Muscle-derived miR-34a increases with age in circulating extracellular vesicles and induces senescence of bone marrow stem cells

Abstract

Extracellular vesicles (EVs) are known to play important roles in cell-cell communication. Here we investigated the role of muscle-derived EVs and their microRNAs in the loss of bone stem cell populations with age. Aging in male and female C57BL6 mice was associated with a significant increase in expression of the senescence-associated microRNA miR-34a-5p (miR-34a) in skeletal muscle and in serum -derived EVs. Muscle-derived, alpha-sarcoglycan positive, EVs isolated from serum samples also showed a significant increase in miR-34a with age. EVs were isolated from conditioned medium of C2C12 mouse myoblasts and primary human myotubes after cells were treated with hydrogen peroxide to simulate oxidative stress. These EVs were shown to have elevated levels of miR-34a, and these EVs decreased viability of bone marrow mesenchymal (stromal) cells (BMSCs) and increased BMSC senescence. A lentiviral vector system was used to overexpress miR-34a in C2C12 cells, and EVs isolated from these transfected cells were observed to home to bone in vivo and to induce senescence and decrease Sirt1 expression of primary bone marrow cells ex vivo. These findings suggest that aged skeletal muscle is a potential source of circulating, senescence-associated EVs that may directly impact stem cell populations in tissues such as bone via their microRNA cargo.

Keywords: exosomes; muscle-bone crosstalk; osteoporosis; sarcopenia; senescence.

Figure 1

miR-34a is increased in mouse skeletal muscle and in serum EVs with age. (A) Expression levels of miR-34a, miR-155, miR-183, and miR-141 in skeletal muscle of young and aged male and female mice. (B) Expression levels of miR-34a, miR-155, miR-183, and miR-141 in serum EVs from young and aged male and female mice. *P<.05, **P<.01, ***P<.001.

Figure 2

miR-34a is increased with age in muscle-derived, alpha-sarcoglycan positive (SGCA+) EVs. (A) Representative particle distribution histograms from nanoparticle tracking analysis showing similar particle sizes between serum EVs and SGCA+ serum EVs. (B) Particle concentrations of SGCA+ EVs are significantly lower than total serum EVs, and SGCA+ EVs in bone marrow are significantly less abundant than SGCA+ EVs in serum. (C) SGCA+ EVs show a significant increase in miR-34a expression with age.

Figure 3

Hydrogen peroxide increases miR-34a in EVs secreted by C2C12 myoblasts and human myotubes, and these EVs can reduce bone stem cell (BMSC) viability and increase senescence. (A) EVs isolated from C2C12 cells treated with hydrogen peroxide show a significant increase in miR-34a. (B) EVs isolated from human myotubes treated with hydrogen peroxide show a significant increase in miR-34a. (C) BMSC viability indicated by MTT assay is significantly reduced after treatment with EVs isolated from these C2C12 cells exposed to hydrogen peroxide. (D) BMSC senescence measured by beta-galactosidase (β-gal) assay is significantly increased after treatment with EVs isolated from these C2C12 cells exposed to hydrogen peroxide. *P<.05, **P<.01.

Figure 4

C2C12 cells overexpressing miR-34a secrete EVs with elevated levels of miR34a. (A) Confocal images of C2C12 cells transfected with a lentivirus overexpressing miR-34a. The virus contains a GFP reporter under a constitutive CMV promoter. Images show GFP expression in transfected cells. Blue staining represents nuclear DAPI staining. Scale bar = 20 µm. (B) Analysis of miR-34a expression in EVs from transfected and non-transfected cells shows a three-fold increase in miR-34a in EVs isolated from conditioned medium of transfected cells.

Figure 5

EVs from C2C12 cells overexpressing miR-34a reduce BMSC viability and increase senescence. (A) Confocal images of BMSCs treated with EVs isolated from conditioned medium of C2C12 cells overexpressing miR-34a. EVs are unlabeled (control, bottom row) or labeled with the membrane dye PKH67 (top row). Images show abundant EVs in cytoplasm of BMSCs. Blue staining represents nuclear DAPI staining. Scale bar = 20 µm. (B) BMSC viability indicated by MTT assay is significantly reduced after treatment with EVs isolated from C2C12 cells overexpressing miR-34a. (C) BMSC senescence measured by beta-galactosidase (β-gal) assay is significantly increased after treatment with EVs isolated from these C2C12 cells overexpressing miR-34a. *P<.05, **P<.01.

Figure 6

EVs from C2C12 cells overexpressing miR-34a home to bone marrow in vivo and reduce Sirt1 expression ex vivo. (A) Mice were injected via tail vein with DiR dye alone (VEH) or EVs from C2C12 cells overexpressing miR-34a labeled with DiR (EVs + DiR) and imaged with AmiX imaging. Mice receiving labeled EVs show high image intensity in the metaphyseal regions of long bones. (B) Bone marrow cells flushed from untreated mice and cultured in the presence of EVs from miR-34a overexpression cells show reduced Sirt1 expression compared to cells cultured with EVs from control C2C12 cells. (C) Bone marrow cells flushed from untreated mice and cultured in the presence of EVs from miR-34a overexpression cells show reduced Sirt1 protein compared to cells cultured with EVs from control C2C12 cells. Top image is from protein of adherent cells, graph includes pooled data from both adherent and non-adherent cells. *P<.05, **P<.01. (D) Working model summarizing changes in muscle and bone with age, and the role of EV-derived miR-34a in muscle and bone senescence. ROS = reactive oxygen species.

Figure 7

Western blot for exosome markers and experimental design for in vivo treatment. (A) SGCA+ EVs isolated from serum of young (Y) and old (O) mice as well as from conditioned medium of normal C2C12 cells and cells overexpressing miR-34a are positive for the exosome markers CD63 and TSG101. (B) Timeline, EV doses, and outcome measures for in vitro BMSC treatments.