Self-transducible LRS-UNE-L peptide enhances muscle regeneration

Abstract

Background: Muscle regeneration includes proliferation and differentiation of muscle satellite cells, which involves the mammalian target of rapamycin (mTOR). We identified the C-terminal unique attached sequence motif (UNE) domain of leucyl-tRNA synthetase (LRS-UNE-L) as an mTORC1 (mTOR complex1)-activating domain that acts through Vps34 and phospholipase D1 (PLD1) when introduced in the form of a muscle-enhancing peptide.

Methods: In vitro Vps34 lipid kinase assay, phosphatidylinositol 3-phosphate (PI(3)P) measurement, in vivo PLD1 assay, and western blot assay were performed in HEK293 cells to test the effect of the LRS-UNE-L on the Vps34-PLD1-mTOR pathway. Adeno-associated virus (AAV)-LRS-UNE-L was transduced in C2C12 cells in vitro, in BaCl₂ -injured tibialis anterior (TA) muscles, and in 18-month-old TA muscles to analyse its effect on myogenesis, muscle regeneration, and aged muscle, respectively. The muscle-specific cell-permeable peptide M12 was fused with LRS-UNE-L and tested for cell integration in C2C12 and HEK293 cells using FACS analysis and immunocytochemistry. Finally, M12-LRS-UNE-L was introduced into BaCl₂ -injured TA muscles of 15-week-old Pld1^+/+ or Pld1^-/- mice, and its effect was analysed by measurement of cross-sectional area of regenerating muscle fibres.

Results: The LRS-UNE-L expression restored amino acid-induced S6K1 phosphorylation in LRS knockdown cells in a RagD GTPases-independent manner (421%, P = 0.007 vs. LRS knockdown control cells). The LRS-UNE-L domain was directly bound to Vps34; this interaction was accompanied by increases in Vps34 activity (166%, P = 0.0352), PI(3)P levels (146%, P = 0.0039), and PLD1 activity (228%, P = 0.0294) compared with amino acid-treated control cells, but it did not affect autophagic flux. AAV-delivered LRS-UNE-L domain augmented S6K1 phosphorylation (174%, P = 0.0013), mRNA levels of myosin heavy chain (MHC) (122%, P = 0.0282) and insulin-like growth factor 2 (IGF2) (146%, P = 0.008), and myogenic fusion (133%, P = 0.0479) in C2C12 myotubes. AAV-LRS-UNE-L increased the size of regenerating muscle fibres in BaCl₂ -injured TA muscles (124%, P = 0.0279) (n = 9-10), but it did not change the muscle fibre size of TA muscles in old mice. M12-LRS-UNE-L was preferentially delivered into C2C12 cells compared with HEK293 cells and augmented regeneration of BaCl₂ -injured TA muscles in a PLD1-dependent manner (116%, P = 0.0022) (n = 6).

Conclusions: Our results provide compelling evidence that M12-LRS-UNE-L could be a muscle-enhancing protein targeting mTOR.

Keywords: M12-LRS-UNE-L; Muscle differentiation; Muscle regeneration; Muscle-enhancing protein; Self-transducible peptide; mTOR.

Figure 1

LRS‐UNE‐L enhances amino acid‐induced Vps34‐PLD1‐mTORC1 activity. (A) HEK293 cells were transduced with lentivirus expressing shLRS or shScramble (control), selected with puromycin for 3 days, and transfected with Flag‐LRS‐WT or Flag‐LRS‐UNE‐L. The cells were serum‐starved overnight, amino acid‐deprived for 2 h, stimulated with amino acids (AA) for 30 min, and then lysed and subjected to western blot analysis (n = 3). (B) HEK293 cells were transfected with Flag‐LRS‐UNE‐L, serum‐starved overnight, and amino acid‐deprived for 2 h. Amino acid stimulation for 30 min was performed in the presence or absence of 30 μM BC‐LI‐0186 before the cells were lysed and subjected to western blot analysis (n = 3). (C) HEK293 cells were transfected with Flag‐LRS‐UNE‐L. Anti‐Flag immunoprecipitation was conducted, followed by western blot analysis. (D) HEK293 cells were co‐transfected with myc‐Vps34/V5‐Vps15 and Flag‐LRS‐WT or Flag‐LRS‐UNE‐L. The cells were serum‐starved overnight, amino acid‐deprived for 2 h, and stimulated with amino acids for 30 min. Anti‐myc immunoprecipitates of cell lysates were subjected to in vitro kinase assay (n = 3). (E) HEK293 cells were transfected with Flag‐LRS‐WT or Flag‐LRS‐UNE‐L and treated as in (D). PI(3)P levels of lipid extracts were measured by quantitative PI(3)P ELISA assay (n = 3). (F) HEK293 cells were co‐transfected with HA‐PLD1 and Flag‐LRS‐WT or Flag‐LRS‐UNE‐L and treated as in (D), followed by in vivo PLD1 assays (n = 4). (G) HEK293 cells were transfected with Flag‐LRS‐UNE‐L, serum‐starved overnight, and then subjected to western blot analysis (n = 3). All controls were transfected with empty vector. All data shown are the mean ± SD or representative blots from 3–5 independent experiments. The intensities of western blots were quantified using ImageJ software (A, B, G). Data were normalized to amino acid‐stimulated shScramble transduced cells (A), amino acid‐stimulated control (B), amino acid‐starved control (D, E, F), or empty vector transfected control (G). *P < 0.05, **P < 0.01 vs. amino acid‐stimulated control (A, B, D, E, F); ∆P < 0.05, ∆∆P < 0.01 vs. amino acid‐stimulated shLRS‐transduced cells (A);##P < 0.01 vs. amino acid + BC‐LI‐0186 (B); $P < 0.05 vs. amino acid + Flag‐LRS‐UNE‐L (B); §P < 0.05, §§P < 0.01 vs. amino acid‐starved control (D, E, F); *P < 0.05 vs. empty vector transfected control (G) using GraphPad Prism version 9.0.

Figure 2

Adeno‐associated virus (AAV)‐delivered UNE‐L domain of LRS augments myogenic differentiation in C2C12 cells. C2C12 myoblast cells were differentiated for 48 h, transduced with AAV‐UNE‐L or AAV‐EGFP for 24 h, and then incubated in the differentiation media for another 24 h. (A) The cell lysates were analysed using quantitative real‐time PCR (qRT‐PCR) (n = 3). (B) The cell lysates were subjected to western blot analysis (n = 3). (C) The cells were stained with MHC (green) and DAPI (red). Scale bar = 50 μm. Differentiation index (the number of nuclei in the MHC positive cells/total nuclei), fusion index (the number of nuclei in MHC positive myotubes (>2 myonuclei)/total nuclei), and average myotube size (number of myonuclei/myotube) were quantified (n = 3). Data were normalized to AAV‐EGFP‐transduced cells except myotube size. *P < 0.05 **P < 0.01 vs. AAV‐EGFP transduced cells by GraphPad Prism version 9.0.

Figure 3

Adeno‐associated virus (AAV)‐delivered UNE‐L domain of LRS enhances regenerating muscle fibres in BaCl₂‐induced muscle injury. (A) Tibialis anterior (TA) muscles from 10‐week‐old male mice were injected with BaCl₂ or saline, collected at the indicated days after injury (AI), and subjected to quantitative real‐time PCR (qRT‐PCR) (n = 6). (B) TA muscles from 10‐week‐old mice were transduced with AAV‐EGFP or AAV‐UNE‐L, injected with BaCl₂ 8 weeks after transduction, and isolated 5 days AI (n = 9–10). (C) TA muscles of 10‐week‐old mice were transduced with AAV‐EGFP or AAV‐UNE‐L, isolated 8 weeks after injection, and subjected to qRT‐PCR. (D) Representative haematoxylin and eosin (H&E) images of muscle from (B). Scale bar = 60 μm. (E) The cross‐sectional area (CSA) of all centrally nucleated regenerating myofibres in TA muscles from (B) were measured. (F) TA muscles from (B) were subjected to qRT‐PCR. *P < 0.05 **P < 0.01 vs. AI 0 day (A), or AAV‐EGFP group (C, E, F) by Mann–Whitney U test. All data shown are mean ± SD or representative images. Data were normalized to AI 0 day group (A) or AAV‐EGFP group (C, F).

Figure 4

AAV‐delivered UNE‐L domain of LRS does not affect muscle fibre size of aged TA muscles. (A) Tibialis anterior (TA) muscles from 18‐month‐old male mice were injected with AAV‐EGFP or AAV‐UNE‐L and collected 8 weeks after injection (n = 9–10). (B) TA muscles from (A) were lysed and analysed by quantitative real‐time PCR (qRT‐PCR). (C) Representative haematoxylin and eosin (H&E) images of TA muscles from (A). (D) The cross‐sectional area (CSA) of TA muscles from (A) were measured. (E) The lysates of TA muscles from (A) were analysed by qRT‐PCR. (F) TA muscles from young group (10‐week‐old) and old group (18‐month‐old) male mice were isolated, lysed, and subjected to qRT‐PCR (n = 6). (G, H) Gastrocnemii from young group (10‐week‐old) and old group (18‐month‐old) male mice were isolated. (G) The lysates of gastrocnemii were measured for PLD activity according to the manufacturer's procedure (n = 4–5). (H) The gastrocnemii were lysed and subjected to western blot analysis (n = 6–7). *P < 0.05, **P < 0.01 vs. AAV‐EGFP group (B) or young group (F, G, H) by Mann–Whitney U test. All data shown are mean ± SD or representative images. Data were normalized to AAV‐EGFP group (B, E) or young group (F, H).

Figure 5

Characterization of the MTP or M12‐conjugated LRS‐UNE‐L domain. (A) C2C12 myoblast cells were pretreated with or without the indicated concentration of MTP‐LRS‐UNE‐L or M12‐LRS‐UNE‐L for 24 h and subjected to a CCK‐8 assay (n = 3). (B) MTP‐LRS‐UNE‐L or M12‐LRS‐UNE‐L (1 or 5 μM) was incubated with C2C12 myoblasts for 1 h. The cells were washed with PBS twice and cultured for an additional 6 h. After the cells were fixed and permeabilized, the intracellular level of MTP‐LRS‐UNE‐L or M12‐LRS‐UNE‐L was quantified by flow cytometry with anti‐Flag APC stained cells (n = 7). (C) The cells were treated as in (B), fixed, and stained with anti‐Flag APC without permeabilization of the cell membrane (n = 3). (D) The cells were incubated with 10 μM M12‐LRS‐UNE‐L or LRS‐UNE‐L for 1 h and washed with PBS twice. The cells were stained with anti‐Flag (green) and DAPI (blue) and visualized with a Laser Scanning Confocal Microscope 700. Scale bar = 20 μm. **P < 0.01 vs. IgG control using GraphPad Prism version 9.0.

Figure 6

M12‐LRS‐UNE‐L domain augments muscle regeneration of BaCl₂‐injured TA muscles in a PLD1‐dependent manner. (A, B) Tibialis anterior (TA) muscles of 15‐week‐old male Pld1 ^+/+ (n = 7) and Pld1 ^−/− mice (n = 7) were injected with BaCl₂ and isolated on Day 5 after injury. (A) Representative haematoxylin and eosin (H&E) staining images. (B) Myofibre cross‐sectional area (CSA) was measured using ImageJ software. Scale bar = 60 μm. (C, D) TA muscles of 15‐week‐old male Pld1 ^+/+ and Pld1 ^−/− mice were co‐injected with BaCl₂ and LRS‐UNE‐L or M12‐LRS‐UNE‐L and collected on Day 5 after injury (n = 6). (C) Representative H&E staining images. (D) The CSA of regenerating myofibres was measured. **P < 0.01 compared with Pld1 ^+/+ (A), or Pld1 ^+/+ with LRS‐UNE‐L (B) by Mann–Whitney U test.