Mononucleated Muscle Cells Research Articles

Mature adipocytes inhibit differentiation of myogenic cells but stimulate proliferation of fibro-adipogenic precursors derived from trout muscle in vitro

Interactions between tissues and cell types, mediated by cytokines or direct cell–cell exchanges, regulate growth. To determine whether mature adipocytes influence the in vitro growth of trout mononucleated muscle cells, we developed an indirect coculture system, and showed that adipocytes (5 × 106 cells/well) derived from perivisceral adipose tissue increased the proliferation (BrdU-positive cells) of the mononucleated muscle cells (26% vs. 39%; p < 0.001) while inhibiting myogenic differentiation (myosin+) (25% vs. 15%; p < 0.001). Similar effects were obtained with subcutaneous adipose tissue-derived adipocytes, although requiring more adipocytes (3 × 107 cells/well vs. 5 × 106 cells/well). Conditioned media recapitulated these effects, stimulating proliferation (31% vs. 39%; p < 0.001) and inhibiting myogenic differentiation (32 vs. 23%; p < 0.001). Adipocytes began to reduce differentiation after 24 h, whereas proliferation stimulation was observed after 48 h. While adipocytes did not change pax7+ and myoD1/2+ percentages, they reduced myogenin+ cells showing inhibition from early differentiation stage. Finally, adipocytes increased BrdU+ cells in the Pdgfrα+ population but not in the myoD+ one. Collectively, our results demonstrate that trout adipocytes promote fibro-adipocyte precursor proliferation while inhibiting myogenic cells differentiation in vitro, suggesting the key role of adipose tissue in regulating fish muscle growth.

The Functional Role of Myogenin in Cardiomyoblast H9c2 Cells Treated with High Glucose and Palmitic Acid: Insights into No-Rejection Heart Transplantation.

Various pathological alterations, including lipid-deposition-induced comparative cardiac lipotoxicity, contribute to cardiac aging in the failing heart. A decline in endogenous myogenin proteins can lead to the reversal of muscle cell differentiation and the creation of mononucleated muscle cells. Myogenin may be a specific regulator of adaptive responses to avoid pathological hypertrophy in the heart. Hence, it is important to understand the regulation of myogenin expression and functions in response to exposure to varied stresses. In this study, we first examined and verified the cytotoxic effect of palmitic acid on H9c2 cells. The reduction in myogenin mRNA and protein expression by palmitic acid was independent of the effect of glucose. Meanwhile, the induction of cyclooxygenase 2 and activating transcription factor 3 mRNAs and proteins by palmitic acid was dependent on the presence of glucose. In addition, palmitic acid failed to disrupt cell cycle progression when H9c2 cells were treated with no glucose. Next, we examined the functional role of myogenin in palmitic-acid-treated H9c2 cells and found that myogenin may be involved in palmitic-acid-induced mitochondrial and cytosolic ROS generation, cellular senescence, and mitochondrial membrane potential. Finally, the GSE150059 dataset was deposited in the Gene Expression Omnibus website and the dataset was further analyzed via the molecular microscope diagnostic system (MMDx), demonstrating that many heart transplant biopsies currently diagnosed as no rejection have mild molecular-antibody-mediated rejection-related changes. Our data show that the expression levels of myogenin were lower than the average level in the studied population. Combining these results, we uncover part of the functional role of myogenin in lipid- and glucose-induced cardiac cell stresses. This finding provides valuable insight into the differential role of fatty-acid-associated gene expression in cardiovascular tissues. Additionally, the question of whether this gene expression is regulated by myogenin also highlights the usefulness of a platform such as MMDx-Heart and can help elucidate the functional role of myogenin in heart transplantation.

Myomaker mediates fusion of fast myocytes in zebrafish embryos

Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they were unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.

Generation of a monoclonal antibody reactive to prefusion myocytes

We established a novel monoclonal antibody, Yaksa that is specific to a subpopulation of myogenic cells. The Yaksa antigen is not expressed on the surface of growing myoblasts but only on a subpopulation of myogenin-positive myocytes. When Yaksa antigen-positive mononucleated cells were freshly prepared from a murine myogenic cell by a cell sorter, they fused with each other and formed multinucleated myotubes shortly after replating while Yaksa antigen-negative cells scarcely generated myotubes. These results suggest that Yaksa could segregate fusion-competent, mononucleated cells from fusion–incompetent cells during muscle differentiation. The Yaksa antigen was also expressed in developing muscle and regenerating muscle in vivo and it was localized at sites of cell–cell contact between mono-nucleated muscle cells and between mono-nucleated muscle cells and myotubes. Thus, Yaksa that marks prefusion myocytes before myotube formation can be a useful tool to elucidate the cellular and molecular mechanisms of myogenic cell fusion.Electronic supplementary materialThe online version of this article (doi:10.1007/s10974-011-9247-8) contains supplementary material, which is available to authorized users.

Larval convergence in a colonial tunicate: the organization of the sarcotubular complex in Ecteinascidia turbinata (Perophoridae, Phlebobranchiata, Tunicata, Chordata)

Ecteinascidia turbinata is a colonial ascidian that as an adult shares characters with phlebobranch ascidians, whereas the larvae are similar to aplousobranch ascidian larvae. The sarcotubular complex consists of invaginations of the sarcolemma that contact the sarcoplasmatic reticulum via dyads or triads. If present, the invaginations of the sarcolemma in tunicates have been characterized as laminar or tubular. We comparatively investigated the sarcotubular complex of E. turbinata and seven other tunicate species using 3D-reconstruction techniques based on electron micrographs of serial sections. The mononucleate muscle cells in E. turbinata possess intermediate and close junctions and contain several layers of peripheral myofibrillae. The myofibrillae are surrounded by continuous cisternae of the sarcoplasmic reticulum that forms interconnected rings around the z-bands. The invaginations of the sarcolemma are laminar, contacting the sarcoplasamatic reticulum at the height of the z-bands via dyads and triads. We present a clear definition of character states encountered in Tunicata: laminar invaginations are characterized by a width to length ratio of smaller than 1:20, tubular invagination by a width to length ratio of larger than 1:10. Laminar invaginations are found in stolidobranch ascidians and E. turbinata. Tubular invaginations are present in aplousobranch ascidians and appendicularians. This character state distribution across taxa supports the hypothesis that E. turbinata should be included in Phlebobranchiata as suggested by adult characters and that the larval similarities with Aplousobranchiata arose by convergent evolution.

Drosophila Lame duck, a novel member of the Gli superfamily, acts as a key regulator of myogenesis by controlling fusion-competent myoblast development.

A hallmark of mature skeletal muscles is the presence of multinucleate muscle fibers. In Drosophila, the formation of muscle syncytia requires the cooperative participation of two types of myoblasts, founder cells and fusion-competent myoblasts. We show that a newly identified gene, lame duck (lmd), has an essential regulatory role in the specification and function of fusion-competent myoblasts. Embryos that lack lmd function show a loss of expression of two key differentiation and fusion genes, Mef2 and sticks-and-stones, in fusion-competent myoblasts and are completely devoid of multinucleate muscle fibers. By contrast, founder cells are specified and retain their capability to differentiate into mononucleate muscle cells. lmd encodes a novel member of the Gli superfamily of transcription factors and is expressed in fusion-competent myoblasts and their precursors in a Wingless- and Notch-dependent manner. The activity of the Lmd protein appears to be additionally controlled by its differential cytoplasmic versus nuclear localization. Results from an independent molecular screen for binding factors to a myoblast-specific Mef2 enhancer further demonstrate that Lmd is a direct transcriptional regulator of Mef2 in fusion-competent myoblasts.

Sequence and Expression of Amphioxus Alkali Myosin Light Chain (AmphiMLC-alk) Throughout Development: Implications for Vertebrate Myogenesis

The lower chordate amphioxus, widely considered the closest living invertebrate relative of the vertebrates, is a key organism for understanding the relationship between gene duplications and evolution of the complex vertebrate body plan. In tetrapod vertebrates, the alkali myosin light chain genes (MLC-alk), which code for proteins associated with the globular head of the myosin heavy chain, constitute a large family with stage-, tissue-, and fiber-type-specific expression of different isoforms thought to have arisen by duplication of a single ancestral gene. In protostome invertebrates, e.g., arthropods, molluscs, and nematodes, only one MLC-alk gene has been found, but the number of such genes in deuterostome invertebrates and lower vertebrates is unknown. The present report, describing the sequence and expression throughout development of the amphioxus gene for alkali myosin light chain (AmphiMLC-alk), thus fills a major gap in understanding the relation between gene duplication and increasing diversity of muscle-cell types. A full-length clone (1 kb) of AmphiMLC-alk was isolated from a larval amphioxus cDNA library. It coded for a 149-amino-acid protein most closely related to the vertebrate embryonic form of MLC-alk. Southern blot analysis revealed only one copy of AmphiMLC-alk and suggested that it is the only MLC-alk gene in amphioxus. Northern blot analysis indicated that this gene produces only one transcript, which is expressed at all stages of development and in adults. In situ hybridizations showed expression initially in the myotomes of somites 2-5 of neurula embryos and soon thereafter in the myotomes of somite 1 and of newly forming somites progressively added posteriorly. Myotomal expression continues throughout larval development and into the adult stage as the myotomal cells differentiate into striated, mononucleate muscle cells—unlike vertebrate striated muscle cells, those of amphioxus never become multinucleate. In late larvae and adults myotomal expression of AmphiMLC-alk is localized along the medial edge of the myotome and at the ends of the cells. This is the first demonstration of intracellular localization of MLC transcripts in muscle cells of any animal. Expression of AmphiMLC-alk was also detected in smooth muscles as well as in striated muscles not derived from the myotome. These expression data are consistent with the Southern blot analysis in suggesting that there is only one MLC-alk gene in amphioxus. Thus, duplication of an ancestral vertebrate MLC-alk gene probably occurred after the vertebrate and amphioxus lineages split. We conclude that development of a segmented axial musculature preceded the evolution of multiple MLC-alk isoforms, which evidently arose about the time of multinucleation. Since myogenesis in amphioxus is similar to but far simpler than myogenesis in vertebrates at both the structural and gene levels, an understanding of myogenesis in amphioxus can give insights into both the evolutionary history and the detailed mechanisms of vertebrate myogenesis.

The number of Na+ channels in cultured chick muscle is increased by ARIA, an acetylcholine receptor-inducing activity

ARIA is a glycoprotein purified from chick brain on the basis of its ACh receptor-inducing activity (ARIA). In this study we present evidence that ARIA increases the number of voltage-gated sodium channels in chick muscle as well as the number of ACh receptors (AChRs). Exposure of chick myotubes to ARIA increased by twofold the number of 3H-saxitoxin binding, an effect that is comparable to the increase of AChRs assayed by 125I-alpha-bungarotoxin (125I-alpha-BTX) binding. We also documented effects of ARIA on myoblasts: the number of 125I-alpha-BTX binding sites in the mononucleated muscle cells was increased by 1.5-fold, and the peak TTX-sensitive inward currents increased by the same amount. No change was detected in the voltage dependence of channel activation, in mean channel current, or in mean channel open time. Thus, the Na+ channel is the first molecule, other than AChR subunits, whose expression has been shown to be induced by ARIA. Since sodium channels are concentrated at motor end plates, our results provide circumstantial evidence that ARIA may regulate several genes expressed at developing neuromuscular junctions. Moreover, the finding that ARIA's effects extend to mononucleated myoblasts suggests that this protein may be important during the earliest stages of muscle formation and innervation.

A role for the Ca 2+-dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis

The formation of multinucleate skeletal muscle cells (myotubes) is a Ca 2+-dependent process involving the interaction and fusion of mononucleate muscle cells (myoblasts). Specific cell-cell adhesion precedes lipid bilayer union during myoblast fusion and has been shown to involve both Ca 2+-independent (CI) 2 2 Abbreviations used: CI, Ca 2+-independent; CD, Ca 2+-dependent; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; EE, embryo extract; EGTA, ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; HHBSS, 10 m M Hepes-buffered Hanks' balanced salt solution; NP-40, Nonidet P-40; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; TE cells, cells exposed to 0.01% trypsin/ 2.5 m M EDTA; TC cells, cells exposed to 0.01% trypsin/2.5 m M Ca 2+; LTE cells, cells exposed to 0.0005% trypsin/2.5 m M EDTA. and Ca 2+ dependent (CD) mechanisms. In this paper we present evidence that CD myoblast adhesion involves a molecule similar or identical to two known CD adhesion glycoproteins, N-cadherin and A-CAM. These molecules were previously identified by other laboratories in brain and cardiac muscle, respectively, and are postulated to be the same molecule. Antibodies to N-cadherin and A-CAM immunoblotted a similar band with a molecular weight of approximately 125,000 in extracts of brain, heart, and pectoral muscle isolated from chick embryos and in extracts of muscle cells grown in vitro at Ca 2+ concentrations that either promoted or inhibited myotube formation. In assays designed to measure the interaction of fusion-competent myoblasts in suspension, both polyclonal and monoclonal anti-N-cadherin antibodies inhibited CD myoblast aggregation, suggesting that N-cadherin mediates the CD aspect of myoblast adhesion. Anti-N-cadherin also had a partial inhibitory effect on myotube formation likely due to the effect on myoblast-myoblast adhesion. The results indicate that N-cadherin/A-CAM plays a role in myoblast recognition and adhesion during skeletal myogenesis.

Ultrastructure of the caudal muscle cells in the larva of a polyclinid ascidian

Two paraxial bands of somatic striated muscle occur in the tail of the larva of the compound ascidian Aplidium ?constellatum. The mononucleate muscle cells of each band align in longitudinal rows between the epidermis and the notochord. The cross-striated myofibrils, originating and terminating at intermediate junctions on the transverse cellular boundaries, are indiscrete. They follow a spiral course through the subcortical and medullary sarcoplasm, bypassing the nucleus and the other organelles and inclusions in the center of the cell. Cisternae of the sarcoplasmic reticulum envelop the myofibrils, forming compact fenestrated sheets that are continuous between the sarcomeres and locally undifferentiated with respect to the myofibrillar striations. Cisternae of the perifibrillar sarcoplasmic reticulum near each sarcomeric Z-line establish dyadic interior couplings with a network of tubular invaginations of the sarcolemma. The sarcolemmal tubules can originate from any surface, including the transverse cellular boundaries. Near the half I-bands of the terminal sarcomeres at the intermediate junctions, the perifibrillar cisternae frequently leave the fenestrated sheets and extend to the overlying sarcolemma, becoming the sub-sarcolemmal cisternae of dyadic peripheral couplings.

Synthesis of rat myosin light chains in heterokaryons formed between undifferentiated rat myoblasts and chick skeletal myocytes.

The control of gene expression during terminal myogenesis was explored in heterokaryons between differentiated and undifferentiated myogenic cells by analyzing the formation of species specific myosin light chains of chick and rat skeletal muscle. Dividing L6 rat myoblasts served as the biochemically undifferentiated parent. The differentiated parental cells were mononucleated muscle cells (myocytes) that were obtained from primary cultures of embryonic chick thigh muscle by blocking myotube formation with EGTA and later incubating the postimitotic cells in cytochalasin B. Heterokaryons were isolated by the selective rescue of fusion products between cells previously treated with lethal doses of different cell poisons. 95-99% pure populations of heterokaryons formed between undifferentiated rat myoblasts and differentiated chick myocytes were obtained. The cells were labeled with [35S]methionine, and whole cell extracts were analyzed on two-dimensional polyacrylamide gels. These heterokaryons synthesize the light chain of chick myosin and both embryonic and adult light chains of rat skeletal myosin. Control homokaryons formed by fusing undifferentiated cells to themselves did not synthesize skeletal myosin light chains. Control heterokaryons formed between undifferentiated rat myoblasts and chick fibroblasts also failed to synthesize myosin light chains. These results indicate that differentiated chick muscle cells provide some factor that induces L6 myoblasts to synthesize rat myosin light chains. This system provides a model for investigating the processes by which differentiated cell functions are induced.

Atypical myogenesis in hamster hereditary polymyopathy. An in vitro study.

An in vitro study of dissociated muscle cells from normal and myopathic neonatal hamsters was undertaken in an attempt to characterize, by morphological means, a possible abnormal myogenesis in the hereditary polymyopathy of the hamster. There were fundamental structural differences between mononucleated muscle cells before and during myogenic cell fusion, as well as between multinucleated myotubes of normal and myopathic cultures. With reference to conventional morphologic features of normal muscle cultures, more numerous "atypical" foci of myogenesis, some with a bizarre appearance, were seen in myopathic hamsters, as compared to the controls. Because the cultured cells developed in the absence of vascular and neurogenic influences, a myogenic origin may be inferred for the different structural base found in vitro in the myopathic hamster.

The nature of 5-bromodeoxyuridine inhibition of muscle enzyme development in vitro

Although it is generally believed that 5-bromodeoxyuridine (BrdUrd) can suppress the phenotypic expression of a wide variety of cell types by virtue of its incorporation into DNA, clear quantitative evidence has been lacking. We have employed the enzymes (creatine phosphokinase and glycogen phosphorylase) which are important in energy provision for contraction and linked with the morphological differentiation of muscle, as markers for examining the mechanism of BrdUrd inhibition. Several findings lead us to conclude that BrdUrd most likely prevents the synthesis of these enzymes as a result of its presence in DNA: (1) BrdUrd is most effective as an inhibitor while mononucleated muscle cells are synthesizing DNA; (2) the reversal of inhibition requires times of the order of a division(s) and is 5-fluorodeoxyuridine (FdUrd) sensitive; (3) loss of enzyme activity can be correlated with increasing [ 3H]BrdUrd incorporation into DNA; and (4) altering the extent of [ 3U]BrdUrd incorporation into DNA by addition of thymidine also decreases the inhibitory effectiveness of BrdUrd. As noted by others, BrdUrd appears to preferentially inhibit those enzymes which are usually associated with muscle. In addition, we find from our studies with BrdUrd that the development of these enzymes and myotube formation may be separate events. There are no apparent altered forms of other enzymes produced in BrdUrd treated cells. In muscle, BrdUrd does not appear to act by exclusively inhibiting inducible enzymes. The manner by which BrdUrd appears to preferentially inhibit “tissue specific” products can still only be treated speculatively. However, the muscle isoenzymes of creatine phosphokinase and glycogen phosphorylase serve as experimentally amenable markers for probing the molecular details involved in BrdUrd action.