Inhibition of the formation of myotubes in vitro by inhibitors of transglutaminase

The effects of competitive inhibitors of transglutaminase on the formation of myotubes by the fusion of myoblasts in vitro has been investigated. Myotube formation was inhibited when myoblasts from 11-day-old chick embryos were cultured in vitro in the presence of 10 mM histamine or 0.2 mM dansyl cadaverine. The inhibitions observed were reversed when the treated cells were subsequently cultured in normal medium. Glycine methyl ester also inhibited myotube formation but sarcosine methyl ester, which is not a competitive inhibitor of transglutaminase, had little if any inhibitory action. The formation of myotubes was not inhibited by cultivation in normal medium adjusted to pH 8.0-8.1, indicating that the observed effects of histamine and of dansyl cadaverine were not mediated by a lysosomotropic effect. Inhibition of myotube formation in the presence of histamine was accompanied by the production of abnormal multinucleated cells, indicating that myoblast fusion occurred in the treated cultures but that the fused cells failed to elongate into normal myotubes. Transglutaminase activity has been found in cell-free lysates of embryonic chick myoblasts and it is concluded that a transglutaminase enzyme, activated by an increase in the concentration of intracellular Ca2+, plays an important role in stabilising the cytoskeletal network of developing myotubes.     

Muscle and cartilage differentiation in axolotl limb regeneration blastema cultures

A tissue culture system is described for explants of mesenchyme from Ambystoma mexicanum limb regeneration blastemas. Explants were cultured on collagen substrate for 3 weeks in minimal essential medium supplemented with the hormones insulin, thyroxine, somatotropin, and hydrocortisone, plus beef embryo extract (EE), 2%. This medium supported extensive cell migration onto the substrate followed by cell proliferation and differentiation of both cartilage matrix and myotubes. Cultures on plastic substrate, rather than on collagen, displayed similar cell outgrowth and cartilage formation, but relatively little myotube formation. Differentiation in EE-supplemented medium was compared with that in two defined media: Explants in medium containing only the hormones showed little outgrowth or cartilage development and never formed myotubes; medium containing the hormones plus fibroblast growth factor, 50 ng/ml, supported an intermediate degree of outgrowth and cartilage development and occasional myotube formation. Explant size was also a factor: Smaller explants survived and formed myotubes less frequently, even when on collagen in EE-supplemented medium.     

Mannose receptor regulates myoblast motility and muscle growth

Myoblast fusion is critical for the formation, growth, and maintenance of skeletal muscle. The initial formation of nascent myotubes requires myoblast-myoblast fusion, but further growth involves myoblast-myotube fusion. We demonstrate that the mannose receptor (MR), a type I transmembrane protein, is required for myoblast-myotube fusion. Mannose receptor (MR)-null myotubes were small in size and contained a decreased myonuclear number both in vitro and in vivo. We hypothesized that this defect may arise from a possible role of MR in cell migration. Time-lapse microscopy revealed that MR-null myoblasts migrated with decreased velocity during myotube growth and were unable to migrate in a directed manner up a chemoattractant gradient. Furthermore, collagen uptake was impaired in MR-null myoblasts, suggesting a role in extracellular matrix remodeling during cell motility. These data identify a novel function for MR during skeletal muscle growth and suggest that myoblast motility may be a key aspect of regulating myotube growth.     

Combination of agrin and laminin increase acetylcholine receptor clustering and enhance functional neuromuscular junction formation In vitro

Clustering of acetylcholine receptors (AChR) at the postsynaptic membrane is a crucial step in the development of neuromuscular junctions (NMJ). During development and after denervation, aneural AChR clusters form on the sarcolemma. Recent studies suggest that these receptors are critical for guiding and initiating synaptogenesis. The aim of this study is to investigate the effect of agrin and laminin‐1; agents with known AChR clustering activity; on NMJ formation and muscle maturation. Primary myoblasts were differentiated in vitro on collagen, laminin or collagen and laminin‐coated surfaces in the presence or absence of agrin and laminin. The pretreated cells were then subject to innervation by PC12 cells. The number of neuromuscular junctions was assessed by immunocytochemical co‐localization of AChR clusters and the presynaptic marker synaptophysin. Functional neuromuscular junctions were quantitated by analysis of the level of spontaneous as well as neuromuscular blocker responsive contractile activity and muscle maturation was assessed by the degree of myotube striation. Agrin alone did not prime muscle for innervation while a combination of agrin and laminin pretreatment increased the number of neuromuscular junctions formed and enhanced acetylcholine based neurotransmission and myotube striation. This study has direct clinical relevance for treatment of denervation injuries and creating functional neuromuscular constructs for muscle tissue repair. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 551–565, 2016     

Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX-/-) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro.     

Cortisone and dexamethasone inhibit myogenesis by modulating the AKT/mTOR signaling pathway in C2C12

Myogenesis occurs in both the prenatal and postnatal periods and the prenatal myogenesis is related to the postnatal myogenesis and the incidence of disease later in life. Glucocorticoids used as therapeutic agents for many diseases, but cause adverse effects on muscle homeostasis, including defects in fetal muscle development. The action of glucocorticoids on differentiated skeletal muscle was well studied, but their effects on myotube formation have not been well investigated. Dexamethasone (DEX) and cortisone (COR), two synthetic therapeutic glucocorticoids, suppress myotube formation in C2C12 cells. Both COR and DEX attenuated myotube formation through modulation of myogenic regulatory factors. In addition, they affected the IGF/PI3K/AKT/mTOR signaling pathway, resulting in increased proteolytic protein (atrogin-1 and MURF1) for muscle degradation and decreased ribosomal S6 phosphorylation. The current results conclude that COR and DEX inhibit myotube formation in C2C12 cells by modulating both the myogenic program via MRFs and protein metabolism via IGF/PI3K/AKT/mTOR signaling pathway.     

Influence of fibroblast growth factor, insulin-like growth factor I, and transforming growth factor beta on satellite cell type I collagen expression and localization during differentiation

Expression, and temporal and spatial distribution of type I collagen were investigated in chicken satellite cell cultures during differentiation. There was no difference in the relative amounts of type I collagen after treatment with basic fibroblast growth factor (FGF), insulin-like growth factor-I (IGF-I), or transforming growth factor beta 1 (TGF-beta 1). However, myotube morphology was influenced by the presence of the growth factors. The temporal and spatial distribution of type I collagen was also modified. Control cultures maintained a predominant distribution of type I collagen surrounding the cellular area until approximately 48 h after the initiation of fusion whereas cultures with FGF or IGF-I maintained a cellular localization of type I collagen throughout the fusion process. TGF-beta 1 resulted in the early formation of an extracellular network of type I collagen preceding control cultures by approximately 24 h. These results suggest that type I collagen expression but not localization is independent of satellite cell proliferation and differentiation.     

A laminin substrate promotes myogenesis in rat skeletal muscle cultures: analysis of replication and development using antidesmin and anti-BrdUrd monoclonal antibodies

Cells from newborn rat hindlimb show greatly enhanced myogenicity when grown on surfaces coated with poly-L-lysine followed by laminin (PLL/Lam) instead of the collagens routinely used. Coating with poly-L-lysine (PLL) alone or with PLL followed by collagen does not enhance myogenicity. Both myogenic and nonmyogenic cells, as distinguished by a monoclonal antibody specific for desmin, attach equally well to collagen- and laminin-coated surfaces, but there is a two- to five-fold increase in the number of myogenic cells on PLL/Lam by 72 hr, followed by increased myotube formation. To determine whether this increase in myogenic cells was a consequence of a selective increase in proliferation on PLL/Lam, incorporation of 5-bromodeoxyuridine into DNA followed by labeling with anti-BrdUrd antibody was used as an index of cell proliferation. The results indicate that desmin is expressed in replicating rat myoblasts, and that replication of myogenic cells is greatly enhanced on laminin compared to collagen. The rate of replication of nonmyogenic cells is the same on both substrates. Addition of 10 micrograms/ml laminin to the medium of cells seeded on PLL or collagen has no effect on myogenicity. We conclude that a laminin substrate enhances skeletal myogenesis in vitro by promoting selectively the replication of myoblasts. Cultures prepared from fetuses at 17 and 19 days gestation also show enhanced myogenicity when grown on PLL/Lam, while those from 15-day fetuses do not. Growth and development of fetal myoblasts on collagen were very poor, whereas myoblasts from the newborn rat do proliferate and differentiate on this substrate. Thus myogenic cells at different stages of fetal and neonatal development may require and respond to different extracellular environments. Myotube formation in the E63 clone of L8 rat myoblasts is inhibited by PLL/Lam.     

Regulation of myoblast motility and fusion by the CXCR4-associated sialomucin, CD164

Myoblast fusion is fundamental to the development and regeneration of skeletal muscle. To fuse, myoblasts undergo cell-cell recognition and adhesion and merger of membranes between apposing cells. Cell migration must occur in advance of these events to bring myoblasts into proximity, but the factors that regulate myoblast motility are not fully understood. CD164 is a cell surface sialomucin that is targeted to endosomes and lysosomes via its intracellular region. In hematopoietic progenitor cells, CD164 forms complexes with the motility-stimulating chemokine receptor, CXCR4, in response to the CXCR4 ligand, CXCL12/SDF-1 (Forde, S., Tye, B. J., Newey, S. E., Roubelakis, M., Smythe, J., McGuckin, C. P., Pettengell, R., and Watt, S. M. (2007) Blood 109, 1825-1833). We have previously shown that CD164 stimulates myotube formation in vitro. We report here that CD164 is associated with CXCR4 in C2C12 myoblasts. Cells in which CD164 levels are increased or decreased via overexpression or RNA interference-mediated knockdown, respectively, show enhanced or reduced myotube formation and cell migration, the latter both basally and in response to CXCL12/SDF-1. Furthermore, expression of CD164 cytoplasmic tail mutants that alter the endosome/lysosome targeting sequence and, consequently, the subcellular localization in myoblasts, reveals a similar correlation between cell motility and myotube formation. Finally, Cd164 mRNA is expressed in the dorsal somite (the early myogenic compartment of the mouse embryo) and in premuscle masses. Taken together, these results suggest that CD164 is a regulator of myoblast motility and that this property contributes to its ability to promote myoblast fusion into myotubes.     

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.     

Novel method for measuring active tension generation by C2C12 myotube using UV‐crosslinked collagen film

We have developed a novel method for measuring active tension generated by cultured myotubes using UV‐crosslinked collagen film. Skeletal myoblasts cell line C2C12 or human primary skeletal myoblasts were seeded onto a thin (35 µm) collagen film strip, on which they proliferated and upon induction of differentiation they formed multinucleated myotubes. The collagen film–myotube complex contracted upon electric pulse stimulation which could be observed by light microscope. When collagen film–myotube complex were attached to force transducer, active tension generation was observed upon electric pulse stimulation. Measurement of active tension was possible for multiple times for more than 1 month with the same batch of collagen film–myotube complex. Active tension generation capability of C2C12 myotubes increased with progression of differentiation, reaching maximal value 6 days after induction of differentiation. Using this method, we measured the effect of artificial exercise induced by electric pulse on active tension generation capability of C2C12 myotubes. When the electric pulses of 1 Hz were continuously applied to induce artificial exercise, the active tension augmentation was observed. After 1 week of artificial exercise, the active tension reached ～10× of that before the exercise. The increased active tension is attributable to the formation of the sarcomere structure within the myotubes and an increased amount of myotubes on the collagen film. The increased amount of myotubes is possibly due to the suppressed atrophy of myotubes by enhanced expression of Bcl‐2. Biotechnol. Bioeng. 2010; 106: 482–489. © 2010 Wiley Periodicals, Inc.     

A role for phospholipase D3 in myotube formation

Phospholipase D3 (PLD3) is a non-classical, poorly characterized member of the PLD superfamily of signaling enzymes. PLD3 is a type II glycoprotein associated with the endoplasmic reticulum, is expressed in a wide range of tissues and cells, and undergoes dramatic upregulation in neurons and muscle cells during differentiation. Using an in vitro skeletal muscle differentiation system, we define the ER-tethering mechanism and report that increased PLD3 expression enhances myotube formation, whereas a putatively dominant-negative PLD3 mutant isoform reduces myotube formation. ER stress, which also enhances myotube formation, is shown here to increase PLD3 expression levels. PLD3 protein was observed to localize to a restricted set of subcellular membrane sites in myotubes that may derive from or constitute a subdomain of the endoplasmic reticulum. These findings suggest that PLD3 plays a role in myogenesis during myotube formation, potentially in the events surrounding ER reorganization.     

Static magnetic fields enhance skeletal muscle differentiation in vitro by improving myoblast alignment.

Static magnetic field (SMF) interacts with mammal skeletal muscle; however, SMF effects on skeletal muscle cells are poorly investigated. The myogenic cell line L6, an in vitro model of muscle development, was used to investigate the effect of a 80 +/- mT SMF generated by a custom-made magnet. SMF promoted myogenic cell differentiation and hypertrophy, i.e., increased accumulation of actin and myosin and formation of large multinucleated myotubes. The elevated number of nuclei per myotube was derived from increased cell fusion efficiency, with no changes in cell proliferation upon SMF exposure. No alterations in myogenin expression, a modulator of myogenesis, occurred upon SMF exposure. SMF induced cells to align in parallel bundles, an orientation conserved throughout differentiation. SMF stimulated formation of actin stress-fiber like structures. SMF rescued muscle differentiation in the presence of TNF, a muscle differentiation inhibitor. We believe this is the first report showing that SMF promotes myogenic differentiation and cell alignment, in the absence of any invasive manipulation. SMF-enhanced parallel orientation of myotubes is relevant to tissue engineering of a highly organized tissue such as skeletal muscle. SMF rescue of muscle differentiation in the presence of TNF may have important therapeutic implications.     

AMPK activation by AICAR inhibits myogenic differentiation and myostatin expression in Cattle

AMP-activated protein kinase (AMPK) regulates metabolism in skeletal muscle, and myostatin (MSTN) negatively regulates skeletal muscle development and growth. In the present study, AMPK activation and the relationship between AMPK and MSTN during myogenic differentiation were investigated in cultures derived from bovine skeletal muscle. Myoblasts capable of forming myotubes were obtained from bovine skeletal muscle and treated with AICAR to activate AMPK, resulting in suppressed myotube formation. AICAR treatment significantly reduced the expression of MSTN mRNA during myogenic differentiation. Combined treatment with AICAR and MSTN suppressed myotube formation to a greater extent than AICAR alone. SB431542, an inhibitor of MSTN signaling, promoted myotube formation during myogenic differentiation. However, simultaneous treatment with AICAR blocked this effect of SB431542. Therefore, AMPK activation inhibits myogenic differentiation but may suppress MSTN expression to balance muscle development.     

Effects of cis-4-hydroxy- -proline, an inhibitor of Schwann cell differentiation, on the secretion of collagenous and noncollagenous proteins by Schwann cells

The proline analog cis-4-hydroxy- -proline (CHP) was previously shown to inhibit both Schwann cell (SC) differentiation and extracellular matrix (ECM) formation in cultures of rat SCs and dorsal root ganglion neurons. We confirmed that CHP inhibits basal lamina formation by immunofluorescence with antibodies to laminin, type IV collagen, and heparan sulfate proteoglycan. In order to test the hypothesis that CHP inhibits SC differentiation by specifically inhibiting the secretion of collagen, cultures grown in the presence or absence of CHP were metabolically labeled with [³H]leucine and the media were analyzed for relative amounts of (a) collagenous and noncollagenous proteins by assay with bacterial collagenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or (b) triple-helical collagen by pepsin digestion followed by SDS-PAGE. The results indicate that although CHP inhibited the accumulation of secreted collagen in the culture medium and disrupted collagen triple-helix formation, it also significantly inhibited the accumulation of secreted noncollagenous proteins in the medium. CHP had no significant effect on either total protein synthesis (medium plus cell layer) or cell number. We conclude that CHP does not act as a specific inhibitor of collagen secretion in this system, and thus data from these experiments cannot be used to relate SC collagen production to other aspects of SC differentiation. We discuss the evidence for and against specificity of CHP action in other systems.     

Neogenin Regulates Skeletal Myofiber Size and Focal Adhesion Kinase and Extracellular Signal-regulated Kinase Activities In Vivo and In Vitro

A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.     

Effects of cis-4-hydroxy-L-proline, and inhibitor of Schwann cell differentiation, on the secretion of collagenous and noncollagenous proteins by Schwann cells

The proline analog cis-4-hydroxy-L-proline (CHP) was previously shown to inhibit both Schwann cell (SC) differentiation and extracellular matrix (ECM) formation in cultures of rat SCs and dorsal root ganglion neurons. We confirmed that CHP inhibits basal lamina formation by immunofluorescence with antibodies to laminin, type IV collagen, and heparan sulfate proteoglycan. In order to test the hypothesis that CHP inhibits SC differentiation by specifically inhibiting the secretion of collagen, cultures grown in the presence or absence of CHP were metabolically labeled with [3H]leucine and the media were analyzed for relative amounts of (a) collagenous and noncollagenous proteins by assay with bacterial collagenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or (b) triple-helical collagen by pepsin digestion followed by SDS-PAGE. The results indicate that although CHP inhibited the accumulation of secreted collagen in the culture medium and disrupted collagen triple-helix formation, it also significantly inhibited the accumulation of secreted noncollagenous proteins in the medium. CHP had no significant effect on either total protein synthesis (medium plus cell layer) or cell number. We conclude that CHP does not act as a specific inhibitor of collagen secretion in this system, and thus data from these experiments cannot be used to relate SC collagen production to other aspects of SC differentiation. We discuss the evidence for and against specificity of CHP action in other systems.     

cis-4-Hydroxy-L-proline and ethyl-3,4-dihydroxybenzoate prevent myogenesis of C2C12 muscle cells and block MyoD1 and myogenin expression.

cis-4-Hydroxy-L-proline (cis-OH-Pro) and ethyl-3,4-dihydroxybenzoate (EDHB), two distinct inhibitors of collagen synthesis, prevented myogenesis in C2C12 mouse skeletal muscle cells. Both inhibitors blocked myotube formation and the expression of sarcomeric myosin heavy chain. Northern blot analysis showed that cis-OH-Pro- and EDHB-treated C2C12 muscle cells did not express the myogenic regulatory genes, MyoD1 and myogenin, but continued to express non-muscle isoforms of actin (beta and gamma) and alpha-tropomyosin. 10TFL2-3B cells, a C3H10T1/2 cell line permanently transfected with myogenin cDNA, constitutively expressed exogenous myogenin in the presence of cis-OH-Pro but failed to activate endogenous myogenin and to undergo myogenesis. These results demonstrate that commitment to terminal differentiation and activation of myogenic regulatory genes requires active synthesis of the extracellular matrix component collagen.     

The calcium-dependent myoblast adhesion that precedes cell fusion is mediated by glycoproteins

Presumptive myoblasts from explants of chick embryo pectoral muscle proliferate, differentiate, and fuse to form multinucleate myotubes. One event critical to multinucleate cell formation is the specific adhesion of myoblasts before union of their membranes. In the studies reported here five known inhibitors of myotube formation--trifluoperazine, sodium butyrate, chloroquine, 1,10 phenanthroline, and tunicamycin--were tested for their effect on the Ca++-dependent myoblast adhesion step. The first four inhibitors of myotube formation do not perturb myoblast adhesion but rather block fusion of aggregated cells, which suggests that these agents perturb molecular events required for the union of the lipid bilayers. By contrast, tunicamycin exerts its effect by inhibiting the myoblast adhesion step, thereby blocking myotube formation. The effect of tunicamycin can be blocked by a protease inhibitor, however, which implies that the carbohydrate residues protect the glycoproteins from proteolytic degradation rather than participate directly in cell-cell adhesion. Whereas trypsin treatment of myoblasts in the absence of Ca++ destroys the cells' ability to exhibit Ca++-dependent adhesion, the presence of Ca++ during trypsin treatment inhibits the enzyme's effect, which suggests that myoblast adhesion is mediated by a glycoprotein(s) that has a conformation affected by Ca++. Finally, myoblast adhesion is inhibited by an antiserum raised against fusion-competent myoblasts. The effect of the antiserum is blocked by a fraction from the detergent extract of pectoral muscle that binds to immobilized wheat germ agglutinin, which again suggests that glycoproteins mediate Ca++-dependent myoblast adhesion.     

Simple micropatterning method for enhancing fusion efficiency and responsiveness to electrical stimulation of C2C12 myotubes

Cultured myotubes induced in vitro from myoblast cell lines have been widely used to investigate muscle functional properties and disease‐related biological phenotypes. Until now, several cell patterning techniques have been applied to regulate in vitro myotube structures. However, these previous studies required specific geometry patterns or soft materials for inducing efficient myotube formation. Thus, more simple and easy handling method will be promising. In this study, we aimed to provide a method to form C2C12 myotubes with regulated sizes and orientations in simple line patterns. We used a poly(dimethylsiloxane) (PDMS) stamp and a 2‐methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to fabricate line patterns for myotube formation onto a culture dish. We confirmed that C2C12 myotubes of well‐defined size and orientation were reproducibly formed. In particular, myotubes formed in the micropatterned lines showed the increased fusion efficiency. Then, functional dynamics in the micropatterned myotubes were detected and analyzed using a calcium imaging method. We confirmed micropatterning in line patterns enhanced the responsiveness of myotubes to external electrical stimulations. These results indicate that micropatterning myoblasts with the MPC polymer is a simple and effective method to form functional myotube networks. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:220–225, 2015