Mouse Skeletal Myoblasts Research Articles

Relaxin Favors the Morphofunctional Integration between Skeletal Myoblasts and Adult Cardiomyocytes in Coculture

We have investigated the interaction between mouse skeletal myoblasts and rat cardiomyocytes in coculture and the influence of relaxin. Connexin43 expression, Lucifer yellow microinjection, Ca2+ propagation, and electrophysiological analysis have shown that myoblasts and cardiomyocytes are coupled by functional gap junctions. Cardiomyocytes and relaxin upregulated connexin43 expression and gap junctional communication in myoblasts. Relaxin also increased transjunctional current between myoblasts and between myoblasts and cardiomyocytes. In conclusion, relaxin potentiates the intercellular coupling, upregulating the transcellular exchange of regulatory molecules between myoblasts and cardiomyocytes, including Ca2+. This could favor the transdifferentiation of myoblasts toward a cardiac phenotype.

686. Bone Formation Inhibitors in Animal Sera

Based on previous gene therapy studies in bone formation induced by BMP adenoviral vectors, the osteogenic potentials of different BMP adenoviral vectors (ADhBMP-2, -4, -6, -7, and -9) are very high in immunodeficient animals but vary significantly among immunocompetent animals. We have demonstrated that the amounts of bone formation in immunocompetent animals were not significantly affected by use of different generations of adenoviral vectors and different species of BMPs. The similar protein expression curves between immunodeficient and immunocompetent rats indicated that reduced amount of BMP expression was not the main factor limiting the bone formation. Nevertheless, some components of sera may specifically block the signal transduction pathways of particular BMPs, thus interrupting bone formation. We use the term |[ldquo]|bone formation inhibitors (BFIs)|[rdquo]| to describe these immune factors to distinguish them from |[ldquo]|BMP antagonists|[rdquo]|, which are mainly secreted by bone tissue. A mouse skeletal muscle myoblast cell line (C2C12) was used as a model. Reduction of alkaline phosphatase (AP) expression was used as an index to evaluate the inhibiting activity of serum. The distribution of BFIs among different animal species and strains was investigated. Sera from rabbit, rat, and mice were tested. Sera from two strains of rabbit included 14 normal control sera and five adenovirus-vaccinated sera. Most rabbit sera did not exhibit significant inhibiting activity on cell response to any BMP. Only one serum from adenovirus-vaccinated rabbit displayed inhibiting activity on cell response to BMP6. Two sera from NOD SCID mice did not show any inhibiting activity on cell response to BMP2, -6 and -9. Forty-four sera obtained from athymic nude, SD, and Wistar rats were tested. From athymic nude rats we obtained five normal and seven adenovirus-vaccinated sera. All the rat sera inhibited response of cells to BMP6. Two sera obtained from adenovirus (ADLUC)-vaccinated athymic nude rats 16 months after the vaccination inhibited cell response to BMP2, -6, and -9. Four sera from untreated Wistar rats demonstrated inhibiting activities to all three BMPs. Sera from 13 normal, and 15 adenovirus-vaccinated SD rats, all displayed inhibiting activity to BMP6. Some sera also showed inhibition of cell response to BMP2 and -9. The results indicate that BFIs are widely distributed among different animal species and strains. Adenovirus-vaccinated sera may have higher titers than normal sera. This finding will provide a new point of view to assess the relationship between the immune system and BMPs.

Basic Progress and Future Therapeutic Perspectives of Relaxin in Ischemic Heart Disease

Relaxin has been validated as a cardiotropic hormone, being produced by the heart and acting on specific heart receptors. Evidence is accumulating that it could hamper the pathophysiologic mechanisms of ischemic heart disease. Time is ripe to study relaxin as a cardiotropic drug, as recombinant human relaxin (hrRLX) is now available and previous clinical trials have shown a virtual lack of toxicity and adverse side effects, even at high doses. Our recent observations suggest that relaxin, besides being a preventative agent, may also be effective in the treatment of acute myocardial infarction and may be an adjuvant for precursor cell grafting to repair postinfarct myocardium. In a swine model of myocardial infarction currently used to test cardiotropic drugs due to its similarities with human ischemic heart disease, hrRLX, given at reperfusion upon 30 min of ischemia, markedly reduced serum and tissue markers of myocardial injury, cardiomyocyte apoptosis and leukocyte recruitment, resulting in overall improvement in cardiac performance compared with the controls. In in vitro mixed cultures of mouse skeletal myoblasts and adult rat cardiomyocytes, relaxin increased gap junction formation and potentiated gap junction-mediated intercellular exchanges and signaling between the coupled cells. In view of the therapeutic use of myoblast grafting for cardiac repair, relaxin could hence favor the electromechanical coupling of grafted myoblasts with the resident cardiomyocytes and facilitate their transdifferentiation towards a cardiac phenotype. Relaxin, therefore, shows promising therapeutic potential in cardiology and cardiac surgery.

Contribution of human bone marrow stem cells to individual skeletal myotubes followed by myogenic gene activation

Much attention is focused on characterizing the contribution of bone marrow (BM)-derived cells to regenerating skeletal muscle, fuelled by hopes for stem cell-mediated therapy of muscle degenerative diseases. Though physical integration of BM stem cells has been well documented, little evidence of functional commitment to myotube phenotype has been reported. This is due to the innate difficulty in distinguishing gene products derived from donor versus host nuclei. Here, we demonstrate that BM-derived stem cells contribute via gene expression following incorporation to skeletal myotubes. By co-culturing human BM-derived mesenchymal stem cells (MSC) with mouse skeletal myoblasts, physical incorporation was observed by genetic lineage tracing and species-specific immunofluorescence. We used a human-specific antibody against the intermediate filament protein nestin, a marker of regenerating skeletal muscle, to identify functional contribution of MSC to myotube formation. Although nestin expression was never detected in MSC, human-specific expression was detected in myotubes that also contained MSC-derived nuclei. This induction of gene expression following myotube integration suggests that bone marrow-derived stem cells can reprogram and functionally contribute to the muscle cell phenotype. We propose that this model of myogenic commitment may provide the means to further characterize functional reprogramming of MSC to skeletal muscle.

Cellular Fate of Truncated Slow Skeletal Muscle Troponin T Produced by Glu180 Nonsense Mutation in Amish Nemaline Myopathy

A nonsense mutation at codon Glu180 in exon 11 of slow skeletal muscle troponin T (TnT) gene (TNNT1) causes an autosomal-recessive inherited nemaline myopathy. We previously reported the absence of intact or prematurely terminated slow TnT polypeptide in Amish nemaline myopathy (ANM) patient muscle. The present study further investigates the expression and fate of mutant slow TnT in muscle cells. Intact slow TnT mRNA was readily detected in patient muscle, indicating unaffected transcription and RNA splicing. Sequence of the cloned cDNAs revealed the single nucleotide mutation in two alternatively spliced isoforms of slow TnT mRNA. Mutant TNNT1 cDNA is translationally active in Escherichia coli and non-muscle eukaryotic cells, producing the expected truncated slow TnT protein. The mutant mRNA was expressed at significant levels in differentiated C2C12 myotubes, but unlike intact exogenous TnT, truncated slow TnT protein was not detected. Transfective expression in undifferentiated myoblasts produced slow TnT mRNA but not a detectable amount of truncated or intact slow TnT proteins, indicating a muscle cell-specific proteolysis of TnT when it is not integrated into myofilaments. The slow TnT-(1-179) fragment has substantially lower affinity for binding to tropomyosin, in keeping with the loss of one of two tropomyosin-binding sites. Our findings suggest that inefficient incorporation into myofilament is responsible for the instability of mutant slow TnT in ANM muscle. Rapid degradation of the truncated slow TnT protein, rather than instability of the nonsense mRNA, provides the protective mechanism against the potential dominant negative effect of the mutant TnT fragment.

Requirement of the transmembrane semaphorin Sema4C for myogenic differentiation

Semaphorins constitute a large family of signaling proteins that contribute to axonal guidance. Here we demonstrate that the transmembrane semaphorin Sema4C is up-regulated both in the early stage of differentiation of C2C12 mouse skeletal myoblasts into myotubes and during injury-induced muscle regeneration in vivo. Depletion of Sema4C in C2C12 cells resulted in marked attenuation of myotube formation. A fusion protein containing the extracellular Sema domain and a peptide corresponding to the intracellular COOH-terminal region of Sema4C each inhibited the differentiation of C2C12 cells. These findings indicate that Sema4C-mediated interaction among myoblasts plays an important role in terminal myogenic differentiation.

CSX/Nkx2.5 Modulates Differentiation of Skeletal Myoblasts and Promotes Differentiation into Neuronal Cells in Vitro

CSX/Nkx2.5 transcription factor plays a pivotal role in cardiac development; however, its role in development and differentiation of other organs has not been investigated. In this study, we used C2C12 myoblasts and human fetal primary myoblasts to investigate the function of Nkx2.5 in skeletal myogenesis. The expression levels of Nkx2.5 decreased as C2C12 myoblasts elongated and fused to form myotubes. The expression of human NKX2.5 in C2C12 myoblasts inhibited myocyte differentiation and myotube formation, and up-regulated Gata4 and Tbx5 expression. The expression of NKX2.5 in terminally differentiated C2C12 myotubes resulted in a change in morphology and breakdown into smaller myotubes. Furthermore, overexpression of NKX2.5 in C2C12 cells and primary cultures of human fetal myoblasts led to differentiation of myoblasts into neuron-like cells and expression of neuronal markers. This study sheds light on the previously unknown non-cardiac functions of Nkx2.5 transcription factor.

Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment

The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C2C12 cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca2+ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca2+, in the modulation of myoblast differentiation is discussed.

Electrophysiological changes of mouse myoblasts during culture

Background and Purpose: Heart failure patients who had been transplanted myoblasts caused lethal ventricular tachyarrhythmias (VT/VF). To clarify the proarrhythmic mechanism and establish safe myoblast transplantation, we studied electrophysiological properties of myoblast and myotube during culture. Methods: Mouse skeletal myoblasts were dispersed by enzyme and cultured for 2weeks. To differentiate from myoblast to myotube, FBS concentration was lowered at 9days from 20% to 5%. Action potential (AP), I Na, I Ca,S, I K1, I to, and I Kur were measured by patch-clamp technique. Results: In myoblasts cultured for less than 8days, resting membrane potential was in hyperpolarized level (−102 ± 5mV, n = 6). AP could not be evoked by current injection. No inward or outward currents other than IK1 were recorded. After 9days culture, cells were divided into 3 groups: myoblasts, non-beating myotubes and beating myotubes. In myoblasts, resting potentials were elevated to −49 ± 2mV (n = 5), but AP was not elicited. In non-beating myotubes, resting potential was −41 ± 2mV (n = 5). In beating myotubes, resting potentials was −35 ± 2mV (n = 4). Both types of myotubes exhibit narrow APD (less than 30msec) and express all currents examined in this study. Though diastolic potential was observed in beating-myotubes, there were no differences about current expressions. Conclusions: Heterogeneous electrical properties between developed myotubes and adjacent cardiomyocytes could be arrhythmogenic by increasing the propensity for re-entry or abnormal focal activity.

IGF-1 receptor mediates differentiation of primary cultures of mouse skeletal myoblasts

Studies involving immortalized myoblasts suggested that insulin-like growth factors (IGFs) promote differentiation of skeletal muscle, but gene targeting experiments in mice did not provide support for this hypothesis. To address this discrepancy, we examined differentiation of primary cultures of mouse myoblasts. Differentiation was normally unaffected by addition of IGFs to the differentiation medium. However, when we interrupted IGF-mediated signaling, by incubating myoblasts with suramin or with a monoclonal antibody to the IGF-I receptor, differentiation was inhibited. Inhibition was reversed by exogenous IGF-I or IGF-II, but not by insulin. Differentiation was enhanced in myoblasts that were incubated with an inhibitor of the mitogen-activated protein kinase signaling pathway (PD098059) and such cells were responsive to exogenous IGF-I. Our results demonstrate that IGF action contributes to the differentiation of non-immortalized mouse myoblasts and that these cells represent a model system that can be experimentally manipulated to study the molecular events involved in skeletal muscle differentiation.

Growth factor stimulation of matrix metalloproteinase expression and myoblast migration and invasion in vitro.

We investigated the role of growth factors and fibronectin on matrix metalloproteinase (MMP) expression and on migration and invasion of mouse skeletal myoblasts in vitro. None of the growth factors tested significantly affected MMP-1 or MMP-2 activity as revealed by gelatin zymography, but both basic FGF (bFGF) and tumor necrosis factor (TNF)-alpha significantly increased MMP-9 activity (10- and 30-fold, respectively). The increase in secreted MMP-9 activity with TNF-alpha stimulation was due at least in part to an increase in MMP-9 gene transcription, because an MMP-9 promoter construct was approximately fivefold more active in TNF-alpha-treated myoblasts than in control myoblasts, as well as an increase in MMP-9 proteolytic activation. However, whereas fibronectin, bFGF, hepatocyte growth factor, and TGF-beta1 significantly augmented migration of mouse myoblasts, TNF-alpha did not, nor did PDGF-BB or IGF-I. Fibronectin and bFGF also significantly augmented invasion of myoblasts across a Matrigel barrier, and plasmin cotreatment potentiated whereas N-acetyl cysteine suppressed the effects of bFGF and fibronectin on myoblast migration and invasion. Finally, transient transfection with an MMP-9 overexpression construct had only minimal effects on myoblast migration/invasion, whereas overexpression of either MMP-2 or MMP-1 significantly augmented myoblast migration and invasion. These observations support the hypothesis that MMP activity is a necessary component of growth factor-mediated myoblast migration but suggest that other consequences of growth factor signaling are also necessary for migration to occur.

Gene expression changes during mouse skeletal myoblast differentiation revealed by transcriptional profiling.

Studies described here utilize high-density oligonucleotide arrays to characterize changes in global mRNA expression patterns during proliferation, cell cycle withdrawal, and terminal differentiation in mouse C2C12 myoblasts. Statistical analyses revealed 629 sequences differentially regulated between proliferating and differentiating myoblasts. These genes were clustered using self-organizing maps to identify sets of coregulated genes and were assigned to functional categories that were analyzed for distribution across expression clusters. Clusters were identified with statistically significant enrichment of functional categories including muscle contraction, cell adhesion, extracellular matrix function, cellular metabolism, mitochondrial transport, DNA replication, cell cycle control, mRNA transcription, and unexpectedly, immune regulation. In addition, functional category enrichment data can be used to predict gene function for numerous differentially regulated expressed sequence tags. The results provide new insight into how genes involved in these cellular processes may play a role in skeletal muscle growth and differentiation.

Peroxisome Proliferator-activated Receptor γ Ligands Differentially Modulate Muscle Cell Differentiation and MyoD Gene Expression via Peroxisome Proliferator-activated Receptor γ-dependent and -independent Pathways

The effects of distinct classes of peroxisome proliferator-activated receptor gamma (PPARgamma) ligands on myogenesis and MyoD gene expression were examined in mouse skeletal muscle C2C12 myoblasts. Treatment of C2C12 cells with the PPARgamma ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), repressed morphologically defined myogenesis and reduced endogenous mRNA levels of the myogenic differentiation markers MyoD, myogenin, and alpha-actin. In contrast, two synthetic PPARgamma ligands, L-805645 and ciglitazone, exhibited no effects. In transient transfection assays, 15d-PGJ2 specifically inhibited the expression of a MyoD promoter-luciferase reporter gene (MyoDLuc) in a cell type- and promoter-specific manner, indicating that 15d-PGJ2 functions in part by repressing MyoD gene transcription. The inhibition of MyoD gene expression by 15d-PGJ2 is mediated by the distal region of the MyoD gene promoter. PPARgamma on its own also inhibited MyoDLuc expression and further augmented the 15d-PGJ2 response. In contrast, L-805645 and ciglitazone did not inhibit MyoDLuc expression on their own but did so in the presence of ectopically expressed PPARgamma. Interestingly, a transdominant inhibitor of PPARgamma (hPPARgamma2Delta500) had no effect on the 15d-PGJ2-dependent repression of MyoDLuc expression but overcame L-805645/PPARgamma-dependent repression. Finally, saturating concentrations of L-805645, which did not affect myogenesis, failed to ablate 15d-PGJ2-mediated repression of the myogenic program. Thus, distinct PPARgamma ligands may repress MyoD gene expression through PPARgamma-dependent and -independent pathways, and 15d-PGJ2 can inhibit the myogenic program independent of its cognate receptor, PPARgamma.

Protease-activated receptor-2 mediates proliferative responses in skeletal myoblasts.

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by proteases within the N terminus, exposing a new tethered ligand that binds and activates the receptor. Activators of PAR-2 include trypsin and mast cell tryptase. Skeletal myoblasts are known to express PAR-1, a thrombin receptor. The current study was undertaken to determine whether myoblasts express PAR-2. Primary neonatal rat and mouse skeletal myoblast cultures were shown to express PAR-2 in polymerase chain reaction and immunocytochemical studies. Expression of PAR-2 was also demonstrated by immunohistochemistry in developing mouse skeletal muscle in vivo. Trypsin or a synthetic peptide corresponding to the rat PAR-2 tethered ligand caused a dose-dependent elevation in intracellular calcium in cultured rat myoblasts, with an EC(50) of 13 nM or 56 microM, respectively. Studies aimed at identifying the function of PAR-2 in myoblasts demonstrated no effect of the receptor-activating peptide on survival or fusion in serum-deprived myoblasts. The PAR-2-activating peptide did, however, stimulate proliferation of serum-deprived myoblasts. These results demonstrate that skeletal muscle cells express PAR-2, activation of which leads to stimulation of myoblast proliferation.

Radiation retards muscle differentiation but does not affect osteoblastic differentiation induced by bone morphogenetic protein-2 in C2C12 myoblasts

Purpose : Effects of radiation on the differentiation of C2C12 myoblasts were studied using cell cultures in which the cells differentiated into either muscle cells or osteoblasts. Materials and methods : A mouse skeletal myoblast cell line, C2C12, was cultured and irradiated at confluence. Muscle differentiation was estimated by counting the number of myosin heavy chain (MHC)-positive multinucleated myotubes. Osteoblast differentiation was analysed by measuring alkaline phosphatase (ALP) activity and osteocalcin production in C2C12 cells treated with recombinant human bone morphogenetic protein-2 (rhBMP-2). Results : Myotube formation in C2C12 cells was inhibited by 2 and 4 Gy irradiation, but recovered to non-irradiated control levels following further culture. In irradiated cultures, the number of MHC-positive mononucleated cells increased significantly. Irradiation did not, however, alter the induction of osteoblastic phenotypes induced by rhBMP-2 in C2C12. Irradiation also did not affect the expression of MyoD mRNA and myogenin mRNA in myogenic differentiation and induction of osteocalcin mRNA in osteoblastic differentiation. Conclusions : During differentiation of C2C12 cells, myotube formation was delayed after irradiation, while induction of osteoblasts with BMP-2 was unaffected.

Alginate hydrogels as synthetic extracellular matrix materials

Alginate hydrogels are used extensively in cell encapsulation, cell transplantation, and tissue engineering applications. Alginates possess many favorable properties required in biomaterials, but are unable to specifically interact with mammalian cells. We have therefore covalently modified alginate polysaccharides with RGD-containing cell adhesion ligands utilizing aqueous carbodiimide chemistry. The chemistry has been optimized and quantified with reaction efficiencies reaching 80% or greater. The concentration of peptide available for reaction was then varied to create hydrogels with a range of ligand densities. Mouse skeletal myoblasts were cultured on alginate hydrogel surfaces coupled with GRGDY peptides to illustrate achievement of cellular interaction with the otherwise non-adhesive hydrogel substrate. Myoblasts adhere to GRGDY-modified alginate surfaces, proliferate, fuse into multi-nucleated myofibrils, and express heavy-chain myosin which is a differentiation marker for skeletal muscle. Myoblast adhesion and spreading on these GRGDY-modified hydrogels was inhibited with soluble ligand added to the seeding medium, illustrating the specificity of adhesion to these materials. Alginate may prove to be an ideal material with which to confer specific cellular interactive properties, potentially allowing for the control of long-term gene expression of cells within these matrices.

Type‐1 insulin‐like growth factor receptor overexpression produces dual effects on myoblast proliferation and differentiation

Using a retroviral vector, we developed a line of C2 mouse skeletal myoblasts, C2-LISN, which expressed high levels of the human type-1 insulin-like growth factor (IGF) receptor. When switched to low serum medium, C2-LISN myoblasts underwent terminal differentiation extremely rapidly compared to control C2 myoblasts. In high serum conditions which were not permissive for differentiation, C2-LISN myoblasts expressed ten-fold higher levels of the myogenic transcription factor myogenin than did control C2 myoblasts. When cultured in low serum medium with both transforming growth factor-beta (TGF-beta) and high concentrations of IGF-I, C2-LISN myoblasts failed to differentiate and grew to very high saturation densities, forming multilayers. Upon removal of TGF-beta, multilayered C2-LISN myoblasts differentiated within 2 days. These results demonstrate that overexpression of the type-1 IGF receptor can amplify signals which stimulate myogenic differentiation. Overexpressed type-1 IGF receptors can also mediate strong mitogenic signals if differentiation is inhibited by TGF-beta. The C2-LISN myoblast cell line may be a useful model to investigate the intracellular pathways which stimulate myogenic differentiation. Additionally, overexpression of the type-1 IGF receptor could provide a strategy to expand populations of differentiation-competent myoblasts for experimental or clinical applications.

Alternative extracellular and cytoplasmic domains of the integrin alpha 7 subunit are differentially expressed during development.

Examination of cDNAs for the laminin-binding alpha 7 integrin subunit identified two different sequences (designated X1 and X2) coding for the variable region between the III and IV homology repeat domains near the putative ligand-binding site. Sequencing of a mouse alpha 7 genomic clone established that the X1 and X2 regions are derived by mutually exclusive alternative mRNA splicing. Reverse transcriptase-polymerase chain reaction analysis of alpha 7 mRNA indicated that the X1 and X2 isoforms were present in equal amounts in mouse skeletal myoblasts and adult heart. However, in adult skeletal muscle, the X2 variant was exclusively expressed. Amino acid sequence homologies in the III/IV segment suggest that alpha 3 and alpha 6 are also alternatively spliced at this site. We identified alternatively spliced exons in a human alpha 6 genomic clone that encode X1- and X2-like segments. Analysis of the alpha 7 cytoplasmic domain indicated that this region was also alternatively spliced and like alpha 3 and alpha 6 could exist as the A or B form. In mouse skeletal and cardiac muscle the B form of alpha 7 was strongly expressed. However, we identified alpha 7A in neonate and adult skeletal muscle but not in cardiac tissue. High levels of alpha 7A were detected in differentiating myotubes, but in proliferating myoblasts only the alpha 7B isoform was present. These results indicate that alternative splicing of alpha 7 mRNA is differentially regulated during development and generates variant integrin chains with structurally and presumably functionally unique ligand-binding and cytoplasmic domains.

FGF-mediated aspects of skeletal muscle growth and differentiation are controlled by a high affinity receptor, FGFR1

Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) play major roles in vertebrate embryogenesis, including control of skeletal muscle growth and differentiation. Understanding their roles requires delineating the specific FGF and FGFR isoforms involved. This study analyzes the FGFR transcripts found in a model mouse skeletal myoblast cell line (MM14) during growth and terminal differentiation. MM14 cells express transcripts for FGFR1 ( flg) but not FGFR2 ( bek). The predominate FGFR1 transcript contains three immunoglobulin (Ig)-like domains in the extracellular ligand binding region. Approximately one-fourth of the three Ig-like domain transcripts possess a 6-nt deletion between the first and second Ig-like domains which after translation would result in deletion of an Arg-Arg pair. Cloning of mouse genomic DNA surrounding the region of the FGFR1 6-nt deletion indicates that the deletion is derived by alternative splicing of FGFR1 transcripts. Transcripts containing two Ig-like domains account for less than 5% of total FGFR1 mRNA in MM14 cells. A survey of RNA from mouse tissues indicated that two Ig-like domain FGFR1 transcripts are rare in all tissues except in lung, in which the two Ig-like domain form accounts for roughly 70% of the lung FGFR1 mRNA. PCR RACE cloning studies disclosed 162 nt of additional FGFR1 5′-flanking RNA which was highly GC-rich. FGFR1 transcripts decline 8- to 10-fold during low serum, (−)FGF-mediated differentiation of MM14 cultures. The kinetics of the FGFR1 mRNA decline is similar to the previously described differentiation-dependent decrease in cell surface FGF receptors.

Expression of human factor IX in mice after injection of genetically modified myoblasts.

Hemophilia B is an X chromosome-linked recessive bleeding disorder. To develop a somatic gene therapy for this disease, we have examined whether mouse skeletal myoblasts can serve as efficient vehicles for systemic delivery of recombinant factor IX. When mouse myoblasts (C2C12) transduced with a Moloney murine leukemia virus-based vector containing the bacterial beta-galactosidase gene were injected into mouse skeletal muscles, they fused with the existing and regenerating myofibers and continued to express beta-galactosidase. C2C12 myoblasts that were infected with recombinant retroviruses containing a human factor IX cDNA secreted biologically active human factor IX cDNA secreted biologically active human factor IX into the culture medium at a rate of 2.6 micrograms per 10(6) cells per day. Myotubes derived from these cells in culture continued to express human factor IX (0.68 micrograms/day from myotubes derived from 10(6) C2C12 cells). After injection of the transduced C2C12 myoblasts into skeletal muscles of mice, the systemic level of recombinant human factor IX was found to be as high as approximately 1 microgram/ml of serum. These results provide the rationale for using skeletal myoblasts as an efficient gene delivery vehicle in the somatic gene therapy for hemophilia B.