Characterization of the Skeletal Muscle Secretome Reveals a Role for Extracellular Vesicles and IL1α/IL1β in Restricting Fibro/Adipogenic Progenitor Adipogenesis

Figure 1. Analysis of cytokine expression during muscle regeneration. (A) The diagram illustrates the experimental procedure. Mononuclear cell fractions from uninjured muscles (UN) and at four different times after damage (CTX 1d, CTX 3d, CTX 5d, and CTX 12d) were isolated and lysed to determine the abundance of 120 cytokines by a multiplex ELISA array approach. Created with BioRender.com (B) PCA analysis using the entire dataset. Each dot represents a single replicate of three independent experiments. (C) Secreted protein expression patterns were clustered using a k-mean algorithm. In each graph the thin lines represent the single protein patterns while the thick one represents the average of all the expression patterns within the cluster. The shaded area indicates the variance within the cluster. (D) Heat maps of clusters illustrating the variation in concentration of all cytokines modulated during muscle regeneration. Each row represents a cytokine while the five columns are, from left to right, the five conditions UN, CTX 1d, CTX 3d, CTX 5d, and CTX 12d. Statistical significance was assessed by a One-Way ANOVA test: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2. Inferred cytokine–receptor interactions in FAPs. (A) Representation of the experimental workflow. Three days after cardiotoxin injury, muscle–resident cell populations were sorted using MACS technology. The obtained cell fractions were immediately used to purify RNA. Created with BioRender.com (B) PCA analysis using the entire dataset and (C) only the mRNA coding secreted proteins. Each dot represents a replica of three independent experiments. (D) Schematic representation of the inferred cytokine–receptor pair resulting from the analysis of Multiplex ELISA and RNAseq data. Only receptors upregulated in FAPs, in comparison with the other cell types, are shown.

Figure 3. Modulation of FAPs adipogenesis and proliferation. (A–E) FAPs isolated 3 days after injury with cardiotoxin were plated and treated with 12 cytokines. (A) Experimental design of cell treatment. GM = Growth Medium. AIM = Adipogenic Induction Medium. MM = Maintenance Medium. Created with BioRender.com (B) Representative images showing anti-SMA immunofluorescence (green), ORO-staining (red) and nuclei staining with Hoechst 33,342 (blue). Scale Bar = 100 μm. (C,D) Quantification of adipogenic differentiation, represented as percentage of ORO-positive cells and ORO-positive area. (E) Nuclei count at the end of adipogenic differentiation. (F–J) FAPs isolated from 3 days injured mice were plated and treated with 1 pg, 10 pg, 100 pg, 1 ng, and 10 ng of IL1α or IL1β. (E) Representative anti-PPARγ immunofluorescence (green), ORO-staining (red), and nuclei staining with Hoechst 33,342 (blue). Scale Bar = 100 μm. (F–I) Quantification of adipogenic differentiation, represented as percentage of PPARγ positive nuclei and ORO-positive area, after treatment with IL1α and IL1β. (K) Experimental design of cell treatment. GM = Growth Medium. Created with BioRender.com (L–N) FAPs isolated from 3 days…

Figure 4. Modulation of FAPs Fibrogenesis. (A–E) FAPs isolated 3 days after injury with cardiotoxin were plated and treated with 12 cytokines to influence TGFb-induced fibrogenesis. (A) Experimental design of cell treatment. GM = Growth Medium. Created with BioRender.com (A) Representative images showing anti-SMA immunofluorescence (green) and nuclei staining with Hoechst 33,342 (blue). Scale Bar = 100 μm. (B,C) Quantification of fibrogenic differentiation, represented as SMA-positive area (pixel2). (D) Nuclei count at the end of in vitro differentiation. (E–G) FAPs isolated from 3 days injured mice were plated and treated with TGFβ or IL1α and IL1β. (E) Experimental design of cell treatment. GM = Growth Medium. Created with BioRender.com F) Representative anti-SMA immunofluorescence (green) and nuclei staining with Hoechst 33,342 (blue). Scale Bar = 100 μm. (G) Quantification of fibrogenic differentiation, represented SMA-positive area (pixel2). Statistical analysis was performed by One-Way Anova test: * p < 0.5; ** p < 0.01; *** p < 0.001.

Figure 5. Muscle-derived EVs characterization. (A) Size distribution analysis of muscle-derived EVs. Size ranges 0–100 nm, 100–160 nm, 160–200 nm, 200–240 nm, 240–300 nm, 300–500 nm, and 500–900 nm are based on Gigamix fluorescent beads. (B) Overlay of UN, 1d, 3d, and 5d EVs size distributions. For each sample 100,000 EVs are displayed. (C) Quantitation of EVs labelled with Syto RNASelect and (D) calcein, represented as percentage of positive EVs. (E) Protein quantification of the EVs sample with the Lowry method normalized over the number of lysed EVs. (F) Representative western blot of proteins extracted from UN and 3d EVs. Ponceau staining was used to show the protein loading. (G) Densitometric quantification of bands in panel F. Data are presented as means ± SD of three independent experiments. Statistical analysis was performed by a One-Way Anova test. * p < 0.05; ** p < 0.01; **** p < 0.000.

Figure 6. Interaction between EVs and mononuclear muscle cell populations. (A) Representative immunofluorescence of SCs and FAPs incubated with labelled EVs. Syto RNASelect signal and nuclei staining with Hoechst 33,342 are shown, respectively, in green and blue. (B) Percentage of CD45+ cells derived from uninjured muscles and (C) Signal intensity in cells incubated with EVs previously labelled with Syto RNASelect. (D) Percentage of CD45+ cells derived from 3d injured muscles and (E) Signal intensity in cells incubated with EVs previously labelled with Syto RNASelect. Data are presented as means ± SD of three independent experiments. Statistical analysis was performed by a One-Way Anova test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 7. Muscle-derived EVs affect FAPs differentiation. (A,C,D). FAPs isolated from 3d injured muscles were plated and treated with EVs for 72 h. (A) Representative anti-Ki67 immunofluorescence (red) and nuclei staining with Hoechst 33,342 (blue). Scale Bar = 100 μm. (C) Quantification of Ki67 expression in FAPs treated with vesicles. (D) Nuclei count represented as Fold Change compared to the untreated control. (B,E,F) FAPs isolated from 3d injured muscles were plated induced to differentiate and treated with EVs. (D) Representative anti-PPARγ immunofluorescence (green), ORO-staining (red) and nuclei staining with Hoechst 33,342 (blue). Scale Bar = 100 μm. (E) Percentage of PPARγ-positive nuclei. (F) Ratio between the total stained area with ORO and nuclei number. Data are presented as means ± SD of three independent experiments. Statistical analysis was performed by a One-Way Anova test: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 8. Muscle-derived EVs affect SCs proliferation and differentiation. (A,C,D) SCs isolated from uninjured muscles were plated for 2 days and then treated with EVs for 24 hours in GM. (A) Representative anti-Ki67 immunofluorescence (red) and nuclei staining with Hoechst 33,342 (blue). Scale Bar = 100 μm. (C) Quantification of Ki67 expression in SCs treated with vesicles. (D) Nuclei count represented as Fold Change compared to the untreated control. (B,E) SCs isolated from uninjured muscles were plated for 3 days and then treated for 24 h in DM. (B) Representative anti-MYOG immunofluorescence (red) and nuclei staining with Hoechst 33,342 (blue). Scale Bar = 100 μm. (E) Percentage of MYOG-positive nuclei. Data are presented as means ± SD of three independent experiments. Statistical analysis was performed by a One-Way Anova test: * p < 0.05; ** p < 0.01; *** p < 0.001.