Genetic ablation of TWEAK augments regeneration and post-injury growth of skeletal muscle in mice

Abstract

Impairment in the regeneration process is a critical determinant for skeletal muscle wasting in chronic diseases and degenerative muscle disorders. Inflammatory cytokines are known to cause significant muscle wasting, however, their role in myofiber regeneration is less clear. In this study we have investigated the role of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in skeletal muscle regeneration in vivo. Our results show that expression levels of TWEAK and its receptor Fn14 are significantly increased in skeletal muscles of mice after injury. Genetic deletion of TWEAK increased the fiber cross-sectional area and levels of embryonic isoform of myosin heavy chain in regenerating tibial anterior muscle. Conversely, muscle-specific transgenic overexpression of TWEAK reduced the fiber cross-sectional area and levels of the embryonic myosin heavy chain in regenerating muscle. TWEAK induced the expression of several inflammatory molecules and increased interstitial fibrosis in regenerating muscle. Genetic ablation of TWEAK suppressed, whereas overexpression of TWEAK increased, the activation of nuclear factor-kappa B without affecting the activation of Akt or p38 kinase in regenerating myofibers. Primary myoblasts from TWEAK-null mice showed enhanced differentiation in vitro, whereas myoblasts from TWEAK-Tg mice showed reduced differentiation compared with wild-type mice. Collectively, our study suggests that TWEAK negatively regulates muscle regeneration and that TWEAK is a potential therapeutic target to enhance skeletal muscle regeneration in vivo.

Figure 1

Expression of TWEAK and Fn14 in regenerating skeletal muscle. A: TA muscle of mice was injected with cardiotoxin (CTX); five days later, mRNA levels of TWEAK and Fn14 were measured by QRT-PCR. Data show a significant increase in the transcript levels of TWEAK and Fn14 in CTX-injected TA muscle (n = 4 in each group). B: Quantification using enzyme-linked immunosorbent assay showed a significant increase in protein levels of TWEAK in CTX-injected TA muscle compared with contralateral control TA muscle (n = 4). C: Immunoblotting with anti-Fn14 showed increased protein levels of Fn14 in CTX-injected TA muscle compared with contralateral control TA muscle injected with PBS alone. *P < 0.05, values significantly different from contralateral PBS-injected TA muscle.

Figure 2

Histological analyses of regenerating TA muscle of TWEAK-KO and TWEAK-Tg mice. TA muscle of TWEAK-KO and TWEAK-Tg mice and the corresponding control mice were given intramuscular injections of CTX followed by isolation of TA muscle at different time points and H&E staining. Representative images of H&E-stained sections demonstrate increased fiber diameter in TA muscle of TWEAK-KO and its reduction in TWEAK-Tg mice compared with the corresponding control mice (n = 6 in each group at indicated time point). Scale bar = 50 μm.

Figure 3

Quantification of fiber cross section area (CSA) in regenerating TA muscle of TWEAK-KO and TWEAK-Tg mice. H&E-stained TA muscle sections from TWEAK-KO and wild-type and TWEAK-Tg and littermate wild-type mice 10 days post-CTX injections were used to calculate the fiber CSA. A: Data show that fiber CSA was increased in TWEAK-KO (n = 6) compared with wild-type mice (n = 6). B: A reduction in fiber CSA was noticed in TWEAK-Tg mice (n = 6) compared with littermate wild-type mice (n = 6). C: Analysis of H&E-stained TA muscle sections 10 days post-CTX injection showed significantly increased number of myofibers containing more than two central nuclei in TWEAK-KO mice compared with wild-type mice. This count was significantly reduced in TWEAK-Tg mice compared with littermate wild-type mice. n = 6 in each group. *P < 0.05, values significantly different from wild-type mice.

Figure 4

Expression of embryonic myosin heavy chain (E-MyHC) and myogenin in regenerating TA muscle of TWEAK-KO and TWEAK-Tg mice. A: TA muscle of TWEAK-KO, TWEAK-Tg, and the corresponding control mice were given an intramuscular injection of CTX. After 5 days, muscle sections were prepared and immunostained with anti-E-MyHC and average fiber cross-sectional area of E-MyHC-positive fibers was measured. *P < 0.05, values significantly different from wild-type mice. B: Representative immunoblots show protein levels of E-MyHC in CTX-injected TA muscle of TWEAK-KO and its reduced levels in TWEAK-Tg as compared with the corresponding control mice. C: Quantification of Western blot (normalized for tubulin levels) shows a significant increase in protein levels of E-MyHC in CTX-injected TA muscle of TWEAK-KO compared with wild-type mice. The levels of E-MyHC were significantly reduced in CTX-injected TA muscle of TWEAK-Tg compared with littermate wild-type mice. n = 4 in each group. D: Transcript levels of myogenin in CTX-injected TA muscle of TWEAK-KO and TWEAK-Tg mice. n = 3 in each group. *P < 0.01, values significantly different from wild-type mice.

Figure 5

Expression of pro-inflammatory molecules in regenerating TA muscle of TWEAK-Tg and TWEAK-KO mice. A: Fold difference in mRNA levels of TNF-α, IL-1β, MMP-9, IL-6, and CCL-2 in TA muscle of TWEAK-KO and wild-type mice measured five days after CTX injection by QRT-PCR. n = 4 in each group. B: Expression levels of TNF-α, IL-1β, MMP-9, IL-6, and CCL-2 in CTX-injected TA muscle of TWEAK-Tg and littermate wild-type five days post CTX injections. n = 4 in each group. *P < 0.05, values significantly different form wild-type mice. WT, wild-type.

Figure 6

Fibrosis in regenerating TA muscle of TWEAK-KO and TWEAK-Tg mice. A: Muscle sections prepared from TA muscle ten days post CTX injection were stained with Sirius red dye. Representative photomicrographs show that the level of fibrosis is reduced in regenerating TA muscle of TWEAK-KO mice and increased in TWEAK-Tg mice compared with CTX-injected TA muscle of the corresponding wild-type mice. B: Transcript levels of collagen I measured by QRT-PCR in TA muscle ten days post CTX injection were comparable in wild-type and TWEAK-KO mice but were significantly increased in TWEAK-Tg mice compared with littermate wild-type mice. *P < 0.05, values significantly different form wild-type mice. WT, wild-type.

Figure 7

Activation of Akt, p38 MAPK, and NF-κB in regenerating TA muscle of control, TWEAK-Tg, and TWEAK-KO mice. A: Representative immunoblots show no apparent differences in levels of phospho-Akt and phospho p38MAPK in regenerating TA muscle of control, TWEAK-Tg, and TWEAK-KO mice. B: DNA-binding activation of NF-κB measured by EMSA was found to be increased in regenerating TA muscle of TWEAK-Tg mice and reduced in TWEAK-KO mice as compared with control mice.

Figure 8

Differentiation of satellite cells prepared from control, TWEAK-Tg, and TWEAK-KO mice. A: Representative MF-20 antibody (for MyHC) stained photomicrographs of primary myoblast cultures after incubation in differentiation medium for 72 hours. Arrow points to a typical and representative myotube in each culture. Analyses of these cultures showed that the (B) fusion index and (C) myotube diameter were significantly reduced in TWEAK-Tg and significantly increased in TWEAK-KO, as compared with wild-type mice. *P < 0.05, values significantly different from that of wild-type mice. D: DNA-binding activity of NF-κB transcription factor was considerably higher in TWEAK-Tg cultures compared with control mice. EMSA, electrophoretic mobility shift assay.