Controlling the Orientation and Synaptic Differentiation of Myotubes with Micropatterned Substrates

Micropatterned poly(dimethylsiloxane) substrates fabricated by soft lithography led to large-scale orientation of myoblasts in culture, thereby controlling the orientation of the myotubes they formed. Fusion occurred on many chemically identical surfaces in which varying structures were arranged in square or hexagonal lattices, but only a subset of patterned surfaces yielded aligned myotubes. Remarkably, on some substrates, large populations of myotubes oriented at a reproducible acute angle to the lattice of patterned features. A simple geometrical model predicts the angle and extent of orientation based on maximizing the contact area between the myoblasts and patterned topographic surfaces. Micropatterned substrates also provided short-range cues that influenced higher-order functions such as the localization of focal adhesions and accumulation of postsynaptic acetylcholine receptors. Our results represent what we believe is a new approach for musculoskeletal tissue engineering, and our model sheds light on mechanisms of myotube alignment in vivo.     

Micropatterned polyelectrolyte nanofilms promote alignment and myogenic differentiation of C2C12 cells in standard growth media

Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro‐patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi‐nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer‐by‐layer (LbL) polyelectrolyte multilayer deposition was combined with a micro‐molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self‐assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4‐styrene‐sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly‐L ‐arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)₅‐coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro. Biotechnol. Bioeng. 2013; 110: 586–596. © 2012 Wiley Periodicals, Inc.     

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.     

The roles of RGD and grooved topography in the adhesion, morphology, and differentiation of C2C12 skeletal myoblasts

Both chemical and topographic cues are crucial for the development of skeletal muscle. In this study, the relative roles of both signals in regard to cell adhesion, morphology, and differentiation of C2C12 skeletal myoblasts were investigated. Grooved polystyrene substrates containing grooves with approximately 900 nm in width with 600 nm ridge spans and 665 nm in depth were conjugated with the cell adhesion peptide arginine-glycine-aspartic acid (RGD). RGD conjugation significantly enhanced the adhesion, growth and differentiation of C2C12 cells. On the other hand, anisotropic topography primarily directed the direction and alignment of myoblasts and myotubes. The results in this study provide information regarding the relative roles of chemical and topographic cues in musculoskeletal myogenesis, and are of interest to applications in muscle tissue engineering.     

Micropatterning and Alignment of Skeletal Muscle Myoblasts Using Microflowed Plasma Process

ObjectivesThe primary objective of the study was to optimize micropatterning environments using the microchannel flowed plasma process for controlling the orientation and behaviour of skeletal muscle cells. We have studied the cellular patterning and alignment of skeletal myoblast cells on the various micropattern widths developed on glass substrates.Materials and MethodsIn this method, we have utilized the microchannel flowed plasma process to create micropatterned self-assembled monolayers of octadecyltrichlorosilane and 3-aminopropyltrichlorosilane for creating cell adhesive widths of 20, 200 and 1000 microns on the glass substrates. The micropatterned substrates were characterized by using fluorescein 5(6)-isothiocyanate. Thereafter, the substrates were used to culture and pattern C2C12 and primary rat skeletal muscle cells. Further, we have studied the spatiotemporal variation in the orientation of the cells by using bright field and fluorescence microscopy. The microscopic images were analysed by using orientation order parameter and orientation distribution analysis.ResultsFITC based characterization of micropatterns reveals that the adopted process for micropatterning can effectively create cell adhesive widths with dimensions comparable to the diameter of myofiber. Microscopic observations and the orientation order parameter analysis reveal the precise alignment and specific orientation of myoblasts along the designated cell adhesive widths that closely mimics the physiological scenario. Both the cells showed immediate alignment within smaller cell adhesive widths of 20 and 200 μm. Actin cytoskeletal staining and its orientation distribution analysis of micropattrned C2C12 cells emphasises the influence of micropatterned environment on cytoskeletal actin orientation.ConclusionThis study corroborates the alignment of the myoblasts using surface cues facilitated by changing surface chemistry of the glass substrates. The study promotes the application of a simple micropatterning technique as a useful tool to regulate the orientation and behaviour of skeletal muscle cells. Also, the study emphasizes the role of spatial topography created by surface modification and its effect on cell adhesion and communication of alignment information across the micropatterns. The microchannel flowed plasma process could be applied to selectively pattern different adherent cell types, which could prove to be a useful platform for the exploration of various cellular processes.     

Prion infection of muscle cells in vitro

The prion agent has been detected in skeletal muscle of humans and animals with prion diseases. Here we report scrapie infection of murine C2C12 myoblasts and myotubes in vitro following coculture with a scrapie-infected murine neuroblastoma (N2A) cell line but not following incubation with a scrapie-infected nonneuronal cell line or a scrapie brain homogenate. Terminal differentiation of scrapie-infected C2C12 myoblasts into myotubes resulted in an increase in the expression of the disease-specific prion protein, PrP(Sc). The amount of scrapie infectivity or PrP(Sc) in C2C12 myotubes was comparable to the levels found in scrapie-infected N2A cells, indicating that a high level of infection was established in muscle cells. Subclones of scrapie-infected C2C12 cells produced high levels of PrP(Sc) in myotubes, and the C-terminal C2 polypeptide fragment of PrP(Sc) was found based on deglycosylation and PrP(Sc)-specific immunoprecipitation of cell lysates. This is the first report of a stable prion infection in muscle cells in vitro and of a long-term prion infection in a nondividing, differentiated peripheral cell type in culture. These in vitro studies also suggest that in vivo prion infection of skeletal muscle requires contact with prion-infected neurons or, possibly, nerve terminals.     

Optically controlled contraction of photosensitive skeletal muscle cells

As the skeletal muscle cell is an efficient force transducer, it has been incorporated in bio-microdevices using electrical field stimulation for generating contractile patterns. To improve both the spatial and temporal resolutions, we made photosensitive skeletal muscle cells from murine C2C12 myoblasts, which express channelrhodopsin-2 (ChR2), one of archaea-type rhodopsins derived from green algae Chlamydomonas reinhardtii. The cloned ChR2-expressing C2C12 myoblasts were made and fused with untransfected C2C12 to form multinucleated myotubes. The maturation of myotubes was facilitated by electrical field stimulation. Blue LED light pulse depolarized the membrane potential of a ChR2-expressing myotube and eventually evoked an action potential. It also induced a twitch-like contraction in a concurrent manner. A contraction pattern was thus made with a given pattern of LED pulses. This technique would have many applications in the bioengineering field, such as wireless drive of muscle-powered actuators/microdevices.     

Changes in mitochondrial reactive oxygen species synthesis during differentiation of skeletal muscle cells

Myogenesis is accompanied by an intensive metabolic remodeling. We investigated the mitochondrial reactive oxygen species (ROS) generation at different levels of skeletal muscle differentiation: in C2C12 myoblasts, in C2C12 myotubes and in adult mouse skeletal muscle. Differentiation was accompanied by an increase in mitochondrial content and respiratory chain activity. The detected ROS production levels correlated with mitochondrial content, being the lowest in the myoblasts. Unlike the adult skeletal muscle, myoblast ROS production was significantly stimulated by the complex I inhibitor rotenone. Our results show that mitochondria are an important ROS source in skeletal muscle cells. The substantial changes in mitochondrial ROS synthesis during skeletal muscle differentiation can be explained by intensive bioenergetic remodeling.     

Transient production of alpha-smooth muscle actin by skeletal myoblasts during differentiation in culture and following intramuscular implantation

alpha-smooth muscle actin (SMA) is typically not present in post-embryonic skeletal muscle myoblasts or skeletal muscle fibers. However, both primary myoblasts isolated from neonatal mouse muscle tissue, and C2C12, an established myoblast cell line, produced SMA in culture within hours of exposure to differentiation medium. The SMA appeared during the cells' initial elongation, persisted through differentiation and fusion into myotubes, remained abundant in early myotubes, and was occasionally observed in a striated pattern. SMA continued to be present during the initial appearance of sarcomeric actin, but disappeared shortly thereafter leaving only sarcomeric actin in contractile myotubes derived from primary myoblasts. Within one day after implantation of primary myoblasts into mouse skeletal muscle, SMA was observed in the myoblasts; but by 9 days post-implantation, no SMA was detectable in myoblasts or muscle fibers. Thus, both neonatal primary myoblasts and an established myoblast cell line appear to similarly reprise an embryonic developmental program during differentiation in culture as well as differentiation within adult mouse muscles.     

Fabrication of skeletal muscle constructs by topographic activation of cell alignment

Skeletal muscle fiber construction for tissue-engineered grafts requires assembly of unidirectionally aligned juxtaposed myotubes. To construct such a tissue, a polymer microchip with linearly aligned microgrooves was fabricated that could direct myoblast adaptation under stringent conditions. The closely spaced microgrooves fabricated by a modified replica molding process guided linear cellular alignment. Examination of the myoblasts by immunofluorescence microscopy demonstrated that the microgrooves with subcellular widths and appropriate height-to-width ratios were required for practically complete linear alignment of myoblasts. The topology-dependent cell alignment encouraged differentiation of myoblasts into multinucleate, myosin heavy chain positive myotubes. The monolayer of myotubes formed on the microstructured chips allowed attachment, growth and differentiation of subsequent layers of linearly arranged myoblasts, parallel to the primary monolayer of myotubes. The consequent deposition of additional myoblasts on the previous layer of myotubes resulted in three-dimensional multi-layered structures of myotubes, typical of differentiated skeletal muscle tissue. The findings demonstrate that the on-chip device holds promise for providing an efficient means for guided muscle tissue construction.     

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.     

Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms

Group II phospholipase A(2) (PLA(2)) myotoxins isolated from Viperidae/Crotalidae snake venoms induce a rapid cytolytic effect upon diverse cell types in vitro. Previous studies suggested that this effect could be more pronounced on skeletal muscle myotubes than on other cell types, including undifferentiated myoblasts. This study utilized the murine skeletal muscle C2C12 cell line to investigate whether differentiated myotubes are more susceptible than myoblasts, and if this characteristic is specific for the group II myotoxic PLA(2)s. The release of lactic dehydrogenase was quantified as a measure of cytolysis, 3 h after cell exposure to different group II PLA(2)s purified from Bothrops asper, Atropoides nummifer, Cerrophidion godmani, and Bothriechis schlegelii venoms. In addition, susceptibility to lysis induced by synthetic melittin and group III PLA(2) from bee (Apis mellifera) venom, as well as by anionic, cationic, and neutral detergents, was comparatively evaluated on the two cultures. Myotubes were significantly more susceptible to group II PLA(2) myotoxins, but not to the other agents tested, under the same conditions. Moreover, the increased susceptibility of myotubes over myoblasts was also demonstrated with two cytolytic synthetic peptides, derived from the C-terminal region of Lys49 PLA(2) myotoxins, that reproduce the action of their parent proteins. These results indicate that fusion and differentiation of myoblasts into myotubes induce changes that render these cells more susceptible to the toxic mechanism of group II PLA(2) myotoxins, but not to general perturbations of membrane homeostasis. Such changes are likely to involve myotoxin acceptor site(s), which remain(s) to be identified.     

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)     

Role of laminin and fibronectin in selecting myogenic versus fibrogenic cells from skeletal muscle cells in vitro

Growth of embryonic skeletal muscle occurs by fusion of multinucleated myotubes with differentiated, fusion-capable myoblasts. Selective recognition seems to prevent fusion of myotubes with nonmyogenic cells such as muscle fibroblasts, endothelial cells, or nerve cells, but the nature of the signal is as yet unknown. Here we provide evidence that one of the selection mechanisms may be the enhanced affinity for laminin of myogenic cells as compared to fibrogenic cells. Growing myotubes in myoblast cultures accumulate laminin and type IV collagen on their surface in patches and strands as the first step in assembling a continuous basal lamina on mature myofibers (U. Kühl, R. Timpl, and K. von der Mark (1982), Dev. Biol. 93, 344-359). Fibronectin, on the other hand, assembles into an intercellular fibrous meshwork not associated with the free myotube surface. Over a brief time period (10-20 min) myoblasts from embryonic mouse thigh muscle adhere faster to laminin than do fibroblasts from the same tissue; these adhere faster to fibronectin. When a mixture of the cells is plated for 20 min on laminin/type IV collagen substrates, only myogenic cells adhere, giving rise to cultures with more than 90% fusion after 2 weeks; on fibronectin/type I collagen in the same time primarily fibroblastic cells adhere, giving rise to cultures with less than 10% nuclei in myotubes. The differential affinities of myoblasts for basement membrane constituents and of fibroblasts for interstitial connective tissue components may play a role in sorting out myoblasts from fibroblasts in skeletal muscle development.     

Simvastatin impairs ADP-stimulated respiration and increases mitochondrial oxidative stress in primary human skeletal myotubes

Statins, the widely prescribed cholesterol-lowering drugs for the treatment of cardiovascular disease, cause adverse skeletal muscle side effects ranging from fatigue to fatal rhabdomyolysis. The purpose of this study was to determine the effects of simvastatin on mitochondrial respiration, oxidative stress, and cell death in differentiated primary human skeletal muscle cells (i.e., myotubes). Simvastatin induced a dose-dependent decrease in viability of proliferating and differentiating primary human muscle precursor cells, and a similar dose-dependent effect was noted in differentiated myoblasts and myotubes. Additionally, there were decreases in myotube number and size following 48 h of simvastatin treatment (5 μM). In permeabilized myotubes, maximal ADP-stimulated oxygen consumption, supported by palmitoylcarnitine+malate (PCM, complex I and II substrates) and glutamate+malate (GM, complex I substrates), was 32-37% lower (P<0.05) in simvastatin-treated (5 μM) vs control myotubes, providing evidence of impaired respiration at complex I. Mitochondrial superoxide and hydrogen peroxide generation were significantly greater in the simvastatin-treated human skeletal myotube cultures compared to control. In addition, simvastatin markedly increased protein levels of Bax (proapoptotic, +53%) and Bcl-2 (antiapoptotic, +100%, P<0.05), mitochondrial PTP opening (+44%, P<0.05), and TUNEL-positive nuclei in human skeletal myotubes, demonstrating up-regulation of mitochondrial-mediated myonuclear apoptotic mechanisms. These data demonstrate that simvastatin induces myotube atrophy and cell loss associated with impaired ADP-stimulated maximal mitochondrial respiratory capacity, mitochondrial oxidative stress, and apoptosis in primary human skeletal myotubes, suggesting that mitochondrial dysfunction may underlie human statin-induced myopathy.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

Variation in ACE activity affects myogenic differentiation in C2C12 cells

Variation in ACE activity is related to affect the skeletal muscle function. To elucidate the mechanism by which ACE affects skeletal muscle function, we examined the effects of loss and gain of ACE activity on myogenic differentiation in C2C12 myoblasts. The treatment of captopril, an ACE inhibitor, in differentiating cells significantly induced the up-regulation of myosin heavy chain, and the hypertrophic myotubes. In addition, an AT2 antagonist PD123319, not AT1 antagonist losartan, induced the up-regulation of myosin heavy chain. On the other hand, overexpression of ACE induced the down-regulation of myosin heavy chain. These results suggest that ACE negatively regulate the myogenesis through the mechanism at least in part via production of angiotensin II followed by its binding to AT2 receptor.     

Extracellular matrix (ECM) synthesis in muscle cell cultures: quantitative and qualitative studies during myogenesis

When the synthesis of extracellular matrix components was examined in G8-1 murine skeletal muscle cells as a function of differentiation, non-collagen and to an even greater extent collagen synthesis was increased. Specifically, collagen types I, III, IV, laminin and fibronectin were identified by SDS-PAGE. Immunoprecipitation, with specific antibodies revealed that both the cell layer and medium of differentiated multinucleated myotubes contained increased levels of type IV collagen and laminin, decreased levels of type III collagen and fibronectin and equivalent levels of type I collagen compared to mononuclear myoblasts.     

Extracellular ATP signaling during differentiation of C2C12 skeletal muscle cells: role in proliferation

Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation. We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and high in myotubes, consistent with the increased expression of extracellular enzymes during differentiation. Stimulation of cells with extracellular nucleotides produces substantial Ca(2+) transients, whose amplitude and shape changed during differentiation. Consistently, C2C12 cells express several P2X and P2Y receptors, whose level changes along with maturation stages. Supplementation with either ATP or UTP stimulates proliferation of C2C12 myoblasts, whereas excessive doses were cytotoxic. The data indicate that skeletal muscle development is accompanied by major functional changes in extracellular ATP signaling.