Spatiotemporal expression of connexin 39 and -43 during myoblast differentiation in cultured cells and in the mouse embryo

Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C(2)C(12) mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C(2)C(12) cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo.     

Spatiotemporal Expression of Connexin 39 and -43 During Myoblast Differentiation in Cultured Cells and in the Mouse Embryo

Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C₂C₁₂ mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C₂C₁₂ cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo.     

Expression of the Extracellular Fatty Acid Binding Protein (Ex-FABP) during Muscle Fiber Formationin Vivoandin Vitro

We report that Ex-FABP, an extracellular protein belonging to the lipocalin family and involved in the extracellular transport of long-chain fatty acids, is expressed in the forming myotubes bothin vivoandin vitro.The presence of the protein and of the mRNA was observed in newly formed myotubes at early stages of chick embryo development by immunohistochemistry and byin situhybridization. At later stages of development myofibers still expressed both the mRNA and the protein. Ex-FABP expression was observed also in the developing myocardium and the muscular layer of large blood vessels. In agreement with these findings, an initial expression of the mRNA and protein secretion by cultured chicken myoblasts were observed only after the onset of myoblast fusion. Double-immunofluorescence staining of these cultured cells revealed that multinucleate myotubes were stained by antibodies directed against both the Ex-FABP and the sarcomeric myosin, whereas immature myotubes and single myoblasts were not. When added to cultured myoblasts, antibodies against the Ex-FABP induced a strong enhancement of the production of the same protein. In all experiments some cell sufferance and a transient impairment of myotube formation were also observed. The finding that the continuous removal of the Ex-FABP from the culture medium of myoblasts, due to the formation of immune complexes, resulted in an overproduction of the protein suggests a feedback (autocrine) control during myotube differentiation and maturation. We propose that the requirement for increased transport and metabolism of free fatty acid released from the membrane phospholipids and storage lipids, mediated by Ex-FABP, may be essential during differentiation of multinucleated myotubes or that an increased local demand of fatty acids and metabolites may act as a local hormone in tissues differentiating and undergoing morphogenesis.     

Formation and characterization of spontaneously formed heterokaryons between quail myoblasts and 3T3-L1 preadipocytes: correlation between differential plasticity and degree of differentiation

Skeletal muscle cells and adipose cells have a close relationship in developmental lineage. Our previous study has shown that the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells (preadipocytes) normally differentiated into myotubes, whereas the heterokaryons between myoblasts and differentiated 3T3-L1 cells (adipocytes) failed myogenic differentiation. These results suggest differences between preadipocytes and adipocytes. The purpose of this study was to clarify whether preadipocytes have flexibility in differentiation before terminal adipose differentiation. Presumptive quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and mouse 3T3-L1 cells (either preadipocytes or adipocytes) were co-cultured for 48 h under conditions allowing myogenic differentiation. On co-culture between myoblasts and undifferentiated 3T3-L1 cells, heterokaryotic myotubes formed spontaneously, but not on co-culture with differentiated 3T3-L1 cells. In addition, the heterokaryotic myotubes expressed mouse myogenin derived from the 3T3-L1 cell gene. Our previous study indicated that the fusion sensitivity of differentiating myoblasts change with decreasing cholesterol of the cell membrane during myoblast fusion. Thus we compared the level of membrane cholesterol between undifferentiated and differentiated 3T3-L1 cells. The result showed that the level of membrane cholesterol in 3T3-L1 cells increases during adipose differentiation. Corresponding to the increase in membrane cholesterol content, differentiated 3T3-L1 cells had lower sensitivity to HVJ (Sendai virus)-mediated cell fusion than undifferentiated 3T3-L1 cells. This study demonstrated that 3T3-L1 cells at an undifferentiated state have a capacity for spontaneous fusion with differentiating myoblasts following myogenic differentiation, and that the capacity is lost after terminal adipose differentiation.     

Inhibition of Notch signaling induces myotube hypertrophy by recruiting a subpopulation of reserve cells

During muscle differentiation, a population of quiescent undifferentiated myoblasts (reserve cells) emerges among mature muscle cells. However, the molecular mechanisms underlying such cell segregation and the characterization of this subpopulation of myoblasts remain to be determined. Notch is known to control the behavior and fate of murine muscle stem cells. In this study, we examined the role of Notch in myoblast segregation. We showed that inhibition of Notch activity by either overexpressing Numb or by using a pharmacological gamma-secretase inhibitor (DAPT) enhanced differentiation of murine and human myoblasts. This effect was not restricted to in vitro culture systems since DAPT-treated zebrafish embryos also showed increased differentiation. Using C2.7 myoblasts as a model, we showed that inhibition of Notch induced myotube hypertrophy by recruiting reserve cells that do not normally fuse. We further showed that endogenous Notch-signaling components were differentially expressed and activated in reserve cells with respect to Notch 1 and CD34 expression. We identified CD34 negative reserve cells as the subpopulation of myoblasts recruited to fuse into myotubes during differentiation in response to Notch inhibition. Therefore, we showed here that the activation of Notch 1 is important to maintain a subpopulation of CD34 negative reserve cells in an undifferentiated state.     

The HIF-1 response to simulated ischemia in mouse skeletal muscle cells neither enhances glycolysis nor prevents myotube cell death

Hypoxia-inducible factor (HIF) plays an important role in regulating gene expression in response to ischemia. Although activation of HIF-1 in muscle tissue was found during ischemia in vivo, the meaning and mechanisms in isolated cells are still incompletely understood. We studied activation of HIF-1 in skeletal muscle cells cultured in either their undifferentiated myoblast state or differentiated into myotubes. HIF-1 was activated in myoblasts and myotubes by hypoxia and simulated ischemia. Induction of adrenomedullin mRNA and, to a lesser extent, VEGF mRNA correlated well with the induction of HIF-1alpha protein in both cell types. Enzymes of glycolysis-like lactate dehydrogenase and pyruvate kinase showed upregulation of their mRNA only under hypoxic conditions but not during simulated ischemia. Phosphofructokinase mRNA showed no significant upregulation at all. Although HIF-1 was activated in myotubes during simulated ischemia, myotubes died preceded by a loss of ATP. Myoblasts survived simulated ischemia with no decrease in ATP or ATP turnover. Furthermore, pharmacological inhibition of HIF-1 hydroxylases by dimethyloxalylglycine (DMOG) increased HIF-1alpha accumulation and significantly upregulated the expression of adrenomedullin, VEGF, lactate dehydrogenase, and pyruvate kinase in myoblasts and myotubes. However, DMOG provided no protection from cell death. Our data indicate that HIF-1, although activated in myotubes during simulated ischemia, cannot protect against the loss of ATP and cell viability. In contrast, myoblasts survive ischemia and thus may play an important role during regeneration and HIF-1-induced revascularization.     

Expression of glucocorticoid receptors in the regenerating human skeletal muscle

Many stress conditions are accompanied by skeletal muscle dysfunction and regeneration, which is essentially a recapitulation of the embryonic development. However, regeneration usually occurs under conditions of hypothalamus-pituitary-adrenal gland axis activation and therefore increased glucocorticoid (GC) levels. Glucocorticoid receptor (GR), the main determinant of cellular responsiveness to GCs, exists in two isoforms (GRalpha and GRbeta) in humans. While the role of GRalpha is well characterized, GRbeta remains an elusive player in GC signalling. To elucidate basic characteristics of GC signalling in the regenerating human skeletal muscle we assessed GRalpha and GRbeta expression pattern in cultured human myoblasts and myotubes and their response to 24-hour dexamethasone (DEX) treatment. There was no difference in GRalpha mRNA and protein expression or DEX-mediated GRalpha down-regulation in myoblasts and myotubes. GRbeta mRNA level was very low in myoblasts and remained unaffected by differentiation and/or DEX. GRbeta protein could not be detected. These results indicate that response to GCs is established very early during human skeletal muscle regeneration and that it remains practically unchanged before innervation is established. Very low GRbeta mRNA expression and inability to detect GRbeta protein suggests that GRbeta is not a major player in the early stages of human skeletal muscle regeneration.     

Irreversible reduction in potassium fluxes accompanies terminal differentiation of human myoblasts to myotubes

Potassium and sodium fluxes believed to be important in the cellular response to serum and growth factors have not been widely investigated in cells which have undergone terminal differentiation. In this study we have analyzed two main K+ transport systems--the ouabain-sensitive Na+/K+ pump and the bumetanide-sensitive transporter--in human muscle in vitro at two developmental stages: proliferating myoblasts and differentiated myotubes. Myoblast differentiation to myotubes was accompanied by a marked decrease in both the ouabain-sensitive and the bumetanide-sensitive K+ (Rb+) influxes. The addition of serum to the terminally differentiated myotubes had no effect on these K+ transporters. However, serum addition to serum-deprived, undifferentiated myoblasts produced a marked stimulation of these K+ fluxes. The bumetanide-sensitive K+ transporter in human myoblasts and myotubes has the following properties: (1) It carries 30% and 40% of the total K+ influx in myoblasts and myotubes, respectively. (2) It performs net efflux of K+ in the undifferentiated myoblasts and zero net flux (self-exchange) in terminally differentiated myotubes. (3) It is dependent on extracellular Na+ and Cl- in addition to K+. (4) In myoblasts, the Km value for K+ is 1.36 mM, similar to the Km for K+ of the Na+/K+ pump. (5) It is resistant to ouabain (up to 2 mM) and sensitive to furosemide (K0.5 = 5 X 10(-6) M) and bumetanide (K0.5 = 10(-7) M). These data indicate that following terminal differentiation of proliferating myoblasts to mitotically inactive myotubes there is an irreversible reduction of K+ fluxes with a change in the net flux of K+ carried by the bumetanide-sensitive transporter.     

Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells

We examined the time course of mRNA expression of myogenic cell differentiation- and muscle proteolytic system-related genes in cultures of C2C12 cells during differentiation from myoblasts to myotubes. Furthermore, we treated C2C12 myotubes with dimethyl sulphoxide (DMSO) and dexamethasone (Dex), and examined changes in these mRNA levels. Myogenin (Myog), Atrogin1, forkhead box O1 (Foxo1) and Capn1 mRNA levels increased in C2C12 cells differentiating from myoblasts to myotubes, whereas Myf5 mRNA levels decreased. Although genes such as MRF4, Foxo3a, UbB, Capn1 and MuRF1 mRNAs in the myotubes were affected by DMSO exposure, mRNA levels of other genes were not markedly affected by exposure to 0.02% or 0.5% DMSO. Myf5, MRF4, Atrogin1, Foxo3 and MuRF1 mRNA levels were elevated by Dex at all time points, Cbl and Capn1 mRNA levels were significantly elevated by Dex at 8 h, and Myog mRNA levels were significantly elevated by Dex at 24 h. However, CtsH mRNA levels decreased significantly with Dex at 24 h. This study provides a useful database of gene profiles that are differentially expressed throughout myogenic cell differentiation and the muscle proteolytic system.