Abstract
The formation of skeletal muscle fibers is an intricate process controlled by a multitude of signaling pathways, including Wnt, Shh, and FGF. However, the role of the Hippo pathway during vertebrate myofiber formation has conflicting reports, which we decided to address in chick muscle cultures. We found that the transcriptional regulator Yes-associated protein (YAP) was highly concentrated within the nuclei of myoblasts. As cells differentiate into myotubes, YAP localization shifted to the cell cytoplasm in more mature myotubes. Treatment of cultures with XMU-MP-1 (XMU), a MST1/2 inhibitor, stimulated the nuclear localization of YAP in myoblasts and in myotubes, upregulated myogenin, and promoted myoblast fusion, ultimately resulting in the formation of large and fully striated multinucleated myotubes. The XMU-induced phenotype was blocked by the protein kinase C (PKC) inhibitor calphostin, which raises the possibility that the Hippo pathway controls the growth of skeletal muscle fibers through a PKC-dependent mechanism.
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The datasets generated for this study can be found within the manuscript figures and supplementary material.
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Funding
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Funding Number 302115/2017-0 for CM, 301443/2018-1 for MLC) and Fundação de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Funding Number E-26/202.920/2019 for CM and E26/210.220/2018 for MLC). A.D.J. is a fellow of the Estácio de Sá University (UNESA) Research Productivity Program and Institutos Nacionais de Ciência e Tecnologia (INCT/CAPES, Number 88887.568853/2020-00).
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CM, GG and MLC conceived the work. GG, IAR and KMB contributed to the acquisition of data. ADJ, CM, GG, KMB and MLC contributed to the analysis and interpretation of data. CM wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.
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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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The use of chick embryos was approved by the Ethics Committee for Animal Care and Use in Scientific Research from the Federal University of Rio de Janeiro and received the Approval Number: 069/19.
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10974_2021_9608_MOESM1_ESM.tif
Supplementary Figure 1 Activation of YAP by XMU leads to an increase in the size of myotubes. Chick muscle cells grown for 24 hours were treated for 48 hours with DMSO or 1 μM of XMU, and triple labelled with an antibody against desmin (magenta), the filamentous actin fluorescent probe Phalloidin (green) and DAPI (cyan). Some cultures were left untreated (control). Merged images are shown in the right column. Note the increase in the size of myotubes after XMU treatment. Scale bars = 20 μm (TIF 4973 kb)
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Gomes, G., Bagri, K.M., de Andrade Rosa, I. et al. Activation of YAP regulates muscle fiber size in a PKC-dependent mechanism during chick in vitro myogenesis. J Muscle Res Cell Motil 43, 73–86 (2022). https://doi.org/10.1007/s10974-021-09608-8
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DOI: https://doi.org/10.1007/s10974-021-09608-8