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Deletion of phosphatidylserine flippase β-subunit Tmem30a in satellite cells leads to delayed skeletal muscle regeneration

Kuan-Xiang Sun Xiao-Yan Jiang Xiao Li Yu-Jing Su Ju-Lin Wang Lin Zhang Ye-Ming Yang Xian-Jun Zhu

Kuan-Xiang Sun, Xiao-Yan Jiang, Xiao Li, Yu-Jing Su, Ju-Lin Wang, Lin Zhang, Ye-Ming Yang, Xian-Jun Zhu. Deletion of phosphatidylserine flippase β-subunit Tmem30a in satellite cells leads to delayed skeletal muscle regeneration. Zoological Research, 2021, 42(5): 650-659. doi: 10.24272/j.issn.2095-8137.2021.195
Citation: Kuan-Xiang Sun, Xiao-Yan Jiang, Xiao Li, Yu-Jing Su, Ju-Lin Wang, Lin Zhang, Ye-Ming Yang, Xian-Jun Zhu. Deletion of phosphatidylserine flippase β-subunit Tmem30a in satellite cells leads to delayed skeletal muscle regeneration. Zoological Research, 2021, 42(5): 650-659. doi: 10.24272/j.issn.2095-8137.2021.195

卫星细胞中敲除磷脂酰丝氨酸翻转酶β亚基Tmem30a延迟骨骼肌再生

doi: 10.24272/j.issn.2095-8137.2021.195

Deletion of phosphatidylserine flippase β-subunit Tmem30a in satellite cells leads to delayed skeletal muscle regeneration

Funds: This study was supported by the National Natural Science Foundation of China (81770950, 81970841), Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (2019-12M-5-032), and Department of Science and Technology of Sichuan Province (21ZDYF4279, 2020JDZH0026, 2021JDZH0022)
More Information
    Corresponding author: E-mail: xjzhu@uestc.edu.cn
  • #Authors contributed equally to this work
  • 摘要: 真核细胞质膜中的磷脂酰丝氨酸(PS)是不对称分布的。磷脂酰丝氨酸翻转酶(flippase)能将PS从质膜双分子层的外叶转移到膜的内叶,维持PS的不对称分布。Flippase的β亚基TMEM30A决定其正确的细胞定位及细胞功能。先前研究报道ATP11A和TMEM30A复合物是控制肌管形成的分子开关,然而Tmem30a在骨骼肌再生过程中的作用尚不明确。在该研究中,我们发现Tmem30a在肌肉营养不良小鼠和BaCl2诱导骨骼肌损伤模型小鼠的胫骨前肌中都有高表达。进一步我们构建了在卫星细胞中特异性敲除Tmem30a的小鼠模型来研究Tmem30a在骨骼肌再生过程中的作用。通过H&E和天狼星红染色,分析BaCl2损伤骨骼肌后再生卫星细胞的数量和直径,发现敲除小鼠的骨骼肌再生被延迟。紧接着通过免疫组织化学实验发现,条件性敲除小鼠的再生骨骼肌区域中Pax7阳性和MYH3阳性的卫星细胞数量减少,表明卫星细胞增殖受到一定的影响。随后发现在敲除小鼠的再生骨骼肌区域中肌肉调节因子(MYOD和MYOG)表达也减少,表明骨骼肌再生中成肌细胞的增殖受损。综上所述,这些结果证明Tmem30a在骨骼肌再生过程中发挥着重要作用。
    #Authors contributed equally to this work
  • Figure  1.  Tmem30a was up-regulated during skeletal muscle regeneration

    A, D: Immunoblot analysis of TMEM30A protein expression in 3-month-old wild-type (WT) and mdx mice. Relative protein density was calculated by ImageJ. B, F: Immunoblot analysis of TMEM30A protein expression in uninjured WT mice and in WT mice at 5 days post-injury (dpi). Relative protein density was calculated by ImageJ. C: mRNA expression analysis of Tmem30a in 3-month-old WT and mdx mice using qRT-PCR. E: mRNA expression analysis of Tmem30a in uninjured WT mice and in WT mice at 3 dpi using qRT-PCR. Data are mean±SEM. n=3 in each group. Significance was calculated using two-tailed Student’s t-test. *: P<0.05; **: P<0.01. G: Immunofluorescence analysis of TMEM30A expression in TA muscles of uninjured WT mice and of WT mice at 5 dpi. TMEM30A immunostaining is shown in green, and nuclei are shown in blue (counterstained with DAPI). Scale bar: 50 μm.

    Figure  2.  Generation of Tmem30a conditional knockout (cKO) mice

    A: Breeding strategy of cKO mice by crossing Tmem30aloxP/loxP with Pax7CreER mice. B: Genotyping of mice by PCR and agarose gel electrophoresis. C: Axis of tamoxifen intraperitoneal injection, BaCl2 injection, and TA muscle harvesting in mice. D: In 7 dpi cKO mice, expression of Pax7CreER was monitored by ROSA-tdTomato reporter (red). SCs were labeled with Pax7 antibody (green). Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. E: TMEM30A protein from 7 dpi WT and cKO mice was labeled with TMEM30A antibody (green) and DAPI (blue). Scale bar: 50 μm. F, G: Immunoblot analysis of TMEM30A protein expression in 7 dpi WT and cKO mice. Relative protein band density was calculated by ImageJ. Data are mean±SEM. n=3 in each group. Significance was calculated by two-tailed Student’s t-test. *: P<0.05.

    Figure  3.  Myoblast regeneration was impaired in Tmem30a cKO mice after injury

    A: H&E staining of TA muscles from uninjured WT and cKO mice and WT and cKO mice at 3, 5, 7, 10, and 14 dpi. Representative H&E-stained images of TA muscles from WT and cKO mice are shown. Blue arrows represent inflammatory cells; black arrows represent necrotic myofibers; yellow arrows represent proliferating SCs. Scale bar: 50 μm. B: Average number of regenerating myofibers per field in uninjured mice and mice at 3, 5, 7, 10, and 14 dpi. Reduced number of regenerative myofibers was observed in cKO mice. C: Average diameter of regenerating myofibers in uninjured mice and mice at 3, 5, 7, 10, and 14 dpi. Reduced diameter of regenerative myofibers was observed in cKO mice. Data are mean±SEM. n=3 in each group. Significance was calculated by two-tailed Student’s t-test. *: P<0.05; **: P<0.01; ***: P<0.001; ns: Not significant.

    Figure  4.  Pax7 expression was decreased in Tmem30a cKO mice

    A, B: Immunohistochemical results of TA muscles in uninjured WT and cKO mice and in WT and cKO mice at 3, 5, 7, 10, and 14 dpi. Pax7 immunostaining is shown in green, and nuclei are shown in blue (counterstained with DAPI). Scale bar: 50 μm. C: Average number of Pax7+ cells per field in uninjured mice and in mice at 3, 5, 7, 10, and 14 dpi. Data are mean±SEM. n=3 in each group. Significance was calculated by two-tailed Student’s t-test. **: P<0.01, ***: P<0.001; ns: Not significant.

    Figure  5.  Deletion of Tmem30a impaired SC proliferation

    A, B: Expression of MYH3 in uninjured WT and cKO mice and in WT and cKO mice at 3, 5, 7, and 14 dpi by immunohistochemical staining. MYH3 was labeled with MYH3 antibody (green). Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. C: Immunohistochemical staining analysis of size and number of regenerating myofibers in TA muscles of uninjured WT and cKO mice and of WT and cKO mice at 5, 7, 10, and 14 dpi. Laminin immunostaining is shown in green, and nuclei are shown in blue (counterstained with DAPI). Scale bar: 50 μm. D: Average number of MYH3+ cells per field in uninjured mice and in mice at 3, 5, 7, and 14 dpi. E: Average number of regenerating myofibers per field in uninjured mice and in mice at 5, 7, 10, and 14 dpi. F: Average diameter of regenerating myofibers in uninjured mice and in mice at 5, 7, 10, and 14 dpi. Data are mean±SEM. n=3 in each group. Significance was calculated by two-tailed Student’s t-test. *: P<0.05; **: P<0.01; ***: P<0.001.

    Figure  6.  Specific deletion of Tmem30a in SCs impaired differentiation of myoblasts

    A: Immunofluorescence analysis of MYOD expression in TA muscles of WT and cKO mice at 7 dpi. MYOD immunostaining is shown in green, and nuclei are shown in blue (counterstained with DAPI). Arrows depict cells positive for MYOD. Scale bar: 50 μm. B: Average number of MYOD+ cells per field at 7 dpi. C: Immunofluorescence analysis of MYOG expression in TA muscles of WT and cKO mice at 7 dpi. MYOG immunostaining is shown in green, and nuclei are shown in blue (counterstained with DAPI). Arrows depict cells positive for MYOG. Scale bar: 50 μm. D: Average number of MYOG+ cells per field at 7 dpi. Data are mean±SEM. n=3 in each group. Significance was calculated by two-tailed Student’s t-test. ***: P<0.001. E: H&E and Sirius Red staining of TA muscles from WT and cKO mice at 14 dpi. Arrows depict abnormal myofiber fusion. n=3 in each group. Scale bar: 50 μm.

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  • 收稿日期:  2021-08-03
  • 录用日期:  2021-08-30
  • 网络出版日期:  2021-08-31
  • 刊出日期:  2021-09-18

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