IL-6 Deficiency Attenuates Skeletal Muscle Atrophy by Inhibiting Mitochondrial ROS Production through the Upregulation of PGC-1 α in Septic Mice

Abstract

Current evidences indicate that both inflammation and oxidative stress contribute to the pathogenesis of sepsis-associated skeletal muscle atrophy. However, the interaction between inflammation and oxidative stress has not been completely understood in sepsis-associated skeletal muscle atrophy. Here in the present study, a murine model of sepsis has been established by cecal ligation and puncture (CLP) with wild-type and interleukin- (IL-) 6 knockout (KO) mice. Our results suggested that IL-6 KO largely attenuated skeletal muscle atrophy as reflected by reduced protein degradation, increased cross-sectional area (CSA) of myofibers, and improved muscle contractile function (all P < 0.05). In addition, we observed that IL-6 KO promoted the expression of peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α) and inhibited CLP-induced mitochondrial reactive oxygen species (ROS) production in skeletal muscles (all P < 0.05). However, the knockdown of PGC-1α abolished the protective effects of IL-6 KO in CLP-induced skeletal muscle atrophy and reversed the changes in mitochondrial ROS production (all P < 0.05). Ex vivo experiments found that exogenous IL-6 inhibited PGC-1α expression, promoted mitochondrial ROS production, and induced proteolysis in C2C12 cells (all P < 0.05). Together, these results suggested that IL-6 deficiency attenuated skeletal muscle atrophy by inhibiting mitochondrial ROS production through the upregulation of PGC-1α expression in septic mice.

Figure 1

IL-6 KO reduced IL-6 expression in plasma and EDL. (a)–(c) ELISA assays were performed to detect the protein expression of IL-1, TNF-, and IL-6 in plasma of mice; (d)–(f) ELISA assays were performed to detect the protein expression of IL-1, TNF-, and IL-6 in EDL of mice; CLP: cecal ligation and puncture; EDL: extensor digitorium longus. ^∗P < 0.001 (ANOVA followed by unpaired Student t-test, n = 6).

Figure 2

IL-6 KO attenuated CLP-induced skeletal muscle atrophy and weakness. (a) Immunoblots for Atrogin-1 and MuRF-1 in wild-type mice and IL-6 KO mice received sham operation or CLP; immunofluorescence staining (b) was performed to measure the CSA (c) of myofibers (green: anti-laminin was used to outline the myofibers); the maximum tetanic forces (d) and the force-frequency curves (e) were generated to evaluate muscle force-generating capacity. CLP: cecal ligation and puncture; CSA: cross-sectional area. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 (ANOVA followed by unpaired Student t-test, n = 6).

Figure 3

IL-6 KO inhibited mitochondrial ROS production and upregulated PGC-1ɑ expression in septic mice. (a) Mitochondrial ROS production in EDL was measured at the end of experiment; (b) correlation analysis revealed a positive association between IL-6 mRNA expression and mitochondrial ROS production; RT-PCR (c) and western blots (d) were performed to measure the mRNA and protein expressions of PGC-1ɑ in EDL; (e) correlation analysis revealed a negative association between IL-6 mRNA expression and PGC-1ɑ mRNA expression; (f) mitochondria-targeted antioxidant MitoTempol (MitoT) abolished CLP-induced mitochondrial ROS production; (g) MitoTempol treatment increased CSA of myofibers of septic mice; (h) MitoTempol treatment improved muscle force-generating capacity of mice subjected to CLP. CLP: cecal ligation and puncture. ^∗P < 0.05, ^∗∗P < 0.01; ^∗∗∗P < 0.001 (ANOVA followed by unpaired Student t-test, n = 6).

Figure 4

The knockdown of PGC-1ɑ abolished the protective effects of IL-6 KO in CLP-induced skeletal muscle atrophy. RT-PCR (a) and western-blots (b) were performed to determine the mRNA and protein expressions of PGC-1ɑ in skeletal muscle of septic mice after PGC-1ɑ knockdown; (c) the immunoblots for the protein expressions of atrophy-related genes Atrogin-1 and MuRF-1; (d) mitochondrial ROS production was measured after PGC-1ɑ knockdown in septic mice; the cross-sectional area (CSA) of myofibers (e) and the maximum tetanic forces (f) were measured to evaluate muscle atrophy and contractile capacity. CLP: cecal ligation and puncture; CSA: cross-sectional area. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 (ANOVA followed by unpaired Student t-test, n = 6).

Figure 5

Exogenous IL-6 induced mitochondrial ROS-mediated proteolysis by inhibiting PGC-1ɑ expression ex vivo. RT-PCR (a) and western blots (b) were performed to determine the mRNA and the protein expressions of PGC-1ɑ, respectively; (c) changes in mitochondrial ROS production after IL-6 treatments; (d) correlation analysis revealed a negative associated between PGC-1ɑ expression and mitochondrial ROS production; (e) western blots for the protein expressions of atrophy-related genes Atrogin-1 and MuRF-1 in C2C12 cells after IL-6 treatment; ^∗P < 0.001 (ANOVA followed by unpaired Student t-test, n = 6).