MSCs-derived apoptotic extracellular vesicles promote muscle regeneration by inducing Pannexin 1 channel-dependent creatine release by myoblasts

Animals

C57BL/6 J mice were purchased from the Animal Center of the Air Force Medical University. All animal experiments were performed by following protocols approved by the Animal Care Committee of the Air Force Medical University (IRB-REV-2021027). 8-week-old C57BL/6 J mice were used for MSCs isolation, ApoEVs extraction, and the establishment and treatment of CTX injured TA models. All mice were maintained in a specific pathogen-free condition with 12:12-h day/night illumination cycle.

Isolation and characterization of bone marrow-derived MSCs

MSCs were isolated from the tibia and femur of 8-week-old female C57BL/6 J mice according to Huang’s protocol49 and cultured in alpha minimum essential medium (α-MEM) (Gibco, USA) supplemented with 20% fetal bovine serum (Gibco, USA), 2 mmol·L−1 L-glutamine, 100 U·mL−1 penicillin, and 100 g·mL−1 streptomycin (all from Sigma, USA). The MSCs were cultured at 37 °C with a humidified atmosphere of 5% CO2, and the culture medium was changed every 3 days. The MSCs were digested and passaged by using 0.25% trypsin (Gibco, USA) when reached 80%–90% confluence.

The characterization of surface markers on MSCs based on flow cytometry and characterization of multidirectional differentiation ability of MSCs were referred to the previous procedure19. Phycoerythrin (PE)-conjugated anti-mouse CD105, CD90, CD73, and CD45 were all purchased from Biolegend (USA). Alizarin red staining was performed on MSCs after 28 days’ osteogenic induction while Oil Red O staining was performed on MSCs after 14 days’ adipogenic induction. Besides that, MSCs-colonies were stained by 0.2% crystal violet after 14 day’s culture at a low seeding density. Alizarin red, Oil Red O, and crystal violet were all purchased from Sigma (USA).

Isolation and characterization of apoptotic extracellular vesicles (ApoEVs)

The procedure of isolating MSCs-ApoEVs is based on our previous work20. Briefly, 0.5 μmol·L−1 STS with completed culture medium containing EVs-depleted FBS (pre-ultracentrifugation at 100 000 g for 18 h) was used to induce P2 MSCs undergoing apoptosis. After 12 h induction, the supernatants of apoptotic MSCs were collected and prepared for centrifugation. The procedure for isolating MSCs-ApoEVs is using 800 g, 10 min to remove cell debris of apoptotic supernatants and then using 16 000 g, 30 min to obtain MSCs-ApoEVs. The apoptosis-related protein Bcl-2 (CST, 3498), Caspase-3 (CST, 9662), Cleaved-Caspase-3 (CST, 9661), and GAPDH (CST, 2118) were examined of apoptotic MSCs at different STS-induced time by western blot. Morphology identification of MSCs-ApoEVs was measured by SEM (Hitachi, Japan) after fixation, gradient dehydration, and gold spraying. Apoptosis-related phosphatidylserine on the surface of MSCs-ApoEVs was stained by FITC-conjugated Annexin V Apoptosis Detection Kit (BD Biosciences, 556547), and DNA fragments inside MSCs-ApoEVs were stained by Hoechst 33342 (Invitrogen, H21492) according to manufactures’ protocols and detected by laser scanning confocal microscope (Nikon, Japan). EV-enriched protein TSG101 (Abcam, ab125011) and Flotillin-1 (CST, 3253) with apoptosis-related protein caspase-3 (CST, 9662) were examined of MSCs and MSCs-ApoEVs by western blot. The size distribution of MSCs-ApoEVs was measured by Dynamic Light Scattering (DLS) using Zetasizer Nano ZSE (Malvern, UK) according to the manufacturer’s protocol.

The establishment and treatment of cardiotoxin (CTX) induced TA injury

The procedure of establishment of CTX-induced TA injury was referred to the previous work34. Briefly, 50 μL 1 × 10−5 mol·L−1 CTX (Merck, USA) was injected into the left and right TA muscles of C57BL/6 J mice on day 0. After 3 days, 30 μg MSCs-ApoEVs (20 μL) was injected into the left TA muscle while PBS (20 μL) was injected into the right TA muscle for control. The TA muscles were collected and fixed by 4% paraformaldehyde on day 3, day 7, and day 14 for histological analysis. For H&E and Masson staining, 4 μm sections were stained and at least three fields were captured by microscope (Nikon, Japan) for analysis which referred to the previous study10,50. Besides that, Image J software (NIH, USA) was used to calculate the cross-sectional area as well as the number of multinuclear cells of H&E-stained myofiber and the collagen volume area for each field.

Myoblast fusion assay

C2C12 murine myoblasts were purchased from Fuheng Biotechnology (China) and used for myoblast fusion assay. C2C12 myoblasts were maintained in DMEM (Gibco, USA) with 10% FBS for growth and induced for fusion in DMEM supplemented with ITS (Sigma, USA) and 0.1% FBS. For C2C12 fusion assay, C2C12 myoblasts were seeded in CellCarrier Ultra 96-well plates (PerkinElmer, USA) and induced to fusion by changing medium from growth medium to fusion medium when cells reached 70%–80% confluency. Moreover, the fusion medium was replaced every 24 h for a total of 72 h unless specified otherwise.

Z-VAD-FMK (MCE, HY-16658B) was added into the fusion medium on day 1 for apoptosis inhibition, while BB-FCF (MCE, HY-D0915) was added into the fusion medium on each day for Pannexin 1 channel inhibition. C2C12 myoblasts-derived ApoEVs were isolated according to the separation procedure of MSCs-ApoEVs. MSCs-ApoEVs or Myo-ApoEVs were added to the fusion medium to treat C2C12 myoblasts on day 1 and changed to new fusion medium after 24 h. Creatine (Sigma, C3630) was used to rescue the fusion of C2C12 myoblasts by being added into the fusion medium each day.

The analysis of myoblasts fusion

For measuring fusion, C2C12 myoblasts were washed and fixed with 4% paraformaldehyde for 20 min and then permeabilized in 0.1% Triton X-100 for 10 min. After blocking with goat serum (Boster, China), C2C12 myoblasts were incubated with mouse anti-myosin antibody (R&D, MAB4470) at 1:250 dilution overnight at 4 °C and then incubated with Alexa Fluor 594 labeled secondary antibody (Yeasen, 33212ES60) at 1:500 dilution for 1 h at room temperature and stained with Hoechst 33342 (Invitrogen, H21492) for 5 min at room temperature. The fluorescently stained C2C12 myoblasts were captured by Operetta CLS (PerkinElmer, USA) for analysis. The fusion index and the number of multinuclear myotubes were quantitated based on myosin+ cells per field by Image J software referred to the previous study34.

Phagocytosis experiment in vivo and in vitro

For in vivo phagocytosis experiment, CTX-induced TA injury was established as mentioned above while MSCs-ApoEVs were pre-stained by PKH26 (Sigma, USA) according to the manufacturer’s protocol. The PKH26 labeled MSCs-ApoEVs were injected into left TA muscles 3 days after TA injury models were established. The TA muscles were collected and fixed, dehydrated, and embedded in optimal cutting temperature compound (Leica, Germany) 24 h after PKH26 labeled MSCs-ApoEVs injection. For immunofluorescence staining, 10 μm sections were incubated with mouse anti-myosin antibody (R&D, MAB4470) at 1:250 dilution overnight at 4 °C and then incubated with Alexa Fluor 488 labeled secondary antibody (Yeasen, 33112ES60) at 1:500 dilution for 2 h at room temperature and finally stained with Hoechst 33342 (Invitrogen, H21492) for 10 min at room temperature, all operations are carried out in dark environments. The fluorescence images were captured by laser scanning confocal microscope (Nikon, Japan).

For in vitro phagocytosis experiment, PKH26 labeled MSCs-ApoEVs were added to C2C12 myoblasts and incubated for 3 h. After that, C2C12 myoblasts were fixed, permeated, and then stained by 488-conjugated phalloidin (AAT Bioquest, 23115) for 1 h and Hoechst 33342 for 5 min at room temperature. The fluorescence images were captured by laser scanning confocal microscope (Nikon, Japan).

Observation of ApoEVs generated during myoblasts fusion

To dynamic observe the ApoEVs generated during C2C12 myoblasts fusion, C2C12 cells were seeded in CellCarrier Ultra 96-well plates and cultured in the growth medium until cells reached 70%–80% confluency. After that, C2C12 myoblasts were changed into the fusion medium with 1 μmol·L−1 TO-PRO-3 (Thermo, T3605) to label ApoEVs through activated Pannexin 1 channel25. The time-lapse images were captured by Operetta CLS every 15 min at 37 °C and 5% CO2. Besides that, to measure the diameter of ApoEVs generated during C2C12 myoblasts fusion, the supernatants were collected from C2C12 myoblasts cultured in growth or fusion medium every day when changing the medium. The diameter of particles in supernatants of different groups was measured by DLS. The myoblasts-derived ApoEVs were isolated from the supernatants of C2C12 myoblasts in the fusion medium according to the protocol for obtaining MSCs-derived ApoEVs and observed by SEM (Hitachi, Japan). Moreover, EV-enriched protein TSG101 (Abcam, ab125011) and Flotillin-1 (CST, 3253) with apoptosis-related protein caspase-3 (CST, 9662) were examined of MSCs and MSCs-ApoEVs by western blot.

Flow cytometric analysis for apoptosis

PE-Annexin V Apoptosis Detection Kit I (BD Biosciences, 559763) was used to measure the apoptosis ratio of C2C12 myoblasts in different groups according to the manufacturer’s protocol. Briefly, C2C12 myoblasts from different groups were digested, collected, washed, and finally resuspended in the annexin V-binding buffer and stained with PE-conjugated annexin V for 15 min. The apoptosis ratio was assessed by flow cytometry (Beckman Coulter, USA) based on the annexin V-stained C2C12 myoblasts.

The detection of creatine release

Untargeted metabolomics analysis service was provided by QLBio (China). In brief, the supernatants were collected from normal and apoptotic C2C12 myoblasts and precipitated in methanol for analysis. Metabolite analysis was conducted by Rapid Separation LC (Thermo, USA) and Q Exactive (Thermo, USA). Besides that, the mass spectrometry data were processed based on METLIN Database, Human Metabolome Database, and ChemSpider Database.

Creatine Assay Kit (Abcam, ab65339) was used to detect the relative expression of creatine in the supernatants of C2C12 myoblasts according to the manufacturer’s protocol in the different groups. The supernatants of C2C12 myoblasts were collected from C2C12 myoblasts cultured in different determined groups and centrifuged at 1 000 g, 4 °C to remove cell debris. The fluorometric detection of creatine from different groups was measured by EnSight Multimode Plate Reader (PerkinElmer, USA).

The analysis of Pannexin 1 channel activation

The activation of Pannexin 1 channel of the total protein lysis as well as the membrane protein lysis of normal C2C12 myoblasts, apoptotic C2C12 myoblasts, and Myo-ApoEVs was measured by Western Blot. The total proteins from C2C12 myoblasts were extracted by RIPA buffer (Beyotime, China) with protease inhibitor (MCE, USA) while the membrane proteins were extracted by Membrane Protein Extraction Kit (Thermo, 89842) following the manufacturer’s protocol. Anti-mouse Pannexin 1 antibody was purchased from CST (91137). The cleaved Pannexin 1 protein fragment was considered to be a marker of irreversible activation of the Pannexin 1 channel51.

Statistical analysis

All statistical data were represented as the mean ± standard deviation (SD). Comparisons of two groups were performed by two-tailed Student’s t-tests and multiple groups were performed by one-way ANOVA with Tukey correction, P < 0.05 was considered statistically significant. All statistical analyses were performed by SPSS 23.0. All statistical graphs were drawn by GraphPad Prism 8.0.2.

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