Ashwani Mittal

The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice

The TNF-related cytokine TWEAK promotes skeletal muscle atrophy that is associated with classical disuse syndromes.

Matrix metalloproteinase-9 inhibition ameliorates pathogenesis and improves skeletal muscle regeneration in muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disorder of skeletal muscle caused by mutation in dystrophin gene. Although the degradation of skeletal muscle extracellular matrix, inflammation and fibrosis are the common pathological features in DMD, the underlying mechanisms remain poorly understood. In this study, we have investigated the role and the mechanisms by which increased levels of matrix metalloproteinase-9 (MMP-9) protein causes myopathy in dystrophin-deficient mdx mice. The levels of MMP-9 but not tissue inhibitor of MMPs were drastically increased in skeletal muscle of mdx mice. Besides skeletal muscle, infiltrating macrophages were found to contribute significantly to the elevated levels of MMP-9 in dystrophic muscle. In vivo administration of a nuclear factor-kappa B inhibitory peptide, NBD, blocked the expression of MMP-9 in dystrophic muscle of mdx mice. Deletion of Mmp9 gene in mdx mice improved skeletal muscle structure and functions and reduced muscle injury, inflammation and fiber necrosis. Inhibition of MMP-9 increased the levels of cytoskeletal protein beta-dystroglycan and neural nitric oxide synthase and reduced the amounts of caveolin-3 and transforming growth factor-beta in myofibers of mdx mice. Genetic ablation of MMP-9 significantly augmented the skeletal muscle regeneration in mdx mice. Finally, pharmacological inhibition of MMP-9 activity also ameliorated skeletal muscle pathogenesis and enhanced myofiber regeneration in mdx mice. Collectively, our study suggests that the increased production of MMP-9 exacerbates dystrophinopathy and MMP-9 represents as one of the most promising therapeutic targets for the prevention of disease progression in DMD.

Tumor Necrosis Factor-related Weak Inducer of Apoptosis Augments Matrix Metalloproteinase 9 (MMP-9) Production in Skeletal Muscle through the Activation of Nuclear Factor-κB-inducing Kinase and p38 Mitogen-activated Protein Kinase A POTENTIAL ROLE OF MMP-9 IN MYOPATHY

Destruction of skeletal muscle extracellular matrix is an important pathological consequence of many diseases involving muscle wasting. However, the underlying mechanisms leading to extracellular matrix breakdown in skeletal muscle tissues remain unknown. Using a microarray approach, we investigated the effect of tumor necrosis factor-related weak inducer of apoptosis (TWEAK), a recently identified muscle-wasting cytokine, on the expression of extracellular proteases in skeletal muscle. Among several other matrix metalloproteinases (MMPs), we found that the expression of MMP-9, a type IV collagenase, was drastically increased in myotubes in response to TWEAK. The level of MMP-9 was also higher in myofibers of TWEAK transgenic mice. TWEAK increased the activation of both classical and alternative nuclear factor-κB (NF-κB) signaling pathways. Inhibition of NF-κB activity blocked the TWEAK-induced production of MMP-9 in myotubes. TWEAK also increased the activation of AP-1, and its inhibition attenuated the TWEAK-induced MMP-9 production. Overexpression of a kinase-dead mutant of NF-κB-inducing kinase or IκB kinase-β but not IκB kinase-α significantly inhibited the TWEAK-induced activation of MMP-9 promoter. The activation of MMP-9 also involved upstream recruitment of TRAF2 and cIAP2 proteins. TWEAK increased the activity of ERK1/2, JNK1, and p38 MAPK. However, the inhibition of only p38 MAPK blocked the TWEAK-induced expression of MMP-9 in myotubes. Furthermore the loss of body and skeletal muscle weights, inflammation, fiber necrosis, and degradation of basement membrane around muscle fibers were significantly attenuated in Mmp9 knock-out mice on chronic administration of TWEAK protein. The study unveils a novel mechanism of skeletal muscle tissue destruction in pathological conditions.

TNF-Like Weak Inducer of Apoptosis (TWEAK) Activates Proinflammatory Signaling Pathways and Gene Expression through the Activation of TGF-β-Activated Kinase 1

TWEAK, TNF-like weak inducer of apoptosis, is a relatively recently identified proinflammatory cytokine that functions through binding to Fn14 receptor in target cells. Although TWEAK has been shown to modulate several biological responses, the TWEAK-induced signaling pathways remain poorly understood. In this study, we tested the hypothesis that TAK1 (TGF-β-activated kinase 1) is involved in TWEAK-induced activation of NF-κB and MAPK and expression of proinflammatory protein. TWEAK increased the phosphorylation and kinase activity of TAK1 in cultured myoblast and fibroblast cells. The activation of NF-κB was significantly inhibited in TAK1-deficient (TAK1−/−) mouse embryonic fibroblasts (MEF) compared with wild-type MEF. Deficiency of TAK1 also inhibited the TWEAK-induced activation of IκB kinase and the phosphorylation and degradation of IκBα protein. However, there was no difference in the levels of p100 protein in TWEAK-treated wild-type and TAK1−/− MEF. Furthermore, TWEAK-induced transcriptional activation of NF-κB was significantly reduced in TAK1−/− MEF and in C2C12 myoblasts transfected with a dominant-negative TAK1 or TAK1 short interfering RNA. TAK1 was also required for the activation of AP-1 in response to TWEAK. Activation of JNK1 and p38 MAPK, but not ERK1/2 or Akt kinase, was significantly inhibited in TAK1−/− MEF compared with wild-type MEF upon treatment with TWEAK. TWEAK-induced expression of proinflammatory genes such as MMP-9, CCL-2, and VCAM-1 was also reduced in TAK1−/− MEF compared with wild-type MEF. Furthermore, the activation of NF-κB and the expression of MMP-9 in response to TWEAK involved the upstream activation of Akt kinase. Collectively, our study demonstrates that TAK1 and Akt are the important components of TWEAK-induced proinflammatory signaling and gene expression.

Genetic Ablation of TWEAK Augments Regeneration and Post-Injury Growth of Skeletal Muscle in Mice

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.

TWEAK causes myotube atrophy through coordinated activation of ubiquitin-proteasome system, autophagy, and caspases†

Proinflammatory cytokine TWEAK has now emerged as a key mediator of skeletal muscle‐wasting in many catabolic conditions. However, the mechanisms by which TWEAK induces muscle proteolysis remain poorly understood. Here, we have investigated the role of ubiquitin‐proteasome system, autophagy, and caspases in TWEAK‐induced muscle wasting. Addition of TWEAK to C2C12 myotubes stimulated the ubiquitination of myosin heavy chain (MyHC) and augmented the expression of E3 ubiquitin ligase MuRF1. Pretreatment of myotubes with proteasome inhibitors MG132 or lactacystin or knockdown of MuRF1 by RNAi blocked the TWEAK‐induced degradation of MyHC and myotube atrophy. TWEAK increased the expression of several autophagy‐related molecules. Moreover, the inhibitors of autophagy improved the levels of MyHC in TWEAK‐treated myotubes. TWEAK also increased activity of caspases in C2C12 myotubes. Pan‐caspase or caspase 3 inhibitory peptide inhibited the TWEAK‐induced loss of MyHC and myotube diameter. Our study demonstrates that nuclear factor‐kappa B (NF‐κB) transcription factor is essential for TWEAK‐induced expression of MuRF1 and Beclin1. Furthermore, our results suggest that caspases contribute, at least in part, to the activation of NF‐κB in response to TWEAK treatment. Collectively, the present study provides novel insight into the mechanisms of action of TWEAK in skeletal muscle. J. Cell. Physiol. 227: 1042–1051, 2012.

α1-Adrenergic receptor subtype function in fetal and adult cerebral arteries

In the developing fetus, cerebral artery (CA) contractility demonstrates significant functional differences from that of the adult. This may be a consequence of differential activities of alpha(1)-adrenergic receptor (alpha(1)-AR) subtypes. Thus we tested the hypothesis that maturational differences in adrenergic-mediated CA contractility are, in part, a consequence of differential expression and/or activities of alpha(1)-AR subtypes. In CA from fetal ( approximately 140 days) and nonpregnant adult sheep, we used wire myography and imaging, with simultaneous measurement of tension and intracellular Ca(2+) concentration ([Ca(2+)](i)), radioimmunoassay, and Western immunoblots to examine phenylephrine (Phe)-induced contractile responses. The alpha(1A)-AR antagonists (5-MU and WB-4101) completely inhibited Phe-induced contraction in adult but not fetal CA; however, [Ca(2+)](i) increase was reduced significantly in both age groups. The alpha(1D)-AR antagonist (BMY-7378) blocked both Phe-induced contractions and Ca(2+) responses to a significantly greater extent in adult compared with fetal CA. In both age groups, inhibition of alpha(1A)-AR and alpha(1B)-AR, but not alpha(1D)-AR, significantly reduced inositol 1,4,5-trisphosphate responses to Phe. Western immunoblots demonstrated that the alpha(1)-AR subtype expression was only approximately 20% in fetal CA compared with the adult. Moreover, in fetal CA, the alpha(1D)-AR was expressed significantly greater than the other two subtypes. Also, in fetal but not adult CA, Phe induced a significant increase in activated ERK1/2; this increase in phosphorylated ERK was blocked by alpha(1B)-AR (CEC) and alpha(1D)-AR (BMY-7378) inhibitors, but not by alpha(1A)-AR inhibitors (5-MU or WB-4101). In conclusion, in the fetal CA, alpha(1B)-AR and alpha(1D)-AR subtypes play a key role in contractile response as well as in ERK activation. We speculate that in fetal CA alpha(1B)-AR and alpha(1D)-AR subtypes may be a critical factor associated with cerebrovascular growth and function.