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Muscle Stem Cell Function Is Impaired in β2-Adrenoceptor Knockout Mice

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A Correction to this article was published on 14 April 2022

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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.

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Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgments

The authors are grateful to Chao Yun Irene Yan for providing cryosections of cephalic regions from HH19/20 chick embryos, Luiz C. Navegantes for stimulating discussions, and Anselmo S. Moriscot for allowing the use of the cryostat from his laboratory. This study was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant Nos. 14/23391-8 and 18/24946-4) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Fellowship/Grant No. 312142/2018-8). Tatiana E. Koike received a Ph.D. fellowship from FAPESP (Grant No. 17/09069-4).

Funding

This study was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant Nos. 14/23391–8 and 18/24946–4) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Fellowship/Grant No. 312142/2018–8). Tatiana E. Koike received a Ph.D. fellowship from FAPESP (Grant No. 17/09069–4).

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Authors and Affiliations

  1. Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, Avenida Professor Lineu Prestes, 2415. CEP, São Paulo, SP, 05508-000, Brazil

    Tatiana E. Koike & Elen H. Miyabara

  2. Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil

    Cesar S. Fuziwara & Edna T. Kimura

  3. School of Physical Education and Sport, University of São Paulo, São Paulo, SP, Brazil

    Patricia C. Brum

  4. Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA, USA

    Thomas A. Rando

  5. Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA

    Thomas A. Rando

  6. Broad Stem Cell Research Center, University of California Los Angeles, Los Angeles, CA, USA

    Thomas A. Rando

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  1. Tatiana E. Koike
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Contributions

Conceptualization: Tatiana E. Koike, Elen H. Miyabara. Experimental performance and/or data analysis: Tatiana E. Koike, Cesar S. Fuziwara, Patricia C. Brum, Edna T. Kimura, Thomas. A. Rando, Elen H. Miyabara. Funding acquisition: Thomas. A. Rando, Elen H. Miyabara. Project administration: Tatiana E. Koike, Elen H. Miyabara. Supervision: Elen H. Miyabara. Manuscript preparation: Tatiana E. Koike, Elen H. Miyabara. Manuscript review: Tatiana E. Koike, Cesar S. Fuziwara, Patricia C. Brum, Edna T. Kimura, Thomas. A. Rando, Elen H. Miyabara.

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Correspondence to Elen H. Miyabara.

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All applicable international, national, and institutional guidelines for the care and use of animals were followed. All animal experiments were approved by the Animal Research Ethics Committee of the Institute of Biomedical Sciences of the University of São Paulo under Protocol No. 106/2017.

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The original online version of this article was revised: Figure 5 was updated only in HTML, but not in the PDF version. In addition, the Supplementary Information for Tables S1 and S2 (or ESM 3) has to be replaced and updated.

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ESM 1

(PNG 595 kb)

12015_2022_10334_MOESM1_ESM.tif

Fluorescence-activated cell sorting gating strategy of myogenic progenitors. Gates were defined based on unstained or single-stained samples. Cells were gated on side scatter (SSC) and forward scatter (FSC), and dead cells were excluded by DAPI dilactate staining. Representative FACS of CD45– CD31– Sca1– VCAM1+ cells. (TIF 9746 kb)

ESM 2

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A

Adrβ2 expression in FACS-purified activated satellite cells isolated from wild-type (WT) mice at 3 dpi and cultured for 24 h in wash medium. Blue: DAPI; White: Synd-4; Green: MyoD; Red: Adrβ2. Adrβ2 expression in early differentiating myoblasts (FACS-purified quiescent satellite cells cultured in wash medium for 24 h and in differentiation medium for 24 h) from WT mice. Blue: DAPI; White: Synd-4; Red: myogenin (MyoG); Green: Adrβ2. Bar: 50 μm. B Cryosections obtained from the cephalic regions from HH19/20 chick embryos and incubated with antibodies against MyoD and MyoG and Adrβ2 and DAPI are negative controls of these antibodies. Syndecan-4 is a marker of quiescent and activated satellite cells. Once activated, satellite cells maintain the expression of syndecan-4 for at least 96 h (Cornelison et al. 57). Therefore, satellite cells were stained with the antibody against syndecan-4 (Cornelison et al. 58). Myogenic stages—activation/proliferation or early differentiation— were identified using MyoD or MyoG, respectively. C Cross-sections of uninjured TA muscles from WT and β2ko mice stained with Pax7 (red), laminin (white), and DAPI (blue). Arrows indicate Pax7+ satellite cells. Bar: 20 μm. D Number of Pax7+ satellite cells per mm2 of uninjured TA muscles from WT and β2ko mice (four fields per animal were analyzed). Mean ± SEM of three different biological replicates per group. *p ≤ 0.05 vs. WT. E mRNA levels of Nur-77 in proliferating myoblasts (cultured in growth medium for 24 h) from WT and β2ko mice. Mean ± SEM of four biological replicates per group. *p ≤ 0.05 vs. WT. (TIF 9686 kb)

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Koike, T.E., Fuziwara, C.S., Brum, P.C. et al. Muscle Stem Cell Function Is Impaired in β2-Adrenoceptor Knockout Mice. Stem Cell Rev and Rep 18, 2431–2443 (2022). https://doi.org/10.1007/s12015-022-10334-y

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