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

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

Activation of Pax7-positive cells in a non-contractile tissue contributes to regeneration of myogenic tissues in the electric fish S. macrurus

The ability to regenerate tissues is shared across many metazoan taxa, yet the type and extent to which multiple cellular mechanisms come into play can differ across species. For example, urodele amphibians can completely regenerate all lost tissues, including skeletal muscles after limb amputation. This remarkable ability of urodeles to restore entire limbs has been largely linked to a dedifferentiation-dependent mechanism of regeneration. However, whether cell dedifferentiation is the fundamental factor that triggers a robust regeneration capacity, and whether the loss or inhibition of this process explains the limited regeneration potential in other vertebrates is not known. Here, we studied the cellular mechanisms underlying the repetitive regeneration of myogenic tissues in the electric fish S. macrurus. Our in vivo microinjection studies of high molecular weight cell lineage tracers into single identified adult myogenic cells (muscle or noncontractile muscle-derived electrocytes) revealed no fragmentation or cellularization proximal to the amputation plane. In contrast, ultrastructural and immunolabeling studies verified the presence of myogenic stem cells that express the satellite cell marker Pax7 in mature muscle fibers and electrocytes of S. macrurus. These data provide the first example of Pax-7 positive muscle stem cells localized within a non-contractile electrogenic tissue. Moreover, upon amputation, Pax-7 positive cells underwent a robust replication and were detected exclusively in regions that give rise to myogenic cells and dorsal spinal cord components revealing a regeneration process in S. macrurus that is dependent on the activation of myogenic stem cells for the renewal of both skeletal muscle and the muscle-derived electric organ. These data are consistent with the emergent concept in vertebrate regeneration that different tissues provide a distinct progenitor cell population to the regeneration blastema, and these progenitor cells subsequently restore the original tissue.     

Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the alpha-vascular but not the gamma- enteric isoform in differentiating striated myocytes

The cellular distribution of the alpha-vascular and gamma-enteric smooth muscle actin isoforms was analyzed in rat embryos from gestational day (gd) 8 through the first neonatal week by in situ antigen localization using isoactin specific monoclonal antibodies. The alpha-vascular actin isoform was first detected on gd 10 in discrete cells lining the embryonic vasculature. By gd 14, this isoform was also present in the inner layers of mesenchymal cells condensing around the developing airways and gut. The gamma-enteric actin, however, was not detected until gd 15 when cells surrounding the developing aorta, airways, and gut labeled with the gamma-enteric-specific probe. There was continued expression of these two actin isoforms in regions of developing smooth muscle through the remainder of gestation and first neonatal week at which time their distribution coincided with that found in the adult. In addition to developing smooth muscle, the alpha- vascular actin isoform was expressed in differentiating striated muscle cells. On gd 10, there was intense labeling with the alpha-vascular specific probe in developing myocardiocytes and, within 24 h, in somitic myotomal cells. Although significant levels of this smooth muscle actin were present in striated myocytes through gd 17, by the end of the first postnatal week, alpha-vascular actin was no longer detectable in either cardiac or skeletal muscle. Thus, the normal developmental sequence of striated muscle cells includes the transient expression of the alpha-vascular smooth muscle actin isoform. In contrast, the gamma-enteric smooth muscle actin was not detected at any time in embryonic striated muscle. The differential timing of appearance and distribution of these two smooth muscle isoforms indicates that their expression is independently regulated during development.     

The transformational potential of striated muscle in hydromedusae.

Isolated striated muscle tissue of the Anthomedusa Podocoryne carnea participates in the regeneration of a functional manubrium (the feeding organ of medusae) when it is combined homoclonally with endodermal cells of the medusa umbrella. The morphogenetic potential of striated muscle cells in this regeneration process was evaluated by combining nuclear labeled striated muscle cells with some unlabeled endoderm cells. Histological and autoradiographical results demonstrate that transformation of striated muscle cells into smooth muscle cells of the ectoderm and also into endoderm cells must have occurred in the regenerate. The potential for cell transformation of isolated striated muscle cells of Podocoryne carnea is discussed and it is postulated that under appropriate conditions all cell types necessary for the regeneration of a manubrium can be formed from striated muscle cells.     

Efficient in vitro myogenic reprogramming of human primary mesenchymal stem cells and endothelial cells by Myf5

Background information. The identification of a source of stem cells able to regenerate skeletal muscle was the goal of numerous studies with the aim to develop new therapeutic approaches for genetic muscle diseases or muscle injuries. A series of studies have demonstrated that stem cells derived from various tissues may have a role in the regeneration of damaged muscles, but this contribution is always very weak. Thus we established a project aiming to reprogramme non‐muscle cells into the skeletal striated differentiation pathway. Results. We transduced several human primary adult stem or progenitor cells using a recombinant lentivirus containing the coding sequence of the Myf5 gene considered as a master gene for the determination of skeletal striated muscle. These original results are the first demonstration of a myogenic conversion of human mesenchymal and endothelial cells by Myf5. Conclusions. The procedure described in the present paper could be used to develop new research protocols with the prospect of using these cells as therapeutic agents.     

Coexpression of α-sarcomeric actin, α-smooth muscle actin and desmin during myogenesis in rat and mouse embryos I. Skeletal muscle

Expression of vimentin, desmin, alpha-sarcomeric and alpha-smooth muscle actins in embryonic tissues of rat and mice was examined using an immunohistochemical approach. The results showed a similarity in the expression of desmin and alpha-actin isoforms (alpha-sr and alpha-sm) in skeletal muscle cells during murine feto-embryonic development. In the two species, coexpression of alpha-sr and alpha-sm actins has been observed in cardiomyoblasts, myotomal myoblasts and myotubes. The intensity of alpha-sm actin expression decreased during the terminal steps of myogenesis and disappeared completely in mature cardiomyocytes and myofibres. Desmin was expressed in all prefusion myoblasts (type 1 and 2 myoblasts), myotubes, and in myofibres. The appearance of desmin in myoblasts of somites preceded by a few hours the expression of the alpha-actins (alpha-sr and alpha-sm). Our study on vimentin expression, limited to rat embryos, revealed that somite premyoblasts expressed only vimentin, type 1 myoblasts expressed vimentin and desmin, and type 2 myoblasts (rhabdomyoblasts) expressed desmin and alpha-actins (alpha-sr and alpha-sm). Our study demonstrates the resemblance between feto-embryonic myogenesis and myogenic neoplastic differentiation: desmin appears before the alpha-actins in embryonic myoblasts, and can be considered as a marker of an initial step in myogenic differentiation. alpha-sm actin, considered as a striated muscle cell feto-embryonic actin, is expressed transiently in skeletal myoblasts and cardiomyoblasts during development and reappears during neoplastic transformation of skeletal muscle.     

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.     

Development of acetylcholine sensitivity during myogenesis

The development of acetylcholine (ACh) sensitivity during myogenesis has been studied by iontophoretic application of ACh and intracellular recording from myogenic cells from rat forelimbs cultured in vitro. The fine structure of the cells was then examined by electron microscopy. The development of ACh sensitivity is correlated with the appearance of thick and thin filaments and precedes myofibril formation. All myotubes are sensitive to ACh. Myogenic cells arising by cell division in vitro can become sensitive to ACh and construct myofibrils without cell fusion. When cell fusion is inhibited by calcium ion deficiency or when cell division is blocked by FUdR, many mononucleate, striated, ACh-sensitive cells appear in culture. While ACh sensitivity appears at the onset of muscle differentiation, ACh receptors seem to play no role in the early events of myogenesis, as evidenced by the failure of receptor block or of desensitization to interfere with myogenesis. The concurrent appearance of myofilaments and ACh sensitivity is discussed in relation to the early events and control mechanisms of myogenesis.     

Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes

Quiescent satellite cells sit on the surface of the muscle fibres under the basal lamina and are activated by a variety of stimuli to disengage, divide and differentiate into myoblasts that can regenerate or repair muscle fibres. Satellite cells adopt their parent's fibre type and must have some means of communication with the parent fibre. The mechanisms behind this communication are not known. We show here that satellite cells form dynamic connections with muscle fibres and other satellite cells by F‐actin based tunnelling nanotubes (TNTs). Our results show that TNTs readily develop between satellite cells and muscle fibres. Once developed, TNTs permit transport of intracellular material, and even cellular organelles such as mitochondria between the muscle fibre and satellite cells. The onset of satellite cell differentiation markers Pax‐7 and MyoD expression was slower in satellite cells cultured in the absence than in the presence of muscle cells. Furthermore physical contact between myofibre and satellite cell progeny is required to maintain subtype identity. Our data establish that TNTs constitute an integral part of myogenic cell communication and that physical cellular interaction control myogenic cell fate determination. J. Cell. Physiol. 223: 376–383, 2010. © 2010 Wiley‐Liss, Inc.     

Changes in proliferation,differentiation, fibroblast growth factor 2 responsiveness and expression of syndecan‐4 and glypican‐1 with turkey satellite cell age

Myogenic satellite cells are heterogeneous multipotential stem cells that are required for muscle repair, maintenance, and growth. The membrane‐associated heparan sulfate proteoglycans syndecan‐4 and glypican‐1 differentially regulate satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) signal transduction, and expression of the myogenic regulatory factors MyoD and myogenin. The objective of the current study was to determine the effect of age on syndecan‐4 and glypican‐1 satellite cell populations, proliferation, differentiation, FGF2 responsiveness, and expression of syndecan‐4, glypican‐1, MyoD, and myogenin using satellite cells isolated from the pectoralis major muscle of 1‐day‐old, 7‐week‐old and 16‐week‐old turkeys. Proliferation was significantly reduced in the 16‐week‐old satellite cells, while differentiation was decreased in the 7‐week‐old and the 16‐week‐old cells beginning at 48 h of differentiation. Fibroblast growth factor 2 responsiveness was highest in the 1‐day‐old and 7‐week‐old cells during proliferation; during differentiation there was an age‐dependent response to FGF2. Syndecan‐4 and glypican‐1 satellite cell populations decreased with age, but syndecan‐4 and glypican‐1 were differentially expressed with age during proliferation and differentiation. MyoD and myogenin mRNA expression was significantly decreased in 16‐week‐old cells compared to the 1‐day‐old and 7‐week‐old cells. MyoD and myogenin protein expression was higher during proliferation in the 16‐week‐old cells and decreased with differentiation. These data demonstrate an age‐dependent effect on syndecan‐4 and glypican‐1 satellite cell subpopulations, which may be associated with age‐related changes in proliferation, differentiation, FGF2 responsiveness, and the expression of the myogenic regulatory factors MyoD and myogenin.     

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.     

Divergent and conserved roles of Dll1 signaling in development of craniofacial and trunk muscle

Craniofacial and trunk skeletal muscles are evolutionarily distinct and derive from cranial and somitic mesoderm, respectively. Different regulatory hierarchies act upstream of myogenic regulatory factors in cranial and somitic mesoderm, but the same core regulatory network – MyoD, Myf5 and Mrf4 – executes the myogenic differentiation program. Notch signaling controls self-renewal of myogenic progenitors as well as satellite cell homing during formation of trunk muscle, but its role in craniofacial muscles has been little investigated. We show here that the pool of myogenic progenitor cells in craniofacial muscle of Dll1^LacZ/Ki mutant mice is depleted in early fetal development, which is accompanied by a major deficit in muscle growth. At the expense of progenitor cells, supernumerary differentiating myoblasts appear transiently and these express MyoD. The progenitor pool in craniofacial muscle of Dll1^LacZ/Ki mutants is largely rescued by an additional mutation of MyoD. We conclude from this that Notch exerts its decisive role in craniofacial myogenesis by repression of MyoD. This function is similar to the one previously observed in trunk myogenesis, and is thus conserved in cranial and trunk muscle. However, in cranial mesoderm-derived progenitors, Notch signaling is not required for Pax7 expression and impinges little on the homing of satellite cells. Thus, Dll1 functions in satellite cell homing and Pax7 expression diverge in cranial- and somite-derived muscle.     

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

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

Myogenic potential of human alveolar mucosa derived cells

Difficulties related to the obtainment of stem/progenitor cells from skeletal muscle tissue make the search for new sources of myogenic cells highly relevant. Alveolar mucosa might be considered as a perspective candidate due to availability and high proliferative capacity of its cells. Human alveolar mucosa cells (AMC) were obtained from gingival biopsy samples collected from 10 healthy donors and cultured up to 10 passages. AMC matched the generally accepted multipotent mesenchymal stromal cells criteria and possess population doubling time, caryotype and immunophenotype stability during long-term cultivation. The single myogenic induction of primary cell cultures resulted in differentiation of AMC into multinucleated myotubes. The myogenic differentiation was associated with expression of skeletal muscle markers: skeletal myosin, skeletal actin, myogenin and MyoD1. Efficiency of myogenic differentiation in AMC cultures was similar to that in skeletal muscle cells. Furthermore, some of differentiated myotubes exhibited contractions in vitro. Our data confirms the sufficiently high myogenic potential and proliferative capacity of AMC and their ability to maintain in vitro proliferation-competent myogenic precursor cells regardless of the passage number.