Deletion of TECRL promotes skeletal muscle repair by up-regulating EGR2

Abstract

Myogenic regeneration relies on the proliferation and differentiation of satellite cells. TECRL (trans-2,3-enoyl-CoA reductase like) is an endoplasmic reticulum protein only expressed in cardiac and skeletal muscle. However, its role in myogenesis remains unknown. We show that TECRL expression is increased in response to injury. Satellite cell-specific deletion of TECRL enhances muscle repair by increasing the expression of EGR2 through the activation of the ERK1/2 signaling pathway, which in turn promotes the expression of PAX7. We further show that TECRL deletion led to the upregulation of the histone acetyltransferase general control nonderepressible 5, which enhances the transcription of EGR2 through acetylation. Importantly, we showed that AAV9-mediated TECRL silencing improved muscle repair in mice. These findings shed light on myogenic regeneration and muscle repair.

Keywords: ischemia; muscle regeneration; satellite cell.

Fig. 1.

TECRL expression in skeletal muscle is mainly restricted to satellite cells. (A) The mRNA level of TECRL was increased in IR injured gastrocnemius muscles (n = 6, values are mean ± SD, unpaired t test with Welch’s correction, ***P < 0.001). (B) The mRNA levels of TECRL in the gastrocnemius muscles of 3 and 5-wk-old mdx mice (n = 6, values are mean ± SD, unpaired t test with Welch’s correction, ***P < 0.001). (C) The mRNA levels of TECRL in BaCl₂-injured TA muscles (n = 6, values are mean ± SD, Kruskal–Wallis test with Dunn’s multiple comparisons, ***P < 0.001, *P < 0.05). (D) RT-qPCR analysis of mRNA level of TECRL in gastrocnemius muscles and satellite cells (n = 6, values are mean ± SD, unpaired t test with Welch’s correction, ***P < 0.001). (E) Immunostaining of TECRL (green) and Pax7 (red) in primary myogenic cells. Nuclei were labeled by DAPI (blue). (Scale bar: 50 μm.) (F) Cryosections from uninjured tibialis anterior muscles from wild-type C57BL/6J mice were stained for TECRL (green), Pax7 (red), and Dystrophin (gray). Nuclei were stained with DAPI (blue). The secondary antibodies used for detecting TECRL, Pax7, and Dystrophin were Alexa Fluor 568 goat anti-rabbit IgG (H+L), Alexa Fluor 488 goat anti-mouse IgG1 and Alexa Fluor 647 goat anti-mouse IgG2b, respectively. (Scale bar: 5 μm.) (G) Western blot analysis of the levels of TECRL, Pax7, MyoD, and MyHC in primary myogenic cells cultured in differentiation medium containing 2% horse serum. GAPDH was used as loading control. (H and I) The mRNA levels of TECRL and Pax7 in primary myoblasts cultured in differentiation medium during a 3-d period (n = 3, values are mean ± SD, Kruskal–Wallis test with Dunn’s multiple comparisons, *P < 0.05).

Fig. 2.

Loss of TECRL enhances skeletal muscle regeneration. (A) Representative images of injured and uninjured tibialis anterior muscles from TECRL^flox/flox and TECRL^scko mice. (B) Average wet weight of TA muscles (n = 6, values are mean ± SD, ANOVA with post hoc Tukey’s multiple comparisons, ***P < 0.001, **P < 0.01). (C) H&E staining of TA muscles from TECRL^scko and TECRL^flox/fox littermates after BaCl₂ injection and uninjured muscles. (Scale bar: 50 μm.) (D) Number of centronucleated fibers (CNF) per field (0.04 mm²) on day 5 post injury (n = 6, values are mean ± SD, unpaired t test, ***P < 0.001). (E) The number of muscle fibers containing more than one central nucleus per field (0.04 mm²) (n = 6, values are mean ± SD, unpaired t test, ***P < 0.001). (F) The average CSA of CNF of regenerated muscle fibers (n = 6, values are mean ± SD, unpaired t test, ***P < 0.001). (G) H&E staining of TA muscles from TECRL^scko and TECRL^flox/flox at day 5 after BaCl₂ injection. The black-dotted line shows necrosis. (Scale bar: 50 μm.)

Fig. 3.

Deletion of TECRL in satellite cells increased expression of eMyHC. (A) Immunostaining of eMyHC (green) for regenerating myofibers in TA muscles from TECRL^flox/flox and TECRL^scko mice at different time points after BaCl₂-induced injury and uninjured muscles. The nuclei were stained with DAPI (blue). (Scale bar: 50 μm.) (B and C) Quantification of the numbers (n = 6, values are mean ± SD, unpaired t test, ***P < 0.001) and CSA of eMyHC⁺ fibers per field in TA muscles 5 d after injury (n = 6, values are mean ± SD, unpaired t test with Welch’s correction, ***P < 0.001). (D) RT-qPCR analysis of relative mRNA levels of Myh3 (eMyHC mRNA) in TA muscles 3 d after injury (n = 6, values are mean ± SD, Brown–Forsythe and Welch ANOVA tests with post hoc Dunnett’s T3 multiple comparisons, ***P < 0.001, **P < 0.01, *P < 0.05). (E) Western blot analysis of eMyHC in TA muscles of TECRL^flox/flox and TECRL^scko mice at different time points after BaCl₂ injection.

Fig. 4.

Deletion of TECRL enhances the expression of Pax7. (A) Immunostaining with Pax7 (red) and laminin (green) showing an increased number of satellite cells (white arrows) in uninjured and BaCl₂-injured TA muscles of TECRL^scko mice compared to TECRL^flox/flox mice. (Scale bar: 50 μm.) (B) Quantification of the average number of Pax7⁺ cells per myofiber in TA muscles of TECRL^flox/flox and TECRL^scko mice (n = 6, values are mean ± SD, Brown–Forsythe and Welch ANOVA tests with post hoc Dunnett’s T3 multiple comparisons, ***P < 0.001, **P < 0.01). (C) RT-qPCR analysis of relative mRNA levels of Pax7 in uninjured and injured TA muscles (n = 6, values are mean ± SD, Brown–Forsythe and Welch ANOVA tests with post hoc Dunnett’s T3 multiple comparisons, ***P < 0.001, **P < 0.01). (D) Western blot analysis of Pax7 in TA muscles. U: uninjured, I: injured. (E and F) RT-qPCR analysis of mRNA levels of Pax7 and TECRL in TECRL^+/+ and TECRL^−/− primary myogenic cells (n = 3, values are mean ± SD, unpaired t test, ***P < 0.001, *P < 0.05). (G) Western blot analysis of protein levels of Pax7 and TECRL in TECRL^+/+ and TECRL^−/− primary myogenic cells. (H) Densitometric quantification of relative protein levels of Pax7 and TECRL (n = 3, values are mean ± SD, unpaired t test, ***P < 0.001).

Fig. 5.

TECRL regulates satellite cell proliferation. (A) Immunostaining of Pax7 (green) and Ki67 (red) in TECRL^+/+ and TECRL^−/− primary myoblasts cultured for 3 d. The cells were incubated with anti-Ki67 (1:50 dilution) at 4 °C overnight. (Scale bar: 50 μm.) (B) The percentage of Pax7⁺Ki67⁺ cells from TECRL^+/+ and TECRL^−/− myoblasts (n = 3, values are mean ± SD, unpaired t test, ***P < 0.01). (C) Cell proliferation was performed by plating TECRL^+/+ and TECRL^−/− primary myoblasts at the same density and culturing for 5 d (n = 3, values are mean ± SD, unpaired t test, **P < 0.01). The cells were enumerated at different time points. (D) Colony forming assay for freshly isolated TECRL^+/+ and TECRL^−/− primary myogenic cells. (Scale bar: 100 μm.) (E) Quantification of the number of satellite cell colonies after 5 d in culture (n = 3, values are mean ± SD, unpaired t test with Welch’s correction, *P < 0.05). (F) Quantification of the number of satellite cell colonies after 10 d in culture (n = 3, values are mean ± SD, unpaired t test, ***P < 0.001). (G) Single myofibers were isolated from the EDL muscle of TECRL^flox/flox and TECRL^scko mice. The myofibers collected either immediately after isolation or after 72 h of culture were immunostained for Pax7 (red) or MyoD (green). Nuclei were identified by staining with DAPI (blue). (Scale bar: 100 μm.) (H) Quantification of the number of Pax7⁺ cells in each myofibers immediately after isolation (n = 6, values are mean ± SD, unpaired t test, ***P < 0.001). (I) Quantification of the number of Pax7⁺/MyoD⁻ cells in each myofibers immediately after isolation (n = 6, values are mean ± SD, unpaired t test, **P < 0.01). (J) Quantification of the number of cells in each cluster on cultured myofibers (n = 6, values are mean ± SD, unpaired t test, ***P < 0.001). (K) Quantification of the number of Pax7⁺MyoD⁺ cells on cultured myofibers (n = 6, values are mean ± SD, unpaired t test, ***P < 0.001). Analysis was performed on 6 to 10 myofibers for each mouse at each time point.

Fig. 6.

TECRL deletion led to activation of the ERK1/2 signaling pathway, which then activates EGR2 to induce the expression of Pax7 and the proliferation of satellite cells. (A) Immunostaining of Pax7 (green) and Ki67 (red) in primary myoblasts transduced with the empty vector or shRNA-EGR2 lentiviral construct cultured for 3 d. (Scale bar: 50 μm.) (B) The mRNA level of Pax7 in TECRL^+/+, TECRL^−/−, TECRL^−/−-EGR2-shRNA, and TECRL^−/−-NC-shRNA in primary myoblasts (n = 3, values are mean ± SD, one-way ANOVA tests with post hoc Tukey’s multiple comparisons, ***P < 0.001). (C) The mRNA level of Pax7 in TECRL^+/+, TECRL^−/−, and TECRL^+/+-EGR2-cDNA in primary myoblasts (n = 3, values are mean ± SD, one-way ANOVA tests with post hoc Tukey’s multiple comparisons, ***P < 0.001). (D) Western blot analysis of phosphorylated and total ERK1/2 in TECRL^+/+ and TECRL^−/− cultures. (E) Ratio of phospho-ERK1/2 and total ERK1/2 in TECRL^+/+ and TECRL^−/− primary myoblasts (n = 3, values are mean ± SD, unpaired t test with Welch’s correction, *P < 0.05). (F) Western blot analysis of Pax7 and EGR2 in primary myoblasts in the presence of ERK1/2 inhibitors (PD184352) or vehicle. (G) The mRNA level of EGR2 in TECRL^+/+, TECRL^−/−, and TECRL^−/− primary myoblasts in the presence of PD184352 (n = 3, values are mean ± SD, one-way ANOVA tests with post hoc Tukey’s multiple comparisons, ***P < 0.001). (H) Single myofibers isolated from EDL muscle of WT mice were cultured in satellite cell growth medium in the presence of ERK1/2 inhibitors (PD184352) or vehicle for 72 h. The single myofibers were stained for Pax7 (red) and MyoD (green). Nuclei were identified by staining with DAPI (blue). (Scale bars: 100 μm.) (I) Quantification of the number of cell clusters on each myofiber (n = 6, values are mean ± SD, unpaired t test, ***P < 0.001). (J) Quantification of the number of Pax7⁺MyoD⁺ cells per cluster (n = 6, values are mean ± SD, unpaired t test, ***P < 0.001). Analysis was performed using 3-6 myofibers for each mouse in each group. (K) Primary myoblasts were processed for ChIP assay. The semiquantitative RT-qPCR showing EGR2 was enriched at the indicated sites of the Pax7 promoter. The numbers represent the location of the consensus sequence upstream of the first ATG of the Pax7 gene. (L) RT-qPCR analysis of ChIP product to determine the percentage of EGR2 input enrichment at specific sites in the Pax7 promoter in TECRL^+/+ and TECRL^−/− cultures (n = 3, values are mean ± SD, unpaired t test, ***P < 0.001, **P < 0.01). (M) Dual-luciferase reporter assay of Pax7 promoter activation by EGR2. WT myoblasts were cotransfected with 250 ng empty Vector or EGR2-cDNA, 250 ng Pax7-Luc, and 20 ng pRL-TK plasmid by Lipofectamine 2000 (n = 3, values are mean ± SD, unpaired t test with Welch’s correction, **P < 0.01). (N) RT-qPCR analysis of GCN5 in TECRL^+/+ and TECRL^−/− cultures (n = 3, values are mean ± SD, unpaired t test, *P < 0.05). (O) Western blot analysis of GCN5, Pax7, and Ac-EGR2 levels in TECRL^+/+, TECRL^−/−, and TECRL^−/−-CPTH2 cultures. EGR2 proteins in the lysates of TECRL^+/+, TECRL^−/−, and TECRL^−/−-CPTH2 cultures were immunoprecipitated with the EGR2 antibodies, followed by immunoblotting with an anti-acetyl lysine–specific antibody to detect acetylated proteins. 1: TECRL^+/+, 2: TECRL^−/−, 3: TECRL^−/−-CPTH2. CPTH2 (0.05M) was added to the cell culture medium for 12 h.