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  • CC-115 Previous reports indicate that the expression of AChR

    2024-02-21

    Previous reports indicate that the expression of AChRs and their clustering on myotubes are critical factors required to induce contacts on muscle fibers in a natural biological system [14], [15], [16], [17], [18], [19]. For instance, in NMJ development in the mouse, AChRs are pre-patterned at sites that are used by the outgrowing motor neurons to form synaptic contacts [20]. On each muscle fiber, one synaptic site (often contacted by several different nerves) becomes stabilized, CC-115 and the mature endplate pattern is established at a later embryonic stage. During postnatal development, further structural and functional changes are observed. Multiple innervations of muscle fibers are reduced until motor neurons form single synaptic contacts, and embryonic AChRs are replaced by CC-115 of adult AChRs that acquire a characteristically shaped “prezel-like” pattern. During the process of maturation of NMJs between muscle fibers and motor neurons, the formation of embryonic AChR clusters on muscle fibers clearly appears to be a prerequisite step that controls the overall process leading to mature innervations of muscle fibers [14], [17]. Although this well-organized process of pre-forming AChR clusters on embryonic muscle fibers is present in normal vertebrate systems, tissue engineered muscle tissue is not able to provide such a sophisticated process, and this is a major concern in terms of whether functional innervations of engineered muscle tissue can be achieved in vivo. In this study, we hypothesized that pre-fabrication of AChR clusters on engineered muscle tissue using molecular and cellular factors that are involved in the NMJ formation in other systems would accelerate innervation in vivo. We utilized a neural-released trophic factor [21], [22], [23], [24], [25], [26], agrin, to pre-pattern AChR clusters on engineered muscle tissues in a 3-D fibrin gel scaffold system. Agrin, which is a 400-kDa heparan sulfate proteoglycan expressed and released by motor neurons, has a critical role in inducing AChR expression and their clustering on muscle cells [16], [21], [27]. Neural agrin stimulates the rapid phosphorylation of a muscle-specific kinase, MuSK, which has been shown to be necessary for the formation of the NMJ, and this leads to the redistribution of previously unlocalized proteins, including AChRs, to synaptic sites [28]. This function of agrin was established by generating agrin-deficient mice, which fail to form functional endplates [29], and by gene transfer experiments and injection of recombinant agrin [30]. In these previous studies, agrin was shown to induce in vivo ectopic clusters of AChRs and postsynaptic-like differentiation of the muscle membrane [21], [25]. In the present study we examined whether agrin treatment would promote AChR clusters formation on cultured myotubes obtained by differentiating muscle precursor cells (C2C12), and whether it would enhance established contacts with the dorsal root ganglion (DRG). Also, we investigated whether pre-treatment of C2C12 myotubes with agrin would enhance contacts of the implanted myotubes with host nerve using a rat model (Fig. S1).
    Materials and methods
    Results
    Discussion The essential components in building functional skeletal muscle tissue include construction of organized muscle tissue capable of appropriate contraction in vitro and adequate vascularization and established contacts with the host's nervous system following implantation in vivo[4], [9], [10]. Various methods have been developed to provide contractile function and vascularization to engineered muscle tissue; however, limited approaches are available to address the problem of establishing innervations in engineered muscle tissue in vivo. In this study we investigated whether pre-fabrication of AChR clusters on engineered myotubes would facilitate acceleration of nerve contacts for rapid recovery of muscle function in vivo. We showed that agrin treatment significantly increased the formation of AChR clusters on myotubes, and that this enhanced neural contacts in vitro and in vivo in a rat ectopic nerve transposition model. Our data support previous findings that stimulated nerve-muscle constructs result in better contractility characteristics than muscle-only constructs, indicating that skeletal muscle constructs can be better developed in the presence of neural cells and NMJs [19], [34]. Other evidence suggests that the interactions of myofibers with neural cells in a co-culture system are necessary to induce NMJ formation on myofibers and that these NMJs are required for the development of functional properties of myofibers [35]. However, neural cell isolation for in vitro use is difficult, largely because the current sources of neural tissue are insufficient. In addition, neural cell isolation requires extensive cell manipulation, and this process as well as the subsequent co-culture with muscle cells are time and cost ineffective processes, which can hamper the development of efficient clinical applications. To overcome these limitations, it is necessary to develop alternative methods for inducing AChR clusters formation on skeletal muscle fibers, which may eliminate the need for donor neural cell procurement and subsequent in vitro cell manipulation.