IL-6 induced STAT3 signalling is associated with the proliferation of human muscle satellite cells following acute muscle damage

Abstract

Background: Although the satellite cell (SC) is a key regulator of muscle growth during development and muscle adaptation following exercise, the regulation of human muscle SC function remains largely unexplored. STAT3 signalling mediated via interleukin-6 (IL-6) has recently come to the forefront as a potential regulator of SC proliferation. The early response of the SC population in human muscle to muscle-lengthening contractions (MLC) as mediated by STAT3 has not been studied.

Methodology/principal findings: Twelve male subjects (21±2 y; 83±12 kg) performed 300 maximal MLC of the quadriceps femoris at 180°•s(-1) over a 55° range of motion with muscle samples (vastus lateralis) and blood samples (antecubital vein) taken prior to exercise (PRE), 1 hour (T1), 3 hours (T3) and 24 hours (T24) post-exercise. Cytoplasmic and nuclear fractions of muscle biopsies were purified and analyzed for total and phosphorylated STAT3 (p-STAT3) by western blot. p-STAT3 was detected in cytoplasmic fractions across the time course peaking at T24 (p<0.01 vs. PRE). Nuclear total and p-STAT3 were not detected at appreciable levels. However, immunohistochemical analysis revealed a progressive increase in the proportion of SCs expressing p-STAT3 with ∼60% of all SCs positive for p-STAT3 at T24 (p<0.001 vs. PRE). Additionally, cMyc, a STAT3 downstream gene, was significantly up-regulated in SCs at T24 versus PRE (p<0.05). Whole muscle mRNA analysis revealed induction of the STAT3 target genes IL-6, SOCS3, cMyc (peaking at T3, p<0.05), IL-6Rα and GP130 (peaking at T24, p<0.05). In addition, Myf5 mRNA was up-regulated at T24 (p<0.05) with no appreciable change in MRF4 mRNA.

Conclusions/significant findings: We demonstrate that IL-6 induction of STAT3 signaling occurred exclusively in the nuclei of SCs in response to MLC. An increase in the number of cMyc+ SCs indicated that human SCs were induced to proliferate under the control of STAT3 signaling.

Figure 1. Serum measures and Pax7 positivity.

(1a) Average serum CK response in U/L. (1b) Average serum IL-6 response in pg/mL; note the similar serum responses between IL-6 and CK. (1c) Myf5 mRNA expression relative to GAPDH, expressed as fold change from PRE. (1d) Pax7⁺ cells as a percentage of total myonuclei over the time-course. (1e) Representative image at 40× magnification of a Pax7/Laminin stain with Pax7 in red, Laminin in green and DAPI in blue. Values are reported as mean ± S.E.M. *p<0.05 vs. PRE; † p<0.05 vs. T1, ‡ vs. T3.

Figure 2. IL-6/Pax7 co-localization.

(2a) Representative merged image of a muscle cross-section at 40× magnification stained for IL-6 in green, Pax7 in red, and nuclei in blue being DAPI⁺. The scale bar is 50 µm. (2b–e) 100× magnification image of the inset box in 2a. Scale bar is 10 µm (2b) DAPI⁺ nuclei. (2c) Pax7⁺ nuclei. Note only one of the nuclei is Pax7 positive showing the specificity of the Pax7 stain (2d) IL-6⁺ nuclei. Note that both nuclei are IL-6 positive while there is less intense IL-6 staining in the fiber itself. (2e) Merged image showing the co-localization of DAPI, IL-6 and Pax7. (2f) IL-6⁺ cells as a percentage of Pax7⁺ cells. Values are reported as mean ± S.E.M. *p<0.05 vs. PRE.

Figure 3. Nuclear and cytoplasmic expression of STAT3 and JAk2.

(3a) Ratio of phosphorylated to total STAT3 protein in the cytoplasmic fraction. (3b) Representative images of p-STAT3 and t-STAT3 in both the cytoplasmic and the nuclear fraction. (3c) Representative images of p-JAK2 and t-JAK2 in the cytoplasmic fraction. (3d) Ratio of phosphorylated to total JAK2 in the cytoplasmic fraction. Values are reported as mean ± S.E.M. *p<0.05 vs. PRE.

Figure 4. p-STAT3⁺/Pax7⁺ cells.

(4a) Representative merged image of PRE at 40× magnification with inset box showing (a-i) DAPI⁺ nuclei, (a-ii) Pax7⁺ nuclei, (a-iii) no p-STAT3 stained nuclei and (c-iiii) a merged image. (4b) Representatvie merged image of T3 at 40× magnification with inset box showing (b-i) DAPI⁺ nuclei, (b-ii) Pax7⁺ nuclei, (b-iii) p-STAT3⁺ nuclei and (b-iiii) a merged image showing co-localiztion. Note that punctate p-STAT3 is not present at PRE but co-localizes with Pax7 at T3 and that there is an increase in diffuse fiber staining that occurs from PRE to T3. (4c) Percentage of p-STAT3⁺ SC as quantified over the time course peaking at T24. Values are reported as mean ± S.E.M. *p<0.05 vs. PRE.

Figure 5. STAT3 downstream genes.

(5a) IL-6, (5b) IL-6Rα, (5c) GP130 and (5d) SOCS3 mRNA expression relative to GAPDH, expressed as fold change from PRE. Values are reported as mean ± S.E.M. *p<0.05 vs. PRE; † p<0.05 vs. T1, ‡ vs. T3.

Figure 6. cMyc⁺/Pax7⁺ quantification at Pre and T24.

(6a) cMyc mRNA relative to GAPDH, expression represented as fold changes from PRE. (6b) cMyc⁺ SC as a percentage of Pax7⁺ cells between Pre and T24, n = 7. (6c & 6d) Serial sections of the same muscle cross-section with higher magnification (100×) boxes showing the same nuclei in both images. (6c) Representative image at 20× magnification of a merged Pax7/Laminin costain where (c-i) Pax7 is in green, (c-ii) DAPI⁺ nuclei are in blue and (c-iii) laminin in red. Note the two Pax7⁺ nuclei which have been highlighted to show their individual Pax7 positivity, DAPI staining and location within the laminin boarder. These nuclei are highlighted again in (6d) where the same cells have been stained colourometrically for cMyc. (d-i) A magnified image of the inset box showing the brown cMyc staining co-localized to the nuclear marker hematoxalin The brown cMyc positive cells are clearly within the visible fiber boarder. *p<0.05 vs. PRE.