A computerized mechanical cell stimulator for tissue culture: Effects on skeletal muscle organogenesis

Summary A tissue culture system has been developed which can mechanically stimulate cells growing on a highly elastic plastic substratum in a 24-well cell growth chamber. The collagen-coated substratum to which the cells attach and grow in the Mechanical Cell Stimulator (Model I) can be repetitively stretched and relaxed by stepper motor with linear accuracy of 30 μm. The activity controlling unit is an Apple IIe computer interfaced with the cell growth chamber via optical data links and is capable of simulating many of the mechanical activity patterns that cells are subjected to in vivo. Primary avian skeletal myoblasts proliferate and fuse into multinucleated myotubes in this set-up in a manner similar to normal tissue culture dishes. Under static culture conditions, the muscle cells differentiate into networks of myotubes which show little orientation. Growing the proliferating muscle cells on a unidirectional stretching substratum causes the developing myotubes to orient parallel to the direction of movement. In contrast, growing the cells on a substratum undergoing continuous stretch-relaxation cycling orients the developing myotubes perpendicular to the direction of movement. Neither type of mechanical activity significantly affects the rate of cell proliferation of the rate of myoblast fusion into myotubes. These results indicate that during in vivo skeletal muscle organogenesis, when substantial mechanical stresses are placed on skeletal muscle cells by both continuous bone elongation and by spontaneous contractions, only bone elongation plays a significant role in proper fiber orientation for subsequent functional work. Supported by grants NS16753, AR36266, and RR05818 from the National Institutes of Health, Bethesda, MD.     

Maintenance of highly contractile tissue-cultured avian skeletal myotubes in collagen gel

Summary Highly contractile skeletal myotubes differentiated in tissue culture are normally difficult to maintain on collagen-coated tissue culture dishes for extended periods because of their propensity to detach as a sheet of cells from their substratum. This detachment results in the release of mechanical tension in the growing cell “sheet” and, consequently, loss of cellular protein. We developed a simple method of culturing high density contractile primary avian myotubes embedded in a collagen gel matrix (collagel) attached to either a stainless steel mesh or nylon support structure. With this system the cells are maintained in a highly contractile state for extended periods in vitro under tension. Structural integrity of the myotubes can be maintained for up to 10 d in basal medium without serum or embryo extract. Total cellular protein and myosin heavy chain accumulation in the cells can be maintained for weeks at levels which are two to three times those found in timematched controls that are under little tension. Morphologically, the myotubes are well differentiated with structural characteristics of neonatal myofibers. This new collagel culture system should prove useful in the analysis of in vitro gene expression during myotube to myofiber differentiation and its regulation by various environmental factors such as medium growth factors, innervation, and mechanical activity. This work was supported by grant AM 36266 from the National Institutes of Health, Bethesda, MD, and grant NAG2-414 from the National Aeronautics and Space Administration, Washington, D.C. Parts of this work have appeared in abstract form, In Vitro 23:24a; 1987.     

Mechanical signals and IGF-I gene splicing in vitro in relation to development of skeletal muscle

It has been shown that the insulin-like growth factor (IGF-I) gene is spliced in response to mechanical signals producing forms of IGF-I which have different actions. In order to study how mechanical signals influence this gene splicing in developing muscle, C2C12 cells were grown in three-dimensional (3D) culture and subjected to different regimens of mechanical strain. IGF-IEa which initiates the fusion of myoblasts to form myotubes was found to be constitutively expressed in myoblasts and myotubes (held under endogenous tension) and its expression upregulated by a single ramp stretch of 1-h duration but reduced by repeated cyclical stretch. In contrast, mechano growth factor (MGF), which is involved in the proliferation of mononucleated myoblasts that are required for secondary myotube formation and to establish the muscle satellite (stem) cell pool, showed no significant constitutive expression in static cultures, but was upregulated by a single ramp stretch and by cycling loading. The latter types of force simulate those generated in myoblasts by the first contractions of myotubes. These data indicate the importance of seeking to understand the physiological signals that determine the ratios of splice variants of some growth factor/tissue factor genes in the early stages of development of skeletal muscle.     

Myotube Formation on Micro-patterned Glass: Intracellular Organization and Protein Distribution in C2C12 Skeletal Muscle Cells

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. Studies of myocyte differentiation, cell fusion, and muscle repair are limited by an appropriate in vitro muscle cell culture system. We developed a novel cell culture technique [two-dimensional muscle syncytia (2DMS) technique] that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. This technique is based on UV lithography–produced micro-patterned glass on which conventionally cultured C2C12 myoblasts proliferate, align, and fuse to neatly arranged contractile myotubes in parallel arrays. Combining this technique with fluorescent microscopy, we observed alignment of actin filament bundles and a perinuclear distribution of glucose transporter 4 after myotube formation. Newly formed myotubes contained adjacently located MyoD-positive and MyoD-negative nuclei, suggesting fusion of MyoD-positive and MyoD-negative cells. In comparison, the closely related myogenic factor Myf5 did not exhibit this pattern of distribution. Furthermore, cytoplasmic patches of MyoD colocalized with bundles of filamentous actin near myotube nuclei. At later stages of differentiation, all nuclei in the myotubes were MyoD negative. The 2DMS system is thus a useful tool for studies on muscle alignment, differentiation, fusion, and subcellular protein localization. (J Histochem Cytochem 56:881–892, 2008)     

Conditions for isolation and culture of porcine myogenic satellite cells.

Myogenic satellite cells were isolated from semimembranosus muscles of 4-8 week-old pigs. Muscles were ground and incubated in 0.8 mg/ml Pronase solution for 40 min at 37 degrees C. Following enzymatic digestion, cells were separated from muscle debris by differential centrifugation and sequential filtering through 500 and 53 microns nylon mesh. Primary cultures grown in 16 mm diameter cell culture wells were used to evaluate five sera, media, and substrata for their ability to promote satellite cell proliferation and differentiation. Porcine satellite cell proliferation and myotube formation were optimized in cultures grown on gelatin-coated substratum in the presence of Minimum Essential Medium-alpha supplemented with 10% fetal bovine serum (FBS) (P less than 0.01). Maximum fusion was induced by 48 hr exposure to 2% FBS, horse serum, or lamb serum. These data 1) document the first evidence that myogenic satellite cells can be isolated from porcine skeletal muscle, and 2) identify culture conditions which optimize proliferation and myotube formation of porcine satellite cells.     

Overexpression of interleukin-15 induces skeletal muscle hypertrophy in vitro: implications for treatment of muscle wasting disorders

Interleukin-15 (IL-15) is a novel anabolic factor for skeletal muscle which inhibits muscle wasting associated with cancer (cachexia) in a rat model. To develop a cell culture system in which the mechanism of the anabolic action of IL-15 on skeletal muscle could be examined, the mouse C2 skeletal myogenic cell line was transduced with a retroviral expression vector for IL-15 and compared to sister cells transduced with a control vector. Overexpression of IL-15 induced fivefold higher levels of sarcomeric myosin heavy chain and alpha-actin accumulation in differentiated myotubes. Secreted factors from IL-15-overexpressing myogenic cells, but not from control cells, induced increased myofibrillar protein accumulation in cocultured control myotubes. IL-15 overexpression induced a hypertrophic myotube morphology similar to that described for cultured myotubes which overexpressed the well-characterized anabolic factor insulin-like growth factor-I (IGF-I). However, in contrast to IGF-I, the hypertrophic action of IL-15 on skeletal myogenic cells did not involve stimulation of skeletal myoblast proliferation or differentiation. IL-15 induced myotube hypertrophy at both low and high IGF-I concentrations. Furthermore, in contrast to IGF-I, which stimulated only protein synthesis under these culture conditions, IL-15 both stimulated protein synthesis and inhibited protein degradation in cultured skeletal myotubes. These findings indicate that IL-15 action on skeletal myogenic cells is distinct from that of IGF-I. Due to the ability of IGF-I to stimulate cell division and its association with several forms of cancer, controversy exists concerning the advisability of treating cachexia or age-associated muscle wasting with IGF-I. Administration of IL-15 or modulation of the IL-15 signaling pathway may represent an alternative strategy for maintaining skeletal muscle mass under these conditions.     

Entactin promotes adhesion and long-term maintenance of cultured regenerated skeletal myotubes.

The basal lamina protein, laminin, has been shown to promote migration and proliferation of cultured skeletal myoblasts, resulting in increased myotube formation. However, skeletal myotubes adhere poorly to a laminin substrate, and long-term cultures of skeletal myotubes on laminin have not been achieved. We have found that cultured satellite cells from bupivacaine-damaged rat skeletal muscle actively proliferate and differentiate on a diluted Matrigel substrate composed of laminin, type IV collagen, heparan sulfate proteoglycan, and entactin. Myotubes cultured on diluted Matrigel are contractile and have never been observed to detach from the culture dish; rather, myotubes generally atrophy after 2-3 weeks in culture. Antibodies directed against the various protein components of Matrigel were used to determine the role of each component in enhancing muscle differentiation. Anti-laminin impaired satellite cell adhesion, whereas antibodies against either type IV collagen or heparan sulfate proteoglycan had no effect. Anti-entactin did not inhibit attachment, proliferation, or fusion of cultured satellite cells; however, myotubes exposed to anti-entactin failed to adhere to the culture dish after spontaneous myotube contractions began. We conclude that entactin is responsible for long-term maintenance and maturation of contractile skeletal myotubes on a diluted Matrigel substrate. This is the first study to assign a biological function for entactin in myogenesis.     

Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation

This work describes the step-by-step development of a novel, serum-free, in vitro cell culture system resulting in the formation of robust, contracting, multinucleate myotubes from dissociated skeletal muscle cells obtained from the hind limbs of fetal rats. This defined system consisted of a serum-free medium formulation developed by the systematic addition of different growth factors as well as a nonbiological cell growth promoting substrate, N-1[3-(trimethoxysilyl) propyl] diethylenetriamine. Each growth factor in the medium was experimentally evaluated for its effect on myotube formation. The resulting myotubes were evaluated immunocytochemically using embryonic skeletal muscle, specifically the myosin heavy chain antibody. Based upon this analysis, we propose a new skeletal muscle differentiation protocol that reflects the roles of the various growth factors which promote robust myotube formation. Further observation noted that the proposed skeletal muscle differentiation technique also supported muscle–nerve coculture. Immunocytochemical evidence of nerve–muscle coculture has also been documented. Applications for this novel culture system include biocompatibility and skeletal muscle differentiation studies, understanding myopathies, neuromuscular disorders, and skeletal muscle tissue engineering.     

Leukaemia inhibitory factor increases myoblast replication and survival and affects extracellular matrix production: combined in vivo and in vitro studies in post-natal skeletal muscle

Leukaemia inhibitory factor (LIF) has been reported to specifically enhance myoblast proliferation in vitro and increase the number and size of myotubes in regenerating skeletal muscle in vivo. The present study specifically tests the effect of LIF on myoblast replication in vivo. Administration of exogenous LIF by slow release alginate gels in vivo sustained the level of myoblast proliferation at 2 days in regenerating crush-injured muscle. Since the extracellular matrix (ECM) plays an important role in regulating the effects of many growth factors, the hypothesis was tested, both in vivo and in vitro, that some of the beneficial effects of LIF are mediated by modulation of the ECM. The effects of LIF in vivo on the amount and localisation of the ECM molecules, fibronectin, tenascin-C, collagen type IV and laminin were assessed by immunohistochemistry on regenerating skeletal muscle but no influence of LIF on ECM composition was observed. In tissue culture, LIF increased BALB/c myoblast proliferation at day 3 on culture dishes coated with Matrigel and also increased the viability in vitro of BALB/c myoblasts grown under suboptimal conditions. Quantitation of the ECM produced by cultures (enzyme-linked immunosorbent assay) showed that LIF affected the amount of fibronectin, tenascin-C, collagen type IV and laminin produced by fusing myoblasts. No significant affect of LIF was seen on myotube formation either in vitro or in vivo. These combined in vitro and in vivo studies show an effect of LIF on ECM production in vitro, on myoblast survival and on in vivo myoblast replication.     

Oxygen enhances fusion of cultured chick embryo myoblasts

Fusion of mononucleate myoblasts to form multinucleated myotubes increases when skeletal muscle cells are grown in progressively higher oxygen concentrations (5%, 20%, and 40% oxygen). At four days of growth fusion of myoblasts (as expressed by the percent of all muscle nuclei that are located in myotubes) is 57 ± 2% in 5% oxygen, 68 ± 1% in 20% oxygen, and 78 ± 2% in 40% oxygen (P<0.001). However, at a concentration of 40%, oxygen depresses the rate of cell division and thereby affects the number of myoblasts available for fusion. Thus, oxygen concentration significantly modifies growth of skeletal muscle in vitro. Its net effect on myotube formation results from the interaction of its separate effects to enhance cell fusion and to depress cell proliferation.     

Effect of low-intensity magnetic fields on the development of satellite muscle cells of a newborn rat in the primary culture

The influence of Earth magnetic field shielded down to 0.3 microT and static magnetic field (60-160 microT) on the proliferation and differentiation of satellite muscle cells in the primary culture has been investigated. A stimulatory effect of static magnetic fields on the rate of the formation of massive multinucleated myotubes and an increase in the intracellular calcium concentration ([Ca2+]i) have been detected for magnetic fields of the microtesla range. On the other hand, it was shown that the reduction of earth magnetic fields to 0.3 microT leads to the inhibition of proliferation and differentiation of skeletal muscle cells in the primary culture. Since the formation of contractile myotubes during in vitro experiments is similar to the regeneration of skeletal muscle fibers under muscle damage in vivo, it may be concluded that weak magnetic fields have a strong effect on intracellular processes by influencing all phases of muscle fiber formation. It is necessary to take this fact into consideration when forecasting probable complications of skeletal muscle regeneration during long-term exposure of man to low-intensity magnetic fields and also for the potential use of low static magnetic fields as a tool to recover the affected myogenesis.     

Photolithographic patterning of C2C12 myotubes using vitronectin as growth substrate in serum-free medium

The C2C12 cell line is frequently used as a model of skeletal muscle differentiation. In our serum-free defined culture system, differentiation of C2C12 cells into myotubes required surface-bound signals such as substrate-adsorbed vitronectin or laminin. On the basis of this substrate requirement of myotube formation, we developed a photolithography-based method to pattern C2C12 myotubes, where myotubes formed exclusively on vitronectin surface patterns. We have determined that the optimal line width to form single myotubes is approximately 30 mum. To illustrate a possible application of this method, we patterned myotubes on the top of commercial substrate-embedded microelectrodes. In contrast to previous experiments where cell patterning was achieved by selective attachment of the cells to patterned surfaces in a medium that contained all of the factors necessary for differentiation, this study illustrates that surface patterning of a signaling molecule, which is essential for skeletal muscle differentiation in a defined system, can result in the formation of aligned myotubes on the patterns. This technique is being developed for applications in cell biology, tissue engineering, and robotics.     

Cell shape and growth regulation in skeletal muscle: Exogenous versus endogenous factors

Passive stretch (10–12%) of tissue-cultured avian skeletal myotubes in serum-containing medium stimulates myotube growth in a manner analogous to hormonal stimulation of adult muscle. The resulting increase in myotube length is accompanied by marked reduction in the number of surface microvilli seen by scanning electron microscopy. We investigated the possible involvement of exogenous growth factors in the transduction of stretch-induced alterations in cell shape into the concurring biochemical changes that are associated with cell growth. We show that the acute stimulation of myotube amino acid transport and protein synthesis by stretch are independent of serum growth factors in the culture medium by evidence obtained from serum dose-response experiments. The myotubes synthesize and secrete high molecular weight factors into their culture medium, which regulates myotube amino acid transport and protein synthesis. Stretch of the myotubes did not alter the appearance of these factors in the culture medium. The initial growth-related biochemical alterations induced by myotube stretch in vitro thus depend only on events intrinsic to the cells. However, subsequent stretch-induced growth of the myotubes occurs only in serum-containing medium. There are both serum-independent and serum-dependent steps in the transduction of the stretch stimulus into myotube growth.     

IGF-1 induces human myotube hypertrophy by increasing cell recruitment

Insulin-like growth factor-1 (IGF-1) has been shown in rodents (i) in vivo to induce muscle fiber hypertrophy and to prevent muscle mass decline with age and (ii) in vitro to enhance the proliferative life span of myoblasts and to induce myotube hypertrophy. In this study, performed on human primary cultures, we have shown that IGF-1 has very little effect on the proliferative life span of human myoblasts but does delay replicative senescence. IGF-1 also induces hypertrophy of human myotubes in vitro, as characterized by an increase in the mean number of nuclei per myotube, an increase in the fusion index, and an increase in myosin heavy chain (MyHC) content. In addition, muscle hypertrophy can be triggered in the absence of proliferation by recruiting more mononucleated cells. We propose that IGF-1-induced hypertrophy can involve the recruitment of reserve cells in human skeletal muscle.     

Neutrophils injure cultured skeletal myotubes

The purpose of the study was to test the hypothesis that neutrophils can injure cultured skeletal myotubes. Human myotubes were grown and then cultured with human blood neutrophils. Myotube injury was quantitatively and qualitatively determined using a cytotoxicity (51Cr) assay and electron microscopy, respectively. For the 51Cr assay, neutrophils, under non-in vitro-stimulated and N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated conditions, were cultured with myotubes at effector-to-target cell (E:T) ratios of 10, 30, and 50 for 6 h. Statistical analyses revealed that myotube injury was proportional to the E:T ratio and was greater in FMLP-stimulated conditions relative to non-in vitro-stimulated conditions. Transmission electron microscopy, using lanthanum as an extracellular tracer, revealed in cocultures a diffuse appearance of lanthanum in the cytoplasm of myotubes and a localized appearance within cytoplasmic vacuoles of myotubes. These observations and their absence in control cultures (myotubes only) suggest that neutrophils caused membrane rupture and increased myotube endocytosis, respectively. Myotube membrane blebs were prevalent in scanning and transmission electron micrographs of cultures consisting of neutrophils and myotubes (E:T ratio of 5) and were absent in control cultures. These data support the hypothesis that neutrophils can injure skeletal myotubes in vitro and may indicate that neutrophils exacerbate muscle injury and/or delay muscle regeneration in vivo.     

Influence of low-intensity magnetic fields on the development of satellite muscle cells of a newborn rat in primary culture

The influence of Earth magnetic field shielded down to 0.3 μT and static magnetic field (60–160 μT) on the proliferation and differentiation of satellite muscle cells in primary culture has been investigated. A stimulatory effect of static magnetic fields on the rate of the formation of massive multinucleate myotubes and an increase in the intracellular calcium concentration ([Ca²⁺] _i ) have been detected for magnetic fields of the microtesla range. On the other hand, it was shown that the reduction of earth magnetic fields to 0.3 μT leads to inhibition of proliferation and differentiation of skeletal muscle cells in primary culture. Since the formation of contractile myotubes during in vitro experiments is similar to the regeneration of skeletal muscle fibers under muscle damage in vivo, it may be concluded that weak magnetic fields have a strong effect on intracellular processes by influencing all phases of muscle fiber formation. It is necessary to take this fact into consideration when forecasting probable complications of skeletal muscle regeneration during long-term exposure of man to low-intensity magnetic fields and also for the potential use of low static magnetic fields as a tool to recover the affected myogenesis.     

Mechanically induced orientation of adult rat cardiac myocytes in vitro

Summary A population of freshly isolated adult rat cardiac myocytes is spatially oriented using a computerized mechanical cell stimulator device for tissue cultured cells. A continuous unidirectional stretch of the substratum at 60 to 400 μm/min for 120 to 30 min, respectively, during the cell attachment period in serum-free medium induces a significant three-fold increase in the number of rod-shaped myocytes oriented parallel to the direction of movement. The myocytes orient less well with unidirectional substratum stretching after their adhesion to the substratum. In contrast, adult myocytes plated onto a substratum undergoing continuous 10% stretch-relaxation cycling show no significant change in myocyte orientation or cytoskeletal organization. Orientation of rod-shaped myocytes is dependent on several factors other than the type of mechanical activity. These include: a) the speed of substratum movement; b) the final stretch amplitude; and c) the timing between initiation of substratum stretching and adhesion of myocytes to the substratum. Oriented adult rod shaped myocytes representing 65 to 70% of the total myocyte population in this model system can now be submitted to different patterns of repetitive mechanical stimulation for the study of stretch-induced alterations in cell growth and gene expression. This work was supported by grants AR36266, AR39998, and RR05818 from the National Institutes of Health, Bethesda, MD, and grant NAG2-414 from the National Aeronautics and Space Administration, Washington, DC. J.-L. Samuel was a recipient from the Foundation pour la Recherche Médicale.     

ACTH-like peptides in postimplantation mouse embryos: a possible role in myoblast proliferation and muscle histogenesis.

ACTH and related peptides are mitogens for certain mesodermal cell types such as adrenocortical cells, T-lymphocytes, and skeletal myoblasts. In order to postulate a possible physiological role for these peptides in skeletal muscle histogenesis, it is necessary to establish whether they are present in muscle forming anlagens of postimplantation mouse embryos. By radioimmunoassay and immunofluorescence with antibodies specific for ACTH, we have detected these peptides in many areas of mouse embryos including neural tube, limb buds, eye lens, and myotomal muscles. During fetal development, immunoreactivity decreased in muscle tissue and appeared in visceral ganglia. Furthermore, primary myotubes or C2C12 myotubes, but not muscle or 3T3 fibroblasts, release significant levels of ACTH immunoreactive peptides into the culture medium. Using a microassay for mitogen production, primary myotubes or C2C12 myotubes, but not other mesodermal cells (with the exception of dermal fibroblasts) were shown to release factors into the medium which support myoblast proliferation. Neutralizing antibodies against ACTH inhibit myoblast but not fibroblast proliferation in a dose-dependent fashion. Based on these results, we propose that myotube-derived mitogens (including ACTH-like peptides) promote the proliferation of surrounding myoblast during muscle histogenesis in vivo.