Association of Interleukin-6 Signalling with the Muscle Stem Cell Response Following Muscle-Lengthening Contractions in Humans

Abstract

BackgroundThe regulation of muscle stem cells in humans in response to muscle injury remains largely undefined. Recently, interleukin-6 (IL-6) has been implicated in muscle stem cell (satellite cell)-mediated muscle hypertrophy in animals; however, the role of IL-6 in the satellite cell (SC) response following muscle-lengthening contractions in humans has not been studied.Methodology/Principal FindingsEight subjects (age 22±1 y; 79±8 kg) performed 300 maximal unilateral lengthening contractions (3.14 rad.s−1) of the knee extensors. Blood and muscle samples were collected before and at 4, 24, 72, and 120 hours post intervention. IL-6, IL-6 receptor (IL-6Rα), cyclin D1, suppressor of cytokine signling-3 (SOCS3) mRNA were measured using quantitative RT-PCR and serum IL-6 protein was measured using an ELISA kit. JAK2 and STAT3 phosphorylated and total protein was measured using western blotting techniques. Immunohistochemical analysis of muscle cross-sections was performed for the quantification of SCs (Pax7+ cells) as well as the expression of phosphorylated STAT3, IL-6, IL-6Rα, and PCNA across all time-points. The SC response, as defined by an amplification of Pax7+ cells, was rapid, increasing by 24 h and peaking 72 h following the intervention. Muscle IL-6 mRNA increased following the intervention, which correlated strongly (R2 = 0.89, p<0.002) with an increase in serum IL-6 concentration. SC IL-6Rα protein was expressed on the fiber, but was also localized to the SC, and IL-6+ SC increased rapidly following muscle-lengthening contractions and returned to basal levels by 72 h post-intervention, demonstrating an acute temporal expression of IL-6 with SC. Phosphorylated STAT3 was evident in SCs 4 h after lengthening contraction, and the downstream genes, cyclin D1 and SOCS3 were significantly elevated 24 hours after the intervention.Conclusions/SignificanceThe increased expression of STAT3 responsive genes and expression of IL-6 within SCs demonstrate that IL-6/STAT3 signaling occurred in SCs, correlating with an increase in SC proliferation, evidenced by increased Pax7+/PCNA+ cell number in the early stages of the time-course. Collectively, these data illustrate that IL-6 is an important signaling molecule associated with the SC response to acute muscle-lengthening contractions in humans.

Elevated SOCS3 and altered IL-6 signaling is associated with age-related human muscle stem cell dysfunction

“…3C. As demonstrated previously (35), the temporal response of serum IL-6 and muscle IL-6 mRNA was very similar. To investigate if the altered IL-6 response observed systemically and at the level of the gene was also affecting the SC pool following heavy resistance exercise, we examined the SCspecific IL-6 response.…”

“…It has been shown previously that IL-6 is a key regulatory factor for SC proliferation in young men and animals (35,53,56). However, the appearance of elevated IL-6 was transient, with SOCS3 mRNA being induced rapidly (24 h) following muscle damage, likely to inhibit the STAT3 cascade and limit proliferation to allow for differentiation to begin (18,35,56). In healthy older humans, IL-6 has been shown to be chronically elevated (57), which may impair the normal IL-6 response and the sensitivity of the SCs to IL-6.…”

“…RNA was isolated from homogenized muscle samples using the TRIzol/RNeasy method (35). Briefly, ϳ25 mg of each muscle sample were homogenized in a total of 1.0 ml of TRIzol Reagent (Invitrogen) using an electric homogenizer (Pro200 BioGen).…”

“…Individual 25-l reactions were prepared in 0.2-ml Eppendorf twin.tec PCR plates (Eppendorf) and run in duplicate for each time point. Primers are listed in (Table 1) (35,36,42). Briefly, for coimmunofluorescent staining, sections were fixed with 2% paraformaldehyde (Sigma-Aldrich) for 10 min followed by several washes in PBS.…”

“…Recently, IL-6 has been investigated as a positive regulator of SC proliferation in both animals and humans through its interactions with the Janus activated kinase/signal transducer and activator of transcription (JAK/STAT) signaling cascade (35,53,56). Transient increases in local IL-6 concentrations following acute exerciseinduced myotrauma are associated with SC proliferation and increased transcription of cell-cycle regulatory genes as well as increased pSTAT3 and cMyc localized to the SCs during the proliferative phase of the myogenic response (35,56). Furthermore, the genetic loss of IL-6 in a murine model was shown to abolish SC proliferation and inhibit muscle hypertrophy (53).…”