Striated acto-myosin fibers can reorganize and register in response to elastic interactions with the matrix

The remarkable striation of muscle has fascinated many for centuries. In developing muscle cells, as well as in many adherent, nonmuscle cell types, striated, stress fiberlike structures with sarcomere-periodicity tend to register: Based on several studies, neighboring, parallel fibers at the basal membrane of cultured cells establish registry of their respective periodic sarcomeric architecture, but, to our knowledge, the mechanism has not yet been identified. Here, we propose for cells plated on an elastic substrate or adhered to a neighboring cell, that acto-myosin contractility in striated fibers close to the basal membrane induces substrate strain that gives rise to an elastic interaction between neighboring striated fibers, which in turn favors interfiber registry. Our physical theory predicts a dependence of interfiber registry on externally controllable elastic properties of the substrate. In developing muscle cells, registry of striated fibers (premyofibrils and nascent myofibrils) has been suggested as one major pathway of myofibrillogenesis, where it precedes the fusion of neighboring fibers. This suggests a mechanical basis for the optimal myofibrillogenesis on muscle-mimetic elastic substrates that was recently observed by several groups in cultures of mouse-, human-, and chick-derived muscle cells.     

Differences in the Expression and Distribution of Flotillin-2 in Chick,Mice and Human Muscle Cells

Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.     

Soluble age-related factors from skeletal muscle which influence muscle development

Successful regeneration of damaged striated muscle in adult mice is dependent on the regeneration of newly differentiated myofibers from proliferating satellite cells and inhibition of scar tissue formation by fibroblasts. As with most tissues, the ability of skeletal muscle to regenerate decreases in older animals. In this study, we have analysed soluble extracts from intact and regenerating skeletal muscle from mice of different ages for their ability to affect avian myogenesis in tissue culture. We were interested in determining whether an age-dependent difference could be detected with this tissue culture bioassay system. Total cell proliferation in the cultures, measured by [3H]thymidine incorporation was increased equally by muscle extracts from both young and older mice but the resulting cell populations differed in proportion of cell types. The ratio of myoblasts to fibroblasts was significantly greater in cultures exposed to extracts from younger mouse muscle as compared with cultures exposed to extracts from older animals. This age-related activity was found to reside in a low molecular weight (MW) (greater than 12 kD) component of the extract. This fraction had dissimilar effects on myoblasts and fibroblasts. Relative to saline controls, myoblast proliferation was increased and fibroblast proliferation decreased. The low MW fraction from younger mouse muscle extracts stimulated myogenic cell proliferation and myotube formation to a greater extent than the similar fraction prepared from older mouse muscle. Conversely, younger mouse muscle fractions had significantly greater inhibitory activity against fibroblast proliferation than did older mouse muscle fractions.     

Skeletal muscle satellite cell diversity: satellite cells form fibers of different types in cell culture

Following skeletal muscle injury, new fibers form from resident satellite cells which reestablish the fiber composition of the original muscle. We have used a cell culture system to analyze satellite cells isolated from adult chicken and quail pectoralis major (PM; a fast muscle) and anterior latissimus dorsi (ALD; a slow muscle) to determine if satellite cells isolated from fast or slow muscles produce one or several types of fibers when they form new fibers in vitro in the absence of innervation or a specific extracellular milieu. The types of fibers formed in satellite cell cultures were determined using immunoblotting and immunocytochemistry with monoclonal antibodies specific for avian fast and slow myosin heavy chain (MHC) isoforms. We found that satellite cells were of different types and that fast and slow muscles differed in the percentage of each type they contained. Primary satellite cells isolated from the PM formed only fast fibers, while up to 25% of those isolated from ALD formed fibers that were both fast and slow (fast/slow fibers), the remainder being fast only. Fast/slow fibers formed from chicken satellite cells expressed slow MHC1, while slow MHC2 predominated in fast/slow fibers formed from quail satellite cells. Prolonged primary culture did not alter the relative proportions of fast to fast/slow fibers in high density cultures of either chicken or quail satellite cells. No change in commitment was observed in fibers formed from chicken satellite cell progeny repeatedly subcultured at high density, while fibers formed from subcultured quail satellite cell progeny demonstrated increasing commitment to fast/slow fiber type formation. Quail satellite cells cloned from high density cultures formed colonies that demonstrated a similar change in commitment from fast to fast/slow, as did serially subcloned individual satellite cell progeny, indicating that the observed change from fast to fast/slow differentiation resulted from intrinsic changes within a satellite cell. Thus satellite cells freshly isolated from adult chicken and quail are committed to form fibers of at least two types, satellite cells of these two types are found in different proportions in fast and slow muscles, and repeated cell proliferation of quail satellite cell progeny may alter satellite cell progeny to increasingly form fibers of a single type.     

Cytoskeletal heart-enriched actin-associated protein (CHAP) is expressed in striated and smooth muscle cells in chick and mouse during embryonic and adult stages

We recently identified a new Z-disc protein, CHAP (Cytoskeletal Heart-enriched Actin-associated Protein), which is expressed in striated muscle and plays an important role during embryonic muscle development in mouse and zebrafish. Here, we confirm and further extend these findings by (i) the identification and characterization of the CHAP orthologue in chick and (ii) providing a detailed analysis of CHAP expression in mouse during embryonic and adult stages. Chick CHAP contains a PDZ domain and a nuclear localization signal, resembling the human and mouse CHAPa. CHAP is expressed in the developing heart and somites, as well as muscle precursors of the limb buds in mouse and chick embryos. CHAP expression in heart and skeletal muscle is maintained in adult mice, both in slow and fast muscle fibers. Moreover, besides expression in striated muscle, we demonstrate that CHAP is expressed in smooth muscle cells of aorta, carotid and coronary arteries in adult mice, but not during embryonic development.     

Activity-dependent gene regulation in conditionally-immortalized muscle precursor cell lines

Skeletal muscle contractile activity has been implicated in many aspects of muscle cell differentiation and maturation. Much of the research in this area has depended upon costly and labor-intensive cultures of isolated primary muscle cells because widely available immortalized muscle cell lines often do not display a high level of either spontaneous or stimulated contractile activity. We sought to develop conditionally-immortalized skeletal muscle cell lines that would provide a source of myofibers that exhibit robust spontaneous contractile activity similar to primary muscle cultures. Using a tetracycline-regulated retroviral vector expressing a temperature-sensitive T-antigen to infect primary myoblasts, we isolated individual clonal muscle precursor cell lines that have characteristics of activated satellite cells during growth and rapidly differentiate into mature myotubes with spontaneous contractile activity after culture in non-transformation-permissive conditions. Comparison of these cell lines (known as rat myoblast-like tetracycline (RMT) cell lines) to primary cell cultures revealed that they share a wide variety of morphological, physiological, and biochemical characteristics. Most importantly, the time-course and extent of activity-dependent gene regulation observed in primary cell culture for all genes tested, including subunits of the nicotinic acetylcholine receptor (nAChR), muscle specific kinase (MuSK), and myogenin, is reproduced in RMT lines. These immortalized cell lines are a useful alternative to primary cultures for studying muscle differentiation and molecular and physiological aspects of electrical activity in muscle fibers.     

Biological studies of a putative avian muscle-derived neurotrophic factor that prevents naturally occurring motoneuron death in vivo

A series of in vivo studies have been carried out using the chick embryo to address several critical questions concerning the biological, and to a lesser extent, the biochemical characteristics of a putative avian muscle-derived trophic agent that promotes motoneuron survival in vivo. A partially purified fraction of muscle extract was shown to be heat and trypsin sensitive and rescued motoneurons from naturally occurring cell death in a dose-dependent fashion. Muscle extract had no effect on mitotic activity in the spinal cord and did not alter cell number when administered either before or after the normal cell death period. The survival promoting activity in the muscle extract appears to be developmentally regulated. Treatment with muscle extract during the cell death period did not permanently rescue motoneurons. The motoneuron survival-promoting activity found in skeletal muscle was not present in extracts from a variety of other tissues, including liver, kidney, lung, heart, and smooth muscle. Survival activity was also found in extracts from fetal mouse, rat, and human skeletal muscle. Conditioned medium derived from avian myotube cultures also prevented motoneuron death when administered in vivo to chick embryos. Treatment of embryos in ovo with muscle extract had no effect on several properties of developing muscles. With the exception of cranial motoneurons, treatment with muscle extract did not promote the survival of several other populations of neurons in the central and peripheral nervous system that also exhibit naturally occurring cell death. Initial biochemical characterization suggests that the activity in skeletal muscle is an acidic protein between 10 and 30 kD. Examination of a number of previously characterized growth and trophic agents in our in vivo assay have identified several molecules that promote motoneuron survival to one degree or another. These include S100β, brain-derived neurotrophic factor (BDNF), neurotrophin 4/5 (NT-4/5), ciliary neurotrophic factor (CNTF), transforming growth factor β (TGFβ), platelet-derived growth factor-AB (PDGF-AB), leukemia inhibitory factor (CDF/LIF), and insulin-like growth factors I and II (IGF). By contrast, the following agents were ineffective: nerve growth factor (NGF), neurotrophin-3 (NT3), epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF, bFGF), and the heparin-binding growth-associated molecule (HB-GAM). Of those agents that were effective, CDF/LIF, IGF-1 and -2, BDNF, and TGF are reported to be expressed in developing or adult muscle. Studies are underway to determine whether the survival activity found in avian muscle extract can be accounted for by one or more of these growth factors. Of all the tissue extracts and purified proteins tested here, only the neurotrophins—NGF, NT-3, and BDNF (but not NT-4/5)—rescured sensory neurons from naturally occurring cell death. © 1993 John Wiley & Sons, Inc.     

An embryonic pineal body as a multipotent system in cell differentiation

The differentiating potency of pineal cells from 8-day quail embryos was studied with cell culture. It was found that the differentiation of striated muscle fibres occurred abundantly in the pineal cells cultured in hypertonic culture conditions. Muscle nature of these fibres was confirmed by utilizing the antiserum against the striated muscle type creatine kinase (MM-CK). When CO2, NAHCO3, NaCl, KCl and MgCl2 were added in hypertonic concentrations, extensive myogenesis occurred in cultured pineal cells. Myogenesis in pineal cultures began as early as 2 days and, after 3 days in the medium with 75 mM additional NaCl, reached 100-fold when compared with that in the isotonic medium. Muscle fibres from pineal cells in culture were similar in morphology to the skeletal muscle fibres of mesodermal origin in situ. Myogenesis of pineal cells under hypertonic conditions was accompanied by the synthesis of a unique 56 x 10(3) Mr protein, which was not found in the intrinsic muscle cells. Clonal cell culture revealed that about 80% of clonable pineal cells were myogenic precursors. Pineal cells of 8-day quail embryos were not only myogenic but oculopotent (melanogenic and lentoidogenic) in cultures. This study examined whether multipotential progenitor cells with both potentials are present in the pineal or not. The results showed that at least 16% of all clonable pineal cells were multipotent precursors. The embryonic pineal is considered to be a typical multipotent system in parallel with the pigmented and neural retina, the neural crest and the teratocarcinoma.     

Expression of the adrenergic phenotype by dorsal root ganglion cells of the quail in culture in vitro

From the results of previous studies, we have suggested that "autonomic" cell precursors exist in latent form in sensory ganglia of avian embryos. The potentialities can be expressed when the ganglia are transplanted into a young embryo host. In the present study, we have observed a similar transformation in cultures of dissociated dorsal root ganglia taken from quail embryos of 7-15 days of incubation. From the 4th day of culture onward, numerous adrenergic cells appear. They display tyrosine hydroxylase immunoreactivity, synthesise and store catecholamines and generally differ in size and shape from primary sensory neurons. They and/or their precursors can actively proliferate in culture. The differentiation of these catecholaminergic cells, which can not be detected in quail dorsal root ganglia during normal development in vivo, is dependent on one or more factors present in 9-day chick embryo extract.     

Increased viability and differentiation of normal and dystrophic striated muscle in vitro

Summary Primary cultures of muscle from normal (line 412) and dystrophic (line 413) chick embryos were exposed to corticosterone-21-acetate (C-21-A) or sodium ibuprofen (Motrin) for 28 d after myotube formation. Ibuprofen (0.5 to 500 μg/ml) or C-21-A (0.4 to 40 μg/ml)-treated cultures were fixed and assessed semiquantitatively using phase microscopy. On this basis, ibuprofen (50 μg/ml) and C-21-A (40 μg/ml) seemed to be effective in maintaining both normal and dystrophic muscle cultures. Using ibuprofen and C-21-A at these concentrations, experiments were repeated and analyzed quantitatively. Ibuprofen maintained culture viability (up to 68% more myotubes than untreated controls) but had no significant effect on the number of striated cells. C-21-A effectively maintained culture viability (up to 73% increase) and strongly promoted the formation of striated cells in these cultures (up to a sixfold increase). Both normal and dystrophic cultures were affected similarly by these agents, but the dystrophic cultures showed more consistent if not more extensive improvements in the parameters examined here. Thus, it seems that ibuprofen and C-21-A may affect both normal and dystrophic muscle directly to maintain survival and even promote differentiation.     

Differences in the Developmental Fate of Cultured and Noncultured Myoblasts When Transplanted into Embryonic Limbs

Myoblasts from embryonic, fetal, and adult quail and chick muscles were transplanted into limb buds of chick embryos to determine if myoblasts can form muscle fibers in heterochronic limbs and to define the conditions that affect the ability of transplanted cells to populate newly developing limb musculature. Myoblasts from each developmental stage were either freshly isolated and transplanted or were cultured prior to transplantation into limb buds of 4- to 5-day (ED4-5) chick embryos. Transplanted myoblasts, regardless of the age of the donor from which they were derived, formed muscle fibers within embryonic limb muscles. Transplanted cloned myoblasts formed muscle fibers, although there was little evidence that the number of transplanted myoblasts significantly increased following transplantation or that they migrated any distance from the site of injection. The fibers that formed from transplanted clonal myoblasts often did not persist in the host limb muscles until ED10. Diminished fiber formation from myoblasts transplanted into host limbs was observed whether myoblasts were cloned or cultured at high density. However, when freshly isolated myoblasts were transplanted, the fibers they formed were numerous, widely dispersed within the limb musculature, and persisted in the muscles until at least ED10. These results indicate that transplanted myoblasts of embryonic, fetal, and adult origin are capable of forming fibers during early limb muscle formation. They also indicate that even in an embryonic chick limb where proliferation of endogenous myoblasts and muscle fiber formation is rapidly progressing, myoblasts that are cultured in vitro do not substantially contribute to long-term muscle fiber formation after they are transplanted into developing limbs. However, when the same myoblasts are freshly isolated and transplanted without prior cell culture, substantial numbers of fibers form and persist after transplantation into developing limbs. Thus, these studies demonstrate that the extent to which transplanted myoblasts fuse to form fibers which persist in host musculature depends upon whether donor myoblasts are freshly isolated or maintained in vitro prior to injection.     

Cellular and Secreted Forms of Acetylcholinesterase in Mouse Muscle Cultures

When grown in primary cell culture in the absence of neurons, muscle cells from a variety of species synthesize several forms of acetylcholinesterase (AChE), including the collagen-tailed A12 form. A12 AChE has been the subject of much study because it is thought to be a major functional enzyme form normally found in the basal lamina at the neuromuscular junction. In this paper, we show that muscle fibers derived from mouse embryos and neonates are also able to synthesize substantial percentages of their AChE as the A12 form when grown in vitro. This synthesis is modulated by a process associated with spontaneous muscle contractile activity since both total enzyme levels and the proportion of A12 AChE expressed on the cell surface are decreased when the cells are grown in the sodium channel blocker tetrodotoxin, which blocks muscle contraction. On the other hand, when the cells are treated with veratridine, which opens sodium channels, thereby mimicking one aspect of muscle contraction, their AChE levels are comparable to those of untreated cells. Although smaller in magnitude, these changes are similar to those seen in rat muscle cultures. A novel feature of mouse muscle cultures, not seen in those from rat and chick, is the presence of a secreted enzyme form that sediments in the same position as the cellular A12 form (when separated on sucrose density gradients containing high salt) and is also collagenase sensitive.     

Lasp-2 expression, localization, and ligand interactions: a new Z-disc scaffolding protein

The nebulin family of actin-binding proteins plays an important role in actin filament dynamics in a variety of cells including striated muscle. We report here the identification of a new striated muscle Z-disc associated protein: lasp-2 (LIM and SH3 domain protein-2). Lasp-2 is the most recently identified member of the nebulin family. To evaluate the role of lasp-2 in striated muscle, lasp-2 gene expression and localization were studied in chick and mouse tissue, as well as in primary cultures of chick cardiac and skeletal myocytes. Lasp-2 mRNA was detected as early as chick embryonic stage 25 and lasp-2 protein was associated with developing premyofibril structures, Z-discs of mature myofibrils, focal adhesions, and intercalated discs of cultured cardiomyocytes. Expression of GFP-tagged lasp-2 deletion constructs showed that the C-terminal region of lasp-2 is important for its localization in striated muscle cells. Lasp-2 organizes actin filaments into bundles and interacts directly with the Z-disc protein alpha-actinin. These results are consistent with a function of lasp-2 as a scaffolding and actin filament organizing protein within striated muscle Z-discs.     

Electrosensitive polyacrylic acid/fibrin hydrogel facilitates cell seeding and alignment

Three-dimensional cell culture and conditioning is an effective means to guide cell distribution and patterning for tissue engineered constructs such as vascular grafts. Polyacrylic acid is known as an electroresponsive polymer, capable of transforming environmental stimuli like electrical energy to mechanical forces. In this study, we developed an electrosensitive and biocompatible hydrogel-based smart device composed of acrylic acid and fibrin as a tissue engineered construct to mechanically stimulate cells. Structural properties of the hydrogel were assessed by FTIR-ATR, scanning electron microscopy, prosimetry, and swelling measurement. Distribution and alignment of porcine smooth muscle cells (pSMCs) seeded on the surface of lyophilized hydrogels were evaluated and quantified by two-photon laser scanning microscopy. Smooth muscle cell tissue constructs exposed to 2 h of pulsatile electrical stimulation showed significantly enhanced cell penetration and alignment due to dynamic changes produced by alternative swelling and deswelling, in comparison with static samples. On the basis of the results, this hydrogel under electrical stimulation works as a mechanical pump, which can direct SMC alignment and facilitate infiltration and distribution of cells throughout the structure.     

Expression of fast and slow isoforms of the Ca2+-ATPase in developing chick skeletal muscle

The expression of fast and slow isoforms of the sarcoplasmic reticulum Ca2+-ATPase was studied in the developing chick embryo and in tissue-cultured myotubes. Monoclonal antibodies specific for each isoform were used as probes of protein expression. Analysis of expression of Ca2+-ATPase isoforms in chick thigh muscles by immunofluorescence microscopy revealed that all muscle fibers expressed both isoforms during their development. Primary generation muscle fibers expressed predominantly the slow isoform. Secondary generation fibers expressed both isoforms at comparable levels. Loss of the "inappropriate" isoforms occurred late in embryonic development. Immunoblot analysis of embryonic thigh muscle proteins indicated that the expression of the slow isoform varied little from embryonic Day 6 (ED6) to ED19, while expression of the fast isoform increased dramatically just prior to ED19. Tissue-cultured myotubes derived from ED12 chick thigh muscle myoblasts, plated at high density, expressed both isoforms of the Ca2+-ATPase at very similar levels. Clonal analysis of myoblasts taken from early (ED6) and late (ED12) chick thigh muscles showed that all muscle colonies expressed both forms, consistent with in vivo results. Fiber-type specific isoforms of the Ca2+-ATPase and myosin heavy chain are not coordinately expressed in developing chick skeletal muscle.     

Transformation of chick fibroblast cultures with avian myeloblastosis virus

Cellular transformation was induced with avian myeloblastosis virus strain BAI-A (standard AMV) and with a strain of AMV containing subgroup B only. Cultures of muscle tissues from either chick embryo or day old chicks were used for this study. Results were similar in C/O and C/A cells. Leukemogenic virus was continuously produced by these transformed cultures.     

Lack of differentiation following mouse serum treatment of cultured chick limb myoblasts

This investigation was conducted to assess the effects of mouse serum on chick skeletal muscle cell differentiation. In light of earlier findings of altered membrane phospholipid metabolism following mouse serum treatment of Friend erythroleukemic and chick chondrogenic cells, it was of interest to determine whether similar changes would modulate the fusion of mononucleated myoblasts, which is necessary for the formation of multinucleated skeletal muscle fibers. When mouse serum is added to low density cultures of enriched chick myoblasts shortly following cell attachment to the substratum, fusion is inhibited and neutral lipid accumulation ensues. There is an early inhibitory effect on DNA synthesis but not on protein synthesis. There is no increase in the uptake of 2-deoxyglucose following insulin stimulation of the cells, which suggests that while the cells are accumulating large amounts of lipid, they are not being converted into typical adipocytes. Finally, even in cultures of mouse serum-treated cells that undergo significant fusion, one observes thinner myotubes that do not spontaneously contract as do those of control cultures, as well as a disorganization of fluorescently stained actin and myosin myofilaments. These findings demonstrate that mouse serum acts in a dose-dependent manner, is not cytotoxic to the cells, but is capable of modulating normal developmental events of myoblasts as reported for other cell and tissue types.     

Differentiation of myogenic cells in micromass cultures of cells from chick facial primordia

Antibodies to the myosin heavy chains of striated muscle were used to trace myogenic differentiation in the developing face and in cultures of cells from the facial primordia of chick embryos. In the intact face, myogenic cells differentiate first in the mandibular primordia and can be detected at stage 28. The early muscle blocks contain both fast and slow classes of myosin heavy chains. At stages 20 and 24, no myogenic cells are found in any of the facial primordia. However, when the cells are placed in micromass (high density) cultures, myogenic cells differentiate, revealing the presence of potentially myogenic cells in all the facial primordia. The number of myogenic cells bears no consistent relationship to the extent and pattern of chondrogenesis. Therefore the ability of the cell populations of the facial primordia to differentiate into cartilage when placed in culture is independent of the muscle cell lineage. The facial primordia represent a mixed cell population of neural crest and mesodermal cells from at least as early as stage 18.     

Electrogenesis of embryonic chick skeletal muscle cells differentiated in vitro

Electrogenesis of embryonic chick skeletal muscle cells differentiated in monolayer cultures was investigated. Muscle fibers in vitro generate spike potentials similar to those of fibers in vivo. However, other responses, plateaux resembling those in heart muscle, are also elicited. These results suggest that a functional differentiation exists in cultured muscle fibers.