E2 signaling in myofibers promots macrophage efferocytosis in mouse skeletal muscles with cardiotoxin-induced acute injury

doi:10.12122/j.issn.1673-4254.2024.11.16

Abstract

Abstract:

Objective To investigate the effect of E₂ signaling in myofibers on muscular macrophage efferocytosis in mice with cardiotoxin-induced acute skeletal muscle injury. Methods Female wild-type C57BL/6 mice with and without ovariectomy and male C57BL/6 mice were given a CTX injection into the anterior tibial muscle to induce acute muscle injury, followed by intramuscular injection of β-estradiol (E₂) or 4-hydroxytamoxifen (4-OHT). The changes in serum E₂ of the mice were detected using ELISA, and the number, phenotypes, and efferocytosis of the macrophages in the inflammatory exudates and myofiber regeneration and repair were evaluated using immunofluorescence staining and flow cytometry. C2C12 cells were induced to differentiate into mature myotubes, which were treated with IFN-γ for 24 before treatment with β-Estradiol or 4-OHT. The treated myotubes were co-cultured with mouse peritoneal macrophages in a 1:2 ratio, followed by addition of PKH67-labeled apoptotic mouse mononuclear spleen cells induced by UV irradiation, and macrophage efferocytosis was observed using immunofluorescence staining and flow cytometry. Results Compared with the control mice, the female mice with ovariectomy showed significantly increased mononuclear macrophages in the inflammatory exudates, with increased M1 cell percentage, reduced M2 cell percentage and macrophage efferocytosis in the injured muscle, and obviously delayed myofiber regeneration and repair. In the cell co-culture systems, treatment of the myotubes with β-estradiol significantly increased the number and proportion of M2 macrophages and macrophage efferocytosis, while 4-OHT treatment resulted in the opposite changes. Conclusion In injured mouse skeletal muscles, myofiber E₂ signaling promotes M1 to M2 transition to increase macrophage efferocytosis, thereby relieving inflammation and promoting muscle regeneration and repair.

Key words: E₂ signaling, acute skeletal muscle injury, macrophages efferocytosis, inflammation

Qihui CAI, Haiqiang LAN, Bojun XIAN, Lian LIU, Nan WANG, Xiaolei HUANG, Xiaolu NIU, Xinyu HU, Chen LI, Junyi XIE, Zhaohong LIAO. E₂signaling in myofibers promots macrophage efferocytosis in mouse skeletal muscles with cardiotoxin-induced acute injury[J]. Journal of Southern Medical University, 2024, 44(11): 2192-2200.

Figures/Tables 10

Tab.1 Primer sequence for qRT-PCR

Gene

Primer sequence (5'-3')

ERα

For:ACTGGCCAATCTTTCTCTGC

Rev:CAATTCATCCCCAAAGACATGGAC

ERβ

For:TCACTTCTGCGCTGTCTGCAGCG

Rev:CCTGGGTCGCTGTGCCAAG

GAPDH

For:CAATGTGTCCGTCGTGGATCT

Rev:GTCCTCAGTGTAGCCCAAGATG

Tab.2 Primer sequence for qRT-PCR

Gene	Primer sequence(5'-3')
IL-1β	For:GCCCATCCTCTGTGACTC Rev:TGTGCCGTCTTTCATTAC
IL-10	For:TTTCAAACAAAGGACCAG Rev:GGATCATTTCCGATAAGG
iNOS	For:CTTCCGGGCAGCCTGTGAGACG Rev:ATCCCCAGGTGTTCCCCAGGTAGG
TNF-α	For:GCTGTCTCCCCCGAAAGATG Rev:AGGCAGGTGTAGATGTTGTGG
Arg1	For:CTCCAAGCCAAAGTCCTTAGAG Rev:AGGAGCTGTCATTAGGGACA
Mrc1	For:CTCTGTTCAGCTATTGGACGC Rev:TGGCACTCCCAAACATAATTTGA
GAPDH	For:CAATGTGTCCGTCGTGGATCT Rev:GTCCTCAGTGTAGCCCAAGATG

Fig.1 Effect of E2 in myofiber signaling on acute skeletal myositis in mice. A: HE and immunofluorescence staining for detecting inflammation in the tibialis anterior muscle (TA) after CTX injury (Scale bar=50 μm). B: ELISA for detection of serum E2 in male and female mice at different time points after muscle injury (*P<0.05).

Fig.2 Expression of estrogen receptors in acute skeletal myositis. A: Western blotting of ERα and ERβ expression level in mature myotubes derived from C2C12 cells. B: qRT-PCR analysis of ERα and ERβ mRNA levels in the damaged muscle of female mice. C: Immunofluorescence staining of ERβ expression level in injured mouse muscle (Scale bar=50 μm). *P<0.05, **P<0.01.

Fig.3 Effect of E2 signaling in myofibers on exudation of intramuscular mononuclear macrophages after acute skeletal muscle injury. A: ELISA for detecting serum E2 level in female mice after different treatments. B: ELISA for detecting serum E2 levels in female mice at different time points after OVX treatment. C: HE staining for detecting muscular inflammation in female mice with different treatments after CTX injury. D: Immunofluorescence staining for detecting muscular inflammation in female mice after CTX injury with different treatments. *P<0.05, **P<0.01; Scale bar=50 μm.

Fig.4 Effect of E2 signaling in myofibers on phenotype of intramuscular macrophages in mice after acute skeletal muscle injury. A: Flow cytometric analysis of mononuclear macrophages in the damaged muscle of female mice after OVX treatment. B: Flow cytometric analysis of the phenotype of macrophages in the injured muscles of female mice after OVX treatment. *P<0.05, **P<0.01.

Fig.5 Effect of E2 signaling in myofibers on intramuscular inflammatory cytokines after acute skeletal muscle injury. A: qRT-PCR analysis of inflammatory cytokines in female mice after OVX treatment. B: qRT-PCR analysis of inflammatory cytokines in macrophages from the injured muscles isolated by flow cytometry in female mice after OVX treatment. *P<0.05, **P<0.01.

Fig.6 E2 signaling in myofibers drives intramuscular macrophage efferocytosis in acute skeletal myositis. A: Immunofluorescence detection of intramuscular macrophage efferocytosis in the damaged muscle. (scale bar=50 μm). B: Flow cytometry of intramuscular macrophage efferocytosis in the damaged muscle (*P<0.05).

Fig.7 Effect of E2 signaling on macrophage phenotypes and efferocytosis in an in vitro co-culture system. A: Immunofluorescence detection of macrophage phenotypes in the in vitro co-culture system. B: Immunofluorescence detection and flow cytometry analysis of macrophage efferocytosis in the in vitro co-culture system. *P<0.05; Scale bar=50 μm.

Fig.8 E2 signaling in myofibers regulates regeneration and repair of intramuscular myofibers in injured mouse skeletal muscles of female mice after different treatments (Immunofluorescence staining, scale bar=50 μm; *P<0.05).

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E₂signaling in myofibers promots macrophage efferocytosis in mouse skeletal muscles with cardiotoxin-induced acute injury

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