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We recently demonstrated that mesenchymal progenitors play a critical role in regulating satellite cell-dependent myonuclear accretion during overload-induced muscle hypertrophy. Here, we describe the detailed protocol for whole-mount immunofluorescence staining of mesenchymal progenitors in mouse plantaris muscle. Z-stack image reconstruction provides a whole-cell image and enables examination of YAP nuclear translocation in mesenchymal progenitors induced by overload. For complete details on the use and execution of this protocol, please refer to Kaneshige et al. (2022a)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Muscle satellite cells (MuSCs) supply nuclei to existing myofibers in response to mechanical loading. This myonuclear accretion is critical for efficient muscle hypertrophy. Herein, we present protocols for the detection of MuSC-derived new myonuclei in loaded mouse muscle, including procedures for EdU injection to stain myonuclei, followed by surgery and skeletal muscle fixation. We then describe immunostaining for EdU + myonuclei and image acquisition for quantitative analyses. For complete details on the use and execution of this protocol, please refer to Kaneshige et al. (2022)., Competing Interests: The authors declare no competing financial interests., (© 2022 The Author(s).)

Quiescence is a fundamental property of adult stem cells. Recent evidence indicates that quiescence is not a default state but requires active signaling that prevents accidental or untimely activation of stem cells. The calcitonin receptor (CalcR) is critical for sustaining quiescence in muscle satellite (stem) cells (MuSCs). However, the molecular mechanisms by which CalcR signaling regulates quiescence in MuSCs are enigmatic. Here, we demonstrate that transgenic expression of the catalytic domain of protein kinase A (PKA) restores the quiescence of CalcR-mutant MuSCs and delays MuSC activation. Mechanistically, CalcR-activated PKA phosphorylates Lats1/2, the main effector of Hippo signaling, thereby inhibiting the nuclear accumulation of Yap1, which prevents expression of Hippo-target genes, including cell-cycle-related molecules. Importantly, genetic inactivation of Yap1 in CalcR-mutant MuSCs reinstates quiescence in CalcR-mutant MuSCs, indicating that the CalcR-PKA-Lats1/2-Yap1 axis plays a critical role in sustaining MuSC quiescence., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sarcopenia, age-related muscle weakness, increases the frequency of falls and fractures in elderly people, which can trigger severe muscle injury. Rapid and successful recovery from muscle injury is essential not to cause further frailty and loss of independence. In fact, we showed insufficient muscle regeneration in aged mice. Although the number of satellite cells, muscle stem cells, decreases with age, the remaining satellite cells maintain the myogenic capacity equivalent to young mice. Transplantation of young green fluorescent protein (GFP)-Tg mice-derived satellite cells into young and aged mice revealed that age-related deterioration of the muscle environment contributes to the decline in regenerative capacity of satellite cells. Thus, extrinsic changes rather than intrinsic changes in satellite cells appear to be a major determinant of inefficient muscle regeneration with age. Comprehensive protein expression analysis identified a decrease in insulin-like growth factor-II (IGF-II) level in regenerating muscle of aged mice. We found that pro- and big-IGF-II but not mature IGF-II specifically express during muscle regeneration and the expressions are not only delayed but also decreased in absolute quantity with age. Supplementation of pro-IGF-II in aged mice ameliorated the inefficient regenerative response by promoting proliferation of satellite cells, angiogenesis, and suppressing adipogenic differentiation of platelet derived growth factor receptor (PDGFR)α(+) mesenchymal progenitors. We further revealed that pro-IGF-II but not mature IGF-II specifically inhibits the pathological adipogenesis of PDGFRα(+) cells. Together, these results uncovered a distinctive pro-IGF-II-mediated self-reinforcement mechanism of muscle regeneration and suggest that supplementation of pro-IGF-II could be one of the most effective therapeutic approaches for muscle injury in elderly people., (© 2015 AlphaMed Press.)