Trans‐activation of the murine dystrophin gene in human‐mouse hybrid myotubes

Noursadeghi, Mahdad; Walsh, Frank S.; Heiman‐Patterson, Terry; Dickson, George

doi:10.1016/0014-5793(93)80082-6

Cited by 8 publications

(6 citation statements)

References 19 publications

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“…Thus, we can reasonably speculate that advantageous morphological and adhesive features of C2C12 cells would overcome some of the shortcomings of human cells, yielding vigorous contractility development of human-mouse hybrid myotubes under the culture conditions used in this study. Consistent with this interpretation, hybrid myotubes comprised of human-mouse 24 and human-cat 25 cells have also been studied, and malfunctions of human-origin muscle cells such as dystrophin-deficiency could reportedly be partially compensated for by forming hybrid myotubes with the non-human muscle cells.…”

Section: Discussionmentioning

confidence: 74%

In vitro exercise model using contractile human and mouse hybrid myotubes

Chen

Nyasha

Koide

et al. 2019

Sci Rep

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Contraction of cultured myotubes with application of electric pulse stimulation (EPS) has been utilized for investigating cellular responses associated with actual contractile activity. However, cultured myotubes derived from human subjects often exhibit relatively poor EPS-evoked contractile activity, resulting in minimal contraction-inducible responses ( i . e . myokine secretion). We herein describe an “ in vitro exercise model”, using hybrid myotubes comprised of human myoblasts and murine C2C12 myoblasts, exhibiting vigorous contractile activity in response to EPS. Species-specific analyses including RT-PCR and the BioPlex assay allowed us to separately evaluate contraction-inducible gene expressions and myokine secretions from human and mouse constituents of hybrid myotubes. The hybrid myotubes, half of which had arisen from primary human satellite cells obtained from biopsy samples, exhibited remarkable increases in the secretions of human cytokines (myokines) including interleukins (IL-6, IL-8, IL-10, and IL16), CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CC chemokines (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), and IFN-γ in response to EPS-evoked contractile activity. Together, these results indicate that inadequacies arising from human muscle cells are effectively overcome by fusing them with murine C2C12 cells, thereby supporting the development of contractility and the resulting cellular responses of human-origin muscle cells. Our approach, using hybrid myotubes, further expands the usefulness of the “ in vitro exercise model”.

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Section: Discussionmentioning

confidence: 74%

In vitro exercise model using contractile human and mouse hybrid myotubes

Chen

Nyasha

Koide

et al. 2019

Sci Rep

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“…These results demonstrate that embryonic and fetal myoblasts will fuse with each other in vitro to form heterotypic myotubes, insofar as markers for each myoblast population can be found within individual myotubes. However, this should not be taken as proof that these myoblasts represent a common lineage; fusion of genetically distinct myoblasts from different species has previously been reported (Yaffe and Feldman, 1965;Pavlath et al, 1989;Noursadeghi et al, 1993). What this result does suggest is that there may be mechanisms at work in the developing embryo to inhibit the formation of heterotypic myotubes in vivo, because EM analysis of muscle development in vivo has shown that fetal myoblasts do not normally fuse with 1°m yotubes Harris et al, 1989).…”

Section: Discussionmentioning

confidence: 90%

Regionalized expression of myosin isoforms in heterotypic myotubes formed from embryonic and fetal rat myoblasts in vitro

Merrifield

1997

Dev. Dyn.

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The development of mammalian limb muscles involves the appearance and fusion of at least two separate populations of muscle precursor cells. These two populations, termed embryonic and fetal myoblasts, are first detected within the limb bud at different stages of development. We have previously demonstrated that, in the rat, each myoblast population expresses a unique pattern of myosin heavy chains (MyHCs) during differentiation in vitro (Pin and Merrifield [1993] Dev. Genet. 14:356–368). Embryonic myoblasts accumulate embryonic and slow MyHCs, whereas fetal myoblasts accumulate embryonic, neonatal, and adult fast MyHCs but not slow MyHC. To determine if the two populations can fuse with each other and whether the pattern of MyHC expression is altered in the resulting heterokaryons, embryonic and fetal myoblasts were labelled with the lipophilic dye PKH26, [3H]‐thymidine, or 5‐bromodeoxyuridine (BRDU) and cocultured for 24–48 hr. Our results demonstrate that fusion occurs between embryonic and fetal myoblasts in vitro. Moreover, analysis of the resulting heterokaryons revealed regionalized accumulations of MyHC around individual nuclei. Interestingly, these accumulations were typical of the default pattern of expression that individual nuclei would have normally expressed in single culture. Nuclei contributed by embryonic myoblasts were surrounded by localized accumulations of slow MyHC, whereas nuclei from fetal myoblasts were surrounded by neonatal/fast MyHC. The occurrence of such nuclear domains indicates that the myoblast‐specific expression of MyHC isoforms is dictated by cis‐acting factors established prior to fusion. Dev. Dyn. 208:420–431, 1997. © 1997 Wiley‐Liss, Inc.

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“…Even though dystrophin was localized earlier to focal adhesions of cultured Xenopus laevis muscle cells (34) and in web-like surface structures of differentiated chicken myotubes (65), to our knowledge, this is a first report showing dystrophin localization at various adherens-type junctions in cultured muscle cells during reorganization of the actin cytoskeleton accompanying myogenesis. Earlier, it was reported that the original C2 mouse muscle cell line expresses only trace, if any, dystrophin (66,67). On the contrary, we have detected substantial amounts of dystrophin in the C2C12 subclone of C2 cells, therefore, allowing us to localize this protein during myodifferentiation in culture and to search for dystrophin-associated proteins in cultured muscle cells.…”

Section: Discussionmentioning

confidence: 51%

Association of Aciculin with Dystrophin and Utrophin

Belkin

Burridge

1995

Journal of Biological Chemistry

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Aciculin is a recently identified 60-kDa cytoskeletal protein, highly homologous to the glycolytic enzyme phosphoglucomutase type 1, (Belkin, A. M., Klimanskaya, I. V., Lukashev, M. E., Lilley, K., Critchley, D., and Koteliansky, V. E. (1994) J. Cell Sci. 107, 159-173). Aciculin expression in skeletal muscle is developmentally regulated, and this protein is particularly enriched at cell-matrix adherens junctions of muscle cells (Belkin, A. M., and Burridge, K. (1994) J. Cell Sci. 107, 1993-2003). The purpose of our study was to identify cytoskeletal protein(s) interacting with aciculin in various cell types. Using immunoprecipitation from cell lysates of metabolically labeled differentiating C2C12 muscle cells with anti-aciculin-specific antibodies, we detected a high molecular weight band (M(r) approximately 400,000), consistently coprecipitating with aciculin. We showed that this 400 kDa band comigrated with dystrophin and immunoblotted with anti-dystrophin antibodies. The association between aciculin and dystrophin in C2C12 cells was shown to resist Triton X-100 extraction and the majority of the complex could be extracted only in the presence of ionic detergents. In the reverse immunoprecipitation experiments, aciculin was detected in the precipitates with different anti-dystrophin antibodies. Immunodepletion experiments with lysates of metabolically labeled C2C12 myotubes showed that aciculin is a major dystrophin-associated protein in cultured skeletal muscle cells. Double immunostaining of differentiating and mature C2C12 myotubes with antibodies against aciculin and dystrophin revealed precise colocalization of these two cytoskeletal proteins throughout the process of myodifferentiation in culture. In skeletal muscle tissue, both proteins are concentrated at the sarcolemma and at myotendinous junctions. In contrast, utrophin, an autosomal homologue of dystrophin, was not codistributed with aciculin in muscle cell cultures and in skeletal muscle tissues. Analytical gel filtration experiments with purified aciculin and dystrophin showed interaction of these proteins in vitro, indicating that their association in skeletal muscle is due to direct binding. Whereas dystrophin was shown to be a major aciculin-associated protein in skeletal muscle, immunoblotting of anti-aciculin immunoprecipitates with antibodies against utrophin showed that aciculin is associated with utrophin in cultured A7r5 smooth muscle cells and REF52 fibroblasts. Immunodepletion experiments performed with lysates of metabolically labeled A7r5 cells demonstrated that aciculin is a major utrophin-binding protein in this cell type. Taken together, our data show that aciculin is a novel dystrophin- and utrophin-binding protein. Association of aciculin with dystrophin (utrophin) in various cell types might provide an additional cytoskeletal-matrix transmembrane link at sites where actin filaments terminate at the plasma membrane.

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Trans‐activation of the murine dystrophin gene in human‐mouse hybrid myotubes

Cited by 8 publications

References 19 publications

In vitro exercise model using contractile human and mouse hybrid myotubes

In vitro exercise model using contractile human and mouse hybrid myotubes

Regionalized expression of myosin isoforms in heterotypic myotubes formed from embryonic and fetal rat myoblasts in vitro

Association of Aciculin with Dystrophin and Utrophin

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