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Updated: Oct 1, 2025
Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo
Published on: February 15, 2021
Muscle Stem Cell Function Is Impaired in β2-Adrenoceptor Knockout Mice
Tatiana E Koike1, Cesar S Fuziwara2, Patricia C Brum3
1Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, Avenida Professor Lineu Prestes, 2415. CEP, São Paulo, SP, 05508-000, Brazil.
Abstract:
Knockout (ko) mice for the β2 adrenoceptor (Adrβ2) have impaired skeletal muscle regeneration, suggesting that this receptor is important for muscle stem cell (satellite cell) function. Here, we investigated the role of Adrβ2 in the function of satellite cells from β2ko mice in the context of muscle regeneration, through in vivo and in vitro experiments. Immunohistochemical analysis showed a significant reduction in the number of self-renewed Pax7+ satellite cells, proliferating Pax7+/MyoD+ myogenic precursor cells, and regenerating eMHC+ myofibers in regenerating muscle of β2ko mice at 30, 3, and 10 days post-injury, respectively. Quiescent satellite cells were isolated by fluorescence-activated cell sorting, and cell cycle entry was assessed by EdU incorporation. The results demonstrated a lower number of proliferating Pax7+/EdU+ satellite cells from β2ko mice. There was an increase in the gene expression of the cell cycle inhibitor Cdkn1a and Notch pathway components and the activation of Notch signaling in proliferating myoblasts from β2ko mice. There was a decrease in the number of myogenin-positive nuclei in myofibers maintained in differentiation media, and a lower fusion index in differentiating myoblasts from β2ko mice. Furthermore, the gene expression of Wnt/β-catenin signaling components, the expression of nuclear β-catenin and the activation of Wnt/β-catenin signaling decreased in differentiating myoblasts from β2ko mice. These results indicate that Adrβ2 plays a crucial role in satellite cell self-renewal, as well as in myoblast proliferation and differentiation by regulating Notch and Wnt/β-catenin signaling, respectively.
Insights
The β2 adrenoceptor (Adrβ2) is crucial for skeletal muscle regeneration. Knockout mice show impaired satellite cell self-renewal, proliferation, and differentiation due to altered Notch and Wnt/β-catenin signaling.
Area of Science:
- Muscle stem cell biology
- Regenerative medicine
- Molecular signaling pathways
Background:
- Skeletal muscle regeneration relies on satellite cells.
- The role of β2 adrenoceptor (Adrβ2) in satellite cell function is not fully understood.
- Previous studies suggest Adrβ2 knockout mice exhibit impaired muscle regeneration.
Purpose of the Study:
- To investigate the role of Adrβ2 in satellite cell function during muscle regeneration.
- To elucidate the molecular mechanisms by which Adrβ2 influences satellite cell self-renewal, proliferation, and differentiation.
Main Methods:
- In vivo and in vitro experiments using β2 adrenoceptor knockout (β2ko) mice.
- Immunohistochemistry to assess cell populations (Pax7+, MyoD+, eMHC+).
- Fluorescence-activated cell sorting (FACS) for satellite cell isolation and EdU incorporation for cell cycle analysis.
- Gene expression analysis of cell cycle inhibitors (Cdkn1a), Notch, and Wnt/β-catenin signaling pathways.
Main Results:
- β2ko mice showed reduced numbers of self-renewed satellite cells, proliferating myoblasts, and regenerating myofibers post-injury.
- Satellite cells from β2ko mice exhibited decreased proliferation and increased cell cycle inhibitor Cdkn1a expression.
- Myoblasts from β2ko mice displayed reduced differentiation, evidenced by lower myogenin expression and fusion index.
- Notch signaling was activated, while Wnt/β-catenin signaling was suppressed in myoblasts from β2ko mice.
Conclusions:
- Adrβ2 is essential for satellite cell self-renewal and myoblast proliferation.
- Adrβ2 regulates myoblast differentiation through modulation of Notch and Wnt/β-catenin signaling pathways.
- Targeting Adrβ2 may offer therapeutic potential for enhancing skeletal muscle regeneration.
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