Multinucleated Muscle Fibers Research Articles

Regulation of Skeletal Muscle Plasticity by Glycogen Synthase Kinase-3β: A Potential Target for the Treatment of Muscle Wasting

Muscle wasting is a prevalent and disabling condition in chronic disease and cancer and has been associated with increased mortality and impaired efficacy of surgical and medical interventions. Pharmacological therapies to combat muscle wasting are currently limited but considered as an important unmet medical need. Muscle wasting has been attributed to increased muscle proteolysis, and in particular ubiquitin 26S-proteasome system (UPS)-dependent protein breakdown. However, rates of muscle protein synthesis are also subject to extensive (patho) physiological regulation, and the balance between synthesis and degradation ultimately determines net muscle protein turnover. As multinucleated muscle fibers accommodate threshold changes in muscle protein content by the accretion and loss of muscle nuclei, myonuclear turnover may additionally determine muscle mass. Current insights in the mechanisms dictating muscle mass plasticity not only reveal intricate interactions and crosstalk between these processes, but imply the existence of signaling molecules that act as molecular switchboards, which coordinate and integrate cellular responses upon conditions that evoke changes in muscle mass. These "master regulators" of skeletal muscle mass plasticity are preferred targets for pharmacological modulation of skeletal muscle wasting. In this review Glycogen synthase kinase-3β (GSK-3β) is highlighted as a master regulator of muscle mass plasticity since, in addition to its role in UPS-mediated muscle protein degradation, it also controls protein synthesis, and influences myonuclear accretion and cell death. Moreover, the regulation of GSK-3β activity as well as currently available pharmacological inhibitors are described and discussed in the context of multimodal treatment strategies aimed at the inhibition of GSK-3β, and optimal exploitation of its potential role as a central regulator of skeletal muscle mass plasticity for the treatment of muscle wasting.

Protein‐Engineered Biomaterials to Generate Human Skeletal Muscle Mimics

Skeletal muscle function, which is essential in the human body, can be lost due to injury, trauma, or diseases such as muscular dystrophy, prompting the need for in vitro models of human skeletal muscle tissue. In vitro tissue models are indispensable in performing quantitative studies of injury and disease progression as well as in providing sources of tissue for potential regenerative medicine therapies. An ideal bioengineered model of skeletal muscle should promote myoblast (muscle cell precursor) alignment as well as subsequent myoblast fusion into mature, multinucleated muscle cells known as myotubes. Accordingly, we report the development of an engineered protein biomaterial that promotes the alignment and fusion of primary human myoblasts into organized and differentiated myotubes. Furthermore, these aligned human myotubes exhibit markers of functional maturation including organization of sarcomeres (the force-generating apparatus within muscle) and induced contractility upon electrical stimulation.

Non Conservation of Function for the Evolutionarily Conserved Prdm1 Protein in the Control of the Slow Twitch Myogenic Program in the Mouse Embryo

Muscles are composed of multinucleated muscle fibers with different contractile and physiological properties, which result from specific slow or fast gene expression programs in the differentiated muscle cells. In the zebra fish embryo, the slow program is under the control of Hedgehog signaling from the notochord and floor plate. This pathway activates the expression of the conserved transcriptional repressor, Prdm1 (Blimp1), which in turn represses the fast program and promotes the slow program in adaxial cells of the somite and their descendants. In the mouse embryo, myogenesis is also initiated in the myotomal compartment of the somite, but the slow muscle program is not confined to a specific subset of cells. We now show that Prdm1 is expressed in the first differentiated myocytes of the early myotome from embryonic day (E)9.5-E11.5. During this period, muscle formation depends on the myogenic regulatory factors, Myf5 and Mrf4. In their absence, Prdm1 is not activated, in apparent contrast to zebra fish where Prdm1 is expressed in the absence of Myf5 and MyoD that drive myogenesis in adaxial cells. However, as in zebra fish, Prdm1 expression in the mouse myotome does not occur in the absence of Hedgehog signaling. Analysis of the muscle phenotype of Prdm1 mutant embryos shows that myogenesis appears to proceed normally. Notably, there is no requirement for Prdm1 activation of the slow muscle program in the mouse myotome. Furthermore, the gene for the transcriptional repressor, Sox6, which is repressed by Prdm1 to permit slow muscle differentiation in zebra fish, is not expressed in the mouse myotome. We propose that the lack of functional conservation for mouse Prdm1, that can nevertheless partially rescue the adaxial cells of zebra fish Prdm1 mutants, reflects differences in the evolution of the role of key regulators such as Prdm1 or Sox6, in initiating the onset of the slow muscle program, between teleosts and mammals.

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

BackgroundMammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations.ResultsCell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.ConclusionsOur results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations. Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres. Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.

Modulation of alignment and differentiation of skeletal myoblasts by biomimetic materials

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. We developed a novel cell culture technique that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. Alignment and fusion of myoblasts into parallel arrays of multinucleated myotubes are critical in skeletal muscle tissue engineering and more micro-patterning techniques and surface engineering have been tested by switching differentiating myotubes from growth medium (GM) to differentiative media (DM). One of the goals of tissue engineering is to develop tools allowing in vitro construction and mimicking of the final tissue architectures. The fabrication of polyelectrolyte multilayers (PEMs) may represent a promising approach for recreating physiological nanometer-sized cell environments in vitro. In this study we describe a method for generating biomimetic microstructured surfaces that promote cell adhesion and differentiation of parallel arrays of mature C2C12 myotubes continuously maintained in GM for 7 days. The structure consists of a 'double-sheet' PDMS structure that provides 'compliant' or 'stiff' microdomains to guide the cell self-patterning, coupled to layer-by-layer (LbL) self-assembled multilayers of biocompatible polyelectrolytes that promote C2C12 myoblasts alignment and differentiation. Our findings have relevance to the interpretation of in vitro data as well as to the study of cellular interactions with biomaterials.

Cytodiagnosis of sternocleidomastoid tumor of infancy

Sterocleidomastoid tumor of infancy (SCMI), also known as fibromatosis colli of infancy, is a benign, self limiting disease of new born characterised by its classical history and clinical presentation of firm to hard fusiform mass in the lower and middle portion of sternocleidomastoid. SCMI often appears during early perinatal period between second to fourth weeks of life. A well recognized association between SCMI and primiparous birth, breech presentation, prolonged difficult labor and forceps deliveries is found. Cytology shows spindle shaped mature fibroblastic cells scattered singly along with degenerated and multinucleated giant muscle cells in a clean background. It is important to differentiate this lesion from different forms of infantile fibromatosis. Fine-needle aspiration cytology (FNAC), as a time saving, rapid and reliable diagnostic procedure, has got bigger role to play in reassurance of anxious parents, guiding for conservative management and avoiding surgery.

CKIP-1 REGULATES MAMMALIAN AND ZEBRAFISH MYOBLAST FUSION

Multinucleated muscle fibres arise by fusion of precursor cells called myoblasts. We previously showed that CKIP-1 ectopic expression in C2C12 myoblasts increased cell fusion. In this work, we report that CKIP-1 depletion drastically impairs C2C12 myoblast fusion in vitro and in vivo during zebrafish muscle development. Within developing fast-twich myotome, Ckip-1 localises at the periphery of fast precursor cells, closed to the plasma membrane. Unlike wild-type myoblasts that form spatially arrayed multinucleated fast myofibres, Ckip-1-deficient myoblasts show a drastic reduction in fusion capacity. A search for CKIP-1 binding partners identified the ARPC1 subunit of Arp2/3 actin nucleation complex essential for myoblast fusion. We demonstrate that CKIP-1, through binding to plasma membrane phosphoinositides via its PH domain, regulates cell morphology and lamellipodia formation by recruiting the Arp2/3 complex at the plasma membrane. These results establish CKIP-1 as a regulator of cortical actin that recruits the Arp2/3 complex at the plasma membrane essential for muscle precursor elongation and fusion.

Clinical signs and histopathology of brain, spinal cord and muscle of the pelvic limb of rats experimentally infected with Trypanosoma evansi

The aim of this study was to investigate whether rats infected with Trypanosoma evansi had neurological and locomotor signs, as well as histological lesions in the central nervous system (CNS) and pelvic muscles. To carry out this study, 52 rats were used and divided into two groups. The animals in Group A ( n = 40) were infected with T. evansi, and the rats in Group B ( n = 12) were used as negative controls (non-infected). Neurological examination was performed at Days 5, 15, 30 and 150 post-infection (PI) with eventual euthanasia of the rats. Samples of brain, spinal cord and skeletal muscle (biceps femoris and gastrocnemius muscles) were collected. The neurological tests evaluated motor capacity, balance and pain sensitivity. At Day 5 PI in Subgroup A1, the rats showed high parasitemia, became apathetic and presented with slow movements and signs of disorientation. After Day 15 PI in Subgroup A2 and Day 30 PI in Subgroup A3, no more clinical abnormalities were observed. Histologically, there was no damage to the CNS in these three subgroups, but within Subgroup A3, mononuclear infiltration of the muscle was observed. Rats chronically infected (Subgroup A4 – Day 150 PI) showed muscle atrophy, walking dysfunction and paralysis of the hind limbs. Mild mononuclear inflammatory infiltrates and perivascular cuffs were observed in the CNS of some of the animals in Subgroup A4. In these rats, severe muscle damage was observed in the skeletal muscle which included atrophy and loss of muscle fibers, multinucleated giant muscle cells, mononuclear myositis, Wallerian degeneration of the innervating fibers and mononuclear inflammatory infiltrate in the perineurium and adipose tissue. Based upon these findings, we conclude that infection by T. evansi in rats leads to muscle damage, which is probably the cause of the paralysis of hind limbs.

Stem cells for skeletal muscle repair

Skeletal muscle is a highly specialized tissue composed of non-dividing, multi-nucleated muscle fibres that contract to generate force in a controlled and directed manner. Skeletal muscle is formed during embryogenesis from a subset of muscle precursor cells, which generate both differentiated muscle fibres and specialized muscle-forming stem cells known as satellite cells. Satellite cells remain associated with muscle fibres after birth and are responsible for muscle growth and repair throughout life. Failure in satellite cell function can lead to delayed, impaired or failed recovery after muscle injury, and such failures become increasingly prominent in cases of progressive muscle disease and in old age. Recent progress in the isolation of muscle satellite cells and elucidation of the cellular and molecular mediators controlling their activity indicate that these cells represent promising therapeutic targets. Such satellite cell-based therapies may involve either direct cell replacement or development of drugs that enhance endogenous muscle repair mechanisms. Here, we discuss recent breakthroughs in understanding both the cell intrinsic and extrinsic regulators that determine the formation and function of muscle satellite cells, as well as promising paths forward to realizing their full therapeutic potential.

Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells

Proper muscle function is dependent on spatial and temporal control of gene expression in myofibers. Myofibers are multinucleated cells that are formed, repaired and maintained by the process of myogenesis in which progenitor myoblasts proliferate, differentiate and fuse. Gene expression is dependent upon proteins that require facilitated nuclear import, however little is known about the regulation of nucleocytoplasmic transport during the formation of myofibers. We analyzed the role of karyopherin alpha (KPNA), a key classical nuclear import receptor, during myogenesis. We established that five karyopherin alpha paralogs are expressed by primary mouse myoblasts in vitro and that their steady-state levels increase in multinucleated myotubes, suggesting a global increase in demand for classical nuclear import during myogenesis. We used siRNA-mediated knockdown to identify paralog-specific roles for KPNA1 and KPNA2 during myogenesis. KPNA1 knockdown increased myoblast proliferation, whereas KPNA2 knockdown decreased proliferation. In contrast, no proliferation defect was observed with KPNA4 knockdown. Only knockdown of KPNA2 decreased myotube growth. These results identify distinct pathways involved in myoblast proliferation and myotube growth that rely on specific nuclear import receptors suggesting that regulation of classical nuclear import pathways likely plays a critical role in controlling gene expression in skeletal muscle.

Myotube vs myoblast sensitivity to apoptosis induction by chemical triggers

Apoptosis is necessary for skeletal muscle tissue development and homeostasis, where it plays a multifaceted role. Muscle cell death increase is associated to disuse, denervation and to several muscle myopathies (Siu et al., Life Sci, 2009), while apoptosis resistance is typical of muscle differentiation (Xiao et al., Apoptosis, 2011) The aim of this study is to evaluate in vitro apoptotic response in differentiated myotubes exposed to chemical triggers and to compare apoptosis susceptibility to that of proliferant myoblasts. (Salucci et al., Micron, 2010). C2C12 myotubes, differentiated as previously described (Curci et al, Micron, 2008), were exposed to etoposide (ETO), cisplatin (CP), hydrogen peroxide (H2O2) and staurosporine (SP) and apoptosis was evaluated by flow cytometry and transmission electron microscopy. Flow cytometry analysis revealed a certain subdiploid peak after ETO and CP exposure, lower, however, than that evidenced in myoblasts. Differently, DNA cleavage after H2O2 or SP treatments, was absent, in contrast with undifferentiated cells. At ultrastructural analysis, characteristic chromatin condensation was observed after ETO and CP treatments, but few apoptotic cells were detected, differently from what evidenced at undifferentiated stage. Moreover, autophagic cell death appeared. Cells exposed to ETO presented a particular behaviour: myonuclei with condensed chromatin coexisted with normal nuclei, in the same myotube, as previously demonstrated in UVB-induced apoptosis (D’Emilio et al., Histol Histopathol, 2010). On the contrary, and differently from myoblasts, apoptosis was absent in myotubes after H2O2 exposure, being necrosis and autophagy the most common cell death processes. After SP treatment all cells showed features of secondary necrosis, a response present also in myoblasts. In addition, autophagic vacuoles were observed, exclusively in differentiated cells. These results show a certain resistance to apoptosis in myotubes, if compared to myoblasts, that could be associated to an increase in autophagic cell death and in a probable upregulation of anti-apoptotic pathways. Particularly intriguing appears the response to ETO treatment where apoptosis could be correlated to a localized gene expression within each myonuclear domain, suggesting that this process works to selectively eliminate the designated nuclei in multinucleated skeletal muscle fibers.

Plasticity and Physiological Role of Stem Cells Derived from Skeletal Muscle Interstitium: Contribution to Muscle Fiber Hyperplasia and Therapeutic Use

Stem cells other than satellite cells that can give rise to primary myoblasts, which are able to form additional new fibers postnatally, are present in the interstitial spaces of skeletal muscle. These cells are sorted into CD34(+)/45(-) (Sk-34) and CD34(-)/45(-) (Sk-DN) cell fractions, and they are wholly (>99%) negative for Pax7 at initial isolation. Colony-forming units of these cells typically include non-adherent type myogenic cells, while satellite cells are known to be adherent in cell culture. In addition, both Pax7(-) and Pax7(+) cells are produced, depending on asymmetric cell division. A large number of myotubes are also formed in each colony, thus suggesting that putative Pax7(+) satellite cells also present in each colony. Interestingly, interstitial myogenic cells show basal lamina formation at early stages of myogenesis in response to various types of stimulation in compensatory enlarged muscle, a property that satellite cells do not possess in the parent fiber basal lamina cylinder. Basal lamina formation and production of satellite cells are essential before muscle fiber establishment in vivo. It is therefore likely that myogenic cells in skeletal muscle can be divided into two populations: 1) basal lamina-producing myogenic cells; and 2) basal lamina-non-producing myogenic cells. The latter population may be Pax7(+) satellite cells showing adherent capacity in cell culture, while the lamina-producing myogenic population derived from interstitial multipotent stem cells, which is predominant among Sk-34 and Sk-DN cells, plays a role in primary myoblast generation and shows non-adherent behavior in culture. Therefore, the physiological role of interstitial myogenic cells is as a source for new postnatal muscle fiber formation, and multinucleated muscle fibers (cells) are potentially formed clonally.

BMP regulation of myogenesis in zebrafish

In amniotes, BMP signaling from lateral plate and dorsal neural tube inhibits differentiation of muscle precursors in the dermomyotome. Here, we show that BMPs are expressed adjacent to the dermomyotome during and after segmentation in zebrafish. In addition, downstream BMP pathway members are expressed within the somite during dermomyotome development. We also show that zebrafish dermomyotome is responsive to BMP throughout its development. Ectopic overexpression of Bmp2b increases expression of the muscle precursor marker pax3, and changes the time course of myoD expression. At later stages, overexpression increases the number of Pax7+ myogenic precursors, and delays muscle differentiation, as indicated by decreased numbers of MEF2+ nuclei, decreased number of multi-nucleated muscle fibers, and an increased myotome angle. In addition, we show that while BMP overexpression is sufficient to delay myogenic differentiation, inhibition of BMP does not detectably affect this process, suggesting that other factors redundantly inhibit myogenic differentiation.

Physical inactivity and muscle weakness in the critically ill

Patients in the intensive care unit commonly develop muscle weakness. In part, this reflects loss of mechanical loading due to physical inactivity, bed rest, or immobilization. Mechanical unloading stimulates a complex adaptive response that results in muscle atrophy and loss of specific force. One element of this response is slowing of protein synthesis, which is regulated by signaling pathways downstream of mammalian target of rapamycin and insulin-like growth factor-1. In parallel, protein degradation is accelerated via three coordinate processes: calcium-dependent proteolysis, adenosine triphosphate-dependent proteolysis, and lysosomal proteolysis. Finally, unloading stimulates apoptosis of a subset of myonuclei within multinucleated muscle fibers. This helps to stabilize the relationship between nuclear number and cell volume during atrophy. Each of these responses is promoted by concurrent development of oxidative stress caused by increased production of reactive oxygen species in unloaded muscle fibers. Countermeasures that lessen the effects of unloading include physical activity, nutritional supplements, hormone therapy, and antioxidant administration. Targeted research is needed to define the role of mechanical unloading in intensive care unit-associated weakness and develop countermeasures to preserve muscle function, lessen illness, and hasten the recovery of critically ill patients.

Determination of intracellular glutathione and cysteine using HPLC with a monolithic column after derivatization with monobromobimane

An optimized high-performance liquid chromatography (HPLC) method is used to show that, as myoblasts differentiate into multinucleated muscle fibers, there is a shift to a more oxidized cell redox state. The HPLC method incorporated derivatization with monobromobimane for the determination of the reduced (GSH) and oxidized (GSSG) forms of glutathione and the reduced (Cys) and oxidized (CysSS) forms of cysteine. The derivatization was optimized to improve the sensitivity of the approach; the limits of detection for glutathione and cysteine were 3 x 10(-8) and 5 x 10(-8) M, respectively.

Sns and Kirre, theDrosophilaorthologs of Nephrin and Neph1,direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytes

The Immunoglobulin superfamily (IgSF) proteins Neph1 and Nephrin are co-expressed within podocytes in the kidney glomerulus, where they localize to the slit diaphragm (SD) and contribute to filtration between blood and urine. Herein, we demonstrate that their Drosophila orthologs Kirre (Duf) and Sns are co-expressed within binucleate garland cell nephrocytes (GCNs) that contribute to detoxification of the insect hemolymph by uptake of molecules through an SD-like nephrocyte diaphragm (ND) into labyrinthine channels that are active sites of endocytosis. The functions of Kirre and Sns in the embryonic musculature, to mediate adhesion and fusion between myoblasts to form multinucleate muscle fibers, have been conserved in the GCNs, where they contribute to adhesion of GCNs in the ;garland' and to their fusion into binucleate cells. Sns and Kirre proteins localize to the ND at the entry point into the labyrinthine channels and, like their vertebrate counterparts, are essential for its formation. Knockdown of Kirre or Sns drastically reduces the number of NDs at the cell surface. These defects are associated with a decrease in uptake of large proteins, suggesting that the ND distinguishes molecules of different sizes and controls access to the channels. Moreover, mutations in the Sns fibronectin-binding or immunoglobulin domains lead to morphologically abnormal NDs and to reduced passage of proteins into the labyrinthine channels for uptake by endocytosis, suggesting a crucial and direct role for Sns in ND formation and function. These data reveal significant similarities between the insect ND and the SD in mammalian podocytes at the level of structure and function.

MG53 Regulates Membrane Budding and Exocytosis in Muscle Cells

Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis. We have identified a tripartite motif (TRIM72) family member protein named MG53 and defined its role in mediating the dynamic process of membrane fusion and exocytosis in striated muscle. MG53 is a muscle-specific protein that contains a TRIM motif at the amino terminus and a SPRY motif at the carboxyl terminus. Live cell imaging of green fluorescent protein-MG53 fusion construct in cultured myoblasts showed that although MG53 contains no transmembrane segment it is tightly associated with intracellular vesicles and sarcolemmal membrane. RNA interference-mediated knockdown of MG53 expression impeded myoblast differentiation, whereas overexpression of MG53 enhanced vesicle trafficking to and budding from sarcolemmal membrane. Co-expression studies indicated that MG53 activity is regulated by a functional interaction with caveolin-3. Our data reveal a new function for TRIM family proteins in regulating membrane trafficking and fusion in striated muscles.

Engineering a multi‐nucleated myotube, the role of the actin cytoskeleton

Myoblasts in vitro form characteristic arrays of bipolar-shaped cells prior to fusion. We have shown that the actin cytoskeleton re-organizes in these fusing cells and that the interaction of non-muscle myosin 2A with actin at the plasma membrane helps to generate the bipolar shape of myoblasts, which is key for fusion. Here we discuss how fusion occurs, and in particular how the actin cytoskeleton is involved. Myoblast fusion is essential to form the multi-nucleated muscle fibres that make up the skeletal muscle. Skeletal muscle fibres contain many nuclei, roughly one nucleus to every 15 sarcomeres (35 microm) in adult muscle, although this varies with muscle type (Bruusgaard et al., 2006). Thus a muscle fibre 30 cm long contains about 8000 nuclei and is formed by the fusion of about 8000 cells during development. The formation of multi-nucleated myotubes has been intensively studied for many years using a number of different systems. Many different proteins have been identified using Drosophila as a model system (e.g. see reviews by Taylor, 2000, 2002) that have given an insight into what happens in mammals. However, the process of fusion of mammalian cells is less well understood, and this paper will cover some of the aspects of mammalian myoblast fusion, with a particular focus on the role of the actin cytoskeleton.

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.