Myogenic Cell Line Research Articles

Insulin-like growth factor I (IGF-I) receptor overexpression abolishes the IGF requirement for differentiation and induces a ligand-dependent transformed phenotype in C2 inducible myoblasts.

Insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cell lines, and these actions are mostly mediated through the type I IGF receptor (type I IGF-R). To further investigate the role of this receptor in phenotypic characteristics of C2 murine myoblasts, we overexpressed the human type I IGF-R in the inducible clone of C2 cells, which requires IGFs in the differentiation medium to undergo terminal differentiation. Inducible myoblasts were transfected with either the eukaryotic expression vector pNTK or pNTK containing the human type I IGF-R complementary DNA, and we isolated two clones named Ind-Neo and Ind-R, respectively. Binding and autophosphorylation experiments indicate that Ind-R cells express about 10 times as much type I IGF-R compared with Ind-Neo control cells and that the transfected type I IGF-R is functional in Ind-R cells. We show that overexpression of the human type I IGF-R makes inducible myoblasts able to differentiate spontaneously, as assessed by expression of the myogenic transcription factors MyoD and myogenin, detection of the muscle-specific protein troponin T, and myotube formation. Moreover, when exposed to IGF-I, Ind-R cells lose contact inhibition, grow in the presence of a low level of growth factors and form colonies in soft agar, which is characteristic of a ligand-dependent transformed phenotype. It emerges from this study that 1) the type I IGF-R is strongly involved in the phenotypic differences between inducible and permissive cells with respect to the differentiation program; and 2) overexpression causes this receptor to act as a ligand-dependent transforming protein in muscle cells. We suggest that type I IGF-R abundance and level of activation may determine the efficiency of the autocrine mode of action of IGFs and discriminate their biological functions.

Developmental Regulation of Mac25/Insulin-like Growth Factor-Binding Protein-7 Expression in Skeletal Myogenesis

Mac25 is a newly discovered member of the insulin-like growth factor-binding protein (IGFBP) family, recently assigned the name IGFBP-7. Mac25/IGFBP-7 is hypothesized to have growth-suppressing activity, since mac25/IGFBP-7 mRNA is down-regulated in several tumor cell lines and is highly expressed in senescent mammary epithelial cells. In this study, mac25/IGFBP-7 mRNA expression was characterized in the C2 skeletal myogenic cell line, which undergoes a transition from actively dividing, undifferentiated myoblasts to nondividing, differentiated myotubes. Mac25/IGFBP-7 mRNA levels were 2.5-fold higher in dividing C2 myoblasts than in nondividing myotubes. The inverse correlation between mac25/IGFBP-7 expression and myogenic differentiation was further examined by treating myogenic cultures with transforming growth factor-β (TGF-β) or insulin-like growth factor-I (IGF-I). TGF-β inhibited myogenic differentiation by 98% and stimulated mac25/IGFBP-7 mRNA expression 2-fold. IGF-I stimulated differentiation by 50% and inhibited mac25/IGFBP-7 expression 2- to 3-fold. These findings indicate that, in contrast to other cell systems examined so far, expression of this new member of the IGFBP family is not always correlated with a nonproliferative state.

Degradation of the inducible cAMP early repressor (ICER) by the ubiquitin-proteasome pathway.

The inducible cAMP early repressor (ICER) is a powerful transcriptional inhibitor that plays an important role in the regulation of the cAMP-dependent transcriptional response in the neuroendocrine system. ICER activity is primarily determined by its intracellular concentration, rather than by post-translational modifications, such as phosphorylation. We investigated the mechanisms that regulate the levels of ICER transcript and polypeptides in cardiocytes, myogenic (C2C12) and pituitary-derived (GH3) cell lines. We show that in primary cardiocytes and GH3 cells ICER was inducible by cAMP but not by membrane depolarization. Moreover, lactacystin, a specific proteasome inhibitor, decreased the rate of ICER degradation. This effect was associated with the accumulation of ICER-ubiquitin conjugates. We conclude that the intracellular levels of ICER are controlled by the ubiquitin-proteasome pathway for protein breakdown.

The glucocorticoid receptor and AP-1 are involved in a positive regulation of the muscle regulatory gene myf5 in cultured myoblasts.

The muscle regulatory factor, myf5, is involved in the establishment of skeletal muscle precursor cells. Little is known, however, about the control of the expression of the gene encoding this basic helix-loop-helix (bHLH) factor. We have addressed this question in the mouse myogenic cell line, C2, and in a derivative of this cell line where the myf5 gene is the only muscle-specific bHLH factor to be expressed at the myoblast stage. We present evidence that the synthetic glucocorticoid dexamethasone, and the pharmacological agent anisomycin, act synergistically to rapidly up-regulate the levels of myf5 transcript and protein. The glucocorticoid antagonist RU 486 abolishes this synergy, demonstrating the involvement of the glucocorticoid receptor. The expression of a dominant negative mutant of c-jun which interferes with the transactivating properties of all AP-1 family members also blocks the induction of myf5 by anisomycin and dexamethasone. An activator of protein kinase C (PKCs), 12-O-tetradecanoyl phorbol 13-acetate (TPA), abolishes the up-regulation of myf5 gene expression by dexamethasone and anisomycin, and its effect is counteracted by an inhibitor of PKCs, GF 109203X. These results point to the possible involvement of PKCs in the negative control of myf5. Evidence that both positive and negative regulation of myf5 transcripts, described here, does not require the fresh synthesis of transcription factors suggests that myf5 may behave like an immediate early gene.

Simultaneous overexpression of two stress proteins in rat cardiomyocytes and myogenic cells confers protection against ischemia-induced injury.

Mitochondria are known to be a major target during ischemic cardiac injury. Previous studies have shown that in rodent myogenic cells and in the hearts of transgenic mice in which the heat shock or stress protein 70 is increased, there is a marked tolerance to ischemia/reperfusion injury. Two other heat shock proteins (HSP60 and HSP10) are known to form, within the mitochondria, a chaperonin complex that is important for mitochondrial protein folding and function. We were then interested in investigating whether increased expression of these two stress proteins is able to protect myogenic cells against ischemia/reperfusion injury. We generated recombinant adenoviral vectors containing HSP60, HSP10, or a combination of the two genes. These adenoviral constructs overexpress significant amounts of these stress proteins in both rat neonatal cardiomyocytes and the myogenic H9 c2 cell line. Cells infected with an adenoviral construct overexpressing both HSP60 and HSP10 were found to be protected against simulated ischemia, whereas cells infected with adenoviral constructs overexpressing only HSP60 or HSP10 alone were not rendered tolerant to simulated ischemic injury. These results suggest that the simultaneous expression of these two proteins that form a chaperonin complex in the mitochondria plays an important role in the survival of myogenic cells after ischemia/reperfusion injury.

Actin Cytoskeleton as a Target for 2-Chloro Adenosine: Evidence for Induction of Apoptosis in C2C12 Myoblastic Cells

Recent results for various cell types have shown that adenosine is capable of inducing relevant cytophatological alterations which can eventually lead to apoptosis. No data are available regarding the involvement of adenosine in apoptosis of muscle cells. In this work, we studied the effect of the relatively hydrolysis-resistant adenosine analog 2-chloro adenosine on a cultured myogenic cell line, C2C12, which is able to differentiatein vitro,leading to the formation of syncythia, giant multinucleated cells called myotubes. Results indicated that 2-chloro adenosine induces apoptotic cell death in both myoblasts and myotubes; this was preceded by a derangement of actin microfilaments, which represent the main cytoskeletal component and play a pivotal role in the physiology of such cell line. The time-dependency of cytoskeletal alterations suggested a causal relationship between these changes and the induction of apoptosis. These results implicate adenosine as an endogenous regulator of apoptosis in muscle cells and validate this cell model system as a useful tool for studying human muscle cell pathologies.

Induction of heat shock proteins by tyrosine kinase inhibitors in rat cardiomyocytes and myogenic cells confers protection against simulated ischemia.

Previous studies have shown that in rodent myogenic cells and in the hearts of transgenic mice in which heat shock protein expression is increased there is a marked tolerance to ischemic/reperfusion injury. Furthermore, a recent study has shown that the benzoquinoid ansamycin antibiotic and tyrosine kinase inhibitor, herbimycin A, is capable of inducing the expression of heat shock proteins in fibroblasts. Our intention, in the present study, was to investigate if exposure of rat cardiomyocytes and the myogenic cell line H9c2 to herbimycin A would induce these proteins and, thus, confer protection against ischemic stress. For this purpose, we exposed both rat neonatal cardiomyocytes and H9c2 cells to herbimycin A and another related benzoquinoid ansamycin antibiotic, geldanamycin. We found that cells exposed to these compounds overexpressed heat shock proteins and are also rendered more tolerant to simulated ischemia as measured by the release of cytoplasmic enzymes. In addition, we found that the mechanism of induction of heat shock proteins by these compounds is similar, if not identical, to that of a heat shock (42 degrees C, 60 min). These results suggest that these benzoquinoid ansamycin antibiotics, or closely related analogues, may offer a pharmacological means of increasing the level of heat shock proteins in cardiac tissue and thus protect the heart against ischemic/reperfusion injury.

A natural hepatocyte growth factor/scatter factor autocrine loop in myoblast cells and the effect of the constitutive Met kinase activation on myogenic differentiation.

As a rule, hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells, while its receptor, the tyrosine kinase encoded by the met proto-oncogene, is expressed by the neighboring epithelial cells in a canonical paracrine fashion. In the present work we show that both HGF/SF and met are coexpressed by undifferentiated C2 mouse myoblasts. In growing cells, the autocrine loop is active as the receptor exhibits a constitutive phosphorylation on tyrosine that can be abrogated by exogenously added anti-HGF/SF neutralizing antibodies. The transcription of HGF/SF and met genes is downregulated when myoblasts stop proliferating and differentiate. The coexpression of HGF/SF and met genes is not exclusive to C2 cells since it has been assessed also in other myogenic cell lines and in mouse primary satellite cells, suggesting that HGF/SF could play a role in muscle development through an autocrine way. To analyze the biological effects of HGF/SF receptor activation, we stably expressed the constitutively activated receptor catalytic domain (p65(tpr-met)) in C2 cells. This active kinase determined profound changes in cell shape and inhibited myogenesis at both morphological and biochemical levels. Notably, a complete absence of muscle regulatory markers such as MyoD and myogenin was observed in p65(tpr-met) highly expressing C2 clones. We also studied the effects of the ectopic expression of human isoforms of met receptor (h-met) and of HGF/SF (h-HGF/SF) in stable transfected C2 cells. Single constitutive expression of h-met or h-HGF/SF does not alter substantially the growth and differentiation properties of the myoblast cells, probably because of a species-specific ligand-receptor interaction. A C2 clone expressing simultaneously both h-met and h-HGF/SF is able to grow in soft agar and shows a decrease in myogenic potential comparable to that promoted by p65(tpr-met) kinase. These data indicate that a met kinase signal released from differentiation-dependent control provides a negative stimulus for the onset of myogenic differentiation.

Chimeric calsequestrin and its targeting to the junctional sarcoplasmic reticulum of skeletal muscle.

Calsequestrin (CS) is the junctional sarcoplasmic reticulum (jSR) Ca2+ binding protein responsible for intraluminal Ca2+ storage. The targeting mechanisms of CS to the jSR are yet to be unraveled. The nine-amino acid epitope of the influenza virus hemoagglutinin (referred to as HA1) was added at the COOH-terminal of CS by polymerase chain reaction cloning. The HA1-tagged CS cDNA was transiently transfected in either HeLa cells, myogenic cell lines, such as C2 and L8 cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of chimeric CS-HA1 were monitored by epifluorescence and confocal microscopy using either anti-CS antibodies or anti-HA1 antibodies. About 30% of transfected HeLa cells and 20-40% of myogenic cells expressed CS-HA1 into intracellular compartments, such as the perinuclear cisternae of endoplasmic reticulum (ER). Myoblasts of newborn rat skeletal muscles were first transfected and subsequently stimulated to differentiate into myotubes. CS-HA1 was detected in approximately 20% of transfected myotubes and did not affect CS distribution in myotubes. In the soleus muscle of adult rat, intramuscular injection of bupivacaine induced necrosis followed by regeneration. In vivo transfection of HA1-tagged CS cDNA in regenerating skeletal muscles determined expression in a few skeletal muscle fibers; CS-HA1 was localized only in jSR, as judged by confocal microscopy of longitudinal sections. The present results show that chimeric CS-HA1 is correctly sorted to ER/SR compartments and that the free COOH-terminal is not requested for sorting, retention, and segregation of CS to the SR.

Gene transfer and expression of human alpha-galactosidase from mouse muscle in vitro and in vivo.

Lysosomal storage disorders are amenable to treatment by enzyme replacement. Genetic modification of muscle via direct injection of expression vectors might represent an alternative method of providing the defective enzymes, if adequate and long-lasting expression levels can be achieved in muscle. We have used the C2C12 mouse myogenic cell line to study the effect of combination of muscle-specific regulatory elements on the expression of the human lysosomal enzyme alpha-galactosidase (alpha-gal). In differentiated myotubes, a construct containing the myosin light chain 1/3 enhancer in combination with the human cytomegalovirus promoter resulted in higher expression than constructs combining the same enhancer with the rabbit beta-myosin heavy chain promoter, or containing the CMV promoter only. Increased enzymatic activity was detectable both in cell extracts and in supernatants. Furthermore, human fibroblasts deficient in alpha-gal were able to take up the enzyme from medium conditioned by transfected myoblasts. This did not occur in the presence of mannose-6-phosphate which indicates that the uptake was via mannose-6-phosphate receptors. To our knowledge, this is the first report in which a correctly processed form of human alpha-gal was expressed and secreted from differentiated muscle cells. Direct injection of a plasmid expression vector into mouse tibialis anterior muscle showed significantly increased levels of alpha-gal 7 days after injection.

Inhibition by xipamide of amiloride-induced acidification in cultured rat cardiocytes

The diuretic drug xipamide improves myocardial relaxation in hypertensive patients with left ventricular hypertrophy, but its mechanism of action is unknown. Here, xipamide was tested in cultured rat heart myogenic H9c2 cells and newborn cardiomyocytes for its effects on cell acidification (and Ca 2+ mobilization). In H9c2 cells, blocking Na +/H + exchange with amiloride (2 mM) provoked cell acidification with rate=0.82±0.17 pH units/h ( n=6). Xipamide 1 μM maximally inhibited 50±7% ( n=9) of cell acidification. The action of xipamide required the presence of HCO 3 − and was antagonized by the HCO 3 −-transport blocker DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid). Conversely, the carbonic anhydrase (EC 4.2.1.1) inhibitor acetazolamide failed to prevent xipamide action. Finally, xipamide was without significant effect on the Ca 2+ signals induced by endothelin-1, vasopressin or the Ca 2+ ionophore ionomycin. In newborn rat cardiomyocytes, xipamide reduced amiloride-induced cell acidification at similar concentrations as in H9c2 cardiocytes, but with a slightly higher extent of maximal inhibition (70–80%). In conclusion, xipamide reduced amiloride-dependent cell acidification in the rat heart myogenic H9c2 cell line and in newborn rat cultured cardiomyocytes. This action of xipamide seems to be related to a complex interaction with DIDS-sensitive HCO 3 − movements. Prevention of cell acidification by xipamide could be involved in the beneficial effects of this compound in myocardial relaxation and left ventricle filling in hypertensive patients with left ventricular hypertrophy. © 1997 Elsevier Science B.V.

Increase in the proliferative capacity of human myoblasts by using the T antigen under the vimentin promoter control.

Normal myoblasts have a strictly limited growth potential and senesce after a defined number of population doubling. The objective of this study was to determine whether the proliferative capacity of human myoblasts could be extended without inhibiting myogenic differentiation. We have established a stable transfected human myoblast cell line that expresses the SV 40 large T antigen under the control of the human vimentin promoter. We show that these cells have an increased proliferative capacity compared with that of normal myoblasts. Indeed, the final proliferative capacity was increased to 19 passages (5 for normal myoblasts). Moreover, they retained their capacity to differentiate fully, as indicated by their morphology and electrophysiological properties as well as by the expression of different markers of differentiation. The generation of human myogenic cell lines with the ability to proliferate for a longer period of time than primary myoblasts and while retaining the capacity to differentiate into myotubes could provide a valuable tool for the derivation of cell lines from human diseased muscle cells.

Increase in the proliferative capacity of human myoblasts by using the T antigen under the vimentin promoter control

Normal myoblasts have a strictly limited growth potential and senesce after a defined number of population doubling. The objective of this study was to determine whether the proliferative capacity of human myoblasts could be extended without inhibiting myogenic differentiation. We have established a stable transfected human myoblast cell line that expresses the SV 40 large T antigen under the control of the human vimentin promoter. We show that these cells have an increased proliferative capacity compared with that of normal myoblasts. Indeed, the final proliferative capacity was increased to 19 passages (5 for normal myoblasts). Moreover, they retained their capacity to differentiate fully, as indicated by their morphology and electrophysiological properties as well as by the expression of different markers of differentiation. The generation of human myogenic cell lines with the ability to proliferate for a longer period of time than primary myoblasts and while retaining the capacity to differentiate into myotubes could provide a valuable tool for the derivation of cell lines from human diseased muscle cells. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 437–445, 1997

Modulation of the specificity and activity of a cellular promoter in an adenoviral vector.

Most gene therapy studies with recombinant adenoviruses employ viral promoters and lack tissue specificity. To determine whether a tissue-specific cellular promoter inserted into the adenoviral genome can direct the expression of a reporter gene in a tissue-specific manner, recombinant adenoviruses containing a nuclear lacZ gene driven by a human ventricular/slow muscle myosin light chain 1 promoter with and without a muscle creatine kinase enhancer were constructed. The ability of these viruses to express the reporter genes in infected myogenic and nonmyogenic cell lines was studied. Intramuscular injection of these viruses into mice showed that little or no reporter gene expression occurred in muscle fibers, although a relatively high level of lacZ gene expression was observed in surrounding connective tissue. Insertion of adenovirus sequences from the 5' inverted terminal repeat (ITR) region and/or the protein IX region into plasmids resulted in decreased reporter gene expression from myosin light chain 1 promoter in transfected C2C12 myotubes and 293 cells, as well as in injected muscles. These results suggested that negative elements are present in the adenoviral genome. This negative effect seems neither tissue nor species specific. Adenovirus cis-elements that may affect the specificity and activity of a cellular promoter are discussed.

The anti-cancer agent distamycin A displaces essential transcription factors and selectively inhibits myogenic differentiation.

The anticancer drug, distamycin A, alters DNA conformation by binding to A/T-rich domains. We propose that binding of the drug to DNA alters transcription factor interactions and that this may alter genetic regulation. We have analyzed the effects of distamycin A upon expression of the muscle-specific cardiac and skeletal alpha-actin genes which have A/T-rich regulatory elements in their promoters. Distamycin A specifically inhibited endogenous muscle genes in the myogenic C2 cell line and effectively eliminated the myogenic program. Conversely, when 10T1/2C18 derived pleuripotential TA1 cells were induced to differentiate in the presence of distamycin A, adipocyte differentiation was enhanced whereas the numbers of cells committing to the myogenic program decreased dramatically. Using the mobility shift assay distamycin A selectively inhibited binding of two important transcription factors, SRF and MEF2, to their respective A/T-rich elements. The binding of factors Sp1 and MyoD were not affected. The inhibition of factor binding correlated with a repression of muscle-specific promoter activity as assayed by transient transfection assays. Co-expression of the myoD gene, driven by a distamycin A-insensitive promoter, failed to relieve the inhibition of these muscle-specific promoters by distamycin A. Additionally, SRF and MEF2 dependent promoters were selectively down regulated by distamycin A. These results suggest that distamycin A may inhibit muscle-specific gene expression by selectively interfering with transcription factor interactions and demonstrate the importance of these A/T-rich elements in regulating differentiation of this specific cell type.

Constitutive and variable regions of Z-disk titin/connectin in myofibril formation: a dominant-negative screen.

The giant muscle protein titin (connectin) is assumed to play a crucial role in the control of Z-disk assembly. Analysis of the Z-disk region of titin/connectin revealed a novel 45 residue repeat that is spliced in variable copy numbers. The repeat region is coexpressed in normal human myocardium in size variants corresponding to between 5 and 7 repeats. Smaller isoforms can be detected in muscles with thinner Z-disks like M. psoas. Sequence analysis of chicken breast muscle titin/connectin reveals that in this tissue, where Z-disks are thin, only two Z-repeats are expressed. The greatly variable thickness of Z-disks is therefore correlated to a region of variable length in titin/connectin, constructed from a novel protein building block. Transfection experiments in myogenic cell lines demonstrate that overexpression of the entire integral Z-disk region of titin leads to a disruption of sarcomere assembly. The differentially expressed titin/connectin regions, however, show no dominant-negative effects on myofibril assembly and are not targeted to Z-disks. This supports the idea that the Z-repeat region of titin/connectin is responsible for the control Z-disk thickness as an element of highly variable length, due to extensive differential splicing.

Two-step delivery of retroviruses to postmitotic, terminally differentiated cells.

Recombinant replication-defective retroviral vectors are currently the most commonly used vectors for introducing foreign genes into human cells in gene therapy protocols. Their genomes stably incorporate in the host chromosomes of mitotic cells, thus ensuring stable expression. However, the applications of retroviruses to gene therapy are limited by their inability to infect postmitotic cells such as muscle fibers. In an attempt to overcome such limitations, we have developed a novel two-step transduction protocol that allows integration and expression of retroviral genes in differentiated cells. We induced DNA synthesis in terminally differentiated cultured mouse myotubes derived from both established myogenic cell lines and from primary myoblasts. We infected the postmitotic cells with a recombinant replication-defective adenoviral vector encoding the SV40 large T antigen as a mitogen. Subsequently we transduced the adenovirus-infected cells with a Moloney retroviral vector bearing the LacZ gene. Histochemical analysis revealed the coincident expression of LacZ gene in those myotubes that had been induced to synthesize DNA.

Rabbit slow and fast skeletal muscle‐derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin‐like growth factor system

The insulin-like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow-twitch muscle-derived satellite myoblasts (SSM), which differ phenotypically from fast-twitch muscle-derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF-I and IGF-II were similar in SSM and FSM as well as their concentrations measured in cell-conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37–40, 32, 30–31, 28, and 24 kDa. The 30–31 kDa doublet was visible in SSM-conditioned medium only and associated with the presence of a 22-kDa protein, which may represent a proteolytic fragment. In contrast, the 32-kDa band was observed in FSM-conditioned medium only. The other IGFBP moieties were present in both SSM- and FSM-conditioned media. Cross-linking experiments revealed the presence of the M6P/IGF-II receptor on both SSM and FSM membranes. We also observed an IGF-I receptor form bearing unusual high affinity for IGF-II: the binding of [125I]IGF-I on this receptor was preferentially displaced by IGF-I but that of [125I]IGF-II was mostly inhibited by IGF-II, suggesting that the two tracers did not bind on the same epitopes. [125I]IGF-II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA-purified receptors revealed an ∼400-kDa band after SDS-PAGE under nonreducing conditions, which corresponded to the α2β2 form of the IGF-I receptor, and two β subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105-kDa moiety was more intensively increased than that of the 101-kDa protein after growth factor stimulation. Basal phosphorylation state of the two β subunits was similarly stimulated by IGF-I and IGF-II and less by insulin. Since both insulin and IGF-I receptors were expressed in FSM and SSM, one of the two β subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system. © 1996 Wiley-Liss, Inc.

Rabbit slow and fast skeletal muscle-derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin-like growth factor system.

The insulin-like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow-twitch muscle-derived satellite myoblasts (SSM), which differ phenotypically from fast-twitch muscle-derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF-I and IGF-II were similar in SSM and FSM as well as their concentrations measured in cell-conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37-40, 32, 30-31, 28, and 24 kDa. The 30-31 kDa doublet was visible in SSM-conditioned medium only and associated with the presence of a 22-kDa protein, which may represent a proteolytic fragment. In contrast, the 32-kDa band was observed in FSM conditioned medium only. The other IGFBP moieties were present in both SSM- and FSM-conditioned media. Cross-linking experiments revealed the presence of the M6P/IGF-II receptor on both SSM and FSM membranes. We also observed an IGF-I receptor form bearing unusual high affinity for IGF-II: the binding of [125I]IGF-I on this receptor was preferentially displaced by IGF-I but that of [125I]IGF-II was mostly inhibited by IGF-II, suggesting that the two tracers did not bind on the same epitopes. [125I]IGF-II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA-purified receptors revealed an approximately 400-kDa band after SDS-PAGE under nonreducing conditions, which corresponded to the alpha 2 beta 2 form of the IGF-I receptor, and two beta subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105-kDa moiety was more intensively increased than that of the 101-kDa protein after growth factor stimulation. Basal phosphorylation state of the two beta subunits was similarly stimulated by IGF-I and IGF-II and less by insulin. Since both insulin and IGF-I receptors were expressed in FSM and SSM, one of the two beta subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system.

Immunocytochemical localization of a full-length myotonin protein kinase in rat L6 myoblasts

Expansion mutation of CTG-repeat motifs within myotonin protein kinase (MtPK) gene is responsible for pathological changes in myotonic dystrophy (DM). To explore its pathological role in skeletal muscle, a full-length human MtPK cDNA was transfected into rat L6 myogenic cell line. Recombinantly expressed human MtPK protein in L6 cell line has a predicted molecular mass of 70 kDa. We have raised a polyclonal antibody against a synthetic peptide in the deduced sequence of the C-terminal portion of MtPK. MtPK in L6 cell is localized to perinuclear region, that resembles with sarcoplasmic reticulum. The MtPK-transfected myoblast cells established in this study will allow us to elucidate the molecular pathomechanism of muscle manifestations in DM.