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. 2019 Jan 18;27(3):571–583. doi: 10.1016/j.ymthe.2019.01.008

Figure 3.

Figure 3

Exo/miR-29 Attenuated UUO-Induced Muscle Loss

Mice were injected with exosomes carrying either miR-control or miR-29. (A) Mice were injected in the left TA with Exo/miR29 labeled with 1 μmol/L fluorescent lipophilic tracer DiR at the same time as UUO surgery. The injection was repeated once per week. The fluorescence was assessed in muscle at 14 days after injection. Panels from left to right: sham operated with no exosome injection, UUO mouse at 14 days (twice injection) after DiR∼Exo/miR29 injection, and sham operated with DiR∼exosome injection. In each pair, the left TA received the Exo/miR29 injection and the right did not. Fluorescent images were acquired using a Bruker Small Animal Optical Imaging System (In-Vivo Xtreme II). The white color indicates fluorescence level over maximal measurement limits. The picture with all organs compared is shown in Figure S1. The bar graph compares fluorescence intensity from each muscle (bars: mean ± SE; n = 3/group; *p < 0.05 versus sham injected muscle; #p < 0.05 versus sham non-injected muscle). (B) Total RNA was isolated from TA of sham plus Exo/miR-ctrl (sham), sham plus Exo/miR29 (miR29), UUO plus Exo/miR-ctrl (UUO), and UUO plus Exo/miR-29 mice (UUO/29). The expressions of miR-29a-3p, miR-29b-3p, and miR-29c-3p were assayed by real-time qPCR. The bar graph shows expression levels of the three miRs in each treatment group compared with levels in sham mice (represented by a line at 1-fold). Results are normalized to U6 (bars: mean ± SE; n = 9/group; *p < 0.05 versus sham; #p < 0.05 versus UUO). (C) The representative cross-sectional area of TA of sham plus Exo/miR-ctrl (sham), UUO plus Exo/miR-ctrl (UUO), and UUO plus Exo/miR-29 mice (UUO/29). Cryosections of TA were immunostained with anti-laminin antibody. The bar graph shows the frequency distribution of fiber cross-sectional areas in sham (blue), UUO (orange), and UUO/29 (green) mice (data are mean ± SE; n = 6/group; *p < 0.05 versus sham; #p < 0.05 versus UUO). (D) Shown are representative western blots of the muscle regeneration- and atrophy-related proteins myoD, myogenin, eMyHC, YY1, PTEN, MuRF1, and atrogin1 in muscle lysates from the different treatment groups of mice. All blots were also probed for GAPDH, and all protein band densities have been normalized to their corresponding GAPDH loading control. The bar graph shows the fold change of each protein band compared with levels in sham mice (represented by a line at 1-fold) (bars: mean ± SE; n = 9/group; *p < 0.05 versus sham; #p < 0.05 versus UUO).