Evidence on the participation of protein kinase C alpha in the proliferation of cultured myoblasts.

There is evidence involving protein kinase C (PKC) in the signal transduction pathways that regulate the differentiation of myoblasts into mature multinucleated muscle cells (myotubes). In order to obtain information on the possible role of individual PKC isozymes in myogenesis, in the present work we investigated the differential expression of PKC isoforms alpha, beta, delta, epsilon, and zeta during muscle cell development in vitro. Chick embryo myoblasts cultured from 1 to 6 days were used as experimental model. Morphological characterization and measurement of specific biochemical parameters in cultures, e.g., DNA synthesis, creatine kinase activity, and myosin levels, revealed a typical muscle cell developmental pattern consisting of an initial proliferation of myoblasts followed by their differentiation into myotubes. PKC activity was high at the proliferation stage, decreased as myoblasts elongated and fused, and increased again in differentiated myotubes. In proliferating myoblasts, the PKC inhibitors calphostin C and bisindolylmaleimide I decreased DNA synthesis whereas in myoblasts undergoing differentiation they exerted the opposite effect, suggesting that PKC plays a role at both stages of myogenesis. Western blot analysis of changes in the expression of PKC isoforms during muscle cell development showed high levels of PKC alpha in the proliferating phase which markedly decreased as myoblasts differentiated. Treatment with TPA of proliferative myoblasts inhibited DNA synthesis and selectively down-regulated PKC alpha, suggesting that this isozyme may have an important role in maintaining myoblast proliferation. On the other hand, an increase in the expression of PKC beta, delta, and epsilon was detected during myogenesis, suggesting that one or more of these isoforms may participate in the differentiation process of myoblasts.     

Regulation of tropomyosin gene expression during myogenesis.

In skeletal muscle, tropomyosin has a critical role in transduction of calcium-induced contraction. Presently, little is known about the regulation of tropomyosin gene expression during myogenesis. In the present study, qualitative and quantitative changes in the nucleic acid populations of differentiating chicken embryo muscle cells in culture have been examined. Total nucleic acid content per nucleus increased about fivefold in fully developed myotubes as compared to mononucleated myoblasts. The contribution of deoxyribonucleic acid to the total nucleic acid population decreased from 24% in myoblasts to 5% of total nucleic acid in myotubes. Concomitant with the decrement in deoxyribonucleic acid contribution to total nucleic acid was an increase in polyadenylated ribonucleic acid (RNA) content per cell which reached levels in myotubes that were 17-fold higher than those of myoblasts. Specific changes in the RNA population during myogenesis were further investigated by quantitation of the synthetic capacity (messenger RNA levels) per cell for alpha- and beta-tropomyosin. Cell-free translation and immunoprecipitation demonstrated an approximately 40-fold increase in messenger RNA levels per nucleus for alpha- and beta-tropomyosin after fusion in the terminally differentiated myotubes. Indirect immunofluorescence with affinity-purified tropomyosin antibodies demonstrated the presence of tropomyosin-containing filaments in cells throughout myogenesis. Thus, the tropomyosin genes are constitutively expressed during muscle differentiation through the production of tropomyosin messenger RNA and translation into tropomyosin protein.     

Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy

During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/beta-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of beta-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/beta-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts.     

The multiple ADP/ATP translocase genes are differentially expressed during human muscle development.

The expression of the genes encoding the three isoforms of the human ADP/ATP translocase (T1, T2, and T3) has been analyzed at different stages of myogenic differentiation in an in vitro muscle cell system and compared with that in mature muscle. The results indicate that the three stages of muscle differentiation corresponding to myoblast proliferation, myotube formation, and mature muscle fibers are characterized by a different pattern of expression of the ADP/ATP translocase genes. In particular, the two T2-specific mRNAs are present at high, similar levels in myoblasts and myotubes and markedly decrease in amount in mature adult muscle. By contrast, the T3-specific mRNA is present in high amount in growing myoblasts, decreases markedly in myotubes, and is barely detectable in adult muscle. Finally, the T1-specific mRNA is present at a high level in adult muscle and is not detectable in either myoblasts or myotubes. Therefore, T1 gene expression appears to be a marker of a late stage in myogenesis. A parallel investigation of expression of the myosin heavy chain mRNA revealed absence of hybridization with the specific probe in RNA from proliferating myoblasts, a significant hybridization in myotube RNA, and a strong signal in adult muscle RNA.     

Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts

Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA-encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase beta subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and triose-phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (normoxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3- to 5-fold during commitment and exist from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3- to 6-fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.     

Emerin expression at the early stages of myogenic differentiation

Emerin is an ubiquitous protein localized at the nuclear membrane of most cell types including muscle cells. The protein is absent in most patients affected by the X-linked form of Emery-Dreifuss muscular dystrophy, a disease characterized by slowly progressive muscle wasting and weakness, early contractures of the elbows, Achilles tendons, and post-cervical muscles, and cardiomyopathy. Besides the nuclear localization, emerin cytoplasmic distribution has been suggested in several cell types. We studied the expression and the subcellular distribution of emerin in mouse cultured C2C12 myoblasts and in primary cultures of human myoblasts induced to differentiate or spontaneously differentiating in the culture medium. In differentiating myoblasts transiently transfected with a cDNA encoding the complete emerin sequence, the protein localized at the nuclear rim of all transfected cells and also in the cytoplasm of some myoblasts and myotubes. Cytoplasmic emerin was also observed in detergent-treated myotubes, as determined by electron microscopy observation. Both immunofluorescence and biochemical analysis showed, that upon differentiation of C2C12 cells, emerin expression was decreased in the resting myoblasts but the protein was highly represented in the developing myotubes at the early stage of cell fusion. Labeling with specific markers of myogenesis such as troponin-T and myogenin permitted the correlation of increased emerin expression with the onset of muscle differentiation. These data suggest a role for emerin during proliferation of activated satellite cells and at the early stages of differentiation.     

Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis

During terminal differentiation of skeletal myoblasts, cells fuse to form postmitotic multinucleated myotubes that cannot reinitiate DNA synthesis. Here we investigated the temporal relationships among these events during in vitro differentiation of C2C12 myoblasts. Cells expressing myogenin, a marker for the entry of myoblasts into the differentiation pathway, were detected first during myogenesis, followed by the appearance of mononucleated cells expressing both myogenin and the cell cycle inhibitor p21. Although expression of both proteins was sustained in mitogen-restimulated myocytes, 5- bromodeoxyuridine incorporation experiments in serum-starved cultures revealed that myogenin-positive cells remained capable of replicating DNA. In contrast, subsequent expression of p21 in differentiating myoblasts correlated with the establishment of the postmitotic state. Later during myogenesis, postmitotic (p21-positive) mononucleated myoblasts activated the expression of the muscle structural protein myosin heavy chain, and then fused to form multinucleated myotubes. Thus, despite the asynchrony in the commitment to differentiation, skeletal myogenesis is a highly ordered process of temporally separable events that begins with myogenin expression, followed by p21 induction and cell cycle arrest, then phenotypic differentiation, and finally, cell fusion.     

Intracellular localisation and expression of mammalian CDC2 protein during myogenic differentiation

Myogenic differentiation involves withdrawal of myoblasts from the cell cycle and fusion to form multinucleate myotubes. To examine the role that cell cycle control genes may play in this process, we investigated the steady state levels of CDC2 protein and RNA during myogenesis of L6E9 rat myoblasts. Indirect immunofluorescence using a CDC2 affinity-purified antibody showed that this protein is localised exclusively in the cytoplasm with a higher concentration perinuclearly. Both protein and RNA levels were down-regulated to similar extents early in the differentiation process, as cells became quiescent. There was a further down-regulation of protein after fusion to form myotubes. Autonomous expression of CDC2 protein in L6E9 cells, after stable transfection with a metallothionein: CDC2 gene construct, failed to inhibit the differentiation process. This suggests that, although there is down-regulation in levels of CDC2 RNA and protein during myogenesis, this phenomenon per se does not play a primary role in controlling the differentiation process. If CDC2 is involved in control of differentiation, this must depend on post-translational modification of the protein.     

Development regulation of the subcellular distribution and glycosylation of GLUT1 and GLUT4 glucose transporters during myogenesis of L6 muscle cells.

L6 myoblasts spontaneously undergo differentiation and cell fusion into myotubes. These cells express both GLUT1 and GLUT4 glucose transporters, but their expression varies during myogenesis. We now report that the subcellular distribution and the protein processing by glycosylation of both glucose transporter isoforms also change during myogenesis. Crude plasma membrane and light microsome fractions were isolated from either myoblasts or myotubes and characterized by the presence of two functional proteins, the Na+/K(+)-ATPase and the dihydropyridine receptor (DHPR). Immunoreactive alpha 1 subunit of the Na+/K(+)-ATPase was faint in the crude plasma membrane fraction from myoblasts, but abundant in both membrane fractions from myotubes. In contrast, the alpha 1 subunit of the DHPR, which is expressed only in differentiated muscle, was detected in crude plasma membrane from myotubes but not from myoblasts. Therefore, crude plasma membrane fractions from myoblasts and myotubes contain cell surface markers, and the composition of these membranes appears to be developmentally regulated during myogenesis. GLUT1 protein was more abundant in the crude plasma membrane relative to the light microsome fraction prepared from either myoblasts or myotubes. The molecular size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the GLUT1 transporters in myotubes was smaller than that in myoblasts (Mr 47,000 and 53,000, respectively). GLUT4 protein (Mr 48,000) was barely detectable in the crude plasma membrane fraction and was almost absent in the light microsome fraction prepared from myoblasts. However, GLUT4 protein was abundant in myotubes and was predominantly located in the light microsome fraction. Treatment with endoglycosidase F reduced the molecular size of the transporters in all fractions to Mr 46,000 for GLUT1 and Mr 47,000 for GLUT4 proteins. In myotubes, acute insulin treatment increased the crude plasma membrane content of GLUT1 marginally and of GLUT4 markedly, with a concomitant decrease in the light microsomal fraction. These results indicate that: (a) the subcellular distribution of glucose transporters is regulated during myogenesis, GLUT4 being preferentially sorted to intracellular membranes; (b) both GLUT1 and GLUT4 transporters are processed by N-linked glycosylation to form the mature transporters in the course of myogenesis; and (c) insulin causes modest recruitment of GLUT1 transporters and marked recruitment of GLUT4 transporters, from light microsomes to plasma membranes in L6 myotubes.     

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.     

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.     

Zika virus disrupts gene expression in human myoblasts and myotubes: Relationship with susceptibility to infection

The tropism of Zika virus (ZIKV) has been described in the nervous system, blood, placenta, thymus, and skeletal muscle. We investigated the mechanisms of skeletal muscle susceptibility to ZIKV using an in vitro model of human skeletal muscle myogenesis, in which myoblasts differentiate into myotubes. Myoblasts were permissive to ZIKV infection, generating productive viral particles, while myotubes controlled ZIKV replication. To investigate the underlying mechanisms, we used gene expression profiling. First, we assessed gene changes in myotubes compared with myoblasts in the model without infection. As expected, we observed an increase in genes and pathways related to the contractile muscle system in the myotubes, a reduction in processes linked to proliferation, migration and cytokine production, among others, confirming the myogenic capacity of our system in vitro. A comparison between non-infected and infected myoblasts revealed more than 500 differentially expressed genes (DEGs). In contrast, infected myotubes showed almost 2,000 DEGs, among which we detected genes and pathways highly or exclusively expressed in myotubes, including those related to antiviral and innate immune responses. Such gene modulation could explain our findings showing that ZIKV also invades myotubes but does not replicate in these differentiated cells. In conclusion, we showed that ZIKV largely (but differentially) disrupts gene expression in human myoblasts and myotubes. Identifying genes involved in myotube resistance can shed light on potential antiviral mechanisms against ZIKV infection.