Extracellular vesicle-derived miRNAs improve stem cell-based therapeutic approaches in muscle wasting conditions

Abstract

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells.

Keywords: aging; extracellular vesicle; hypertrophy; miRNA; muscular dystrophy; skeletal muscle.

Figure 1

Characterization of extracellular vesicles derived from the plasma of different mouse models. (A) NanoSight analysis of the obtained extracellular vesicles (EVs), representative graphs of particle sizes in wild type (WT)-, hypertrophic (Hyp)-, dystrophic (Dys)-, and aged-derived EVs are shown. (B) Average size (upper graph) and concentration (lower graph) of EVs. One-way ANOVA was used and results are displayed as mean ± s.e.m (n=4 samples/group, EVs from 2-3 mice/sample; *p < 0.05). (C) Transmission electron microscopy analysis of EVs. Scale bars: 100 nm. (D) Western blot analysis of common positive (CD9, CD63, CD81) and negative (GM130, HSP90, Calnexin) markers for EVs and/or exosomes.

Figure 2

Effect of extracellular vesicle treatment on myogenic differentiation of progenitor cells. (A) Red fluorescent protein-marked extracellular vesicle (EV) uptake (red) from mouse myoblast cell line C2C12 after 4 hours of EV treatment. Representative confocal images of cells treated with wild type (WT)- and hypertrophic (Hyp)-derived EV samples are shown. Hoechst was counterstained in blue. (B) Quantification of EV uptake in C2C12 myoblasts after 4 hours of EV treatment (n=4 per group). (C) Myogenic differentiation of C2C12 cells incubated with WT-, Hyp-, dystrophic (Dys)-, and aged-derived EVs. Immunofluorescence analysis for myosin heavy chain (MyHC) (red). Hoechst was counterstained in blue. (D) Fusion index of differentiated C2C12 cells at days 3 and 5 of myogenic differentiation (n = 5). (E) Myogenic differentiation of human mesoangioblasts (hMABs) treated with EVs. Immunofluorescence analysis for MyHC (red) and human lamin A/C (green). (F) Fusion index of differentiated hMABs at day 12 (n = 3). (G) Example of a western blot for MyHC protein level at day 12 of myogenic differentiation. GAPDH was used as a loading control. (H) Quantification of MyHC western blot. For a, c, and e, scale bars: 50 µm. For b, d, f, and h one-way ANOVA was used and results are displayed as mean ± s.e.m (n=3-5, *p < 0.05).

Figure 3

miR-1 and miR-208a increase the myogenic differentiation of human mesoangioblasts in vitro. (A) TaqMan qPCR analysis for the quantification of the selected microRNAs (miRNAs) miR-1, miR-133a, miR-206, and miR-208a in wild type (WT)-, hypertrophic (Hyp)-, dystrophic (Dys)-, and aged-derived extracellular vesicles (EVs). One-way ANOVA was used and results are displayed as mean ± s.e.m (n = 5, *p < 0.05). (B) Fusion index of differentiated human mesoangioblasts (hMABs) treated with combinations of miRNA mimics and antagomirs for miR-1, miR-133a, miR-206, and miR-208a (mimics are denoted by a + sign, antagomirs by a – sign). One-way ANOVA was used and results are displayed as mean ± s.e.m (n = 4, *p < 0.05). (C) Representative images of immunofluorescence analysis for myosin heavy chain (MyHC) (red) and human lamin A/C (green) in differentiated hMABs after miRNA treatments with miRNA mimics and antagomirs for miR-1 and miR-208. Hoechst was counterstained in blue. Scale bars: 100 µm. (D–F) Differentiation of hMABs treated with miR-1 and miR-208 mimics resulted in higher levels of miR-1 and miR-208 (D) and an increase in mRNA expression (E) and protein level (F) of myogenic markers. Two-tailed Student’s t-test was used and results are displayed as mean ± s.e.m (n = 5, *p < 0.05).

Figure 4

Cell transplantation of microRNA-treated and non-treated human mesoangioblasts in aged mice after acute injury. (A) Bioluminescence of human mesoangioblasts (hMABs) cultured in vitro transduced with the enhanced green fluorescent protein (eGFP)/firefly luciferase vector (left) and immunofluorescence analysis for myosin heavy chain (MyHC) (red) and GFP (green) in differentiated hMABs with and without transduction. Hoechst was counterstained in blue. Scale bars: 50 µm. (B) In vivo bioluminescence imaging at days 7, 14, and 21 in injected mice (left) and signal quantification (right). Left limbs received microRNA-treated MABs, right limbs received non-treated MABs. (C) Weight of hindlimb muscles after mice were sacrificed at day 21. TA: Tibialis anterior, GM: Gastrocnemius. (D) Functional analysis of extensor digitorum longus via evaluation of tetanic force. One-way ANOVA was used and results are displayed as mean ± s.e.m (n = 5-6 muscles/group, *p < 0.05).

Figure 5

Histological analysis of aged muscles after acute injury and cell transplantation of microRNA-treated and non-treated human mesoangioblasts. (A) Localization of human mesoangioblasts (hMABs) in the injected skeletal muscle at day 21 after cell transplantation. Immunofluorescence analysis for laminin (red) and lamin A/C (green). Hoechst was counterstained in blue. Scale bars: 25 µm. (B) Representative images of haematoxylin and eosin staining in transplanted muscles with microRNA-treated hMABs, non-treated hMABs, and cardiotoxin control at day 21 (d21). Scale bars: 100 µm. (C) Quantification of fiber size (mean cross-sectional area (CSA)) at day 21. (D) Quantitative frequency distribution analysis of the cross-sectional area of fibers at day 21. (E) Representative images of haematoxylin and eosin staining in transplanted muscles with microRNA-treated hMABs, non-treated hMABs, and cardiotoxin control at day 35 (d35). Scale bars: 100 µm. (G) Quantification of fiber size (mean CSA) at day 35. (F) Percentage of centronucleated fibers at day 35. (H, I) Masson’s tricrome staining of injected hindlimb muscles at day 21 (d21) and quantification of area of fibrosis. One-way ANOVA was used, and results are displayed as mean ± s.e.m (*p < 0.05, n = 4-5 muscles/group).