STAT3 signaling controls satellite cell expansion and skeletal muscle repair

Abstract

The progressive loss of muscle regenerative capacity with age or disease results in part from a decline in the number and function of satellite cells, the direct cellular contributors to muscle repair. However, little is known about the molecular effectors underlying satellite cell impairment and depletion. Elevated levels of inflammatory cytokines, including interleukin-6 (IL-6), are associated with both age-related and muscle-wasting conditions. The levels of STAT3, a downstream effector of IL-6, are also elevated with muscle wasting, and STAT3 has been implicated in the regulation of self-renewal and stem cell fate in several tissues. Here we show that IL-6-activated Stat3 signaling regulates satellite cell behavior, promoting myogenic lineage progression through myogenic differentiation 1 (Myod1) regulation. Conditional ablation of Stat3 in Pax7-expressing satellite cells resulted in their increased expansion during regeneration, but compromised myogenic differentiation prevented the contribution of these cells to regenerating myofibers. In contrast, transient Stat3 inhibition promoted satellite cell expansion and enhanced tissue repair in both aged and dystrophic muscle. The effects of STAT3 inhibition on cell fate and proliferation were conserved in human myoblasts. The results of this study indicate that pharmacological manipulation of STAT3 activity can be used to counteract the functional exhaustion of satellite cells in pathological conditions, thereby maintaining the endogenous regenerative response and ameliorating muscle-wasting diseases.

Figure 1

Stat3 promotes myogenic lineage progression in cultured satellite cells. (a) Left, representative images of satellite cells isolated by FACS, cultured in vitro and analyzed 5 d after isolation (green, pSTAT3; red, Myod1; blue, nuclei). Scale bar, 50 µm. Right, quantification of the percentage of satellite cells positive for pStat3 and Myod1 (n = 3). (b) Left, representative images of Myod1⁺ satellite cells treated with shStat3 lentivirus or control shRNA (shCtrl) 4 d after infection. Scale bar, 50 µm. Right, quantification of the percentage of Myod1⁺ satellite cells after lentiviral infection (n = 3). (c) Left, representative images of myogenin (Myog)⁺ satellite cells infected with shStat3 or shCtrl and cultured in growth medium for 72 h. Scale bar, 50 µm.. Right, quantification of the percentage of Myog⁺ cells after lentiviral infection (n = 3). (d) Left, representative images of Pax7⁺EdU⁺ satellite cells treated with shStat3 or shCtrl lentiviruses 4 d after infection. Scale bar, 50 µm. Right, quantification of the percentage of EdU⁺ satellite cell after lentiviral infection (n = 5). (e) Quantification of Myod1 mRNA levels in satellite cells infected with shStat3 or shCtrl lentivirus and maintained in culture in growth medium in either the absence or presence of 100 ng ml⁻¹ IL-6 for 96 h (n = 4). (f) Left, representative images of differentiated myosin heavy chain (MyHC)⁺ myotubes (green, MyHC; blue, nuclei) in satellite cells that were infected with shStat3 or shCtrl lentivirus and analyzed 72 h after terminal differentiation was induced. Scale bar, 50 µm. Right, quantification of differentiation index in satellite cells after shStat3 treatment (n = 3). All data are represented as the average ± s.e.m. Student’s t test was used for all statistical analyses (***P < 0.001, **P < 0.01, *P < 0.05) except in e, where one-way analysis of variance (ANOVA) with Tukey’s post test was used.

Figure 2

Stat3 gene deletion enhances satellite cell expansion after skeletal muscle injury. (a) Schematic representation of Tmx treatment in male and female 2-month-old Pax7-CreER; Stat3^flox/flox and Pax7-CreER; Stat3^+/+ mice. EdU was administered daily for 5 d before harvesting. (b) Stat3, Myod1 and Socs3 mRNA levels in satellite cells isolated from the uninjured skeletal muscle of Tmx-treated Pax7-CreER; Stat3^flox/flox and Pax7-CreER; Stat3^+/+ mice after culture in growth medium for 96 h (n = 3). (c) Representative images of uninjured skeletal muscle of Tmx-treated Pax7-CreER; Stat3^flox/flox and Pax7-CreER; Stat3^+/+ mice (green, Pax7; red, EdU; white, laminin; blue, nuclei). Scale bar, 20 µm. (d) Schematic representation of Tmx or vehicle treatment and skeletal muscle injury in male and female 2-month-old Pax7CreER; Stat3^flox/flox mice. (e) Left, representative images of Pax7⁺ satellite cells within the muscles of Tmx- or vehicle (Ctrl)-treated Pax7-CreER; Stat3^flox/flox mice 5 d after injury (green, Pax7; white, laminin; blue, nuclei). Scale bar, 50 µm. Right, quantification of Pax7⁺ satellite cell numbers in regenerating muscles at 5 and 25 d after injury (n = 3). (f) Left, representative images of H&E staining of the muscles of Tmx- or vehicle (Ctrl)-treated Pax7-CreER; Stat3^flox/flox mice 25 d after injury. Scale bar, 50 µm. Right, quantification of average myofiber cross-sectional area 25 d after injury (n = 3). All data are represented as the average ± s.e.m. Student’s t test was used for all statistical analyses (***P < 0.001, **P < 0.01, *P < 0.05).

Figure 3

Transient inhibition of Stat3 promotes satellite cell expansion and enhances skeletal muscle tissue repair. (a) Schematic representation of Stat3 inhibitor (Stat3i) treatment and skeletal muscle injury in 2- and 24-month-old wild-type mice. (b) Left, representative images of EdU⁺ satellite cells isolated from wild-type mice treated with Stat3 inhibitor or vehicle (PBS) at 3 d after injury and cultured in growth medium for 2 h. Scale bar, 50 µm. Right, quantification of the percentage of EdU⁺ satellite cells (n = 3). (c) Left, representative images of H&E staining in regenerating muscles of 2- and 24-month-old mice treated with the Stat3 inhibitor or vehicle control at 5 d after injury. Scale bar, 50 µm. Right, quantification of average myofiber cross-sectional area (CSA) in regenerating muscles of 2- and 24-month-old mice 5 d after injury (n = 3). (d) Schematic representation of Stat3 inhibitor treatment in skeletal muscles of dystrophic 2-month-old mdx/mTR^G2 mice. (e) Quantification of the percentage of EdU⁺ satellite cells isolated from mice treated with the Stat3 inhibitor or vehicle control (n = 3). (f) Left, representative images of H&E staining of muscles of mdx/mTR^G2 mice treated with the Stat3 inhibitor or vehicle control at 28 d. Scale bar, 50 µm. Right, quantification of average myofiber cross-sectional area at 28 d (n = 3). All data are represented as the average ± s.e.m. Student’s t test was used for all statistical analyses (**P < 0.01, *P < 0.05).

Figure 4

The effects of STAT3 manipulation are conserved in human myoblasts. (a) STAT3 and MYOD1 mRNA levels in human myoblasts infected with shSTAT3 lentivirus or shControl (shCtrl) at 96 h after infection (n = 4). (b) Left, representative images of human myoblasts infected with shSTAT3 lentivirus or shCtrl and cultured for 96 h (blue, nuclei). Scale bar, 50 µm. Right, quantification of total human myoblast numbers 96 h after infection (n = 3). (c) Left, representative images of human myoblasts treated with the STAT3 inhibitor or vehicle control for 72 h (green, MYOD1; red, myogenin (MYOG); white, nuclei). Scale bar, 50 µm. Right, quantification of the percentage of MYOD1⁺ and MYOG⁺ human myoblasts after STAT3 inhibitor treatment (n = 4). All data are represented as the average ± s.e.m. Student’s t test was used for all statistical analyses (***P < 0.001, **P < 0.01, *P < 0.05).