Myoblast proliferation and syncytial fusion both depend on connexin43 function in transfected skeletal muscle primary cultures

Muscles are formed by fusion of individual postmitotic myoblasts to form multinucleated syncytial myotubes. The process requires a well-coordinated transition from proliferation, through migratory alignment and cycle exit, to breakdown of apposed membranes. Connexin43 protein and cell-cycle inhibitor levels are correlated, and gap junction blockers can delay muscle regeneration, so a coordinating role for gap junctions has been proposed. Here, wild-type and dominant-negative connexin43 variants (wtCx43, dnCx43) were introduced into rat myoblasts in primary culture through pIRES-eGFP constructs that made transfected cells fluoresce. GFP-positive cells and vitally-stained nuclei were counted on successive days to reveal differences in proliferation, and myotubes were counted to reveal differences in fusion. Individual transfected cells were injected with Cascade Blue, which permeates gap junctions, mixed with FITC-dextran, which requires cytoplasmic continuity to enter neighbouring cells. Myoblasts transfected with wtCx43 showed more gap-junctional coupling than GFP-only controls, began fusion sooner as judged by the incidence of cytoplasmic coupling, and formed more myotubes. Myoblasts transfected with dnCx43 remained proliferative for longer than either GFP-only or wtCx43 myoblasts, showed less coupling, and underwent little fusion into myotubes. These results highlight the critical role of gap-junctional coupling in myotube formation.     

Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a "myosheet," was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.     

Is Intercellular Communication Via Gap Junctions Required for Myoblast Fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-β-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.     

Is Intercellular Communication Via Gap Junctions Required for Myoblast Fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-β-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.     

Transient upregulation of connexin43 gap junctions and synchronized cell cycle control precede myoblast fusion in regenerating skeletal muscle in vivo

The spatio-temporal expression of gap junction connexins (Cx) was investigated and correlated with the progression of cell cycle control in regenerating soleus muscle of Wistar rats. Notexin caused a selective myonecrosis followed by the complete recapitulation of muscle differentiation in vivo, including the activation, commitment, proliferation, differentiation and fusion of myogenic cells. In regenerating skeletal muscle, only Cx43 protein, out of Cx-s 26, –32, –37, –40, –43 and –45, was detected in desmin positive cells. Early expression of Cx43 in the proliferating single myogenic progenitors was followed by a progressive upregulation in interacting myoblasts until syncytial fusion, and then by a rapid decline in multinucleate myotubes. The significant upregulation of Cx43 gap junctions in aligned myoblasts preceding fusion was accompanied by the widespread nuclear expression of cyclin-dependent kinase inhibitors p21^waf1/Cip1 and p27^kip1 and the complete loss of Ki67 protein. The synchronized exit of myoblasts from the cell cycle following extensive gap junction formation suggests a role for Cx43 channels in the regulation of cell cycle control. The potential of Cx43 channels to stimulate p21^waf1/Cip1 and p27^kip1 is known. In the muscle, proving the involvement of Cx43 in either a direct or a bystander cell cycle regulation requires functional investigations.     

Drosophila dumbfounded: a myoblast attractant essential for fusion

Aggregation and fusion of myoblasts to form myotubes is essential for myogenesis in many organisms. In Drosophila the formation of syncytial myotubes is seeded by founder myoblasts. Founders fuse with clusters of fusion-competent myoblasts. Here we identify the gene dumbfounded (duf) and show that it is required for myoblast aggregation and fusion. duf encodes a member of the immunoglobulin superfamily of proteins that is an attractant for fusion-competent myoblasts. It is expressed by founder cells and serves to attract clusters of myoblasts from which myotubes form by fusion.     

Effects of C-reactive protein on adipokines genes expression in 3T3-L1 adipocytes

Transplantation of skeletal myoblasts (SMs) has been investigated as a potential cardiac cell therapy approach. SM are available autologously, predetermined for muscular differentiation and resistant to ischemia. Major hurdles for their clinical application are limitations in purity and yield during cell isolation as well as the absence of gap junction expression after differentiation into myotubes. Furthermore, transplanted SMs do not functionally or electrically integrate with the host myocardium. Here, we describe an efficient method for isolating homogeneous SM populations from neonatal mice and demonstrate persistent gap junction expression in an engineered tissue. This method resulted in a yield of 1.4 × 10(8) high-purity SMs (>99% desmin positive) after 10 days in culture from 162.12 ± 11.85 mg muscle tissue. Serum starvation conditions efficiently induced differentiation into spontaneously contracting myotubes that coincided with loss of gap junction expression. For mechanical conditioning, cells were integrated into engineered tissue constructs. SMs within tissue constructs exhibited long term survival, ordered alignment, and a preserved ability to differentiate into contractile myotubes. When the tissue constructs were subjected to passive longitudinal tensile stress, the expression of gap junction and cell adherence proteins was maintained or increased throughout differentiation. Our studies demonstrate that mechanical loading of SMs may provide for improved electromechanical integration within the myocardium, which could lead to more therapeutic opportunities.     

Syncytin-1 in differentiating human myoblasts: relationship to caveolin-3 and myogenin

Myoblasts fuse to form myotubes, which mature into skeletal muscle fibres. Recent studies indicate that an endogenous retroviral fusion gene, syncytin-1, is important for myoblast fusions in man. We have now expanded these data by examining the immunolocalization of syncytin in human myoblasts induced to fuse. Additionally, we have compared the localization of syncytin with the localization of caveolin-3 and of myogenin, which are also involved in myoblast fusion and maturation. Syncytin was localized to areas of the cell membrane and to filopodial structures connecting myoblasts to each other and to myotubes. Weaker staining was present over intracellular vesicles and tubules. Caveolin-3 was detected in the sarcolemma and in vesicles and tubules in a subset of myoblasts and myotubes. The strongest staining occurred in multinucleated myotubes. Wide-field fluorescence microscopy indicated a partial colocalization of syncytin and caveolin-3 in a subset of myoblasts. Super-resolution microscopy showed such colocalization to occur in the sarcolemma. Myogenin was restricted to nuclei of myoblasts and myotubes and the strongest staining occurred in multinucleated myotubes. Syncytin staining was observed in both myogenin-positive and myogenin-negative cells. Antisense treatment downmodulated syncytin-1 expression and inhibited myoblast cell fusions. Importantly, syncytin-1 antisense significantly decreased the frequency of multinucleated myotubes demonstrating that the treatment inhibited secondary myoblast fusions. Thus, syncytin is involved in human myoblast fusions and is localized in areas of contact between fusing cells. Moreover, syncytin and caveolin-3 might interact at the level of the sarcolemma.     

Cell-to-cell communication and myogenesis

Cell-to-cell communication was characterized in prefusion chick embryo myoblast cultures, and it was determined that the prefusion myoblasts can interact via gap junctions, ionic coupling, and metabolic coupling. The biological relevance of this communication was supported by the detection of gap junctions between myoblasts in embryonic muscle. Communication was also examined in fusion-arrested cultures to determine its potential relationship to fusion competency. In cultures that were fusion arrested by treatment with either 1.8 mM ethyleneglycolbis-(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), 3.3 X 10(-6) M 5-bromodeoxyuridine (BUdR), or 1 microgram/ml cycloheximide (CHX), both gap junctions and ionic coupling were present. Therefore, it is possible to conclude that cell communication is not a sufficient property by itself, to generate fusion between myob- asts. The potential role of communication in myogenesis is discusssed with respect to these observations.     

The origin of syncytial muscle fibres in the myotomes of Xenopus laevis--a revision

During the early stages of myogenesis in X. laevis, the primary myoblasts (of mesodermal origin) differentiate simultaneously, in each myotome, into mononucleate myotubes. At later stages mesenchymal cells appear in intermyotomal fissures and then in the myotomes between myotubes and contribute to the formation ofsyncytial muscle fibres. The pathway of mesenchymals cell during myogenesis was described in X laevis by monitoring the incorporation of 3H-thymidine. 3H-thymidine was incorporated in the nuclei of mesenchymal cells in intermyotomal fissures of younger myotomes and then in those of older myotomes between the myotubes revealing the proliferation of mesenchymal cells. As expected, nuclei of differentiating mononucleate myotubes did not incorporate 3H-thymidine. At later stages of myogenesis the myotubes were found to contain two classes of nuclei: large nuclei of the primary myoblasts (of myotomal origin) and smaller nuclei originating from secondary myoblasts ofmesenchymal origin. TEM and autoradiographic analyses confirm that mulinucleate myotubes in X. laevis arise through fusion of secondary myoblasts with mononucleate myotubes.     

The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion

Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where--as in myoblast fusion--membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells are unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane.     

Electromechanical coupling between skeletal and cardiac muscle. Implications for infarct repair

Skeletal myoblasts form grafts of mature muscle in injured hearts, and these grafts contract when exogenously stimulated. It is not known, however, whether cardiac muscle can form electromechanical junctions with skeletal muscle and induce its synchronous contraction. Here, we report that undifferentiated rat skeletal myoblasts expressed N-cadherin and connexin43, major adhesion and gap junction proteins of the intercalated disk, yet both proteins were markedly downregulated after differentiation into myo-tubes. Similarly, differentiated skeletal muscle grafts in injured hearts had no detectable N-cadherin or connexin43; hence, electromechanical coupling did not occur after in vivo grafting. In contrast, when neonatal or adult cardiomyocytes were cocultured with skeletal muscle, approximately 10% of the skeletal myotubes contracted in synchrony with adjacent cardiomyocytes. Isoproterenol increased myotube contraction rates by 25% in coculture without affecting myotubes in monoculture, indicating the cardiomyocytes were the pacemakers. The gap junction inhibitor heptanol aborted myotube contractions but left spontaneous contractions of individual cardiomyocytes intact, suggesting myotubes were activated via gap junctions. Confocal microscopy revealed the expression of cadherin and connexin43 at junctions between myotubes and neonatal or adult cardiomyocytes in vitro. After microinjection, myotubes transferred dye to neonatal cardiomyocytes via gap junctions. Calcium imaging revealed synchronous calcium transients in cardiomyocytes and myotubes. Thus, cardiomyocytes can form electromechanical junctions with some skeletal myotubes in coculture and induce their synchronous contraction via gap junctions. Although the mechanism remains to be determined, if similar junctions could be induced in vivo, they might be sufficient to make skeletal muscle grafts beat synchronously with host myocardium.     

Antibodies to 100- and 60-kDa surface proteins inhibit substratum attachment and differentiation of rodent skeletal myoblasts

A rabbit polyclonal antiserum was raised against membrane vesicles shed from the surface of fusing L6 rat myoblasts. In immunoblots the antiserum recognized fibronectin, a protein of approximately 100,000 Da (100-kDa), and a protein of approximately 60,000 Da (60 kDa). If added prior to cellular alignment, immunoglobulins from this serum inhibited fusion of both rat (L6) and mouse (C2) myoblasts in a dose-dependent fashion. To determine which component of this serum was responsible for fusion inhibition, antibodies against fibronectin, the 100- and 60-kDa proteins were microaffinity purified and tested, individually, for their effects on myoblast fusion. Antibodies against fibronectin had no effect on fusion. Antibodies against the 100-kDa protein released most cells from the substratum. Antibodies against the 60-kDa protein completely inhibited fusion. Fusion inhibition was accompanied by a corresponding inhibition of expression of two differentiation markers, creatine phosphokinase and the acetylcholine receptor. The 60-kDa protein was found, by immunoblot analysis, in smooth muscle-like cells (BC3H1 cells) and in variant L6 cells that do not differentiate and do not fuse. However, in the differentiation incompetent cells, the 60-kDa antigen appeared to be present in reduced amount. Indirect immunofluorescence of unpermeabilized L6 cells revealed alterations in the distribution of all three antigens during development. Fibronectin first appeared in long fibrillar arrays above the surface of cells that were beginning to align and fuse; fibronectin was not present on myotubes. The 100-kDa protein was seen initially in prominent fibrillar projections at the tips of prefusion myoblasts. During fusion the antigen was observed at sites of cell-cell contact and on extracellular vesicles. The 100-kDa protein appeared to be less abundant on myotubes. The 60-kDa protein first appeared in regions of cell-cell contact on cells that were beginning to align and fuse. As. fusion progressed, the 60-kDa protein was also found in extracellular vesicles. The 60-kDa protein was not observed on myotubes. As a result of this study we have identified two previously undescribed cell surface proteins involved in rodent skeletal myogenesis. The first is an approximately 100-kDa protein involved in early interactions of skeletal myoblasts with their substratum. The second is an approximately 60-kDa protein involved in myoblast differentiation. Both proteins are shed from the myoblast surface during myotube formation.     

Expression and synthesis of high mobility group chromosomal proteins in different rat skeletal cell lines during myogenesis.

The synthesis, turnover, and expression of all the major high mobility group (HMG) chromosomal proteins was studied in different rat skeletal myogenic cell lines. Whereas pulse-chase experiments revealed a similar half-life (greater than 2 cell generations) for all the HMG proteins in both L8 myoblasts and myotubes, [3H]lysine incorporation data indicated a 2- to 4-fold greater incorporation of the label in the HMG proteins in proliferating myoblasts relative to the nondividing myotubes. Analysis of the HMG-1, -14, and -17 mRNAs during myogenesis showed a significant down-regulation in L6 and L8 myotubes compared to the myoblasts. However, the timing of the shift and the extent of down-regulation was cell type-dependent, being more pronounced in L6 myotubes at fusion compared to 4 days postfusion in L8 myotubes. By contrast, L8-derived fusion-defective fu-1 cells over the same period of growth showed no change in HMG-14/17 mRNA levels. HMG-I(Y) protein isoforms, noted for the first time in rat myoblasts, like their counterparts, seemed to be stable and showed a precipitous reduction in their mRNAs during myogenesis. The results suggest a cell type-specific correlation between HMG expression and cell proliferation; they also argue for their role in maintenance of the cell's state of differentiation.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

A new technique to identify hybrid myotubes in vitro without culture fixation.

Fluorescent latex microspheres (FLMs) were used to label myoblasts and to permit the observation of hybrid myotubes before culture fixation. This type of labeling did not affect survival, development, or fusion of these cells. The FLMs were retained for several weeks. Labeled mouse myoblasts were co-cultured with unlabeled rat myoblasts to verify whether the marker was released and spread from labeled to unlabeled cells. The nuclear stain Hoechst 33258 was used to distinguish the myoblasts from both species and permitted the demonstration that there was virtually no re-uptake. Hybrid myotubes were also obtained by co-culturing mouse myoblasts containing rhodamine FLMs and rat myoblasts containing green FLMs. These mixed cultures were observed repeatedly with a fluorescent microscope without any cytotoxic effect. Several myotubes were observed before fixation of the cultures to contain both types of fluorescent labels. Subsequent fixation and staining with Hoechst dye confirmed that these myotubes were hybrids.     

Role of laminin and fibronectin in selecting myogenic versus fibrogenic cells from skeletal muscle cells in vitro

Growth of embryonic skeletal muscle occurs by fusion of multinucleated myotubes with differentiated, fusion-capable myoblasts. Selective recognition seems to prevent fusion of myotubes with nonmyogenic cells such as muscle fibroblasts, endothelial cells, or nerve cells, but the nature of the signal is as yet unknown. Here we provide evidence that one of the selection mechanisms may be the enhanced affinity for laminin of myogenic cells as compared to fibrogenic cells. Growing myotubes in myoblast cultures accumulate laminin and type IV collagen on their surface in patches and strands as the first step in assembling a continuous basal lamina on mature myofibers (U. Kühl, R. Timpl, and K. von der Mark (1982), Dev. Biol. 93, 344-359). Fibronectin, on the other hand, assembles into an intercellular fibrous meshwork not associated with the free myotube surface. Over a brief time period (10-20 min) myoblasts from embryonic mouse thigh muscle adhere faster to laminin than do fibroblasts from the same tissue; these adhere faster to fibronectin. When a mixture of the cells is plated for 20 min on laminin/type IV collagen substrates, only myogenic cells adhere, giving rise to cultures with more than 90% fusion after 2 weeks; on fibronectin/type I collagen in the same time primarily fibroblastic cells adhere, giving rise to cultures with less than 10% nuclei in myotubes. The differential affinities of myoblasts for basement membrane constituents and of fibroblasts for interstitial connective tissue components may play a role in sorting out myoblasts from fibroblasts in skeletal muscle development.     

Changes in plasma membrane glycoproteins during differentiation of an established myoblast cell line and a non-fusing α-amanitin-resistant mutant

The glycoproteins of purified plasma membranes from mononucleated myoblasts and from myotubes of the L₆ line were characterized according to their apparent molecular weight (MW) and to their ability to bind concanavalin A (conA). We identified 25 proteins in membranes from mononucleated myoblasts and fused myotubes which specifically bound the lectin. Comparison with the pattern of membrane glycoproteins of a non-fusing mutant allowed us to identify developmentally regulated changes in the accumulation of 8 proteins with an apparent MW of 160, 80, 60, 51.5, 43, 40, 38, and 27 Kilodalton (kD), and changes in the glycosylation of six others which migrate at 215, 150, 135, 90, 85, and 32 kD. Two of them (160 and 38 kD) appeared at fusion, whereas the 51.5 kD band could not be identified in plasma membrane from myotubes. As conA inhibits fusion of myoblasts, it is suggested that at least some of these proteins might be involved in this process.     

The interaction of myotropic and macrophagotropic strains of Trypanosoma cruzi with myoblasts and fibers of skeletal muscle

The process of interaction of bloodstream trypomastigotes from the myotropic CL and Colombiana strains and the macrophagotropic Y strain of Trypanosoma cruzi with mouse myoblasts and myotubes was analysed. After 24 h of parasite-host cell interaction, parasites from the CL and Colombiana strains appeared to be more infective to myoblasts than those from the Y strain. Parasites from the Colombiana strain were more infective for myotubes than those from the Y strain, while those from the CL strain showed very a low ability to infect the cells. For all strains the infectivity was low for short periods of interaction, increasing with time. Myoblasts infected with parasites from the Y strain fused with other infected and uninfected cells to form myotubes. However, the process of fusion was blocked when the myoblasts were infected with parasites from the CL and Colombiana strains. These data indicate a different behavior of muscle cells when in contact with myotropic or non-myotropic strains of T. cruzi.     

Differentiation of quail myoblasts transformed with a temperature sensitive mutant of Rous sarcoma virus. II. Relationship of myoblast fusion with calcium and temperature.

The effects of calcium and temperature on fusion of quail embryonic myoblasts were examined using cells transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). The transformed quail myoblasts (QM-RSV) fused to form myotubes at 41 degrees C, the non-permissive temperature, but not at 35.5 degrees C, the permissive temperature. On incubation at 41 degrees C, a period of more than 10 hr was needed for the myoblasts to become fusion-competent, but calcium was not needed for development of fusion-competence. Once the cells had become competent, fusion proceeded even at 35.5 degrees C. These results suggest that the src gene product expressed at 35.5 degrees C may control the fusion of cells in the competent stage by inactivating a component(s) that is associated with fusion-competence. However, fusion of even myoblasts in the competent stage was blocked in calcium-deficient medium, suggesting that calcium is essential for the fusion, probably at a step immediately before membrane union. Unlike fusion, other biochemical processes of differentiation proceeded even in calcium-deficient medium, indicating a distinction of fusion from these other processes during myoblast differentiation.