Role of CD38 and NAD+ metabolism in macrophages during recovery of muscle after injury in aged mice

2020 Fiscal Year Research-status Report

• Project/Area Number: 20K22733
• Research Institution: University of Toyama
• Principal Investigator: NAWAZ ALLAH 富山大学, 学術研究部医学系, 助教 (80881482)
• Project Period (FY): 2020-09-11 – 2022-03-31
• Keywords: Aging / NAD metabolism / Muscle regeneration / Immune cells / Myoblast

Outline of Annual Research Achievements

To know whether deletion of CD38 affects recovery process, we utilized 8 and 12 weeks of young CD38 KO and WT mice. After cardiotoxin (CTX) administration, the tibialis anterior (TA) and gastrocnemius (Gc) muscles were harvested, and histological analysis was performed to evaluate the muscle recovery. Gene expression, and immunohistochemical analysis partial improvement in recovery of muscle following injury in muscle of CD38 KO mice compared with littermate control WT mice at age of 8 and 12 weeks. This data suggested that even in young mice, deletion of CD38 boosted recovery process. Satellite cells required sufficient amount of NAD+ to sustain mitochondrial functions and its differentiation potential. We presumed that CD38 may affect muscle stem/satellite cells differentiation. To examine whether CD38 affects satellite cells differentiation, we performed in vitro study. Interestingly, 78c (CD38i) promotes C2C12 myoblast differentiation, indicating that CD38 might contributes to the inhibition of myoblast differentiation, thereby delaying the recovery process. Upregulated expression of CD38 massively decreases NAD+ levels that affects satellite cells differentiation. Of note, CD38 KO mice showed 3-4 fold increase in NAD+ levels in muscle of injured mice, suggesting that satellite cells uses NAD+ pools to sustain its regenerative potential.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

We hypothesized that the immune cells (CD38 macrophages) contributes the muscle recovery in aged mice via regulation of satellite cells proliferation or differentiation into myoblast and myofibers, hence mediate the recovery process.
Our initial results show that even in young CD38 KO mice, at least in part, recovery was enhanced. Genetic deletion of CD38 or inhibition of CD38 via 78c (CD38i) both increases NAD+ levels and also enhances satellite cells differentiation. Aging-related decline in NAD+ levels is reported to involve in delaying the recovery process. We presumed that reduction of CD38 in aged mice leads to enhancement in NAD+ and improves satellite cells functions, thereby boosting the recovery process.

Strategy for Future Research Activity

In future, we will perform ex vivo study to see effect of inhibition of CD38 on primary myoblast. For this, muscle will be harvested from cardiotoxin administered mice and processed for collagenase digestion. Primary myoblast will be seeded and 78c will be added in differentiation medium. Gene expression and LC/MS analyses will be performed.
We will utilize aged 38 KO mice and introduce injury to the muscle using cardiotoxin administration. Gene expression, immunohistochemistry, flow cytometry analyses will be performed to assess the effect of deletion of CD38 on recovery process. We will also perform LC-MS to check any change in NAD metabolites in these mice.

Research Products

• [Journal Article] Generation and characterization of a Meflin-CreERT2 transgenic line for lineage tracing in white adipose tissue (2021)
  • Author(s): Kuwano Takahide、Izumi Hironori、Aslam Muhammad Rahil、Igarashi Yoshiko、Bilal Muhammad、Nishimura Ayumi、Watanabe Yoshiyuki、Nawaz Allah、Kado Tomonobu、Ikuta Koichi、Yamamoto Seiji、Sasahara Masakiyo、Fujisaka Shiho、Yagi Kunimasa、Mori Hisashi、Tobe Kazuyuki
  • Journal Title: PLOS ONE Volume: 16 Pages: e0248267
  • DOI: 10.1371/journal.pone.0248267
• [Journal Article] Gut microbiota, determined by dietary nutrients, drive modification of the plasma lipid profile and insulin resistance (2021)
  • Author(s): Watanabe Yoshiyuki、Fujisaka Shiho、Ikeda Kazutaka、Ishikawa Masaki、Yamada Takahiro、Nawaz Allah、Kado Tomonobu、Kuwano Takahide、Nishimura Ayumi、Bilal Muhammad、Liu Jianhui、Yagi Kunimasa、Hase Koji、Tobe Kazuyuki
  • Journal Title: iScience Volume: 24 Pages: 102445～102445
  • DOI: 10.1016/j.isci.2021.102445
• [Journal Article] CD206+ macrophage is an accelerator of endometriotic-like lesion via promoting angiogenesis in the endometriosis mouse model (2021)
  • Author(s): Ono Yosuke、Yoshino Osamu、Hiraoka Takehiro、Sato Erina、Furue Akiko、Nawaz Allah、Hatta Hideki、Fukushi Yoshiyuki、Wada Shinichiro、Tobe Kazuyuki、Hirota Yasushi、Osuga Yutaka、Unno Nobuya、Saito Shigeru
  • Journal Title: Scientific Reports Volume: 11 Pages: 853
  • DOI: 10.1038/s41598-020-79578-3
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] NAD+ Metabolism Regulates Preadipocyte Differentiation by Enhancing α-Ketoglutarate-Mediated Histone H3K9 Demethylation at the PPARγ Promoter (2020)
  • Author(s): Okabe Keisuke、Nawaz Allah、Nishida Yasuhiro、Yaku Keisuke、Usui Isao、Tobe Kazuyuki、Nakagawa Takashi
  • Journal Title: Frontiers in Cell and Developmental Biology Volume: 8 Pages: 586179
  • DOI: 10.3389/fcell.2020.586179
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] CD206+ M2-Like Macrophages Are Essential for Successful Implantation (2020)
  • Author(s): Ono Yosuke、Yoshino Osamu、Hiraoka Takehiro、Sato Erina、Fukui Yamato、Ushijima Akemi、Nawaz Allah、Hirota Yasushi、Wada Shinichiro、Tobe Kazuyuki、Nakashima Akitoshi、Osuga Yutaka、Saito Shigeru
  • Journal Title: Frontiers in Immunology Volume: 11 Pages: 557184
  • DOI: 10.3389/fimmu.2020.557184
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Regulatory mechanisms between macrophages and Fibro-adipogenic progenitors during muscle recovery. (2021)
  • Author(s): Nawaz A, Kado T, Bilal M, Okabe K, Watanabe Y, Aslam MR, Nishimura A, Igarashi Y, Kuwano T, Liu J, Yagi K, Fujisaka S, Nakagawa T, Tobe K.
  • Organizer: 第41回日本肥満学会・第38回日本肥満症治療学会
  • Int'l Joint Research
• [Presentation] Depletion of CD206 M2-like macrophages ameliorates insulin resistance. (2021)
  • Author(s): Nawaz A, Kado T, Bilal M, Okabe K, Watanabe Y, Aslam MR, Nishimura A, Igarashi Y, Kuwano T, Liu J, Yagi K, Fujisaka S, Nakagawa T, Tobe K.
  • Organizer: 第41回日本肥満学会・第38回日本肥満症治療学会
  • Int'l Joint Research / Invited
• [Presentation] Depletion of CD206 M2-like macrophages induces adipocyte progenitors proliferation and promotes insulin sensitivity (2020)
  • Author(s): Nawaz A, Nakagawa T, Tobe K.
  • Organizer: 2020年度富山大学杉谷地区第2回基礎・ C-CAM・臨床研究発表会
• [Presentation] Astaxanthin ameliorate insulin resistance in skeletal muscle of obese mice via activation of the AMPK pathway. (2020)
  • Author(s): Nawaz A, Nishida Y, Nakagawa T, Tobe K.
  • Organizer: The 19th International Symposium on Carotenoids
  • Int'l Joint Research / Invited
• [Presentation] Physiological role of M2-macrophages in adipose tissue and muscle. (2020)
  • Author(s): Nawaz A, Nishida Y, Kado T, Bilal M, Okabe K, Watanabe Y, Nishimura A, Igarashi I, Kuwano T, Liu J, Yagi K, Fujisaka S, Nakagawa T, Tobe K.
  • Organizer: 第63回日本糖尿病学会年次学術集会
  • Int'l Joint Research / Invited