphery of the nuclei. The redistribution of nucleating material was shown to take place early after myoblast fusion. Such a phenomenon appears to be specific to myogenic differentiation in that artificially induced polykaryons behaved differently: the centrosomes aggregated to form only one or a few giant nucleating centers and the nuclei did not participate directly in the nucleation of microtubules. The significance of these results is discussed in relation to the possible role of the centrosome in establishing cell polarity.     

Switching of filamin polypeptides during myogenesis in vitro

During chicken skeletal myogenesis in vitro, the actin-binding protein filamin is present at first in association with actin filament bundles both in myoblasts and in myotubes early after fusion. Later in mature myotubes it is found in association with myofibril Z disks. These two associations of filamin are separated by a period of several days, during which the protein is absent from the cytoplasm of differentiating myotubes (Gomer, R., and E. Lazarides, 1981, Cell, 23:524-532). To characterize the two classes of filamin polypeptides we have compared, by two-dimensional peptide mapping, 125I-labeled filamin immunoprecipitated from myoblasts and fibroblasts to filamin immunoprecipitated from mature myotubes and adult skeletal myofibrils. Myoblast filamin is highly homologous to fibroblast and purified chicken gizzard filamins. Mature myotube and adult myofibril filamins are highly homologous but exhibit extensive peptide differences with respect to the other three classes of filamin. Comparison of peptide maps from immunoprecipitated 35S-methionine-labeled filamins also shows that fibroblast and myoblast filamins are highly homologous but show substantial peptide differences with respect to mature myotube filamin. Filamins from both mature myotubes and skeletal myofibrils exhibit a slightly higher electrophoretic mobility than gizzard, fibroblast, and myoblast filamins. Short pulse-labeling studies show that mature myotube filamin is synthesized as a lower molecular weight variant and is not derived from a higher molecular weight precursor. These results suggest that myoblast and mature myotube filamins are distinct gene products and that during skeletal myogenesis in vitro one class of filamin polypeptides is replaced by a new class of filamin polypeptides, and that the latter is maintained into adulthood.     

Histones and histone phosphorylation during quail myogenesis in vitro

Cultured quail myoblasts were labelled with 32Pi and nuclear proteins extracted before and after myoblast fusion. Histones H1, H4 and the H1-H2B-H2A complex were all phosphorylated in proliferating prefusion cultures, while histone phosphorylation was absent in B1-arrested postfusion cultures except for minor phosphorylation of the H3-H2B-H2A complex. Postfused cultures were distinguished by the appearance of the histone-like protein whch migrated slightly faster than H1. Histone phosphorylation is therefore correlated with cell proliferation, while the appearance of the new histone-like protein is associated with G1 arrest and the absence of cell division.