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.     

Interleukin-10 expression after intramuscular DNA electrotransfer: kinetic studies

A set of monoclonal antibodies that recognizes a Trypanosoma cruzi 45-kDa protein was produced and used to characterize this molecule and study its role in trypanosome adhesion to heart myoblasts. We found that the 45-kDa protein is a surface mucin, is expressed only in invasive trypomastigotes, but not in noninvasive epimastigotes or amastigotes, and is released by the trypanosome in culture medium. One of the monoclonal antibodies (Mab B5) from this set inhibits the attachment of trypomastigotes to heart myoblasts preventing trypanosome entry, whereas the others (Mabs B4 and F1) do not. This inhibition was seen with the B5 hybridoma culture supernatant, with the purified Mab B5 IgG or with Mab B5 Fab fragments. These novel findings identify the 45-kDa mucin as a new T. cruzi ligand that is used by invasive forms of this organism to adhere to heart myoblasts.     

Coordinate accumulation of contractile protein mRNAs during myoblast differentiation.

The synthesis of contractile protein mRNAs has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Eight contractile protein mRNAs were identified by translation of total cellular RNA isolated from differentiated myofibers in wheat germ and reticulocyte lysates. Products of the translation systems were fractionated by two-dimensional gel electrophoresis, and incorporation of [³⁵S]methionine into individual contractile proteins was quantitated by computerized densitometry of autoradiograms. These translation assay systems were used to quantitate levels of contractile protein mRNAs in cultures of myoblasts undergoing highly synchronous differentiation. Our results show that dividing myoblasts contain very little, if any, translatable contractile protein mRNA. The mRNAs coding for myosin heavy chain, the musclespecific actin, three myosin light chains, two tropomyosin subunits, and one troponin subunit begin to coordinately accumulate at fusion, when contractile protein synthesis is activated. Their levels increase 50- to 200-fold during the next 30 hr, paralleling increases in the rates of contractile protein synthesis. These results indicate that the contractile protein mRNAs accumulate coordinately during myoblast differentiation and that contractile protein synthesis is regulated by changes in the levels of these mRNAs.     

Induction of myogenic differentiation in serum-free medium does not require DNA synthesis

Cells of the myogenic rat cell line L6 can be obtained as a confluent, quiescent population of undifferentiated myoblasts after growth in F12 medium supplemented with fetal calf serum. Myogenic differentiation can be induced in these cells by changing to Dulbecco's modified Eagle's (DME) medium containing insulin as the only protein component. Labeling of the cells with [3H]thymidine demonstrates that this induction of fusion occurs in the absence of DNA synthesis in about 85% of the cells. This result was confirmed using cytosine arabinoside: fusion of quiescent L6 cells was induced in the presence of this inhibitor of DNA synthesis. The myotubes formed in DME + insulin medium, with or without cytosine arabinoside, synthesize or accumulate proteins characteristic of differentiated muscle cells including myosin heavy and light chains, alpha-actin, alpha- and beta-tropomyosins, and the acetylcholine receptor. These experiments represent a direct demonstration that DNA synthesis is not required for the induction of myogenic differentiation in undifferentiated quiescent cells.     

Trypanosoma cruzi: inhibition by spirogermanium hydrochloride

Spirogermanium is an antineoplastic agent that has been shown to be useful for the treatment of a variety of solid tumors and Plasmodium falciparum infection. We found that this agent, at concentrations of 1-10 micrograms/ml, markedly inhibited the growth of epimastigotes of Trypanosoma cruzi. This inhibition of growth was seen in liver infusion tryptose cultures as well as on agar where colonial growth was inhibited markedly. Ultrastructural studies demonstrated that affected organisms were round and swollen and contained vacuoles, lamellar structures, and multivesicular bodies. Spirogermanium also significantly decreased the growth of intracellular amastigotes in myotubes. Pretreatment of myotubes with the agent protected them from infection with trypomastigotes but tachyzoites of Toxoplasma sp. readily infected pretreated cells. These data suggest that spirogermanium may be useful as a chemotherapeutic agent against T. cruzi.     

Strain-Dependent Thermosensitivity Influencing Intracellular Differentiation of Trypanosoma cruzi in Cell Culture*

SYNOPSIS. Temperature strongly influenced morphogenesis of intracellular trypomastigotes in cell culture infected with 2 different strains of T. cruzi. With the Gilmar strain the amastigote-to-trypomastigote differentiation readily occurred at 33 and 37 C, whereas with the CL strain differentiation took place at 33 C but was inhibited at 37 C. The possibility of this selective thermosensitivity resulting from mutational adaptation of the parasite is discussed.     

Transient production of alpha-smooth muscle actin by skeletal myoblasts during differentiation in culture and following intramuscular implantation

alpha-smooth muscle actin (SMA) is typically not present in post-embryonic skeletal muscle myoblasts or skeletal muscle fibers. However, both primary myoblasts isolated from neonatal mouse muscle tissue, and C2C12, an established myoblast cell line, produced SMA in culture within hours of exposure to differentiation medium. The SMA appeared during the cells' initial elongation, persisted through differentiation and fusion into myotubes, remained abundant in early myotubes, and was occasionally observed in a striated pattern. SMA continued to be present during the initial appearance of sarcomeric actin, but disappeared shortly thereafter leaving only sarcomeric actin in contractile myotubes derived from primary myoblasts. Within one day after implantation of primary myoblasts into mouse skeletal muscle, SMA was observed in the myoblasts; but by 9 days post-implantation, no SMA was detectable in myoblasts or muscle fibers. Thus, both neonatal primary myoblasts and an established myoblast cell line appear to similarly reprise an embryonic developmental program during differentiation in culture as well as differentiation within adult mouse muscles.     

The CLIC5 (chloride intracellular channel 5) involved in C2C12 myoblasts proliferation and differentiation

CLIC5 (chloride intracellular channel 5) is a CLIC (chloride intracellular channel) with various functions. Its high expression in skeletal muscle and association with actin‐based cytoskeleton suggests that it may play an important role in muscle tissue. This study was conducted to examine whether CLIC5 regulates the proliferation and differentiation of C2C12 myoblasts into myotubes. Differentiation of C2C12 myoblasts induced by switching to a differentiation culture medium was accompanied by a significant increase of CLIC5 protein expression level. Constitutive overexpression of CLIC5 was associated with reduced cell proliferation and more cells from G2/M phase into G0/G1 phase, followed by increased number and size of myotubes and up‐regulation of muscle‐specific proteins of myosin heavy chain, myogenin and desmin. These results demonstrate that CLIC5 is involved in C2C12 proliferation and myogenic differentiation in vitro.     

Polyomavirus genome and polyomavirus enhancer-driven gene expression during myogenesis.

The mRNAs for myogenic functions are coordinately transcribed with polyomavirus (Py) early mRNA during in vitro differentiation of mouse C2 myoblast cells. Sequence analysis shows that the A domain of the Py enhancer includes an E1A-like consensus sequence that is also found in the 5' upstream region of two genes expressed during myoblast differentiation: alpha-actin and myosin light chain. Therefore, the coordinate expression of such genes with Py early mRNA may be activated by a common cellular regulatory factor. In the present work, we report that C2 cells surviving Py infection are unable to differentiate and do not express alpha-actin and myosin light-chain mRNAs. Hybrids between such Py-resistant myoblast cells and the parental cells exhibited dominance of the permissibility to Py growth and of the expression of myogenic mRNAs. In C2 cells transiently transfected with a chimeric plasmid (pSVPy12CAT) harboring the bacterial chloramphenicol acetyltransferase (CAT) gene driven by the Py enhancer-promoter region, the CAT gene was expressed irrespective of their stage of differentiation. Moreover, undifferentiated stably transfected cells expressing the CAT gene restricted viral growth. Py-resistant C2 myoblasts transiently transfected with pSVPy12CAT also expressed the CAT gene driven by the Py enhancer. This contradictory finding is similar to results previously obtained by other investigators with cloned genes specific for myogenic functions, and it may be explained by a structural difference between the pSVPy12CAT and the Py genomic organizations in which the viral enhancer operates.     

Biochemical characterization of a factor produced by trypomastigotes of Trypanosoma cruzi that accelerates the decay of complement C3 convertases

Infective- and vertebrate-stage trypomastigotes of Trypanosoma cruzi resist serum killing by the alternative complement pathway, whereas noninfective vector-stage epimastigotes, from which trypomastigotes derive, are serum-sensitive. This form of developmental preadaption is commonly observed in protozoan parasites, but its mechanisms are poorly understood. We have demonstrated previously that trypomastigotes spontaneously shed molecules which interfere with formation and accelerate the intrinsic decay of complement C3 convertases, a finding which may explain the evasion of complement lysis by trypomastigotes. We now describe the partial purification and characterization of the T. cruzi C3 convertase inhibitor from the supernatant of culture metacyclic and tissue culture trypomastigotes. Decay-accelerating activity for both classical and alternative pathway C3 convertases copurifies on anion-exchange fast protein liquid chromatography and chromatofocusing with 35S-labeled molecules of 87-93 kDa, pI 5.6-5.8. The labeled components are destroyed by papain and retained on concanavalin A-Sepharose, procedures which remove functional decay-accelerating activity from the supernatant. The 87-93-kDa components are immunoprecipitated by sera from patients chronically infected with T. cruzi, but not by antisera to any known regulatory proteins of the human complement cascade. Lytic activity for tissue culture trypomastigotes in chagasic sera is associated with antibody reactivity against the 87-93-kDa 35S-labeled components and with inhibition of decay-accelerating activity. The T. cruzi factor is the first developmentally regulated microbial complement inhibitor to be biochemically characterized.     

Identification of a regulatory function for an orphan receptor in muscle: COUP-TF II affects the expression of the myoD gene family during myogenesis.

COUP-TF II is an 'orphan steroid receptor' that binds a wide variety of AGGTCA repeats and represses thyroid hormone (T3) and retinoid dependent trans-activation; however, very little is known of its functional and/or developmental role during mammalian cell differentiation. T3 and retinoids have been demonstrated to promote terminal muscle differentiation via activation of the muscle specific myoD gene family (myoD, myogenin, myf-5 and MRF-4). The myoD gene family can direct the fate of mesodermal cell lineages, repress proliferation, activate differentiation and the contractile phenotype. Hence, we investigated the expression and functional role of COUP-TF II during muscle differentiation. Proliferating C2C12 myoblasts expressed COUP-TF II mRNA which was repressed when cells were induced to differentiate into post-mitotic multinucleated myotubes by serum withdrawal. Concomitant with the decrease of COUP-TF II mRNA was the appearance of muscle specific mRNAs (e.g. myogenin, alpha-actin). We show that Escherichia coli expressed full length and truncated COUP-TF II bound in a sequence specific manner to the T3 response elements (TREs) in the myoD and myogenin regulatory HLH genes [Olson (1992) Dev. Biol. 154, 261-272]; and the TRE in the skeletal alpha-actin contractile protein gene. COUP-TF II diminished the homodimeric binding of the thyroid hormone receptor and the heterodimeric binding of thyroid hormone and retinoid X receptor complexes to these TREs. Constitutive over-expression of COUP-TF II cDNA in mouse C2C12 myogenic cells suppressed the levels of myoD mRNA and blocked the induction of myogenin mRNA, whereas constitutive expression of anti-sense COUP-TF II cDNA significantly increased the steady state levels of myoD mRNA and hyper-induced myogenin mRNA. These studies demonstrate for the first time (i) that COUP-TF II, functions as a physiologically relevant antagonistic regulator of myogenesis via direct effects on the myoD gene family and (ii) direct evidence for the developmental role of COUP-TF II during mammalian cell differentiation.     

The insulin signal initiating cellular differentiation is preserved by chick embryo myoblasts incubated at 2 degrees C

Insulin pulse treatment, lasting for 1 to 3 min, stimulates differentiation of chick embryo myoblasts to myotubes and myofibers in a serum-free medium. The use of serum-free medium supplemented with 2,2'-thiodiethanol permits terminal differentiation of chick embryo myoblasts to cross-striated, spontaneously contracting myofibers. The experiments carried out showed that incubation of chick embryo myoblasts after the insulin pulse treatment for 10 days at 2 degrees C does not inhibit the progress of their differentiation. The data demonstrate that differentiation can be interrupted at any time by transferring cells to 2 degrees C and resumed without delay after returning to 37 degrees C.     

Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes. Evidence for TWEAK-independent functions of Fn14 during myogenesis

Fibroblast growth factor-inducible 14 (Fn14), distantly related to tumor necrosis factor receptor superfamily and a receptor for TWEAK cytokine, has been implicated in several biological responses. In this study, we have investigated the role of Fn14 in skeletal muscle formation in vitro. Flow cytometric and Western blot analysis revealed that Fn14 is highly expressed on myoblastic cell line C2C12 and mouse primary myoblasts. The expression of Fn14 was decreased upon differentiation of myoblasts into myotubes. Suppression of Fn14 expression using RNA interference inhibited the myotube formation in both C2C12 and primary myoblast cultures. Fn14 was required for the transactivation of skeletal alpha-actin promoter and the expression of specific muscle proteins such as myosin heavy chain fast type and creatine kinase. RNA interference-mediated knockdown of Fn14 receptor in C2C12 myoblasts decreased the levels of myogenic regulatory factors MyoD and myogenin upon induction of differentiation. Conversely, overexpression of MyoD increased differentiation in Fn14-knockdown C2C12 cultures. Suppression of Fn14 expression in C2C12 myoblasts also inhibited the differentiation-associated increase in the activity of serum response factor and RhoA GTPase. In addition, our data suggest that the role of Fn14 during myogenic differentiation could be independent of TWEAK cytokine. Collectively, our study suggests that the Fn14 receptor is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes.